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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: m.i.gibson@warwick.ac.uk; 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

Polym. Chem., 2011, 2, 975–982 | 975

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.

This journal is ª The Royal Society of Chemistry 2011

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,

Polym. Chem., 2011, 2, 975–982 | 977

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

This journal is ª The Royal Society of Chemistry 2011

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

Polym. Chem., 2011, 2, 975–982 | 979

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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This journal is ª The Royal Society of Chemistry 2011