chapter - 3shodhganga.inflibnet.ac.in/bitstream/10603/42182/8/08... · 2018-07-03 · the targeting...

Chapter - 3

Quercetin and Thymoquinone Loaded Fe3O

4 Nanoparticles and

their In-Vitro Anticancer Activities in Breast Cancer Cells

This chapter includes:

Introduction

Structural characterization

Morphological analysis by FESEM and TEM

Surface charge analysis by Zeta potential

Thermal analysis

Magnetic characterization

Drug releasing mechanism

Cytotoxicity studies

Cellular morphological study and Et/Br staining

Apoptosis analysis by flow cytometry

Discussion

Conclusion

References

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CHAPTER - 3 Quercetin and Thymoquinone Loaded Fe O Nanoparticles and their 3 4

In-Vitro Anticancer Activities in Breast Cancer Cells

53

3.1 Introduction

Cancer is one of the most serious diseases for humans worldwide caused by

abnormal growth of cells. Primary stage of the cancer is easily curable but diagnosing the

pathogen at particular target site is very complicated. So, the chemotherapeutic drugs are

widely used to treat the final stages of the cancer cells, but it induces many side effects

and increases the number of deaths. Hence, the innovative drug delivery scheme is

immediately required to cure the final stage of the cancer cells and also reduce the side

effects. So, the researchers are trying to utilize the suitable nanocarriers for targeting

anticancer drugs to particular sites using nanotechnology. The binding of magnetic

nanoparticles with anticancer drugs via suitable surfactants or bio-linkers might be one

solution. These magnetic carriers were used to deliver the anticancer drugs to specific

place of the body by using externally applying magnetic field and hence reduces the side

effects for the normal tissue and cells. The targeting performance of the magnetic

nanoparticles depends on their mean size, structure, bioavailability, magnetization and

magnetic anisotropy [1]. In addition, the monodispersed iron oxide nanoparticles have a

natural tendency to accumulate in cancer tissues [2].

Among many types of the nanoparticles, superparamagnetic Fe3O4 nanoparticles

have more advantages for recent biomedical applications. The applications such as drug

delivery, tissue repairing, magnetic resonance imaging (MRI), magnetic hyperthermia,

water treatment etc., [3,4,5], which requires the spherical shaped monodispersed

superparamagnetic nanoparticles with the size of less than 50 nm [6]. Preparing these

smaller sizes and agglomeration free magnetic nanoparticles with high colloidal stability

is a challenging task for the synthesis methodology. Polymer coating can reduces the

aggregation and improve the colloidal stability of magnetic nanoparticles [7].

Among many polymers, polyethylene glycol (PEG) acts as both structure directing and

stabilizing agent during the preparation of Fe3O4 nanoparticles and enables the growth of

monodispersed particles [8]. But, the mixed surfactants containing PEG and cross-linked

starch coated Fe3O4 nanoparticles produces agglomerated nanoparticles with a diameter

of 100 nm [9]. Similarly, the addition of polyvinyl pyrrolidone (PVP) leads to the

formation of spherical shape magnetic nanoparticles with high stabilization that can be

used for MRI contrast agents [10]. PVP enables the oriented assembly of Fe3O4 primary




54

nanoparticles due to the change in surface energy [11] and it acts like a space block to

form spherical aggregations. Therefore, many PEG and PVP bonded superparamagnetic

nanoparticles were prepared for safe biological applications. Also, these polymers have been

used to change the intrinsic properties of magnetic nanoparticles such as size, surface charge,

reactivity, water dispersiblity and bio-distribution. Similarly, hexamine is also used to

synthesize magnetite nanoparticles with uniform shape and good size distribution.

Hexamine is a chemically inert, cost effective, non-ionic tertiary amine derivative. It is stable

and very good structure directing agent that avoids the aggregation [12].

Till now, the systematic administration of the nanoparticle based targeted

anticancer drug delivery to tumor site in control manner was the severe bottleneck

problem [13]. Many researchers found commercial anticancer drug to be loaded with iron

oxide nanoparticles (magnetic carriers) for successful chemotherapy agents [14]. But,

there were many problems such as solubility, blood circulation, drug clearance, side

effects etc. So, it is required a new drug delivery scheme for targeted cancer therapy to

overcome and understand these problems. Quercetin (3,3‟,4‟,5‟-7-penta-hydroxy flavone)

is a well-known natural bioflavonal that present in edible fruits, vegetables and medicinal

plants [15,16]. It has potent chemotherapy activities for many diseases such as anti-

cancer, anti-inflammatory, anti-viral and anti-oxidant characters [17]. However, the

strong anti-cancer activity of quercetin were found to be in colon, breast, ovarian and

lung cancer cells respectively [18,19]. In recent years, quercetin was not only gained

much attention in cancer cells but also active in other diseases such as malarial, HIV and

cardiovascular diseases [20-21]. But, the major limitation of the quercetin was due to

poorly soluble in aqueous media, poor permeability, low oral bioavailability and

biodegradation [23-25]. So, it hampers the clinical application and needs to overcome

these problems for drug delivery. Conversely, the encapsulation of quercetin molecule on

suitable nanocarriers of dextran coated Fe3O4 nanoparticles is one of the potential

techniques to circumvent these problems and enhanced bioavailability.

Also, thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) is one of the

predominant bioactive constituent of volatile oil derived from the Nigella sativa (NS)

seeds which has been used against many diseases. It possesses multi-health beneficial

activities including antibacterial, anti-inflammatory, antidiabetic, antioxidant, analgesic,




55

antiulcerogenic, anticarcinogenic, antiarthritic, antineoplastic, antimutagenic and antitumor

actions through different mechanisms [26,27]. In normal cells, thymoquinone acts as a

strong antioxidant and reduces the assembly of superoxide radicals and lipid

peroxidation, or enhances the activities of superoxide dismutase (SOD), catalase,

glutathione, glutathinone transferase and quinone reductases [28]. Also, it exhibits multi-

tumor activity against lungs, breast, pancreatic, colon, prostate and liver cancers [29].

Thus, thymoquinone acts as dual role depends on the cellular microenvironment.

In cancer cells, the thymoquinone have the multiple activities such as cell cycle arrest,

DNA damage, preventing NFκB activation and induced apoptosis [30]. Therefore, the

natural source of anticancer agents plays a vital role in biomedical applications.

This motivates to synthesis quercetin and thymoquinone loaded magnetic nanoparticles

and study their anticancer activity using in-vitro method.

3.2 Results and Discussion

3.2.1 Structural analysis by XRD

The crystal structure and phase purity were studied by measuring the XRD pattern

for the pristine, polymer coated (hexamine, PEG, PVP, dextran) and drug loaded

(quercetin and thymoquinone) Fe3O4 nanoparticles were shown in Fig 3.1(a-g). All the

XRD peaks were well indexed with face centered cubic (fcc) spinel structure

corresponding to Fe3O4 nanoparticles. The intensity of the diffraction peak of (311) plane

is stronger than the other peaks and it was used to estimate the average crystal sizes.

The Scherrer‟s formula was used to calculate the average crystal/grain size for the

crystals [31]. The average crystal size of the pristine nanoparticles was found to be 28 nm.

The decrease in the average crystal sizes were observed in the range from 28 nm to

14 nm with the addition of surfactants such as hexamine and various polymers.

The in-situ coating of the polymers can significantly reduce the crystalline sizes. But, the

intensity of the XRD pattern decreases to 50 % as compared with the pristine Fe3O4

nanoparticles as shown in Fig 3.11(f,g). This may be due to the reduction in the grain size

and amorphous materials (drugs) on the surface of magnetite nanoparticles [32].

The calculated cell volume, X-ray density and lattice parameters were similar with the

standard [JCPDS#:89-3854]. However, the polymer coated magnetite nanoparticles




56

shows broadened XRD peaks without any peak shift compared to pristine nanoparticles

and it confirms the reduction in crystal size. The XRD result confirms the amine

functionalized, polymer coated and drug loaded magnetite nanoparticles do not stimulate

any phase transition. This phenomenon ensures the high purity of the prepared materials.

The smaller variation in the lattice constants compared to its bulk counterparts may be

due to the partial oxidizations during the reaction [33].

20 30 40 50 60 70

(g)

(f)

(e)

(d)

(c)

(b)

Inte

nis

ty (

arb

.un

its)

2(degrees)

(a)

Fig 3.1. XRD pattern for the nanostructured Fe3O4 (a) pristine and surface modified

with (b) hexamine, (c) PEG, (d) PVP, (e) dextran and loaded with (f)

quercetin, (g) thymoquinone




57

3.2.2 FTIR analysis

The FTIR spectra of pristine and surfactant coated Fe3O4 nanoparticles were

analyzed in the range of 400-4000 cm-1

as shown in Fig 3.2 (a-e). The FTIR spectrum of

pristine Fe3O4 in Fig 3.2a shows the broad and strong absorption peak at 574 cm-1

is due

to the presence of Fe-O bond of Fe3O4 nanoparticles. A broad peak at 3405 cm-1

represents the O-H stretching vibration with the presence of water molecules. No other

extra peaks were observed and this confirms the high purity of uncoated magnetite

nanoparticles. Figure 3.2b corresponds to the FTIR spectrum of hexamine functionalized

Fe3O4 nanoparticles. The characteristic peaks at 1074 and 1638 cm-1

corresponding to the

C-N stretching vibration and N-H deformation vibration modes attributed to the characteristic

4000 3500 3000 2500 2000 1500 1000 500

573

Wavenumber (cm-1

)

5743405

3412 16131040

587

1166

3421

16323421

1074

800 557

485

1624

3409 16248621047

Tra

ns

mit

tan

ce

1024894

790581

29982910

(a)

(b)

c

d

e

a

b

(c)

(d)

(e)

Fig 3.2. FTIR spectra for the nanostructured Fe3O4 (a) pristine and surface modified

with (b) hexamine, (c) PEG, (d) PVP and (e) dextran




58

frequencies of residual organic materials. The broad peak at 1074 cm-1

supports the

presence of tertiary amines of hexamine molecules which do not undergo degradation at

high temperature [12]. Also, the above mentioned two peaks confirm the presence of

hexamine molecules on the surface of magnetite nanoparticles. In Fig 3.2c, the peak at

1040 cm-1

represents the stretching vibration of C-O-C group which confirms the

presence of PEG molecule on the surface of Fe3O4 nanoparticles and this observation is

well matches with the previously reported value [34]. It further confirms the modification

of the surface on magnetite nanoparticles by hydrophilic molecules which facilitate the

anisotropic crystal growth. In PVP coated magnetic nanoparticles, the peak at 862 cm-1

represents the CH2 rocking vibration and another peak at 1047 cm-1

corresponds to the

C-H stretching vibration mode which demonstrates the coating effect of the PVP

molecule on the surface of the Fe3O4 nanoparticles in Fig 3.2d and the values matches

well with the existing values [35]. The sharp characteristic peak at 1624 cm-1

obtained

from the stretching vibration of C=O corresponds to a strong bond between the PVP

molecules and the Fe3O4 nanoparticles. The intense peak at 1682 cm-1

shifted to 1624 cm-1

is

due to the red shift in C=O stretching vibrations. The reduction in electron density is

responsible for this shift and it leads to a stronger interaction between PVP and Fe3O4

nanoparticles. In Fig 3.2e, the characterized absorption bands at 1024 and 1166 cm-1

represents the stretching vibration of alcoholic hydroxyl C-OH and bending vibration of

C-H bond respectively. These absorption peaks was strong evidences the dextran coating

on the magnetite nanoparticles. In all the four samples, the broad peak at 3400-3450 cm-1

belongs to the O-H stretching vibration of hydroxyl groups, which concludes the higher

hydrophilic nature of the surface of Fe3O4 nanoparticles. The slight shifts in Fe-O bond

were observed in the range of 480-590 cm-1

for the amine and different polymers coated

magnetite nanoparticles respectively. This may be due to the hexamine or polymers

binding to the nanoparticles with the stabilization through some physical interaction on

the surface of Fe3O4. This result confirms the successful wrapping of hexamine and of the

polymers on the surface of the Fe3O4 nanoparticles.

Figure 3.3a shows the FTIR spectrum for the pure quercetin molecule and the

major peaks were observed in the rage of 900-1700 cm-1

. All these peaks represent the

hydroxyl, carboxylic and aromatic group of quercetin molecule [15]. The broad peaks at




59

3390 and 1664 cm

-1 are assigned to stretching and bending vibration of hydroxyl group.

Also, the prominent peaks at 1028 and 945 cm-1

represents the C=O stretching vibration

and C-H bending vibration of aromatic group. All the quercetin peaks were well matched

with existing literature reports [18,36]. In Fig 3.3b, the peak at 560 cm-1

represents the

shift in Fe-O bond compared to pristine nanoparticles due to encapsulation of quercetin

molecules and it influences the insignificant change in electronic structure of Fe3O4

nanoparticles [37]. The carboxylic group present in the peak at 1655 cm-1

represents the

C=O stretching vibration and peak at 1453 cm-1

shows the C-N stretching vibrational

peak that confirms the amide group present in the surface of the Fe3O4 nanoparticles with

the addition of EDC and NHS molecules respectively. Therefore, the zero length linker‟s

such as carbonyl and amine groups were used to conjugate the quercetin on the magnetic

nanoparticles via hydroxyl group. The weak characterize peak in the range of 2800- 3000 cm-

1 attributes to the symmetric and asymmetric CH2 stretching vibration. The major peaks at

945 and 1028 cm-1

were exactly matches with pure quercetin peaks indicates the linkage

4000 3500 3000 2500 2000 1500 1000 500

586

3427

28572923

Wavenumber (cm-1

)

945

1028560

1317

14531655

28902998

3410

1742

16421387

1031890794

Tra

nsm

itta

nce

(d)

(c)

(b)

(a)

Fig 3.3. FTIR spectra for (a) quercetin, (b) QLMNPs, (c) thymoquinone and (d) TLMNPs




60

between drug and magnetite nanoparticles. Thus, it is clear that these peaks clearly

represent the successful loading of quercetin on the dextran coated Fe3O4 nanoparticles.

Similarly, Fig 3.3c shows the FTIR spectrum of pure thymoquinone and the major

peaks were present in the range of 900-1700 cm-1

. The CH3 stretching and bending mode

appears in the region of 1300-1500 cm−1

, which shows several bands and shoulders.

The band located at 1230-1290 cm-1

assigned to bending and stretching mode of C-C

confirms the presence of benzoquinone. The strong peaks at 1642 cm-1

represents

the stretching band of carbonyl group. The intense and weak bands present at 2936 and

3020 cm-1

represents the C-H stretching of aliphatic and vinylic groups of thymoquinone

which is well matches with the reported value [38,39]. In Fig 3.3d, the broad peak at

586 cm-1

represents the Fe-O bond and it is slightly shifted as compared from pristine

nanoparticles due to the strong binding of thymoquinone on the surface of the Fe3O4

nanoparticles. The other corresponding peaks of aliphatic, vinylic, carbonyl, hydroxyl

and benzoquinona were well matches with the free thymoquinone. The matching peaks

were highlighted with round circle and it clearly confirms the successful wrapping of the

thymoquinone on the surface of PVP coated Fe3O4 nanoparticles.

3.2.2 Morphological Analysis

3.2.2.1 Field emission scanning electron microscope (FESEM) analysis

The FESEM analysis was carried out to investigate the size and morphology of

the prepared samples. The typical FESEM images with different magnification of the

pristine, PVP coated and thymoquinon loaded Fe3O4 nanoparticles were shown in

Fig.3.4. The FESEM image of the pristine nanoparticles in Fig 3.4 (a-c) shows the

monodispersed agglommoration free spherical shapes with smooth surface in the size

range of 30-50 nm and it is clearly visible in magnified images. The pristine

nanoparticles were shattered into small-sized nanocrystals and it was packed again due to

strong magnetic dipole interaction to form the monodispersed mesoporous spherical

nanostructure with the in-situ addition of PVP as shown in Fig 3.4(d-f). The size of the

spherical shape obtained as >150 nm and it had a number of ultra-small pores and voids

present on the surface. These pores are produced due to the uniform nucleation growth of

the organic-inorganic assemblies [40]. Thus, the higher surface energy would lead to the




61

Fig 3.4. Low and higher magnification FESEM micrographs of the (a-c) pristine (d-f)

PVP coated and (g-i) thymoquinone loaded Fe3O4 nanoparticles.

aggregation of smaller crystals to form larger mesoporous nanocrystals due to the well

known Ostwald ripening process [41]. Furthermore, the FESEM images of thymoquinone

incorporated magnetite nanoparticles with mesoporous structure were shown in

Fig 3.2(g-i). Here, the PVP coated Fe3O4 nanoparticles act as core and thymoquinone act

as shell to form a core-shell structure and it is highlighted in the micrograph as shown in

Fig 3.4i. The thymoquinone loaded Fe3O4 nanoparticles doesn‟t show noteworthy

changes in shape or size when thymoquinone drug was embedded. It indicates the Fe3O4

nanoparticles were not significantly changed during the encapsulation processes such as

the dissolution or growth of nanocrystals [42].

3.2.2.2 Transmission electron microscope (TEM) analysis

The micro-structural features of the magnetite nanoparticles were further studied

by both TEM and HRTEM. Figure 3.5 shows the TEM images of pure and surfactant




62

coated Fe3O4 nanoparticles. The TEM image of pristine Fe3O4 in Figure 3.5(a,b) consists

of high quality polyhedral nanocrystals with few tiny nanoparticles. The size and shape of

these nanocrystals were not uniform and it shows more shapes like spherical, cubic,

polyhedric, hexagonal, triangular etc., and the maximum particles were lying below 25 nm.

Figure 3.5c represents the histogram of the Fe3O4 nanoparticles with the average particle

size of 24 nm that are calculated among more than hundred particles. The surface

smoothness can be attributed to the uniform arrangement of the lattice points without any

lattice imperfection. Figure 3.5(d-f) shows the TEM image and histogram for the

hexamine functionalized magnetite nanoparticles with the uniform distribution of

spherical shape and the average size calculated was 21 nm. The particles were well

separated from the controlled nucleation, growth and crystal orientation of the magnetite

nanoparticles. The non-covalent bond between the amine and the surface of the magnetite

nanoparticles are important to obtain the colloidal stability, which are useful for

biodegradability. Also, the amine terminated Fe3O4 nanoparticles has the best binding

ability when compared with other functional groups [43], and the functionalization

controls the growth of the nanoparticles in smaller size and spherical shape. In addition,

the hexamine molecules on the surface of Fe3O4 act as a soft template to enhance the

reaction rate for fine orientation as well as the formation of individual spherical

nanoparticles due to controlled magnetic dipole-dipole interaction. It was found that the

primary driving force is responsible to reduce the surface energy and enhances the

formation of monodispersed spherical shape nanoparticles.

The TEM image and the histogram of PEG coated Fe3O4 magnetite nanoparticles

in Fig 3.5(g-i) also shows the monodispersed spherical nanoparticles were in the size

range of 25 nm. The small increase in the particle size may be due to the high

chemisorptions of PEG molecules. The larger narrow sized spherical nanoparticles were

obtained with the expenses of smaller particles by the Ostwald ripening process. The

kinetics of crystal growth leads to the uniform distribution of agglomeration free

nanoparticles [44]. When the particles exceeds their critical size, the PEG molecules acts

as shape controlling agents but also as a stabilizing agents to control the growth of the

magnetite nanoparticles. The micrograph of PVP coated magnetite nanoparticles in Fig

3.5 (j-k) show an exceptionally agglomerated morphology with an average size of 14 nm.




63

50nm

50nm

20nm

20nm

10 15 20 25 30 35 400

5

10

15

20

Nu

mb

er

of

part

icle

s

Particle Size (nm)

10 12 14 16 18 20 22 24 26 28 300

5

10

15

20

Nu

mb

er

of

part

icle

s

Particle Size (nm)

a

ed

b c

f

50nm

50nm

20nm

20nm

15 20 25 30 35 40 45 500

5

10

15

20

Nu

mb

er

of

pa

rtic

les

Particle Size (nm)

6 8 10 12 14 16 18 20 220

2

4

6

8

10

12

14

Nu

mb

er

of

pa

rtic

les

Particle Size (nm)

hg

j k

i

l

Fig 3.5. Low and higher magnification of TEM images and histograms of Fe3O4

magnetic nanostructures for (a-c) pristine and the surface modified with

(d-f) hexamine, (g-i) PEG and (j-l) PVP.




64

a b

Fig 3.6. Low and higher magnification of TEM images of dextran coated Fe3O4

magnetic nanostructures

This may be due to the PVP molecule adsorbed on the particular crystallographic

facets of Fe3O4 nanoparticles due to surfactant [11]. However, the PVP molecule bridges

the surface of Fe3O4 nanoparticles [45] and reduces the surface energy of the system due

to strong inter-particle attractive forces (i.e. Van der Waals forces) on the magnetite

nanoparticles at higher temperatures. The PVP plays an important role in the aggregation

via self assembly process and it cannot break the balance between the individual particles

due to the higher surfactant energy on the surface of Fe3O4 nanoparticles. Similarly, the

mesoporous silica nanoparticles were aggregated due to the existence of protein or lipid

layers [46]. The reaction rate and diffusion of Fe3+

ions decreases considerably between

polyol medium with the addition of PVP and it leads to the aggregation of small spherical

nanoparticles.

The dextran coated Fe3O4 nanoparticles displays monodispersed prism shape in

the size range of > 20 nm that significantly avoid the aggregation of the particles as

shown in Fig 3.6 (a,b). The dextran was attached to the specific crystal planes that direct

to orient anisotropic growth to fabricate Fe3O4 nanoprisms [47]. Here, the dextran may

play a dual role as altering the shape of aggregated Fe3O4 nanoparticles and also it protect

the phase change by shielding effect on the surface of the magnetite nanoparticles. Also,

this biocompatible polymer is important to provide hydrophilic surface that makes to




65

improve the dispersion in water, act as a capping agent to load the quercetin on the

surface and it is used to sustain drug release in bio-molecules.

3.2.2.3 High resolution transmission electron microscope (HRTEM) analysis

The HRTEM image for the pristine Fe3O4 as in Fig. 3.7a shows the cubic

structured with defects free surface. It clearly indicates the well aligned and single

crystalline structure with the d spacing of 0.258 and 0.293 nm corresponding to (220) and

(311) plans [48]. Thus, HRTEM of pristine Fe3O4 shows the dissimilar shape of crystal

growth to facilitate the nanoparticles to be free from preferred orientation due to different

lattice arrangement. Without adding the surfactants like polymers or amines, the

Fe nuclei in the magnetite nanoparticles try to assemble in different orientations.

The hexamine functionalized magnetite nanoparticles show a single crystalline structure.

The measured lattice fringe distance corresponds to the d spacing of (311) plane of Fe3O4

nanoparticles and it also confirms the fcc inverse spinel structure of Fe3O4 as shown in

Fig. 3.7c. Consequently, the hexamine are selectively adsorbed on the (111) facet to

reduce its surface energy and to avoid the aggregation of magnetite nanoparticles.

Therefore (311) plane direction of Fe3O4 crystals may exhibit a higher activity of crystal

growth to generate monodispersed spherical nanoparticles within electrostatic interaction.

Accordingly, the PEG coated nanoparticles in Fig. 3.7e represents the growth of bigger

spherical nanoparticles and the top plane corresponds to (111) direction, exhibiting clear

lattice fringes which shows their high crystalline nature without agglomeration. Thus, the

surfactant PEG is bound on the surface of magnetite nanoparticles for the formation of

monodispersed spherical nanoparticles and reduces the growth rate along this direction to

form bigger size nanoparticles. Similarly, the PVP coated magnetite nanoparticles also

show parallel fringes with the d spacing of 0.258 nm corresponding to the crystal plane of

(311) direction. It is clearly visible with the size of 10 nm as shown in Fig. 3.7 g.

Therefore, PVP coated magnetite nanoparticles demonstrate the close packed structure to

form agglomeration with small molecules of Fe atoms.

HRTEM results are compared with Fast Fourier Transform (FFT) data as shown

in Fig 3.7(b,d,f,h) and indexed as (311), (511) and (440) plane of cubic Fe3O4

nanoparticles. The set of spots with the highest contrast could be indexed to (311) reflection,




66

Fig 3.7. HRTEM and fast Fourier transform (FFT) pattern for nanostructured Fe3O4

for (a,b) pristine and coated with (c,d) hexamine, (e,f) PEG and (g,h) PVP




67

Fe3+ 3OH- Fe OH

OH

HO

Fe(OH)3 Fe O

OH

H2O

2Fe(OH)3 Fe(OH)2 Fe3O4 4H2O

indicating the assembled spherical magnetite nanoparticles with a good single crystalline

shape with a {111} basal plane. The XRD analysis also indicates the same cubic Fe3O4

nanoparticles, supporting the role of the amine and polymer molecules to control the

growth direction along (311) plane on the surface of the magnetite nanoparticles.

The observed uniform and well oriented spots in the FFT supports the good resolution of

the monodispersed spherical nanoparticles having the same orientation due to sub-unit

particles assembly to form single crystalline Fe3O4 structures. These results confirm the

important role of amine and polymers that leads to the formation of small and large sized

spherical nanocrystals via self assembly. Time and temperature were constant in all the

reported experiments and cannot be claimed responsible for the shape and size.

3.2.3 Formation mechanism

The possible reaction equation for the formation of Fe3O4 nanoparticles is given

below:

Initially, the Fe3+

ions react with ethylene glycol to form iron hydroxide with the

addition of KOH at room temperature. The ethylene glycol acts both as solvent and

reducing agents which plays an important role in the formation of magnetite. Potassium

hydroxide is used as alkali medium to induce the reaction and make a deprotonation to

reduce Fe2+

and Fe3+

ions [49]. Finally, the iron hydroxides are converted into Fe3O4 at

210˚C with continuous flow of Ar gas during the polyol process.

The schematic illustration for the plausible growth mechanisms of spherical Fe3O4

nanoparticles prepared with different polymers is shown in Fig 3.8. The pristine Fe3O4

nanoparticles at equilibrium produces agglomeration free Fe3O4 nanoparticles. The

addition of hexamine in pristine Fe3O4 nanoparticles provides the uniform distribution of

spherical shape nanoparticles due to adsorption of chemical species that dramatically




68

Fig 3.8. Schematic illustration for the formation mechanism of Fe3O4 nanoparticles.

impinge on the surface energies. Since, hexamine controls the growth of magnetic

nanoparticles due to its strong electrostatic force [50] and prevents the aggregation with

adjacent nanoparticles. Moreover, the amine molecule converts the surface from

hydrophobic to hydrophilic, facilitating water solubility and target binding. Similarly, the

addition of PEG contributes to the larger spherical shape of the magnetic nanoparticles.

Here, PEG acts as surface capping agent to increase the size to 30 nm. Thus, PEG covers

the surface of the Fe3O4 nanoparticles and controls the growth of particles stabilizing the

reaction system due to effective confinement of their random Brownian motion [51].

The steric repulsive force minimizes the agglomeration and produces the uniform size of

the particles [52]. In contrast, non-uniform distribution of pure Fe3O4 nanoparticles is

converted into the uniform spherical nanoparticles, solitary by changing the concentration

of Fe3+

ions under the confinement of surfactant molecules. The PVP coated Fe3O4

nanoparticles shows agglomerated spherical particles due to the high chemisorption on

the surface of magnetic nanoparticles. Also, the PVP acts as a mortar to hold the

individual magnetite nanoparticles to agglomerate via hydrogen bond. The constant

temperature is maintained in all these experiments and thereby, the metal ions nucleate at




69

crystal planes, closely packed in three dimensions to form a smooth surface of the

spherical nanoparticles or self assembly. The homogeneous distribution of spherical

shaped Fe3O4 magnetic nanoparticles was obtained by minimizing the interfacial energy

on the surface of nanoparticles by using polymers or amines.

3.2.4 Surface charge analysis by zeta potential measurements

The stability and dispersibility of the Fe3O4 nanoparticles are mostly depends on

their surface chemistry and it is studied by zeta potential measurements. Generally, the

surface charge polarity of the magnetite nanoparticles were changed by deprotonation

and protonation effects, according to the following chemical equations [53]

[protonation] (1)

[deprotonation] (2)

If the surface of the Fe3O4 nanoparticles is hydrophobic, the protonation reaction

takes place and get positive charged surface at low pH values. Whereas, the hydrophilic

nature of the Fe3O4 nanoparticles were obtained due to the change in the negative charged

surface at high pH values. Similarly, the point of zero charge was obtained due to the neutral

magnetite surface. On the basis of the above reaction principle, the surface charges of the

magnetite nanoparticles were analyzed and the interaction of bio-molecules was studied.

The zeta potential graph to study the surface charges of the dextran, PVP coated

and drugs (quercetin and thymoquinone) loaded Fe3O4 nanoparticles were shown in

Fig 3.9(a-d). The zeta potential was used to study not only the surface charge but also

implies the physical stability of the magnetic nanoparticles, cellular uptake, bio-

distribution and interaction with bio-molecules. The potential value of the dextran and

PVP coated magnetite nanoparticles has the negative charge of -25.6 and -13.6 mV due to

the deprotonated surface. Similarly, the quercetin and thymoquinone loaded magnetite

nanoparticles illustrate the positive charge of 6.14 and 20.7 mV due to electrostatic

interaction between drug and magnetite nanoparticles. The comparison graph between

zeta potential value and various types of Fe3O4 nanoparticles were shown in Fig 3.10.

The positive charged surface was attributed to the protonation of the surface groups via

carboxylic/amine molecules and presence of anionic ions from the drug molecules. The




70

0

100000

200000

300000

400000

500000

600000

(a)

(b)

(c)

(d)

50 100 2001500-50-100

Zeta Potential (mV)

-150-200

To

tal C

ou

nts

Fig 3.9. Zeta potential graph for (a) dextran, (b) PVP coated and (c) quercetin loaded,

(d) thymoquinone loaded Fe3O4 nanoparticles

Dextran coated PVP coated QLMN TLMN

-30

-20

-10

0

10

20

30

Z

eta

Po

ten

tia

l (m

V)

Fe3O

4 nanoparticles

Fig 3.10. The graph shows the relation between zeta potential and different types of

Fe3O4 nanoparticles.




71

positive charged surface quickly interacts to the cell surface and supply efficient delivery

of the quercetin or thymoquinone drug. Also, it enhances the deep penetration to tumor

cells to attain a homogeneous allocation of drugs [54]. Therefore, nanoparticles with

positive surface charges have more cellular interaction than negative charged magnetite

nanoparticles. The result shows the efficiency of the anticancer activity increases by

using positive charged magnetite nanoparticles. The change in surface charge from

negative to positive may be due to more hydroxyl residuals is binding on the surface of

the magnetite nanoparticles. Subsequently, the polymer coated and drug loaded Fe3O4

nanoparticles have different size, shape and surface properties. This may influences the

change in surface reactivity of breast cancer cells due to change in radical species and

oxidation sites. The change in the surface charge from positive to negative surface

confirms the successful loading of organics or drugs material on the surface of the

magnetite nanoparticles.

3.2.5 Thermogravimetric analysis

The amount of organic molecules adsorbed on the surface of the pristine and

thymoquinone loaded Fe3O4 nanoparticles were quantified by thermogravimetric analysis

and the corresponding thermogravimetric curves were shown in Fig 3.11(a,b). The pristine

nanoparticles have single weight loss of about 4 % due to the adsorption of the water

molecules. There was no weight loss occured by increasing the temperature and it confirms

the high stability of the pristine Fe3O4 nanoparticles. The drug loaded Fe3O4 nanoparticles

shows two weight losses corresponding to the strong binding of thymoquinone and water as

shown in Fig. 3.11b. The first weight loss of 7 % can exhibit at the temperature of 50-250 °C

due to the endothermic loss of water molecules and the presence of surface hydroxyl group of

the magnetite nanoparticles [55]. Further increasing the temperature above 250 °C, the

second weight loss was obtained for 18 % and it was ended at 375 °C. The high percentage

of the weight loss was observed due to the strong wrapping of thymoquinone. Because, the

thermal degradation of organic molecules could be found above 250 °C. The result indicates

the thymoquinone drug could be stably attached to the surface of Fe3O4 nanoparticles and

could not be removed during the dialysis process. The, increasing the temperature to above

400 °C, there was no weight loss and it leads to strong Fe-O bond force constant, therefore it

requires more energy to fracture.




72

0 200 400 600 800

70

80

90

100

(b)

(a)

We

igh

t (%

)

Temperature (o

C)

Fig 3.11. Thermogravimetric graph of (a) pristine and (b) thymoquinone loaded Fe3O4

nanoparticles

-20 -10 0 10 20

-100

-80

-60

-40

-20

0

20

40

60

80

100 (a)

(b)

(c)

(d)

(e)

(f)

(g)

Mag

neti

zati

on

(em

u/g

m)

Applied field (KOe)

Fig 3.12. Room temperature magnetic hysteresis loops for the nanostructured Fe3O4

(a) pristine and surface modified with (b) hexamine, (c) PEG, (d) PVP, (e)

Dextran and loaded with (f) quercetin and (g) thymoquinone




73

3.2.6 Magnetic measurements

The magnetic properties of the pristine, surface modified with various polymers

and drug loaded Fe3O4 nanoparticles were studied by recording the hysteresis loop by

using vibrating sample magnetometer at room temperature with an applied magnetic field of

2T as shown in Fig 3.12. The superparamagnetism occurs barely when the thermal energy

exceeds the volume energy and randomized the magnetic moment. The superparamagnetic

nanoparticles do not have permanent magnetic moments in the absence of an applied

magnetic field but can respond to an external magnetic field. The saturation

magnetization of the pristine nanoparticles has 89 emu/g, which is close to the bulk value

of 92 emu/g [56]. When the energy of particles was compared to the thermal energy, thermal

fluctuation significantly reduces the total magnetic moments at a given field [57].

This might be a reason for decreasing saturation magnetization of pristine nanoparticles

when compared to the bulk counterpoints. The hexamine and different polymers (dextran,

hexamine, PEG and PVP) coated magnetic nanoparticles show a small decrease in the

magnetization values such as 48, 59, 60 and 62 emu/g. This reduction in magnetization

may be due to the formation of surface dead layers that produces the shielding effect and

reducing the energy of the spin moment compared to pure Fe3O4 nanoparticles [58].

The electron exchange between surface atoms and polymer ligands also influences the

changes in the saturation magnetization with the influence of applied magnetic field.

Moreover, surface spin canting effect might also reduces the total magnetic moment of the

nanoparticles. For quercetin loaded Fe3O4 nanoparticles have low saturation magnetization of

30 emu/g. It may be due to the higher density of quercetin loaded on the surface of the

magnetite nanoparticles [59,60]. This quercetin molecule produces a magnetic dead layer on

the surface that quenched the magnetic moment [61]. Similarly, the saturation magnetizations

of the thymoquinone loaded Fe3O4 nanoparticles also have low value of 26 emu/g. It happens

due to the strong binding of amorphous thymoquinon drug shielded on the Fe3O4

nanoparticles that might decreased in the effective magnetic moment by increasing the

surface spin disorientation [62]. Therefore, the chemisorptions of non-magnetic layer quench

the saturation magnetization values and confirms that the surface entrapment of the magnetite

nanoparticles. These results confirm the change in magnetic properties strongly depends on

the size, shape and surface effects of the Fe3O4 nanoparticles.




74

Fig 3.13. Drug release profile of quercetin loaded Fe3O4 nanoparticles and the values

are mean ± SD (n = 3)

3.2.7 Drug loading and drug releasing profile

The drug loading and encapsulation efficiency was found to be 6.08 ± 0.3 and

81.6 % respectively. Generally, the drug releasing depends on size, surface character,

internal force between drug and nanoparticles, the rate of hydration and dehydration of

polymers, etc. The in-vitro drug release behavior of quercetin loaded Fe3O4 nanoparticles

was evidenced in PBS buffer at pH 7.4 and temperature of 37 ºC in order to maintain the

experimental condition similar to body fluids. More importantly, the response of dextran

can regulate the release of quercetin from the magnetite nanoparticles in intracellular and

extracellular environments as shown in Fig 3.13. The two stage release profile was

exhibited in the drug loaded Fe3O4 nanoparticles i.e. initial burst release at pH 7.4 and

further fast release rate at pH 5.5 respectively. Generally, the drug release rate was

initially fast and becomes slower as time progressed. Initially, 25 % of drug was released




75

gradually upto 4 days with the pH 7.4 and decreasing the pH to 5.5, the same percentage

was obtained for 2 days. Further increasing the time, the drug release gradually increases

and reach maximum of 69 % and 81.6 % in 16 days at two different pH levels of 7.4 and

5.5 respectively. However, it may be due to the feeble interaction between the Fe3O4

nanoparticles and quercetin molecules to enhance the drug release at higher percentage

rate. The report available for maximum release of quercetin obtained at 14.5 % after 96 h

due to strong interaction between the nanoparticles and drug molecules [25]. In the

present studies, the maximum percentage of quercetin release happens in the acidic

conditions. At the weak basic condition (pH-7.4), the carboxylic bond between the

dextran and Fe3O4 nanoparticles was tightly bonded and it is not easy to break for the fast

release of drug. Also, the water molecules may form a barrier for hydrophobic molecules

of quercetin to decrease the degradation from Fe3O4 nanoparticles [63]. However, the

decrease in pH to 5.5, the solution environment changes and the quercetin was released from

the magnetite nanoparticles quickly. The higher percentage of releasing was obtained due to

the deprotonation of carboxylic and amine groups linkage between dextran and quercetin

molecules. The in-vitro drug release is not only depending on the pH of magnetite

nanoparticles but also depends on the physicochemical properties of stabilizer.

3.2.8 In-vitro toxicity studies

The cytotoxicity of pristine and surfactant (hexamine, PEG and PVP) coated

magnetic nanoparticles for human breast carcinoma (MCF-7) cells was studied with the

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, neutral red

uptake and propidium assay after 72 h of cells exposure in complete medium. Figure 3.14

shows the concentration dependent cytotoxic effects of magnetic nanoparticles in the

concentration range of 5-50 µg/mL for the MTT assay. Generally, MCF7 cells exposed to

the low concentrations of 5-10 µg/mL shows no significant reduction of their metabolic

activity. Increasing the concentration of 15-50 µg/mL causes a weak reduction of

mitochondrial function that induces a mild cytotoxicity to MCF-7 cells. The surface

modified Fe3O4 nanoparticles were good biocompatible when compared to those of

pristine nanoparticles and the phenomenon corresponds with reported literature [64,65].

Therefore, the percentage of inhibition of mitochondrial activity observed in surfactant




76

Fig 3.14. Cytotoxicity of Fe3O4 nanoparticles (a) pristine, coated with (b) hexamine, (c)

PEG and (d) PVP at different concentrations (exposure time 72 h). Results

are mean values ± SEM from six independent experiments; (***) p<0.001 vs

control, Student-Newman-Keuls Multiple Comparisons Test, ANOVA.

Fig 3.15. PI uptake in human MCF7 cells exposed for 72 h for Fe3O4 (a) pristine, coated

with (b) hexamine, (c) PEG and (d) PVP at different concentrations of

nanoparticles. Ctrl – (negative control): cells not exposed to nanoparticles

without addition of ethanol 70%; Ctrl + (positive control): cells not exposed

to nanoparticles plus ethanol 70%. Independent experiments performed in

triplicate.




77

coated nanoparticles is due to the cell adhesive interactions of the nanoparticles.

Our results confirmed that superparamagnetic Fe3O4 nanoparticles cause low cytotoxicity

due to their monodispersed shape and surface properties.

The study of cell cytotoxicity using MTT assay was sometimes reported to

interfere with the relevant measures, when using various nanoparticles, leading to

misleading data [66]. Therefore, the neutral red uptake (NRU) assay system is a means of

measuring living cells via the uptake of the vital dye neutral red. An increase or decrease

in the number of cells or their physiological state results in a concomitant change in the

amount of dye incorporated by the cells in the culture. This indicates the degree of

cytotoxicity caused by the magnetic nanoparticles.

The results of NRU do not exhibit a concentration dependent decline in the

survival of cells exposed to 72 h for the magnetic nanoparticles. Untreated and treated

cells incorporate the same amount of dye revealing that all nanoparticles are not toxic

(data not shown). The results obtained with NRU further confirmed, by the study of cell

cytotoxicity with a complementary assay called Propidium Iodide (PI) assay. It was

commonly used for identifying dead cells because it can penetrate only in death cell

membranes. Under the same conditions used in the previous tests (MTT and NRU), no

evidence of induction of necrotic events (PI assay) was found as in Fig 3.15. Detection of

PI fluorescent is an index of the presence of necrotic cells since PI binds to DNA and

RNA by intercalating between the bases with little or no sequence preference. Again

these results indicate the absence of cytotoxicity for the tested nanomaterials which

indicates there is a relative safety for living cells. These data strongly confirms the

importance of verifying the cytotoxicity data with at least two or more independent test

systems for these nanomaterials.

The pure quercetin, dextran coated and quercetin loaded Fe3O4 nanoparticles were

used to incubate with MCF-7 cells to study the inhibition of the cell growth rate using MTT

assay and their corresponding cell viability graph for different concentrations were shown in

Fig 3.16. The absorption percentage of treated cells compared to that of control cells was

used to calculate the cell survival. In the case of the dextran coated magnetite nanoparticles,

the obtained cell viability was found to be 78-90 % in a broad concentration range of




78

a

b

c

d

Fig 3.16. Cytotoxicity of (a) Control, (b) dextran coated, (c) quercetin and (d) quercetin

loaded Fe3O4 nanoparticles at different concentrations

Fig 3.17. Total number of apoptotic cells measured. *Significantly different from

control data were expressed as mean ±SD. *p < 0.05.




79

1-100 µg/ml for 24 h compared with the control cells. It clearly demonstrates the

magnetite carrier anchored with dextran had good biocompatibility due to strong

electrostatic attraction [67]. The small increase in the toxicity was well known for the

magnetite nanoparticles expose to the cells. These magnetite particles interact the

plasma membrane over a period of time and causes lysis of the cells to induce

cell death as compared to control [68]. Significantly, the quercetin loaded magnetite

nanoparticles (QLMNPs) exhibits a cell viability of below 25 % at the same

concentration which could be much higher inhibition of cell growth rate compared to free

quercetin (50%). This may be attributed to the strong binding between the MCF-7 cells

and quercetin molecules due to the partial release of quercetin from the magnetite

nanoparticles and this also confirmed from the drug releasing studies with different pH.

The toxicity of the breast cancer cells may happens due to the damaged DNA via drug

release from magnetic oxidation. Figure 3.17 shows the IC50 and IC75 value was found to

be 31 and 73 μg/ml respectively. The result shows the high therapeutic effects of the

quercetin which induce the anticancer activity.

Similarly, the MTT assay was applied to evaluate the anti-proliferative effect of PVP

coated Fe3O4, thymoquinone and thymoquinone loaded Fe3O4 nanoparticles (TLMNPs)

with a cell incubation time of 24 h in MCF-7 cells (data not shown). The maximum

concentrations of PVP coated magnetite nanoparticles have no cytotoxic effect and it

confirms the biocompatiblity. The IC50 value was obtained as 30 µg/ml for TLMNPs

whereas 60 µg/ml for free thymoquinone. The growth inhibition caused by thymoquinon

loaded Fe3O4 nanoparticles was high compared with pure thymoquinon due to the

intercalation of the nanoparticles. It happens on their surface stems due to the strong

electrostatic interaction between the positively charged TLMNPs and the negatively

charged cell membranes and it enhances the toxicity to cancerous cells. Therefore, the

TLMNPs cause high cytotoxicity to MCF-7 cells in a dose dependent manner.

The obtained result clearly confirms the increase in the cytotoxicity of the thymoquinone

was due to the sustained release of the drug molecules from the PVP-coated Fe3O4

nanoparticles and/or their more effective uptake by cells.




80

Fig 3.18. Phase contrast microscopic images of cellular morphology, AO/EtBr

staining and DAPI of (a,d,g) control and (b,e,h) 31 µg/ml, (c,f,i) 73 µg/ml

of quercetin loaded Fe3O4 treated with MCF-7 cells.

3.2.9 Morphological study and Et/Br staining

Together with the cytotoxicity assay, the studies on cell morphology and

the integrity of the membrane were used to estimate the toxicity or biocompatibility.

This information was relevance to estimate the cleavage of cells, which depends on the

surface characteristics, size and shape of the nanoparticles. Figure 3.18(a-f) shows the




81

optical microscopic images of MCF-7 cells treated with QLMNPs. The large number of

cells became rounded and shrunk which implies the adherence between the breast cancer

cells and magnetite nanoparticles. This was a very high reduction in cell counts due to

release and interaction of quercetin. The obtained results shows that the treatment with 73

μg/ml of QLMNPs shows more survival rate when compared to cells treated with 31

μg/ml. The cells were stained with AO and EB dyes and the apoptotic cells were

observed by red nucleus due to the DNA binding capacity of EB and viable cells had a

green nuclear fluorescence. The AO can penetrate the normal cell membrane, the cells

were observed as green fluorescence, while in apoptotic cells and apoptotic bodies were

formed as a result of nuclear shrinkage, blebbing and were observed as orange colored

bodies. Whereas, the necrotic cells were also observed due to loss of membrane integrity

which further inhibits the growth of the breast cancer cells.

Similarly, the TLMNPs also have significant morphological changes observed

from MCF-7 cells treated after 24 h is shown in Fig 3.19 (a-f). The phase-contrast

micrograph shows the thymoquinone induces more membrane blebbing, disintegration of

nuclear membrane and forming the floating cells in a dose-dependent way. The control

AO/EtBr image shows that there was no obvious change in the quantity and shape of

MCF-7 cells, which shows the cell viability. The treatment of MCF-7 using TLMNPs

causes a reduction of cell volume, nuclear condensation (a hallmark of apoptotic cells),

and increases non-adherence of the cells to the culture surface. Orange apoptotic cells

containing apoptotic bodies whereas the necrotic cells were observed to be in red color.

After treatment with their respective IC50 concentrations of tested compounds for a period

of 24 h, the induced apoptotic changes observed in the cells (Fig. 3.19(e,f)). The total

number of apoptotic cells (green to orange and red color) increases by increasing the

TLMNPs concentrations. Therefore, the quality of the contrast images clearly represents

the thymoquinone loaded magnetite nanoparticles induce the anticancer activity very

efficiently compared to quercetin molecules.

3.2.10 Fluorescence microscopy of the MCF7 breast cancer cells

The fluorescent microscopy study on MCF-7 breast cancer cells treated with

QLMNPs exhibits the bright blue color emission (DAPI) as shown in Fig 3.17(g-i). The




82

Fig 3.19. Phase contrast microscopic images of cellular morphology of AO/EtBr

staining and DAPI of (a,d,g) control and (b,e,h) 31 µg/ml, (c,f,i) 60 µg/ml

of thymoquinone loaded Fe3O4 treated with MCF-7 cells.

fluorescent image shows the nuclear fragmentation and chromatin condensation of the

cancerous cells which leads to the apoptosis while the normal cell exhibits round intact

nucleus. When the concentration of QLMNPs increases, the bright blue color contrast

was also enhances due to the increasing apoptosis cells or cell death. Recently, the

quercetin shows an induced apoptosis and halts the cell cycle mechanism due to strong

electrostatic attraction and it also observed in our present study [69]. This further

confirms the loading of quercetin in the nanoparticles exhibit the potential anticancer

agent in the breast cancer cells.




83

Similarly, the fluorescence of control cells shows no evidence of fragmentation

and segmentation. The nuclei of untreated MCF-7 cells were shown in Fig 3.19g. A very

few cell death happens due to the internalization of particles for the thymoquinone loaded

Fe3O4 nanoparticles and it is reflected in the blue color contrast in the image.

The increase in the concentration of the thymoquinone loaded Fe3O4 nanoparticles were

improved it property and it was confirmed by the increase in the blue color in the image.

This confirms the segregation of cell nuclei in segments and the DNA condensation

which represents fast cell death.

3.2.11 Apoptosis analysis by flow cytometry

The nanoparticles uptake by MCF-7 cells was investigated using microscopy in

combination with flow cytometry to probe the apoptosis effects in detail. The flow

a b c

0

20

40

60

80

100

120

Control Pro-Apoptotic Late-Apoptotic Necrotic

Nu

mb

er o

f ce

lls

Florescent cells

d

Fig 3.20. Flow cytometric analysis of apoptotic detection of (a) total count of cells, (b)

control, (c) thymoquinone loaded Fe3O4 nanoparticles, (d) analyzing

apoptotic cells




84

cytometry analysis of the total count, control and drug loaded Fe3O4 are shown in Fig

3.20(a-c). An elevated index of apoptosis was observed in cells treated with TLMNPs

and compared with control cells are shown in Fig 3.20(b,c). The TLMNPs shows more

pro and late apoptotic cell than necrotic cells and it was more significant to inhibit the

breast cancer cells. Generally, there are two pathways were involved in apoptosis: the

extrinsic pathway, activated by cell death receptors and the intrinsic or mitochondrial

pathway, which is usually activated by DNA damage and it was controlled by the tumor

suppressor gene [70]. The present study implies the reactive species and oxidative stress

of the thymoquinone loaded Fe3O4 nanoparticles confirms the intrinsic pathway to induce

the apoptosis triggered by DNA damage [71].

3.3 Discussion

The already available literatures reveal that the uncontrolled growth of tumor is

not only due to over proliferation but also due to the loss of natural cell apoptosis.

Therefore, exploring the new medicine that could induce tumor cell apoptosis would be

helpful for this kind of treatment. So, in the present study, we have chosen two natural

anticancer compounds such as quercetin and thymoquinone loaded in superparamagnetic

Fe3O4 nanoparticles due to their bio-availability.

3.3.1 Quercetin loaded Fe3O4 nanoparticles (QLMNPs)

The schematic reaction mechanism for the overall synthesis of the QLMNPs is

shown in Fig 3.21. The pristine and dextran coated magnetite nanoparticles were

prepared in the size range of less than 50 nm. The synthesized nanoparticles were cubic

phase without any impurities have been confirmed from XRD analysis. The in-situ

coating of dextran molecules could improve the agglomeration free nanoprism shape

from agglomerated spherical particles (pristine nanoparticles) due to strong driving

repulsive force and also provides biocompatibility. Additionally, the biocompatible

dextran coating enhances the blood circulation time for making it as a promising

candidate for targeted delivery. Afterwards, the hydrophobic quercetin molecules were

conjugated with dextran coated magnetite nanoparticles via the addition of water soluble

NHS and EDC and it is confirmed from FTIR analysis. The obtained nanoparticles have

superparamagnetic behavior with positive surface charge to improve the suspension




85

O

OHOH

O

CH2O

O

OHOH

OH

CH2O

m

n+

OH

COOHHOOC

HO

COOH

OH

Fe3O4Fe3O4

Qu

erce

tin

O

OOH

OH

HO

OH OH

COOH

COOH

OH

Fe3O4CO

O

NH

S/ E

DC

Fig 3.21. Schematic illustration for the proposed formation mechanism of dextran and

quercetin loaded Fe3O4 nanoparticles

stability. The saturation magnetization was decreases due to the loading of drug

molecules and it induces the non-collinear spins at the particles surface compared with

pristine nanoparticles. The positive surface charge was obtained due to the anionic

surface of quercetin molecules which enhances the cell interaction due to strong

electrostatic force. So, the quercetin drug releasing mechanism could depend on the

different pH conditions. In the basic condition, the drug releasing and releasing rate is

less compared with acidic. However, the acidic environment could have more plausible

effects to interact with cancerous cells than normal cells. So, we get the deliberate drug

release rate of 81.6 % due to the increasing circulation time and higher involvement of

the magnetite nanoparticles. The high percentage of the drug release rate was obtained

due to the formation of ester bond between drug and dextran coated magnetite

nanoparticles as shown in Fig 3.21. The ester bond is easily breakable in biological

environment due to enhanced nucleophilic reaction. The dextran coated magnetite

nanoparticles did not show any induced cytotoxicity due to the biocompatible coating and

the absence of anticancer drugs which shows cytocompatible to MCF-7 cells. In the case

of QLMNPs, the cytotoxicity was gradually increased upto above 75 % when increasing




86

the concentration from 10-100 µg/ml. It happens only the release rate of the quercetin is

soaring due to breakable of ester bond from Fe3O4 nanoparticles and interacts with

particular cancer cells in acidic environment. Thus, it was confirmed that the loss of cell

viability is due to the enormous release rate of the quercetin from Fe3O4 nanoparticles.

The results confirm that the Fe3O4 nanoparticles could be considered as a sustained drug

delivery system with quercetin to improve bioavailability for normal cells and toxic to

breast cancer cells. The colored cell images observed from optical microscope further

confirms the nuclear fragmentation, cell shrinkage and membrane bleebing that undergo

apoptosis in MCF-7 cells. The DAPI image shows the high bright blue concentration and

fragmentation of cells in 73 µg/ml. So, the higher concentration of the QLMNPs induces

rapid apoptosis due to good internalization and specific binding of drug molecules

compared to the lower concentration [75]. Therefore, the result clearly exhibits the

potential anticancer activity of breast cancer cells due to fast release of drug, acidic

environment, concentration dependent and apoptosis mechanism using quercetin loaded

Fe3O4 nanoparticles.

3.3.2 Thymoquinone loaded Fe3O4 nanoparticles (TLMNPs)

Fabrication of thymoquinone loaded Fe3O4 nanoparticles involves three steps.

Initially, the ferric chloride decomposes at high temperature (200 °C) in polyol medium.

It provides the Fe species which was exclusively react with KOH medium in the closed

reaction system to form Fe3O4 nanoparticles. Then, the ordered self assembly of the

primary seed particles influences the building blocks of monodispersed hierarchical shape

by tuning of homogeneous nucleation via in-situ addition of PVP. The in-situ surface

modification could enhances the water solubility, prolonged circulation time and

imparted a stealth-shielding shell. Then, the surface of the magnetite nanoparticles has

been modified with active site with the addition of NHS and EDC through ultra-

sonication process. Because, these amino and carboxyl groups were used to convert the

surface as hydrophilic behavior and tendency to conjugate with drug molecules. Finally,

the thymoquinon drug was permeated into the mesoporous magnetite nanoparticles with

the aid of the nanoprecipitate method. The physio-chemical characterization shows the

successful wrapping of thymoquinone in mesoporous Fe3O4 nanoparticles and exhibits




87

Fig 3.22. Illustration of cellular interaction mechanism for the thymoquinone loaded

Fe3O4 nanoparticles and breast cancer cells

superparamagnetic behavior suitable for targeted drug delivery. The schematic diagram

in Fig. 3.22 shows the enhanced mechanism of the cellular uptake and in-vitro inhibitory

activity of breast cancer cells using thymoquinone loaded Fe3O4 nanoparticles. Initially,

the TLMNPs were entered into the cellular system through endocytosis process. Then,

the TLMNPs directly interact with the mitochondrial membrane and also found to

agglomerate around the nuclear membrane into nucleus [76]. In this biological

environment, the thymoquinone drug was released from Fe3O4 nanoparticles through

weak Van der Waals force. These thymoquinone molecules will accelerate apoptosis

progression through DNA damages or membrane blebbing and resulting cellular

decompositions. Therefore, the anti-proliferation activity of TLMNPs was analyzed by

in-vitro method and the MTT result describes high cytotoxicity at low concentration of

30µg/ml. It was ascribed owing to its enhanced permeation and prolonged localization

inside the cellular cytoplasm [77]. The drug releasing from TLMNPs to the particular

target site was responsible to improve the cytotoxicity than pure thymoquinone.

Therefore, the mesoporous shape and positive zeta potential values plays an important




88

role in tumor uptake and intracellular distribution. In addition, the DAPI staining

provides further evidence for thymoquinone loaded magnetite nanoparticles uptake and

predominant amount of drug accumulation in the nucleus of MCF-7 cells after

internalization into the cytoplasm, which causes the cellular damage and the onset of

apoptosis. It may happen due to the interaction of thymoquinone and proteins on the

breast cancer cells through NH2 functional group [77]. So, it adversely affects the cellular

function and induces cell death and cancerous tissue apoptosis. Therefore, the

thymoquinone loaded Fe3O4 nanoparticles have water soluble, good stability and anti-

proliferation activity on targeting of tumor cell to enhance the drug index of the targeted

agents and reducing the side effects.

3.4 Conclusion

The surface functionalized monodispersed superparamagnetic Fe3O4 nanoparticles

were successfully synthesized by solvothermal method. Quercetin and thymoquinone

were chosen as model anticancer drugs and loaded on these polymer coated magnetite

nanoparticles by nanoprecipitation method. The morphological studies confirm the

surface modified magnetite nanoparticles prevent the agglomeration and it forms

mesoporous, nanoprism and ultra-small nanospheres with the size range of 10-100 nm.

Corresponding, HRTEM and FFT pattern analysis further confirms the single

crystallinity of the nanoparticles. The amine, PEG and PVP plays an important role in

producing the monodispersed spherical shape when compared to pristine magnetite

nanoparticles. Furthermore, the room temperature magnetic properties confirm the

superparamagnetic behavior. The presence of polymer could enhances the water

solubility, drug releasing and increasing the stability during administration of Fe3O4

nanoparticles. It could deliver the pre-determined encapsulated drug more rapidly at

acidic pH compared to basic, shows the efficiency for rapid tumoricidal action.

A comparative cytotoxicity study demonstrates that there is no toxic effect in MCF-7

cells caused by free drug and pristine Fe3O4 nanoparticles. Whereas the magnetite

nanoparticles carried with quercetin or thymoquinon have higher cytotoxicity. Thus, the

magnetite particle size plays a vital role as nanocarriers for accurate targeting and the

releasing drug molecules to improve the anticancer activity in breast cancer cells. It was

also observed from the cellular study that quercetin/thymoquinone loaded Fe3O4




89

nanoparticles accumulated more in tumor cells. It shows the significant tumor regression

effect due to the cellular decomposition and apoptosis of breast cancer cells. Hence, the

developed method shows an effective manner in delivering water insoluble compounds to

target cancer cells and enhanced biological applications.

In future, the systematic clinical correlations (in-vivo), metabolism and depth

mechanism are required to consider in both quercetin and thymoquinone loaded

magnetite nanoparticles to study the potential function against several types of cancers.




90

References

1. C. M. baubeta, K. Simeonidis, A. Makridis et al., Scientific Reports, 3 (2013) 1-8.

2. A. M. Nowicka, A. Kowalczyk, A. Jarzebinska, M. Donten, P. Krysinski, Z. Stojek,

Biomacromolecules, 14 (2013) 828-833.

3. L. Xiao, J. Li, D. F. Brougham, E. K. Fox, N. Feliu, A. Bushmelev, A. Schmidt,

N. Mertens, F. Kiessling, M. Valldor, B. Fadeel, S. Mathur, ACS nano., 5 (2011)

6315-6324.

4. R. G. Chaudhuri, S., Chem. Rev., 112 (2012) 2373–2433.

5. C. C. Huang, K. Y. Chuang, C. P. Chou, M. T. Wu, H. S. Sheu, D. B. Shieh,

C. Y. Tsai, C. H. Su, H. Y. Lei, C. S. Yeh, J. Mater. Chem., 21 (2011) 7472-7479.

6. H. T. Hai, H. Kura, M. Takahashi, T. Ogawa, Journal of Colloid and Interface

Science, 341 (2010) 194–199.

7. L. H. Reddy, J. L. Arias, J. Nicolas, P. Couvreur, Chem. Rev., 112 (2012)

5818−5878.

8. S. Liu, Y. Han, R. Qiao, J. Zeng, Q. Jia, Y. Wang, M. Gao, J. Phys. Chem. C, 114

(2010) 21270-21276.

9. A. J. Cole, A. E. David, J. Wang, C. J. Galban, H. L. Hill, V. C. Yang,

Biomaterials, 32 (2011) 2183-2193.

10. N. Arsalani1, H. Fattahi, M. Nazarpoor, eXPRESS Polymer Letters, 4 (2010) 329-338.

11. D. H. Zhang, G. D. Li, J. X. Li, J. S. Chen, Chem. Commun., 29 (2008) 3414-3416.

12. A. Sugunan, H. C. Warad, M. Boman, J. Dutta, J Sol-Gel SciTechn., 39 (2006) 49-56.

13. J. M. Shen, T. Yin, X. Z. Tian, F. Y. Gao, S. Xu, ACS Appl. Mater. Interfaces,

5 (2013) 7014-7024.

14. G. Ding, Y. Guo, Y. Lv, X. Liu, L. Xu, X. Zhang, Colloids and Surfaces B:

Biointerfaces , 91 (2012) 68-76.

15. A. Kumari, S. K. Yadav, Y. B. Pakade, B. Singh, S. C. Yadav, Colloids and

Surfaces B: Biointerfaces, 80 (2010) 184-192.




91

16. T. H. Wu, F. L. Yen, L. T. Lin, T. R. Tsai, C. C. Lin, T. M. Cham, International

Journal of Pharmaceutics, 346 (2008) 160-168.

17. S. Chakraborty, S.Stalin, N. Das, S. T. Choudhury, S. Ghosh, S. Swarnakar,

Biomaterials, 33 (2012) 2991-3001.

18. M. Kakran, N.G. Sahoo, L. Li, Colloids and Surfaces B: Biointerfaces, 88, (2011)

121-130.

19. P. Wang, D. Heber, S. M. Henning, Food Funct., 3 (2012) 3, 635-642.

20. Y. Gao, Y. Wang, Y. Ma, A. Yu, F. Cai, W. Shao, G. Zhai, Colloids and Surfaces

B: Biointerfaces, 71 (2009) 306-314.

21. H. Patir, S. K. S Sarada, S. Singh, T. Mathew, B. Singh, A. Bansal, Quercetin

Free Radical Biology and Medicine, 53 (2012) 659-668.

22. M. Y. Wong, G. N.C. Chiu, Nanomedicine: Nanotechnology, Biology, and

Medicine, 7 (2011) 834-840.

23. A. R. Patel, P. C.M Heussen, J. Hazekamp, E. Drost, K. P. Velikov, Food

chemistry, 133, (2012), 423-429.

24. R. Khonkarn, S. Mankhetkorn, W. E. Hennink, S. Okonogi, European Journal of

Pharmaceutics and Biopharmaceutics, 79 (2011) 268-275.

25. A. C. H. Barreto, V. R. Santiago, S. E. Mazzetto, J. C. Denardin, R. Lavin,

Giuseppe Mele, M. E. N. P. Ribeiro, Icaro G. P. Vieira, Tamara Goncalves, N. M.

P. S. Ricardo, P. B. A. Fechine, J Nanopart Res., 13 (2011) 6545-6553.

26. S. Ahmad, Z. H. Beg, Food Chemistry, 138 (2013) 1116-1124.

27. J. N. Losso, H. A. Bawadi, M. Chintalapati, Food Chemistry 128 (2011) 55-61.

28. R. S. Stock, I. H. Fakhoury, A. M. Zaki, C. O. El-Baba, H. U. G. Muhtasib, Drug

Discovery Today 19, (2014) 18-30.

29. M. Yusufia, S. Banerjee, M. Mohammad, S. Khatal, K. V. Swamy, E. M. Khan,

A. Aboukameel, F. H. Sarkar, S. Padhye, Bioorganic & Medicinal Chemistry

Letters, 23 (2013) 3101-3104.




92

30. K. M. Sutton, C. D. Doucette, D. W. Hoskin, Biochemical and Biophysical

Research Communications, 426 (2012) 421-426.

31. B. Du, A. Mei, P. Tao, B. Zhao, Z. Cao, J. Nie, J. Xu, Z. Fan, J. Phys. Chem. C, 113

(2009) 10090-10096.

32. F. M. Kievit, F. Y. Wang, C. Fang, H. Mok, K. Wang, J. R. Silber, R. G. Ellenbogen,

M. Zhang, Journal of Controlled Release, 152 (2011) 76-83.

33. J. Topfer, A. Angermann, Materials Chemistry and Physics, 129 (2011) 337-342.

34. B. J. Xie, C. Xu, N. Kohler, Y. Hou, S. Sun, Adv. Mater., 19 (2007) 3163-3166.

35. S. Kayal, R.V. Ramanujan, Materials science and Engineering C, 30 (2010) 484-490.

36. T. H. Wu, F. L. Yen, L. T. Lin, T. R. Tsai, C. C. Lin, T. M. Cham, International

Journal of Pharmaceutics, 346 (2008) 160-168.

37. H. Sun, X. Jiao, Y. Han, Z. Jiang, D. Chen, Eur. J. Inorg. Chem., (2013) 109-114.

38. S. Pagola, A. Benavente, A. Raschi, E. Romano, M.A.A. Molina, P.W. Stephens,

AAPS Pharm SciTech 5 (2003) 1-7.

39. A.B. Raschi, E. Romano, A.M. Benavente, A. Ben Altabef and M.E.

Tuttolomondo, Spectrochimica Acta Part, A77 (2010) 497-505.

40. B. Kong, J. Tang, Z. Wu, J. Wei, H. Wu, Y. Wang, G. Zheng, D. Zhao, Angew.

Chem., 126 (2014) 1-6.

41. H. Qu, D. Caruntu, H. Liu, C. J. O‟Connor, Langmuir, 27 (2011) 2271-2278.

42. C. S. Lee, H. H. Chang, P. K. Bae, J. Jung, B. H. Chung, Macromol. Biosci. 13

(2013) 321-331.

43. E. Maltas, M. Ozmen, H. C. Vural, S. Yildiz, M. Ersoz, Materials letter, 65 (2011)

3499-3501.

44. X. Wang, G. Xi, Y. Liu, Y. Qian Crystal Growth & Design 8 (2008) 1406-1411.

45. M. Mahmoudia, A. Simchi, M. Imani, P. Stroeve, A. Sohrabi, Thin Solid Films

518 (2010) 4281-4289.




93

46. Y. Yang, Y. Jia, L. Gao, J. Fei, L. Dai, J. Zhao, J. Li, Chem. Commun., 47 (2011)

12167-12169.

47. X. Wang, J. Qiu, J. Qu, Z. Wang, D. Su, RSC Advances, 2 (2012) 4329-4334.

48. H. Yang, C. Zhang, X. Shi, H. Hu, X. Du, Y. Fang, Y. Ma, H. Wu, S. Yang,

Biomaterials, 31 (2010) 3667-3673.

49. H. Qu, D. Caruntu, H. Liu, C. J. O‟ Connor, Langmuir, 27 (2011) 2271-2278.

50. G. Xi, C. Wang, X. Wang, Eur. J. Inorg. Chem., 3 (2008) 425-431.

51. B. Wang, B. Wang, P. Wei, X. Wang, W. Lou, Dalton Trans., 41 (2012) 896-899.

52. Z. Pu, M. Cao, J. Yang, K. Huang, C. Hu, Nanotechnology, 17 (2006) 799-804.

53. P. Papaphilippou, M. Christodoulou, O. M. Marinica, A. Taculescu, L. Vekas,

K. Chrissafis, T. K. Christoforou, ACS Appl. Mater. Interfaces, 4 (2012) 2139-2147.

54. F. M. Kievit, F. Y. Wang, C. Fang, H. Mok, K. Wang, J. R. Silber, R. G.

Ellenbogen, M. Zhang, Journal of Controlled Release, 152 (2011) 76-83.

55. J. Zheng, Z. Q. Liu, X. S. Zhao, M. Liu, X. Liu, W. Chu, Nanotechnology, 23

(2012) 165601-165608.

56. R. Turcu, O. Pana, A. Nan, I. Craciunescu, O. Chauvet, C. Payen, J. Phys. D:

Appl. Phys., 41 (2008) 245002-245011.

57. K.V.P.M. Shafi, A. Gedanken, Chem. Mater. 10 (1998) 3445-3450.

58. G. Utkan, F. Sayar, P. Batat, S. Ide, M. Kriechbaum, E. Piskin, Journal of colloids

and Interface Science, 353 (2011) 372-379.

59. W. Fua, H. Yang, H. Bala, S. Liu, M. Li, G. Zou, Materials Chemistry and

Physics, 100 (2006) 246-250.

60. B. Du, A. Mei, P. Tao, B. Zhao, Z. Cao, J. Nie, J. Xu, Z. Fan, J. Phys. Chem. C,

113 (2009) 10090-10096.

61. B. Aslibeiki, P. Kameli, I. Manouchehri, H. Salamati, Current Applied Physics,

12 (2012) 812-816.




94

62. S. Ghosh, A.Z.M. Badruddoza, M.S. Uddin, K. Hidajat, Journal of Colloids and

Interface Science, 354 (2011) 483-492.

63. D. Li, J. Tang, J. Guo, S. Wang, D. Chaudhary, C.Wang, Chem. Eur. J., 18 (2012)

16517-16524.

64. Y. Yang, W. Song, A. Wang, P. Zhu, J. Fei, J. Li, Phys. Chem. Chem. Phys., 12

(2010) 4418-4422.

65. Y. Yang, X.H. Yan, Y. Cui, Q. He, D.X. Li, A.H. Wang, J.B. Fei, J.B. Li, J.

Mater. Chem., 18 (2008) 5731-5737.

66. N. A. M. Riviere, A.O. Inman, L.W. Zhang, Toxicology and Applied

Pharmacology, 234 (2009) 222-235.

67. J. M. Shen, T. Yin, X. Z. Tian, F. Y. Gao, S. Xu, ACS Appl. Mater. Interfaces, 5

(2013) 7014-7024.

68. A.B. Salunkhe, V.M. Khot, N.D. Thorat, M.R. Phadatare, C.I. Sathish, D.S.

Dhawaleb, S.H. Pawar, Applied Surface Science, 264 (2013) 598-604.

69. D.W. Nicholson, Nature, 407 (2000) 810-816.

70. E. S. A. Arafa, Q. Zhu, Z. I. Shah, G. Wani, B. M. Barakat, I. Racoma, M.A. E.

Mahdy, A. A. Wani, Mutation Research, 706 (2011) 28-35.

71. S. C. Wuang, K. G. Neoh, E. T. Kang, D. W. Pack, D. E. Leckband, J. Mater.

Chem., 17 (2007) 3354-3362.

72. Y. Xu, A. Karmakar, D. Wang, M. W. Mahmood, J. Phys. Chem. C, 114, (2010)

5020-5026.

73. M. Shaha, N. Ullah, M. H. Choi, M. O. Kim, S. C. Yoon, European Journal of

Pharmaceutics and Biopharmaceutics, 80 (2012) 518-527.

74. B. Ankamwar, T. C. Lai, J. H. Huang, R. S. Liu, M. Hsiao, C. H. Chen, Y. K.

Hwa, Nanotechnology, 21 (2010) 075101-075109.


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