Review

Stimuli-responsive interfaces and systems for the controlof protein–surface and cell–surface interactions

Martin A. Cole a,*, Nicolas H. Voelcker b, Helmut Thissen c, Hans J. Griesser a

aUniversity of South Australia, Ian Wark Research Institute, Mawson Lakes Campus, Adelaide SA 5095, Australiab Flinders University, School of Chemistry, Physics and Earth Sciences, Adelaide SA 5042, AustraliacCSIRO Molecular and Health Technologies, Melbourne VIC 3168, Australia

a r t i c l e i n f o

Article history:Received 2 September 2008Accepted 7 December 2008Available online 13 January 2009

Keywords:Surface modificationCell cultureProtein adsorptionThermally responsive polymerpH sensitiveStimuli-responsive polymers

a b s t r a c t

Real-time control over and reversibility of biomolecule–surface interactions at interfaces is an increas-ingly important goal for a range of scientific fields and applications. The field of stimuli-responsive, smartor switchable systems has generated much research interest due to its potential to attain unprecedentedlevels of control over biomolecule adsorption processes and interactions at engineered interfaces,including the control over reversibility of adsorption. Advances in this field are particularly relevant toapplications in the areas of biosensing, chromatography, drug delivery and regenerative medicine. Thecontrol over biomolecule adsorption and desorption processes at interfaces is often used to controlsubsequent events such as cell–surface interactions. Considerable research interest has been directed atsystems that can be reversibly switched between interacting and non-interacting states and used thus forswitching, on and off, bio-interfacial interactions such as protein adsorption. Such switchable coatingsoften incorporate features such as temporal resolution, spatial resolution and reversibility. Here wereview recent literature on switchable coatings that employ stimuli such as light, temperature, electricpotential, pH and ionic strength to control protein adsorption/desorption and cell attachment/detach-ment en route to the development of next-generation smart bio-interfaces.

! 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Rationally designed micro- and nano-engineered materials anddevices are becoming an integral part of many scientific fields andapplications. Many of these devices, particularly in the biomedicaland biotechnological arena, would benefit if effective control over

molecular and cellular interactions at material surfaces could beachieved.

Major goals include the control over surface adsorption ofbiomolecules such as DNA and proteins and also the control oversubsequent interfacial interactions between materials bearingadsorbed biomolecules, such as cell-adhesive proteins, and cellularentities such as mammalian cells and bacteria [1–4]. Proteinsadsorbed on synthetic materials surfaces are of particular interestas they are involved in cell attachment, various cellular signallingpathways, and cell functions. Cellular responses such as attachmentto materials surfaces, changes in morphology, proliferation, andphenotypic changes have been shown to be linked to the concen-tration, composition and conformation of the layer of proteins thatadsorbs rapidly on synthetic materials [5–9]. Protein–protein andsignalling molecule–protein interactions at biological interfacesalso regulate countless other biological functions. This reflects thenormal interactions of cells with their environment in vivo, wherecells reside in and interact with the extracellular environment.

Reported approaches towards control over protein adsorption inthe literature describe modifications of the surface chemistry toachieve either adsorbing or non-adsorbing properties towardsproteins. Generally proteins adsorb irreversibly to surfaces with

Abbreviations: AA, acrylic acid; ATR, attenuated total reflection; ATRP, atomtransfer radical polymerisation; BCAm, benzo-18-crown-6-acrylamide; BIS, N, N-methylene-bis-acrylamide; BMA, butylmethacrylate; BSA, bovine serum albumin;CA, contact angle; CP-AFM, colloid probe atomic force microscopy; DP, degree ofpolymerisation; ECM, extracellular matrix; EG-12A, endoglucanase 12A; ELP,elastin-like polypeptide; FN, fibronectin; FT-IR, Fourier transform infrared; HRP,horse-radish peroxidase; LCST, lower critical solution temperature; NIPAM, N-iso-propylacrylamide; NRs, nanorods; OEG, oligo ethylene glycol; PAA, poly(acrylicacid); PBS, phosphate buffered saline; pDEAM, poly(N,N-diethylacrylamide); PDI,polydispersity index; PEG, poly(ethylene glycol); pI, isoelectric point; PLGA, poly(DL lactic acid-co-glycolic acid); pNIPAM, poly(N-isopropylacrylamide); ppNIPAM,plasma polymerised NIPAM; P2VP, poly(2-vinylpyridine); QCM, quartz crystalmicrobalance; RGDS, arginine–glycine-aspartic acid–serine; RGD-Cp, arginine–glycine–aspartic acid–cyclopentadiene; SCF, self-consistent field; SFA, surface forceapparatus; SPR, surface plasmon resonance; TCPS, tissue culture polystyrene.* Corresponding author. Fax: !61 8 8302 3683.

E-mail address: martin.cole@unisa.edu.au (M.A. Cole).

Contents lists available at ScienceDirect

Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

0142-9612/$ – see front matter ! 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2008.12.026

Biomaterials 30 (2009) 1827–1850

only a few synthetic materials capable of markedly retarding orpreventing adsorption (for example [7,10–13]). The adsorption ofproteins at interfaces is undesirable in many cases as it can lead tobiofouling, thrombosis, reduced dynamic range of biosensors, andother adverse events [14–16]. On the other hand, the adsorption ofcell-adhesive proteins is essential for cell and tissue colonisationof materials surfaces [5–9].

Recently, there has been considerable progress in the design ofmaterials and interfaces that can be described as having bothadsorbent and adsorption-resistant properties. This progress isdriven by a number of applications where it may be desirable toattract a protein, cell or DNA layer temporarily, followed by releasefrom the surface at a specific time point [17,18]. Such control overbio-interfacial interactions requires that a material surface can beswitched in real time from an interacting, protein adsorbing state toa non-interactive, protein-repellent state or vice versa. Advances inthe control over protein adsorption and desorption at syntheticinterfaces are expected to benefit many applications and areasincluding diagnostic microarrays, biomaterials and regenerativemedicine, biosensors, chromatography and drug delivery as well asfundamental biological research [16,19–21]. In some cases a one-offswitching behaviour may suffice; in other cases it would beadvantageous to be able to execute reversible switching betweeninteracting and non-interacting states for a number of cycles.

Biomedical applications of thermally reversible polymerhydrogels started attracting interest in the 1980s, particularly onthe basis of work by the group of Hoffman [22]. Bio-interfaces withstimuli-responsive/switchable bio-affinity have been of consider-able recent research interest, with a rapidly increasing number ofreports. Reviews are available [1,23–25] and the use of switchablesystems has been discussed for cell culture, drug delivery and tissueengineering [4,24,26–29]. A particular focus has been the use ofreversible bio-interfaces for cell sheet culture [30–34]. Less atten-tion has been devoted to reviewing the fundamental aspects suchas the modes and mechanisms of switching; accordingly, in thisreview we will focus mostly on molecular and physico-chemicalaspects of reversibly switchable bio-interfaces and the achieve-ments and current directions of this rapidly expanding field.

While switchable materials surfaces also have application infields such as microfluidics, microactuators, bio-catalysts, DNAtransfection, and drug release, it is not our intention to review indetail the large body of studies devoted to such applications. This isbecause the fundamental aspects of the interfacial interactionsbetween the biomaterials or devices and adsorbing/desorbingbiomolecules are essentially the same and the understandinggained while exploring one application often translates well toanother application. Instead, we will review literature on thefundamental aspects and selected applied studies of switchablebiomaterials or systems that enable controlled, real-time switchingof interfacial properties and interactions. By the term ‘‘systems’’ wemean cases where the effects on bio-interfacial interactions arisenot from interfacial re-organisation of a surface, but from otherexternally controllable interactions such as electrical fields.

2. Switchable materials and systems

A wide range of stimuli are available for application in switch-able systems (Table 1). Of these triggers, light, temperature, pH,ionic strength and electric field based methods are the mostcommonly used [35–39]. Other triggers include solvent, pressure,ultrasound and magnetic fields [40–44]. Varied research has alsobeen conducted into switchable systems that are sensitive tospecific chemical analytes or biomolecules themselves. This latterarea of research has been recently reviewed and will not be a majorfocus of this review [45–48].

In principle, there are two main mechanisms by which bio-interfacial interactions between material surfaces and biomole-cules can be switched. Firstly, a stimulus can be applied to act uponthe material surface to elicit a change in the interfacial properties.This in turn alters interactions between biomolecules coming incontact with the interface, ideally adsorbing or repelling them asdesired. Secondly, a stimulus can be applied to the biomoleculesthemselves to attract or repel them to or from the interface.Also, certain stimuli may alter the state or configuration of thebiomolecules to alter their binding affinity or to allow adsorbent ornon-adsorbent properties to dominate [41,49]. The latter approachis based on the fact that biomolecules are complex and containspecific properties by which they may be manipulated or triggeredby stimuli such as metals, ions, drugs, electric fields andtemperature.

Important limiting factors for the function and application ofboth switchable materials and switchable systems are the degree ofchange and the rate at which the change occurs [50]. The revers-ibility and number of cycles a switchable material or system cansustain must also be considered.

The following chapters discuss the most common switchablematerials and switching methods applied to biomolecule adsorp-tion/desorption and cell attachment/detachment, with a particularfocus on stimuli-responsive materials.

3. Thermal switching

3.1. Thermally actuated polymers

Thermal switching of suitable materials results in a change inmolecular conformation in response to a temperature change in thesurrounding environment. Polymer chains exhibit temperaturedependent solvation behaviour in solvents; in many cases polymermolecules become more soluble and better solvated as thetemperature increases, due to enthalpic effects. However, somepolymer structures exhibit the inverse behaviour in that theybecome less solvated with increasing temperature and eventuallyprecipitate from solution at the so-called lower critical solutiontemperature (LCST). This temperature dependence of solvationprovides a convenient handle for changing the properties ofbiomaterials surfaces: when grafting such polymer molecules ontomaterials or devices, surface layers can be produced whosemolecular conformations expand and collapse in response to thetemperature dependent solubility. Given the aqueous environmentapplicable to biomaterials and biomedical purposes, hydrogelpolymers with temperature dependent solvation in water arerequired for thermal switching of interfacial interactions.

Whilst there are many polymers that exhibit some thermalswitching ability, few exhibit sharp transitions at useful tempera-tures suitable for bio-applications. N-alkyl substituted poly-acrylamides are well known thermosensitive polymers andinclude poly(N-isopropylacrylamide), poly(N,N-diethylacrylamide),poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) and

Table 1Stimuli that have been employed in switchable applications.

Energy based Chemical based

Light pHTemperature Ionic strengthElectric field SolventsMagnetic field Metal ionsUltra sonic radiation Chemical analytes

Biochemical analytesBiomolecules

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many others with derivatised substituents [51–54]. Other ther-mosensitive polymers have been reported including thoseof poly(N-substituted amino acids), poly(N-vinylalkylamides),poly(N-vinylcaprolactam) and polyalcohols such as poly-(2-hydroxyethylvinylether), poly(2-isopropyl-2-oxazolines) andpoly(4-hydroxybutyl vinyl ether) [52,55–59]. The polymers listedin Fig. 1 can be reversibly switched between collapsed/insolubleand soluble states above and below their LCST.

In addition to these synthetic polymers, natural and geneticallyengineered polypeptides can also possess thermal switchingproperties [60–63]. Advantageous to these naturally inspiredswitchable polymers is that they can be engineered with a highlevel of specificity, contain functional groups for further modifi-cation, and can be made to be biodegradable or non-biodegradable. Elastin-like polypeptides (ELPs) are a good exampleof natural responsive polymers [41,49,55,64]. For example, thedevelopment of stimuli-responsive ELPs with a specific transitiontemperature of around 39 "C allowed better circulation at physi-ological temperature (37 "C) but accumulation at the site of locallyheated cancerous tissue. This enhanced uptake of the ELP bytumour cells [65,66]. The field of naturally inspired switchablematerials shows great promise due to the potential for enhancedbiocompatibility and the ability to switch in response to a changein pH or temperature [67]. This area of stimuli-responsive poly-peptides could particularly benefit from further research in theengineering of amino acid sequences to fine-tune specificswitching properties.

Also of interest for application in the biomedical arena arepolymers that exhibit thermal switching above and below an uppercritical solution temperature (UCST) [68–70]. These polymersbecome more soluble at temperatures above the UCST. Althoughnot typically applied to controlling protein adsorption or cellattachment, these polymers are expected to be particularly usefulfor biomaterial and drug delivery applications wherein theymay beheated to above their UCST, where they form a sol and then may beinjected into the body to cool to 37 "C, solidify and form a gel [71].Solutions of triblock copolymer PLGA–PEG–PLGA exhibiting sol–

gel–sol properties over a temperature range of 5–60 "C havepotential for application as injectable bandages for corneal woundrepair [72]. Injection of the soluble polymer below room temper-ature followed by gel formation in vivo could facilitate woundrepair and minimise scarring. A common concept for use of thetemperature-responsive phase transition in UCST, LCST and copoly-mers of UCST/LCST materials is to generate a suitable matrix tocontain or trap drugs or proteins to be released or implanted intothe body [69,73–75]. At this stage this area of research is focused ondelivery mechanisms rather than specifically inducing theadsorption and release of biomolecules to and from a solidbiomaterials interface. Further information on drug release appli-cations of stimuli-responsive materials can be found elsewhere[24,25,71,76–79].

The most commonly and successfully studied thermoresponsivemodel by far is that of poly(N-isopropylacrylamide) (pNIPAM). Thesharp transition range [53] as well as the close proximity of thepNIPAM transition temperature (approximately 32 "C) to physio-logically relevant temperatures has made it an attractive ther-moresponsive material to study for potential bio-applications[22,37,80]. A particularly useful characteristic of the pNIPAM phasetransition is the reported transformation of the surface propertiesfrom a low-fouling (cell and protein resistive) state to a collapsedand biologically adhesive surface [81–84]. While the reversibility ofthe pNIPAM phase transition is now well established, the revers-ibility of its fouling/low-fouling behaviour has proved to be morecomplicated. There are reports of thermo-reversible attachment/detachment of proteins, mammalian cells and bacterial cells[30,81,85,86]. However, this is not observed in every case and somereports indicate only partial cycling of protein attachment/detachment, as discussed later (Section 3.3). Incomplete revers-ibility would affect the useful lifetime of many applications. It isalso not clear whether the detachment of cells sheets is accompa-nied by complete desorption of adhesion proteins; some reports,discussed later, have found incomplete protein removal upon celllifting whilst others have found that proteins are largely associatedwith removed cells (Sections 3.2, 3.3).

It is essential to gain an improved understanding of the factorsthat determine whether or not a pNIPAM surface is fully proteinresistant below the LCST, and of the factors that might prevent fullreversibility of protein adsorption and desorption. It is well knownfor PEG graft coatings that chain packing density, chain length, andthe extent of chain stretching affect interfacial interactions withproteins [87–89], by analogy it may be the case that the detailedstructure and properties of graft pNIPAM surfaces might markedlyinfluence interfacial properties, and hence different pNIPAM coat-ings may show different behaviours. Thus, there is a clear need tocontrol and characterise the architecture of pNIPAM coatings and togain improved understanding of how the microstructure affectsinteractions with proteins.

A number of methods have been reported for the generation ofpNIPAM surface coatings, among them the techniques of surfaceinitiated radical polymerisation (grafting from), covalent attach-ment of end functionalised chains (grafting to), plasma polymeri-sation and electron beam irradiation polymerisation are the mostpopular [5,30,90–93]. However, few methods or reports providecomparisons of coatings produced by different methods in terms oftheir degree of hydration, microstructure, segment density distri-bution, and other properties that may be important for regulatingprotein/surface interactions.

Various copolymers and derivatives of NIPAM have beeninvestigated. Incorporation of more polar or ionic co-monomerswith NIPAM causes the LCST of the resulting copolymer to shift tohigher temperatures due to the increased solubility from the polargroups [94–96]. In the reverse case, incorporation of less polar or

Fig. 1. Molecular structures of some polymers that can exhibit LCST behaviour: (a)poly(N-isopropylacrylamide), (b) poly(ethylene glycol), (c) poly(propylene glycol), (d)poly(hydroxybutyl vinyl ether), (e) poly[2-(2-ethoxy)ethoxyethyl vinyl ether], and (f)poly(2-isopropyl-2-oxazoline).

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hydrophobic monomers into NIPAM copolymers causes the LCST toshift to lower temperatures [85,97]. Random incorporation of othermonomers in quantities greater than approximately 5%may shift the LCST to regions where it is no longer useful (polarco-monomers) or remove the LCST altogether (non-polar co-monomers). Here, the use of block copolymers is advantageousover random copolymers due to the ability to retain individualcharacteristics of either block, therefore not significantly affectingthe responsive nature of the switchable component [96].

Another approach to tune the phase transition behaviour hasbeen to synthesise different N-substituted acrylamides in order togenerate new polymers with different phase transition tempera-tures based upon the replacement of the N-isopropyl group witha more polar or less polar group to increase or decrease theLCST. Examples are 2-carboxyisopropylacrylamide and N-butylacrylamide [85,98]. N-substituted derivatives of NIPAM havealso enabled incorporation of functional groups for covalentattachment of further molecules [98,99]. The design of specificmonomers such as 2-carboxyisopropylacrylamide with similarproperties to NIPAM allows that the sensitivity and position of theLCST are not significantly altered. It should be noted that polymerscomprising other N-substituted acrylamides may not have ananalogously sharp LCST as pure pNIPAM, and so are most oftenapplied as co-monomers with NIPAM as the major component.

However, when thermosensitive polymers are used as surfacegraft coatings to control interfacial interactions, it is not only thechemical composition that matters; the graft density and otherfactors may also affect the interfacial properties. It is thereforeessential to perform extensive characterisation of coatings inorder to disentangle effects arising from changes in chemistry(such as different alkyl substituents, or co-monomers) from thosearising from physical properties such as polymer chain packingdensity.

Chemical and elemental surface characterisation by X-rayphotoelectron spectroscopy (XPS) is a convenient and sensitivemethod to investigate polymer coatings. The percentage ofelements present in the outermost w10 nm of the dry materialprobed by XPS (depending on the electron escape depth and takeoff angle) can provide information on coating thickness, uniformity,impurities and composition (such as, does the ratio of elementspresent match the theoretical ratio of the polymer coating). High-resolution XPS can provide information on the chemical structureof polymer coatings based on known shifts for chemical groups.XPS has been used in a number of studies for the characterisation ofpNIPAM coatings; four examples of high-resolution C1s spectra arereproduced in Fig. 2. The first spectrum (Fig. 2A) is from a graftpolymerised coating and shows that the spectrum can be fittedwith the expected three contributions (‘‘neutral’’ C, C–N, and amideC) such that relative intensities close to theoretical values (4:1:1)are obtained, verifying the presence of the intended graft coatingwith no contaminants [83]. The plasma polymerised NIPAM(ppNIPAM) spectrum recorded by Pan et al. [100] (Fig. 2B) is alsoclose to that expected for pure pNIPAM. For the ppNIPAM coatingproduced by Canavan et al. [101] (Fig. 2C), it is evident that there isa higher hydrocarbon signal, indicating abstraction of some amidesduring plasma polymerisation. The high binding energy region(>286 eV) shows a broader, indistinct structure, consistent with anextent of scrambling of the chemical structure of the plasma film,which is common in plasma polymerisation. The last spectrum(Fig. 2D) was recorded onmagnetic nanoparticles putatively coatedwith a pNIPAM graft layer [102]. However, clearly the spectrumdoes not match expectations at all; the absence of an amidecomponent in the spectrum indicates that the organic layer on themagnetic nanoparticles is not pNIPAM; this was not recognised ordiscussed.

3.2. Thermosensitive coatings for the control of cell and cell sheetadhesion

One of the most studied and successful demonstrations ofswitching in biomedical applications concerns the growth andrelease of cells and in particular, cell sheets, on thermosensitivesurfaces, as shown schematically in Fig. 3 [37,103,104]. Since itsconception, much research into thermally switchable polymers hasbeen devoted to this field known as ‘cell sheet engineering’[30,33,105,106]. The concept is based on the observation thatsurface coatings of pNIPAM are able to support the culture ofa variety of cells to confluence at 37 "C. The collapsed state of thepolymer provides similar surface properties as tissue culturepolystyrene (TCPS), allowing adsorption of serum proteins andsubsequent cell attachment and spreading required for the prolif-eration of cells. Reducing the temperature below the LCST causesthe thermoresponsive coating to swell and hydrate, initiating therelease of living cells, or if confluence was achieved, an intact cellsheet [30,31,33]. Studies of single cell detachment from pNIPAMsurfaces have found, however, that cell release was not just due tochanges in the pNIPAM polymer but also affected by cell morpho-logical changes and metabolic activity [107,108].

Cell culture on pNIPAM surfaces and the subsequent release ofcell sheets upon lowering the temperature is often discussed interms of ‘‘hydrophobic’’ and ‘‘hydrophilic’’ surface properties, withcells able to attach onto pNIPAM at 37 "C because of its hydro-phobicity and being released because of increased hydrophilicity.The contact angle (CA) results reported for pNIPAM surfaces atT> LCST are typically below 90" [5,31,83,109], and thus the surfaceis not truly hydrophobic. Moreover, human endothelial cells andfibroblasts can attach and grow on solid substrate surfaces with CAspanning the range 33–96" [110], a range that encompasses the CAvalues reported for pNIPAM coatings under both expanded andcollapsed conditions. Thus, it is clear that it is not the change in thecontact angle per se that matters; instead, a more appropriateinterpretation is that it is the change in hydration of the pNIPAMpolymer that renders the pNIPAM surface adhesive or nonadhesiveto cells. Above the LCST the pNIPAM assumes a collapsed confor-mation of polymer chains, and this is considered to be akin toconventional solid polymer surfaces, such as TCPS, that support cellattachment via the adsorption of cell-adhesive glycoproteins. In thewell-hydrated state, on the other hand, it appears reasonable thatentropic repulsion of protein adsorption prevents cell attachment,in the same way as for other hydrated hydrogel polymer surfaces,such as PEG, pHEMA and hyaluronan, which also resist theattachment of cells [111]. However, fluorescently labelled fibro-nectin (FN) has been found not to adsorb onto pNIPAM surfacesabove the LCST in some cases [112]. Even if a small amount ofprotein adsorption takes place, the material may be too soft toenable cell spreading. Therefore, it is physically moremeaningful toview the change in the bio-adhesiveness of pNIPAM as a conse-quence of considerable changes in the hydration and consequentmechanical properties of the polymer as it passes through the LCST.

Removal of cells via cooling below the LCST of thermallyreversible polymer coatings is advantageous compared to alterna-tive methods such as mechanical (scraping) or enzymatic removal(proteolytic degradation of cell–cell and cell–surface junctions) ofcells, which can have an adverse impact on cell vitality[31,101,113,114]. More importantly, the ability to gently detach cellsand cell sheets with their associated extracellular matrix (ECM)proteins has important implications for tissue engineering andregenerative medicine. The technique has now been appliedsuccessfully to a variety of cell types including smoothmuscle cells,hepatocytes, endothelial cells, fibroblasts, keratinocytes, epithelialcells, macrophages, microglial cells and stem cells [44,82,106,115].

M.A. Cole et al. / Biomaterials 30 (2009) 1827–18501830

Furthermore, commercially available pNIPAM modified cell cultureproducts have reached the market [116] and target applicationswhere trypsinisation or other methods of cell release cannot beused.

The application of cell sheet technology, particularly from theground-breaking work of Okano et al. has resulted in a number ofmedical advances, such as the treatment of corneal epitheliumdysfunction through the transplantation of cultured cell sheets[32,115]. Transplantation of corneal endothelial cell sheets has alsobeen investigated [117]. Single cell sheet transplantation is alsouseful for the regeneration of regions other than the cornea, such asthe urothelium and periodontal ligament [32]. Cell sheet tech-nology has also been applied to reconstruction and repair ofmyocardial tissue [104,118]. Furthermore, microtextured poly-styrene substrates with coatings of pNIPAM have also beenproduced in order to achieve defined structural organisation of cellsheets [119].

Cell sheet engineering is advancing further with demonstrationsof co-cultured cell sheets. The work, which progressed frompatterning of pNIPAM on TCPS [106], has evolved to utilise the

patterning of two thermoresponsive polymers with different LCSTs,allowing further control over the system [97]. Cell culture dishesweremodified with pNIPAM, followed by patterning with a copoly-mer of NIPAM and n-butylmethacrylate (pNIPAM–BMA). The resultof incorporating the hydrophobic BMA into the polymer was toreduce the LCST of the patterned area [97]. When the resultingsurface was cultured with cells at 27 "C they were seen to attachand proliferate on the collapsed pNIPAM–BMA areas. Following thisa different cell type could then be grown at 37 "C causing them toadhere to the remaining pNIPAM coated areas of the thermo-sensitive culture surface (Fig. 4A). Upon cooling the system tobelow the LCST of the two polymers the patterned sheet could beremoved with the ECM still intact (Fig. 4B) [97].

Other advances in cell sheet engineering have been under-pinned by immobilisation of cell-adhesion peptides such as RGDSand cell growth factors such as insulin onto pNIPAM, allowingaccelerated cell culture or culture under serum free conditions[99,120]. While the reported system did require additional growthfactors in solution in order to grow cells to confluence, thisapproach shows potential for tissue engineering applications.

Fig. 2. (A) High-resolution XPS C1s spectrum of pNIPAM graft coating on silicon wafer. Fitted C1s components corresponding to aliphatic hydrocarbon (C1), secondary amine (C2)and amide (C3) are present in amounts approximately according to the theoretical ratio of 4:1:1. (B) C1s spectrum of ppNIPAM with fitted C1s components. (C) C1s spectrum ofppNIPAM. The spectrum shows a broader distribution in comparison to that of linear grafted pNIPAM coatings due to the variety of functional groups formed in plasma depositedcoatings. (D) C1s of magnetic nanoparticles after pNIPAM grafting. The absence of amide component at w288.0 eV however is questionable. Adapted with permission from: (A)Ref. [83] ! SPIE; (B) Ref. [100] ! American Chemical Society; (C) Ref. [101] ! Wiley-VCH Verlag GmbH & Co. KGaA; (D) Ref. [102] ! Wiley-VCH Verlag GmbH & Co. KGaA.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–1850 1831

Furthermore, successful engineering of 2D cell sheets into otherdevices and/or 3D shapes, or layers shows that cell sheet engi-neering has the potential to be used in sophisticated devices such asa pump actuated by cardiomyocyte cell sheets or in regenerativemedicine for tissue regeneration treatment [32,118,121–123].

Observational correlations between thickness/density of pNI-PAM coatings and their performance with respect to cell attach-ment/detachment have been reported in a number of pNIPAM/cellsheet related articles [2,31,37,112]. Very thin pNIPAM films werefound to be superior. However, in some studies cells and cellsheets were prepared and lifted from films not following thesedesign constraints [5,82,124]. This apparent discrepancy in theliterature is consistent with our comments above that the coatingmicrostructure may play a key role but has not been characterised

systematically; it would seem that the properties measured inthese studies were not sufficient to provide a full interpretation ofinterfacial interactions with cells. Improved understanding of therelevant properties required for cell release, such as thickness,degree of swelling/deswelling, change in mechanical modulus andability to release proteins, is required before the rational design ofoptimal graft pNIPAM coatings can be done with confidence.

Low temperature cell lift-off from flat surfaces has beenachieved from ATRP grown pNIPAM coatings and coatings ofpNIPAM/PEG copolymers [92,94,112] but electron beam irradiationand plasma polymerisation coating techniques have mainlybeen reported. Particles in the form of hydrogel beads of poly(N-isopropylacylamide-co-N-aminoethylmethacrylate) with RGDattached have also been investigated for thermo-reversible cell

Fig. 3. A model for thermosensitive cell attachment. (A) Below the transition temperature the surface resists protein adsorption and cell attachment. (B) Raising the temperatureabove the transition temperature allows adsorption of serum proteins and cell attachment. (C) During extended cell culture, secretion of ECM proteins along with cell proliferationand communication results in confluent cell sheets. (D) Low seeding density or short cell incubation times results in adherent cells without extensive cell–cell junction formation. (E& F) Cooling to below the transition temperature allows release of intact cells or cell sheets along with associated ECM proteins.

Fig. 4. (A) Sequentially seeded bovine carotid artery endothelial cells (ECs) adhere to pNIPAM regions and co-culture with pre-seeded rat primary hepatocytes (HCs) at 37 "C intoorganised patterns. (B) Macroscopic view of detaching co-cultured cell monolayer from patterned dishes after reducing culture temperature to 20 "C. Scale bars: (A)# 0.5 mm,(B)# 1 cm. Adapted from Ref. [97], ! Elsevier Ltd.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–18501832

attachment/lift-off [125]. Furthermore, pNIPAM coated membraneshave been used to demonstrate a cell separation/enrichmentconcept. Here, pNIPAM coated poly(propylene) membranes withadsorbed antibodies specific to one of two cell types were able topreferentially attach the cell type expressing the complementaryantibody [126]. Release of captured cells from the membrane intofreshmedium at 4 "C allowed enrichment of the desired cell type toup to 72% (starting from a 50:50% mixture) [126].

One aspect lacking in many studies of cell support applicationsis an assessment of cytotoxicity [39,57,127]. This data is essential fora number of applications since residual monomer and short poly-mer segments may have high toxicity towards cells and hamper theuse of many polymers. Further investigation of whether the act ofswitching polymer conformation (free in solution or surfaceimmobilised) has adverse effects on cell vitality would also bebeneficial [57,128]. Finally, while much work has focused on theadhesion and detachment of cells and cell sheets, few studies haveconsidered whether or not the entire ECM lifts off the surface alongwith the cells, despite a report by Canavan et al. that on a ppNIPAMcoating some of the ECM proteins remained on the surface after lowtemperature cell lift-off [33].

3.3. Thermosensitive coatings for the control of protein–surfaceinteractions

Polymers and copolymers containing NIPAM and other ther-moresponsive components have been applied to numerous surfacearchitectures for the control of switchable interactions withproteins, cells, lipid vesicles and bacteria as well as DNA forexpression and biosensors/chemical sensors [30,81,85,91,129–131].In most cases where cells are grown on synthetic surfaces, proteinadsorption is an important precursor to cell attachment and thusshould be characterised extensively before cell growth results canbe interpretedwith confidence. Protein adsorptionwill either occurupon contact with serum or cell media, or can be performed viaexposure to the desired protein solutions prior to cell growth.

For thermosensitive polymer graft coatings that enable cellculture and detachment, it would appear reasonable to assume thatcell-adhesion proteins would adsorb above the LCST and therebysupport cell attachment, butwould be incapable of adsorbing belowthe LCST. Alternatively, however, it is also conceivable that someprotein adsorption might occur below the LCST but cells cannotattach because the mechanical properties of the hydrated hydrogelare unsuitable (too soft). As protein adsorption ontopNIPAMsurfacemight depend on the graft architecture, such as segment density,

analogous to findings with PEG graft coatings [87–89], the adsorp-tion of proteins should be investigated for each specific pNIPAMcoating, both above and below the LCST, until the factors governingprotein adsorption onto these coatings are better understood.

Surface analysis of protein adsorption onto pNIPAM coatings hasbeen performed using a number of methods including antibodylabelling, time of flight secondary ion mass spectrometry (ToF-SIMS), and XPS. However, XPS can be hampered by spectral simi-larities between the polymer and adsorbed proteins, making itdifficult to obtain conclusive results. Appropriate sensitivity limitsof techniques used to probe protein adsorption are of crucialimportance since it is known that minute amounts of adsorbedproteins can trigger a significant biological response. For example,less than 10 ng/cm2 of adsorbed fibrinogen has been shown toinduce platelet adhesion [132].

Studies of the mechanisms of protein adsorption and theinterfacial forces involved between proteins and pNIPAM coatingshave been evenmore demanding. In some reports the use of colloidprobe atomic force microscopy (CP-AFM) has provided an assess-ment of the intermolecular interactions between protein coatedprobes and pNIPAM surfaces above and below the LCST [133,134].

While topographical and mechanical properties [3,135,136] alsoaffect cell attachment onto surfaces, understanding the extent andmechanism of protein adsorption on pNIPAM and similar stimuli-responsive coatings is imperative not only for biomolecule basedstudies but also for studies of the mechanisms of interactions withcells and bacteria. In particular, sensitive analytical techniques arerequired to detect whether any biomolecules remain on the surfaceafter stimuli controlled release of cells. Unfortunately, in manystudies this has not been investigated.

Depending on the goals of the study, care should be taken to usemultiple, complementary techniques rather than make inferencesbased on either surface or solution analysis alone. A good exampleis the report by Cheng et al. on protein adsorption to plasmapolymerised NIPAM (ppNIPAM) surfaces bymeans of radiolabellingand ToF-SIMS [84]. It was established that the coatings were lowfouling below the LCST with less than 25 ng/cm2 of three differentproteins adsorbed at room temperature. At 37 "C the coatings wereprotein retentive with >100 ng/cm2 adsorbed at the ppNIPAMsurface. Furthermore, experiments were carried out to investigatethe reversibility of adsorption and it was found that with thesecoatings, adsorption was irreversible with the exception of bovineserum albumin (BSA) adsorption, which was partially reversible(Fig. 5) [84]. An interesting result in that studywas that anti-ferritinantibody seemed to adsorb with temperature dependent

Fig. 5. Amount of protein remaining on ppNIPAM surfaces after different incubation conditions as determined by measurement of radioactivity of radiolabelled proteins. Eachcondition was repeated with PET control surfaces (not shown here). Adapted with permission from Ref. [84], ! AVS The Science & Technology Society.

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orientation/conformation on the ppNIPAM coatings. Althoughthese were preliminary findings, the notion that in the future,stimuli-responsive coatings could be used to control the orienta-tion of surface-immobilised proteins and thus secondary bindingevents is an exciting possibility.

It is interesting to note that despite the clear demonstration ofirreversible protein adsorption by Cheng et al. [82], manyresearchers in cell sheet engineering seem to assume that on lift-offof the cell sheet, the ECM is also detached in its entirety anda pristine pNIPAM surface regenerated. This may be so for somecoating architectures, but the point is that this needs to be verifiedrather than simply assumed.

One aspect that has not been sufficiently investigated is thepossibility that protein adsorptionmay not just involve the pNIPAMsurface. When a thermosensitive polymer is grafted to a surface, itscollapse will expose substrate surface areas in-between thecollapsed chains (Fig. 6) unless the polymer chains are grafted atsufficiently high brush density, which is difficult to achieve due tohydrodynamic exclusion volumes of the hydrated chains. The factthat CA measurements were still found to change even when themeasured dry coating thickness increased [112,137] suggests thateither the surface roughness is changing or that the grafted chainswere sparse enough in the thinner coatings for the CA to be influ-enced by a contribution from incompletely covered substratesurface area. The use of plasma polymerisation, which createsa three-dimensional polymer network rather than grafted linearchains, may be advantageous in that there is less likelihood ofexposing substrate surface areas upon hydrophobic collapse. Thequestion arises whether proteins that do not desorb uponre-hydration of a thermosensitive polymer coating are irreversiblyadsorbed onto the graft surface, onto exposed substrate surfaceareas, or enmeshed within the graft layer. The molecular weights(MWs) of the proteins may play a role; low density hydratedcoatings may be able to prevent large proteins from reaching thesubstrate but may be susceptible to in-diffusion of smaller proteins.As protein adsorption is usually irreversible onto non-hydratedsubstrate materials, incomplete coverage of the substrate bycollapsed graft chains is expected to be a key issue, causingincomplete reversal of interactions between proteins and coatedmaterials.

The amount of protein that can no longer be desorbed froma thermosensitive polymer surface may be determined not only bythe chemical and microstructural properties of the polymer surfacebut also by the properties of the protein, such as the ease and rate of

denaturation on the particular surface, and the residence time[81,138]. Advances in this area of research relate to the moregeneral topic of understanding the mechanisms of proteinadsorption and of non-fouling materials and surfaces.

Temperature dependence of protein interactions appears not tobe confined to pNIPAM and its relatives and copolymers. A study ofmixed surfaces of methyl (CH3) terminated and oligo(ethyleneglycol) (OEG) self-assembled monolayers (SAMs) found interestingtemperature-responsive characteristics. It was observed thata sharp temperature dependent relationship existed for proteinadsorption to these SAMs, which incorporate the non-fouling OEGtermini along with the protein adsorbing CH3 termini [139].Adsorption to the OEG or the CH3 terminated SAMs alone showedeither non-fouling or fouling behaviour, respectively, regardless oftemperature. The mixed SAM of 95:5 OEG:CH3 showed proteinresistive behaviour at temperatures less than 32 "C, whereasproteins adsorbed above 32 "C, based on ellipsometry measure-ments. It was thought that conformational changes of the OEG headgroup occurring at T> 32 "C reduce electrostatic repulsion and/orexpose hydrophobic moieties to allow protein adsorption [126].Washing at 4 "C was shown to release adsorbed protein from thesurface after thermally induced adsorption. Preliminary insight intothe mechanism was provided via vibrational sum frequencygeneration; however, protein adsorption data would benefit fromadditional analytical techniques such as SPR or QCM.

Through careful preparation of lateral gradients of differentpNIPAM copolymers it has been shown that lateral control over thesurface topography can be achieved with temperature [140]. Theconcept here was to have immobilised thermoresponsive copoly-mers to form lateral LCST gradients and patterns. Swelling/desw-elling of different spatial regions (each having different LCST) acrossthe gradient coating could be controlled over the range of 19–55 "C.Furthermore the control over deswelling/swelling could be used toexpose or shield kinesin motor proteins embedded in the gradientcoatings to/from solution and thus spatially direct gliding micro-tubules onto collapsed (kinesin exposed) regions and remove themvia swelling the area [140]. On–off control over the interactions ofimmobilised bioactive molecules via exposing or shielding withresponsive polymers is an exciting advancement with implicationsfor cell differentiation, attachment/detachment and ligand receptorinteractions [140,141].

Investigation of whether surface adsorbed proteins associatedwith growing cells or cell sheets remain on the stimuli-responsivesurface or are lifted off with the cells or cell sheets during low

Fig. 6. Schematic depiction of grafted polymer chains in the mushroom conformation (A, B) and a brush conformation (C, D), each in the hydrated state (A, C) and the collapsed state(B, D). The mushroom conformation occurs when the grafting separation distance (d) is greater than the radius of gyration (Rg) of the polymer chains, whereas d< Rg for the brushconformation. In the mushroom conformation the low density of polymer chains may lead to exposure of the underlying substrate particularly in the collapsed state.

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temperature lift-off has been the subject of only a few reports [30].The cell-adhesion mediator fibronectin was investigated viaimmunoblotting and a fluorescent antibody/microscopy procedure,showing FN associated with the cell sheet released below the LCST[30]. After cell sheet lift-off, the pNIPAM surface was probed forremaining FN via a fluorescent antibody, but, given the detectionlimits of this method, the matter should be re-investigated usingmore sensitive surface analysis techniques. In a later report,investigation of cell release found that FN associated with the cellswas predominantly removed during lift-off below the LCST but FNin other areas remained on the pNIPAM surface [142]. ToF-SIMs andradiolabelling studies of ppNIPAM surfaces have shown thatadsorption of fibrinogen (Fg) and anti-ferritin antibody from solu-tion was irreversible whilst BSA adsorption was partially reversible[84]. When ppNIPAM coatings were used as stimuli-responsive cellculture surfaces it was found that, amongst other proteins, FN waspredominantly removed during cell sheet lift-off, indicating that itremained part of the ECM of the cell sheet [33,143]. Neverthelesssmall amounts of proteins or protein fragments still remained onthe ppNIPAM surface after cell lift-off [33,143]. In these studies ToF-SIMS and principal component analysis (PCA) provided excellentinformation of the presence of and differences between minuteamounts of proteins present at the interfaces. The ToF-SIMS limit ofdetection for adsorbed proteins on plasma polymers can be lessthan a few ng/cm2 [144] and in some cases can provide informationon protein orientation [145].

It is generally agreed that protein adsorption to pNIPAM coat-ings below the LCST is unfavourable, as demonstrated by minimalor zero interaction forces in several CP-AFM studies [5,133,134].However, even with a small initial attraction, the ability for someproteins to unfold and thereby become more tightly bound meansthat some fouling below the LCSTmight occur if sufficient residencetime is available for the initially loosely adsorbed proteins. Abovethe LCST, adsorption is stronger and denaturation of some proteinslikely to be faster and more pronounced, which might result insufficiently tight binding such that they can no longer be releasedupon lowering the temperature below the LCST. Such unfolding isusually a slow process and hence the reversibility of proteinadsorption and desorption upon temperature cycling is likely tovary with the cycling speed, or, more precisely, the residence timeof a protein on the surface above the LCST. Clearly, with someproteins and some conditions, protein adsorption might becomeirreversible and thus prevent repeated switching. A key factor ofthe reversibility of protein adsorption on pNIPAM coatings istherefore whether the phase transition back to the hydratedprotein resistant state is energetically more favourable than theinteractions formed between chains and the protein in thecollapsed state [84,134]. The question of denaturation of proteinson thermosensitive polymer surfaces, and its significance forreversibility of adsorption/desorption, is in need of furtherinvestigation.

Thermosensitive coatings have generated particular attentionfor application in protein purification, concentration and separa-tion. For chromatography applications, thermal switching ofadsorbed poly(N-vinylcaprolactam) [146] or grafted pNIPAM [147]to their collapsed states was used to expose bound affinity ligandsto their binding partners in solution. Lowering of the temperaturebelow the LCST allowed the bound proteins to be released andeluted [146,147]. Furthermore these systems were able to separate/purify targeted proteins from crude muscle extract [146] or humansera [147] by this method.

In another approach thermosensitive poly(N,N-diethyl-acrylamide) (pDEAM) has been used to reversibly expose or shieldthe active site or binding pocket of streptavidin. Here, pDEAM ofcontrolled molecular weight was conjugated to streptavidin

tetramer which is immobilised on magnetic beads [148]. Theaccessibility of biotinylated proteins to a binding site on thestreptavidin approximately 2 nm away from the polymer anchorwas screened by the swollen pDEAM below the LCST and exposedabove the LCST when the pDEAM collapsed [148]. This approachalso allowed that binding of biotinylated proteins could be tunedbased on their size with the small B-protein G (6.2 kDa) able to bindwhen the pDEAM was swollen or collapsed whilst larger B-BSA(66 kDa) could only bind when pDEAM was collapsed and bindingof B-IgG (150 kDa) was prevented in both states [148].

Using pNIPAM coated latex particles modified with PEG-biotinMalmstadt et al. reported the preparation and use of a stimuli-responsive affinity chromatography matrix. The modified beadswere capable of binding streptavidin whilst immobilised ona microfluidic column under flow conditions [149]. Subsequentlowering of the temperature induced release of the beads alongwith captured streptavidin from the columnwall. This basic systemhas the potential to separate target proteins from complexmixtureswith the use of immobilised antibodies and the ability to form andrelease the matrix allows reuse of the microfluidic column [149].Other work on stimuli-responsive systems has also combined theuse of beads or particles with thermoresponsive pNIPAM forprotein studies [150,151]. The ability to incorporate a magnetic coreinto particles may also benefit processing and purification appli-cations whereby a magnetic field may separate particles fromprotein solutions for reuse [150,152–155]. Coatings of pNIPAM onparticles may be used to switch protein adsorption and allowrelease into different solutions or a smaller volume, thus allowingtransfer or concentration of high value biomolecules [150].Furthermore, the use of antibodies attached to, and controlled viaa suitable switchable material may allow directed entrapment ofspecific proteins or cells from a complex mixture [126].

An excellent demonstration of a processing device based on theaction of a stimulus-responsive polymer was reported by Huberet al. A microheater was used to switch a pNIPAM film to above itsLCST in less than 1 ms [81]. Although cooling was passive, themicrofluidic nature of the device (fluid volume w3 mL) allows forthe rapid desorption of a cloud of protein into the fluid chamberjust seconds after the heater was turned off [81]. It is not clearwhether some protein still remains at the surface but the highrelease efficiency reported is a considerable achievement. It ispossible that this is related to the short incubation time above theLCST [138].

Methods to enhance or assist the desorption of proteins havealso been achieved via manipulation of pH and/or ionic strength ofthe buffer solution to favour desorption [138,156,157]. Although notdifficult in principle, changing of buffer, pH or ionic strength forprotein removal may not be suitable for some applications ofswitchable coatings and is relatively slow unless microfluidics areused. For biomedical applications, one needs to consider thesensitivity of many proteins to changes in pH or ionic strengths.Thus, means for complete control over adsorption/desorption viaexternal triggers such as temperature, light or electric potentialwould be more widely applicable.

3.4. Characterisation of surface phase transitions

Research and literature on pNIPAM based switchable systemsdominate the field of switchable systems for biomedical andbiotechnological applications. Therefore it is necessary to discusstechniques and studies focused on gaining a better understandingof this model temperature-responsive polymer. Although thissection will focus on the thermal transition of pNIPAM, the tech-niques discussed here have also been employed for investigating

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the physical chemistry of other stimuli-responsive materials[41,43,158,159].

Switching the phase transition of LCST type surface-graftedpolymers by increasing the temperature is commonly associatedwith a change in surface wettability but also a thickness decrease(deswelling) and a concomitant increase in rigidity. An increase inthe water contact angle is generally observed upon increasing thetemperature above the LCST. This is often more pronounced withlow polymer cross-linking and can be enhanced through engi-neering the surface topography (lotus effect) and/or control ofsolution pH/ionic strength [83,93,160]. Other observations such assignificant swelling/deswelling are also commonly associated withthe phase transition of these polymers. This section will discusstechniques and studies that probe the phase transition of pNIPAMand investigate bio-interfacial interactions in the two states forgreater understanding of the properties of these coatings.

Fundamental studies of the structural changes in stimuli-responsive materials such as pNIPAM in the two states are difficultto carry out and suitable techniques are few. Surface force appa-ratus (SFA) and CP-AFM are two techniques that can be used toinvestigate the interactions between a probe surface and a stimuli-responsive material [134,161]. Importantly these techniques canaccommodate fluid environments so that samples may be investi-gated under conditions similar to their intended biomedicalapplications. Investigation of adsorbed pNIPAM based copolymerbetween two mica sheets using SFA showed that below the LCSTthere was no adhesion, but repulsion between the coated surfaces.This repulsion was attributed to electrostatic and steric effectsdepending on the separation distance [161]. In agreement with theexpected phase change, attractive forces were observed attemperatures above the LCST during approach to and retractionfrom the surfaces. This confirmed the expected nature of thestimulus-responsive coating. The LCST of adsorbed pNIPAMcopolymer (28 "C) was lower than that of the free copolymer insolution (33.7 "C) as determined by measurement of opticalabsorbance [161].

Another SFA investigation of pNIPAM coatings resulted indifferent observations. Increasing the temperature above the LCSTresulted in decreased repulsion but not an attractive force betweenpNIPAM andmica surfaces. Advancing water CA and swelling of thegrafted pNIPAM coating was observed to change at temperaturesabove the LCST, but the net interfacial force, investigated fora number of graft densities and molecular weights, remainedrepulsive [137]. SFA studies by Zhu et al. showed that graftedpNIPAM coatings of lower molecular weights (MW w2500 and10,000) do not display thermally mediated chain collapse but,instead, remain swollen at temperatures up to 35 "C [162]. It ispossible that in this case the experiment required investigationover a larger temperature range as Xia et al. have reported pNIPAMof similar MW to have LCSTs of greater than or equal to 35 "C[163,164]. These literature discrepancies reinforce the notion thatthe properties and behaviour of pNIPAM and similar polymers areincompletely understood, and may vary considerably with themicrostructure of graft coatings, as a consequence of fabricationprotocols.

A number of studies have utilised CP-AFM to probe the forcesbetween pNIPAM surfaces [5,90,133,134,165,166]. A recent studyby Ishida et al. utilised silica particles modified with alkyl silane toprobe the interaction between pNIPAM and a hydrophobic particle[167]. It was noted that significant attractive forces existedbetween the hydrophobic probe and the pNIPAM surface at highertemperatures (30–40 "C) whilst at low temperatures (15–25 "C)negligible or small interaction forces were observed [167].Importantly, the study found that cycling of the colloid probe intoand out of contact with the surface often increased the adhesion

force, presumably due to the mechanical collapse of the coating bythe probe.

Careful adjustment and consideration of parameters in CP-AFMis therefore required to minimise analysis-induced collapse of thecoating. If parameters include repeated cycling of approach/retractat high rates and with large forces, the mechanical compression ofthe polymer is likely to disturb the hydration shell, expel waterfrom the coating, and encourage intermolecular H-bonding ofchains. These effects may take some time to relax and would thusproduce hysteresis in probe–surface interactions during the nextcycle.

CP-AFM has provided further insight into the temperaturecontrolled protein adsorption onto pNIPAM surfaces. Adsorption ofproteins is modelled by measuring the force between a proteincoated probe sphere and a pNIPAM surface at various temperaturesin buffer solutions. However, differences in preparation of coatingsand conditions used in CP-AFM analysis have given rise to differentresults in the literature [134,165]. Again it is essential to be aware ofuncertainties and possible artefacts when performing and inter-preting CP-AFM measurements with protein coated probes. Arte-facts can again arise from physical factors, such as excessivecompression of the surface layers, or from uncertainties about thedensity and orientation of proteins on the probe. Nevertheless it isclear that the forces involved in the attraction of biomolecules suchas proteins are complicated and more research is required beforewell supported interpretations can be made on the relationshipsbetween surface properties/structure and the forces involved inprotein adsorption and desorption.

Studies of pNIPAM grafted on surfaces using the techniques ofsurface plasmon resonance (SPR) and quartz crystal microbalance(QCM) have demonstrated that the phase transition of tetheredpNIPAM coatings is more gradual than that of free polymer chains[109,168,169]. This important finding suggests that geometricconstraints arising from surface binding adversely affect the abilityof polymer chains to adopt a more ordered, collapsed conforma-tion. The conformational freedom of the grafted polymer chainswill also be affected by the grafting density, which is of particularinterest where the brush to globule transition of dense pNIPAMchains is a focus [168]. Clearly, the thermal behaviour of graftedthermoresponsive polymers may depend on a number of factorsincluding grafting density and chain length [137], which may bea reason for some of the inconsistencies in the literature. As shownschematically in Fig. 6, the effects of the thermal transition mightbe quite different for sparse chains compared with dense chains,and even the extent of collapse/dehydration will be affected bychain/chain interactions. Thus, with some combinations of chainlength and density, despite partial collapse the surface of the graf-ted layer may not change to an extent sufficient to markedly affectthe surface properties relevant to protein adsorption. In addition tothe questions regarding the extent and geometric effects of chaincollapse, an important question is the rates/kinetics of chaincollapse and re-hydration. Of particular note is the hysteresis ofre-swelling observed by Liu et al. where it was thought that H-bonding expected to have formed in the collapsed, dehydrated statewas not reversed at the same rate during re-hydration compared tothe rate of formation during dehydration [168]. QCM and SPR arewell suited to the study of such kinetic phenomena in aqueoussolutions.

Quantitative analysis of QCMdata on hydrated polymer chains isdifficult due to variation in the manner in which the mass of thesoft/flexible polymer and associated solvent molecules couplemechanically to the resonating crystal. The interpretation ofviscoelastic QCM data in terms of mass and dissipation is modeldependent, and users need to be aware of the assumptions madein the software of commercial QCM instruments. Care is also

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necessary with respect to assumptions made on coating homoge-neity, thickness, roughness and the like. Although not all studies arein agreement [168,170], it appears that for the phase transition ofsurface-attached pNIPAM chains monitored by QCM, a loss of massis observed upon increasing the temperature to above the LCST[168,169,171]. This was attributed to the loss of solvent (water) fromthe hydrated coating as the chains collapse [168,169]. A concomi-tant increase in rigidity during the phase transitionwas inferred bya decrease in the dissipation factor.

For PEG the LCST is known to vary significantly with molecularweight, whereas for pNIPAM the effect of molecular weight is lessclear. Some studies have found that the LCST of pNIPAM in solutiondepends little on molecular weight [172–174], whilst others haveshown a pronounced effect [163]. For surface tethered pNIPAM ithas been reported that the molecular weight does play a role[162,175]. The solution LCST varies with polymer concentration[53,172,176,177], accordingly, the surface LCST can be predicted tovary with packing density. The change in thermal properties ofsurface-grafted pNIPAM chains may depend on various interrelatedfactors and there is a need for further study to unravel the factorsbehind apparently contradictory observations. The chain packingdensity may also affect whether or not a clearly visible structuralrearrangement/collapse can occur; if chains are grafted too densely,theymay have insufficient mobility to manifest a clearly observableLCST, and chain length may playmore of a role on a surface when inclose confinement than it does in solution. Differences in prepa-ration and coating techniques are known to cause differences insurface coverage, density, molecular weight and substrate–polymerinteractions, all of which affect switching properties [178,179]. Boththe onset and the width of the phase transition of surface-graftedswitchable polymers may be affected by these factors.

Whilst there may be advantages in designing surfaces witha broad transition gradient, for other applications a narrowswitching range may be advantageous or essential. Higher perfor-mance (for adsorption/desorption applications) may be obtainedfrom coatings with sharper, more distinct transitions. Currently,design criteria are not well established for producing surface-grafted thermoresponsive coatings that enable prediction andcontrol of the position of the LCST, the width of the transition, andthe extent of switching. It is also unclear how these physico-chemical parameters affect performance in the adsorption andrelease of proteins and cells or how critical some of these para-meters are.

3.5. Theoretical studies and mechanistic insights

Because of the interest in pNIPAM and the relevance tobiomedical applications, theoretical studies of LCST have focusedon this polymer. The mechanism of pNIPAM switching has not beenfully established. However, it is thought that heating alters theentropic contribution to solvation, making inter- and intra-molecular hydrogen bonding between polymer chain segmentsmore favourable and thus causing dehydration [180]. The samepolymer physics principle of reduced solvation that causes thephenomenon of LCST in solution, with aggregation at T> LCST, isthought to lead to interactions between, and reduced solvation of,surface-grafted polymer chains. Extending into the aqueous phaseat T< LCST, surface-anchored polymer chains lose solvating waterand collapse into denser coatings at T> LCST, as shown for exampleby a study using temperature dependent ellipsometry [181]. Here itwas found that a spin-cast coating of poly(N-isopropylacrylamide-co-acrylic acid) microgels on silicon wafer changed in thicknessfromw400 nm tow170 nm (Fig. 7). Yet, even the collapsed coatingabove the LCST still appeared to contain appreciable amounts ofwater, as indicated by a dry thickness of 30 nm [181]. It has been

postulated that hydrophobic interactions [182,183] and interactionswith the surface [31] play a role in the collapse.

The formation of intra- and intermolecular hydrogen bonds atdifferent points of the pNIPAM phase transition has been investi-gated with infrared techniques in a number of reports [54,184,185].ATR/FT-IR studies of pNIPAM in water have shown that intermo-lecular H-bonding between pNIPAM and water is predominantbelow the LCST whilst above the LCST intra-molecular bondingwithin pNIPAM dominates [185]. Reported data also indicates theexposure of hydrophobic isopropyl groups (normally shielded atT< LCST) above the LCST, which is thought to enhance theswitching effect of pNIPAM [185]. The ability to discern differentpeaks such as those between C]O and solvent (H-bonded H2O,D2O) and C]O and N–H (inter- or intra-molecular H-bonding) hasalso provided the ability to monitor the relative amount ofsecondary bonding during the transition [184].

In addition to optical methods the transition of pNIPAM andother stimuli-responsive polymers has been investigated vianuclear magnetic resonance [183,186–188]. This technique has theability to investigate the signal of particular protons in a solublepolymer and monitor changes in specific peaks with temperature.The key findings from NMR studies have provided information onwhich parts of the pNIPAM repeating unit are interacting with eachother or with solvent molecules above and below the LCST.

Collapse/aggregation of solvated pNIPAM chains and otherswitchable materials in solution is often regarded as a sharp ordiscontinuous coil to globule transition. The transition of graftedchains on particles has been found to have a similar sharp transitionin some instances, but broadening of the transition has also been

Fig. 7. Thickness vs. temperature and refractive index vs. temperature curves forcoatings made from PNIPAM-co-PAA microgels with 2% BIS (A) and 10% BIS (B). Fullsymbols and empty symbols represent data obtained during heating and coolingcycles, respectively. Reprinted from Ref. [181], ! Elsevier Ltd.

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reported [60,189]. Contact angle data of surface-grafted pNIPAMbrushes often exhibits a sharp transition [109,112,179,190]. Butmore recently real-time investigations have shown pNIPAM coat-ings to have a more gradual phase transition [109,168,169,191]. Thisis likely a consequence of surface confinement, which limits theentropic space and the segmental mobility in the ordering of chainsas they dehydrate. The question then arises as to the kinetics ofordering versus the time scale of the experiment.

A number of efforts towards modelling polymer chains inaqueous environments have been made in recent years. Modellingand theoretical approaches to modelling the coil to globule tran-sition of free pNIPAM and other hydrophilic polymers in solution ishas been reported and reviewed [180,192] and self-consistent field(SCF) theory is one approach which has been applied to immobilisedhydrophilic polymer chains such as poly(ethylene oxide) [193,194].SCF has also been applied to the modelling of thermosensitivepolymers including pNIPAM [195]. The problem of modelling thecoil to globule transition for a single molecule in dilute solution iswell understood, but when polymer chains are grafted ontoa surface, issues of confinement, surface wetting, and cooperativityarise. The phase behaviour of polymer chains end-grafted to solidwalls is still poorly understood even though recent theoretical workhas provided additional insights into the phase transitions of freeand grafted polymers [196].

The experimental solution phase diagramwas used as one of theinputs in theoretical studies of tethered pNIPAM chains in water[197]; an interesting outcome was that the polymer surfacecoverage has a marked effect [195]. The model predictions indi-cated a transition occurring over a broad range, with an onset wellbelow the LCST, in agreement with experimental data, and anaccelerating rate of change near the LCST. The model also predicteda strong dependence on graft density; with increasing surfacecoverage the LCST shifted to lower values. Interestingly, the tran-sition also depended on chain length [195], in contrast to reports ofpNIPAM in solution where the molecular weight has practically noeffect. This latter point nicely illustrates the possible dangers ofinferring the behaviour of surface-grafted polymer chains fromtheir solution behaviour, disregarding surface confinement. Men-dez et al. [195] also modelled the relative magnitude of the thermalchange and found that it was greatest at intermediate surfacecoverage, and the surface coverage required for maximum changevaried inversely with molecular weight. The theoretical modellingstudies illustrate the interactions between various factors involvedin thermoresponsive behaviour and can provide guidance toexperimental studies.

While it is often thought that the concentration of pNIPAMchains in solution affects the LCST such that more concentratedsolutions will switch from the soluble to the insoluble state at lowertemperatures, some studies have reported little effect of increasingmolecular weight, whereas others reported that very high molec-ular weight pNIPAM polymers have a lower LCST whilst low MWpNIPAM has been reported by some to lack switchable behaviour[162,175]. For surface-immobilised pNIPAM, a number of studieshave shown that a distinct correlation exists between the MW ofgrafted chains and their ability to undergo a thermally actuatedphase transition [162,175,198]. In addition, low MW pNIPAMcovalently attached to lipid bilayers enabled the use of SFA toinvestigate the interactions between two mica-lipid-pNIPAMsurfaces as well as interactions between a mica-lipid and a mica-lipid-pNIPAM surface at different temperatures. With low MW(w10,000) pNIPAM grafted at low density there was no significantchange in adhesion at temperatures above and below 32 "C [162].

On the other hand, Akiyama et al. found that very thin surface-grafted pNIPAM coatings were required for switchable cell adhe-sion [31]; while a coating of 15 nm dry thickness gave good cell

culture results, a coating of 29 nm dry thickness gave inferior cellattachment in contrast to the results of Xu et al. where oppositebehaviour was observed [124]. It was postulated that the TCPSsubstrate underneath the pNIPAM layer drove the hydrophobiccollapse of pNIPAM above the LCST [31], and that at 37 "C thepNIPAM layer consisted, if the dry thickness was >15 nm, ofa dehydrated structure near the TCPS interface and a less dehy-drated structure further away. It was argued that ‘‘hydrophobicinteraction at TCPS interfaces is also likely to promote aggregationand enhanced dehydration of the covalently bound chains. Thehydrophobic and immobile TCPS interfaces restrict molecularmotion of the grafted chains.’’ [31].

An interesting aspect of this study is that it implicitly raisesa more widely applicable question concerning the effect a substratesurface might have on thermally induced collapse and the nature ofthe interfacial forces involved. If the end group on a linear chain canaffect the LCST [163]; what effect might a SAM, lipid bilayer orsurface of polymeric or crystalline substrate have on the phasetransition? A number of questions are raised in the analysis of theTCPS example from Ref. [31]. The interpretation implies thatwithout a force emanating from the TCPS substrate, the pNIPAMlayer would not undergo dehydration sufficient to enable adsorp-tion of cell-adhesive proteins and subsequent cell colonisation.However, modelling and real-time investigation have shown thatsurfaces tend to impede rather than assist the phase transition dueto surface constraint. Furthermore, what force emanating from theTCPS substrate would have a range of the order of the thickness ofthe hydrated thinner coating (whose hydrated thickness was notdetermined, but must be [15 nm) such as to drive its collapsewhen the temperature is raised to 37 "C? Dispersion forces and‘hydrophobic’ solvation forces have much smaller ranges [199]. Wenote that the two coatings (15 nm and 29 nm dry) were preparedby electron beam grafting from solutions of differing concentra-tions. It is possible that not just the thickness but also the chaindensity was different, which might yield different collapse behav-iours, as predicted by modelling [195]. While the substrate surfacemay play a role, a full mechanistic picture may be more complex,and the (extent of) collapse of surface-attached chains is likely to beaffected by a number of factors such as the MW and the graftdensity, which affects segmental mobility, as predicted by theo-retical modelling [163]. The substrate influence should be investi-gated by using substrates with different surface properties, but itmay not be trivial to ensure that coatings deposited onto differentsubstrates are identical with regard to chain length, density, andpolydispersity; a ‘‘grafting-to’’ approach may be more controllable.

A further relevant aspect is raised by the findings of Beines et al.[200], who observed a refractive index gradient across pNIPAM-co-methacrylic acid films and attributed this to a dense outer skinlayer. Upon swelling and deswelling, a porous structure wasobserved by AFM. It is not known whether these observationsmight apply only to their specific films, and if so, why, or whetheranalogous structures might also exist in films produced by othersbut perhaps not readily detected. Internal microstructures havebeen shown to be an important factor for the volume phase tran-sition of bulk hydrogels [201]. Why some pNIPAM surfaces showgood switching ability while others show (partial or complete)absence of thermal switching is unclear. In addition to molecularmechanistic models, the kinetics of switching, and its dependenceonmicrostructural factors of grafted coatings, also need to be betterunderstood. For some applications, switching response time maybe essential, whilst for others the magnitude of change in thematerials property of interest (e.g. volume, wettability, andmodulus) may be important.

Clearly, the segment density of grafted coatings is a key factor[137], as it is for PEG grafts [10,87]. Neutron reflectivity studies have

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covered a range of MW and graft densities of surface-attachedpNIPAM chains. It was reported that an intermediate graftingdensity combined with high MW pNIPAM (MWw 152,000) gavethe greatest conformational change with increasing temperature.On the other hand, coatings of low MW pNIPAM (MW< 75,000)showed little or no conformational change [198]. The calculatedsegment density distribution suggested that pNIPAM chains weremore concentrated at the substrate surface at both high and lowtemperatures. This was more pronounced on gold/thiol substratesthan on silicon/silane substrates and was attributed to differingaffinities of the pNIPAM chains to the two substrates [198].However, Zhu et al. [162] also reported an unusual segment densitystructure; while pNIPAM chains did not adsorb onto the lipidbilayer, suggesting no substrate surface affinity, there was never-theless a denser structure of the pNIPAM layer near the lipidsupport. The authors attributed this to an inherent structure ofpNIPAM graft layers rather than as arising from the substratesurface. In contrast, Xue et al. [202] and Beines et al. [200] postu-lated a denser skin layer at the outer surface of the coating.

The literature suggests that attachment of low MW pNIPAMbrushes to surfaces results in dampening or removal of theirswitching behaviour. However, in the case of low average MWcoatings with less control and high PDI, switching of wettability hasbeen observed [179]. Discrepancies in the observations from QCMstudies [168–170] particularly emphasise that a detailed under-standing of the various factors involved in thermal switching oftethered polymer chains has not been achieved yet, whichcontrasts with the current understanding of the solution behaviourfor pNIPAM of similar MWs [163,164]. Two reports from Xia et al.provide the most comprehensive studies of the MW dependence ofthe pNIPAM phase transition (for free polymers in solution) thusfar. An increase in molecular weight from 2.8 kDa to 26.5 kDashowed a decrease in the LCST from 43.0 "C to 33.3 "C [164].Although higher MW polymers were not investigated, the positionof the ‘‘cloud point’’, or LCST, was consistently inversely dependenton the MW [164] in contrast to the results of Tong et al. where49.4 kDa pNIPAM had a lower cloud point (31.2 "C) than 101 kDapNIPAM (31.7 "C) [177]. Xia et al. paid particular attention topreparing polymers with low PDI (w1.1–1.2) using atom transferradical polymerisation [163,164]. Furthermore, variability of theend group via initiator selection was shown to have a pronouncedeffect on the LCST with polar end groups increasing and non-polargroups decreasing the LCST, respectively. Finally, small NIPAMoligomers (DP$ 10) were investigated and still found to exhibitcloud points, albeit at higher temperatures of 50.9 "C for oligoNIPAM-10 and 70.4 "C for oligo NIPAM-4 [163].

Clearly, there is still a need for further insight and carefulfundamental studies on surfaces grafted with stimuli-responsivepolymers. Theoretical modelling studies such as those discussedearlier are valuable for such advances as they can investigatesegment density issues and constraints arising from close prox-imity of grafted chains and from restricted translational and rota-tional entropy. A key issue is the question of the influence of theproperties of the substrate on the solubility, conformation andcollapse of grafted chains.

4. Other switchable systems

4.1. Switching with pH and/or ionic strength

One of the most important considerations for the interfacialcontrol of biomolecule–surface interactions is electrostatic inter-action between the surface and the biomolecules in solution.Biomolecules can be attracted or repelled from a surface dependingon the overall charge of the surface as well as their own charge

(Section 4.2). Furthermore, the ionic strength of the solution hasa significant influence on biomolecule–surface interactions, withhigher ionic strength solutions reducing the range of electrostaticinteractions. Due to the fact that most biologically relevant solu-tions contain mixtures of biomolecules with different overallcharges, surfaces that are designed to reduce or preventbiomolecule–surface interactions are generally based on neutralcoatings.

The pH of an aqueous solution affects the properties of manysurfaces due to protonation/deprotonation of ionisable groups andthe consequent interactions between these charged surface groups,ions and biomolecules. Although less commonly used for theinterfacial control of biomolecule–surface interactions in compar-ison to thermal switching, the use of pH as a trigger to alter theproperties of stimuli-responsive polymers shows considerablepromise for a number of applications, particularly in drug delivery[63,77,96,203–205]. However, in vitro and in vivo cell based appli-cations are restricted to a narrow range of pH and ionic strengthconditions required for normal biological functions. For otherapplications such as processing devices and some bio-diagnostics,the pH and ionic strength requirements may be less restrictive andpH sensitive polymers and copolymers can find diverse usages.

One difficulty faced by the use of pH or salt induced switching isthat for a system to exhibit reversibility, the aqueous solutionenvironment must be changed in order to elicit a response. That is,the solution needs to be either removed and replaced, or added to,in order to trigger the system, which makes this a more compli-cated approach comparedwith ‘‘remote’’ switchingmodalities suchas light, temperature or electric potential. For this reason, it is morelikely that switching pH or ionic strength will find applicationmainly in one-off switching where a change in the solutionconditions allows release/delivery of a payload, or for microfluidicchips where programmed pH/ionic strength gradients may enablecontrolled separation of biomolecules through control over inter-actions with stimuli-responsive surfaces.

A number of polymers, including those based on poly(N-alkylamides) and poly(N-alkylacrylamides) respond not only tostimuli such as temperature but also to changes in pH or salt.Copolymerisation of an uncharged monomer with monomerscontaining ionisable groups is a commonly used approach toenhance the pH-responsive behaviour of hydrogel systems[58,86,160,204,206,207]. An early concept for pH based switchingwas described in 1991; an electric stimulus caused electrolysis ata polymer coated electrode. The localised pH change was thoughtto alter hydrogen bonding within the polymer gel, causing erosionof the hydrogel [208]. It was shown that hydrogel degradationcould be switched on and off and thereby insulin release. Althoughelectrolysis is not ideal in many situations, this early study hasencouraged further research into such systems that enable ‘ondemand’ switching.

Switching via changes in ionic strength may shift the LCST[39,209,210]; the effect of anions relates to the Hofmeister series.When intending to switch bio-interfacial interactions however, thecapacity for ionic strength to affect aggregation and/or adsorptionof proteins must be considered.

Whilst pH switching for the control over protein adsorption orcell attachment has not beenwidely reported, biomolecule deliveryusing smart materials is more common in the literature. Work onintra-cellular delivery has demonstrated that pH-responsive poly-mers may be used to deliver fragile biomolecules such as peptidesor oligonucleotides to the cytoplasm of macrophages [77]. In thatwork, pH cleavable acetal linkages were used to remove PEG graftsthat mask the delivery vehicle and disrupt the endosome causingthe delivery of biomolecules into the cytoplasm before they aredegraded in the lysosomes [77].

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The inherent sensitivity of some materials to the pH and/or saltcontent of the surrounding environment could be employed insensing applications. The conformational change of surface-graftedelastin-like polypeptide upon alteration of the pH and ionicstrength conditions was found to exert a mechanical stress uponAFM micro-cantilever [211]. Such micro-cantilever based sensorsincorporating stimuli-responsive materials can be designed forspecific sensing ranges and environmental conditions (tempera-ture, pH and ionic strength) [211].

Other smart systems using pH as a trigger or tuning mechanisminclude surfaces decorated with polyelectrolyte brushes. Tetheredpolyelectrolyte coatings change their conformation in response topH and/or ionic strength, as shown schematically in Fig. 8. Elec-trostatic repulsion between like charges along polymer chains iscontrolled by the degree of ionisation and the ionic strength of themedium. As ionised functional groups repel each other, thisrepulsion causes chain stretching and swelling of the coating,whereas neutral chains can adopt a denser conformation. The lowerthe ionic strength of the aqueous solution, the greater the Debyelength of electrostatic interactions [199] and hence the distancethat charged groups will try to adopt. The result is an extended coilor stretched brush conformation of the polymer chains.

For example, surfaces grafted with diblock copolymers of acrylicacid and 2-vinylpyridine or 4-vinylpyridine were investigated withrespect to pH-responsive control over coating thickness [212]. Atthe isoelectric point (pI), where the negative charge of the anionicblock equals the positive charge of the cationic block, the leastchain stretching was obtained. Stretching of either block (poly-electrolyte behaviour) exists when there is charge asymmetry[212]. The concept behind the use of weak negative and weakpositive polymer blocks is that at high and low pH ranges therespective anionic and cationic blocks will exhibit polyelectrolytebehaviour and thus an extended conformation. In a study of similarmaterials, control over protein adsorption at high and low saltconcentrations was investigated [213]. Binary brushes of poly(2-vinylpyridine) (P2VP, weakly positive) and poly(acrylic acid) (PAA,weakly negative) were prepared, with the resultant surface havinga pI of w4.9. The combination of an amphiphilic surface withamphiphilic proteins (a-chymotrypsin, pIw 8.1 and a-lactalbumin,pIw 4.3) led to interesting protein adsorption behaviours [213].One observation was that negatively charged a-chymotrypsinadsorbed to the negatively charged surface under low salt condi-tions, overcoming repulsive steric and electrostatic forces. This wasthought to arise from the protein’s ability to reverse its charge uponentry into the PAA along with entropically based forces fromcounter ion release to drive the adsorption [213].

Generation of switchable surface micropatterns of grafted PAAand P2VP have recently been investigated for reversibly switchingwettability, topography and charge using a pH stimulus [214]. AtpH> 5.5 the PAA areas were swollen and negatively charged whilstat pH< 2.3 P2VP was swollen and positively charged. At pH 2.3–5.5

both polymers were considered to be collapsed. Fluorescenceinvestigation of the adsorption of FITC-Casein (pI 7.63) clearlyshowed that at high pH (8.6), when negatively charged, the FITC-Casein was adsorbed to the collapsed P2VP areas but electrostati-cally repelled from swollen, negative PAA. At low pH (1.2),adsorption of FITC-Casein (now positively charged) was directed tothe collapsed PAA areas and repelled from the P2VP pattern [214].

Changes in pH and ionic strength can alter protein adsorptionregardless of whether the substrate material is stimuli-responsive.This is because proteins themselves can be switched between netnegative, positive and neutral overall charge according to their pI[214–216]. Thus, it can be expected that a given switchable materialcould show different switching conditions for different proteins.Proteins and nucleic acids are routinely separated via electropho-resis and isoelectric focusing with the aid of hydrogel networks.However, advanced control over electrostatic interactions withnext-generation micro- and nanofluidic chip devices shows greatpromise for biomolecule separation in the future [215,217].

4.2. Electrostatic manipulation and switching

A common procedure used in biology and other fields today isgel electrophoresis. In this technique, protein or DNA is loaded ontoa permeable gel such as agarose or polyacrylamide and an electricfield is applied. The inherent net charge of the biomolecules causesthem to diffuse through the gel towards the electrode of oppositecharge at a rate that is based upon their size, allowing them to besorted and isolated [218]. This concept has also been applied tosubstrate surfaces, whereby an attractive electric potential is usedto increase or induce the adsorption of biomolecules such asproteins, enzymes and DNA, with the biomolecule of interest withan inherent net charge opposite to that of the electrode[35,219–221].

Application of an electric potential to manipulate properties ofa surface coating, rather than to attract charged species fromsolution, is an alternative approach that shows promise in appli-cations such as control over wettability. A study by Lahann et al.focused on the ability to attract charged head groups of a lowdensity alkanethiolate coating towards the substrate, thus bendingthe chains and exposing the alkyl chain section, as shown in Fig. 9[222]. This approach could reversibly induce surface rearrangementto expose the hydrophilic head groups or hydrophobic alkyl chains[222]. Switching materials with surface re-organisation triggeredby an applied potential are of particular interest for microfluidicsand microactuators, and such approaches may also find applicationin biomedical engineering.

Ferapontova et al. showed that the surface charge of a metalelectrode surface can significantly alter the adsorption of proteinssuch as horse-radish peroxidase (HRP) [219]. Of primary interestwas improvement of direct electron transfer between the hemegroup of HRP and the electrode. Control over the adsorption and

Fig. 8. Polyelectrolyte brush coating exhibiting neutral collapsed (A) and ionised expanded state (B).

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orientation of HRP is therefore of great consequence. Manipulationof the surface charge such that it was opposite to that of the enzymewas shown to enhance adsorption, whereas adsorption was greatlyreduced when surface and enzyme had like charges. Alteration ofthe charge or the isoelectric point of the enzyme was alsodemonstrated to be an effective means to control the adsorption.Although this was not a switchable process, the report providessupport for the use of electrostatic attraction and repulsion forcontrol over protein deposition onto surfaces.

Although not a focus of this review, it should be mentioned thatconducting surfaces have been utilised successfully for electricpotential assisted adsorption and desorption of DNA [223]. Varioussystems have been demonstrated whereby functionalised DNA,linear DNA, plasmid DNA and DNA/lipid complexes (lipoplexes)could be switched via an electric potential applied to gold elec-trodes [35,223,224]. The inherent negative charge of DNA allowsreversible attraction and repulsion by applying alternating poten-tials upon surface electrodes. This work has been extended inanother study, wherein attractive and repulsive potentials appliedto semi-conducting silicon substrates modified with thin polymericcoatings were able to assist the deposition of biomolecules viaapplication of an electric potential [220]. Modification of conduc-tive substrates with polymer coatings can enable more favourablesurface properties for adsorption of active proteins and subsequentcell attachment. Incorporating switchable systems that can act oncharged biomolecules adsorbed atop polymeric coatings areexpected to benefit applications such as transfected cell micro-arrays [225]. Research along similar lines has focused on theattraction of enzymes from buffer to surface-modified semi-conducting silicon substrates [221]; charged enzymes could bedeposited at the interface of an otherwise low-fouling PEG coatingvia the application of a small electric bias of opposite charge. Acolorimetric assay confirmed activity of the deposited enzymes[221]. Such research may enable that biomolecules of interest areattracted to the surface where their activity or function can beutilised, followed by subsequent release.

A molecular modelling study on controlling adsorption/desorption of proteins via surface potentials was reported by Fanget al. The study, which focused on the controlled release of proteinsfrom polymermodified electrode surfaces, showed the feasibility ofa switchable system based on control over electrostatic and stericsurface–protein interactions [226]. Theoretical studies such as this

may prove instrumental for the rational design of switchablesurfaces.

Other investigations into electric potential assisted/enhanceddeposition of proteins and subsequent cell growth have showninteresting responses such as an increase in neurite extension fromgrowing cells [227]. Electroactive substrates have also been studiedfor the purpose of controlling the immobilisation of cell-adhesionpeptides such as the commonly used RGD peptide [228]. Yeo et al.took an electrochemical approach that enabled release of RGD (E*-RGD) from the surface via electrochemical oxidation, leavingbenzoquinone groups that could in turn enable the chemicalattachment of cyclopentadiene tagged RGD (RGD-Cp) througha Diels–Alder reaction. Thus the system has a mechanism to ‘switchoff’ cell attachment and ‘switch on’ or refresh, the cell-adhesivesurface. This was demonstrated by the electrochemical release ofE*-RGD and subsequent release of the cells growing atop thepatterned E*-RGD areas (Fig.10A, B). Furthermore, the surface couldbe refreshed without harming remaining cells via the attachmentof RGD-Cp in a serum free step. Replacement with serum con-taining medium allowed cells growing on surrounding areas tomigrate back to the areas where initial cleavage of the RGD hadoccurred (Fig. 10C) [228].

Other examples of electric switching of cells concern the elec-trochemical desorption of oligo ethylene glycol (OEG) SAMs fromgold surfaces [229]. Cells were first allowed to adhere to 2D proteinadsorbing patterns where they remained confined by surroundinglow-fouling OEG terminated regions. Application of an electricpotential triggered desorption of the SAMs from the gold andinitiated spreading of the confined cells across the surface [229].Whilst not a reversible switching system, this approach has on-demand control, does not require extensive chemical processing,and may allow controlled studies of cell motility and growth.

In summary, electric stimuli have contributed to the field ofswitchable interfaces and systems for control over biomoleculesand cells through release of surface materials (along with drugs,biomolecules, cells) and attraction of charged biomolecule speciesto electrodes.

4.3. Photo-activated switching

The use of light to study and control chemical and biologicalsystems is a well established area of research. Photosensitive

Fig. 9. Schematic diagram of electrically induced surface re-organisation via switching of surface polarity. Adapted from Ref. [222]. Reprinted with permission from AAAS.

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molecules such as photocleavable protecting groups have beenapplied extensively to conventional and solid phase synthesis. Theyhave also been applied in biomolecular research [230]. However,the use of photosensitive molecules to control the adsorption anddesorption of biomolecules at interfaces has been less studied.

Studies incorporating photocleavable groups on surfaces havebeen used in similar protective fashions as they are utilised insynthetic chemistry. Control over the chemistry and subsequentcovalent attachment of peptides for control over adsorption andattachment of cells has been demonstrated; however, this is not anactive form of switching [231,232]. A different approach, alsoutilising a photocleavage strategy, focused on the photocleavablegroup 2-nitrobenzyl, which was attached to glass slides viaa silane linker. It was found that BSA protein could be adsorbed onthe modified surface and then removed through the photo-cleavage of the underlying surface with radiation at 365 nm [233].This is a simple and effective example of one-off switchingwhereby light-stimulated removal of BSA enabled subsequentcontrol over the localised adsorption of cell-adhesive FN [233].Due to the low-fouling nature of BSA, ensuing cell attachment andgrowth was confined to the FN coated areas. Furthermore, byrepeating photocleavage on areas not previously exposed to light,spatially directed attachment of other strains of cells could beachieved during cell cultivation without damaging the alreadygrowing cells. [233].

Another switchable approach based on light as a trigger involvesphotoisomerisation. Although less applied to biochip andbiomedical devices than photocleavable groups and linkers, pho-toisomerisation has received some attention. Photoisomerisationhas been demonstrated to be able to induce changes in surface

wettability [234]; however, surface wettability by itself is a poordiscriminant of biomolecule adsorption and, moreover, onceadsorbed onto hydrophobic surfaces, proteins generally cannot bedesorbed again except on hydrated layers such as pNIPAM.

One study that utilised a photoisomerisable azobenzene deriv-ative reported partial increases or decreases in cell attachmentbased upon the conformation of the chromophore. Cell-adhesiveRGD peptides were attached to the azobenzene derivative and theiravailability to growing cells was controlled via photo-switching[235].

Elsewhere a copolymer of methyl methacrylate with photo-isomerisable spirobenzopyran (Fig. 11) was investigated for itsability to induce detachment of fibrinogen, platelets and cells viaphotoisomerisation of the chromophore [236]. The study showedthat UV illumination decreased the amount of previously adsorbedfibrinogen, presumably through switching of the surface. However,other experiments found that approximately 20% more fibrinogenwas adsorbed following UV stimulation than prior to illumination[236]. These results should help stimulate research into photo-switchable systems. The potential for molecular rearrangements,such as cis–trans isomerisation, to induce a sufficient change in thesurface binding of adsorbed biomacromolecules, and thus alter cellgrowth, is certainly intriguing. It is, however accepted that photo-isomerisation on its own is generally less appropriate for controlover biomolecule adsorption/desorption, mainly due to the limitedrange of changes in physical properties that can be achieved.

Photoisomerisable constituents have been combined withstimuli-responsive polymers such as pNIPAM through copoly-merisation of photoisomerisable chromophores [36,237,238]. Thishas enabled the blending of the unique switchable properties of

Fig. 10. (A, B) Release of E*-RGD and subsequent release of cells. Cells remaining after E*-RGD cleavage are confined on fibrinogen patterned areas surrounded by nonadhesivecleaved areas. (C) Coupling of RGD-Cp to benzoquinone groups in cleaved regions allows cells to spread across the surface from the confined fibrinogen patterns. Reproduced withpermission from Ref. [228], ! American Chemical Society.

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both materials. In studies discussed later, spirobenzopyran chro-mophores were incorporated with pNIPAM hydrogels for duallight/temperature control over the switching of the phase transi-tion [36,237]. Although the introduction of these less polarmolecules can often cause broadening of the LCST and alterwettability properties, further research may overcome theseissues, increasing the potential of photo-thermal switchingmaterials in applications such as chromatography or optical cellsorting [237].

Investigations of copolymers of spirobenzopyran and NIPAMhave reported that switching of the copolymer containing only1.1 mol% of the chromophore could be achieved by photo-isomerisation or thermal manipulation [238]. Subsequent researchhas included development of a photo-controllable membranewhereby the permeability of the hydrogel can be altered accord-ing to the photo-modulated collapse and expansion of thehydrogel within a porous poly(tetrafluoroethylene) membrane[239].

A different approach to photo-switching was based on theunique properties of gold nanorods. Photo-irradiation of goldnanorods (NRs) of specific size and structure results in conver-sion of light energy to heat through non-radiative relaxationprocesses [240]. It was found that the photo-thermal heatgenerated at the NRs localised within thermosensitive copolymermicrogels composed of NIPAM and maleic acid monomers wassufficient to induce deswelling of the surrounding hydrogel.A volume reduction of 78% 4% was obtained for microgels con-taining NRs using laser light with a wavelength of 809 nm asa trigger (Fig. 12). Unexpectedly, microgels free of NRs exhibiteda volume reduction of 25%1% under the same irradiation(Fig. 12) [240].

The high degree of control, non-invasive application, and speedof switching are some of the advantages to using electromagneticradiation as a trigger in the switching of responsive systems. Asidefrom photocleavage of surfaces for patterning and/or release ofbiomolecules, progress made in this field with biological applica-tions has predominantly been through the combined use of

hydrogels or other stimuli-responsive materials. Such approachesmay offer photo-tuneable drug delivery from responsive particlesor control over antibodies or enzymes attached to responsivesurfaces or materials [241].

4.4. Dual or multi-triggered switchable systems

An exciting development in the field of stimuli-responsivematerials is the incorporation of multiple switching mechanismsinto coatings to enable switching via a number of different triggers.These systems have unique abilities, often allowing finer controlover material properties and interfacial switching behaviour, whichas a result can give enhanced control over interactions withbiomolecules and cells at surfaces. The extra level of sophisticationin these systems may assist in enhancing reversibility; allowswitching of multiple biomolecules and increase switching

Fig. 11. Schematic of photoisomerisation of nitrospyrobenzopyran chromophore within copolymer of (poly(NSP-co-MMA)) upon irradiation via UV. Adapted with permission fromRef. [236], ! American Chemical Society.

Fig. 12. Swelling/deswelling exhibited by the hydrogel (diamonds) and hydro-gel! nanorod hybrids (squares), showing repeatability of the phase transition and themagnitude of the volume change during swelling/deswelling cycles. Reproduced withpermission from Ref. [240], ! American Chemical Society.

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performance. Also the ability to pattern the deposition of biomole-cules and cells with light and another suitable stimulus, as opposedto chemical patterning of surfaces and use of one stimulus, isexpected to be highly advantageous.

By combining the thermal switching properties of pNIPAM withphotoisomerisable spiropyran chromophores Edahiro et al. wereable to design a photoresponsive culture surface upon which theattachment and growth of cells could be spatially directed by UVlight and could then either be removed or retained on the surface inthe presence or absence of visible light with low temperaturewashing (Fig. 13) [36]. This dual trigger system affords control overthe spatial attachment and detachment of cells on demand. Thesystem has the key advantage that any form of pattern can beapplied quickly, easily and reversibly. [36] The concept has thepotential to form complex patterns of different types of cells andcell sheets.

Another dual switching concept applied to cell culture studies hasbeen described where dead cells attached to a thermally reversiblepolymer surface are selectively released from the coating and livecells remain [242]. In that study, incorporation of a crown etherreceptor, benzo-18-crown-6-acrylamide (BCAm) into a pNIPAMcopolymer enabled shifting of the LCST upon binding of potassiumions toBCAm.The shift of theLCST tohigher temperatures, attributedto disruption of H-bonding of the BCAm upon guest–host binding,allows localised swelling of the surface (in the presence of potassiumions)withouta change in temperature [242]. This concept is basedonreceiving the signal or trigger directly from the cells, since upon celldeath, intra-cellular potassium is released andmay be bound by theBCAm receptors. Furthermore, live cells could also be removed bycooling to below the LCST in the conventionalmanner but also by theaddition of potassium ions to the aqueous culture solution to stim-ulate the BCAm receptor mechanism [242]. This approach showsswitchablematerials could find use in screening applications similarto the smart petri dish concept [243].

Work by Garcia et al. has included investigation of multi-triggered switching of copolymers [244], focusing on the prepara-tion of tuneable microgels that could be manipulated via light,temperature, and pH stimuli. Although it is known that bothphotochromic molecules and thermoresponsive polymers inher-ently have a degree of pH dependence, a number of studies have

mentioned the potential benefit of the design of multi-stimuli-responsive materials [238,244,245]. On one hand, the presence ofmore than two triggers is likely to introduce limitations especiallyin the working conditions of engineered materials. On the otherhand, systems responsive to multiple triggers which work ina cooperativemannermay add further capabilities or insights to thefield of switchable materials.

Attachment of photo- and temperature-responsive polymerchains to the enzyme endoglucanase 12A (EG-12A) conferred theability to block enzymatic activity through shifting of the LCST ofthe polymer via photo-switching [241]. The LCSTs of two stimuli-responsive polymers incorporating photoisomerisable azobenzenemolecules could be shifted by UV or visible photo-irradiation, thusenabling the polymer to be switched between collapsed andexpanded states while keeping the external system temperatureconstant at 45 "C. Subsequent enzymatic activity under UV orvisible light illumination could be prevented when polymer chainswere switched to the collapsed state by steric blocking of the EG-12A active site. Switching the EG-12A conjugated polymer chains tothe expanded state yielded 60% enzymatic activity as compared tothe native enzyme without attached polymer [241].

Dual switching of pH and temperature is an attractive area ofresearch due to the concept that smart materials can be tuned toform a gel matrix at physiological temperatures, which they retainuntil they reach a physiological region that has a pH that allowsswelling and release of a desired payload. In pioneering work byChen and Hoffman, graft copolymers were produced that incor-porated thermosensitive pNIPAM and pH sensitive PAA such thatthe LCST of the polymer could be controlled or tuned by changingthe pH [96]. Other studies have also investigated this category ofsmart materials [38,246,247].

Whilst solely temperature-responsive [248] or pH-responsive[249] surfaces have been used to control wettability, temperatureand pH stimuli have also been combined in order to control thewettability of hydrogel coated micro-engineered substrates as wellas the solution properties of polymeric micelles [125,160]. In onesuch study, acrylic acid (AA) was used to develop a pNIPAM-co-AAcopolymer sensitive to pH and temperature [160]. Use of the dualstimuli-responsive coating combined with surface micro-structuring enabled the water CA of the surface to be controlledover a range of 150" (Fig. 14) [160]. On a flat surface, however, theCA change was only 13" and 9.6" for a temperature range of21–45 "C and a pH range of 2–11, respectively. Although not appliedto control biomolecule adsorption, in this case the device haspotential applications in drug delivery and microfluidics.

Investigation of a dual-responsive system using ionic strengthand temperature as triggers for control over stimuli-responsiveelastin-like polypeptide (ELP) has proved rewarding. ImmobilisedELP that had been patterned on a surface collapsed to a hydro-phobic state from a swollen hydrophilic state following theaddition of 1.5 M NaCl [41]. Furthermore, free proteins in solutionthat were labelled with the ELP could be induced to bind withsurface-immobilised ELP when the LCST of the polypeptides wasswitched. ELP labelled proteins could then be released by rinsingwith PBS at 4 "C, which caused the ELP to return to its swollenhydrophilic state [41].

5. Conclusions and future outlook

Switchable interfaces that are suitable for the control ofbiomolecule–surface and cell–surface interactions have alreadybeen demonstrated for a variety of biomedical applications.Advantages that are expected from such switchable interfacesinclude speed, ease, reversibility, temporal control and spatialcontrol over bio-interfacial interactions and events. Whilst there

Fig. 13. Living cell pattern generated on light/temperature-responsive surface viaphoto-patterning and thermally controlled cell release. Enhanced cell adhesion wasachieved by UV irradiation in the shape of a question mark and allowed cells on theirradiated area to remain attached whilst those on surrounding non-irradiated areaswere removed by low temperature washing. Reproduced with permission fromRef. [36], ! American Chemical Society.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–18501844

Fig. 14. (A) Manipulation of temperature and/or pH allows tuning of the surface wettability. (B) Cycling the dual-responsive surface between hydrophilic and hydrophobic statesshows the reproducibility and reversibility of switching. Adapted from Ref. [160], ! Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–1850 1845

has beenmuch excitement generated in this area, in many cases theunderstanding of the underlying mechanisms is still limited andneeds to be extended, so that improved, rational design of closelycontrollable switchable systems can be accessed. As knowledgeincreases in regard to the fundamentals of switchable interfaces,researchers can expand studies on the design of responsivematerials and systems for use in combination and in moresophisticated systems to expand the capabilities of medical diag-nostics, biological research and biotechnological/biomedicaldevices.

From the literature it is also clear that much of the success todate on switching the adsorption and desorption of biomolecules toand from surfaces has been for the control of either cell–surface orDNA–surface interactions. This has provided progress for implantand tissue growth applications, solid phase transfection, biochips,microarrays, and a range of other biomedical and biotechnologicalapplications. However, there has been less progress in the area ofswitchable protein–surface interactions in comparison to theachievements with DNA and cells. One reason for this is that unlikeDNA, properties of proteins are highly diverse owing to the diver-sity of their functions. Proteins also have a 3D structure sensitive tofolding/unfolding and often exist and function as complex entities,or associated with other molecules such as lipids and othercomponents. Although research into the switching of biomoleculeshas been highly successful, breakthroughs in understanding andspecifically tailored systems still need to be made to allow therealisation of many potential switching applications.

We certainly expect that the importance of the ability to controlbio-interfacial interactions will continue to grow not only forbiomedical applications but also for other purposes (such as marinebiofouling, bacterially resistant surfaces and water purification).We predict that in the near future, specific areas of enhanced focuswill include the design and application of truly reversible switchingmechanisms and interfaces due to the large number of applicationsthat would benefit from this. Furthermore, we predict that the areaof switchable interfaces that display biologically active signals orligands on demand will have an impact on the field of biomaterialsand other cell based applications. For example, such interfaces mayenable automated cell culture protocols. Finally, current advancesin this field are leading to in vitro and in vivo applications whereswitching is triggered internally by cell or tissue derived signals andmay be further controlled with the use of external stimuli inconcert.

Acknowledgements

This work was supported by the Australian CommonwealthGovernment under the ARC Special Research Centres Scheme(Special Research Centre for Particle and Materials Interfaces). Wethank Deborah Leckband for stimulating discussions.

References

[1] Alexander C, Shakesheff KM. Responsive polymers at the biology/materialsscience interface. Adv Mater 2006;18(24):3321–8.

[2] Kikuchi A, Okano T. Nanostructured designs of biomedical materials: appli-cations of cell sheet engineering to functional regenerative tissues andorgans. J Control Release 2005;101(1–3):69–84.

[3] Siebers MC, ter Brugge PJ, Walboomers XF, Jansen JA. Integrins as linkerproteins between osteoblasts and bone replacing materials. A critical review.Biomaterials 2005;26(2):137–46.

[4] Ratner BD, Bryant SJ. Biomaterials: where we have been and where we aregoing. Annu Rev Biomed Eng 2004;6:41–75.

[5] Cheng XH, Canavan HE, Stein MJ, Hull JR, Kweskin SJ, Wagner MS, et al.Surface chemical and mechanical properties of plasma-polymerized N-isopropylacrylamide. Langmuir 2005;21(17):7833–41.

[6] Allen LT, Tosetto M, Miller IS, O’Connor DP, Penney SC, Lynch I, et al.Surface-induced changes in protein adsorption and implications for

cellular phenotypic responses to surface interaction. Biomaterials2006;27(16):3096–108.

[7] Nath N, Hyun J, Ma H, Chilkoti A. Surface engineering strategies for control ofprotein and cell interactions. Surf Sci 2004;570(1–2):98–110.

[8] Keselowsky BG, Collard DM, Garcia AJ. Surface chemistry modulates fibro-nectin conformation and directs integrin binding and specificity to controlcell adhesion. J Biomed Mater Res A 2003;66A(2):247–59.

[9] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric controlof cell life and death. Science 1997;276(5317):1425–8.

[10] Kingshott P, Thissen H, Griesser HJ. Effects of cloud-point grafting, chainlength, and density of PEG layers on competitive adsorption of ocularproteins. Biomaterials 2002;23(9):2043–56.

[11] Kane RS, Deschatelets P, Whitesides GM. Kosmotropes form the basis ofprotein-resistant surfaces. Langmuir 2003;19(6):2388–91.

[12] Norde W, Gage D. Interaction of bovine serum albumin and human bloodplasma with PEO-tethered surfaces: influence of PEO chain length, graftingdensity, and temperature. Langmuir 2004;20(10):4162–7.

[13] Wang RLC, Kreuzer HJ, Grunze M. Molecular conformation and solvation ofoligo(ethylene glycol)-terminated self-assembled monolayers. J Phys Chem B1997;101(47):9767–73.

[14] Haimovich B, Difazio L, Katz D, Zhang L, Greco RS, Dror Y, et al. A newmethod for membrane construction on ePTFE vascular grafts: effect onsurface morphology and platelet adhesion. J Appl Polym Sci1997;63(11):1393–400.

[15] McMillan R, Meeks B, Bensebaa F, Deslandes Y, Sheardown H. Cell adhesionpeptide modification of gold-coated polyurethanes for vascular endothelialcell adhesion. J Biomed Mater Res 2001;54(2):272–83.

[16] Sung WJ, Bae YH. A glucose oxidase electrode based on polypyrrole withpolyanion/PEG/enzyme conjugate dopant. Biosens Bioelectron2003;18(10):1231–9.

[17] Fuentes M, Pessela BCC, Maquiese JV, Ortiz C, Segura RL, Palomo JM, et al.Reversible and strong immobilization of proteins by ionic exchange onsupports coated with sulfate-dextran. Biotechnol Prog 2004;20(4):1134–9.

[18] Kikuchi A, Okano T. Intelligent thermoresponsive polymeric stationary pha-ses for aqueous chromatography of biological compounds. Prog Polym Sci2002;27(6):1165–93.

[19] Lee JH, Jeong BJ, Lee HB. Plasma protein adsorption and platelet adhesiononto comb-like PEO gradient surfaces. J Biomed Mater Res1997;34(1):105–14.

[20] McArthur SL, McLean KM, Kingshott P, St John HAW, Chatelier RC,Griesser HJ. Effect of polysaccharide structure on protein adsorption. ColloidsSurf B Biointerfaces 2000;17(1):37–48.

[21] Wang J, Pan CJ, Huang N, Sun H, Yang P, Leng YX, et al. Surface charac-terization and blood compatibility of poly(ethylene terephthalate) modi-fied by plasma surface grafting. Surf Coating Technol 2005;196(1–3):307–11.

[22] Hoffman AS. Applications of thermally reversible polymers and hydrogels intherapeutics and diagnostics. J Control Release 1987;6:297–305.

[23] Hoffman AS, Stayton PS. Bioconjugates of smart polymers and proteins:synthesis and applications. Macromol Symp 2004;207:139–51.

[24] Kikuchi A, Okano T. Pulsatile drug release control using hydrogels. Adv DrugDeliv Rev 2002;54(1):53–77.

[25] Kost J, Langer R. Responsive polymeric delivery systems. Adv Drug Deliv Rev2001;46(1–3):125–48.

[26] Jagur-Grodzinski J. Polymers for tissue engineering, medical devices, andregenerative medicine. Concise general review of recent studies. Polym AdvTechnol 2006;17(6):395–418.

[27] Langer R, Peppas NA. Advances in biomaterials, drug delivery, and biona-notechnology. AICHE J 2003;49(12):2990–3006.

[28] Gupta P, Vermani K, Garg S. Hydrogels: from controlled release to pH-responsive drug delivery. Drug Discov Today 2002;7(10):569–79.

[29] Yasuda A, Kojima K, Tinsley KW, Yoshioka H, Mori Y, Vacanti CA. In vitroculture of chondrocytes in a novel thermoreversible gelation polymer scaf-fold containing growth factors. Tissue Eng 2006;12(5):1237–45.

[30] Kushida A, Yamato M, Konno C, Kikuchi A, Sakurai Y, Okano T. Decrease inculture temperature releases monolayer endothelial cell sheets together withdeposited fibronectin matrix from temperature-responsive culture surfaces. JBiomed Mater Res 1999;45(4):355–62.

[31] Akiyama Y, Kikuchi A, Yamato M, Okano T. Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control. Langmuir 2004;20(13):5506–11.

[32] Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, et al. Cell sheetengineering: recreating tissues without biodegradable scaffolds. Biomaterials2005;26(33):6415–22.

[33] Canavan HE, Cheng XH, Graham DJ, Ratner BD, Castner DG. Surface charac-terization of the extracellular matrix remaining after cell detachment froma thermoresponsive polymer. Langmuir 2005;21(5):1949–55.

[34] Da Silva RMP, Mano JF, Reis RL. Smart thermoresponsive coatings andsurfaces for tissue engineering: switching cell-material boundaries. TrendsBiotechnol 2007;25(12):577–83.

[35] Wang J, Jiang M, Mukherjee B. On-demand electrochemical release of DNAfrom gold surfaces. Bioelectrochemistry 2000;52(1):111–4.

[36] Edahiro J, Sumaru K, Tada Y, Ohi K, Takagi T, Kameda M, et al. In situ controlof cell adhesion using photoresponsive culture surface. Biomacromolecules2005;6(2):970–4.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–18501846

[37] Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y. Thermo-responsive polymeric surfaces; control of attachment and detachment ofcultured cells. Makromol Chem Rapid Commun 1990;11(11):571–6.

[38] Zhu X, De Graaf J, Winnik FM, Leckband D. Tuning the interfacial propertiesof grafted chains with a pH switch. Langmuir 2004;20(4):1459–65.

[39] Panayiotou M, Freitag R. Influence of the synthesis conditions and ionicadditives on the swelling behaviour of thermo-responsive poly-alkylacrylamide hydrogels. Polymer 2005;46(18):6777–85.

[40] Ainslie KM, Sharma G, Dyer MA, Grimes CA, Pishko MV. Attenuation ofprotein adsorption on static and oscillating magnetostrictive nanowires.Nano Lett 2005;5(9):1852–6.

[41] Hyun J, Lee WK, Nath N, Chilkoti A, Zauscher S. Capture and release ofproteins on the nanoscale by stimuli-responsive elastin-like polypeptide‘‘switches’’. J Am Chem Soc 2004;126(23):7330–5.

[42] Kwok CS, Mourad PD, Crum LA, Ratner BD. Self-assembled molecularstructures as ultrasonically-responsive barrier membranes for pulsatile drugdelivery. J Biomed Mater Res 2001;57(2):151–64.

[43] Lemieux M, Usov D, Minko S, Stamm M, Shulha H, Tsukruk VV. Reorgani-zation of binary polymer brushes: reversible switching of surfacemicrostructures and nanomechanical properties. Macromolecules2003;36(19):7244–55.

[44] Roy I, Gupta MN. Smart polymeric materials: emerging biochemical appli-cations. Chem Biol 2003;10(12):1161–71.

[45] Holtz JH, Asher SA. Polymerized colloidal crystal hydrogel films as intelligentchemical sensing materials. Nature 1997;389(6653):829–32.

[46] Miyata T, Asami N, Uragami T. A reversibly antigen-responsive hydrogel.Nature 1999;399(6738):766–9.

[47] Rawsterne RE, Gough JE, Rutten FJM, Pham NT, Poon WCK, Flitsch SL, et al.Controlling protein retention on enzyme-responsive surfaces. Surf InterfaceAnal 2006;38(11):1505–11.

[48] Ulijn RV, Bibi N, Jayawarna V, Thornton PD, Todd SJ, Mart RJ, et al. Bio-responsive hydrogels. Mater Today 2007;10(4):40–8.

[49] Megeed Z, Winters RM, Yarmush ML. Modulation of single-chain antibodyaffinity with temperature-responsive elastin-like polypeptide linkers. Bio-macromolecules 2006;7(4):999–1004.

[50] Crowe JA, Genzer J. Creating responsive surfaces with tailored wettabilityswitching kinetics and reconstruction reversibility. J Am Chem Soc2005;127(50):17610–1.

[51] Ebara M, Yamato M, Hirose M, Aoyagi T, Kikuchi A, Sakai K, et al. Copoly-merization of 2-carboxyisopropylacrylamide with N-isopropylacrylamideaccelerates cell detachment from grafted surfaces by reducing temperature.Biomacromolecules 2003;4(2):344–9.

[52] Eeckman F, Moes AJ, Amighi K. Synthesis and characterization of thermo-sensitive copolymers for oral controlled drug delivery. Eur Polym J2004;40(4):873–81.

[53] Heskins M, Guillet JE. Solution properties of poly(N-isopropylacrylamide). JMacromol Sci Chem 1968;2(8):1441–55.

[54] Plate NA, Lebedeva TL, Valuev LI. Lower critical solution temperature inaqueous solutions of N-alkyl-substituted polyacrylamides. Polym J1999;31(1):21–7.

[55] Ohya Y, Toyohara M, Sasakawa M, Arimura H, Ouchi T. Thermosensitivebiodegradable polydepsipeptide. Macromol Biosci 2005;5(4):273–6.

[56] Sugihara S, Kanaoka S, Aoshima S. Double thermosensitive diblock copoly-mers of vinyl ethers with pendant oxyethylene groups: unique physicalgelation. Macromolecules 2005;38(5):1919–27.

[57] Vihola H, Laukkanen A, Valtola L, Tenhu H, Hirvonen J. Cytotoxicity of ther-mosensitive polymers poly(N-isopropylacrylamide), poly(N-vinyl-caprolactam) and amphiphilically modified poly(N-vinylcaprolactam).Biomaterials 2005;26(16):3055–64.

[58] Yamamoto K, Serizawa T, Muraoka Y, Akashi M. Synthesis and functionalitiesof poly(N-vinylalkylamide). 13. Synthesis and properties of thermal and pHstimuli-responsive poly(vinylamine) copolymers. Macromolecules2001;34(23):8014–20.

[59] Park JS, Akiyama Y, Winnik FM, Kataoka K. Versatile synthesis of end-functionalized thermosensitive poly(2-isopropyl-2-oxazolines). Macromole-cules 2004;37(18):6786–92.

[60] Nath N, Chilkoti A. Interfacial phase transition of an environmentallyresponsive elastin biopolymer adsorbed on functionalized gold nanoparticlesstudied by colloidal surface plasmon resonance. J Am Chem Soc2001;123(34):8197–202.

[61] Chilkoti A, Dreher MR, Meyer DE, Raucher D. Targeted drug delivery bythermally responsive polymers. Adv Drug Deliv Rev 2002;54(5):613–30.

[62] Maskarinec SA, Tirrell DA. Protein engineering approaches to biomaterialsdesign. Curr Opin Biotechnol 2005;16(4):422–6.

[63] Mart RJ, Osborne RD, Stevens MM, Ulijn RV. Peptide-based stimuli-responsive biomaterials. Soft Matter 2006;2(10):822–35.

[64] Di Zio K, Tirrell DA. Mechanical properties of artificial protein matricesengineered for control of cell and tissue behavior. Macromolecules2003;36(5):1553–8.

[65] Meyer DE, Kong GA, Dewhirst MW, Zalutsky MR, Chilkoti A. Targetinga genetically engineered elastin-like polypeptide to solid tumors by localhyperthermia. Cancer Res 2001;61(4):1548–54.

[66] Meyer DE, Shin BC, Kong GA, Dewhirst MW, Chilkoti A. Drug targeting usingthermally responsive polymers and local hyperthermia. J Control Release2001;74(1–3):213–24.

[67] Tachibana Y, KurisawaM, Uyama H, Kobayashi S. Thermo- and pH-responsivebiodegradable poly(alpha-N-substituted gamma-glutamine)s. Bio-macromolecules 2003;4(5):1132–4.

[68] Yoshioka H, Mori Y, Tsukikawa S, Kubota S. Thermoreversible gelation oncooling and on heating of an aqueous gelatin-poly(N-isopropylacrylamide)conjugate. Polym Adv Technol 1998;9(2):155–8.

[69] Jeong B, Bae YH, Lee DS, Kim SW. Biodegradable block copolymers asinjectable drug-delivery systems. Nature 1997;388(6645):860–2.

[70] Chu LY, Li Y, Zhu JH, Chen WM. Negatively thermoresponsive membraneswith functional gates driven by zipper-type hydrogen-bonding interactions.Angew Chem Int Ed 2005;44(14):2124–7.

[71] Jeong B, Kim SW, Bae YH. Thermosensitive sol–gel reversible hydrogels. AdvDrug Deliv Rev 2002;54(1):37–51.

[72] Pratoomsoot C, Tanioka H, Hori K, Kawasaki S, Kinoshita S, Tighe PJ, et al. Athermoreversible hydrogel as a biosynthetic bandage for corneal woundrepair. Biomaterials 2008;29(3):272–81.

[73] Jeong B, Bae YH, Kim SW. Thermoreversible gelation of PEG–PLGA–PEG tri-block copolymer aqueous solutions. Macromolecules 1999;32(21):7064–9.

[74] Burillo G, Bucio E, Arenas E, Lopez GP. Temperature and pH-sensitive swellingbehavior of binary DMAEMA/4VP grafts on poly(propylene) films. MacromolMater Eng 2007;292(2):214–9.

[75] Bae SJ, Suh JM, Sohn YS, Bae YH, Kim SW, Jeong B. Thermogellingpoly(caprolactone-b-ethylene glycol-b-caprolactone) aqueous solutions.Macromolecules 2005;38(12):5260–5.

[76] Hatefi A, Amsden B. Biodegradable injectable in situ forming drug deliverysystems. J Control Release 2002;80(1–3):9–28.

[77] Murthy N, Campbell J, Fausto N, Hoffman AS, Stayton PS. Bioinspired pH-responsive polymers for the intracellular delivery of biomolecular drugs.Bioconjug Chem 2003;14(2):412–9.

[78] Kim MS, Kim SK, Kim SH, Hyun H, Khang G, Lee HB. In vivo osteogenicdifferentiation of rat bone marrow stromal cells in thermosensitive MPEG–PCL diblock copolymer gels. Tissue Eng 2006;12(10):2863–73.

[79] Kretlow JD, Klouda L, Mikos AG. Injectable matrices and scaffolds fordrug delivery in tissue engineering. Adv Drug Deliv Rev 2007;59(4–5):263–73.

[80] Langer R. Drug delivery and targeting. Nature 1998;392(6679):5–10.[81] Huber DL, Manginell RP, Samara MA, Kim BI, Bunker BC. Programmed

adsorption and release of proteins in a microfluidic device. Science2003;301(5631):352–4.

[82] Cheng XH, Wang YB, Hanein Y, Bohringer KF, Ratner BD. Novel cell patterningusing microheater-controlled thermoresponsive plasma films. J BiomedMater Res Part A 2004;70A(2):159–68.

[83] Cole MA, Jasieniak M, Voelcker NH, Thissen H, Horn R, Griesser HJ. Switch-able surface coatings for control over protein adsorption. Proc SPIE – Int SocOpt Eng 2007;6416:641606/1–641606/10 (Biomedical Applications of Micro-and Nanoengineering III).

[84] Cheng X, Canavan HE, Graham DJ, Castner DG, Ratner BD. Temperature-dependent activity and structure of adsorbed proteins on plasma polymer-ized N-isopropyl acrylamide. Biointerphases 2006;1(1):61–72.

[85] Cunliffe D, Alarcon CD, Peters V, Smith JR, Alexander C. Thermoresponsivesurface-grafted poly(N-isopropylacrylamide) copolymers: effect of phasetransitions on protein and bacterial attachment. Langmuir2003;19(7):2888–99.

[86] Kurkuri MD, Nussio MR, Deslandes A, Voelcker NH. Thermosensitive copoly-mer coatings with enhanced wettability switching. Langmuir2008;24(8):4238–44.

[87] Pasche S, Textor M, Meagher L, Spencer ND, Griesser HJ. Relationshipbetween interfacial forces measured by colloid-probe atomic force micros-copy and protein resistance of poly(ethylene glycol)-grafted poly(L-lysine)adlayers on niobia surfaces. Langmuir 2005;21(14):6508–20.

[88] Pasche S, Voros J, Griesser HJ, Spencer ND, Textor M. Effects of ionic strengthand surface charge on protein adsorption at PEGylated surfaces. J Phys ChemB 2005;109(37):17545–52.

[89] Cole MA, Thissen H, Losic D, Voelcker NH. A new approach to the immobi-lisation of poly(ethylene oxide) for the reduction of non-specific proteinadsorption on conductive substrates. Surf Sci 2007;601(7):1716–25.

[90] Jones DM, Smith JR, Huck WTS, Alexander C. Variable adhesion of micro-patterned thermoresponsive polymer brushes: AFM investigations of poly(N-isopropylacrylamide) brushes prepared by surface-initiated polymeriza-tions. Adv Mater 2002;14(16):1130–4.

[91] Theato P, Zentel R. Alpha, omega-functionalized poly-N-isopropylacrylamides: controlling the surface activity for vesicle adsorptionby temperature. J Colloid Interface Sci 2003;268(1):258–62.

[92] Xu FJ, Zhong SP, Yung LYL, Tong YW, Kang ET, Neoh KG. Thermoresponsivecomb-shaped copolymer-Si(100) hybrids for accelerated temperature-dependent cell detachment. Biomaterials 2006;27(8):1236–45.

[93] Bullett NA, Talib RA, Short RD, McArthur SL, Shard AG. Chemical and thermo-responsive characterisation of surfaces formed by plasma polymerisation ofN-isopropyl acrylamide. Surf Interface Anal 2006;38(7):1109–16.

[94] Schmaljohann D, Oswald J, Jorgensen B, Nitschke M, Beyerlein D, Werner C.Thermo-responsive PNiPAAm-g-PEG films for controlled cell detachment.Biomacromolecules 2003;4(6):1733–9.

[95] Takeuchi S, Omodaka I, Hasegawa K, Maeda Y, Kitano H. Temperature-responsive graft-copolymers for immobilization of enzymes. MakromolChem 1993;194(7):1991–9.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–1850 1847

[96] Chen GH, Hoffman AS. Graft-copolymers that exhibit temperature-inducedphase-transitions over a wide-range of pH. Nature 1995;373(6509):49–52.

[97] Tsuda Y, Kikuchi A, Yamato M, Nakao A, Sakurai Y, Umezu M, et al. The use ofpatterned dual thermoresponsive surfaces for the collective recovery asco-cultured cell sheets. Biomaterials 2005;26(14):1885–93.

[98] Aoyagi T, Ebara M, Sakai K, Sakurai Y, Okano T. Novel bifunctional polymerwith reactivity and temperature sensitivity. J Biomater Sci Polym Ed2000;11(1):101–10.

[99] Hatakeyama H, Kikuchi A, Yamato M, Okano T. Bio-functionalized thermor-esponsive interfaces facilitating cell adhesion and proliferation. Biomaterials2006;27(29):5069–78.

[100] Pan YV, Wesley RA, Luginbuhl R, Denton DD, Ratner BD. Plasma polymerizedN-isopropylacrylamide: synthesis and characterization of a smart thermallyresponsive coating. Biomacromolecules 2001;2(1):32–6.

[101] Canavan HE, Cheng XH, Graham DJ, Ratner BD, Castner DG. A plasma-deposited surface for cell sheet engineering: advantages over mechanicaldissociation of cells. Plasma Process Polym 2006;3(6–7):516–23.

[102] Wuang SC, Neoh KG, Kang ET, Pack DW, Leckband DE. Heparinized magneticnanoparticles: in-vitro assessment for biomedical applications. Adv FunctMater 2006;16(13):1723–30.

[103] Takezawa T, Mori Y, Yoshizato K. Cell culture on a thermo-responsive poly-mer surface. Biotechnology 1990;8(9):854–6.

[104] Shimizu T, Yamato M, Kikuchi A, Okano T. Cell sheet engineering formyocardial tissue reconstruction. Biomaterials 2003;24(13):2309–16.

[105] Ebara M, Aoyagi T, Sakai K, Okano T. Introducing reactive carboxyl side chainsretains phase transition temperature sensitivity in N-isopropylacrylamidecopolymer gels. Macromolecules 2000;33(22):8312–6.

[106] Yamato M, Konno C, Utsumi M, Kikuchi A, Okano T. Thermally responsivepolymer-grafted surfaces facilitate patterned cell seeding and co-culture.Biomaterials 2002;23(2):561–7.

[107] Okano T, Yamada N, Okuhara M, Sakai H, Sakurai Y. Mechanism of celldetachment from temperature-modulated, hydrophilic–hydrophobic poly-mer surfaces. Biomaterials 1995;16(4):297–303.

[108] Yamato M, Okuhara M, Karikusa F, Kikuchi A, Sakurai Y, Okano T. Signaltransduction and cytoskeletal reorganization are required for cell detach-ment from cell culture surfaces grafted with a temperature-responsivepolymer. J Biomed Mater Res 1999;44(1):44–52.

[109] Balamurugan S, Mendez S, Balamurugan SS, O’Brien MJ, Lopez GP. Thermalresponse of poly(N-isopropylacrylamide) brushes probed by surface plasmonresonance. Langmuir 2003;19(7):2545–9.

[110] Griesser HJ, Chatelier RC, Gengenbach TR, Johnson G, Steele JG. Growth ofhuman-cells on plasma polymers – putative role of amine and amide groups.J Biomater Sci Polym Ed 1994;5(6):531–54.

[111] Kingshott P, Griesser HJ. Surfaces that resist bioadhesion. Curr Opin SolidState Mater Sci 1999;4(4):403–12.

[112] Mizutani A, Kikuchi A, Yamato M, Kanazawa H, Okano T. Preparation ofthermoresponsive polymer brush surfaces and their interaction with cells.Biomaterials 2008;29(13):2073–81.

[113] Canavan HE, Cheng XH, Graham DJ, Ratner BD, Castner DG. Cell sheetdetachment affects the extracellular matrix: A surface science studycomparing thermal liftoff, enzymatic, and mechanical methods. J BiomedMater Res Part A 2005;75A(1):1–13.

[114] Kushida A, Yamato M, Isoi Y, Kikuchi A, Okano T. A noninvasive transfersystem for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes. Eur Cell Mater 2005;10:23–30.

[115] Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, et al.Corneal reconstruction with tissue-engineered cell sheets composed ofautologous oral mucosal epithelium. N Engl J Med 2004;351(12):1187–96.

[116] Hutmacher DW. A commentary on ‘‘Thermo-responsive polymeric surfaces;control of attachment and detachment of cultured cells’’ by N. Yamada, T.Okano, H. Sakai, F. Karikusa, Y. Sawasaki, Y. Sakurai (Makromol Chem RapidCommun, 1990, 11, 571–576). Macromol Rapid Commun 2005;26(7):505–13.

[117] Ide T, Nishida K, Yamato M, Sumide T, Utsumi M, Nozaki T, et al. Structuralcharacterization of bioengineered human corneal endothelial cell sheetsfabricated on temperature-responsive culture dishes. Biomaterials2006;27(4):607–14.

[118] Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al.Monolayered mesenchymal stem cells repair scarred myocardium aftermyocardial infarction. Nat Med 2006;12(4):459–65.

[119] Isenberg BC, Tsuda Y, Williams C, Shimizu T, Yamato M, Okano T, et al. Athermoresponsive, microtextured substrate for cell sheet engineering withdefined structural organization. Biomaterials 2008;29(17):2565–72.

[120] Ebara M, Yamato M, Aoyagi T, Akihiko K, Sakai K, Okano T. A novel approachto observing synergy effects of PHSRN on integrin-RGD binding usingintelligent surfaces. Adv Mater 2008;20(16):3034–8.

[121] Tanaka Y, Sato K, Shimizu T, Yamato M, Okano T, Kitamori T. A micro-spherical heart pump powered by cultured cardiomyocytes. Lab Chip2007;7(2):207–12.

[122] Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, et al. Fabrication ofpulsatile cardiac tissue grafts using a novel 3-dimensional cell sheetmanipulation technique and temperature-responsive cell culture surfaces.Circulation Research 2002;90(3):e40.

[123] Matsuda T. Poly(N-isopropylacrylamide)-grafted gelatin as a thermores-ponsive cell-adhesive, mold-releasable material for shape-engineeredtissues. J Biomater Sci Polym Ed 2004;15(7):947–55.

[124] Xu FJ, Zhong SP, Yung LYL, Kang ET, Neoh KG. Surface-active and stimuli-responsive polymer-Si(100) hybrids from surface-initiated atom transferradical polymerization for control of cell adhesion. Biomacromolecules2004;5(6):2392–403.

[125] Kim MR, Jeong JH, Park TG. Swelling induced detachment of chondrocytesusing RGD-modified poly(N-isopropylacrylamide) hydrogel beads. Bio-technol Prog 2002;18(3):495–500.

[126] Okamura A, Itayagoshi M, Hagiwara T, Yamaguchi M, Kanamori T, Shinbo T,et al. Poly(N-isopropylacrylamide)-graft-polypropylene membranes con-taining adsorbed antibody for cell separation. Biomaterials2005;26(11):1287–92.

[127] Timmer MD, Shin H, Horch RA, Ambrose CG, Mikos AG. In vitro cytotoxicityof injectable and biodegradable poly(propylene fumarate)-based networks:unreacted macromers, cross-linked networks, and degradation products.Biomacromolecules 2003;4(4):1026–33.

[128] Bisht HS, Manickam DS, You YZ, Oupicky D. Temperature-controlled prop-erties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide). Biomacromolecules 2006;7(4):1169–78.

[129] Kurisawa M, Yokoyama M, Okano T. Gene expression control by temperaturewith thermo-responsive polymeric gene carriers. J Control Release2000;69(1):127–37.

[130] Chen JH, Yoshida M, Maekawa Y, Tsubokawa N. Temperature-switchablevapor sensor materials based on N-isopropylacrylamide and calcium chlo-ride. Polymer 2001;42(23):9361–5.

[131] Zintchenko A, Ogris M, Wagner E. Temperature dependent gene expressioninduced by PNIPAM-based copolymers: potential of hyperthermia in genetransfer. Bioconjug Chem 2006;17(3):766–72.

[132] Tsai WB, Grunkemeier JM, Horbett TA. Human plasma fibrinogen adsorptionand platelet adhesion to polystyrene. J Biomed Mater Res 1999;44(2):130–9.

[133] Cho EC, Kim YD, Cho K. Thermally responsive poly (N-isopropylacrylamide)monolayer on gold: synthesis, surface characterization, and protein interac-tion/adsorption studies. Polymer 2004;45(10):3195–204.

[134] Cho EC, Kim YD, Cho K. Temperature-dependent intermolecular forcemeasurement of poly(N-isopropylacrylamide) grafted surface with protein. JColloid Interface Sci 2005;286(2):479–86.

[135] Kasemo B. Biological surface science. Surf Sci 2002;500(1–3):656–77.[136] Wong JY, Leach JB, Brown XQ. Balance of chemistry, topography, and

mechanics at the cell–biomaterial interface: issues and challenges forassessing the role of substrate mechanics on cell response. Surf Sci2004;570(1–2):119–33.

[137] Plunkett KN, Zhu X, Moore JS, Leckband DE. PNIPAM chain collapse dependson the molecular weight and grafting density. Langmuir2006;22(9):4259–66.

[138] Duracher D, Veyret R, Elaissari A, Pichot C. Adsorption of bovine serumalbumin protein onto amino-containing thermosensitive core-shell latexes.Polym Int 2004;53(5):618–26.

[139] Balamurugan S, Ista LK, Yan J, Lopez GP, Fick J, Himmelhaus M, et al.Reversible protein adsorption and bioadhesion on monolayers terminatedwith mixtures of oligo(ethylene glycol) and methyl groups. J Am Chem Soc2005;127(42):14548–9.

[140] Ionov L, Synytska A, Diez S. Temperature-induced size-control of bioactivesurface patterns. Adv Funct Mater 2008;18(10):1501–8.

[141] Ebara M, Yamato M, Aoyagi T, Kikuchi A, Sakai K, Okano T. The effect ofextensible PEG tethers on shielding between grafted thermo-responsivepolymer chains and integrin-RGD binding. Biomaterials 2008;29(27):3650–5.

[142] Yamato M, Konno C, Kushida A, Hirose M, Utsumi M, Kikuchi A, et al. Releaseof adsorbed fibronectin from temperature-responsive culture surfacesrequires cellular activity. Biomaterials 2000;21(10):981–6.

[143] Canavan HE, Graham DJ, Cheng XH, Ratner BD, Castner DG. Comparison ofnative extracellular matrix with adsorbed protein films using secondary ionmass spectrometry. Langmuir 2007;23(1):50–6.

[144] Wagner MS, McArthur SL, Shen MC, Horbett TA, Castner DG. Limits ofdetection for time of flight secondary ion mass spectrometry (ToF-SIMS) andX-ray photoelectron spectroscopy (XPS): detection of low amountsof adsorbed protein. J Biomater Sci Polym Ed 2002;13(4):407–28.

[145] Tidwell CD, Castner DG, Golledge SL, Ratner BD, Meyer K, Hagenhoff B, et al.Static time-of-flight secondary ion mass spectrometry and X-ray photoelec-tron spectroscopy characterization of adsorbed albumin and fibronectinfilms. Surf Interface Anal 2001;31(8):724–33.

[146] Galaev IY, Warrol C, Mattiasson B. Temperature-induced displacement ofproteins from dye-affinity columns using an immobilized polymeric dis-placer. J Chromatogr A 1994;684(1):37–43.

[147] Yoshizako K, Akiyama Y, Yamanaka H, Shinohara Y, Hasegawa Y, Carredano E,et al. Regulation of protein binding toward a ligand on chromatographicmatrixes by masking and forced-releasing effects using thermoresponsivepolymer. Anal Chem 2002;74(16):4160–6.

[148] Ding ZL, Fong RB, Long CJ, Stayton PS, Hoffman AS. Size-dependent control ofthe binding of biotinylated proteins to streptavidin using a polymer shield.Nature 2001;411(6833):59–62.

[149] Malmstadt N, Yager P, Hoffman AS, Stayton PS. A smart microfluidic affinitychromatography matrix composed of poly(N-isopropylacrylamide)-coatedbeads. Anal Chem 2003;75(13):2943–9.

[150] Ding XB, Sun ZH, Zhang WC, Peng YX, Wan GX, Jiang YY. Adsorption/desorption of protein on magnetic particles covered by thermosensitivepolymers. J Appl Polym Sci 2000;77(13):2915–20.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–18501848

[151] Taniguchi T, Duracher D, Delair T, Elaissari A, Pichot C. Adsorption/desorption behavior and covalent grafting of an antibody onto cationicamino-functionalized poly(styrene-N-isopropylacrylamide) core-shell latexparticles. Colloids Surf B Biointerfaces 2003;29(1):53–65.

[152] Schmidt AM. Thermoresponsive magnetic colloids. Colloid Polym Sci2007;285(9):953–66.

[153] Shamim N, Hong L, Hidajat K, Uddin MS. Thermosensitive-polymer-coatedmagnetic nanoparticles: adsorption and desorption of bovine serumalbumin. J Colloid Interface Sci 2006;304(1):1–8.

[154] Shamim N, Hong L, Hidajat K, Uddin MS. Thermosensitive polymer coatednanomagnetic particles for separation of bio-molecules. Sep Purif Technol2007;53(2):164–70.

[155] Ding ZL, Long CJ, Hayashi Y, Bulmus EV, Hoffman AS, Stayton PS. Temperaturecontrol of biotin binding and release with a streptavidin-poly(N-isopropylacrylamide) site-specific conjugate. Bioconjug Chem1999;10(3):395–400.

[156] Elaissari A, Bourrel V. Thermosensitive magnetic latex particles for control-ling protein adsorption and desorption. J Magn Magn Mater2001;225(1–2):151–5.

[157] Ulbricht M, Yang H. Porous polypropylene membranes with differentcarboxyl polymer brush layers for reversible protein binding via surface-initiated graft copolymerization. Chem Mater 2005;17(10):2622–31.

[158] Liu GM, Yan LF, Chen X, Zhang GZ. Study of the kinetics of mushroom-to-brush transition of charged polymer chains. Polymer 2006;47(9):3157–63.

[159] Jhon YK, Bhat RR, Jeong C, Rojas OJ, Szleifer I, Genzer J. Salt-induceddepression of lower critical solution temperature in a surface-graftedneutral thermoresponsive polymer. Macromol Rapid Commun2006;27(9):697–701.

[160] Xia F, Feng L, Wang ST, Sun TL, Song WL, Jiang WH, et al. Dual-responsivesurfaces that switch superhydrophilicity and superhydrophobicity. AdvMater 2006;18(4):432–6.

[161] Schmitt FJ, Park C, Simon J, Ringsdorf H, Israelachvili J. Direct surface forceand contact angle measurements of an adsorbed polymer with a lowercritical solution temperature. Langmuir 1998;14(10):2838–45.

[162] Zhu X, Yan C, Winnik FM, Leckband D. End-grafted low-molecular-weight PNIPAM does not collapse above the LCST. Langmuir2007;23(1):162–9.

[163] Xia Y, Burke NAD, Stover HDH. End group effect on the thermal response ofnarrow-disperse poly(N-isopropylacrylamide) prepared by atom transferradical polymerization. Macromolecules 2006;39(6):2275–83.

[164] Xia Y, Yin XC, Burke NAD, Stover HDH. Thermal response of narrow-dispersepoly(N-isopropylacrylamide) prepared by atom transfer radical polymeriza-tion. Macromolecules 2005;38(14):5937–43.

[165] Kidoaki S, Ohya S, Nakayama Y, Matsuda T. Thermoresponsive structuralchange of a poly(N-isopropylacrylamide) graft layer measured with anatomic force microscope. Langmuir 2001;17(8):2402–7.

[166] He Q, Kuller A, Grunze M, Li JB. Fabrication of thermosensitive polymernanopatterns through chemical lithography and atom transfer radical poly-merization. Langmuir 2007;23(7):3981–7.

[167] Ishida N, Kobayashi M. Interaction forces measured between poly(N-iso-propylacrylamide) grafted surface and hydrophobic particle. J Colloid Inter-face Sci 2006;297(2):513–9.

[168] Liu GM, Zhang GZ. Collapse and swelling of thermally sensitive poly(N-isopropylacrylamide) brushes monitored with a quartz crystal microbalance.J Phys Chem B 2005;109(2):743–7.

[169] Zhang GZ. Study on conformation change of thermally sensitive lineargrafted poly(N-isopropylacrylamide) chains by quartz crystal microbalance.Macromolecules 2004;37(17):6553–7.

[170] Ishida N, Biggs S. Direct observation of the phase transition for a poly(N-isopropylacrylamide) layer grafted onto a solid surface by AFM and QCM-D.Langmuir 2007;23(22):11083–8.

[171] Annaka M, Yahiro C, Nagase K, Kikuchi A, Okano T. Real-time observation ofcoil-to-globule transition in thermosensitive poly(N-isopropylacrylamide)brushes by quartz crystal microbalance. Polymer 2007;48(19):5713–20.

[172] Afroze F, Nies E, Berghmans H. Phase transitions in the system poly(N-isopropylacrylamide)/water and swelling behaviour of the correspondingnetworks. J Mol Struct 2000;554(1):55–68.

[173] Fujishige S, Kubota K, Ando I. Phase-transition of aqueous-solutions ofpoly(N-isopropylacrylamide) and poly(N-isopropylmethacrylamide). J PhysChem 1989;93(8):3311–3.

[174] Schild HG. Poly (N-isopropylacrylamide) – experiment, theory and applica-tion. Prog Polym Sci 1992;17(2):163–249.

[175] Yim H, Kent MS, Mendez S, Lopez GP, Satija S, Seo Y. Effects of graftingdensity and molecular weight on the temperature-dependent conforma-tional change of poly(N-isopropylacrylamide) grafted chains in water.Macromolecules 2006;39(9):3420–6.

[176] Marchetti M, Prager S, Cussler EL. Thermodynamic predictions of volumechanges in temperature-sensitive gels .1. Theory. Macromolecules1990;23(6):1760–5.

[177] Tong Z, Zeng F, Zheng X, Sato T. Inverse molecular weight dependence ofcloud points for aqueous poly(N-isopropylacrylamide) solutions. Macromol-ecules 1999;32(13):4488–90.

[178] Gilcreest VP, Carroll WM, Rochev YA, Blute I, Dawson KA, Gorelov AV. Ther-moresponsive poly(N-isopropylacrylamide) copolymers: contact angles andsurface energies of polymer films. Langmuir 2004;20(23):10138–45.

[179] Yakushiji T, Sakai K, Kikuchi A, Aoyagi T, Sakurai Y, Okano T. Graft architec-tural effects on thermoresponsive wettability changes of poly(N-isopropylacrylamide)-modified surfaces. Langmuir 1998;14(16):4657–62.

[180] Graziano G, Catanzano F, Barone G. On the nature of the temperature-induced transition from the molten globule to the unfolded state of globularproteins. J Therm Anal Calorim 1999;57(1):329–41.

[181] Schmidt S, Motschmann H, Hellweg T, von Klitzing R. Thermoresponsivesurfaces by spin-coating of PNIPAM-co-PAA microgels: a combined AFM andellipsometry study. Polymer 2008;49(3):749–56.

[182] Badiger MV, Lele AK, Bhalerao VS, Varghese S, Mashelkar RA. Moleculartailoring of thermoreversible copolymer gels: some new mechanisticinsights. J Chem Phys 1998;109(3):1175–84.

[183] Deshmukh MV, Vaidya AA, Kulkarni MG, Rajamohanan PR, Ganapathy S. LCSTin poly(N-isopropylacrylamide) copolymers: high resolution proton NMRinvestigations. Polymer 2000;41(22):7951–60.

[184] Keerl M, Smirnovas V, Winter R, Richtering W. Interplay between hydrogenbonding and macromolecular architecture leading to unusual phase behaviorin thermosensitive microgels. Angew Chem Int Ed 2008;47:338–41.

[185] Lin SY, Chen KS, Liang RC. Thermal microATR/FT-IR spectroscopic system forquantitative study of the molecular structure of poly(N-isopropylacrylamide)in water. Polymer 1999;40(10):2619–24.

[186] Tanaka N, Matsukawa S, Kurosu H, Ando I. A study on dynamics of water incrosslinked poly(N-isopropylacrylamide) gel by NMR spectroscopy. Polymer1998;39(20):4703–6.

[187] Andersson M, Maunu SL. Volume phase transition and structure of poly(N-isopropylacrylamide) microgels studied with H-1-NMR spectroscopy in D2O.Colloid Polym Sci 2006;285(3):293–303.

[188] Diez-Pena E, Quijada-Garrido I, Barrales-Rienda JM, Schnell I, Spiess HW.Advanced H-1 solid-state NMR spectroscopy on hydrogels, 2dThe formationof hydrogen bonds in hydrogels based on N-isopropylacrylamide (NiPAAm)and methacrylic acid (MAA). Macromol Chem Phys 2004;205(4):438–47.

[189] Hu TJ, Gao J, Wu C. Swelling and shrinking of poly(N-isopropylacrylamide)chains adsorbed on the surface of polystyrene nanoparticles. J Macromol SciPhys 2000;B39(3):407–14.

[190] Takei YG, Aoki T, Sanui K, Ogata N, Sakurai Y, Okano T. Dynamic contact-anglemeasurement of temperature-responsive surface-properties for poly(N-isopropylacrylamide) grafted surfaces. Macromolecules 1994;27(21):6163–6.

[191] Tu H, Heitzman CE, Braun PV. Patterned poly(N-isopropylacrylamide)brushes on silica surfaces by microcontact printing followed by surface-initiated polymerization. Langmuir 2004;20(19):8313–20.

[192] Baysal BM, Karasz FE. Coil-globule collapse in flexible macromolecules.Macromol Theory Simul 2003;12(9):627–46.

[193] Steels BM, Koska J, Haynes CA. Analysis of brush-particle interactions usingself-consistent-field theory. J Chromatogr B 2000;743(1–2):41–56.

[194] Svensson M, Alexandridis P, Linse P. Phase behavior and microstructure inbinary block copolymer/selective solvent systems: experiments and theory.Macromolecules 1999;32(3):637–45.

[195] Mendez S, Curro JG, McCoy JD, Lopez GP. Computational modeling of thetemperature-induced structural changes of tethered poly(N-isopropylacrylamide) with self-consistent field theory. Macromolecules2005;38(1):174–81.

[196] Binder K, Baschnagel J, Muller M, Paul W, Rampf F. Simulation of phasetransitions of single polymer chains: recent advances. Macromol Symp2006;237:128–38.

[197] Baulin VA, Halperin A. Signatures of a concentration-dependent Flory chiparameter: swelling and collapse of coils and brushes. Macromol TheorySimul 2003;12(8):549–59.

[198] Yim H, Kent MS, Satija S, Mendez S, Balamurugan SS, Balamurugan S, et al.Study of the conformational change of poly(N-isopropylacrylamide)-graftedchains in water with neutron reflection: molecular weight dependence athigh grafting density. J Polym Sci B Polym Phys 2004;42(17):3302–10.

[199] Israelachvili JN. Intermolecular & surface forces. 2nd ed. San Diego: AcademicPress; 1992.

[200] Beines PW, Klosterkamp I, Menges B, Jonas U, Knoll W. Responsive thinhydrogel layers from photo-cross-linkable poly(N-isopropylacrylamide)terpolymers. Langmuir 2007;23(4):2231–8.

[201] Ju XJ, Chu LY, Zhu XL, Hu L, Song H, Chen WM. Effects of internal micro-structures of poly(N-isopropylacrylamide) hydrogels on thermo-responsivevolume phase-transition and controlled-release characteristics. Smart MaterStruct 2006;15(6):1767–74.

[202] Xue W, Hamley IW, Huglin MB. Rapid swelling and deswelling of thermor-eversible hydrophobically modified poly(N-isopropylacrylamide) hydrogelsprepared by freezing polymerisation. Polymer 2002;43(19):5181–6.

[203] Sun TL, Wang GJ, Feng L, Liu BQ, Ma YM, Jiang L, et al. Reversible switchingbetween superhydrophilicity and superhydrophobicity. Angew Chem Int Ed2004;43(3):357–60.

[204] Yuk SH, Cho SH, Lee SH. pH/temperature-responsive polymer composed ofpoly((N,N-dimethylamino)ethyl methacrylate-co-ethylacrylamide). Macro-molecules 1997;30(22):6856–9.

[205] Markland P, Zhang Y, Amidon GL, Yang VC. A pH- and ionic strength-responsive polypeptide hydrogel: synthesis, characterization, andpreliminary protein release studies. J Biomed Mater Res 1999;47(4):595–602.

[206] Schwarte LM, Peppas NA. Novel poly(ethylene glycol)-grafted, cationichydrogels: preparation, characterization and diffusive properties. Polymer1998;39(24):6057–66.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–1850 1849

[207] Liu YY, Shao YH, Lu J. Preparation, properties and controlled release behav-iors of pH-induced thermosensitive amphiphilic gels. Biomaterials2006;27(21):4016–24.

[208] Kwon IC, Bae YH, Kim SW. Electrically erodible polymer gel for controlledrelease of drugs. Nature 1991;354(6351):291–3.

[209] Park TG, Hoffman AS. Sodium chloride-induced phase-transition in nonionicpoly(N-isopropylacrylamide) gel. Macromolecules 1993;26(19):5045–8.

[210] Routh AF, Vincent B. Salt-induced homoaggregation of poly(N-isopropylacrylamide) microgels. Langmuir 2002;18(14):5366–9.

[211] Valiaev A, Abu-Lail NI, Lim DW, Chilkoti A, Zauscher S. Microcantileversensing and actuation with end-grafted stimulus-responsive elastin-likepolypeptides. Langmuir 2007;23(1):339–44.

[212] Ayres N, Cyrus CD, Brittain WJ. Stimuli-responsive surfaces usingpolyampholyte polymer brushes prepared via atom transfer radical poly-merization. Langmuir 2007;23(7):3744–9.

[213] Uhlmann P, Houbenov N, Brenner N, Grundke K, Burkert S, Stamm M. In-situinvestigation of the adsorption of globular model proteins on stimuli-responsive binary polyelectrolyte brushes. Langmuir 2007;23(1):57–64.

[214] Synytska A, Stamm M, Diez S, Ionov L. Simple and fast method for thefabrication of switchable bicomponent micropatterned polymer surfaces.Langmuir 2007;23(9):5205–9.

[215] Schoch RB, Bertsch A, Renaud P. pH-Controlled diffusion of proteins withdifferent pl values across a nanochannel on a chip. Nano Lett 2006;6(3):543–7.

[216] Salim M, O’Sullivan B, McArthur SL, Wright PC. Characterization of fibrinogenadsorptionontoglassmicrocapillarysurfacesbyELISA.LabChip2007;7(1):64–70.

[217] Fu JP, Schoch RB, Stevens AL, Tannenbaum SR, Han JY. A patterned anisotropicnanofluidic sieving structure for continuous-flow separation of DNA andproteins. Nat Nanotechnol 2007;2(2):121–8.

[218] Li C, Yang YN, Craighead HG, Lee KH. Isoelectric focusing in cyclic olefincopolymer microfluidic channels coated by polyacrylamide using a UVphotografting method. Electrophoresis 2005;26(9):1800–6.

[219] Ferapontova E, Dominguez E. Adsorption of differently charged forms ofhorseradish peroxidase on metal electrodes of different nature: effect ofsurface charges. Bioelectrochemistry 2002;55(1–2):127–30.

[220] Hook AL, Thissen H, Hayes JP, Voelcker NH. Spatially controlled electro-stimulated DNA adsorption and desorption for biochip applications. BiosensBioelectron 2006;21(11):2137–45.

[221] Cole MA, Voelcker NH, Thissen H. Electro-induced protein deposition on low-fouling surfaces. Smart Mater Struct 2007;16(6):2222–8.

[222] Lahann J, Mitragotri S, Tran TN, Kaido H, Sundaram J, Choi IS, et al. Areversibly switching surface. Science 2003;299(5605):371–4.

[223] Wang J, Rivas G, Jiang MA, Zhang XJ. Electrochemically induced release ofDNA from gold ultramicroelectrodes. Langmuir 1999;15(19):6541–5.

[224] Jiang M, Ray WW, Mukherjee B, Wang J. Electrochemically controlled releaseof lipid/DNA complexes: a new tool for synthetic gene delivery system.Electrochem Commun 2004;6(6):576–82.

[225] Hook AL, Thissen H, Voelcker NH. Surface manipulation of biomolecules forcell microarray applications. Trends Biotechnol 2006;24(10):471–7.

[226] Fang F, Szleifer I. Controlled release of proteins from polymer-modifiedsurfaces. Proc Natl Acad Sci U S A 2006;103(15):5769–74.

[227] Kotwal A, Schmidt CE. Electrical stimulation alters protein adsorption andnerve cell interactions with electrically conducting biomaterials. Biomate-rials 2001;22(10):1055–64.

[228] Yeo WS, Yousaf MN, Mrksich M. Dynamic interfaces between cells andsurfaces: electroactive substrates that sequentially release and attach cells. JAm Chem Soc 2003;125(49):14994–5.

[229] Jiang XY, Ferrigno R, Mrksich M, Whitesides GM. Electrochemicaldesorption of self-assembled monolayers noninvasively releases patterned

cells from geometrical confinements. J Am Chem Soc2003;125(9):2366–7.

[230] Feringa BL. Molecular switches. Groningen: Wiley-VCH; 2001.[231] DillmoreWS, Yousaf MN, Mrksich M. A photochemical method for patterning

the immobilization of ligands and cells to self-assembled monolayers.Langmuir 2004;20(17):7223–31.

[232] Luo Y, Shoichet MS. A photolabile hydrogel for guided three-dimensional cellgrowth and migration. Nat Mater 2004;3(4):249–53.

[233] Nakanishi J, Kikuchi Y, Takarada T, Nakayama H, Yamaguchi K, Maeda M.Photoactivation of a substrate for cell adhesion under standard fluorescencemicroscopes. J Am Chem Soc 2004;126(50):16314–5.

[234] Rosario R, Gust D, Hayes M, Jahnke F, Springer J, Garcia AA. Photon-modulated wettability changes on spiropyran-coated surfaces. Langmuir2002;18(21):8062–9.

[235] Auernheimer J, Dahmen C, Hersel U, Bausch A, Kessler H. Photoswitchedcell adhesion on surfaces with RGD peptides. J Am Chem Soc2005;127(46):16107–10.

[236] Higuchi A, Hamamura A, Shindo Y, Kitamura H, Yoon BO, Mori T, et al.Photon-modulated changes of cell attachments on poly(spiropyran-co-methyl methacrylate) membranes. Biomacromolecules 2004;5(5):1770–4.

[237] Ivanov AE, Eremeev NL, Wahlund PO, Galaev IY, Mattiasson B. Photosensitivecopolymer of N-isopropylacrylamide and methacryloyl derivative of spyr-obenzopyran. Polymer 2002;43(13):3819–23.

[238] Sumaru K, Kameda M, Kanamori T, Shinbo T. Characteristic phase tran-sition of aqueous solution of poly(N-isopropylacrylamide) functionalizedwith spirobenzopyran. Macromolecules 2004;37(13):4949–55.

[239] Sumaru K, Ohi K, Takagi T, Kanamori T, Shinbo T. Photoresponsive propertiesof poly(N-isopropylacrylamide) hydrogel partly modified withspirobenzopyran. Langmuir 2006;22(9):4353–6.

[240] Das M, Sanson N, Fava D, Kumacheva E. Microgels loaded with gold nano-rods: photothermally triggered volume transitions under physiologicalconditions. Langmuir 2007;23(1):196–201.

[241] Shimoboji T, Larenas E, Fowler T, Kulkarni S, Hoffman AS, Stayton PS.Photoresponsive polymer-enzyme switches. Proc Natl Acad Sci U S A2002;99(26):16592–6.

[242] Okajima S, Sakai Y, Yamaguchi T. Development of a regenerable cell culturesystem that senses and releases dead cells. Langmuir 2005;21(9):4043–9.

[243] Schwartz MP, Derfus AM, Alvarez SD, Bhatia SN, Sailor MJ. The smart petridish: a nanostructured photonic crystal for real-time monitoring of livingcells. Langmuir 2006;22(16):7084–90.

[244] Garcia A, Marquez M, Cai T, Rosario R, Hu ZB, Gust D, et al. Photo-, thermally,and pH-responsive microgels. Langmuir 2007;23(1):224–9.

[245] Desponds A, Freitag R. Synthesis and characterization of photoresponsive N-isopropylacrylamide cotelomers. Langmuir 2003;19(15):6261–70.

[246] Sakamoto C, Okada Y, Kanazawa H, Ayano E, Nishimura T, Ando M, et al.Temperature- and pH-responsive aminopropyl-silica ion-exchange columnsgrafted with copolymers of N-isopropylacrylamide. J Chromatogr A2004;1030(1–2):247–53.

[247] Zhang XZ, Wu DQ, Chu CC. Synthesis and characterization of partiallybiodegradable, temperature and pH sensitive Dex-MA/PNIPAAm hydrogels.Biomaterials 2004;25(19):4719–30.

[248] Wang N, Zhao Y, Jiang L. Low-cost, thermoresponsive wettability of surfaces:poly(N-isopropylacrylamide)/polystyrene composite films prepared by elec-trospinning. Macromol Rapid Commun 2008;29(6):485–9.

[249] Zhang QL, Xia F, Sun TL, Song WL, Zhao TY, Liu MC, et al. Wettabilityswitching between high hydrophilicity at low pH and high hydrophobicity athigh pH on surface based on pH-responsive polymer. Chem Commun2008;10:1199–201.

M.A. Cole et al. / Biomaterials 30 (2009) 1827–18501850