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Thermoresponsive behaviour of poly[(oligo(ethyleneglycol methacrylate)]sand their protein conjugates: importance of concentration and solvent system†
Konstantinos Bebis, Mathew W. Jones, David M. Haddleton and Matthew I. Gibson*
Received 17th December 2010, Accepted 7th January 2011
DOI: 10.1039/c0py00408a
Thermoresponsive poly[oligo(ethyleneglycol) methacrylate]s with a variety of different
oligo(ethyleneglycol) graft lengths were synthesised by reversible-addition fragmentation chain transfer
(RAFT) polymerisation. The lower critical solution temperature (LCST) behaviour of these polymers
was evaluated as a function of the polymer concentration and the concentration of dissolved solutes, in
order to understand their applicability for in vitro and in vivo applications. It was observed that in the
relevant dilute (<1 mg mL�1) concentration range the observed LCSTs increased by approximately 6 �C
compared to higher concentrations. This was confirmed by complimentary dynamic light scattering and
differential scanning calorimetry measurements. The impact of biological solutions on the LCST was
determined using bovine blood plasma, which resulted in observed LCSTs lower than what is found in
traditional buffer or pure aqueous solutions. Finally, a well-defined polymer–protein conjugate was
synthesised by ‘grafting from’ using single-electron transfer (SET) polymerisation. This model
polymer–protein therapeutic also displayed similar concentration dependant behaviour, highlighting
the importance of testing novel ‘smart’ materials and conjugates at both relevant concentration ranges
and in appropriate solvent systems in order to use them in biotechnological applications.
Introduction
Environmentally responsive polymers have received significant
attention due to their rapid conformational changes in response
to an external stimulus.1–4 ‘Smart’ polymers have been syn-
thesised which respond to a wide variety of stimuli including pH,
light, salt concentration, specific ions, carbohydrates and more.
In particular, polymers exhibiting a lower critical solution
temperature (LCST) in aqueous solution are of interest. Upon
heating above the LCST the hydrogen bonds between polymer and
water break leading to demixing and phase separation, which is an
entropy driven process due to the expulsion of water. Upon cooling
this process is reversed, returning to a homogenous mixture and can
be thought of a triggered hydrophilic–hydrophobic switch. This
reversible phase transition has been exploited for a variety of bio-
and nanotechnological applications including: reversible cell
attachment,5 assembly of giant amphiphiles,6 triggered cellular
uptake,7,8 nanoparticle aggregation,9 protein purification10 soluble
sensors11,12 and bacterial aggregation.13 We have demonstrated that
gold nanoparticles coated with a thermoresponsive polymer
undergo diameter-dependent LCST transitions and that binary
mixtures of nanoparticles of different diameters display a single
transition controlled by the mass fraction of each particle.14 This
Department of Chemistry, University of Warwick, Coventry, CV4 7AL,UK. E-mail: [email protected]; Fax: +44 247 652 4112; Tel:+44 247 652 4803
† Electronic supplementary information (ESI) available. See DOI:10.1039/c0py00408a
This journal is ª The Royal Society of Chemistry 2011
cooperative aggregation could be used to immobilise particles
onto complementary surfaces, as has also been shown by
Cameron and co-workers.15 Common examples of polymers
which display LCST behaviour include poly(N-iso-
propylacrylamide) (NIPAM),8,16 poly(2-n-propyl-2-oxazoline)17
and elastin side-chain polymers.18
In 2006 Lutz demonstrated that statistical copolymers of oli-
go(ethyleneglycol methacrylate) (OEGMA) with different side-
chain lengths could display an LCST, which could be tuned by
varying the molar ratio of the different OEGMAs.19 This poly-
mer is appealing as it appears to show high biocompatibility
(inferred from its relationship to linear PEG, but the synthesis
method can also influence this20), prevents protein/cell absorp-
tion21 (like with other PEG-derivatives22), can be prepared by a
variety of controlled radical polymerisation techniques and also
shows limited hysteresis while cycling above/below its LCST.23
Furthermore the degree of polymerisation of the POEGMA does
not strongly influence the LCST,23,24 giving more predictable
properties than e.g. pNIPAM. More detailed studies on the
influence of polymer structure on LCST have been conducted, in
particular highlighting the importance of the polymer end-group
as shown by Theato et al.25 The influence of the end-group is
particularly important for biological applications where it is
common to additionally functionalise polymers with a fluores-
cent dye for in vitro uptake/trafficking analysis. The architecture
of the POEGMAs must also be taken into consideration, as
hyperbranched26 or dendritic27 structures display different LCST
behaviour compared to linear. Another attractive feature of
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Scheme 1 Conditions. [Monomer] : [CTA] : [Initiator]¼ 100 : 1 : 0.2/70�C/90 min. CTA ¼ 2-cyano-2-propyl benzodithioate. Initiator ¼ AIBN.
Fig. 2 Turbidimetry curve showing LCST transition of P3 (5 mg mL�1)
Fig. 1 SEC chromatograms of polymers shown in Table 1, conducted in
DMF (1 g L�1 LiBr) at 60 �C.
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POEGMA is the rather lower dependence of the observed LCST
on concentration compared to, for example, elastin-based poly-
mers in which the LCST increases significantly upon dilution.18,28
This relationship is important for in vivo drug delivery applications
where sub mg mL�1 concentrations of the polymer will be applied
and the concentration of polymer may increase (due to tissue
accumulation) or decrease in the blood stream (due to excretion,
dilution or tissue accumulation). In particular, the targeting of
cancerous tissue due to the increased lipid solubility (and hence
cellular uptake) of thermosensitive polymers above their
LCST,7,8,29 triggered by the increased temperature of tumour
tissue relative to healthy would be particularly dependent on
understanding dilution effects on LCST.
Considering the above, the aim of this work was to investigate
the LCST behaviour of poly[oligo(ethyleneglycol) methacrylate]
across a wide concentration range, including those relevant for
in vivo applications. The influence of salt and blood-plasma
constituents on the LCST is also investigated to determine if
standard testing protocols are sufficient to predict the in vivo
behaviour. Finally, the thermoresponsive behaviour of a well-
defined polymer–protein conjugate as a model drug delivery
system is evaluated. These studies are important to aid in creating
design rules for stimuli responsive materials, and also to aid in the
understanding of in vitro and in vivo behaviour.
during heating in PBS. Normalised absorbance value of 0.5 (cloud point)is defined as being the LCST.
Results and discussion
Poly[oligo(ethyleneglycol) methacrylate] (POEGMA) was syn-
thesised by reversible addition fragmentation chain transfer
(RAFT) polymerisation of the corresponding OEG-methacry-
lates, Scheme 1. Five different polymers were prepared for this
study using [monomer] : [chain transfer agent] ¼ 100 : 1 with
PEG side chain length varying from diethylene glycol to
oligoethyleneglycol475. These were characterised by 1H NMR
and SEC, Fig. 1. All the polymers had narrow polydispersity
indices (<1.2), and the measured Mn agrees with those predicted
Table 1 Polymers used in this studyf
Code Compositiona Conversionb
P1 DEGMA 70%P2 OEGMA475-co-DEGMAe 70%P3 TEGMA 65%P4 OEGMA300 71%P5 OEGMA475 77%
a Indicates the monomer(s) used in the polymerisation. b Determined by 1H Nconversion. d Determined by SEC in DMF using PMMA standards. e Ratif DEGMA ¼ diethylene glycol methacrylate, TEGMA ¼ triethylene glycolthe number average molecular weight of the oligo(ethylene glycol) chain is in
976 | Polym. Chem., 2011, 2, 975–982
by the feed ratio and the conversion of monomer indicating
a controlled polymerisation. The exception was P2, which had
a lower Mn than expected according to SEC, indicating that some
chain transfer or retarded kinetics occurred, Table 1.
With this array of polymers at hand, the LCST behaviour
could be evaluated. A typical turbidimetry plot obtained in
phosphate buffered saline shows an increase in light absorption
due to polymer precipitation as the solution is heated through its
LCST, Fig. 2. Using this curve, the LCST was defined as being
Mn (theo)c/g mol�1 Mn (SEC)
d/g mol�1 Mw/Mnd
13 000 11 400 1.1320 700 9200 1.1513 900 9000 1.2020 700 16 000 1.2136 000 23 300 1.19
MR. c Determined from the [monomer] : [CTA] ratio and the degree ofo of monomers used in polymerisation: PEG9MA : PEG2MA ¼ 8:92.methacrylate, OEGMAxxx ¼ oligo(ethylene glycol)methacrylate wheredicated by XXX.
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Fig. 3 Observed LCST’s (red) and LCST onset (blue) of polymers in Table 1, obtained by turbidimetry in PBS.
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the midpoint of the curve corresponding to a normalised
absorbance of 0.5. Strictly speaking this is the cloud point of the
solution, and the LCST is actually the minimum of the transition
in the phase transition diagram. The quoted LCSTs in this report
are the cloud points, to allow direct comparison with other
literature in the field. The broadness of the transitions was not
considered in this study due to the dependence of this on the test
conditions, (e.g. stirring rate, heating rate, method of analysis). It
should be noted that the curves were often broader than those
shown by Lutz,23 but molecular weights used here are different.
The values of LCST and onset temperature were evaluated for
all polymers in the concentration range of 0.5–5 mg mL�1, which
was the lower limit of sensitivity of the equipment used.
Decreasing the polymer concentration gave an increase in the
This journal is ª The Royal Society of Chemistry 2011
observed LCST, typically 6 �C over the concentration range
tested, Fig. 3. The shape of the curve indicates an exponential
increase at lower concentrations, but these could not be
measured using turbidimetry (vide infra). The onset temperatures
also displayed the same relationship indicating that the whole
process, rather than just the midpoint, is shifted to higher
temperatures by dilution. Commonly, LCST values are evalu-
ated at concentrations above 5 mg mL�1 which is not relevant for
most in vivo applications. If P2 is considered: it was designed to
have an LCST �37 �C (body temperature). At 10 mg mL�1 in
PBS the LCST was 36 �C, but once diluted to 0.5 mg mL�1
(which is still higher than what might be found in vivo) the LCST
transition was 42 �C (Fig. 3). If the target transition was, for
example, 39 �C then the polymer would fail in its application,
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Fig. 4 Determination of the LCST in dilute solution using dynamic light
scattering for P1 in PBS.
Fig. 5 Measured count rate (by dynamic light scattering) of P2 held
isothermally in PBS. [P2] ¼ 1 mg mL�1.
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clearly indicating that concentration is a critical parameter. To
confirm the results from the turbidimetry experiments, dynamic
light scattering (DLS) was used as a complimentary technique,
Fig. 4, monitoring a change in count rate as a function of
temperature as P1 is heated at different concentrations. The
count rate is related to the diameter (or more specifically the
diffusion coefficient) of the species in solution, and an increased
value indicates the formation of larger aggregates (Fig. 5).
At 0.25 mg mL�1 the observed LCST was 28.5 �C and at
0.1 mg mL�1 this increased to 33 �C, which is in good agreement
with the values obtained by turbidimetry. As a final measure of
the LCST, differential scanning calorimetry (DSC) was
employed which can unravel the complicated process of mixing/
demixing,30 but was used here simply to assign a phase transition
temperature. Fig. 6 shows the heat flow through the samples
during heating as a function of polymer (P1) concentration.
Upon heating, all the samples showed an exothermic transition
corresponding to the LCST. The centre of the transition shifted
from 28.5 �C for 0.5 mg mL�1 polymer concentration to 25.4 �C
for 5 mg mL�1. The values agree with those obtained by DLS and
turbidimetry indicating that the shift in the LCST upon dilution
is real and quantifiable. As the LCST is concentration dependent,
this suggests that the transition must involve multiple polymer
chains aggregating together. There may be a kinetic barrier at
low concentrations, i.e. the polymers chains do not ‘find’ each
other as quickly in dilute solution and hence the apparent LCST
with a constant heating rate is increased. Therefore, to rule out
kinetic factors isothermal experiments were conducted using
DLS. P2 was held at either 36 �C or 39 �C (1 mg mL�1) for 20
minutes and the count rate monitored. These temperatures were
carefully chosen: 36 �C is above the onset temperature but below
the LCST and 39 �C is the LCST and thus acts as a reference
sample. If dilution simply slows the aggregation process, the
sample held above the onset temperature, but below the LCST,
would be expected to aggregate and hence show an increase in the
recorded count rate, Fig. 5. At 39 �C, the count rate is extremely
high and does not increase any further during the experiment. At
36 �C there is no increase in the count rate after the first
4 minutes, even though the polymer is heated above its onset
temperature. Heating this sample above the LCST leads to
aggregation and an increase in the count rate. The same
978 | Polym. Chem., 2011, 2, 975–982
experiment conducted using 5 mg mL�1 at 36 �C leads to rapid
aggregation, clearly demonstrating the importance of concen-
tration. Taken together, these data show that concentration is
a critical factor in describing the LCST transition of thermo-
sensitive polymers.
The next step was to evaluate the influence of dissolved
solutes on the LCST, which is again important to be able to
predict in vivo properties. For example, different regions of the
body or cellular compartments have different concentrations of
dissolved solutes. Furthermore, finding the ideal solvent for
performing turbidimetry analysis to demonstrate the potential
of new materials is also essential to allow direct comparison of
the most promising materials. Alexander and co-workers have
previously shown the importance of salt concentration on
POEGMA-based polymers, and their relationship to the Hof-
meister series of ions.31 In the present work the relative influence
of NaCl on the LCST of polymers (P1–P5) with different PEG
side chains was measured. The influence of NaCl on LCST was
measured here and, as expected, increased salt concentration
leads to decreased LCST’s. The rate of change was uniform for
all polymer concentrations and is included in the ESI†. This
strong relationship becomes more important when in vivo
conditions are considered: in the circulation a polymeric ther-
apeutic is likely to be dissolved in blood plasma which has high
concentrations of proteins, amino acids, sugars and salts other
than NaCl. Dried bovine plasma was rehydrated and used as
a model for the blood plasma conditions likely to be encoun-
tered by a polymer therapeutic. P2 was selected as this has an
LCST close to 37 �C and is therefore the most physiologically
relevant.
The measured LCST values of P2 in bovine plasma are typi-
cally 2 �C lower than the same measurement conducted in PBS,
and far lower than those measured in dilute NaCl concentration
and pure water, Fig. 7. The same trend as observed in Fig. 3 was
seen here; reducing polymer concentration increased the LCST.
Control experiments of the plasma solution alone did not indi-
cate any increase in turbidity in the temperature range used,
ruling out false positive results due to aggregation of dissolved
proteins. There are only a few examples of the LCST transition
temperature of thermosensitive polymers being studied in the
dilute concentration range, probably due to the fact that turbi-
dimetry at high (>5 mg mL�1) concentration is a simpler means
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Fig. 6 Differential scanning calorimetry thermograms for P1 during heating.
Fig. 7 Observed LCST for P2 in phosphate buffer saline (black squares)
or bovine plasma (red circles) as a function of polymer concentration.
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of analysis. These results indicate that polymer concentration
and solvent are key factors in the design of POEGMA-based
materials and should be considered more frequently to allow for
comparisons and to attribute observed macroscopic properties
(i.e. cell uptake) correctly.
To exploit LCST transitions for biotechnological or drug
delivery applications it is desirable to have an additional active
component to the thermoresponsive polymer, e.g. a drug mole-
cule.32 We have previously synthesised complex (co)polymers
This journal is ª The Royal Society of Chemistry 2011
and polypeptides33,34 for biotechnological applications by direct
polymerisation of functional monomers or post-polymerisation
modification of preformed polymers.35,36 Alternatively, we
demonstrated the site-specific conjugation of initiators suitable
for controlled radical/single electron transfer (SET) polymerisa-
tion onto reduced cysteine residues via a Michael addition (thiol-
ene ‘click’) process.37 Here salmon calcitonin (sCT), a 32 amino
acid calcitropic hormone currently administered for the treat-
ment of a number of hypercalcemia-related diseases, which can
still function when its disulfide bridge is reduced to free
cystiene38,39 was modified with an initiator for SET-LRP. A
mixture of diethylene glycol methacrylate (DEGMA) and tri-
ethylene glycol methacrylate (TEGMA) was copolymerised by
SET polymerisation to give a polymer–protein conjugate, Scheme
2. The LCST of this conjugate was measured by turbidimetry in
PBS in the range of 5 to 0.5 mg mL�1, Fig. 8. As seen for the
polymers alone, upon decreasing the concentration of the conju-
gate the LCST increased. Temperature-dependent DLS analysis
did not reveal the formation of higher-order structures (micelles,
vesicles), but rather large (>micron sized) agglomerates indicating
aggregation, rather than self-assembly, had occurred. A slight
elevation of the LCST compared to a copolymer of DEGMA/
TEGMA with similar monomer mole fractions (�32 �C) was
observed, but potential differences in copolymer composition
prevent comparisons. Secondly, this conjugate is approximately
10% by weight peptide, meaning the properties of this are strongly
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Scheme 2 Polymer–protein conjugate synthesis. Conditions: (i) TCEP, acryloyloxyethyl 2-bromoisobutyrate; (ii) DEGMA/TEGMA (2 : 1)/Cu(0)/
PMDETA/DMSO/25 �C/180 min/sacrificial initiator.
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controlled by the polymer. Investigations using larger peptides/
proteins would provide an interesting comparison.
These results suggest that when designing new polymeric drug
delivery devices exploiting thermosensitive polymers, the eval-
uation of the LCST properties should be undertaken as a func-
tion of polymer concentration and solvent conditions. This is
essential in order to both fully characterise the material prop-
erties, but also to provide sufficient information to explain/
allow transition into in vivo and in vitro experiments. We are
currently investigating the biological applications of these
responsive materials.
Conclusions
Herein we have evaluated the LCST transition temperatures of
a range of poly[(oligo(ethylene glycol)methacrylate]s as a func-
tion of both the polymer concentration and the aqueous solvent
system with the aim being to better predict their biological
properties. The LCST was evaluated by turbidimetry, DLS and
differential DSC. These measurements indicated that as the
concentration is reduced from 5 to 0.5 mg ml�1 (i.e. close to that
used in therapeutics) the LCST increased by up to 6 �C. The
onset temperature for the LCST transition also shifted to
a similar degree. Similar measurements conducted in bovine
blood plasma indicated a further significant decrease in LCST as
Fig. 8 Observed LCST’s of calcitonin–polymer conjugate in phosphate
buffered saline.
980 | Polym. Chem., 2011, 2, 975–982
compared to either pure water or PBS highlighting the subtle
changes caused by dissolved solutes. A well defined polymer–
protein conjugate based on salmon calcitonin and POEGMA
was synthesised and demonstrated to display similar concentra-
tion dependant behaviour, indicating that these observations
apply to actual drug delivery devices not just the isolated
polymer.
It is important to emphasise that the aim of this work was to
highlight the need to consider physiological conditions when
synthesising new thermosensitive polymer-based conjugates for
biological applications, in particular the dependence of the LCST
of the concentration of the polymer (or conjugate) under
conditions which are relevant for their intended applications.
The large increase in LCST upon dilution could lead to the
failure of polymer delivery systems, simply due to inappropriate
testing protocols. These results will be used in the future to
optimise polymeric drug delivery system for in vivo and in vitro
applications.
Experimental
Materials
Oligo(ethylene glycol)n methacrylates, with an average n ¼ 2, 5
and 9, were all purchased from Sigma-Aldrich and passed
through a column of basic alumni prior to use. Triethylene glycol
methacrylate, initiator functionalised (sacrificial) Wang resin and
the salmon calcitonin macroinitiator were synthesised as previ-
ously reported.37 Azobisisobutyronitrile (AIBN), N,N,N0,N0,N0 0-
pentamethyldiethylenetriamine (PMDETA), Cu(0) wire (0.25
mm diameter) and sodium chloride (>99%) were purchased from
Sigma-Aldrich and used as received. The RAFT agent cyano-2-
propyl benzodithioate was purchased from Aldrich. Ultrahigh
quality water with a resistance of 18.2 MU cm (at 25 �C) was
obtained from a Millipore Milli-Q gradient machine fitted with
a 0.22 mm filter. Pre-formulated, powdered, phosphate buffered
saline was purchased from Sigma-Aldrich and the desired solu-
tion made by addition of ultrahigh quality water to give [NaCl]¼0.138 M, [KCl] ¼ 0.0027 M and pH ¼ 7.4. Lyophilized bovine
plasma was purchased from Sigma-Aldrich and reconstituted
according to the manufacturers specifications using high-quality
water.
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Analytical and physical methods
1H and 13C NMR spectra were recorded on Bruker DPX-300 and
DPX-400 spectrometers using deuterated solvents obtained from
Sigma-Aldrich. SEC was conducted on a Varian 390-LC system
in DMF (1 g L�1 LiBr) at 50 �C, equipped with refractive index
and viscometry detectors, 2 � PLgel 5 mm mixed-D columns
(300� 7.5 mm), 1� PLgel 5 mm guard column (50� 7.5 mm) and
autosampler. Data were analysed using Cirrus 3.2 software.
Molecular weight was determined relative to narrow poly(methyl
methacrylate) standards. Lower critical solution temperatures
were evaluated using OptiMelt MPA100 system from Stanford
Research Systems. The LCST was determined by normalising the
turbidimetry curve such that the values were in the range of 0 to 1,
and the transition temperature was defined as being the tempera-
ture corresponding to a normalised absorbance of 0.5. A constant
heating rate of 2 �C min�1 was used in all experiments. Dynamic
light scattering was conducted using a Nano-Zs from Malvern
Instruments, UK. Scattered light was detected at 173� and the
observed count rates recorded. Hydrodynamic radii (where
appropriate) were determined using the manufacturer’s software.
Procedures
Synthesis of poly[poly(ethylene glycol methyl ether-
methacrylate)]
As an example the polymerisation using di(ethyleneglycol)
methacrylate is given (P2 in Table 1). Diethylene glycol meth-
acrylate (2 g, 10 mmol) was added to a Schlenk tube and 2 mL of
dioxane added. Next, 0.67 mL of a dioxane solution containing
0.16 M 2-cyano-2-propyl benzodithiolate and 24 mM of azobi-
sisobutyronitrile (AIBN) was added to the Schlenk tube by
syringe giving [monomer] : [initiator] : [chain transfer agent] ¼100 : 0.2 : 1. The solution was degassed by 4 freeze–pump–thaw
cycles and back-filled with nitrogen gas. The flask was then
immersed into a thermostated oil bath at 70 �C for 180 minutes.
After this time, a 25 mL sample was removed and diluted with
CDCl3 for NMR analysis. The remainder was rapidly cooled in
an ice-water bath and precipitated into diethyl ether (35 mL).
The polymer was re-precipitated from THF to diethyl ether twice
to yield a waxy pink polymer. Isolated yield: 1.05 g, 53%;
Conversion (NMR): 70%; Mn (theo): 20 700 g mol�1; Mn (SEC):
9200 g mol�1; Mw/Mn ¼ 1.15 (SEC).1H NMR (300 MHz, CDCl3) dppm: 1.41 (3H, backbone-CH3)
1.80–2.00 (2H, backbone-CH2), 3.35 (3H, CH3-PEG), 3.40–3.80
(16H, CH2CH2O), 4.09 (2H, CH2OC(]O)), 7.42 (o-Ar,
end-group), 7.61 (p-Ar, end-group), 7.85 (m-Ar, end-group).
Salmon calcitonin-poly(DEGMA-co-TEGMA) conjugate
The salmon calcitonin macroinitiator (4.2 mg, 1.3 mm) was added
to a Schlenk tube along with an initiator-functional Wang resin
(0.442 g, 1.32 mmol), 5 cm of copper (0) wire (0.25 mm diameter),
copper (II) bromide (29.6 mg, 0.13 mmol) and a magnetic stirrer
bar. The Schlenk was sealed, thoroughly degassed and purged
with nitrogen. In a separate Schlenk tube, diethylene glycol
methacrylate (3.30 g, 17.5 mmol), triethylene glycol methacrylate
(2.05 g, 8.8 mmol), DMSO (10 mL), N,N,N0,N0,N0 0-pentam-
ethyldiethylenetriamine (0.43 mL, 1.99 mmol) and mesitylene
This journal is ª The Royal Society of Chemistry 2011
(0.37 mL, 2.6 mmol) were added and subjected to four freeze–
pump–thaw cycles. The degassed solution was transferred via
cannula to the first Schlenk tube and immersed in an oil bath at
25 �C for 180 min at which point the polymerisation was
quenched in liquid nitrogen and filtered to remove solids. The
remaining solution was dialysed against a 50 : 50 mixture of
water/methanol for 3 days and 1 day against pure water. The
solution was then lyophilised and the conjugate isolated as a
colourless oil. Mn (SEC): 33 000 g mol�1; Mw/Mn ¼ 1.49.
Acknowledgements
Equipment used was supported by the Innovative Uses for
Advanced Materials in the Modern World (AM2), with support
from Advantage West Midlands (AWM) and part funded by the
European Regional Development Fund (ERDF). MIG is a Bir-
mingham Science City Interdisciplinary Research Fellow, sup-
ported HEFCE. We thank EPSRC and Warwick Effect
Polymers for funding (MJ).
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