CHAPTER ndash 4
Inclusion complexation of procainamide hydrochloride and
propafenone hydrochloride with α- and β-cyclodextrinslowast
In this chapter the encapsulation behaviour of two potential cardiovascular drugs
namely procainamide hydrochloride (PCA) and propafenone hydrochloride (PFO) with
α-CD and β-CD nanocavities has been studied by absorption fluorescence and time-
resolved fluorescence techniques The solid inclusion complexes were prepared and
characterized by SEM TEM FTIR DSC XRD and 1H NMR techniques Further the
geometries of these inclusion complexes were proposed with the help of computational
calculations (PM3 method) The chemical structures of PCA and PFO are given below
Chemical structures of (a) PCA and (b) PFO
41 Absorption and fluorescence measurements
In general inclusion complex formation with CD often causes change in the
electronic spectra of the guest molecules [87-93] In this regard the absorption and
fluorescence spectra of PCA and PFO drugs were recorded in various concentrations of
CDs at pH ~7 and the spectral data are given in Table 41 Fig 41 displays the
absorption spectra of the above drug molecules in aqueous solution as a function of
α-CD and β-CD concentrations In water PCA exhibits a strong absorption maximum at
278 nm with a slight shoulder (at 221 nm) whereas PFO shows two absorption maxima
at 303 and 249 nm Addition of various concentrations of β-CD into drug solution caused
blue shift about ~4 nm in the absorption maxima of PCA with gradual increase in the
molar extinction coefficient whereas in PFO the absorbance decreased at the same
wavelength However no significant spectral shift was observed in the presence of α-CD
except an increase in absorbance The above behaviour may be attributed to the
lowast Spectrochimica Acta Part A 115 (2013) 559
(a) (b) HH22NN
NNHH
OONN
OOOO
HHOO HHNN
93
enhanced dissolution of the drugs through hydrophobic interactions between the drugs
and the cavity of CDs These results indicate that both drugs are entrapped in the CD
cavity to form stable inclusion complexes Further the blue shift in β-CD solutions
reveals the less polar environment experienced by the drug molecule which is offered by
β-CD cavity rather than that of α-CD
Fig 42 shows the typical fluorescence spectra of PCA and PFO in different
concentrations of α-CD and β-CD The effect produced by the addition of CD on the
emission spectra of drugs in aqueous solution is more pronounced than the
corresponding effect on the absorption spectra The changes in the emission intensities of
drugs with increasing concentrations of CDs are shown in the inset of Fig 42 In
aqueous solution a single fluorescence emission is observed for both PCA (at 357 nm)
and PFO (at 420 nm) when excited at ~280 nm and 305 nm respectively The
fluorescence characteristics of the drug molecules undergo drastic changes in the
presence of CD With the addition of different concentrations of CD lead to a
considerable enhancement in the emission intensity of PCA especially for β-CD
Moreover the presence of β-CD concentrations produced a slight blue shift (~5 nm) in
PCA In PFO upon the addition of β-CD the fluorescence maxima was significantly red
shifted from 420 nm to 440 nm with a concomitant increase in the fluorescence intensity
whereas no significant emission shift was observed in the α-CD (Fig 42) These
phenomena suggest that the drug molecule is moving more deeply into the nonpolar CD
cavity during the formation of stable inclusion complexes An increase in the
fluorescence intensity after the formation of inclusion complex was also observed in
earlier studies [87-93] Since the CD cavity provided an apolar environment for the
guest and the movement of guest in the cavity was largely confined Further the
enhanced rigidity of the drugs resulted in an increase in their fluorescence quantum
yield
The binding constant (K) and stoichiometric ratio of the inclusion complexes of
drugs with CDs can be determined using the Benesi-Hildebrand (B-H) relation [120]
The K values were obtained from the slope of the linear plots Figs 43 and 44 depict
B-H double-reciprocal plot of 1AndashA0 and 1IndashI0 versus 1[CD] and 1[CD]2 In the case
of PCA according to eqn (2) the plots of 1AndashA0 and 1IndashI0 versus 1[CD] give upward
or downward curves as shown in Fig 43 While the plot of 1AndashA0 or 1IndashI0 versus
1[CD]2 shows a linear correlation However in PFO a good-linear relationship is
obtained when 1AndashA0 andor 1IndashI0 is plotted against 1[CD] (Fig 43) indicating that
the stoichiometry of PFOCD inclusion complex is 11 Further the non-linearity of
94
Table 41
95
Fig 41
96
Fig 42
97
Fig 43
98
Fig 44
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
93
enhanced dissolution of the drugs through hydrophobic interactions between the drugs
and the cavity of CDs These results indicate that both drugs are entrapped in the CD
cavity to form stable inclusion complexes Further the blue shift in β-CD solutions
reveals the less polar environment experienced by the drug molecule which is offered by
β-CD cavity rather than that of α-CD
Fig 42 shows the typical fluorescence spectra of PCA and PFO in different
concentrations of α-CD and β-CD The effect produced by the addition of CD on the
emission spectra of drugs in aqueous solution is more pronounced than the
corresponding effect on the absorption spectra The changes in the emission intensities of
drugs with increasing concentrations of CDs are shown in the inset of Fig 42 In
aqueous solution a single fluorescence emission is observed for both PCA (at 357 nm)
and PFO (at 420 nm) when excited at ~280 nm and 305 nm respectively The
fluorescence characteristics of the drug molecules undergo drastic changes in the
presence of CD With the addition of different concentrations of CD lead to a
considerable enhancement in the emission intensity of PCA especially for β-CD
Moreover the presence of β-CD concentrations produced a slight blue shift (~5 nm) in
PCA In PFO upon the addition of β-CD the fluorescence maxima was significantly red
shifted from 420 nm to 440 nm with a concomitant increase in the fluorescence intensity
whereas no significant emission shift was observed in the α-CD (Fig 42) These
phenomena suggest that the drug molecule is moving more deeply into the nonpolar CD
cavity during the formation of stable inclusion complexes An increase in the
fluorescence intensity after the formation of inclusion complex was also observed in
earlier studies [87-93] Since the CD cavity provided an apolar environment for the
guest and the movement of guest in the cavity was largely confined Further the
enhanced rigidity of the drugs resulted in an increase in their fluorescence quantum
yield
The binding constant (K) and stoichiometric ratio of the inclusion complexes of
drugs with CDs can be determined using the Benesi-Hildebrand (B-H) relation [120]
The K values were obtained from the slope of the linear plots Figs 43 and 44 depict
B-H double-reciprocal plot of 1AndashA0 and 1IndashI0 versus 1[CD] and 1[CD]2 In the case
of PCA according to eqn (2) the plots of 1AndashA0 and 1IndashI0 versus 1[CD] give upward
or downward curves as shown in Fig 43 While the plot of 1AndashA0 or 1IndashI0 versus
1[CD]2 shows a linear correlation However in PFO a good-linear relationship is
obtained when 1AndashA0 andor 1IndashI0 is plotted against 1[CD] (Fig 43) indicating that
the stoichiometry of PFOCD inclusion complex is 11 Further the non-linearity of
94
Table 41
95
Fig 41
96
Fig 42
97
Fig 43
98
Fig 44
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
94
Table 41
95
Fig 41
96
Fig 42
97
Fig 43
98
Fig 44
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
95
Fig 41
96
Fig 42
97
Fig 43
98
Fig 44
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
96
Fig 42
97
Fig 43
98
Fig 44
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
97
Fig 43
98
Fig 44
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
98
Fig 44
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
99
the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of
12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion
complexes are formed between PCA and CDs whereas PFO formed 11 inclusion
complexes with CDs The K values for the inclusion complexes are given in Table 41
The quantitative comparison of binding constants of the two complexes suggests that
β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD
cavity Further it has been observed that the binding constants for 12 PCACD inclusion
systems are almost similar to the previously studied systems For example the binding
constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion
system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12
inclusion system [87]
The thermodynamic parameter free energy changes (∆G) for the binding of
guests within α-CD and β-CD were determined The negative free energy change values
in Table 41 indicate that the binding process is spontaneous and thermodynamically
favoured in the experimental temperature range (303 K) The negative values of ∆G
arose from the van der Waals interaction and the steric barrier caused by molecular
geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest
molecule
42 Effect of solvents
In order to understand the polarity around the drugs the absorption and
fluorescence spectra of PCA and PFO have been recorded in various solvents of different
polarities and hydrogen bonding abilities The absorption and fluorescence spectral
maxima in different solvents are compiled in Table 42 It can be observed from the
Table 42 that PCA shows much more interesting spectral features in different solvents
of various polarities than that of PFO In all solvents the absorption spectra of PCA are
much less structured with a slight shoulder around ~220 nm while the absorption spectra
of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively
Data in Table 42 clearly indicate that the absorption spectra of PCA are red
shifted from cyclohexane to methanol but when compared to methanol blue shift is
observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating
that the observed absorption band corresponds to allowed πrarrπlowast transitions of the
benzene ring with a considerable charge transfer (CT) character Such kind of CT
process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide
group (COndashNH) which is characterized by a high electron accepting character This can
be explained by analyzing the atomic orbital compositions of frontier molecular orbitals
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
100
of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were
performed at PM3 level of theory In the HOMO the charge density is mainly
accumulated on the benzene ring and amino group (Fig 45) However in the case of
LUMO more charge density shifts to the benzene ring and carbonyl group This picture
clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2
group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the
carbonyl group is hidden by an extended π conjugation of the phenyl ring to the
neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red
shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and
benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs
~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which
indicates an apparent perturbation effect of the amino group on the energy levels of the
parent benzamide molecule Further the spectral shifts observed in the absorption
spectrum of the drug molecule in protic and aprotic solvents are consistent with the
characteristic behaviour of aromatic molecules containing the amino group as an
auxochrome [90]
Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts
(cm-1) of PCA and PFO in different solvents
Solvents PCA PFO
λabs log ε λflu Stokes shifts λabs log ε λflu
Stokes shifts
Cyclohexane 280 223
sat 303 2660 300 249
sat 355s 336
5142
14-Dioxane 280 223
401 333
321 4460 300 248
369 367
340 331
3832
Ethyl acetate 285 222
416 353
326 4340 301 248
372 395
353s 339
4872
Acetonitrile 286 221
431 395
344 5871 302 246
377 390
353s 340
4783
2-Propanol 295 429 350 5235 303 248
378 398
354s 339
4667
Methanol 290 421 350 5746 303 249
376 401
419 338
9228
Water (pH ~65)
278 221s
408 303
357 7857 304 249
344 380
420 9176
sat - saturated
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
101
Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations
The fluorescence emission spectra of PCA and PFO in various solvents are
shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is
more pronounced than that on the corresponding effect on the absorption spectra which
reveals the excited state properties of the above drugs differ much from those in the
ground state PCA exhibits a single emission in all of the solvents whereas PFO gives
dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted
with increasing the polarity of solvents from cyclohexane to water The shift in the
emission maxima over absorption maxima indicates that the emitting state of the drug is
more polar than the ground state In PFO both SW and LW fluorescence bands are
affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are
largely red shifted and the half width of the LW fluorescence is significantly increased
(b) (a) H
OM
O
LU
MO
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
102
As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in
the present work can also be explained as hydrogen bond formation between the protic
solvents and electron withdrawing carbonyl group facilitates the formation of the
intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen
bonding seems to make the migration of electron density from benzene ring to the
electron withdrawing group more facile Further if the LW maximum is due to ICT this
should be more red shifted in protic solvents because ICT is more pronounced in protic
solvents than non-polar and aprotic solvents The spectral changes revealed that the LW
emission of PFO appeared as a result of ICT [160 161] which is more pronounced in
protic solvents than in other solvents The large Stokes shift indicates the strong changes
in the geometry of above molecules in the excited state [160 161] Moreover the
decreased emission intensity of PCA in water is likely due to the radiationless decay of
the probe molecule because of hydrogen bonding
Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
700
350
0 290 370 450
1
2
3 4
5
6
PCA λexci = 280 nm
Fluo
resc
ence
inte
nsity
(au
)
Wavelength (nm)
400
200
0 315 4075 500
1
2
3 4
5
PFO λexci = 305 nm
6
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
103
43 Time-resolved fluorescence studies
In order to confirm the interpretation of steady-state fluorescence spectroscopic
measurement data time-resolved fluorescence spectra and fluorescence decay times of
PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very
sensitive indicators for exploring the local environment around the excited state
fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and
CD environments are found to be quite different The typical time-resolved fluorescence
decay profile in different media is presented in Fig 47 and the average lifetimes are
given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly
affected by increasing the concentration of CD The results were judged by the statistical
fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission
wavelength was 355 nm Single exponential decay was observed for PCA in aqueous
solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve
becomes biexponential This indicates that PCA is encapsulated within the hydrophobic
nanocavity of CDs The life time of the inclusion complexes are higher than free drug
molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the
complexation ability of β-CD is higher in other words β-CD has given the privileged
encapsulation The amplitude of the complexed component also increased due to
increase in the complex formation and that of the decreased free species The
enhancement of τ1 and τ2 values with increase in the CD concentration is due to the
encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD
and the nature of the process with regard to short-lived species The decay of PCA is
dependent of the CD This may be due to the vibrational restriction of PCA in the excited
state
The fluorescence decay of PFO in water obtained by monitoring the emission
wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =
343 ns This analysis supports the presence of two emitting species ie LE and ICT
species in water possessing different fluorescence lifetimes However in the presence of
CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in
α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A
consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches
our interpretation of the steady-state fluorescence data indicating the formation of
inclusion complex between PFO and CDs The enhanced lifetimes associated with the
concentration of CD leads to the restriction of rotational degrees of freedom with
consequent impact on depletion of non-radiative decay channels Further when compared
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
104
to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows
the stronger hydrophobic interaction between PFO and β-CD
Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions
Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)
Drugs Medium Lifetime (ns) Pre exponential
factor ltτgt τ1 τ2 τ3 a1 a2 a3
PCA λemis = 355 nm
Water 056 041
α-CD 017 192 038 013 156
β-CD 021 386 032 007 313
PFO λemis = 420 nm
Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486
(a)
(b)
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
105
44 Molecular modeling studies
To scrutinize further the mechanism of inclusion complexation between the drugs
and CDs semiempirical quantum mechanical calculations at PM3 level of theory were
performed for the most favourable 11 inclusion mode This method is a very useful tool
for investigating the CD inclusion complexes in order to calculate the binding energy as
well as the geometrical structure of the inclusion complexes It is already proved that this
method is more reproducible when compared with the experimental data [162]
PM3 calculations for the inclusion process of drugs into the CD cavities were
carried out from the wider rim of the cavity because generally the approach of the guest
molecule to the CD cavity is more favourable for the wider rim side In addition when the
guest molecule is inserted into the cavity of CD the guest may rotate much more freely than
that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is
inserted from the wider rim of the CD cavity The energetically most favourable complex
formation strongly depends on the size and shape of the host and guest molecules From
PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately
~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular
dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit
entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped
within the hydrophobic cavity of CD So the calculations were carried out by inserting the
aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in
11 stoichiometric ratio followed by energy minimization But there was a failure in an
attempt to optimize 12 inclusion complexes of PCACD Hence only the energy
minimized structure of 12 PCACD complexes is given Upon the energy minimization it
was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host
molecules adjust themselves to give the most stable complex with least energy The 11
inclusion complex structure leading to minimum heat of formation shows the aniline moiety
or phenyl ring along with carbonyl group is encapsulated inside the CD cavity
The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD
PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for
12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in
Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the
hydrophobic CD cavity from the wider side and the binding geometries of both complexes
are almost similar Most prominently the simulation results are in good agreement with
the experimental data When compared to β-CD α-CD is closely associated with the drug
molecules consistent with the reduced diameter of the cavity
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
106
Figs 48
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
107
Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method
Fig 49 shows the structures of inclusion complexes of PCACDs in which both the
amino and methyl group protons along with phenyl ring are positioned nearly at narrow
rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and
methyl group protons of the drug in the process of complexation is huge compared with
other protons Further we could observe in the structures of 12 complexes that there are
several hydrogen bonds between the two neighbouring CD molecules (shown as green
dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes
The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that
could get involved in hydrogen bonding interactions Although the binding energies
prove the fact that the drug molecules enters with the aniline group or phenyl ring
through the wider rim of the CD cavity In order to explore the thermodynamic
parameters of the complexation process the statistical thermodynamic calculations were
carried out at 1 atm pressure and 298 K temperature The energy features
thermodynamic characteristics and electronic properties for the most stable 11 inclusion
complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated
drugs Further to quantify the interaction between drugs and CDs in the optimized
geometries we have determined the binding energy (ΔE) using Eqn (26) As expected
the binding energies of the inclusion complexes were more negative than the energies of
the isolated drugs and CDs suggest that the complexation process is more
thermodynamically favourable one The difference in binding energies (ΔE) for the most
stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal
mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD
(b)
2612 Aring
2701 Aring
2878 Aring
2804 Aring
(a)
2851 Aring
2675 Aring
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
108
The negative ΔH and ΔS values revealed that the formation of both inclusion complexes
is exothermic and enthalpy driven process On the other hand more negative ΔH values
observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion
complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG
values for these complexes are positive suggesting that the inclusion reaction was a non-
spontaneous process in vacuum phase Regrettably due to limitations in the calculating
ability of computers available with us the calculations for such a huge molecular system
could not be performed for aqueous phase However the experimental data as stated
earlier indicate that the formation of inclusion complexes in aqueous solution was
spontaneous one These variations may be due to the reason that all the calculations were
carried out in vacuum phase It is also considerable that during the inclusion
complexation in aqueous solution a number of water molecules are released from the
CD cavities
It can be observed from the complex structures that there are several
intermolecular hydrogen bonds between host and guest molecules in the structures (Figs
48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and
OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results
indicate that the intermolecular hydrogen bonding plays a significant role for the binding
energies and stabilizing the drugCD inclusion complexes
The geometric parameters such as bond distances bond angles and dihedral
angles of guest molecules before and after complexation in α-CD and β-CD were
obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion
complexes the geometry of drugs was completely changed The dihedral angles were
significantly altered indicating that guest must adapt a specific conformation to form a
more stable inclusion complex Further after the drug inclusion the geometrical
parameters of optimized structure of free CD also changed from truncated cone to an
oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen
and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that
those atoms played a crucial role in binding the drug molecules
The electronic structure of the inclusion complexes was also investigated in
addition to that of the isolated drug molecules HOMO as an electron donor represents
the capability to donate an electron and LUMO represents the capability to gain an
electron In HOMO of PCA the charge density is mainly accumulated on the benzene
ring and amino group whereas in PFO the charge density is observed on the CndashO and
CndashN bonds However in both cases of LUMO more charge density shifts to the benzene
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
109
ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during
HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC
bond of the benzene ring and carbonyl group It was also found that the charge densities
on each atom of the drugs within the CDs cavity were slightly changed Moreover the
energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical
species [145] The energy gaps for PCA and PFO as well as those for the most stable
complexes with CD were estimated by PM3 method and are shown in Table 44
Evidently the much lower energy gap which explains the fact that ultimate charge
transfer interaction occurs within the drug molecule The energy gap of drugβ-CD
complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD
complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex
is more stable than that of drugα-CD complex which is in good agreement with the
above discussed energy results In addition the EHOMOndashELUMO gap of both complexes
just fall in the range that of the PCA molecule suggesting there will be a blue shift in the
electronic spectrum of the guest molecule during binding and molecular recognition
This agrees well with the experimentally determined results
The polarity of the CD cavity is also taken into consideration for the formation of
most stable inclusion complexes The polarity of CD cavity significantly changed after
the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the
α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex
with respect to that of the dipole moment of optimized isolated hosts Although the
dipole moment of both complexes is lower than the sum of the dipole moments of
optimized isolated host and guest molecules From the above results one can conclude
that the dipole moment values show a strong correlation with the complexation
behaviour of CD
For the most stable complexes the physical parameters such as electronic
chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are
determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)
of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is
evident that the micro values of complexes differ remarkably from the isolated guest and host
molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD
therefore they are expected to act as electron acceptor in the inclusion complexation
interaction while the higher μ values of PCA indicates the drug act as electron donor In
the case of both η and ω values significant differences are observed when compared
with the isolated host (CD) molecule but it lies within the range that of the guest
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
110
Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method
Properties PCA PFO PCA α-CD
PCA β-CD
PFO α-CD
PFO β-CD
E -3427 -9462 -129371 -150783 -135294 -156835
∆E -1182 -1581 -1070 -1598
H 18209 19684 -40016 -49895 -37996 -48526
∆H -1141 -1349 -596 -1455
G 13929 14576 -53942 -65604 -51887 -63619
∆G -234 -581 1174 757
S 0143 0171 0449 0479 0456 0506
∆S -0047 -0073 -0068 -0074
EHOMO (eV) -886 -930 -829 -840 -946 -939
ELUMO (eV) -016 -024 009 004 -033 -019
EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920
Dipole moment (D) 439 168 583 995 784 1366
∆D -99 -673 -518 -031
micro -451 -477 -418 -410 -489 -479
η 435 453 422 419 456 460
S 022 022 023 024 021 021
ω 233 251 207 200 262 249
Unit =kcal mol-1 =kcalmol-Kelvin
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
111
Table 45
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
112
45 Solid inclusion complex studies
451 Scanning Electron Microscopy (SEM)
In order to inspect the influence of complexation on the surface morphology of
nanomaterials SEM photographs were taken for the isolated components ie free drugs
free CDs and their corresponding inclusion complexes The selected SEM photographs
of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A
drastic change in the morphology and shape of the inclusion complexes from the starting
materials was observed In Fig 410a typical crystal of PCA was found to be in irregular
shape with a smooth surface and obtuse corners The SEM images indicate that the PFO
particles appeared as micronised crystals of oval shaped stone-like structure in
agreement with the sharp peaks observed by powder XRD In contrast the solid
containing PCA in the complexed form with α-CD was in quite different size Further
PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO
with CDs are quite distinct from those of pure materials in which the actual
morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes
in Figs 410e and f showed agglomerated particles with irregular shape These changes
in the shape and size parameters of the solid samples are indicative of the formation of
inclusion complexes between the drugs and CDs
452 Transmission Electron Microscopy (TEM)
The morphologies of various kinds of supramolecular self-assemblies are
demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles
were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm
150 nm and 210 nm respectively In the case of PCAβ-CD complex the different
structural features are obtained by supramolecular self-assembly in highly pure water and
characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show
a well-ordered 2D microtube structures about 225-31 μm in width while the length
extends above 20 μm which is consistent with the result of the micro-Raman images
(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm
Also it can be observed in TEM images of microtubes hallow cavities with diameters
ranging approximately from 162 μm to 252 μm A proper matching between the
dimension of the guest and the β-CD cavity size is ascribed to be responsible for the
formation of extended supramolecular structure [163] Interestingly one important
observation was made from the TEM images of β-CD complex that there is a branch of
nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands
of smaller nanotubes in a stack way layer by layer The width of such rods is
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
113
Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(b)
(a)
(c)
(e)
(d)
(f)
50 microm
100 microm
10 microm
50 microm
20 microm
20 microm
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
114
Fig 411
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
115
approximately in the range of 570 nm Further the microtubes could be formed by the
secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as
guest molecule (Fig 413)
The detailed structural characteristics of such microtubes formed from CD
nanotubes have been often reported [164] Recently some researchers reported that a
linear structure was obtained from β-CD inclusion complex by encapsulating linear guest
molecule through the intra and intermolecular hydrogen bonding as well as the
intradimer π-π interactions whereas the wave-type structure was observed other than the
linear structure due to the non-linear molecular structure of the guest molecule [164] For
example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure
evidently demonstrated by Liu et al [164] Moreover they have also reported that a
wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form
with β-CD acting as the host molecule Based on the literature it is proposed that the
single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear
molecular structure of PCA The microtubes are formed by the secondary self-
aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-
assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure
were already reported by Wu et al [165] and Sowmiya et al [166] According to their
inference it can also be explained that even with low concentration of guest molecules
the lesser amount of nanotubes could be formed with β-CD Furthermore in order to
form nanotubes the presence of guest molecule is essential and the aggregation of
nanotubes is dependent on the concentration of guest molecules This reveals that
inclusion complex formation and their extensions to construct nanomicro structures are
induced by hydrophobic interactions between CD and guest molecules
Raman imaging was also performed for further confirmation of the micro-sized
tube structures Large number of such microtubes is found in the morphologies obtained
from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM
images With these images herein reported the bunch of such micro-sized tubes formed
due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a
regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was
not obtained because such particles are present nano in size Two drops of the solution
was slowly evaporated on a clean glass surface at room temperature for Raman imaging
whereas the solution was directly deposited on a carbon coated TEM grid for electron
microscopic analysis Moreover when adjusting the pH value to 130 it was found that
the turbid solution becomes completely transparent since the pKa value of β-CD is 120
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
116
hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)
Also under this condition the breakdown of hydrogen bonding between neighbouring
CD would lead to the collapse of the nanotubular structure [167] Consequently there
was no tubular structure except spherical ones observed by TEM images Therefore
besides hydrophobic interaction and van der Waals interaction hydrogen bonding
between the hydroxyl groups of neighbouring CDs is essential for the formation of the
nanotubular structure
The represented nanomicro tube structures are formed through the secondary
assembly of nanotubes The mechanism for the formation of microtube structures from
the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be
explained as follows (Fig 414) The examination of prepared sample of micro tubular
structures by TEM indicated that rhombus-shaped nano sheets were formed Further a
closer investigation at the cleavage of the wall of the microtube confirmed the presence
of several thin supramolecular level layers which have thickness-dependent contrasts
and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition
to that micro-Raman imaging was performed on the bulk aqueous solutions which were
already prepared for the TEM analysis as same Interestingly in some areas of the
sample a few multilayered bulk sheets were found along with the microtubes
Furthermore the observation of sheet like structure suggests that the microtubes are
formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker
[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through
the sheet rolling mechanism in the presence of water It is also monitored in the present
case the occurrence of the shape-shifts in the absence of any external forces A closer
examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure
This inference is also supported by the closer investigation at the cleavage of the
microtube wall (Fig 411e)
The above results revealed that intermolecular hydrogen bonding plays an
important role in the supramolecular ordering and self-assembly besides the hydrophobic
interactions and van der Waals forces which contribute to the formation of the PCA
β-CD complex Through these intermolecular interactions 12 complexes of CD
aggregate can form a sheet like structure with a thickness of around 14-17 nm since the
width of CD is approximately 137-156 nm The nano rods and supramolecular level
layers in the wall of the microtubes probably originate from the hydrophobic surface
effect on the sheet like structures in the presence of water Further the hydrogen bonding
is the driving force for the secondary self-assembly Therefore it is expected that rod like
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
117
structures can also be formed in solution In addition to this B-H double reciprocal plots
did not show any linear relationship for 11 as like for 12 complexation even at lower
concentration where a good linear correlation was observed in the plot of 1(AndashA0)
andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher
concentration of CDs (4 times 10-4 M) in solution
Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure
Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their
secondary self-assembly
Sheet-like structure
Microtube structure
(a) (b)
(c) (d)
(e)
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
118
Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets
12 complex
(a) Staking
(b)
Multilayer nanosheets
Single nanosheet
Microtube (f)
Rolling force
(c) (e)
(d)
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
119
453 FTIR spectral analysis
FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD
and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of
both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both
CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the
literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and
1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the
primary amine stretching vibrations disappeared in the inclusion complexes and the
secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH
stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the
inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1
diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH
stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes
and the OH stretching peak of CD moved to higher frequency which might be due to the
existence of the intermolecular hydrogen bonding between the drug and CD These
obvious changes in the IR spectra confirmed that the inclusion complexes were formed
and the benzene ring with the secondary amine group of PCA was included into the CD
nanocavity
The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)
The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1
and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1
and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1
and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching
and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending
vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation
The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all
these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in
concert with the disappearance of some of the characteristic bands of PFO For example
the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)
completely disappeared in the inclusion complexes The C=O stretching vibration at
1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending
vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in
α-CD complex whereas it is completely disappeared in the β-CD complex The observed
changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
120
the vibration of free drug molecule upon encapsulation into CD cavity indicating the
formation of inclusion complexes between PFO and CD
Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
400
Tra
nsm
ittan
ce
4000 3000 2000 1000 Wavenumber (cm-1)
(a)
(b)
(c)
3402
74
3215
63
1512
33
3319
79
2978
36
1392
73
1599
13
1545
12
630
33
601
73
2928
21
3377
66 16
396
4
1509
40
2941
71
2577
13
2467
18
1643
50
1294
35
1184
40
1028
15
964
50
767
74
841
05
1608
01
1157
39
1030
08
570
98
844
90
2928
55
3375
73
1640
84
1509
65
1608
78
1157
50
1031
15
578
70
846
83
1413
95
1294
35
1334
85
(d)
(e)
(f)
767
74
854
54
3315
93
1030
08
1662
79
3423
96 65
393
2972
57
1593
35
1452
53
1165
33
2781
60
1304
21
706
15
636
57
3396
95
2930
14
1452
53
1155
46
767
74
707
94
1652
20
1030
08
831
47
1586
76
3393
09
2930
29
1452
86
1155
46
758
57
706
47
1651
89
1031
55
858
40
1329
07
732
45
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
121
454 DSC analysis
The thermal analysis is a preliminary and widely used analytical tool for the
characterization of multicomponent systems such as host-guest inclusion complexes in
the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA
β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp
endothermic peak at 1686 degC corresponding to the melting point of the drug As can be
seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the
peaks of CD which represent water losses are strongly reduced in intensity and shifted to
lower temperature In addition the complete disappearance of PCA thermogram clearly
indicates a strong interaction between the PCA and CD molecules The DSC data
suggested the true inclusion complex formation and the drug is well dispersed in the
hydrophobic CD nanocavities
It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a
crystalline anhydrous substance showed an initial flat profile followed by a sharp
endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug
On the contrary the melting endothermic peaks corresponding to PFO was disappeared
in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was
completely disappeared and other endothermic peaks correspond to the dehydration of
free CDs were shifted to lower temperature in the inclusion complexes The comparative
analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug
and both CDs reveals the strong interactions of PFO with CDs and the formation of
stable inclusion complexes The overall DSC results indicate that usual thermal
properties of the drugs as well as the isolated CDs were altered after inclusion complex
formation
455 XRD analysis
Powder XRD is a useful tool for the detection of CD complexation in powder or
microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their
corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed
a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm
193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm
2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the
drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those
are crystalline substances because of the presence of characteristics peaks at 2θ angles
range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the
crystalline drug in the CD patterns in which the characteristic peaks of drug were still
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
122
Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)
(d)
(e)
1722degC
1313degC 1075degC
1188degC
Exo
ther
mic
(mW
mg)
40 60 80 100 120 140 160 180
(f)
Exo
ther
mic
(mW
mg)
40 Temperature (oC)
(a)
(b)
(c)
1686degC
1258degC 712degC 1048degC
1166degC
60 80 100 120 140 160 180
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
123
Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex
(a)
(b)
(c)
(d)
(e)
(f)
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
124
detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA
β-CD complexes are amorphous and display halo patterns which are evidently different
from the physical mixture suggesting the interaction between the drug and CD In
addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the
diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm
disappeared indicating that the formation of inclusion complex between the PCA and
CD However the diffraction patterns of the PFOCD inclusion complexes
(Figs 417e and f) showed complete disappearance or considerable reduction of
characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which
suggest the formation of an inclusion complex The presence of new peaks at 2θ values
of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for
PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid
phase Further the inclusion complexes showed amorphous halo behaviour as evidenced
by broader diffraction peaks with lower intensity obtained for the solid samples
456 1H NMR spectral analysis
The mechanism of interaction between PCA or PFO and CD was investigated
using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419
illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR
spectra of both CDs possessing six types of protons are very close to those already
reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at
497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H
NMR spectra of inclusion complexes indicates that the addition of drugs influences the
chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)
protons located in the interior of the CD cavity are greatly shifted upfield rather than
those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in
comparison to that of isolated CD suggesting the drug molecules enter into close
proximity with both H-5 and H-6 protons A negligible shift is observed for the signals
of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift
changes suggest interaction of the drugs with the internal protons of CD
The chemical shift values of the different protons of PCA PFO and the changes
in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46
The proton signals of PCA (Fig 418a) are in agreement with the results reported by
Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons
in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA
protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
125
only undergo a downfield shift while the other protons including the aromatic and amino
protons show an upfield shift The signals for all drug protons are shifted from 004 ppm
to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi
protons The signals corresponding to amino protons (He) and amide protons (Hb) are
significantly affected upon inclusion complexation Therefore it can be concluded that
PCA is inserted inside the cavity with these protons closer to the interior protons of CD
(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha
and He protons which indicates the aromatic ring and the amino protons are included
into the CD cavity
Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and
β-CD also revealed that the complex formation which evince by the significant changes
in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR
spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene
protons (Hk and Hm) have shown significant chemical shift changes in the presence of
CDs However in the case of β-CD inclusion complex besides the above chemical shift
changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was
largely shifted to upfield andor downfield respectively The chemical shift changes of
these protons suggest the penetration of PFO involves insertion of the aromatic skeleton
and part of the aliphatic chain along with the carbonyl group This interpretation is
supported by strong frequency changes observed on H-3 and H-5 protons of the CD
Some of the most significant chemical shift changes on the spins of the CD protons come
from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD
or β-CD H-3 and H-5 protons are located inside the CD cavity Among these
H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the
narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other
protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO
the significant upfield shifts was observed only for internal protons of CD These shifts
are indicative of the fact that the aromatic part of guest molecule entered closer to the
internal protons of CD and the stable inclusion complexes are formed between the drugs
and CDs
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
126
Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA
(c)
(b)
(a) a b
c d e
i
g
j
f
a e b
N
C O
NH CH2
CH2 NH
H
H
CH2
CH2
CH3
CH3 c
c
d
d f
g
i
i
j
j Cl +
e
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
127
Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO
(c)
(b)
(a) a
b cd ef
gi
j mn
l
p
q r
s
k
a
b
c
d
e
f
g i
j
g i k
l
m n
p q
r
s O
C
O
CH2 CH2
CH2
OH
CH CH2
HN CH2 CH2 CH3
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
128
Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift
(Δδ) after forming the inclusion complexes
Protons PCA (δ)
PCAα-CD (Δδ)
PCAβ-CD (Δδ)
PFO (δ)
PFOα-CD (Δδ)
PFOβ-CD (Δδ)
Ha 1034 -033 -042 915 -004 -037
Hb 846 -010 -013 595 -005 028
Hc 762 007 -004 754 -004 -016
Hd 653 -012 -015 751 -001 -010
He 568 -007 -009 716 -008 -009
Hf 357 006 004 702 -007 -010
Hg Hi 332-315 721-
727 0001 0002
Hj 122 013 014 715 0001 001
Hk 334
Hl 292 001 005
Hm 432 006 010
Hn 412 -002 -011
Hp 292 0001 004
Hq 273 0001 001
Hr 164 001 -001
Hs 087 0002 0006
-merged with CD protons
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes
129
46 Conclusion
The encapsulation behaviour of α-CD and β-CD with PCA and PFO was
investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR
DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs
did not show any significant spectral shifts in the solvents In aqueous solutions each
CD has been found to form inclusion complexes with PCA and PFO Addition of CD to
aqueous solutions of the above drugs has resulted in the observation of the enhancement
of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas
PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates
the fact that phenyl ring along with carbonyl group is present in the inner part of the
hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the
formation stable inclusion complexes of the drugs with both CDs in the solid state The
results of solid state studies showed the important modifications in the physicochemical
properties of free drug The supramolecular nanomicro structures were also fabricated in
aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug
molecules The analysis of thermodynamics of inclusion process indicates that the drug-
CD complexation is spontaneous and hydrogen bonding plays an important role in the
complexation process Investigations of thermodynamic and electronic properties
confirmed the stability of the inclusion complexes