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By Francisco Fernandez-Trillo, Jan C. M. van Hest, Jens C. Thies,

Thierry Michon, Ralf Weberskirch, and Neil R. Cameron*

Biocompatible surfaces are of considerable interest inbiotechnology, with application in the immobilization of, inter

alia, proteins,[1] (including enzymes[2]) and DNA.[3] Commonlyused substrates for such immobilization include silicon,[4]

mica,[5] gold,[6] and polymeric matrices.[7] Amongst the methodsavailable for enzyme immobilization, adsorption appears attrac-tive because of its simplicity and low cost. However, adsorptiongenerally does not lead to very stable immobilization, andenzymes tend to become desorbed during catalysis and recycling,leading to decreased activity over time. Furthermore, since thereis a lack of control over the enzyme positioning, only a fraction ofthe enzyme may be available for catalysis, reducing significantlythe support efficiency.

Recently, we have described a simple and effective strategy forthe immobilization of Candida Antartica Lipase B (CAL-B) ontohighly porous polymeric supports.[8] N-acryloxysuccinimide wasused to prepare polymerized high internal phase emulsions(PolyHIPE) bearing N-hydroxysuccinimide (NHS) esters to allowcovalent coupling with lysine residues present on the surface ofthe protein. PolyHIPEs are highly porous polymers with awell-defined, open cellular morphology, and excellent flow-through properties,[9] making them ideal supports for solid-phasesynthesis,[10] catalysis,[11] superadsorbents,[12] electrochemicalsensors,[13] or matrices for in vitro cell culture in 3D.[14] Thecombination of PolyHIPE morphology and covalent attachment

[*] Dr. N. R. Cameron, Dr. F. Fernandez-TrilloInterdisciplinary Research Centre in Polymer Science & TechnologyDepartment of Chemistry, University of DurhamSouth Road, Durham DH1 3LE (UK)E-mail: n.r.cameron@durham.ac.uk

Dr. J. C. ThiesDSM Research Campus GeleenPerformance Materials–Chemistry & Technology Lab 094.2.201Urmonderbaan 22, 6167 RD Geleen (The Netherlands)

Prof. J. C. M. van HestRadboud University NijmegenOrganic Chemistry Institute for Molecules and MaterialsToernooiveld 1, 6525 ED Nijmegen (The Netherlands)

Dr. T. MichonINRA, UMR GDPP, IBVM, Interactions Plante VirusBP 81, 33883 Villenave d’Ornon Cedex (France)

Dr. R. WeberskirchDepartment Chemie, Technische Universitat MunchenLehrstuhl fur Makromolekulare StoffeLichtenbergstr. 4, 85747 Garching (Germany)

DOI: 10.1002/adma.200801986

Adv. Mater. 2009, 21, 55–59 � 2009 WILEY-VCH Verlag Gmb

of the enzyme onto the support turned out to be very suitable forthe immobilization of CAL-B. High-biocatalytic activity wasobserved with a relatively low loading of the enzyme, whencompared to commercially available sources of immobilizedCAL-B, as well as stable levels of enzymatic activity afterbiocatalyst reuse.

Despite this, there are several drawbacks to the use of enzymescovalently immobilized onto insoluble supports, for instance,nonlinear kinetic behavior and solvation problems associatedwith the nature of the support. The latter will affect the diffusionof substrates through the matrix and can even cause loss ofactivity due to conformational changes on the surface of thesupport.

As a means to overcome some of these problems, stimu-li-responsive polymers, the solubility of which can be regulated bychanges in pH, ionic strength, or temperature, have been used forthe immobilization of enzymes.[15] These polymers allow thebiotransformation to be performed in solution, thereforeminimizing some of the limitations described above. Thebiocatalyst can be recovered at the end of the reaction bychanging the conditions (pH, salt concentration, or temperature)to promote precipitation of the polymer–enzyme conjugate.

Elastin-like peptides (ELPs) are amongst the most widelystudied thermoresponsive polymers. ELPs are linear peptidescomposed of repeating units of a pentapeptide (VPGXG, where Xcan be any amino acid except proline) that display lower criticalsolution transition (LCST) behavior, that is, below the transitiontemperature (Tt) the polymer is soluble in aqueous solution, butwhen the temperature is raised above Tt the polymer chainsaggregate, leading to an insoluble phase.[16] They have beendescribed in the literature for the reversible immobilization ofenzymes onto flat surfaces such as glass[17] or gold slides.[6] Inthese approaches, ELPs were attached to a surface in order topromote the reversible assembly with ELP–enzyme fusionproteins, a technique the authors termed thermodynamicallyreversible addressing of proteins (TRAP). This could haveapplication in bioanalytical devices, as well as for the preparationof rewritable peptide and protein arrays.

Recently, we described the synthesis of elastin-based side-chain polymers (EBPs),[18–20] in which a methacrylate derivativeof the pentapeptide VPGVG was polymerized using controlledradical polymerization (CRP) techniques, to give well-definedsynthetic polymers with a thermoresponsive behavior similarto linear ELPs. These EBPs can be viewed as chemically accessiblevariants of ELPs. In this paper, we describe the reversible im-mobilization of EBPs onto PolyHIPEs, by harnessing the

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coassembly of EBPs. This could result in an immobilizationplatform that combines the best features of heterogeneous andhomogenous catalysis; the reaction occurs in solution and theenzyme is subsequently sequestered onto the highly porous, highcapacity–substrate by altering conditions.

To prevent non-specific and, possibly, irreversible adsorption ofEBPs onto the porous support, it was reasoned that a hydrophilicPolyHIPE matrix would be required. PolyHIPEs based onhydrophilic monomers such as acrylamide[21] and acrylic acid[22]

have been described recently. A common approach when trying tominimize non-specific protein adsorption onto surfaces isPEGylation,[23] in which the surface of interest is covered withpoly(ethylene glycol) (PEG) chains. The effectiveness of thisstrategy is attributed to PEG’s large excluded volume in aqueoussolution and hydrated chain mobility, which prevents proteinadsorption.

The synthesis of PEGylated PolyHIPEs using commerciallyavailable poly(ethylene glycol) methacrylate (PEGMA), employingoil-in-water (o/w) HIPEs as described by Cooper and coworkersfor the preparation of polyacrylamide PolyHIPE beads, wasinvestigated.[21] Despite its water solubility, methylene bisacry-lamide (MBAM) could not be dissolved easily in the monomer(aqueous) phase, and the emulsions obtained were very unstable(Table 1, entries 1 and 2). This problem was solved partially byusing a cosolvent such as DMF (Table 1, entries 3 and 4). To oursurprise, however, the more hydrophobic cross-linker ethyleneglycol dimethacrylate (EGDMA) gave much better results,probably due to its higher solubility in the monomer phase,which has a high content of chemically similar PEGMA. Thisallowed us to increase themonomer content in the aqueous phaseup to 50% by weight (Table 1, entries 5 and 6).

In general, apart from polyacrylamide PolyHIPE (Fig. 1a), thereverse (i.e., oil-in-water, o/w) HIPEs were fairly unstable overtime, and large voids could be seen after curing, due to oil dropletcoalescence (Fig. 1b and c). In addition, PEGylated PolyHIPEswere found to be quite elastic, and tended to shrink upon drying,making their characterization by standard methods difficult. Infact, when a more flexible cross-linker such as poly(ethyleneglycol) dimethacrylate (PEGDMA) was used (Table 1, entries 7–9),

Table 1. Conditions for the preparation of hydrophilic polyHIPEs based on P

PolyHIPE Monomers [w/v][a] Oil phase[v/v][b] Surfac

1 PEGMA/MBAM 85:15 (30) LMO (76) Triton

2 PEGMA/MBAM 85:15 (30) LMO (77) SD

3 PEGMA/MBAM 80:20 (20) LMO (80) SD

4 PEGMA/MBAM 80:20 (23) LMO (83) SD

5 PEGMA/EGDMA 80:20 (45) LMO (90) SD

6 PEGMA/EGDMA 80:20 (50) LMO (85) SD

7 PEGMA/PEGDMA 80:20 (50) LMO (88) SD

8 PEGMA/PEGDMA 80:20 (50) Cy/LMO 1:1 (90) SD

9 PEGMA/PEGDMA 80:20 (50) Cy (85) SD

[a] PEGMA¼ poly(ethylene glycol) methacrylate (Mw¼ 360 gmol�1); MBAM¼methylen

glycol) dimethacrylate (Mw¼ 875 gmol�1). Weight per volume ratio, defined as the t

parentheses; [b] LMO¼ light mineral oil; Cy¼ cyclohexane; volumetric ratio of oil phas

SDS¼ sodium dodecyl sulfate. Weight per volume ratio, defined as the total mass of s

DMF¼dimethylformamide; PVA¼ polyvinyl alcohol (Mw¼ 22000 gmol�1, 88% hydro

total volume of aqueous phase, shown in parentheses; [e] Polymerization temperatu

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the PolyHIPE shrank completely, and its morphology (voidsize and volume, window size, and surface area) could not becharacterized (Fig. 1d). Despite this shrinking behavior, theobservation that PEGylated PolyHIPEs recover their originalshape, and presumably their pore structure, upon rewettingencouraged us to use them for the reversible immobilization ofEBPs.

Previously, we found that EBPs show an interesting co-operative assembly behavior in that two polymers of sufficientlydifferent molecular weight, with well-separated Tt values,undergo a single transition, at a temperature between the Ttvalues of the individual polymers, when mixed together in thesame solution.[20] This could have important implications in thedesign and application of this kind of polymers, since it allowsco-assembly to be favored over self-assembly. With this co-operation in mind, we prepared two EBPs with identical chemicalstructures but different molecular weights; a ‘‘short’’ polymer,which would be present in solution, and a ‘‘long’’ polymer, whichwould be covalently attached to the support. The molecularweights of the short and long polymers were chosen so that,under the assembly conditions, the short polymer in solutionwould be below its Tt, whereas the long polymer would be aboveits Tt. This would result in co-assembly onto the support, ratherthan self-assembly of the short polymer in solution.

Thus, VPGVG-methacrylate derivative 1 (MA-VPGVG) waspolymerized by the reversible addition fragmentation chaintransfer (RAFT) method using dithioester 2 as the chain transferagent and commercially available 4,40-azobis- (4-cyanopentanoicacid) as the initiator, at 70 8C, to give two sets of well-defined,narrow molecular weight distribution EBPs (Scheme 1). Thepredicted number average degree of polymerization (DPn,th) ofthese polymers was altered by varying the ratio of 1 relative to 2(29:1 or 88:1; see Table 2). An interesting feature of RAFTpolymerization is that the dithioester moieties at one end of eachpolymer chain can be reduced easily under basic conditions,[24] toproduce thiol-ended polymers, which are very suitable forconjugation onto Michael acceptors such as maleimides. Bearingthis in mind and in order to attach the higher molecular weightpolymer (B) to the porous matrix, we decided to transform its

EGMA.

tant[w/v][c] Additives[w/v][d] T(8C)[e] Notes

X-405 (10) PVA (1.4) 70 Emulsion not stable

S (10) PVA (1.4) – Emulsion not stable

S (5) DMF (14) 90 –

S (6.5) DMF (23)þ PVA (1) 54 –

S (10) – r.t. –

S (10) DMF (17) r.t. –

S (10) – r.t. Shrank upon drying

S (10) – r.t. Shrank upon drying

S (18) – r.t. Shrank upon drying

e bisacrylamide; EGDMA¼ ethylene glycol dimethacrylate; PEGDMA¼ poly(ethylene

otal mass of monomers divided by the total volume of aqueous phase, shown in

e shown in parentheses; [c] Triton X-405¼ polyoxyethylene isooctylcyclohexyl ether;

urfactant divided by the total volume of aqueous phase, shown in parentheses; [d]

lyzed); weight per volume ratio, defined as the total mass of additive divided by the

re.

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Figure 1. Scanning electron micrographs of hydrophilic PolyHIPEs.a) In-house prepared polyacrylamide PolyHIPE, shown for comparison.b) PolyHIPE 6. c) PolyHIPE 5. d) PolyHIPE 9. Compositions describedin Table 1.

Table 2.Molecular weight data, thermoresponsive behavior and end groupfunctionalization of EBPs.

Polymer DPn,th[a] DPn[b] Mn[c] [Kg �mol�1] PDII[d] Tt [8C][e] End group

A 29 46 25.1 1.03 – Dithio-ester

B 88 115 62.5 1.31 – Dithio-ester

C 29 43 23.5 1.05 54.1 Acid

D 29 47 26.0 1.06 46.8 (23.0) Acid[f ]

E 88 113 61.6 1.23 37.4 (19.8) Acid

F 88 124 67.1 1.22 – Thiol

[a] Theoretical number-average degree of polymerization (see Supplementary Infor-

mation for calculation formula); [b] number-average degree of polymerization deter-

mined by aqueous size exclusion chromatography; [c] number average molecular

weight, determined by aqueous size exclusion chromatography; [d] polydispersity

index (Mw/Mn), determined by aqueous size exclusion chromatography; [e] transition

temperature, calculated from turbidimetry experiments in phosphate buffer, [poly-

mer]¼ 0.23mgmL�1, pH adjusted to 3.2 (1.5). Values quoted are the inflection

points of the heating curves; [f ] Some of the acid groups in the polymer were esterified

with Hostasol as a fluorescent probe.

dithioester group into a thiol (polymer F, Table 2, Scheme 1).Regarding the lower molecular weight polymer (A), we decided toremove the dithioester end-group by treatment with an excess ofazo initiator (polymer C),[25] in order to avoid possible sidereactions upon heating, as has already been discussed in ourprevious work.[19] In addition, some of the acid groups in thepolymer C were esterified with a hostasol derivative as afluorescent probe (polymer D, Table 2), which would allow us tofollow the immobilization (see Supporting Information fordetailed experimental procedures of all transformations, andGPC traces of representative polymers).

As shown previously,[19] EBPs are not only thermoresponsivebut also pH-responsive, due to the CO2H group of the amino acidat the carboxy terminus of the peptide chain (Gly). Forexperimental convenience, we decided to use changes in pHin order to promote the transition and thus reversibleimmobilization. Firstly, we examined the pH dependence of Ttfor the two sets of polymers. The Tt value was taken as themid-point of the cloud point curve determined by UV–vis

Scheme 1. Structure of EBPs, methacrylate-functionalized VPGVG(MA-VPGVG, 1), and the RAFT agent 2 used in this work.

Adv. Mater. 2009, 21, 55–59 � 2009 WILEY-VCH Verlag Gmb

spectroscopy, for solutions of polymers at different pH and at afixed concentration (0.23mgmL�1). Analysis of the curves (Fig. 2)reveals that, at pH 1.5, the fluorescently labeled low molecularweight polymer (D) will not undergo a transition if thetemperature is kept below 23 8C, while the higher molecularweight polymer (E) will undergo a transition at around 20 8C. Itshould be noted that, due to side reactions of the thiol moietyupon heating, it was not possible to perform turbidimetryexperiments with polymer F.

We decided to use the easily accessible thiol end groups (i.e.,polymer F) for conjugation of the high molecular weight polymeronto the porous support. To achieve this, the PolyHIPE surfaceshould possess a Michael reaction acceptor group. We hypothe-sised that residual C––C bonds from the cross-linker, the presenceof which was confirmed by Raman spectroscopy (see SupportingInformation), could perform this function. To test the viability ofthis immobilization method, we decided to attach cysteine, as amodel thiol, to polyHIPE 6 (Scheme 2). The presence of cysteinecan easily be monitored by its reaction with Fluram1, a selectivefluorescent probe for amines. The attachment of cysteinewas confirmed by fluorescence, and elemental analysis indicatedthe loading to be 0.56mmol � g�1. We then repeated the experi-ment with PolyHIPE 9, obtaining in this case a loading of0.18mmol � g�1. From this set of experiments it was concludedthat thiol moieties can react with the residual C––C bonds in thePolyHIPE.

For the immobilization of thiol-ended poly(MA-VPGVG)(polymer F), the same procedure as that for cysteine immobiliza-tion was used (Scheme 2). A dilute solution of polymer F wasemployed with the intention of achieving a maximum polymerloading of 12.2mmol � g�1, or approximately 1.41mmol � g�1 ofVPGVG groups. The final loading after the reaction, calculated byelemental analysis, was 0.63mmol � g�1 of the polymer, approxi-mately 0.08mmol � g�1 of VPGVG units.

After immobilization of poly(MA-VPGVG) onto the PolyHIPEsurface, the co-assembly of a complementary EBP onto thismatrix was attempted. The PolyHIPE was soaked in a solution offluorescently labeled polymer D at pH 3.2, and the pH was slowlyreduced to 1.5 to induce co-assembly. After allowing the mixture

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Figure 2. Turbidimetry profiles for polymer D (Mn¼ 26.0 kg �mol�1,0.23mgmL�1,�, pH 1.5;�, pH 3.2) and polymer E (Mn¼ 61.6 kg �mol�1,1, 0.23mgmL�1; &, pH 1.5; &, pH 3.2) at different pH values perfomedby UV–Vis spectroscopy (l¼ 480 nm). The turbidimetry profile for fluor-escein isothiocyanate-modified VPGVG (!) is shown for comparison.Experiments were carried out in PBS buffer.

Figure 3. Fluorescent images of noncovalent immobilization. a) Left:poly(VPGVG)-modified PolyHIPE 9þ polymer D, pH 1.5; right: unmodifiedPolyHIPE 9 (control)þpolymer D, pH 1.5. b) left: poly(VPGVG)-modifiedPolyHIPE 9þ polymer D, pH 1.5; right: poly(VPGVG)-modified PolyHIPE9þ polymer D washed with PBS buffer, pH 3.2. Samples illuminated underUV light (l¼ 254 nm).

58

to stir for 1 h, the PolyHIPE was filtered from solution and thepresence of fluorescent polymerDwas confirmed by illuminationwith UV light. As can be seen in Figure 3a, the treated PolyHIPEis strongly fluorescent. Conversely, the control experiment, inwhich an unmodified PolyHIPE is treated with a solution ofpolymer D under identical conditions, shows no fluorescence(there is some autofluorescence on illumination, however).

To recover the reversibly immobilized polymerD, the modifiedPolyHIPE was soaked in a pH 3.2 buffer solution and the mixturewas allowed to equilibrate for 1 h. After filtering from solutionand washing, no fluorescence could be detected on the surface ofthe monolith (Fig. 3b), showing that the immobilization iscompletely reversible and that the starting material could berecovered by simply washing at the correct pH. The assembly andrecovery processes are shown in Scheme 3.

In conclusion, we have shown that EBPs can be covalentlyimmobilized onto the surface of highly porous emulsiontemplated matrices derived from PEGylated monomers. Theability of EBPs to undergo cooperative assembly has beenexploited to enable the reversible immobilization of comple-mentary EBPs from solution onto the surface of the porousmaterial. Our current investigations are extending this metho-

Scheme 2. Surface functionalization of PEG-PolyHIPE.

Scheme 3. Assembly and recovery processes: a) EBP-functionalized poly-HIPE; b) EBP-functionalized polyHIPE soaked in a solution of polymer D atpH 3.2; c) the mixture in (b) after lowering the pH to 1.5; and d) thepolyHIPE in (c) after washing with PBS buffer, pH 3.2.

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Experimental

All details can be found in the Supporting Information. Only criticalexperiments are described here.

Hydrophilic PolyHIPE Synthesis: An aqueous phase consisting ofPEGMA (1.01 g, 2.81mmol), EGDMA (0.25 g, 0.68mmol), ammoniumpersulfate (0.11 g, 0.48mmol), and the surfactant sodium dodecyl sulphate(SDS) (0.23 g, 10% w/w of the aqueous phase) dissolved in water (1mL)and DMF (0.4mL) was added to a 250mL three-necked round bottomedflask. The aqueous phase was stirred continually at 600 rpm using aD-shaped polytetrafluoroethylene (PTFE) paddle connected to an overheadstirrer. An oil phase consisting of light mineral oil or cyclohexane (15mL)was added over a period of 2min, by means of a pressurised droppingfunnel, until a HIPE had formed. After addition of the organic phase wascomplete, N,N,N0,N0-tetramethylethylenediamine (TMEDA) (0.2mL,0.16 g, 1.32mmol) was added and the HIPE was stirred for a furtherperiod of 1min. The HIPE was then transferred to a polycarbonatecentrifuge tube, which was allowed to react at room temperature or placedin an oven at the corresponding temperature (see Table 1) overnight. Theresulting monolith was recovered from the tube then extracted in a soxhletapparatus with propan-2-ol/hexane 3:2, water/propan-2-ol 3:2 andpropan-2-ol for 24 h each and dried in vacuo.

PolyHIPE Functionalization With Poly(MA-VPGVG)-SH: A monolith ofpolyHIPE 9 (25mg) was soaked in a PBS buffered solution of polymer F(22mgmL�1, 5mL, pH 7.2). After allowing the reaction to run overnightwith gentle stirring, the monolith was filtered, washed exhaustively withPBS buffer (pH 7.2), water and ethanol, and dried under vacuum.

Reversible Immobilization: Immobilization: A monolith of poly(MA-VPGVG)-SH functionalized polyHIPE (25mg) was soaked in a PBSbuffered solution of polymer D (0.23mgmL�1, 3mL, pH 3.2). The pH wasthen slowly taken to pH 1.5 with HCl (0.1M) and the mixture was stirredgently for around 1 h. The monolith was then filtered, washed thoroughlywith PBS buffer (pH 1.5), and the presence of polymer D was checkedunder 254 nm UV light.

Recovery: The polyHIPE obtained from the previous experiment wassoaked in PBS buffered solution (3mL, pH 3.2) and the mixture was stirredgently for around 1 h. The monolith was then filtered, washed thoroughlywith PBS buffer (pH 3.2) and the presence of polymerD was again checkedunder UV light.

Acknowledgements

This work was performed within the European Research Training NetworkSMASHYBIO (Smart Assembly of Hybrid Biopolymers) under contract(HPRN-CT-2001-00188). We would like to thank the rest of theSMASHYBIO Network for discussions. Supporting Information is availableonline from Wiley InterScience or from the author.

Received: July 13, 2008

Published online: October 30, 2008

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