This journal is©The Royal Society of Chemistry 2018 Chem. Soc. Rev.

Cite this:DOI: 10.1039/c8cs00508g

An introduction to zwitterionic polymer behaviorand applications in solution and at surfaces†

Lewis D. Blackman, * Pathiraja A. Gunatillake, Peter Cass andKatherine E. S. Locock *

Zwitterionic polymers, including polyampholytes and polybetaines, are polymers with both positive and

negative charges incorporated into their structure. They are a unique class of smart materials with great

potential in a broad range of applications in nanotechnology, biomaterials science, nanomedicine and

healthcare, as additives for bulk construction materials and crude oil, and in water remediation. In this

Tutorial Review, we aim to highlight their structural diversity and design criteria, and their preparation

using modern techniques. Their behavior, both in solution and at surfaces, will be examined under a

range of environmental conditions. Finally, we will exemplify how their unique behaviors give rise to

specific properties tailored to a selection of their numerous applications.

Key learning points1. What are the structures of zwitterionic polymers and how can they be prepared using modern techniques?2. How do zwitterionic polymers behave in solution and respond to changes in their environment?3. How do polyampholytes interact with charged surfaces and what gives rise to their unique behavior?4. What are some of the numerous key applications of these materials?

Introduction

Zwitterionic polymers, encompassing polyampholytes and poly-betaines, are polymers that consist of oppositely chargedcationic and anionic groups along the chain or side chain.1,2

This is in contrast to true polyelectrolytes (polyanions or poly-cations), where only monomers of the same charge are presentin the polymer. The charge stoichiometry between the polyionscan be equal, or can be weighted towards anionic or cationiccharacter. Such polymers are typically stimuli-responsive, andcan show dual-nature properties, switching between antipoly-electrolyte or polyelectrolyte behaviors depending on their environ-ment, as such they can be considered as ‘‘smart’’ adaptive materials.As will be examined, the charge stoichiometry and distributionunder different environmental conditions have a marked effect onthe material’s behavioral properties. Indeed, these factors areessential design parameters to enable natural polyampholytes suchas enzymes and other proteins to function and support life.

Though abundant in nature, the preparation and investiga-tion of synthetic polyampholytes and polybetaines dates backto the 1950s and 1960s. Modern polymerization techniqueshave further allowed for their facile preparation and ease oftunability. As such, these materials have tremendous potentialas vehicles to aid in drug and gene delivery, as anti-foulingcoatings, stabilizers for nanoparticles and proteins, and as(self-healing) hydrogel materials. In this Tutorial Review, thestructure and preparation of polyampholytes and polybetineswill be outlined and their behavioral properties both in solutionand at interfaces discussed. Finally, some of their numerouspotential applications will be exemplified.

Zwitterionic polymer structures andsynthesis

Considering the definition of a zwitterionic polymer as apolymer or macromolecule with oppositely charged sites, thereare a number of different ways in which the charges can bedistributed throughout the chain. For polyampholytes, the oppo-site charges exist on separate monomer repeat units. Commonpolyampholytes found in nature include proteins and peptides,whereby the charge is distributed in a sequence-defined pattern

Manufacturing Business Unit, Commonwealth Scientific and Industrial Research

Organisation, Bayview Avenue, Clayton, VIC 3168, Australia.

E-mail: Lewis.Blackman@csiro.au, Katherine.Locock@csiro.au

† Electronic supplementary information (ESI) available: Additional figures andsuggested reading relating to zwitterionic polymers. See DOI: 10.1039/c8cs00508g

Received 26th September 2018

DOI: 10.1039/c8cs00508g

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along its primary structure. The sequence of charge along thechain (along with distribution of hydrophobic residues, etc.)dictates the higher order 3D structures that the macromoleculecan adopt. For synthetic polymers, the charge can be distributedin a random or statistical manner, or can exist in blocksof positive and negative charge (block polyampholytes) usingoppositely charged monomers. Polyampholytes can also exist ascopolymers of oppositely charged monomers and neutral mono-mers. The charge asymmetry (i.e. alternating vs. diblock as the twoextremes) and charge density has a marked effect on the polymer’sconformational behavior. For an extensive overview of conforma-tional polyampholyte behavioral models, the reader is directed tothe review by Dobrynin et al.3 Aside from polymerization of twooppositely charged monomers, zwitterionic monomers, such as

those containing sulfobetaine, carboxybetaine, or phosphobetaineside chains, can also be employed, giving rise to polybetaines(also known as polyzwitterions or polymeric inner salts). Ratherthan using the side chain functionality, charge can also bedistributed through the backbone of the polymer, althoughowing to their greater synthetic complexity, these structuresare less commonly utilized.

There are multiple synthetic routes towards polyampholytes,either by polymerization of charged or zwitterionic monomers,or by post-polymerization modifications. The preparation ofpolyampholytes and their monomers has been thoroughlyreviewed elsewhere1,2 but a few examples will be highlightedhere to give an overview of the design criteria. Polymerizationof zwitterionic monomers can be achieved using a range of

Lewis D. Blackman

Dr Lewis Blackman obtained afirst class Masters’ degree inChemistry from the University ofSouthampton, UK, in 2012. Hewent on to complete his PhD in2017 under the supervision ofProf. Rachel O’Reilly and Prof.Matthew Gibson at the Universityof Warwick, UK, which focused onunderstanding the formation andphase behavior of block copolymerself-assemblies in aqueoussolution, and the preparation ofhybrid enzyme-loaded polymersome

nanoreactors. In 2017, he joined the Polymeric Biomaterials Team atCSIRO in Melbourne, Australia, as a ResearchPlus PostdoctoralResearch Fellow. Lewis’ research interests include zwitterionic polymersynthesis and behavior, RAFT polymerization, responsive polymersystems, and block copolymer self-assemblies for novel biomaterialapplications.

Pathiraja A. Gunatillake

Dr Pathiraja Gunatillake obtainedhis PhD from City University of NewYork, and is currently an HonoraryFellow (CSIRO Australia) andadjunct Professor (Monash Institutefor Medical Engineering). He hasfocused his research on the designand synthesis of polymers forbiomedical applications andspin-off companies, AorTechBiomaterials and PolyNovo Bio-materials, were established usingtechnology developed under hisleadership. He received the Sir

Ian McLelan Achievement for Industry Award in 2002, CSIROmedals for research achievement in 2003 and 2005. He hasreceived outstanding international recognition as evidenced byhis record of publications in international journals (70+) andinternational patents/applications (23).

Peter Cass

Dr Peter Cass received his PhD inpolymer chemistry from SwinburneUniversity, Australia in 2000. Peterdid his Postdoctoral Fellowship atMelbourne University, Australiafrom 2001 to 2003. He iscurrently working as a ResearchScientist at the CommonwealthScientific and Industrial ResearchOrganisation, Melbourne, Australia.His research interests lie in polymerchemistry including industrialpolymers, composites, drug deliveryand biomaterials.

Katherine E. S. Locock

Dr Katherine Locock is a ResearchScientist in the ManufacturingBusiness Unit of the CSIRO inMelbourne, Australia. Her researchfocuses on the development ofbiologically active polymers, basedon CSIRO’s patented RAFTtechnology. Katherine also holds aposition as a Royal AustralianChemical Institute (RACI) boardmember for 2016–2018 and sits onthe RACI Inclusion and DiversityCommittee. In recognition of hertrack record, Katherine was selected

as the AIPS Victorian Young Tall Poppy of the Year in 2016, received aJulius Career Development award in 2016 and the CSIRO StaffAssociation Women in Science award in 2013.

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polymerization techniques, such as step-growth polymeriza-tion, ionic polymerization, free-radical polymerization (FRP),reversible deactivation radical polymerization (RDRP) methods,or ring-opening metathesis polymerization (ROMP). For exam-ple, a wealth of zwitterionic monomers, such as those depictedin Fig. 1 have been successfully polymerized directly by FRP andRDRP techniques, as well as ROMP and step-growth methods.1

For block and statistical polyampholytes, direct polymerizationof anionic and cationic monomers is possible, however incertain instances they show poor compatibility of the ionicfunctionality with the polymerization technique in question, ordifficulties concerning solubility or characterization. In thesecases, synthesis of a precursor polymer can be achieved, whereone or more of the blocks is protected and neutral, followed bydeprotection and/or functionalization to reveal the chargedblock. This approach is typically performed with protectedcarboxylic acids such as (meth)acrylic acid, whereby tert-butyl(meth)acrylate is polymerized first to yield a precursor block.Hydrolysis of the ester using a strong acid then yields thecarboxylic acid side chain, which exists as an anionic carboxylateat neutral pH. Similarly, for cationic monomers such as quaternaryammonium, pyridinium and phosphonium moieties, often theneutral, tertiary monomer is prepared first, followed by alkylation,either by reaction with an alkyl halide, or by ring opening alkyla-tion to afford the cationic species.1

Non-deprotection based post-polymerization modificationapproaches allow one to obtain zwitterionic side chain func-tionalities. Typically, an amino-side chain is used to anchor anacidic group such as a carboxylic, phosphonic, phosphoric, orsulfonic acid moiety, for instance by nucleophilic substitutionwith a corresponding alkyl halide, or by ring opening, forinstance of sultones (Fig. S1, ESI†). This installs both the acidgroup (anionic at neutral pH), and the cationic quaternaryammonium ion into the side chain of the repeating unit. Inperforming such a betainization reaction, any incompatibilityissues with the polymerization technique can potentially beavoided and a library of compounds can be easily obtainedfrom a single parent polymer scaffold. This is particularlyuseful for investigating structure–property relationships in

these materials, or for functionalizing surfaces, monoliths orcross-linked nanoparticles.

Others have investigated routes towards alternating poly-ampholytes, where the charge precisely alternates between cationicand anionic throughout the chain. Du Prez and co-workersrecently reported the thiol–ene step-growth polymerization ofN-maleamic acid homocysteine thiolactone-based monomers,which had previously been ring opened with a range of aminesto afford the free thiol (Fig. 2A).4 Owing to the carboxylic acidmoiety and the amine moiety being present on the same repeatunit, this gave rise to precisely alternating polyampholytes. Gibsonand co-workers also developed alternating polyampholytes usingthe post-polymerization ring opening of the low-cost commoditypolymer, poly(methyl vinyl ether-alt-maleic anhydride), withprotected amines, followed by deprotection (Fig. 2B).6

Solution behaviorpH- and salt-responsive solution behavior

As discussed previously, the zwitterionic nature of a polyampholyte,or certain polybetaines, usually depends on its environment, with

Fig. 1 Chemical structures of example zwitterionic monomers compati-ble with various polymerization techniques.1,4,5

Fig. 2 Preparation of alternating polyampholytes. (A) Thiol–ene step-growth polymerization of N-maleamic acid homocysteine thiolactone-based zwitterionic monomers. Reprinted with permission from ref. 4Copyright 2017 American Chemical Society. (B) Post-polymerizationring-opening of poly(methyl vinyl ether-alt-maleic anhydride) to affordan alternating polyampholyte structure. Adapted from ref. 6 Published bythe Royal Society of Chemistry 2015.

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the chemical structure of its components dictating its overallbehavior. Typically, the factor dictating its ionization is pH asthe majority of polyampholytes are protic in either one or moreof their ionizable components. The pH dependency of thechain’s ionization can be observed by finding its isoelectricpoint (IEP), the pH at which the polymer is electricallyneutral.2,7 At the IEP, the polymer is generally insoluble inaqueous solution and attractive forces between the polyionsdominate, forcing a globule conformation. Above and below theIEP, when there is a net overall charge, repulsion between thecharged units dominate. As such, a statistical polyampholytebelow or above its IEP would behave similarly to a polyanion orpolycation, adopting an extended coil conformation to minimizeinterchain repulsion and allow for solvation in aqueous solution.7

Note that the solution behavior is also dependent on the chargesequence; those with broad charge distributions (the extreme beingdiblock polyampholytes) exhibit coil-to-globule transitions whenmoving towards the IEP owing to strong attractive interactionsbetween the repeat units, however, those with a narrow chargedistribution (the extreme being alternating polyampholytes) can beswollen and soluble at the IEP. The IEP can be theoreticallypredicted from the ratio (R) of acidic and basic units, and theirrespective pKa and pKb values (eqn (1)).2,7 It can be shown that atR = 1 (equimolar polyampholytes), the IEP is simply the average ofthe pKa and the pKb. At R = 2 (twice as many acids than bases), theIEP is equal to the pKa of the acid and at R = 0.5 (twice as manybases than acids), the IEP is equal to the pKb of the base (see ESI†).

R ¼ 1þ 10pKa�IEP

1þ 10IEP�pKb(1)

The preparation of more permanently charged polyampho-lytes is achieved when the side chain is fully ionized over a largepH range. The ionic groups can be classified by their strengthof interaction, which is largely dictated by its pKa value (or pKaH

in the case of cations) and its charge density. For instance,cations with a high pKaH, such as guanidines (pKaH B 13), orthose that are permanently quaternized such as phosphoniumor ammonium groups can be considered a strong cation. Weakerorganic bases such as amine groups (pKaH B 10.5) are consideredto be weak cations. Similarly, weak anions include carboxylic acids(pKa B 4.5), whereas strong anions include sulfonic acids (RSO3H,pKa B 2) and phosphonic acids (RPO3H, pKa B 2). Clearly, strongcations and strong anions give the most robust polyampholytebehavior across a range of pH values. For example, the use of atertiary amine group (a weak cation) at high pH would result inthe loss of cationic character owing to deprotonation, whereas theuse of a carboxylate as the anion is not appropriate for highlyacidic environments, as protonation would result in neutraliza-tion of the monomer units. However, by carefully designing thepolymer, systems tailored to a particular application can beobtained. For polybetaines, the situation for carboxylate groupsis further complicated because the pKa value is dependent on theproximity of the carboxylate to the cationic group.

Chen and Lin et al. studied the invertible behavior of alinear-dendritic block polyampholyte of poly(L-lysine)-b-D2-poly(L-glutamic acid).8 As the pH was raised from 1.4, the

polymer underwent a phase transition from large compoundmicelles with a cationic poly(L-lysine) corona and neutral poly-(L-glutamic acid) cores, through a worm-like micelle phaseupon partial corona ionization at pH 3.4. An insoluble precipi-tated phase was reached when the ionization of the twooppositely charged blocks was equal between pH 4–6. Here,no excess charge was available to stabilize the nanostructures.Increasing the pH further to 6.4 resulted in inverted largecompound vesicle structures, the membranes of which wereformed of the ion complex of the two blocks, and stabilized bythe slight excess of anionic poly(L-glutamate). A further increasein pH partially deprotonated the charged poly(L-lysine) block,resulting in vesicles with neutralized poly(L-lysine) cores, beforefull deprotonation occurred at pH 12.1 resulting in tubularvesicles comprised of poly(L-lysine) cores and poly(L-glutamate)coronas.8 This study highlights the dual nature of polyampholytesand their dependency of their solution behavior on pH. For amore comprehensive review of invertible micelles (sometimesreferred to as ‘‘schizophrenic’’ micelles), including other blockpolyampholyte assemblies, such as poly(2-(dimethylamino)ethylmethacrylate)-b-poly(acrylic acid) and poly(4-vinylbenzoic acid)-b-poly(2-(diethylamino)ethyl methacrylate), the reader is directedelsewhere.9

In addition to pH changes, zwitterionic polymers are alsohighly responsive to the addition of salt. Contrary to poly-electrolyte behavior, whereby the addition of salt screens thecharges and reduces inter- and intrachain repulsion of like-charges, the addition of salt to a polyampholyte or polybetainesolution screens inter- and intrachain attraction of oppositecharges. Therefore, as opposed to becoming less soluble in saltsolutions at the IEP, such materials become more soluble at acertain salt concentration compared to pure water, owing todestabilization of the polyion complexation, thereby aiding solva-tion. This is known as the antipolyelectrolyte ‘‘salting-in’’ effect.

Bekturov and Schulz et al. studied the effect of potassiumchloride on aqueous solutions of a near equimolar blockpolyampholyte (poly(acrylic acid)-b-poly(4-vinyl pyridiniumchloride) (PAA-b-4VPCl)).10 The authors demonstrated that atboth high and low pH, where the chain had a net negative orpositive charge, respectively, the chain exhibited polyelectrolytebehavior with repulsion between the polyions dominating andaiding solvation (Fig. 3B and D). On addition of salt, thecharges were screened, which reduced the ionic repulsion,and at a critical ionic strength, a precipitate formed as theattractive inter- and intramolecular forces dominated (Fig. 3E).This salting-out effect is typical for polyelectrolyte systems.Close to the IEP, the polyampholyte became insoluble owingto attraction between the polyions (Fig. 3C), but upon additionof salt, the attractive interactions could be effectively screened,which aided solubilization (Fig. 3A).10 Whilst for the majority ofpolyampholytes and polybetaines the ionic strength requiredfor solubilization close to the IEP is relatively low, one class ofampholytes that form very strong polyion complexes are poly-(ammonio alkoxydicyanoethenolate)s (Fig. S2, ESI†), which arehighly dipolar and insoluble in pure water, even up to 100 1C.11

The addition of very strong acids, which partially protonate the

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anionic component, or the addition of very high salt concentra-tions (e.g. 4.90 M ZnCl2 or 3.42 KI at 25 1C), are required tosolubilize the polyampholyte. In this specific example, theauthors suggested that a different solvation mechanism wasnecessary to solubilize such polymers and that possibly ternarymixtures of higher order salt structures (such as Zn4Cl(H2O)2�,which forms in concentrated aqueous ZnCl2 solutions) aid thenotably difficult solubilization.11

Thermoresponsive solution behavior

As the aqueous solubility of zwitterionic polymers is dictated bycharge–charge interactions, typically, their solubility is alsodictated by the temperature of the system. The necessity tobreak the attractive ionic interactions means that generally,polybetaines display upper critical solution temperature (UCST)-type behavior.12 This is where the polymer becomes solubleabove a critical temperature, where the entropic (�TDSmix) termof the Gibbs free energy of mixing, relating to release of thechains into solution and allowing chain mobility, overcomes theenthalpic contribution (DHmix) associated with the attractiveelectrostatic interactions in the collapsed insoluble state.Indeed, the UCST generally increases as a function of increas-ing molecular weight, owing to the increase of the coulombicattractive interactions in the collapsed state, and the decreasingentropy of mixing. Such behavior is very well documented forzwitterionic homopolymer betaines such as poly(2-(N-3-sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate)(PDMAPS),12–14 and can result in temperature dependentprecipitation, swelling, or morphological transitions.O’Reilly and co-workers studied the solution behavior of ABand ABA block copolymers consisting of a neutral, hydro-philic PEG ‘‘A’’ block and a zwitterionic PDMAPS ‘‘B’’block.15 They found that at low temperatures, the insolublePDMAPS block, whose attraction was driven by coulombicinteractions, drove the self-assembly of micelles. Upon heat-ing, the self-assemblies were shown to disassemble intounimer chains (Fig. 4A). By studying the solutions by variabletemperature light scattering and small angle X-ray scattering,

the authors could deduce that the disassembly occurred via amixed phase of micelles and unimers, with the relative propor-tion of each phase dictated by the solution temperature.15

Similar results have also been observed for statistical poly-ampholytes composed of both cationic and anionic monomers,such as poly(2-(dimethylamino)ethyl methacrylate)-b-poly-(methacrylic acid) (PDMAEMA-co-PMAA) copolymers investi-gated by Hoogenboom and co-workers in alcohol/watermixtures.16 Here, the authors observed UCST-type behavior,owing to the breaking of electrostatic interactions uponheating. The transition temperature could also be tuned byadjusting the solution pH, thereby modulating the net chargeof the chain.

Zwitterionic homopolymer blocks have also been incorpo-rated into block copolymers where one of the blocks exhibitslower critical solution temperature (LCST) properties, wheresolubility is promoted at low temperatures, rather than at hightemperatures for UCST polymers. As with the pH-switchableinvertible micelles discussed previously,8 this can yield doubly-hydrophilic thermoresponsive invertible micelles that switchtheir core and corona chemistries in response to temperature.9

For example, Laschewsky formed doubly-hydrophilic poly(N-isopropylacrylamide)-b-poly(3-[N-(3-methacrylamidopropyl)-N,N-dimethyl]ammoniopropane sulfonate) (PNIPAM-b-PSPP)diblock copolymers with an LCST-type PNIPAM block and aUCST-type zwitterionic PSPP block coronas (Fig. 4B).17 At lowtemperatures, the PNIPAM block was soluble and the PSPPblock insoluble, which drove the formation of micelles withPSPP cores stabilized by PNIPAM coronas. Upon increasing thetemperature past the UCST of the PSPP block but below theLCST of PNIPAM, the micelles underwent an order–disordertransition towards molecularly dissolved unimer chains.Increasing the temperature further, past the LCST of PNIPAM,resulted in a disorder–order transition towards the formationof micelles with inverted chemistries; PNIPAM in the core andPSPP in the corona (Fig. 4B).17

Others have utilized the LCST-type thermoresponsive beha-vior of certain amine-containing blocks in block polyampho-lytes to invoke the opposite temperature-dependent solutionbehavior to that of zwitterionic homopolymer blocks. Han andco-workers investigated the LCST behavior of asymmetricPDMAEMA-b-PAA diblock copolymers with relatively longPDMAEMA blocks and relatively short PAA blocks.18 They foundthat the LCST behavior of the PDMAEMA block was pH depen-dent; at the IEP, and at pH values above the IEP, the neutralizedPDMAEMA units were able to undergo aggregation upon heat-ing the solution. Below the IEP, no transition occurred as thePDMAEMA units were partially protonated, with coulombicrepulsion between the cationic chains preventing aggregation(Fig. 4C).18

Self-coacervation behavior of polyampholytes

Coacervation is the separation of a colloidal system into twodistinct liquid phases. This differs from the classic responsivebehavior described in the previous section because the phaseseparation does not result in dehydration and precipitation of

Fig. 3 Schematic representation of the phase diagram of near equimolarPAA-b-4VPCl studied by Bekturov and Schulz et al.,10 with illustrations ofthe behavior observed. Key: red = cationic, blue = anionic, grey =neutralized, yellow and purple = salt ions.

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the polymer to form a solid–liquid two phase system. Instead, ahydrated concentrated phase and a dilute phase, both of whichmake up the liquid–liquid system, is formed. In chargedpolymer systems, this process is driven enthalpically by attrac-tive charge interactions, followed by the additional entropicgain associated with the release of the counterions into thebulk solution.19 When this occurs, a polymer-rich phase (thecoacervate) and a polymer-poor phase (the supernatant) form.This metastable phase forms only under specific environmentalconditions such as concentration, pH, ionic strength, tempera-ture etc. Coacervation between a polyanion and a polycation toform complex coacervates (Fig. 5A(I)) has been widely studiedand utilized in food science, biomedical and personal careapplications. Many naturally occurring systems have also beenexamined, such as the adhesion mechanism of sandcastleworms. The reader is directed to a recent review by Perry et al.for an overview of the phase behavior and applications ofcomplex coacervates,19 and additional reviews listed in the ESI.†

Contrary to complex coacervation observed with oppositelycharged polyelectrolytes, polyampholytes are able to undergoself-coacervation. As the name suggests, this type of coacervationoccurs with just a single polyampholyte component present insolution. Examples of self-coacervation of polyampholytes found

in nature are the adhesive mussel foot protein, Mfp-3S,20 and inproteins located in the beak of the Humboldt squid.21 Both ofthese examples contain acidic and basic residues, as well asaromatic residues (the primary structure of Mfp-3S is shown inFig. 5B). In both systems, self-coacervation was found to bedictated by a careful balance of columbic and hydrophobicinteractions. Accordingly, the self-coacervation of both systemsshowed pH and ionic strength dependencies. Taking the Mfp-3Ssystem, increasing the pH from 3 up to 7 (close to the IEP of theprotein, pH = 7.5) resulted in self-coacervation at low ionicstrengths of 10 mM (Fig. 5A(II)).20 It was rationalized that thelarge degree of repulsion from charged acidic residues at low pHprevented the coacervation, but upon partial neutralization ofthese residues, the anion : cation ratio became closer to unityand attractive interactions dominated. Upon increasing the ionicstrength of the buffer (to 100 mM then to 600 mM), self-coacervates were formed at a wider range of acidic pH values,including at pH 3.

Here, the salt ions could screen the effective charges of theacidic residues to allow for coacervation. At pH values close tothe IEP and high ionic strengths, full precipitation rather thancoacervation occurred.20 It should be noted that self-coacervationis not limited to electrostatically-driven coacervation. For example,

Fig. 4 Illustrations of zwitterionic polymer assemblies able to undergo thermal transitions. (A) UCST-driven micelle to unimer transition of POEGMA-b-PDMAPS. Adapted from ref. 15 published by the Royal Society of Chemistry. (B) Invertible behavior of PNIPAM-b-PSPP with both UCST and LCSTproperties. Reprinted with permission from ref. 17 Copyright 2002 American Chemical Society. (C) pH-dependent LCST aggregation behavior ofPDMAEMA-b-PAA.

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recombinant human tropoelastin, which is not polyamphoteric,can undergo thermally induced self-coacervation in aqueoussolution, driven predominantly by both hydrophobic interactionsand entropic factors (Fig. 5A(III)).20,21 Very recently, the coacerva-tion behavior of the mussel foot protein Mfp-3B was found to bedependent on various environmental factors.22 At low pH andionic strength, as found inside the mussel’s phenol gland, UCST-driven self-coacervation behavior was observed. At high pH andionic strength, as found in the external sea water, temperature-independent coacervation was induced (Fig. 5A(IV)). Low concen-trations of citrate or sulfate ions could also promote complexcoacervation in either environment. This study gives an intriguinginsight into the formation mechanism of adhesive coacervatesfound in nature.22

Polyampholyte behavior in thepresence of charged surfaces

As well as their unique solution behavior, polyampholytesdisplay a range of interesting behaviors when in the presenceof surfaces. Their behavior is dependent on the structure of thepolyampholyte (e.g. sequence and ratio of charges, weak orstrong ionic groups, etc.) as well as the nature of the substrate.Khan and co-workers investigated the effect of charge sequenceof weakly coupled polyampholytes on their adsorption ontonegatively charged surfaces using Monte Carlo simulations.23 Arange of surface charge densities and degrees of polyampholyteionization were investigated. Weakly coupled polyampholytesare those whose adsorption is induced by polarization of thechain by the charged surface (known as the polarization-induced attraction mechanism). A range of net neutral poly-ampholytes with various monomer sequences from blockthrough to alternating were investigated (Fig. 6A). As one would

expect, they found that for neutral block polyampholytes,adsorption of positively charged monomer units was promoted,whereas repulsion resulted from negatively charged monomers,forcing these units into solution (Fig. 6B and C, trace a).Conversely, for neutral alternating polyampholytes, there wasno preference for adsorption regardless of the monomer charge(Fig. 6B and C, trace d). This was explained by considering thatfor block polyampholytes, owing to the charge separation, it iseasy for the chain to arrange such that the positively chargedmonomers adsorb to the negative surface, whilst minimizinglike-charge interactions. For alternating polyampholytes, it isalmost impossible to do so, which resulted in neither adsorp-tion nor repulsion. For neutral random polyampholytes, thebehavior was between the two extremes, in that both thenegative and positive components of the chains adsorbed tothe surface (Fig. 6B and C, trace b and c). One parameter thatwas also of crucial importance was the Gouy–Chapman length(lGC), defined as the distance from the surface at which theelectrostatic interaction between the surface and a monovalention reaches a magnitude on the order of the thermal energy,kBT. lGC is inversely proportional to the surface charge density,s, according to eqn (2).

lGC ¼ekBT2pejsj (2)

where e is the dielectric constant of the media and e is theelementary charge. The authors observed that for overall netneutral polyampholytes, the thickness of the adsorbed layerincreased with decreasing lGC (increasing surface chargedensity) until a critical thickness was reached, whereby thethickness approached lGC. Upon further increasing the surfacecharge density, decreasing lGC, the thickness was found todecrease (Fig. 6D and E).

Fig. 5 (A) Coacervation behavior of complex coacervates formed from pairs of polyelectrolytes (I), the electrostatically-driven pH-dependent self-coacervation of Mfp-3S (II), the thermally induced self-coacervation of the non-polyamphoteric protein, tropoelastin (III), and the environment-dependent coacervation behavior of Mfp-3B. Adapted with permissions from ref. 20. Copyright 2014 Elsevier Ltd. (B) Primary structure of Mfp-3S. Thearomatic and charged residues are indicated.

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Whilst the results concerning the monomer distributionsfor net neutral polyampholytes are somewhat to be expected,the behavior of polyampholytes with a net overall charge donot follow the same rules as polyelectrolytes where only asingle charge is present. In the same study by Khan andco-workers, it was observed that polyampholytes with a netnegative charge adsorbed to the negative surface, and that theamount of adsorbed material increased as a function ofdecreasing lGC and increasing negative surface charge(Fig. 6F).23 This unexpected behavior was observed experimen-tally by Israelachvili and co-workers, who investigated theadsorption behavior of the polyampholyte polypeptide, gela-tin, on a negatively charged mica surface as a function of pHand salt concentration.24 They found that below the IEP,chains with a net positive charge, adopted a flat configurationand strongly adsorbed onto the negative surface. At the IEP, amaximum in the adsorption excess was measured, which wasexplained by the increase in negative charge on the micasurface at this pH, as well as the more crumpled configurationof the chains, owing to lower repulsive forces, which led to agreater adsorption density. Above the IEP, a significantamount of adsorption of net negative polyampholyte wasobserved, which was attributed to interactions between thenegative mica surface and the few remaining positive mono-mer units. The polymer chains were also found to adopt a

thicker layer at this pH, owing to repulsion of like-chargesbetween the net negative chain and the negative mica surface(Fig. 7A–C).24

Three distinct regimes describe the behavior of a singlepolyampholyte chain in the presence of charged surfaces,depending on the strength of the electric field relative to thechain’s thermal energy.3,25 At low field strengths, when theattractive forces between the surface and the chain are lowerthan the chain’s thermal energy, the chain is elongated in thedirection of the field. In the perpendicular direction to thefield, the chain retains its Gaussian conformation in what isknown as the pole regime (Fig. 7D). Here, the attractive forcesdo not warrant the entropic loss associated with adopting anon-Gaussian conformation. At higher field strengths, thechain becomes confined to within lGC and the chain stretchesalong the surface in what is known as the fence regime(Fig. 7E). Increasing the field strength further still, the pancakeregime is reached, lGC is further reduced, and the polymeradopts a conformation where monomers with opposite chargesto the surface reside within lGC and where units with the samecharge as the surface form loops out into the solution tominimize their interaction with the surface (Fig. 7F).3,25

The reader is referred to the review by Dobrynin, Colby andRubinstein for a more detailed discussion of adsorption behavior,including multi-chain models.3

Fig. 6 (A) Schematic representation of each net neutral polyampholyte used in the theoretical adsorption experiment by Khan and co-workers (red =positive, blue = negative, grey = neutral). (B and C) Adsorption population distributions as a function of distance (h) for negative (A) and positive (B)monomer units comprising polyampholyte chains near a negatively charged surface. (D) Adsorption thickness, hz2i1/2, as a function of lGC for differentpolymer lengths. Adsorption at polymer length N = 20 (circles), N = 40 (squares), N = 80 (diamonds), N = 160 (solid triangles), for f = 1.0. N = 160, f = 0.1(unfilled triangles) are also shown where f is the overall fraction of monomers bearing charge. (E) Illustration of the influence of lGC on the adsorbed layerthickness in panel D. (E) Layer thickness of net negative polyampholytes with f = 1.0 on a negative surface with lGC = 2.2 Å (circles), lGC = 4.4 Å (squares),lGC = 6.6 Å (diamonds), lGC = 8.8 Å (triangles) and lGC = 22 Å (stars). The composition of the polymer, f+ (total fraction of positive monomers) is plottedon the x-axis. Adapted with permissions from ref. 23 Copyright 2001 American Chemical Society.

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Applications of zwitterionic polymersZwitterionic polymers as anti-fouling coatings

Aside from their interaction with charged surfaces, polyampholyteshave also been utilized to prepare polymer brushes covalentlytethered to neutral surfaces. These coating materials play a passiverole in preventing biofilm formation by reducing cell adhesion bythe reduction of protein attachment.26–28 Surfaces with anti-foulingproperties are of high importance to both the field of marineengineering and the biomedical industry. Fouling occurs in ahierarchical manner, with smaller fouling agents such as proteinstaking less time to adhere to the surface.28 For a pristine surface,an electrical double layer forms at the liquid–substrate interface,which promotes the attachment of charged proteins. This proteinlayer then acts as a protective conditioning film for the attachmentof microbes over longer time periods. To prevent the initialattachment of proteins, and hence reduce instances of microbeattachment or biofilm formation, the anti-fouling properties ofseveral coatings have been reported. Bernards et al. hasreviewed polyampholyte brushes comprised of copolymers of[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride (TM)and 3-sulfopropyl methacrylate (SA) monomers, as well as thosecomprised of [2-(acryloyloxy)ethyl]trimethyl ammonium chloride(TMA) and 2-carboxyethyl acrylate (CAA).29 In one example, it wasshown that a 1 : 1 ratio of cationic TM and anionic SA was

required in the polymer brush to achieve ultralow-fouling proper-ties (fouling o5 ng cm�2) across the three proteins investigated.30

Deviation from the optimal ratio resulted in significant proteinattachment.

Polybetaines, such as polymers comprised of the monomersshown in Fig. 1 have also gained attention as anti-foulingmaterials and have comparable or better anti-fouling propertiesthan poly(ethylene glycol) (PEG).26,31 Such polymers have a highdegree of hydration owing to their highly charged nature sohave a tendency to resist non-specific protein attachment; thelevels of protein attachment being lower than the detectionlimit of surface plasmon resonance (SPR) measurements incertain cases (o0.3 ng cm�2).32 It should also be noted that themethod of attachment plays a key role in determining thesurface coverage, for instance the ‘‘grafting to’’ method, wherethe polymer is synthesized first, followed by covalent attach-ment to the surface using an anchoring group, results in poorersurface coverage than the ‘‘grafting from’’ method, where thepolymer is grown directly from the surface. The effect of surfacegrafting density has been shown to influence the low-foulingproperties of surfaces. Wu et al. found that membranes func-tionalized with PDMAPS by a ‘‘grafting to’’ technique showedgreater resistance to bovine serum albumin fouling withincreasing grafting density.33 However, both ‘‘grafting to’’ and‘‘grafting from’’ methodologies for attaching polybetaines havebeen reported for preparing ultralow-fouling surfaces.32 It isalso noteworthy that no clear correlation exists between thefouling resistance towards single protein species, and multiplespecies in complex media such as blood, or the attachment ofbacteria or biofilm to a surface.32,34 Whilst reducing proteinadsorption and attractive electrostatic interactions betweenbiological species and the surface are important factors inreducing microbial adhesion, certain microbes also utilize othermodes of surface colonization, such as the presentation ofadhesins. Additionally, factors such as the temperature, incuba-tion time, growth media and surface properties are critical andmust be addressed by the use of a positive control surface whenevaluating the anti-fouling properties of such materials.

Polyampholytes as hydrogel materials

Polyampholytes have been used extensively to form poly-ampholyte hydrogels, which exhibit reversible swelling behaviorin response to salt and pH.35 At high salt concentrations, or atpH ranges away from the IEP, the gel is swollen owing toelectrostatic repulsion, whereas at low salt concentrations, orat the IEP, the gel collapses as attraction and coacervationdominates. Such materials have found broad application inbiomedicine owing to their soft and responsive nature and largedegree of hydration, and can be comprised of both natural andsynthetic polymers. Additionally, these materials exhibit similaranti-fouling behavior as previously discussed and can mimic thenative properties of various tissue types owing to their ease ofmechanical tunability, making them applicable for a wealth oftissue engineering applications.35

Recently, Chung and co-workers developed a polyampholytehydrogel comprised of anionic sodium 4-vinylbenzenesulfonate

Fig. 7 Polyampholyte conformations. Schematics of the gelatin chainconformation both in solution and on a negatively charged mica surfaceat pH values below (A), at (B), and above (C) the IEP. Filled circles indicatepositive charge and empty circles indicate negative charge. Note that thenet charge of the mica surface increases as a function of increasing pH.Reprinted with permissions from ref. 24 Copyright 1992 AmericanChemical Society. (D–F) Schematic representation of the conformationsof a single polyampholyte chain in the pole (D), fence (E) and pancake (F)regime, as described in ref. 3.

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(NaSS) and two cationic monomers, [2-(acryloyloxy)ethyl]-trimethylammonium chloride (DMAEA-Q) and (3-(methacryloyl-amino)propyl)trimethylammonium chloride (MPTC), the structuresof which are shown in Fig. 8A.38 The hydrogel was cross-linked witha bis-acrylamide linker to form a tough, charge-balanced hydrogel.The UCST of the hydrogel was utilized to induce an opaque-to-transparent phase transition upon heating the hydrogel and thetransition temperature could be tuned by varying the ratio ofDMAEA-Q to MPTC. These materials show great potential as con-struction materials for energy efficient smart windows that can altertheir opacity in response to their environment.38

In addition to classical hydrogels, where a covalent cross-linker permanently links the polymer chains comprising thegel, polyampholytes have also been used to form physicalhydrogels, where the crosslinking contact points are heldtogether by attractive electrostatic interactions, which are rever-sible under certain conditions. Gong and co-workers designedsuch a physical hydrogel that comprised cationic DMAEA-Q andMPTC and anionic NaSS and 2-acrylamido-2-methylpropanesulfonic acid (AMPS, Fig. 8A), to form an overall chargebalanced hydrogel.36 The hydrogel formed both strong, closeproximity electrostatic interactions, which were stabilizedfurther by polymer entanglements, as well as weak electrostaticinteractions between distal polyions. The combination of therange of different interaction strengths enabled high toughness

and viscoelasticity; upon stretching the sample, the weak sacri-ficial bonds could be reversibly broken and reformed (Fig. 8B).36

In a later study, the dynamic nature of these sacrificial bondswas shown to also impart self-healing properties (Fig. 8C).37

Zwitterionic polymers as dispersants and stabilizers

Owing to their ‘‘salting-in’’ behavior, zwitterionic polymershave gained attention as dispersants of nanomaterials suchas graphene and colloidal metal nanoparticles under high saltconcentrations.25 Nanomaterials are of great importance toadvancements in nanotechnology, however their environmen-tal impact and toxicity are of growing concern, of which oneaspect relates to their poor colloidal stability under highsalinity environments, such as in seawater. Vasantha andParthiban et al. prepared methacrylamide and methacrylatebased poly(sulfabetaine)s by free radical polymerization andused them to stabilize silver, gold and palladium nanoparticlesthrough binding of the CQO to the metal particles.39 Theyfound that these poly(sulfabetaine)-based dispersants couldstabilize the particles in high salinity (2 M NaCl) for extendedperiods of up to 2 months. Similarly, the pH-responsive beha-vior of a step-growth polycondensation derived polyampholyte,sulfonated cardo poly(arylene ether sulfone), containing pendantaminoethyl groups (PSPES-NH2, Fig. 9), has been exploited forcatalyst recycling applications.40 Briefly, it was shown that the

Fig. 8 (A) Chemical structures of common monomers used to form polyampholyte hydrogels. (B) Structure of the physical hydrogel investigated byGong and co-workers, showing the formation of strong and weak bonds and its behavior upon stretching. Adapted with permission from ref. 36Copyright 2013 Springer Nature. (C) Structure and behavior of the gel upon cutting and self-healing. Adapted with permission from ref. 37 Copyright 1992American Chemical Society.

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polyampholyte could stabilize gold nanoclusters formed in situin a pH range of 2–14, but the particles precipitated uponacidification to pH 0 or with the addition of organic solvents(Fig. 9). Since this behavior was shown to be reversible, the goldnanoclusters could be recycled, as demonstrated by their nearquantitative catalytic oxidation of benzyl alcohol even after 6utilizations. This smart zwitterionic dispersant therefore showedgreat potential for the recovery of expensive or toxic colloidalcatalysts.40 There is also evidence to suggest that polyampholytesconfer advantageous mechanical properties to constructionmaterials such as subgrade soil, through multivalent bindingof the polymer to the partially charged soil particulates, allowingfor greater durability.41

The low-fouling capabilities of polybetaines have also gainedthem considerable interest in bioconjugation for the stabili-zation of proteins. For example, compared to the correspondingfree proteins, the bioconjugates of various proteins with poly-(2-methacryloyloxyethyl phosphoryl choline) have been shown togive enhanced pharmacokinetic profiles and therapeutic effi-cacy, improved uptake into tumors, improved conformationalstability, and increased circulation half-lives.42 Such stabilizersare excellent candidates as alternatives to poly(ethylene glycol)(PEG), over which there is growing concern regarding immuno-genicity issues, however more studies directly comparing theimmunotoxicity of PEG and emerging zwitterionic polymersmust be conducted (see ESI†). A very recent study by Leckbandand Gruebele et al. also demonstrated that unconjugatedpoly(sulfobetaine methacrylate) altered the folding of threeglobular proteins when mixed into solution at concentrationsof up to 5% w/w polymer.43 The polymer was found to increasethe polarity of partially exposed tryptophan residues and have animpact on the melting temperature of the proteins. However,the degree of stabilization or destabilization was found to beprotein-dependent, suggesting that the presence of the

polybetaine could be advantageous or unfavorable dependingon the application in question.43 This study hence presentsevidence for the future role of polybetaines as biologicalmacromolecule stabilisers, along with some future challengesto their utilization.

Zwitterionic polymers as antifreeze materials

Additives that can prevent or modify the freezing behavior ofwater are important for a number of applications. Theseinclude the cryopreservation of blood and tissue, preventionof ice formation on specialty materials such as those intendedfor energy or aerospace, the reduction of freeze–thaw corrosiondamage on construction materials such as steel and concrete,and reducing crop damage in the agricultural industry. Naturalmacromolecules with antifreeze activity include antifreezeproteins and antifreeze glycoproteins (AF(G)Ps).44 These haveboth hydrophilic domains, as well as hydrophobic domainsthought to be capable of binding to ice surfaces, although theirexact mechanism of action varies between macromolecules andis not yet fully understood. An emerging number of syntheticpolyampholyte mimics of AF(G)Ps have also been shown toinhibit and modulate ice formation and growth.44–46 For example,the alternating poly(methyl vinyl ether-alt-maleic anhydride)depicted in Fig. 2B was found to inhibit ice recrystallization whenan equimolar cation : anion ratio was used.6 Interestingly, devia-tion from this ratio resulted in a loss of activity. Hyong andMatsumusa have utilized partially carboxylated poly(L-lysine) togreatly enhance the cell viability of both rodent and mammaliancell lines after freezing and thawing.45 At high concentrations,they observed changes in the morphology of the formed icecrystals and showed the polyampholyte allowed better cellrecovery and lower toxicity than dimethylsulfoxide (DMSO), acurrent state-of-the-art cryoprotectant.45

A study by Matsumura and co-workers investigated theinteraction of an antifreeze polyampholyte, P(DMAEMA-co-MAA),with varying hydrophobicities, as well as poly(sulfobetaine)s andpoly(carboxybetaine)s, with liposomes in order to bring to lightsome mechanistic understanding of these materials.46 It isevident that these materials not only inhibit ice recrystallizationbut also interact with phospholipid bilayers, offering them protec-tion during freezing and thawing to prevent membrane damage.46

Whilst synthetic zwitterionic polymers on the whole currentlyhave poorer cryoprotective properties than AF(G)Ps, or syntheticpoly(vinyl alcohol), it is thought that further development of thisrelatively new class of cryoprotectants may offer more syntheticallyaccessible and tailorable antifreeze materials.

Zwitterionic polymers in drug delivery and gene therapy

Zwitterionic polymers are also emerging materials for potentialdrug and gene delivery applications. Owing to their relativecharge balance, polymer self-assemblies stabilized by zwitter-ionic components often show a lower toxicity than thosestabilized by purely cationic polyelectrolytes. This phenomenonwas studied by Stenzel and co-workers, who demonstrated thatmicelles containing an additional arginine-based zwitterionicblock (P(M-Arg)) in its shell resulted in similarly high cell

Fig. 9 Recycling of a heterogeneous colloidal gold catalyst stabilized by astimuli-responsive polyampholyte in the study by Zhang and co-workers.40

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viabilities as those stabilized with neutral poly(ethylene glycolmonomethyl ester methacrylate) (PEGMA) alone (Fig. 10A).5 Aderivative containing the methyl ester of the carboxylic acidmoiety in the arginine-based block (P(Me-M-Arg)), which con-tained only the cationic component, or those stabilized with acationic poly(2-methacryolyloxy ethyl)trimethyl ammoniumchloride (PTMA) block, showed high toxicity. Owing to theirguanidine functionality, known for enhancing cell attachmentand uptake in vitro, the P(M-Arg)-stabilized particles out-performed both the PEGMA-, PTMA- or P(Me-M-Arg)-stabilizedmicelles in their cellular uptake into epithelial ovarian adenocar-cinoma cells. In this respect, the zwitterionic structure combinedthe low toxicity of neutral polymers, such as PEGMA, with thehigh cell uptake efficiency of cationic guanidine-functionalpolymers.5 The use of zwitterionic polymers as drug deliveryvehicles may also allow for different uptake mechanisms

compared to the free drug. Geckeler and co-workers developedpaclitaxel-loaded nanoparticles stabilized by PDMAEMA-co-MAA, which were able to reverse the multi-drug resistance ofbreast cancer cells.48 The authors showed that the particleswere taken up by adsorptive endocytosis, thereby bypassingthe efflux pump, P-170 glycoprotein, expressed by multidrugresistant cancer cells. In modulating the uptake mechanism,the loaded paclitaxel avoided efflux and could effectively reduceproliferation in a multi-drug resistant breast cancer model.48

Cationic polyelectrolytes have been used extensively to bindnegatively charged DNA to form tight binary polyion complexesas vectors for gene therapy. This stabilizes the DNA and aids itsdelivery to the cell nucleus. However, such non-viral transcrip-tion vectors show poor transcription efficiencies partly becauseof the strong electrostatic attractive forces, which prevents DNAaccessibility and release at the desired target. In nature,

Fig. 10 Potential application of zwitterionic polymers in drug and gene delivery. (A) Rapid uptake of P(M-Arg) and P(Me-M-Arg)-stabilized micelles. Theuse of cationic P(Me-M-Arg) results in cell death but low toxicities were observed using zwitterionic P(M-Arg). Adapted with permission from ref. 5Copyright 2012 American Chemical Society. (B) Illustration of the binding of cationic polyelectrolytes with DNA to form a tight binary complex, asdescribed in ref. 49. Upon addition of a polyampholyte (PEG-AC), a loose ternary complex is formed. (C) Cryomicrophotographs of L292 cell suspensionsdemonstrating freeze concentration using 10% DMSO as a cryoprotectant (left). Upon cooling, the cells reside in the remaining concentrated solution.Right: Uptake of fluorescein isothiocyanate (FITC)-labelled carboxylated poly(L-lysine) polyampholyte nanoparticles and Texas Red (TR)-labelled proteininto cells upon freezing. Adapted with permission from ref. 47 Copyright 2014 Elsevier Ltd.

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loosening of chromatins (packed DNA complexes) is achievedby high mobility group (HMG) proteins. These are amphotericin structure with both cationic domains able to bind the DNA,and an anionic tail domain, both of which are vital for theirfunction. Koyama and co-workers investigated syntheticpolymer mimics of HMG proteins by preparing PEG with bothamino and carboxyl pendants (PEG-AC) via a thiol–ene post-polymerization reaction.49 Cationic poly(ethylene imine) (PEI)was used to form a binary complex with DNA to test thecomplex loosening ability of the various HMG protein mimics.The authors showed that addition of the PEG polyampholyteresulted in the formation of a loose ternary PEI/DNA/PEG-ACcomplex (Fig. 10B). The use of the PEG HMG mimics thereforeachieved up to a 31-fold increase in transcription efficiency,and up to a 3.3-fold increase in transfection efficiency com-pared with the PEI/DNA binary complex alone.49

Owing to their amphoteric nature, synthetic polyampholytesalso interact with proteins and can be used for their therapeuticdelivery. Akashi and co-workers prepared nanoparticles basedon both poly(g-glutamic acid)-graft-(L-arginine) and poly(g-glutamicacid)-graft-(L-lysine). The nanoparticles were able to bind bothcationic and anionic proteins and could retain them for extendedperiods of at least one week.50 Matsumura and co-workers exploitedthis phenomenon further and combined the antifreeze propertiesof hydrophobically modified carboxylated poly(L-lysine) with itsprotein binding ability for cellular protein delivery.47 Briefly, theydemonstrated that the polyampholyte nanoparticles could beloaded with either anionic bovine serum albumin or cationiclysozyme and delivered to the cytoplasm of mouse L292 fibro-blast cells by endocytosis during freezing of the cells. The cellssurvived freezing and resided in pockets of concentratedsolution upon cooling, which aided the uptake of the protein-loaded nanoparticles in a process known as freeze concentration(Fig. 10C, LHS). Labelling of both the polyampholytes and theloaded protein enabled the visualization of their cellular inter-nalization (Fig. 10C, RHS).47

These examples highlight that owing to their highly versatileand controllable nature, zwitterionic polymers show remark-able potential for aiding the delivery of many classes of therapeuticsincluding small molecules, proteins, and nucleic acids.

Conclusion

Zwitterionic polymers are a fascinating class of soft materials,encompassing a wide range of structures and properties. Theirunique behavior in solution, and at the surface of bulk inter-faces, and those of nanoparticles, makes them of great interestfor a wealth of applications in nanomedicine, materialsscience, catalysis and marine applications to name a few. Theirstructures and the preparation of these materials has beenoutlined, illustrated by examples of modern polymerizationand post-polymerization approaches. Their responsive behaviorin solution under a range of different environmental stimulihave been examined, including temperature, pH, salt, anddual-responsive systems, along with their interactions with

charged surfaces. Finally, an overview of their potential appli-cations has been described, highlighting their broad relevanceto a number of scientific and industrial fields. We envisionthat this introductory text will further the understanding anddesign criteria for these materials and allow for their transla-tion into yet more real-world applications.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was funded by a CSIRO Julius Career Award and aCSIRO ResearchPlus Postdoctoral Fellowship.

References

1 A. Laschewsky, Polymers, 2014, 6, 1544.2 A. B. Lowe and C. L. McCormick, Chem. Rev., 2002, 102,

4177–4190.3 A. V. Dobrynin, R. H. Colby and M. Rubinstein, J. Polym. Sci.,

Part B: Polym. Phys., 2004, 42, 3513–3538.4 C. Resetco, D. Frank, N. U. Kaya, N. Badi and F. Du Prez,

ACS Macro Lett., 2017, 6, 277–280.5 Y. Kim, S. Binauld and M. H. Stenzel, Biomacromolecules,

2012, 13, 3418–3426.6 D. E. Mitchell, N. R. Cameron and M. I. Gibson, Chem.

Commun., 2015, 51, 12977–12980.7 A. Ciferri and S. Kudaibergenov, Macromol. Rapid Commun.,

2007, 28, 1953–1968.8 L. Chen, T. Chen, W. Fang, Y. Wen, S. Lin, J. Lin and C. Cai,

Biomacromolecules, 2013, 14, 4320–4330.9 V. Butun, S. Liu, J. V. M. Weaver, X. Bories-Azeau, Y. Cai and

S. P. Armes, React. Funct. Polym., 2006, 66, 157–165.10 E. A. Bekturov, S. E. Kudaibergenov, R. E. Khamzamulina,

V. A. Frolova, D. E. Nurgalieva, R. C. Schulz and J. Zoller,Makromol. Chem., Rapid Commun., 1992, 13, 225–229.

11 M.-L. Pujol-Fortin and J.-C. Galin, Polymer, 1994, 35, 1462–1472.12 J. Seuring and S. Agarwal, Macromol. Rapid Commun., 2012,

33, 1898–1920.13 H. Willcock, A. Lu, C. F. Hansell, E. Chapman, I. R. Collins

and R. K. O’Reilly, Polym. Chem., 2014, 5, 1023–1030.14 Y. Maeda, H. Mochiduki and I. Ikeda, Macromol. Rapid

Commun., 2004, 25, 1330–1334.15 K. E. B. Doncom, A. Pitto-Barry, H. Willcock, A. Lu, B. E.

McKenzie, N. Kirby and R. K. O’Reilly, Soft Matter, 2015, 11,3666–3676.

16 Q. Zhang and R. Hoogenboom, Chem. Commun., 2015, 51,70–73.

17 M. Arotçarena, B. Heise, S. Ishaya and A. Laschewsky, J. Am.Chem. Soc., 2002, 124, 3787–3793.

18 Z. Xiong, B. Peng, X. Han, C. Peng, H. Liu and Y. Hu,J. Colloid Interface Sci., 2011, 356, 557–565.

Chem Soc Rev Tutorial Review

Publ

ishe

d on

14

Dec

embe

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18. D

ownl

oade

d by

Iow

a St

ate

Uni

vers

ity o

n 1/

20/2

019

7:38

:30

PM.

View Article Online

Chem. Soc. Rev. This journal is©The Royal Society of Chemistry 2018

19 W. C. Blocher and S. L. Perry, Wiley Interdiscip. Rev.:Nanomed. Nanobiotechnol., 2017, 9, e1442.

20 W. Wei, Y. Tan, N. R. Martinez Rodriguez, J. Yu, J. N.Israelachvili and J. H. Waite, Acta Biomater., 2014, 10,1663–1670.

21 H. Cai, B. Gabryelczyk, M. S. S. Manimekalai, G. Gruber,S. Salentinig and A. Miserez, Soft Matter, 2017, 13, 7740–7752.

22 J. Wang and T. Scheibel, Biomacromolecules, 2018, 19,3612–3619.

23 M. O. Khan, T. Åkesson and B. Jonsson, Macromolecules,2001, 34, 4216–4221.

24 Y. Kamiyama and J. Israelachvili, Macromolecules, 1992, 25,5081–5088.

25 S. E. Kudaibergenov and A. Ciferri, Macromol. Rapid Commun.,2007, 28, 1969–1986.

26 H. Zhang and M. Chiao, J. Med. Biol. Eng., 2015, 35, 143–155.27 V. B. Damodaran and N. S. Murthy, Biomater. Res., 2016,

20, 18.28 Z. K. Zander and M. L. Becker, ACS Macro Lett., 2018, 7,

16–25.29 M. Bernards and Y. He, J. Biomater. Sci., Polym. Ed., 2014, 25,

1479–1488.30 M. T. Bernards, G. Cheng, Z. Zhang, S. Chen and S. Jiang,

Macromolecules, 2008, 41, 4216–4219.31 Y. Chang, S.-C. Liao, A. Higuchi, R.-C. Ruaan, C.-W. Chu and

W.-Y. Chen, Langmuir, 2008, 24, 5453–5458.32 S. Jiang and Z. Cao, Adv. Mater., 2010, 22, 920–932.33 J.-J. Wu, J. Zhou, J.-Q. Rong, Y. Lu, H. Dong, H.-Y. Yu and

J.-S. Gu, Chin. J. Polym. Sci., 2018, 36, 528–535.34 E. Ostuni, R. G. Chapman, M. N. Liang, G. Meluleni, G. Pier,

D. E. Ingber and G. M. Whitesides, Langmuir, 2001, 17,6336–6343.

35 S. Haag and M. Bernards, Gels, 2017, 3, 41.

36 T. L. Sun, T. Kurokawa, S. Kuroda, A. B. Ihsan, T. Akasaki,K. Sato, M. A. Haque, T. Nakajima and J. P. Gong, Nat. Mater.,2013, 12, 932.

37 A. B. Ihsan, T. L. Sun, T. Kurokawa, S. N. Karobi, T. Nakajima,T. Nonoyama, C. K. Roy, F. Luo and J. P. Gong, Macromolecules,2016, 49, 4245–4252.

38 T.-G. La, X. Li, A. Kumar, Y. Fu, S. Yang and H.-J. Chung,ACS Appl. Mater. Interfaces, 2017, 9, 33100–33106.

39 V. Arjunan Vasantha, C. Junhui, T. B. Ying and A. Parthiban,Langmuir, 2015, 31, 11124–11134.

40 S. Li, Y. Wu, J. Wang, Q. Zhang, Y. Kou and S. Zhang,J. Mater. Chem., 2010, 20, 4379–4384.

41 A. K. Rodriguez, C. Ayyavu, S. R. Iyengar, H. S. Bazzi,E. Masad, D. Little and H. J. M. Hanley, Int. J. PavementEng., 2018, 19, 467–478.

42 K. Ishihara, M. Mu, T. Konno, Y. Inoue and K. Fukazawa,J. Biomater. Sci., Polym. Ed., 2017, 28, 884–899.

43 L. Kisley, K. A. Serrano, C. M. Davis, D. Guin, E. A. Murphy,M. Gruebele and D. E. Leckband, Biomacromolecules, 2018,19, 3894–3901.

44 C. I. Biggs, T. L. Bailey, B. Graham, C. Stubbs, A. Fayter andM. I. Gibson, Nat. Commun., 2017, 8, 1546.

45 K. Matsumura and S.-H. Hyon, Biomaterials, 2009, 30, 4842–4849.46 R. Rajan, F. Hayashi, T. Nagashima and K. Matsumura,

Biomacromolecules, 2016, 17, 1882–1893.47 S. Ahmed, F. Hayashi, T. Nagashima and K. Matsumura,

Biomaterials, 2014, 35, 6508–6518.48 Y. Lee, R. Graeser, F. Kratz and K. E. Geckeler, Adv. Funct.

Mater., 2011, 21, 4211–4218.49 C. Yoshihara, C.-Y. Shew, T. Ito and Y. Koyama, Biophys. J.,

2010, 98, 1257–1266.50 H. Shen, T. Akagi and M. Akashi, Macromol. Biosci., 2012,

12, 1100–1105.

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