Chiang Mai J. Sci. 2012; 39(3) 351

Chiang Mai J. Sci. 2012; 39(3) : 351-372http://it.science.cmu.ac.th/ejournal/Invited Review

Polymer-Peptide Conjugate Hydrogels; TowardsControlled Drug DeliveryArun A. Sohdi, Darren Campbell and Paul D. Topham*Department of Chemical Engineering and Applied Chemistry, Aston University, Aston Triangle,Birmingham B4 7ET, UK*Author for correspondence; e-mail: p.d.topham@aston.ac.uk

Received: 16 March 2012Accepted: 21 April 2012

ABSTRACTPeptide-based materials exhibit remarkable supramolecular self-assembling

behaviour, owing to their overwhelming propensity to form hierarchical structuresfrom α-helices and β-sheets. Coupling a peptide sequence to a synthetic polymer chainallows greater control over the final physical properties of the supramolecular material.So-called ‘polymer-peptide conjugates’ can be used to create biocompatible hydrogelswhich are held together by reversible physical interactions. Potentially, the hydrogelscan be loaded with aqueous-based drug molecules, which can be injected into targetedsites in the body if they can exhibit a gel-sol-gel transition under application and removalof a shear force. In this review, we introduce this topic to readers new to the field ofpolymer-peptide conjugates, discussing common synthetic strategies and their self-assembling behaviour. The lack of examples of actual drug delivery applications frompolymer-peptide conjugates is highlighted in an attempt to incite progress in this area.

Keywords: polymer-peptide conjugates, hydrogels, drug delivery

CONTENTS1. Introduction.................................................................................................................................. 352

1.1 Hydrogels ............................................................................................................................. 3521.2 Polymer-Peptide Conjugates ........................................................................................ 3541.3 Polymer-Peptide Conjugate Hydrogels .................................................................... 3551.4 Drug Delivery ..................................................................................................................... 355

2. Synthetic Strategies .................................................................................................................... 3562.1 Convergent Strategies (Direct Conjugation) ........................................................... 356

2.1.1 Succinimide ............................................................................................................. 3572.1.2 Schiff base ................................................................................................................ 3582.1.3 Click chemistry ..................................................................................................... 3582.1.4 Thiol-maleimide ..................................................................................................... 358

2.2 Divergent Strategies .......................................................................................................... 3592.2.1 Peptide growth from polymers ....................................................................... 359

352 Chiang Mai J. Sci. 2012; 39(3)

2.2.2 Polymer growth from peptides ..................................................................... 3592.2.2.1 Atom transfer radical polymerisation .......................................... 3592.2.2.2 Reversible addition-fragmentation chain transfer .................... 3602.2.2.3 Other polymerisation techniques ................................................... 360

3. Polymer-Peptide Conjugate Hydrogels Suitable for Drug Delivery ................... 3603.1 Poly(ethylene oxide)-Based Conjugates .................................................................... 3613.2 Conjugates Comprising Alternative Polymers ..................................................... 364

4. Summary ..................................................................................................................................... 365References .......................................................................................................................................... 366

1. INTRODUCTIONThere is growing interest in combining

natural polymers (especially proteins andpeptides) with synthetic polymers [1-3]. Theuse of peptides allows for the incorporationof properties such as biocompatibility andself-assembly, whilst the synthetic polymercomponent in these materials allows controlof physical and chemical properties, suchas viscosity and smart behaviour. It hasalso been shown that the use of syntheticpolymers can prolong the lifetimes ofmaterials in the body. Supramolecularorganisation of asymmetric polymer-peptide molecules offers a gateway totailor-made materials suitable for a widerange of applications. So-called polymer-peptide conjugates (PPCs) can be designedto form hydrogels under certain conditions.Industrial viability of such materialsincreases as the number of amino acids inthe peptide sequence decreases (fewersynthetic steps required). Here we will bediscussing such PPCs, where the aminoacid sequences are 25 or less in length, andtheir suitability for use in drug delivery.More specifically, this report highlights thelack of examples of PPCs as drug deliverydevices, even though their properties appearideal for such an application. Thisdiscussion is intended for those researcherswho may be new to the field; as such it isnot meant as a comprehensive review ofthe literature, but rather as an introduction

to the area. Throughout this review thesingle letter abbreviation will be used foramino acids, for example phenylalanine isdenoted simply as F. Table 1 lists theabbreviations used in this review.

1.1 HydrogelsHydrogels are three-dimensional

structures comprised of a water phaseimmobilised by a scaffold [4]. Due to theirsimilarity to hydrated body tissues,hydrogels are widely used as biomedical andpharmaceutical materials. The three-dimensional structures of hydrogels arenetworks of polymer chains which are heldtogether by chemical or physical bonds(Figure 1). Chemically-bonded hydrogelsare held in place by irreversible covalentbonds linking the polymer chains together.Physically-bonded hydrogels, on theother hand, are held together by reversibleinteractions such as molecular entangle-ments, ionic forces, π- π stacking, hydrogenbonding, and van der Waals forces.Self-assembly into such supramolecularconstructs occurs under favourableconditions. Self-assembly formationthrough noncovalent interactions leads tothe spontaneous organisation of moleculesinto well-defined arrangements [5]. Self-assembling hydrogels have advantagesover chemically-bonded hydrogels forapplication in drug delivery. Most notably,

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Table 1. List of abbreviations used throughout this review.

Abbreviations Amino AcidsAGET Activators Generated by Electron Transfer A alanineARGET Activator Regenerated by Electron Transfer D aspartic acidATRP Atom Transfer Radical Polymerisation E glutamic acidCRP Controlled Radical Polymerisation F phenylalanineCTA Chain Transfer Agent G glycineCuBr copper (I) bromide H histidineCuBr2 copper (II) bromide I isoleucineDCC N,N’-dicyclohexylcarbodiimide K lysineDCM dichloromethane L leucinedmG N,N-dimethylglycine N asparagineFmoc Fluorenylmethoxycarbonyl Q glutamineGPC Gel Permeation Chromatography R arginineHEMA 2-(hydroxyethyl)methacrylate S serineICAR Initiators for Continuous Activator Regeneration T threonineLCST Lower Critical Solution Temperature V valinemPEO monomethoxy poly(ethylene oxide) X unspecified

amino acidnBA N-butyl acrylate Y tyrosineNIPAAm N-isopropylacrylamide (also poly[N-isopropylacrylamide])oligoEO oligo(ethylene oxide)PEO poly(ethylene oxide) / poly(ethylene glycol)PHPMA poly[N-(2-hydroxypropyl)methacrylamide]PPC Polymer-Peptide ConjugateRAFT Reversible Addition-Fragmentation Chain TransferSPPS Solid Phase Peptide SynthesisTFA trifluoroacetic acid

Figure 1. Molecular structures of (a) chemically- and (b) physically-bound hydrogels.

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physical bonding is reversible, meaning thatthese hydrogels can be rendered injectable(via a gel-sol-gel transition during injection).

1.2 Polymer-Peptide ConjugatesPPCs are hybrid materials which

covalently combine peptide sequences withsynthetic polymer chains (Figure 2). Therehas been a great deal of interest in the lasttwenty years or so on the combination ofbiological and synthetic polymers with awide range of applications.

Many biological entities, e.g. oligo-nucleotides [6], saccharides [7] and lipids [8],have been investigated for conjugation withsynthetic polymers, but it is amino acid-containing species as conjugates whichhave received the majority of attention [2].While biologically-inspired polymers suchas synthetic proteins and polypeptideshave been conjugated to polymers, thisreview concerns the combination of thesimplest biological units, those of shortamino acid sequences (peptides) withpolymers. The reader is directed elsewherefor work describing protein andpolypeptide-based polymer conjugates [9-19]. Furthermore, the attachment of ahydrophobic alkyl chain to a hydrophilicpeptidic segment to produce ‘peptideamphiphiles’ (PAs) are also not discussed[20]. However, it should be noted that thisis not a reflection on the ability, or lack

thereof, of these materials to act as drugdelivery scaffolds/vehicles, but more tohighlight the desperate need for progressin the area of polymer-peptide conjugates.

There are examples of biologicalpolymer-synthetic polymer conjugates fordrug delivery, where the biological entityitself may be the active species, i.e. a drugor pro-drug. The delivery of the peptidevia this method is the essence of theRingsdorf model of drug delivery [21],which can be traced back to a landmarkpaper in 1975 [22]. Alternatively, thepeptide may be used for therapeutictargeting, for example via biorecognitionof the peptide motif [23, 24]. Both of thesestrategies are beyond the scope of thisreview.

Herein we discuss PPCs which containpeptide sequences of no more than 25amino acids. Generally, the greater thelength of the amino acid chain, the moreexpensive the cost of synthesis. This is dueto the increased number of synthetic and(potentially) purification steps, and is evenmore of a concern when consideringcommercial processes. Therefore control ofself-assembly with the lowest number ofamino acids is favourable [25]. There areseveral reports of hydrogel-forming PPCswith amino acid sequences greater than 25;selected examples include human fibrincoiled-coil sequences conjugated to PEO

Figure 2. A schematic of a polymer-peptide conjugate.

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[26], collagen-mimetic linked peptidesto four-armed stars of PEO [27], andpentaheptad peptides joined to poly[N-(2-hydroxypropyl)methacrylamide](PHPMA) [28, 29].

Jatzekewitz [30] published a report in1954, on what is thought to be the firstsuccessful PPC, which sparked a steadyinterest in the literature for the following30 years. However, in recent years theinterest in PPCs has exploded as theirpotential applications have becomerealistic. This activity was increased furtherwith incredible advances in couplingmechanisms and other synthetic strategies(discussed in Section 2) [31]. New materialsare being produced with greater fidelity,greater purity and novel properties, whichleads to an even broader range of potentialapplications.

One of the most interesting propertiesof these conjugates is their ability to self-assemble to form complex three dimensionalnetworks. These networks can entangle toform an array nanostructures [4], which arephysically-bound owing to the propensityof the peptide units to aggregate.

1.3 Polymer-Peptide Conjugate HydrogelsPPC hydrogels are physically-bonded

networks capable of imbibing largequantities of water. As with proteins, thephysical bonding can lead to secondarystructure formation, e.g. α-helices and β-sheets, which can lead to tertiary structureformation. Advantages of using PPChydrogels for biomaterials include thecombination of the best properties ofpeptides and those of synthetic polymers[1]. The peptide component providesincreased functional control, well-definedhomogeneous hierarchical structures,consistent mechanical properties, andsupportive folding/unfolding transitions

[1, 32]. It is the peptide component thatdrives the self-assembly in these materials.Synthetic polymers can provide enhancedbiocompatibility (e.g. non-reactivity, lowclearance times), enzymatic degradationresistance and adaptability [1, 32]. Thecombination of peptides and syntheticpolymers can provide materials withproperties superior to those of theindividual components [1]. Commonmethods of self-assembly to give PPChydrogels include; solvent exchange, directrehydration, temperature-switch, and salt-triggered processes. Various structures canbe obtained depending upon the conditionsof assembly, e.g. temperature, solvent, ormolar ratio of polymer to peptide.

1.4 Drug DeliveryCertain PPC hydrogels undergo a gel-

sol transition under shear force, yet reforma gel on removal of the force, i.e. they areinjectable [33]. Such behaviour is describedas thixotropic, that is shear-thinning(viscous flow under shear stress) andrecovery when relaxed (self-healing) [34],and is a highly desirable property fordrug delivery applications. This is a majoradvantage over chemically cross-linked gelswhich require precise incision (and sub-sequent removal) for practical applicationin biological environments. Clearly, thisbehaviour is particularly useful for tissueengineering and drug delivery, where theapplication of the hydrogel through a syringeis highly desirable. In drug delivery,therapeutic molecules can be very easilyincorporated by preparation of theprecursor solution prior to injection [35].Furthermore, PPC hydrogels can bedesigned so that they can be easily brokendown into their unimolecular componentsafter use and, as such, can both be passedfrom the body and have higher efficiency

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of delivery of the drug package (comparedto drugs covalently bound to polymers)[36]. A major disadvantage of PPCs is thatcomprehensive design rules remainsomewhat undefined.

With judicious choice of the peptideand polymer components, PPCs can berendered stimuli-responsive. The hydrogelwill thus respond to changes in thesurrounding environment, e.g. physical(temperature, ionic strength and solvent),chemical (pH, salt effect) or biochemical(enzyme presence) [37]. On the appropriatetrigger, stimuli-responsive materials changetheir characteristic properties; thesetransformations are normally reversibleonce the stimulus is removed. This stimuli-responsive behaviour of PPC hydrogelscan be exploited as a mechanism for drugdelivery.

The remainder of this review is dividedinto two parts; (i) a description of themethods commonly used to synthesisePPCs, and (ii) the potential application ofPPC hydrogels for drug delivery where thePPC hydrogel is employed as a scaffold/reservoir for the active drug molecules orcomponents.

2. SYNTHETIC STRATEGIESThe ability to choose a specific sequence

of amino acids, alongside a range ofsynthetic polymers, allows for the designof various morphologies and materials,such as fibres [38-41], three-dimensionalhydrogels [42-44], nanosponges [45, 46],microgels/nanogels [47, 48], vesicles [49] andmicelles [50]. Peptide motifs allow self-assembly to occur in a manner in whichthe structural arrangements are well-defined, and in which the resultingproperties are more controllable.

It should be noted, however, thatamino acid and monomer selection design

rules are not entirely straightforward, asstructural analogues often behave verydifferently and are hugely affected bypurity [51]. There is also a need to considerthe effect of the species which are used inthe synthesis of PPCs as these have beenshown to affect hydrogel formation.For example, the protecting speciesfluorenylmethoxycarbonyl (Fmoc) isknown to impact upon the formation ofhydrogels [52].

There are many methods employedfor the synthesis of the precursor peptides(i.e. amino acid coupling strategies) andpolymers (i.e. polymerisation techniques)for use in PPCs which are beyond thescope of this paper and are detailed in theliterature. Conjugation strategies fall intotwo distinct approaches: convergent anddivergent. A convergent approach involvesboth building blocks synthesised separatelyand then joined via an additional ligationstep. By contrast, a divergent approachentails either the modification of the peptideor polymer block allowing ‘growth’ of theother component from the first functionalunit. Section 2.1 details convergent strategies,whilst Section 2.2 details divergentstrategies. Figure 3a-d illustrates some ofthe most common coupling methods usedto synthesise PPCs via a convergentapproach, whilst Figure 3e demonstratesan example of a divergent approach.

2.1 Convergent Strategies (Direct Conju-gation)

There are a range of methods availablefor introducing peptide sequences tosynthetic polymers (Figure 3). Coupling cantake place either in solution or solid phase.When dealing with peptides, mostexperimentalists tend to favour the solidphase approach for the peptide synthesis,and then will undergo conjugation in either

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the solid or solution phase. The followingsection provides examples of commoncoupling methods used to link polymersto peptides; (i) succinimide, (ii) Schiff base,(iii) click chemistry, and (iv) thiol-maleimide. Complementary group conju-gation is one of most prevalent routes forcoupling reactions. This approach involvesexploitation of the reactivity of the amine(N-terminus) or carboxylic acid group(s)(C-terminus) of the peptide, and the endgroup of the synthetic polymer which canbe functionalised during, or post, polymersynthesis. Further, any side groups withappropriate functionality can be targetedas potential conjugation sites.

2.1.1 SuccinimideA good example of complementary

group conjugation is demonstrated usingN-hydroxysuccinimide functional polymers(Figure 3a). Succinimidyl esters reactreadily with amines and are especiallyuseful as they can couple to either theterminal amine group of a peptide, or bedirected to an amine side group (in lysine-containing amino acid sequences) by usinga protecting group strategy. Polymers canbe functionalised with succinimidyl esters,such as succinimidyl carbonates withrelative ease, and many are commerciallyavailable, with monomethoxypoly-(ethylene oxide) derivatives being the most

Figure 3. Some of the most common convergent strategies used to synthesisepolymer-peptide conjugates; (a) Succinimide coupling, (b) Schiff base coupling,(c) Azide-alkyne click chemistry, and (d) Thiol-maleimide coupling. (e) An example of adivergent approach synthesising a peptide sequence from a polymer support.

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commonly used polymers for PPCs [53].Further, it is also possible to modify thepeptide to include a succinimidyl moietyand then directly couple this to anappropriately functionalised polymer [54].Alternatively, benzotriazoles can be used,as they behave in a similar manner. Whilstthe succinimidyl esters have a wide use,conjugating PEO-succinimidyl succinateintroduces a second ester group in thepolymer backbone [9, 55]. After conju-gation to a peptide this group remains inthe final product and is highly susceptibleto hydrolysis, which would result indetachment of the PEO and the loss of itsbeneficial properties. This drawbackapplies to all conjugates containing readilyhydrolysable groups, and must be consideredduring synthesis and conjugation, dependingon their intended application.

2.1.2 Schiff baseModifying the polymer end group to

an aldehyde affords another potentialconjugation route [31, 34] via reductiveamination (Figure 3b). The coupling of thepolymer-aldehyde derivative and peptideresults in the formation of a Schiff base,which, when treated with a reducing agent(such as sodium cyanoborohydride),produces an amine (or an amide dependingon the R group). Roberts reported workdescribing the partial selectivity of mPEO-propionaldehyde by modifying the pH [9,56]. This resulted in the aldehyde grouppreferentially binding to the N-terminalamine, due to the more acidic nature ofthe α-amine in comparison with othernucleophilic species.

2.1.3 Click chemistryClick chemistry is used to describe a

set of orthogonal reactions amongst whichis the widely reported copper-catalysed

Huisgen reaction between azides andalkynes (Figure 3c) [57-65]. Click chemistryis noted for its robustness, insensitivityto a wide variety of functional groups,reliability, and ease of purification. Thisarea of bioconjugation has received a largeamount of attention. For example, vanDijk and co-workers [66] reported theconjugation between alkyne functionalised-PEO and azide-functionalised alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide, inwhich the conjugation took place in thepresence of sodium ascorbate and copper(II) sulfate. The resulting conjugate was starshaped and formed supramolecularhydrogels. Tzokova et al. reported thesynthesis of short chain of PEO-peptideconjugates using click chemistry, but unlikethe previous example, the peptide wasmodified to include the terminal alkynegroup [67].

The major disadvantage of this approachis the catalyst system, which mostcommonly contains copper. This presentsa problem for a system required to interactin a biological environment and as such,materials need to be extensively purifiedto ensure no toxic contaminants remain.Furthermore, amino acids have an affinityto complex with metals [68], which couldaffect the conjugation rate and makepurification difficult.

2.1.4 Thiol-maleimideAnother common coupling strategy,

which could be considered a “click” reaction,exploits the reactivity of the side group incysteine; the thiol. The allyl or vinyl groupsin maleimides can react with the thiol groupin an addition reaction to form a stablecarbon-sulfur-carbon linkage (Figure 3d).Cysteine, like lysine, can be engineered intothe peptide sequence during synthesis.However, there is the potential problem

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of forming disulfide bridges, which ingeneral, are susceptible to degradation in abiological environment [69]. Further, themaleimide moiety can be hydrolysed andundergo ring opening to form maleic acid.

2.2 Divergent StrategiesIt is possible to use a divergent strategy

to synthesise PPCs where a sequence is builtup from a polymer substrate (Figure 3e)or a polymer grown from a peptidicmacroinitiator. Consequently, the followingsection is divided into two: peptide growthfrom polymers (2.2.1), and polymergrowth from peptides (2.2.2). However, itshould be noted that the latter approach isonly possible (to date) using radicalpolymerisation systems due to theirtolerance of biological groups, with the twomost common CRP techniques beingreversible addition-fragmentation chaintransfer polymerisation (RAFT) [70], andatom transfer radical polymerisation(ATRP) [71, 72].

2.2.1 Peptide growth from polymersThere are a group of commercial resins

that have PEO preloaded via an acid-labilelinker (usually a benzyl ester). The lengthof the peptide chain however, heavilydetermines the solubility of the conjugateand makes isolation difficult [2]. Forexample, Hentschel reported conjugationinvolving bound PEO and free amino acids.Here the PEO was part of a Tentagel-PAPresin, and was functionalised with aterminal amine group [73]. Protected aminoacids were directly conjugated to this groupthrough standard Fmoc and DCCchemistry [74]. The newly formed PPCwas then cleaved from the resin using TFA(Figure 3e). Using a resin makes isolationand purification easier, however scaling upcan be more difficult due to associated costs.

2.2.2 Polymer growth from peptidesMonomers can be polymerised from

a peptidic macroinitiator system, alsoreferred to as a “grafting from” approach.This allows for easier purification as onlyunreacted reagents will need to be removed,as opposed to the “grafting to” method, inwhich the unreacted reagents may includethe presynthesised polymer, peptide andcoupling reagents. Controlled radicalpolymerisation (CRP) provides a usefulapproach toward the formation of well-defined polymers from peptidic initiators.CRP is commonly used in the synthesis ofPPCs, either in the solution or solid phase,with the former favoured due to lackof control in the latter [75]. One of thegreatest advantages of the CRP techniquesarises from the ability to introduce functionalgroups on the chain end of the polymersvia selection of appropriate functionalreagents (e.g. initiators or chain transferagents, CTA) or through post-polymerisationmodification. Whilst such techniques arecommonly exploited to produce functionalpolymers for conjugation (via the“grafting to” method), this part of thereview focuses on CRP using peptidicinitiators/CTAs only. Readers are directedto the work of Le Droumaguet and Nicolasfor further reading on controlled radicaltechniques [31].

2.2.2.1 Atom transfer radical polymerisa-tion

ATRP has been used in the synthesisof a whole host of designer materials,including polymer brushes [76, 77],pH-responsive vesicles [78], hybridnanoparticles [79], macromonomers [80, 81],non-fouling gold surfaces [82] and manymacromolecules used in biomedicalapplications [83]. Since the discovery ofATRP there has been an evolution of the

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technique to overcome some of its initialdrawbacks. These newer protocols includeAGET [84], ARGET [85], and ICAR [86].

There are numerous examples ofpeptide-based ATRP initiators in theliterature. One particular example byRettig et al. [75] focussed on the synthesisof a peptide macroinitiator which was usedto prepare peptide-poly(n-butyl acrylate).Oligopeptide synthesis was performedusing standard SPPS protocols [74] withthe ATRP initiating group introduced atthe terminal amine of the oligopeptide. Thiswas followed by cleavage from the solidsupport. The oligopeptide macroinitiatorinitiated the polymerisation of nBA indegassed DMSO at 60oC, with a CuBr/CuBr2/PMDETA catalyst system. CuBr2

helped to mediate control of the reactionand PMDETA ensured that the aminegroups (which can act as multidentateligands) of the oligopeptide did notassociate with the copper complex. Thisunwanted effect was observed when theauthors increased the peptide concentra-tion; a decrease in the overall rate ofpolymerisation was observed. GPC analysisof the final product revealed a molar massdispersity of 1.19, highlighting a goodlevel of control. Adams and Young [87]used a small family of oligopeptide-basedinitiators to polymerise 2-(diethylamino)ethylmethacrylate and tert-butyl methacry-late. The initiators were prepared from thefree N-terminus of a series of short (1 to 4amino acid) oligo(phenylalanine)s.

2.2.2.2 Reversible addition-fragmenta-tion chain transfer

RAFT polymerisation is a controlledfree radical polymerisation in the presenceof a CTA, typically based on a dithioester.The CTA, commonly referred to as aRAFT agent, allows the polymer chain to

retain a functional end-group post-polymerisation. There are a vast numberof examples where a peptide moiety is usedas the CTA. Hentschel et al. [88] reportedthe synthesis and self-assembly of a PPCvia RAFT polymerisation. The peptidictransfer agent (i.e. the macro CTA) wasprepared in a two-step process. Firstly, aresin-bound peptide was modified with aterminal amine group. The oligopeptidewas then subsequently reacted withdithiobenzoate to form the CTA.Subsequently, the peptidic CTA wascleaved from the support using TFA inDCM and used to polymerise n-butylacrylate. The final PPC had a molar massdispersity of 1.29 indicating relatively goodcontrol of the polymerisation process.Furthermore, this highlights the toleranceof RAFT with respect to complex, multi-functional peptide structures.

2.2.2.3 Other polymerisation techniquesPreparation of thermoresponsive

peptide-PNIPAAm hydrogels has recentlybeen reported by Maslovskis et al. [89]. Theauthors used AIBN as an initiator for thefree radical polymerisation of NIPAAm.Thiol-terminated oligopeptide was addedto the reaction mixture as a chain transferagent. Unreacted peptide was excludedfrom the final conjugate by dialysingagainst water for several days. It was foundthat varying the quantity of peptide offeredcontrol over the composition of theconjugate. However, as a controlledpolymerisation technique was not used, thefinal conjugate was not as well-defined asthose examples described in Sections 2.2.2.1and 2.2.2.2.

3. POLYMER-PEPTIDE CONJUGATE HYDROGELSSUITABLE FOR DRUG DELIVERY

PPCs have a wide range of applications,

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such as bio-sensors, artificial enzymes andphotonic and nano-electronic devices [3].PPC hydrogels have been investigated fora range of applications, including scaffoldsfor tissue engineering/regeneration [90].Hydrogels have long been recognised asvehicles for drug delivery [91, 92], but self-assembling hydrogels may lead the waytowards better hydrogel drug deliveryvehicles [93], owing to the reversibility oftheir structure formation. Here we focuson the use of peptide motifs as structuraldesign tools for the construction ofhydrogels via nanostructure formation(e.g. tubes, fibrils and tapes). Most of theliterature on PPCs discusses materials whichhave potential for application in drugdelivery, but does not demonstrate theiruse as such. Thus, the following examplesof PPC hydrogels have the potential foruse as protective matrices/reservoirs indrug delivery applications. The followingsections are divided into PPC hydrogelsbased on poly(ethylene oxide) (PEO) andPPC hydrogels based on other polymers.

3.1 Poly(ethylene oxide)-Based ConjugatesA wide range of polymers have been

investigated for conjugation to peptides forpharmaceuticals, but those utilisingpoly(ethylene oxide) (PEO) have receivedthe most attention [2, 36]. This is arguablydue to the biocompatible properties ofPEO [9, 53]. PEO is non-toxic, non-immunogenic, water soluble, inexpensive, haswell known physicochemical properties,and is FDA approved [94]. Combining abiological entity with PEO is oftenreferred to as PEGylation [95]. AlthoughPEO has been successfully used by manygroups, the lack of attachment sites is apotential drawback associated with its usefor PPC hydrogel formation. Examples ofPEO-containing PPCs are presented herein.

Tzokova et al. [67] demonstrated thesynthesis of PEO-F4 and the subsequentself-assembly into hydrogels via nanotubeformation. The F4 hydrophobic motif wasshown to control the formation of self-assembly via antiparallel β-sheets and π−πstacking of the phenyl groups. In anextension of this work the same groupattempted to produce a set of design rulesfor PEO-peptide conjugates based onphenylalanine and valine [73]. Differentlength low molecular weight PEO samples(350, 1200, and 1800 Da) were conjugatedto F4 and V4. This work indicated that boththe PEO length and the nature of thepeptide significantly affect the self-assemblyof such PPCs. PEO-F4 conjugates producednanotubes, fibres, and wormlike micellesas the length of the PEO block wasincreased, respectively. For the homologousPEO-V4 series, β-sheet formation dominated,and the self-assembled structures werecloser to those formed by peptides aloneregardless of the PEO chain length. LargerPEO (3000 and 5000 Da)-F4 PPCs weresynthesised by Castelletto and Hamley [96].Low concentration aqueous solutions ofthese PPCs were shown to self-assemble,due to hydrophobic interactions ofaromatic phenylalanine residues. At highersolution concentrations β-sheet formationwas observed, where self-assembled straightfibril structures were seen. However, theanalogous F2-containing PPC (PEO 3000Da), also synthesised by this group, did notexhibit self-assembly behaviour. Thisindicates that not only is a sufficienthydrophilic/hydrophobic balance nedded,but the number of amino acid repeatsmust also be above a critical value for self-assembly to occur [96].

In other work, Hamley et al. [97]synthesised peptide-PEO-peptide PPCswith a central PEO component (1500 Da)

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flanked by Y2 or F2 dipeptide sequences.The hydrophobic dipeptides were selectedfor their simplicity, and allow directcomparison with the F4 sequence. It wasfound that self-assembly was dependent onthe nature of the dipeptides and theterminal-end group used (Fmoc or amine).Water insoluble PPCs were producedwhen both ends were capped with thehydrophobic Fmoc group. By contrast,PPCs end-capped with NH2 groups at bothends were soluble in water. However,neither of these materials gave rise to self-assembling PPCs. The telechelic PPCs(NH2–Y2–PEO–Y2–Fmoc and NH2–F2–PEO–F2–Fmoc) self-assembled viaβ-sheet formation, to produce fibril-basedhydrogels. A balance of hydrophobic andhydrophilic end-capping was required forthis application. NH2–Y2–PEO35–Y2–Fmoc was found to be thermoresponsivewith a switchable transition near bodytemperature. This is clearly a valuablefeature for a drug delivery device.

B rner and co-workers [38, 98] syn-thesised PEO-based (~3150 Da) PPCscontaining the peptide sequence (TV)2. Thispeptide sequence was chosen as it is knownto form β-sheet structures. Here a peptide-PEO-peptide structure was selected, but inthis example a “template” bridged thepeptide and polymer components (Figure4). The template was designed to preorganisethe peptide sequences into the appropriategeometry to enhance the aggregationtendencies of the PPC. Each peptidewas end-capped with dmG, to yield acationic PPC with an overall structure of[dmG-(TV) 2-NH] 2- t empla te -PEO 72

(Figure 4). The PPCs self-assembled toform nanofibres, which aggregated intowell-defined bundles. This work suggestedthat these PPCs can be tailor-made to targetdifferent structures. In this example, it was

noted that the fibres formed were stiff, andso may also have applications in biomaterialfibre-reinforcement. Hentschel et al. [99]expanded the investigation of TV peptidesequences. Here PPCs containing PEO(~3150 Da) and the (TV)5 sequence weresynthesised. These PPCs self-assembled viaβ-sheet formation, but tape structures,rather than fibrils were formed. Secondarygrowth was occasionally noted on top ofthe primary tapes, and this is currentlyunder further investigation. Interestinglythe introduction of ester segmentsdisrupted the amide backbone, which wasre-established via selective rearrangement(O to N acyl switch, as illustrated inFigure 5) post-synthesis. This workdemonstrated that the aggregation tendency(during the synthesis of the PPC) could betemporarily suppressed via the integrationof multiple “switch-peptide” backbonedefects, which further expands the peptidesequences available for synthesis.

A conjugate containing low molecularweight oligoEO (three repeat units) and ashort peptide sequence (I3) was synthesisedby Ganesh et al. [55, 100]. It was foundthat self-assembly occured via β-sheetformation to give fibrils which entangledto produce gel-like aggregates. The PEO-peptide and PEO-peptide-PEO structuresthat were synthesised produced differencesin secondary structure, and the conforma-tion of these polymer-peptides wasdependent on the concentration and theposition of oxyethylene groups.

Substantially longer peptide sequences(>15 amino acids) have also beeninvestigated. R sler et al. [101], forexample, synthesised PEO-peptide andPEO-peptide-PEO PPCs (Figure 6). PEO(2600 Da) was used in the PEO-peptide,with the addition of PEO (750 Da) for thePEO-peptide-PEO conjugates. The peptide

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sequences were composed of alternatinghydrophilic and hydrophobic α-aminoacids (G[KL]3K[Q]3XLQLXLQGG, whereX was D or Q). It was reported that theamphiphilic β-strand sequences mediatedthe self-assembly of these PPCs intolamellar superstructures. Hamley et al. [88]extended this work using a slightly differentalternating sequence of hydrophilic andhydrophobic α-amino acids, which werebased on the GELELELEQQKLKLKLKGsequence, to synthesise PEO-peptide PPCs.

The polymers self-assembled to form rod-like structures in twisted, helical tapes,which further stacked into fibrillar rods.These examples illustrate that the combinationof a longer peptide sequence containingbiological motifs with a synthetic polymerprovides self-assembling materials, with thepotential to interface with biologicalsystems.

Even longer peptide sequences (>20amino acids) have been incorporated intoPPCs. Two PPCs; one comprising of PEO

Figure 4. A peptide-PEO-peptide structure, where a “template” bridges the peptide andpolymer components introduced to control the orientation of self-assembly [38, 98],where V and T represent valine and threonine, respectively.

Figure 5. Reaction mechanism of O to N acyl switch used in the work of Hentschelet al. [99], where G, W, V and T represent glycine, tryptophan, valine and threonine,respectively.

364 Chiang Mai J. Sci. 2012; 39(3)

(750 Da) and peptide sequences containinga folding motif (GEAK[LAEIEAK]2LAEIY),and the second having a longer PEOcomponent (2,000 Da) were investigatedin the work by Vandermeulen et al. [102].These PPCs favoured the formation ofcoiled-coils, and self-assembled into well-defined supramolecular aggregates. Anequilibrium was established between theunimeric, dimeric and tetrameric coiled-coil assemblies, which was found to bedependent upon concentration, temperature,solvent, and the molecular weight of thePEO component. The same group carriedout further examination of PPCs comprisedof PEO and the LAEIEAK-based coiled-coil sequence [54]. This LAEIEAK sequencewas used as a starting point for the workand a series of PPCs were obtained byexchanging amino acid residues in specificheptad repeat positions. These conjugatesretained their ability to form coiled-coils,highlighting the dominant amino acidsrequired for coiled-coil formation, andaggregates were formed under appropriateconditions. A degree of pH-sensitivity couldbe induced by alteration of the amino acidsequence. Further investigation of thesepolymers was proposed as being directed

towards biomedical applications, inparticular as drug delivery vehicles.Burkoth et al. [103, 104] also investigatedPPCs containing peptides over 20 aminoacids in length. Here, the conjugation of asynthetic amyloid peptide sequence(YEVHHQLVFFAEDVGSNKGAIIGL)to PEO (3000 Da) was investigated. ThesePPCs self-assembled via β-sheet formationto form fibrils. In this case, the PEO wasthought to shield the C-terminus hydro-phobic domain of the peptide sequence,preventing extensive fibril-fibril aggre-gation. Manipulation of this materialduring the synthesis (e.g. peptide sequencealteration) should allow substantial tertiarystructural engineering.

3.2 Conjugates Comprising AlternativePolymers

Although PEO has been widely usedin PPCs featured across the literature,other polymers have also have also receivedattention. Poly(N-isopropylacrylamide)(PNIPAAm) has been extensively used forbiomaterials [105]. A PPC comprising ofPNIPAAm and the octapeptide FEFEFKFKwas reported by Tirelli and co-workers[106]. The peptide was selected as it is

Figure 6. PEO-peptide and PEO-peptide-PEO polymer-peptide conjugates designedby R sler et al [101].

Chiang Mai J. Sci. 2012; 39(3) 365

known to form β-sheet rich fibrillarhydrogels. This PPC had a molecularweight of 18.3 kDa composed of 98.5mol% polymer and formed gels, where thegelation effect was attributed to thebehaviour of the peptide. The hydrogelexhibited double thermo-responsivebehaviour, which would be beneficial indrug delivery applications. Tirelli and co-workers [89] expanded the range ofPNIPAAm-FEFEFKFK PPCs by usingdifferent molecular weight polymercomponents. In this work hydrogels wereobtainable when using small amounts ofthe PPCs to dope pure PNIPAAm. Thiswork demonstrated that polymer/PPCmixtures formed temperature-sensitiveself-supporting hydrogels, when above acritical solution concentration. LCSTtransition was controlled by the PNIPAAmcomponent and was not found to besignificantly influenced by the presence ofthe peptide. The properties of the gels weretuneable via variation of the peptide orpolymer component. For example, theelastic modulus of the conjugate wasproportional to the quantity of peptidepresent in the macromolecules. Carefultuning of the molecular composition ofthese materials will allow hydrogels to beproduced with properties specificallydesigned for drug delivery.

2-hydroxyethyl methacrylate (HEMA)is another polymer well known for its usein biomaterials [92]. A PPC comprised ofHEMA and the peptide GRGDS wassynthesised by Mei et al. [107], where theRGD component of this sequence is thewell-studied cell-binding motif. The PPCwas found to form hydrogels, although themechanism of formation was not discussed.This work illustrates that the biocompatiblepolymer, HEMA, can be used in self-assembling PPCs, and potentially for drug

delivery applications.Poly[N-(2-hydroxypropyl)methacry-

lamide] (PHPMA) is a non-immunogenic,neutral, hydrophilic polymer which hasbeen employed in anticancer drug delivery.Radu et al.[108] conjugated PHPMA to theamino acid sequence QQRFQWQFEQQ.This sequence was selected as it is knownto form antiparallel β-sheets which self-organise into fibrils in aqueous solutions.The ability of the peptide to form β-sheetnanostructures was retained uponconjugation to low molecular weightPHPMA, demonstrating that PHPMA canbe utilised as a self-assembling biomaterial,extending the range of polymercomponents available for PPCs.

4. SUMMARYThere is an incredible body of work

behind the synthesis of polymer-peptideconjugates, with relatively recent discoveriesin controlled radical polymerisationprocesses and ligation chemistries, alongsidethe long-standing methods in amino acidcoupling. Similarly, there have been greatadvances in understanding and manipu-lating the self-assembling behaviour ofPPCs. Although this area of researchremains somewhat in its infancy, ourunderstanding is frequently being expandedand a generic set of design rules for PPCswill be available in the near future.Consequently, we are now in a position todesign macromolecules based on polymersand peptides with controllable propertiesin terms of routes to gelation and physicalcharacteristics of the final hydrogel. Thepotential for injection of thixotropicbiocompatible materials, coupled withtheir ability to accommodate copiousvolumes of aqueous media, renders themideal scaffolds for targeted drug delivery.However, as discussed throughout this

366 Chiang Mai J. Sci. 2012; 39(3)

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