PNIPAM grafted surfaces through ATRP and RAFT polymerization: Chemistry and bioadhesion

HAL Id: hal-01625967

https://hal.archives-ouvertes.fr/hal-01625967

Submitted on 30 Oct 2017

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

PNIPAM grafted surfaces through ATRP and RAFT

polymerization: Chemistry and bioadhesion

Guillaume Conzatti, Sandrine Cavalier, Christèle Combes, J. Torrisani, N.

Carrerre, Audrey Tourrette

To cite this version:

Guillaume Conzatti, Sandrine Cavalier, Christèle Combes, J. Torrisani, N. Carrerre, et al.. PNIPAM

grafted surfaces through ATRP and RAFT polymerization: Chemistry and bioadhesion. Colloids

and Surfaces B: Biointerfaces, Elsevier, 2017, 151, pp.143-155. �10.1016/j.colsurfb.2016.12.007�.

�hal-01625967�

O

pen

A

rchive

T

OULOUSE

A

rchive

O

uverte (

OATAO

)

OATAO is an open access repository that collects the work of Toulouse researchers and

makes it freely available over the web where possible.

This is an author-deposited version published in :

http://oatao.univ-toulouse.fr/

Eprints ID : 18213

To link to this article : DOI:

10.1016/j.colsurfb.2016.12.007

URL :

http://dx.doi.org/10.1016/j.colsurfb.2016.12.007

To cite this version : Conzatti, Guillaume and Cavalier, Sandrine and

Combes, Christèle and Torrisani, J and Carrerre, N and Tourrette,

Audrey PNIPAM grafted surfaces through ATRP and RAFT

polymerization: Chemistry and bioadhesion. (2017) Colloids and

Surfaces B Biointerfaces, vol. 151. pp. 143-155. ISSN 0927-7765

Any correspondence concerning this service should be sent to the repository

PNIPAM

grafted

surfaces

through

ATRP

and

RAFT

polymerization:

Chemistry

and

bioadhesion

G.

Conzatti

_,

_S.

_Cavalie

_,

_C.

_Combes

_,

_J.

_Torrisani

_,

_N.

_Carrere

_,

_A.

_Tourrette

a_{CIRIMAT,UniversityofToulouse,CNRS,INPT,UPS,UniversitéPaulSabatier,FacultédePharmacie,35ChemindesMaraichers,31062Toulousecedex9,} France

b_{CIRIMAT,UniversitédeToulouse,CNRS,INPT,UPS,ENSIACET,4alléeEmileMonso,CS44362,31030Toulousecedex4,France}

c_{UniversitéFédéraleMidi-Pyrénées,UniversitédeToulouseIIIPaulSabatier,InsermU1037,CRCTdeToulouse,2avenueHubertCurienCS5371731037} ToulouseCedex1,France

d_{GastrointestinalSurgeryDepartment,PurpanHospital–CHUdeToulouse,PlaceduDrBaylac,31059Toulouse,France}

Keywords:

Surfacefunctionalization

Reversibleadditionfragmentationchain transferpolymerization

Atomtransferradicalpolymerization Poly(N-isopropylacrylamide) Bioadhesion

a

b

s

t

r

a

c

t

Biomaterialssurfacedesigniscriticalforthecontrolofmaterialsandbiologicalsysteminteractions. Beingregulatedbyalayerofmoleculardimensions,bioadhesioncouldbeeffectivelytailoredbypolymer surfacegrafting.Basically,thissurfacemodificationcanbecontrolledbyradicalpolymerization,which isausefultoolforthispurpose.Theaimofthisreviewistoprovideacomprehensiveoverviewofthe roleofsurfacecharacteristicsonbioadhesionproperties.Weplaceaparticularfocusonbiomaterials functionalizedwithabrushsurface,onpresentationofgraftingtechniquesfor“graftingto”and “graft-ingfrom”strategiesandonbrushcharacterizationmethods.Sinceatomtransferradicalpolymerization (ATRP)andreversibleaddition-fragmentationchaintransfer(RAFT)polymerizationarethemost fre-quentlyusedgraftingtechniques,theirmaincharacteristicswillbeexplained.Throughtheexampleof poly(N-isopropylacrylamide)(PNIPAM)whichisawidelyusedpolymerallowingtuneablecelladhesion, smartsurfacesinvolvingPNIPAMwillbepresentedwiththeirmainmodernapplications.

Contents

1. Introduction...143

2. Bioadhesion...144

2.1. Bioadhesionmechanisms...144

2.2. Surfacesandbioadhesion...144

3. Surfacemodification ... 147

3.1. Brushstructure ... 147

3.2. Graftingtechniques...147

3.2.1. Atomtransferradicalpolymerization(ATRP)...147

3.2.2. Reversibleaddition-fragmentationchaintransfer(RAFT)radicalpolymerization...148

3.3. Characterizationofbrushes ... 148

4. Smartbio-surfaces:exampleofpoly(N-isopropylacrylamide)thermosensitivesurfaces...150

5. Conclusion...152

Acknowledgement...152

References...152

1. Introduction

Current developments in medicine have led to a need for newbiomaterialsin applicationsinvolvinginnovativestrategies

∗ Correspondingauthor.

E-mailaddress:audrey.tourrette@univ-tlse3.fr(A.Tourrette).

for disease treatment.These biomaterialshave to fulfilvarious requirementsdependingontheapplicationbutin allcases bio-compatibilityisacrucialpointthathastobeconsideredinearly processingstages.Biocompatibilityisdefinedastheabilitytoact inalivingsystemwithoutanytoxicityorrejection,whether phys-iological or immunological. Although biocompatibility includes “non-toxicity”,thesetwoconceptsstillremaindifferentiated.

Sur-facefunctionalizationofamaterialissometimesnecessarytoavoid stronginflammatoryresponsesandimprovebiocompatibility.

Ontheotherhand,surfacesnotonlyactaspassiveinterfaces betweenthebody(immunesystem,blood,cells)andthe bioma-terialbutalsotakeanactivepartincellspreading,proliferation, differentiationandmigration:allofthesephenomenaare inten-sively linked to surface/cell interactions. Thus, surfaces play a crucialrole inthefunction ofthebiomaterialand canbeused, forexample,asactivatorsofcellsfortissuereconstruction(tissue engineering)[1].Inthisfield,celladhesiontothesurfaceisakey factorthatmustbecarefullyconsidered.Bioadhesionisdefinedas theadhesionbetweenabiologicalentity,e.g.cellsortissues,anda surface.Itisacomplexphenomenonthatinvolvesmany parame-ters.Foralongtime,itwasdifficultforscientiststoclearlyidentify themechanismsunderlyingcelladhesion,buttheyarebecoming increasinglywellunderstood.Theinterestofscientistsfor bioad-hesionis notonly theoreticalbut isalsoessential for thewide spectrumofapplicationsdependingoncell/surfaceaffinity.

Incellsheetengineering,surfacepropertiesareusedtocontrol celladhesion,sothelivingsheetscanbestrippedoffeasily.Itwas shownthatcellsheetsintegratewellintotissues[2]andare promis-ingtoolsfortissuereconstruction.Smartsurfacestunablebetween “on”(adhesive)and“off”(non-adhesive)statesarethenfeasible. Naturally,thestudyofthermosensitivematerialshasbeenwidely reportedintheliterature.Poly(N-isopropylacrylamide)(PNIPAM) isapromisingpolymerasachangeofitshydrophilicinteractions, andindirectlybioadhesivness,takesplacebetweentheroom tem-peratureandbodytemperature.Moreprecisely,itslowercritical solutiontemperature(LCST)occursat32◦C.Whilethemonomeris cytotoxic,thepolymershowsnotoxicityforthevariouscelltypes

[3]andconstitutesagoodexampletohelpunderstandbioadhesion mechanisms.Itscharacteristicsmakethispolymeroneofthemost intensivelystudiedintheliteraturewhethergraftedorcoatedonto materialsurfaces.

Plasmatreatmentisprobably themostcommonprocess for surface modification, both for the introduction of functional groupsorforcoatingthesurfacewithpolymer[4].In the pres-ence of air, oxygenated surfaces will be produced under the plasma, leading to a change in hydrophilicity [5]. This modifi-cation can involve an increase in cell adhesion, as is thecase for polystyrene (PS) [6]. In addition, the plasma can etch the surface, enhancingits roughness.This modificationof topogra-phy, as observed in plasma-treated poly(methyl methacrylate) (PMMA),tendstoincreasethecellaffinitytothesesurfaces[7]. Theintroductionofreactivegroups alsopermitscompoundsor chemical functionsto begrafted onto thematerial surfaces. In thepresence of oxygen, surfacesactivatedvia theintroduction ofhydroxylgroups,canbegraftedwithmonomersorpolymers, such as NIPAM/PNIPAM though N-(3-dimethylaminopropyl)-N-ethylcarbodiimide(EDC)/N-hydroxysuccinimide (NHS) amide coupling[8].Underargonplasma,radicalswillbeproduced.These radicalscanbeuseddirectlyor,afterexpositiontoair,create per-oxidesthatcaninitiatepolymerization[9].

Whileplasmatreatmentpresentstheadvantageofbeingeasy toperformonmaterials,onlya lowlevelof surfacestructuring isobtained.Inaddition,itwasshownthatplasma-deposited PNI-PAMshowsgreatercelladhesionthane-beamcoatedPNIPAM[10]. Theseresultsemphasizedtheimportanceofthemethodusedfor surfacemodification.Controlofthesurfacestructurealsoseems tobeacriticalpoint.Abrushstructure,i.e.aself-assembled close-packedmonolayerofpolymerchains,providesprecisecontrolof thesurfacemorphologicalproperties.Morespecifically,itbecomes possibletocontrolthethicknessandthedensityofthegraftedlayer. Asaresult,thesestructuresarenowextensivelyused,especiallyfor biomedicalapplications,anddisplaygoodperformanceintermsof bioadhesion[11].

Toobtaincontrolledbrushes,awell-definedgraftedpolymeris necessary.Controlledradicalpolymerizationcanbeusedtocontrol polymergraftingusingvarioustechniques.Themostpopularones arering-openingpolymerization,nitroxide-mediated polymeriza-tion,reversibleadditionfragmentationchaintransfer(RAFT)and atomtransferradicalpolymerization(ATRP).Areviewoffunctional polymerbrushesproduced bycontrolledradicalpolymerization waspublishedbyOlivieretal.[12].RAFTpolymerizationandATRP arewidelyusedastheyareversatileandreducepoly-dispersities. Theyrelyontheequilibriumbetweendormantandactivespecies, soaresometimescalled“living”polymerizations.

It is necessary to understand cell adhesion mechanisms to designabiomaterialwithtunablebioadhesionproperties.Theaim ofthisreviewisnottobeexhaustiveonsurfacemodification pro-cessingbuttogiveacomprehensivemultidisciplinaryoverviewof thecell/surfaceadhesionmechanismsaswellasthechemical engi-neeringofsurfaces.Hence,anintroductiontocellularbiologyand bioadhesionwillbemade.Asecondpartwilldealwithbrushesand thechemicalroutesusedtoobtainsuchstructures.Moreprecisely, ATRPandRAFTpolymerizationmethodswillbereviewedalong withthewaysinwhichtheyhavebeencharacterized.Finally, engi-neeringoftunablesurfacesusingPNIPAMwillbepresentedwith itsmainandmostrecentapplications.

2. Bioadhesion

2.1. Bioadhesionmechanisms

Withasizefrom1to100␮m,cellsarecomposedofvarious enti-ties.Somearededicatedtoitsstructure:aphospholipidbilayer, thecellmembrane,maintains theseparationbetweenthe cyto-sol(internalliquidphase)andtheextra-cellularmatrix(ECM)and thecytoskeleton,composedmainlyofmicrotubulesandactin fil-amentnetworks, control the rigidityof the structure [13]. The ECMcompositionvariesdependingonthetissueconcerned.It con-tainsanumberofproteins:fibronectin,collagen,laminin,butalso growthfactorsandalltheproteinsneededforcellsupportand inter-cellcommunication.Actinfilamentsassembletoconstitute thecytoskeleton.Theyareconnectedtointegrins(transmembrane glycoproteins) throughvinculin andtalin (Fig.1)[14,15].These integrinsspecificallybindtoECMproteinssuchasfibronectin(or vitronectin)througharginylglycylasparticacid(contractionof L-arginine,glycine,andL-asparticacid,abbreviatedRGD)coupling. Theseintegrin/ECM proteininteractions areresponsible forcell adhesiontosurfaces(intercellularcohesionisregulatedbyother mechanisms,e.g. cadherin-mediatedhomotypic junctions).This adhesioniscommonlydividedintothedifferentphasesdescribed in Fig. 2a [16]. The first seconds of contact are characterized by the formation of non-specific interactions. Then, biological interactions occur,including adhesionprotein/fibronectin inter-actions.Thissecondstepleadstoacascadeofactionsincluding thereorganizationofthecytoskeletonandclusteringofintegrin receptors.Consequently,cellscontracttheircytoskeletonto main-tainamechanicalstateoftension,alsocalledprestress.Later,cells produceECMtoreinforcetheirintegrationandmaintaina propi-tiousenvironment.Itisthusobviousthattheadsorptionofadhesive proteinsisakeypointforcelladhesion.

2.2. Surfacesandbioadhesion

Twomainparameterswilldeterminethebehaviorofimplanted biomaterials:(1)theirbulkproperties,especiallytherigidity,plays aroleinthequalityoftheimplantationintotissues,and(2)their surface propertiescontrol theimmune systemresponse (called immunogenicity),thedestructionofcellintegrity,andthe

bioad-Fig.1. Theadhesionstructureofacellinamatrix.Thematrixislinkedtothecytoskeletonthroughintegrinsandtalins.ReproducedfromRef.[14]withthepermissionof TheRoyalSocietyofChemistry.

Fig.2.(a)Stagesofcelladhesion:interactionsandkinetics.(b)SEMpictureofamyoblastcellonanartificialsurface,2001.Fig.2bisreprintedfromRef.[17],Copyright (2002),withthepermissionofElsevier.

hesion. As cell membranes are anionic, cationic surfaces have to be carefully used since they can damage cells and tissues, whereasnegativelychargedpolymerswillelectrostaticallyrepel cells[18–21].Theimportanceofsurface/cellinteractionshasbeen investigatedformanyyears,andaninterestingdiscussionwas pub-lishedin2001byCastneretal.[17].Inthisaim,syntheticmaterials andbioadhesionhavebeenstudiedandobservedbyscanning elec-tronmicroscopy(SEM,Fig.2b).

Animportantpointistorealizethatinalivingsystemsurfaces arespontaneouslycoveredbyproteins.Itisnowbetterunderstood

thatcelladhesionisbothruledby(i)specific(biological) interac-tions(receptor/ligand)and(ii)thephysico-chemicalpropertiesof thematerial.Hydrophilicity,mechanicalpropertiesand morphol-ogyareamongtheparametersinvolvedinthebioadhesionprocess. AsmentionedabovecelladhesionbetweentissuesandECMis possiblethroughthespecificbindingofintegrinwithfibronectin (Fig.1)[22].In2011,Peietal.observedtheimportanceof spe-cificinteractions(i.e.fibrinogenRGDcoupling)intheprocessof attachmentbetweencellsandtheirsubstrate[23].Theynotedthe number of humanforeskin fibroblast(hFF) cells and how they

spread on poly(ethylene glycol)(PEG) brush surfaces (see Sec-tion3.1)graftedontoTiO2andcomparedpre-treatedhFF(blocked

integrinbonding-sites)andnon-treatedhFF(freeintegrinbonding sites).

Thenumberofattachedcellsstronglydecreasedwhenthe inte-grinbondingsiteswereblocked.Nevertheless,weaknon-specific interactions occurred when there was a high level of affinity betweenthecellsandthebiomaterial.Thisstudyalsoprovides pre-ciousinformationontheinfluenceofthePEGbrushdensity.PEG isananti-foulingpolymer.Athighgraftingdensity,PEGchainsare collapsedina“brushregime”(L<2Rg,withLthedistancebetween twoneighboringchainsandRgtheradiusofgyrationofpolymer chains),whereasa“mushroomregime”isobservedatlower val-ues(L>2Rg).AgradientofPEGwasusedtoobserveitseffecton proteinadsorptionand celladhesion.For shortexperiments,as thedensityofPEGincreased,astrongdecreaseofthenumberof adheredcellsoccurredwhenthebrushregimewasreachedanda correlationestablishedwiththeproteinadsorptionprofile. Fibro-blastsaturationoccursinthe“brushregime”,meaningthatevena lowamountoffibrinogen(2.2±3.4ng/cm2)issufficienttoactivate adhesion.Heretheimportanceofprotein/cellspecificinteractions isobviouslycrucial.

Amodelinvolvinga proteinlayerbetweenbiomaterialsand cellswasestablishedintheearly2000’s[1,17,24].Accordingtothis model,cell/matrixadhesiondependsontheabilityofthesurfaceto adsorbproteinswithoutmodifyingtheirnativestructure. Denatu-rationisbroughtaboutbythewaterstructurenearthesurface,i.e. thehydrophilicityofthesuperficiallayeronthematerial.Areview, publishedin2011dealtwiththeconceptofnativeimmune sys-temresponse(cascadesystem)[25].Thearticleemphasizedthe importanceof thenon-denaturationof protein structure atthe material/bodyinterfacetoavoidactivationoftheprimaryimmune system.In1998Volgeretal.alreadyunderlinedtheimportance ofsurfacechemistryintermsofhydrophobic/hydrophilic proper-ties[26].Thisgroupshowedthat water/surfaceinteractions are ofinterest.Ascriteriaofhydrophilicity/hydrophobicitytheyused contactanglemeasurements.Astronglyboundwatertoasurface (i.e.hydrophilicsurface)cannotberemoved.Thiswillavoid inter-actionsdirectlybetweena biologicalentity(e.g.a protein),and thesurface,leadingtoloworabsenceofadsorption[27]. More-over,itiswidelyacceptedthatmoderatelyhydrophilicmaterials aresuitableforcelladhesion,withacontactanglearound70–80◦

[28,29]. For example, endothelial cells show good attachment to polycaprolactone-grafted-poly(methyl methacrylate) (PCL-g-PMMA)surfacewithacontactanglearound70◦.Additionally,it isnowacceptedthatsurfaceswhicharetoohydrophobicleadto thedenaturationofproteins[1,28].Moreprecisely,hydrophobic andhydrophilicamino acids,constitutiveof proteins,rearrange theirorganizationdependingonthesurroundingmediaandthus hydrophobicsurfacescanfavortheexternalisationofhydrophobic moieties,leadingtounfoldedproteins[30].Regardingthis assess-ment, post-treatments (e.g. plasma treatments) are sometimes usedtoincreasesurfacehydrophilicitythroughtheintroduction ofpolarfunctionalgroups.Incontrast,itisknownthatthe interac-tionsofhighlyhydrophilicsurfaceswithECMadhesionproteinsare weak[6].Keselowskyetal.characterizedvariousfunctionalgroups on the criteria of fibronectin adsorption (in increasing order): OH<COOH<CH3<NH2.Ontheotherhand,adhesionofosteoblasts

increasesasfollows:CH3<NH2=COOH<OH[31].Thistrend

inver-sion can beexplained by thegeometrical deformation of ECM proteins,i.e.denaturation.Onbrushsurfaces,theoptimalcontact angledependsnotonlyonthenatureofthematrixbutalsoonthe methodofsurfacemodification.Thiscanbeduetotheinfluenceof chainlengthanddensityofthegraftedpolymeronthe conforma-tionoftheproteinadsorbedonthesurface.Forinstance,ithasbeen pointedoutthatwhenFe2+_{isusedasinitiatorforthegraft}

polymer-izationofPMMAonpoly(L-lacticacid)(PLLA)surfaces,maximum chondrocyteattachmentisobtainedwhenthecontactangleis52◦, whereasUV-initiatedsurfacesareoptimalforacontactangleof76◦

[28].Theauthorssuggestthatthedifferenceofbiological proper-tiesbetweenthetwoPLLAgraftedPMMAcouldbeduenotonlyto surfacewettability(contactangle)butalsotothehigherdensity, uniformityandshorterchainsofiron-initiatedpolymerized sur-faces.Thewettabilitycriterionisobviouslystronglylimitedasthe surfacemechanicalpropertiesandstructure,celltypesandcharge densityareignored.Forexample,Bacakovaetal.showedthatsoft matricesare notfavorablefor celladhesion[6].Theyexplained thatECMdepositedonsuchsurfacesisnotabletoresisttheforces involvedduringcellfocalpointformation.

Indeed,specificinteractionsarenotenoughtodescribed adhe-sivephenomenaentirelyandphysico-chemicalpropertieshaveto beaddedtotheequation.Hence,theinternalorganizationofcellsis remodelledthroughouttheirlifeandisstronglyinfluencedbythe surroundingmediumnotonlyviachemical stimulationbutalso bymechanosensing,untilamorphologicalequilibriumisreached

[13,32].Thereby,theshapeofcells, aswellastheirrigidityand motilitydependontheirsupport.Forexample,manycellshavethe abilitytosensethestiffness,byapplyingastress,oftheir exter-nalenvironment.Thesecellsincludebrain,muscle,neuronsand manyothercelltypes[33].Cellsprobethesurfacethroughmyosin andactin filamentcross-bridging anda stiffnesscontrol loopis setup:cytoskeletonand adhesionwilladapt dependingonthe feedback.Asaconsequence,stiffermatricesleadtoanincreasein theelasticityofthecells, andcanbemeasuredbyatomicforce microscopy(AFM)[34]or,moregently,byindentationwith opti-caltweezers[35].However,rigidityscanningofthecellsubstrate isatime-consumingprocess,takingfromminutestohours[13], thusviscositycanbeconsidered,inthecaseofagelforexample. Sometimes,cross-linkingcanimprovecellactivityonagel[36].In addition,mechanosensingcanbeaninitiatorofcelldisplacement onsurfaces(mechanotaxis,discoveredbyLoetal.[37]),fromthe softtothestiff[38]andmotilitywereshowntobelinkedtofocal contactsandthusindirectlytocellularadhesion[39,40].

Moreover,depending ontheirtype, cells willnot behave in the same way depending on the elasticity of the biomaterial. Forinstance,onsoftmatrices,fibroblastsadhereinalabileway whereasonstiffermaterialstheymakestablefocalpoints (adhe-sion)andrigidifytheircytoskeletons.Consequently,themotilityof fibroblastsonstiffmaterialsisreduced[38].Themobilityofthe chainsthatconstitutethebrushescouldalsolead tosuperficial mechanicalinstabilityandthustoloweradhesion[41].

Despitetheadhesionofcells,thebiomaterialcanalsodisturb cellactivity.Aperturbationintheexocytosisresponseofcellsis revelatoryofthisperturbationinwaysthatcan,forinstance,be measuredbyhistologicalstudiesorcarbon-fibremicroelectrode amperometry(CFMA),asobservedbyReedetal.[10].Inthisstudy, theintroductionof PNIPAM(throughplasmadepositionorspin coating)onsurfaceshaveshowntoslowdownthecellexchange betweenvesiclesandextracellularspace.Additionally,spin-coated PNIPAMwereshowntohyper-activatetheexocytosisactivityof cells,whereasplasmadepositedPNIPAMonlyaffectedkinetics.If thishyper-activationbythesurfacecanpresentharmfuleffects, somestudiesseekforacceleratingtissueregenerationby modify-ingthesurface,suchasobservedbytheintroductionoffreeamino grouponPLLAsurfaces[42].Thesurfacearchitectureisalsoakey point.3Darchitecturesarecommonly modulatedinthefieldof tissueengineering,astheyreproduceamorerealisticnatural bio-logicalenvironment.Anicereviewofthistopicwaspublishedby AbbottandKaplanin2015[43].

Inconclusion,thesurfacehydrophilicity(presenceoffunctional groups),thesurfacestructureandstiffnessarecharacteristicsthat must be considered for efficient bioadhesion. However, in the

Fig.3.Twodifferentstrategiesofpolymergrafting(exampleofPNIPAM):thesurfaceinitiatedpolymerizationofamonomer(“graftingfrom”)orthegraftingofthepolymer (“graftingto”)onasurfacetoobtainabrushstructure.

presentreview,weonlyconsidersurfacemodificationthatdoes notinvolveanybulkmodification,inparticularintermof bioma-terialstiffness,andtheeffectofbrushstructuresonmechanical surfacepropertieswillnotbediscussed.Below,wefocusonakey step,i.e.thechoiceofsurfacemodificationmethodusedtoobtain brushstructuresonmaterialsurfacesthatenablegoodcontrolof thesurfacestateandproperties.

3. Surfacemodification

3.1. Brushstructure

Asdiscussedabove,thecontrolofbiomaterialsurfacesisa chal-lengeforscientistsinvolvedinthedevelopmentofmedicaldevices. Brushstructuresinparticularareinterestingintermsofprotein penetrationandcalibrationstudies[44].Thankstorecent devel-opmentsinchemistry,varioustechniquesareavailabletograftor coatbiomaterialswithpolymers.Forbiologicalapplications, cova-lentgraftingseemstobethebestchoicecomparedtophysically graftedsystems,duetorisksofdesorptionwiththislattermethod. Polymerbrushes,whichconsistonathinfilmofself-assembled polymers,areofinterest[10,45].Wettability,butalsothevarietyof possibleend-groupfunctions,thesubstrateandthechemistryare themainpointsthatmakethesesystemsattractive.The modular-ityofthepolymerbrushsynthesisisillustratedbythebroadrange ofsystemsthathavebeendevelopedinrecentyears:uniformed, patterned,orgradient(intermsofdensityorchemical composi-tion)brushlayershavebeenpreparedwithoneorseveralpolymers

[12,45].

Twodifferentapproachescanbeconsidered,namely“grafting to”(i.e.tothesurface)and“graftingfrom”,asshowninFig.3[45,46]. The “grafting to” method consists in coupling an end-functionalizedpolymerandareactivesurface.Thisapproachyields well-definedgraftedpolymer.However,thedepositedlayershave lowdensitiesandtheirthicknessislimited(100nm[47])duetothe sterichindranceduringgraftinganddiffusionprocesses[48–51].

Fig.4. AdvancedATRPmechanismscatalyzedbyCu(I)/Cu(II)complexes.Reprinted fromRef.[54],Copyright(2014),withpermissionofElsevier.

The“graftingfrom”methodconsistsinpolymerizationdirectly fromthesurface. Severaltechniquesare basedonthis process. Surface-initiatedpolymerization(SIP)basedonradicalchemistryis commonlyused[47,51,52].“Graftingfrom”techniquesyieldhigher graftedlayerdensitiesandovercomethicknesslimitations.These advantagesmadethisstrategythemostwidelyadopted.However, thecharacterizationofgraftedchainsismoredifficultand,apart frommodelsurfaces,stillremainsagreatchallenge.

Varioustechniquesarecommonlyusedforgraftingpolymers to surfaces, both for “grafting to” or “grafting from” strategies

[47,53,54].Amongthesetechniques,themostused,i.e.ATRPand RAFT,willbedevelopedinthenextsections.

3.2. Graftingtechniques

3.2.1. Atomtransferradicalpolymerization(ATRP)

Asisgenerally thecase incontrolledradicalpolymerization, ATRPchemistryreliesontheequilibriumbetweenactiveand dor-mantchains[46,47,55,56].ThemechanismisillustratedinFig.4. SurfaceinitiatedATRP(SI-ATRP)isa“graftingfrom”techniquethat

consistsinimmobilizingahalogenatedinitiatoronthesurface, fol-lowedbyATRP.Themainadvantages oftheATRParetheclose controloffilmthicknessandchainlengthwithlowpolydispersity

[47].Itisalsopossibletocontrolthicknessandgraftdensity sep-arately,bymodulatingthereactiontimeandstoichiometricratio, respectively[57,58].Inaddition,ATRPisknowntobeversatileand easytoperform(mildconditions)[51,58,59].Whilehigh temper-aturesmakethereactiontimeshorter,somestudiesalsoshowa slightreductionofthepolydispersity[60].Nevertheless,theneed ofmetalcatalystsisalimitationforbiomedicalapplications. Basi-cally,themetalisoxidized/reduced,andthusgeneratesorabsorbs aradical,leadingtotheactivation/deactivationofpolymerchains, respectivelyas shown inFig.4.Atthepresent time, due toits highcatalyticactivitycopperisusedmost[61,62].Iron catalyzed-ATRPcanbeperformedusinglowamountsofcatalyst,reducing thetoxicologicalrisksasironis consideredlesstoxicand more environmentallyfriendlythancopper[63–65].Ironisalsothemost abundantmetalonearthmakingitrelativelycheap;these charac-teristicshaveinitiatedalotofresearchandinterestinFecatalyzed organicchemistry,includingATRP,inlinewiththeperspectivesof “greenchemistry”[66,67].Thechoiceoftheligandisacomplex question;itdependsonthenatureofthepolymerandthecatalyst used[68]:forexample,pentamethyldiethylenetriamine(PMDETA) canbeusedincombinationwithCu[69],andtris(3,6-dioxaheptyl) amine(TDA)withFe[70].Furthermore,theactivatorsregenerated byelectrontransfer(ARGET)ATRP,developedbyMatyjaszewski etal.diminishtheamountofcatalystneeded(<50ppm)[71,72]. ThisadvancedATRP,derivedfromtheAGET-ATRP(foractivators generatedbyelectrontransferATRP)involvingareducingagentto initiatethereaction,consistsinusinganexcessofreducingagent (e.g.environmentallyfriendlyascorbicacid[73]).Thisinitiatordoes notonlygeneratebutalsomaintainsasufficientamountofCu(I) (inthecaseofcoppercatalyzedATRP)withouttheuseofaradical organiccompoundwhichcouldleadtosidereactions,cross-linking ortheformationofnewchains[55].Anotheradvantageof ARGET-ATRPisthattheoxidizedcatalyst,e.g.Cu(II)orFe(III),canbeused directlywithouttheneedforearly-stagereductionandcareful han-dling.Finally,thistechniqueincreasestheairtoleranceandcan avoidtheneedofacontrolledatmosphere[74,75].Ascorbicacid (alsocalledvitaminC)ispreferabletoclassicSnreducingagents duetoitsnon-toxicitytowardshumanbeingsand the environ-ment.However,ascorbicacidpresentsthedisadvantageofbeing astrongreducingagent,soitsuseinwatercanleadtoanextensive conversionofCu(II)toCu(I)andcandiminishthecontrolofATRP. Onesolutionwouldbetousealessefficientsolvent,suchasanisole, todecreasethereducingactivityoftheascorbicacid[73,75].

Initiatorsforcontinuousactivatorregeneration(ICAR)ATRPuse theadditionofafreeradicalinitiatorsuchasazobisisobutyronitrile (AIBN)to(re)generatetheactivemetal[63,71].

Insupplementalactivatorandreducingagent(SARA)ATRPthe reducing agent is M(0), e.g. Cu(0) for a coppercatalyzed ATRP

[67,76].Usingironpowder,thepolymerizationcanbecatalyzedby Fe(0)[60],withorwithouttheuseofFe(III)saltsbutinthislatter casealesscontrolledpolymerizationovertimecanoccur[63].

Many ATRP elaborations are involved for surface modifica-tion, nonetheless we would like to mention biomedical uses of this chemical route: biofouling surfaces or membranes [77], double responsive cellulose membranes [78,79], cell attach-ment/detachment(throughPNIPAMgrafting)[69,80].Toperform a“graftingfrom”,theideaistochemicallygrafttheinitiatoronthe surfacesothegrowthwillbedirectlyinitiatedonthematerial.This stepisfacilitatedbythefactthatATRPinitiatorsareacylbromides. Aself-assembledmonolayer(SAM)isgenerallyalsograftedbefore theinitiator.ATRPcanbeachievedincombinationwithplasma treatmentstohelpinitiatorimmobilization[81].Afewexamples ofSI-ATRParegiveninTable1.

ATRPcommonlyexhibitsapseudo-first-orderkinetics,atleast below high rates of conversion. The direct characterization of grafted polymer through the “grafting from” method is diffi-cult.Generally, a sacrificial initiator is used,assumingthat the polymer growth is similar on the surface and in the medium

[51,52,58,59,82–85].Anothermethodistousereversibleor break-ablesurfacebondsinordertodetachandstudythegraftedpolymer

[86].Amoredetaileddiscussiononbrushcharacterizationisgiven inSection3.3.

3.2.2. Reversibleaddition-fragmentationchaintransfer(RAFT) radicalpolymerization

SimilarlytoATRP,RAFTpolymerizationisaliving polymeriza-tionbasedontheequilibriumbetweenactive(i.e.bearingradicals) and dormant chains, and also shows pseudo-first-order kinet-ics.Initiationisperformedintraditionalways,e.g.usingthermal initiatorssuchas azobisisobutyronitrile(AIBN)or 4,4 -azobis(4-cyanovalericacid),whichhastheadvantageof beingcarboxylic acidend-functionalized.Achaintransferagent(CTA)(alsocalled RAFTagent)ensuresthisequilibriumduringthepropagationsteps, asshowninFig.5[87,88].Thereductionofactivechain concen-trationresultsinanarrowdistributionofthechainlength,witha polydispersityindex(PDI),forPNIPAM,abletoreachvaluesaround 1.20,but PDIbelow1.10 canbeobtainedinoptimalconditions

[89–91].Additionally,RAFTpolymerizationcanbeachievedwith abroadrangeoftemperatures,fromroomtemperatureto140◦C

[92].Ahighertemperatureallowsashorterreactiontime;lower polydispersitiescansometimesbeexpected.

OneofthemainadvantagesofRAFTpolymerizationcompared toATRPisthatitisametal-freechemicalroute.Incontrast,itcan requirethesynthesisoftheRAFTagent.InmodernRAFT polymer-ization,thisagentclassicallycontainsathiocarbonylthiomoiety, asforthecommonlyusedtrithio-carbonateanddithio-carboxylate type.AstheRAFTpolymerizationprocessreliesonthekineticsof additionand fragmentationofthis agent,thechoiceof its sub-stitute,classicallycalledZandR,iscrucial.Zisdedicatedtothe activationof thedouble bondby stabilizing theadductradical, Risaleaving group.Acompletediscussionaboutthechoiceof theRAFTagentisavailableintheliterature[92].Thesurface initi-atedRAFT(SI-RAFT)polymerizationofPNIPAMcanallowtoobtain polydispersitiesbelow1.3[93].Thankstothesulfurylgroupsof theCTA,elementalanalysiscan,insomecases,beusedto deter-minethequantityofgraftedRAFTagent.Thetheoreticalmolecular massofthepolymer,Mn_th,canbeestimatedthroughthefollowing equation:

Mnth=

[Mono]0

[CTA]0 ×MMono×con

v

whereM_Monocorrespondstothemolecularweightofthemonomer, [Mono]0toitsinitialconcentration,conv.istheconversionrateof

themonomer,M_CTAisthemolecularweightoftheCTAand[CTA]0

itsinitialconcentration[91,93].

InordertoperformSI-RAFTpolymerization,theinitiator[90]

ortheCTA[93]havetobepreviouslygraftedtothesurface.Inthe firstcasethehomolyticcleavageoftheinitiatorwillleadtogrowth eitheronthesurfaceorinthemedium,bothwiththefreeCTA.In thecaseofCTAgraftedsurfaces,theinitiatorandanotheramount oftheRAFTagentareintroducedtopermitthepolymerizationof freechainsandtheircharacterization.Fewexamplesof SI-RAFT polymerizationaregivenTable1.

3.3. Characterizationofbrushes

Characterizationofthe“graftedfrom”polymerbrushisa chal-lengingtask.Inspecificcases,thegraftedchainscanberemoved fromtheirsubstrate[86].Inothercases,freechainsaregenerally

Table1

ExamplesofsurfacefunctionalizationwithPNIPAMbrushesusingATRPorRAFTpolymerizationprocess.

Radicalpolymerization Technic Substrate Solvent Refs.

ATRP graftingfrom polyethyleneterephthalate(PET) water [94]

Au water/methanol [95]

graphene water [96]

poly(␧-caprolactone)(PCL) water/methanol [69]

paryleneC DMF/water [80]

Si water/methanol [97]

cellulose varioussolvents [98,99]

mesoporousSifilms water [100]

RAFTpolymerization graftingto Aunanoparticles water [89]

graftingfrom mesoporousSinanoparticle DMF [101]

aminatedpolyHIPE(highinternalphaseemulsions) DMF [93]

cellulose varioussolvents [98,102]

glass dioxane [90]

Fig.5.MechanismofaRAFTpolymerization.TheCTAplaystheroleofactivator/deactivator.ReproducedfromRef.[86],Copyright(2002),withpermissionofJohnWileyand Sons.

produced(seeSections3.2.1and3.2.2).Iffreepolymerisgenerated, themolecularweightcanbeeasilydeterminedbysize-exclusion chromatography (SEC)or viscosimetry.Fromoptical waveguide lightmodespectroscopy(OWLS)themassofadepositedpolymer canalsobeobtained[23].

Moreover,themorphology,graftdensity and thicknesshave tobedetermineddirectlyonthesurface.AFMisapowerfultool tostudymorphology[103,104](Fig.6).Forexample,inthecase ofPNIPAM,achangeinconformationaccompanyingachangein temperaturecanbeobservedbyAFM[103].

Surfaceplasmonresonance(SPR)givesthewetthicknessofthe layer,i.e.itsthicknessinaliquidenvironment,takingintoaccount swellingphenomena.Thedrythickness,i.e.intheabsenceofwater, ofthegraftedlayercanbemeasuredbyellipsometry[103].AFM canalsobeusedtodeterminethedrythickness,butsystematic errorswerereportedduetoAFMtipsbeingattractedbythePNIPAM layer[105].PNIPAMgraftingdensitycanbededucedfromthedry thicknessvalue,throughthefollowingequation:

␴=h_M␳NA

n

with␴thegraftdensity,hthedrythickness,NAAvogadro’s

num-berandMnthemolecularweight.ThedensityofdryPNIPAM,␳,is

sometimesarbitrarilytakenequalto0.95g/cm3_{bycertainauthors}

[106],buttheactualdensitycanbemeasuredbyU-tubeoscillation

[107]orbyX-rayreflectometry[108].Fouriertransforminfrared (FTIR)spectroscopycanalsobeused,insomecases,todetermine thegraftdensity,asreportedbyMizutanietal.[109].

Chemical analysis can beperformed through classic surface analysis:X-rayphotoelectronspectrometry(XPS),attenuatedtotal reflectionFTIRspectroscopy(ATR-FTIR),Ramanspectroscopy.For nanolayerstudies,XPSispreferabletoATR-FTIRspectroscopydue toitslowerpenetrationdepth(afewnmforXPSagainstupto1␮m forATR-FTIRspectroscopy)[28].Secondaryionmassspectroscopy (SIMS)techniquescanbeevenmoresurfacelocalized.Inthis tech-nique,thesurfaceisetchedbyanionbeamandsputteredmaterial iscollectedbyadetector.Elementsandchemicalstructurecanbe determined.

Quartzmicrobalance(QCM)isaninterestingtoolforthe eval-uationoftheamountofadepositedlayer.Thedeterminationof thequantityofproteinsadsorbedonabiomaterialsurfacecanbe obtainedbymeasuring,insitu,thefrequencyshiftofthequartz

Fig.6. RelationshipofPNIPAMfilmmorphologytolocalgraftingdensityastrackedthroughtheinitiatordensity.Thegraftingdensityincreases,fromadiscontinuous mushroomstructure(left,lowgraftingdensity),toaheterogeneouspatchystructure(middle,intermediategraftingdensity).Athighgraftingdensity(right),asmoother, presumablymoreextended,structureisobtained.ReprintedfromRef.[102].Copyright(2006),AmericanChemicalSociety.

[24].Quartzmicrobalancewithdissipationmonitoring(QCM-D)is usefultoobtaintheswellingbehaviorofpolymerbrushes[97].

Finally,carbonfibremicroelectrodeamperometry(CMFA)can giveinformationaboutbiochemicalexocytosisofcells(kineticsand amountofrelease)andthusevaluatehowthesubstrateimpactsthe excretionactivityofcells[10].

Mostofthesetechniquesarenotapplicableto“brushgraftedon polymer”systemsduetotherelativelyhighroughnessofthistype ofsurface,whichexplainsthelackofreliablebrushcharacterization techniquesintheliterature.

4. Smartbio-surfaces:exampleof

poly(N-isopropylacrylamide)thermosensitivesurfaces

Poly(N-isopropylacrylamide)isathermo-responsivepolymer. Its structure is shown in Fig. 3. Indeed, PNIPAM changes its water affinityaccordingto thetemperatureof thesurrounding medium,turningfromhydrophilic tohydrophobic.Thischange occursat around 32◦C, whose temperature is calledthe lower criticalsolutiontemperature(LCST),andleadstoachangeinits conformation.Abovethistemperature,PNIPAMcollapsesin solu-tion.Thiscoiltoglobuletransitionisendothermicandisrelated towater/polymerand polymerinterand intra-molecule hydro-genbonding.Inotherwords,below theLCSTPNIPAMisbound towaterthroughamide/water(C O···H O)hydrogenbonding.As thetemperatureincreasesaboveLCST,thepolymerbecomes dehy-dratedandamide/amine(C O···H N)hydrogenbondingappears

[110,111].InthecaseofsurfacesgraftedwithPNIPAM,itmeans thata“brush”systemcanbeturnedintoa“mushroom” conforma-tionabovetheLCST.TheLCSTofPNIPAMwasreportedtodependon thechainlengthandthegraftingdensitywhileremainingbetween roomandphysiologicaltemperaturemakingthispolymer interest-ingforvariousbiomedicalapplications[105].Additionally,theuse ofPNIPAMinacopolymersystem[112],aswellasthepresenceof

saltscanstronglyinfluencethisLCST[113,114].Cl−andCH3COO−

haveaparticularlystronginfluenceaspredictedbythe Holfmeis-terseries.Moreover,ionconcentrationsaregenerallylow(below 0.15M)inbothculturemediaandbodyfluids,itsinfluencethus hastoberelativized.Itisnoteworthytomentionthatproteins,if concentrated,canalsoaffecttheLCSTofPNIPAMfromadecrease of2.6◦Ctoanincreaseof1.5◦C,dependingontheproteininvolved

[111].

Surfaceinteractionscanbemodulatedbythetemperatureof thePNIPAM.Thisvariationoftheinteractionsisclearlyobservedby AFMmeasurements[105,115].Bovineserumalbumin(BSA)bound toAFMtipswasusedtostudythevariation ofprotein/PNIPAM surface interactions. It appeared that interactions are tempera-turedependent,withaproteinadsorptionphenomenonoccurring aboveLCST [116].Thisphenomenon wasalsoobservedusing a QCM.ThemechanismisrelatedtoPNIPAMhydrationbutisnot wellunderstoodatthepresenttime. Variousstudieswere per-formedtoevaluatetheabilityofPNIPAMtotriggercellattachment. InadditiontotheirslightcontrolofLCST,itappearsthatthegrafting densityandthechainlengthalsoplayanimportantroleon bioad-hesion.Thus,thematerialseemstoberesistanttotheadsorption ofeitherproteinsorcellswhenchaindensityandchainlengthare bothhigh[117].Thisisexplainedbythedifficultyforproteinsto enterthePNIPAMlayerduetosterichindrancewhenchaingrafting isdense.Halperinetal.proposedatheoreticalapproachof mech-anismsforharvestingcellsculturedonthermoresponsivePNIPAM polymerbrushes[118].

First,two interaction modeshave tobeexaminedwhen we considerparticles(e.g.proteins)andabrushstructure.The com-pressivemode,wherethebrushesarecompressedbyaparticle, occurswhenitssizeisgreaterthanthespaceavailablebetween chains,takingintoaccounttheabilityofchainstorearrange them-selvesaroundtheparticle.Thisistypicallythecaseforcells.The other mode, theinsertive mode, occursfor smallparticles, e.g.

Fig.7. Thethreedifferentmodesofproteinadsorptionthroughbrushes:(A)primary adsorption,(B)ternaryadsorptionand(C)secondaryadsorption.AdaptedfromRef.

[117],Copyright(2012),withpermissionofElsevier.

extracellularproteinssuchasfibronectin.Here,weseethe impor-tanceofthegraftingdensity:highdensitylimitsproteininsertion. Then,thedepthoftheinsertedproteincanalsovary,formingthree modes:primary,secondaryandternaryadsorption(Fig.7).High affinitybetweenECM proteinsandsubstrate promotesprimary adsorption,whereasahighgraftingdensitytendstosuppress pri-maryandmaybeternaryadsorption[117,118].Primaryandternary adsorptionfavorbioadhesion,whereasatheoreticalmodelpredicts that secondary adsorption preferably occurs for large cylindri-calproteins[44,119].Nonetheless,ifternaryadsorptionmediated bioadhesionwasshowntobepossible,itseffectonprotein denatu-rationisstilltobedemonstrated[118].Finally,athinlayer(i.e.low molecularweight)facilitatesprimaryandternaryadsorbed pro-teins/cellsinteractions,leadingtoanincreaseofcelladhesion.In addition,proteinadsorptionisalsoconcentrationdependentand, athighconcentrations,theadsorptionratebelowandaboveLCST canbecomeclose[120].Then,thegraftdensitywasshownto influ-encethebrush structure. Ahighgraft density cancause phase separation[121],andmoregenerallythedensitywillmodulatethe proteinadsorptionratewithinthebrushes,asforBSA[44].Malham etal.showedthatchainrearrangementsovertimecouldslightly increaseinter-chainadhesionandrelated thisto−NHand C O hydrogenbonding[121].Itisobviousthatinter-chainattractive interactionswouldplayaroleonproteininclusion.

VariouscellsadheretoheatedPNIPAMsurfacesandarereleased during cooling [122]. Typically, cell detachmentis achieved at T=20◦C[118].Thispropertyallowscellstobeseededand gen-tlydetachedthem,withoutaneedoftrypsin:itisusedinthefield ofcellsheetengineering[123].

Monoormultilayercellsheetsarethusproducedandusedfor tissueregeneration[2].Thesebiologicallayersdemonstrategood integrationintissues.Invivostudieswereperformedtotreat vari-ousdiseases:cartilagedegeneration[124],damagedcornealtissues

[125]orcardiactissues[126].Interestingly,itappearedthatafter celllift-off,alayerofECMremainsattachedtothesurface.Research hastried todeterminethecompositionofthis remnantprotein layer[127]anditwasshownthatmostofthefibronectinleaves thesurfacewiththecells.Nonetheless,thisremnantlayerpromotes newcellgrowth,showingitsviability.

Alotofsystemshavealready beendevelopedusingPNIPAM brushsurfaces.PNIPAMbrushesweresuccessfullygraftedthrough ATRP[69,128]and RAFTpolymerization[90,93].Byintroducing reactivegroupsthroughplasmatreatments,PNIPAMcanbegrafted via“graftingto”amidebinding[8]orsurfaceinitiatedATRP[81]. ATRPproducedSi-PNIPAMbrushhybridswhichwereshowntobe efficientinthethermo-triggeredadhesion/de-adhesionof fibro-blastcells[128].InthiscasethethickerthePNIPAMlayeris,the moreprofitablethesurfaceisforcellproliferationafter2days.In allcases,noadhesionisobservedfor temperaturesbelowLCST independently of the thickness(3nm, 11nm or 31nm).It also appearedthattheantifoulingpropertiesofthepoly(ethylene gly-col) monomethacrylate (PEGMA) in combination with PNIPAM increasesthecellreleaseabilitiesofPNIPAM.Forbovine endothe-lialcells,athicknessofPNIPAMbrushontissueculturepolystyrene

around15nmshowedoptimaladhesion/de-adhesionproperties

[129].Theyalsoreportednoadhesionabove30nm,whereas Mitzu-tanietal.observedthatendothelialcelladhesiononpolystyrene ATRPgraftedPNIPAMsurfacesissuppressedforthicknessgreater than60nm[109].Moreover,thebestadhesionwasobtainedfor thinnerPNIPAMlayers(1.8nm).Takahashietal.developed surface-initiated RAFT polymerization brushes on glass coverslips and studiedboth graftdensity andmolecularweightof PNIPAMon reversiblebioadhesion[90].Thestudyshowedthattheamountof cellsalsoincreasedonloweringthegraftdensity.Inaddition, bet-terbioadhesionisobservedforshorterbrushesbutde-adhesion needsathickenoughlayer.Theexplanationisrelatedtothe neces-sitytopushcellsfromthesurface,asthePNIPAMbrushesbecome extendedonreducingthetemperature.Thiscanbethegeneral con-clusion,ifpossible,ofthicknessconsiderations:abalancebetween theabilityforcellstoattach(thinbrushlayer)anddetach(thick brushlayer)astobefound.Consequently,athickPNIPAMlayer canbeusefultoproduceproteinresistantsurfaces.

Zhao et al. studied the anti-fouling properties of PNIPAM graftedpolyurethanesurfacesagainstfibrinogenandhumanserum albumin (HAS) proteins at 37◦C [131]. It appeared that the thermosensitivity of the hydrophilicity was not significant on lowPNIPAMthickness,andthattheproteinadsorptionstrongly decreased asthis thicknessincreased.Thiseffectcanbedue to higherhydrophilicityofthickerlayers.Asaconsequence,cellsdo notadheretothickbrushesandthusanti-adhesionsurfacescanbe producedbytheuseofPNIPAM.Yuetal.showedthickness depen-dent thermo-sensitivity of PNIPAMgrafted Si (surface initiated ATRP)surfacesandmanagedtoproduceHSArepellent,evenwith thinPNIPAMlayers(<15nm)[132].Thevariationofcontactangle andHSAadsorptionbetween27and37◦Cisnotsonotableatlow PNIPAMthickness.However,greatertemperaturesensitivitywas observedathighergraftthicknessbothoncontactangleandHSA proteinadsorption.Moreinterestingly,at37◦CHSAadsorptionis notlinearlydependentwithPNIPAMthicknessand,asthe thick-nessincreases,adecreaseofsensitivityfollows.Thisobservation wasattributedtopossibleadsorptionontheSi-initiatorsurfaceat lowgraftthickness.Ascontactangleshowedhydrophobicsurfaces (higherthantraditionalanti-foulingpolymer),theauthorsdeduced thattheanti-foulingpropertiesoflowPNIPAMthicknesswerenot duetothehydrophilicityofthePNIPAMsurface,buttothe inter-actionsbetweenPNIPAMandthesubstrate.Indeed,shortPNIPAM brushendchainscanalsointeractwiththesubstrateandreducethe freedomofconformationchanges,reducingthetemperatureeffect

[132].Thisstudyalsoshowedtheimportanceoftheproteinsizeon adsorption.Indeed,thesizeoftheproteinmoleculeisofimportance asthepenetrationwillbedependentonsterichindrance phenom-ena.Inaddition,itcanbenotedthatthethreeproteinsstudied,HSA, fibrinogenproteinandlysozymealsohavedifferentcharge char-acteristics.Thesmallestprotein,thelysozymes,adsorbedwhether ornotthePNIPAMwasincollapsedorextendedregime.An expla-nationcanbetheabilityofthissmallproteintopassthroughthe PNIPAMbrushesandthentointeractwiththesubstrate(primary adsorption).Astheproteinsizeincreased,theproteinswereno longerabletoefficientlygothroughPNIPAMchainsbelowtheLCST (extendedregime),butareable,abovetheLCST,tointeractwiththe outermostregionofPNIPAMwhenhydrophobicandmaybewith thesubstrate(collapsedregime).

Comparingthesetwolastresults,itappearsthatinthecaseof polyurethanesubstratethehydrophilicitytendstoincreasewith thethickness,leadingtoa decreaseofproteinadsorption[131], whereastheSisubstrategraftledtoanincreaseofprotein adsorp-tion, as the hydrophobicity increased [132]. Thus, we see the importanceofthesubstrate,andtheresultingsurfaceproperties willdependontheabilityofitssubstratetoallowprimarybinding andonthehydrophilicity/hydrophobicitybalanceoftheresulting

surface.Infact,adsorptionofHSAwasofthesameorderof magni-tudewhateverthesubstrate,forthickerPNIPAMlayers,thelatter havingalsothesamehydrophilicity.

Nanostructuredorpatternedsurfaceswerealsoinvestigated. Silicon nanowires were thus used as a substrate for SI-ATRP

[133].Theadditionof PNIPAMstrongly reducedplatelet activa-tionandadhesion,bothaboveandbelowtheLCST.Asexpected, thenanostructurationofPNIPAMsurfaces(i.e.theincreaseof sur-facearea)involvesanexacerbationofthehydrophilic/hydrophobic surfacestate.Infact,Chenetal.highlightedthatthese nanostruc-tures,whichpresentahighaspectratio,tendtotrapwater.This entrappedwaterledtoareductionoftheplateletprotein/surface interactions,whateverthecoilorglobulestateofPNIPAMbrushes. Theseresultsopennewfieldsofapplicationasplateletactivation andadhesioncanleadtobloodcoagulationandthrombosis.More recently,siliconnanopillarswereshowntobeabletoreversibly attach/detachto/from breast cancer cells, through specific and selectiveinteractions[134].ComparedtoflatSi-PNIPAMsurfaces, theintroductionof nanopillar architecturewidenedthe overall potentialcontactsurfacebutlimitedtheavailablespacefor inter-actions betweencells and surfaces when adhered. Asa result, the3Darchitectureofthesesurfacesenhanced cellcapture, but diminishedthetendencyofcellstospread,makingreleaseeasier. NanopatternedPNIPAMsurfaceswerealsousedtotrap,killand deliverbacteria[135].Inthiswork,biocidesweregraftedbetween patternedSI-ATRPPNIPAMbrushes.Additionally,nanopatterning isapotentialsolutiontoovercomethicknesslimitations:eventhick brushesallowscellstoattach,sothenecessitytohavethickenough brushesinordertodetachcellscanbemoreeasilyfulfilled[136]. OwingtothefactthatthickPNIPAMbrushesdonotsupportcell attachmentbut becomebioadhesive when nanopatterned, con-trolledspatializationofcellcultureispossible[136].

Inthefieldofbodyimplantsandsurgicalbiomaterials,Chenet al.grafted(“graftingto”)PNIPAM-COOHontochitosanthroughan amidebondresultinginacomb-likepolymerstructure(branched PNIPAMonachitosanbackbone)whichformsagel[137].The sur-facefunctionalizationwasfollowedbyastudyofchondrocytesand meniscuscellsbioadhesion.Thethermosensitivebehaviorof PNI-PAM(brushtomushroomthermotriggeredconformationchange) wasshowntoprovokeaphasetransition,liquidtosolid-like hydro-gel.Thegelificationwouldoccurinsidethebodyafterinjection. Fibronectinadsorptionwasobservedbyfluorescenceusing rho-daminelabelledfibronectin.Polypropylene-g-chitosan-g-PNIPAM wasperformedthrougha“graftingto”processwithaviewto eas-ilystrippingoffoftheskinwounddressing[138].Non-toxicityand temperature-responsivenessbehaviorwerefulfilled.

Whilebrushesdo present someinteresting properties,other non-brushsystemshavebeenusedtodevelopthesamekindof functionalities. Ignacio et al. made a wound dressing using UV graftedPNIPAM polyurethane membranes [139]. New subcuta-neousconnectivetissue grewbutnotoxicitywasobserved.The detachmentonmiceskinwoundswastriggeredbythereduction oftemperature below theLCST.We can alsomentionthe easy removalofretinalimplantsachievedwithPNIPAMsurfaces[140]. Inthisstudy,bioadhesion,measuredbyapull-offtest,appearedone minuteafterpassingthroughtheLCST.Thecorrelationbetweenthe cellculturebehaviorandthermo-sensitivetissueadhesionclearly indicatesthat bioadhesionontissues isrelated totheability of PNIPAMtoadsorbproteinsandthuscatchcells.

5. Conclusion

Bioadhesionisacharacteristicofinteractionsbetween materi-alsandcells.Thisphenomenonisnowbetterunderstoodandgives risetointerestingfieldsinbiomedicalsciencesuchascellsheet

engineering.Brushstructureshavebeenshowntobeefficientfor celladhesion,andtooffertheadvantageofawell-controlled prepa-rationprocess.The“graftingfrom”approachenablesdensebrush layerstobemadewithoutsterichindrancelimitationsandthus leadstohomogeneouslayers,especiallyforroughsurfaces.Living polymerizationprovidesawaytocontrolthegrowthofthese lay-ers.Varioustechniquesexist,suchasFe-catalyzedARGET-ATRPor RAFTpolymerizations.Itisknownthatthegraftingmethodcan leadtodifferentproperties(e.g.cross-linking),andimpactthe sur-faceinteractionwithcells.PNIPAM,asathermo-sensitivepolymer, is widelystudied and is a promising polymer inthe cellsheet generationarea, butitsapplicationscanbewider, including,for example,implants.Thus,severalparameterssuchasgraftdensity, layerthicknessandgraftingmethodhavetobestudied, character-izedandcomparedintermsofcytotoxicityandbioadhesion.Asyet, nosolutionshavebeenfoundtothoroughlycharacterizeandstudy brushesdirectlyonpolymersubstrates,this challengewillhave tobeovercomeinthefuture.Thislimitationputsabrakeonthe controlofthesurfacestate,whichisakeypointforthe prospec-tiveworkinbioadhesionandcanallowtheinvestigationofnew insightsinthebioadhesionfield,boththeoreticallyandinterms ofapplications.Insomecases,(i.e.biomedicalimplants) antifoul-ingsurfacesaresoughtinordertolimitthebiologicalcolonization oranyimmunologicalresponse.Onthecontrary,tissue engineer-ingneedsgoodintegration,andthusbioadhesion,ofcellswithin biomaterials.Strongeffortsareneededtofurtherinvestigatethe effectsofthephysio-chemicalparametersofsurfaces: hydrophilic-ity,roughness,mechanicalproperties,patterns.Thedevelopment ofinnovativebiomaterialswillbedependantoftheseadvances.

Acknowledgement

Authors would like to thank the French National Research Agency for its financial support (ANR-14-CE17-0002-01, FP-BioPrevproject).

References

[1]B.Kasemo,Biologicalsurfacescience,Surf.Sci.500(2002)656–677,http://

dx.doi.org/10.1016/S0039-6028(01)01809-X.

[2]J.Yang,M.Yamato,C.Kohno,A.Nishimoto,H.Sekine,F.Fukai,etal.,Cell sheetengineering:recreatingtissueswithoutbiodegradablescaffolds, Biomaterials26(2005)6415–6422,http://dx.doi.org/10.1016/j.biomaterials.

2005.04.061.

[3]M.A.Cooperstein,H.E.Canavan,Assessmentofcytotoxicityof(N-isopropyl acrylamide)andpoly(N-isopropylacrylamide)-coatedsurfaces,

Biointerphases8(2013)19,http://dx.doi.org/10.1186/1559-4106-8-19. [4]A.Tourrette,N.DeGeyter,D.Jocic,R.Morent,M.M.C.G.Warmoeskerken,C.

Leys,Incorporationofpoly(N-isopropylacrylamide)/chitosanmicrogelonto plasmafunctionalizedcottonfibresurface,ColloidsSurf.APhysicochem. Eng.Aspects352(2009)126–135,http://dx.doi.org/10.1016/j.colsurfa.2009.

10.014.

[5]H.Lim,Y.Lee,S.Han,Y.Kim,J.Song,J.Kim,Wettabilityof

poly(styrene-co-acrylate)ionomersimprovedbyoxygen-plasmasourceion implantation,J.Polym.Sci.PartBPolym.Phys.41(2003)1791–1797,http://

dx.doi.org/10.1002/polb.10536.

[6]L.Bacakova,E.Filova,M.Parizek,T.Ruml,V.Svorcik,Modulationofcell adhesion,proliferationanddifferentiationonmaterialsdesignedforbody implants,Biotechnol.Adv.29(2011)739–767,http://dx.doi.org/10.1016/j.

biotechadv.2011.06.004.

[7]A.M.G.Borges,L.O.Benetoli,M.a.Licínio,V.C.Zoldan,M.C.Santos-Silva,J. Assreuy,etal.,Polymerfilmswithsurfacesunmodifiedandmodifiedby non-thermalplasmaasnewsubstratesforcelladhesion,Mater.Sci.Eng.C 33(2013)1315–1324,http://dx.doi.org/10.1016/j.msec.2012.12.031. [8]A.Sdrobis¸,G.E.Ioanid,T.Stevanovic,C.Vasile,Modificationof

cellulose/chitinmixfiberswithN-isopropylacrylamideand

poly(N-isopropylacrylamide)undercoldplasmaconditions,Polym.Int.61 (2012)1767–1777,http://dx.doi.org/10.1002/pi.4268.

[9]Y.M.Lee,J.K.Shim,PreparationofpH/temperatureresponsivepolymer membranebyplasmapolymerizationanditsriboflavinpermeation, Polymer(Guildf)38(1997)1227–1232,

http://dx.doi.org/10.1016/S0032-3861(96)00548-4.

[10]J.A.Reed,S.A.Love,A.E.Lucero,C.L.Haynes,H.E.Canavan,Effectofpolymer depositionmethodonthermoresponsivepolymerfilmsandresulting

cellularbehavior,Langmuir28(2012)2281–2287,http://dx.doi.org/10.

1021/la102606k.

[11]L.Moroni,M.KleinGunnewiek,E.M.Benetti,Polymerbrushcoatings regulatingcellbehavior:passiveinterfacesturnintoactive,ActaBiomater. 10(2014)2367–2378,http://dx.doi.org/10.1016/j.actbio.2014.02.048. [12]A.Olivier,F.Meyer,J.-M.Raquez,P.Damman,P.Dubois,Surface-initiated

controlledpolymerizationasaconvenientmethodfordesigningfunctional polymerbrushes:fromself-assembledmonolayerstopatternedsurfaces, Prog.Polym.Sci.37(2012)157–181,http://dx.doi.org/10.1016/j.

progpolymsci.2011.06.002.

[13]G.Bao,S.Suresh,Cellandmolecularmechanicsofbiologicalmaterials,Nat. Mater.2(2003)715–725,http://dx.doi.org/10.1038/nmat1001.

[14]M.Gao,M.Sotomayor,E.Villa,E.H.Lee,K.Schulten,Molecularmechanisms ofcellularmechanics,Phys.Chem.Chem.Phys.8(2006)3692–3706,http://

dx.doi.org/10.1039/b606019f.

[15]W.Xu,H.Baribault,E.D.Adamson,Vinculinknockoutresultsinheartand braindefectsduringembryonicdevelopment,Development125(1998) 327–337http://www.ncbi.nlm.nih.gov/pubmed/9486805.

[16]K.Anselme,L.Ploux,A.Ponche,Cell/materialinterfaces:influenceofsurface chemistryandsurfacetopographyoncelladhesion,J.Adhes.Sci.Technol.24 (2010)831–852,http://dx.doi.org/10.1163/016942409X12598231568186. [17]D.G.Castner,B.D.Ratner,Biomedicalsurfacescience:foundationsto

frontiers,Surf.Sci.500(2002)28–60,

http://dx.doi.org/10.1016/S0039-6028(01)01587-4.

[18]J.H.Lee,J.W.Lee,G.Khang,H.B.Lee,Interactionofcellsonchargeable functionalgroupgradientsurfaces,Biomaterials18(1997)351–358,http://

dx.doi.org/10.1016/S0142-9612(96)00128-7.

[19]A.Tamura,M.Oishi,Y.Nagasaki,EfficientsiRNAdeliverybasedon PEGylatedandpartiallyquaternizedpolyaminenanogels:enhancedgene silencingactivitybythecooperativeeffectoftertiaryandquaternaryamino groupsinthecore,J.Control.Release.146(2010)378–387,http://dx.doi.

org/10.1016/j.jconrel.2010.05.031.

[20]A.Tamura,M.Nishi,J.Kobayashi,K.Nagase,H.Yajima,M.Yamato,etal., Simultaneousenhancementofcellproliferationandthermallyinduced harvestefficiencybasedontemperature-responsivecationic

copolymer-graftedmicrocarriers,Biomacromolecules13(2012)1765–1773,

http://dx.doi.org/10.1021/bm300256e.

[21]D.Fischer,Y.Li,B.Ahlemeyer,J.Krieglstein,T.Kissel,Invitrocytotoxicity testingofpolycations:influenceofpolymerstructureoncellviabilityand hemolysis,Biomaterials24(2003)1121–1131,http://dx.doi.org/10.1016/

S0142-9612(02)00445-3.

[22]H.Lodish,A.Berk,C.A.Kaiser,M.Krieger,A.Bretscher,H.Ploegh,etal.,

L’intégrationcellulairedansdestissus,in:DeBoeck(Ed.),Biol.Moléculaire

LaCell,4eed.,2014,pp.925–975.

[23]J.Pei,H.Hall,N.D.Spencer,Theroleofplasmaproteinsincelladhesionto PEGsurface-density-gradient-modifiedtitaniumoxide,Biomaterials32 (2011)8968–8978,http://dx.doi.org/10.1016/j.biomaterials.2011.08.034. [24]J.Andersson,K.N.Ekdahl,J.D.Lambris,B.Nilsson,BindingofC3fragments

ontopofadsorbedplasmaproteinsduringcomplementactivationona modelbiomaterialsurface,Biomaterials26(2005)1477–1485,http://dx.doi.

org/10.1016/j.biomaterials.2004.05.011.

[25]K.N.Ekdahl,J.D.Lambris,H.Elwing,D.Ricklin,P.H.Nilsson,Y.Teramura, etal.,Innateimmunityactivationonbiomaterialsurfaces:amechanistic modelandcopingstrategies,Adv.DrugDeliv.Rev.63(2011)1042–1050,

http://dx.doi.org/10.1016/j.addr.2011.06.012.

[26]E.A.Vogler,Structureandreactivityofwateratbiomaterialsurfaces,Adv. ColloidInterfaceSci.74(1998)69–117,

http://dx.doi.org/10.1016/S0001-8686(97)00040-7.

[27]A.J.Pertsin,M.Grunze,Computersimulationofwaternearthesurfaceof oligo(ethyleneglycol)-terminatedalkanethiolself-assembledmonolayers, Langmuir16(2000)8829–8841,http://dx.doi.org/10.1021/la000340y. [28]Z.Ma,Z.Mao,C.Gao,Surfacemodificationandpropertyanalysisof

biomedicalpolymersusedfortissueengineering,ColloidsSurf.B Biointerfaces60(2007)137–157,http://dx.doi.org/10.1016/j.colsurfb.2007.

06.019.

[29]T.Suzuki,Y.Mizushima,Characteristicsofsilica-chitosancomplex membraneandtheirrelationshipstothecharacteristicsofgrowthand adhesivenessofL-929cellsculturedonthebiomembrane,J.Ferment. Bioeng.84(1997)128–132,

http://dx.doi.org/10.1016/S0922-338X(97)82541-X.

[30]C.D.Tidwell,D.G.Castner,S.L.Golledge,B.D.Ratner,K.Meyer,B.Hagenhoff, etal.,Statictime-of-flightsecondaryionmassspectrometryandx-ray photoelectronspectroscopycharacterizationofadsorbedalbuminand fibronectinfilms,Surf.InterfaceAnal.31(2001)724–733,http://dx.doi.org/

10.1002/sia.1101.

[31]B.G.Keselowsky,D.M.Collard,A.J.García,Surfacechemistrymodulates fibronectinconformationanddirectsintegrinbindingandspecificityto controlcelladhesion,J.Biomed.Mater.Res.A66(2003)247–259,http://dx.

doi.org/10.1002/jbm.a.10537.

[32]M.Cockerill,M.K.Rigozzi,E.M.Terentjev,MechanosensitivityoftheIInd kind:tGFbmechanismofcellsensingthesubstratestiffness,PLoSOne (2015)1–11,http://dx.doi.org/10.1371/journal.pone.0139959. [33]D.E.Discher,P.Janmey,Y.Wang,Tissuecellsfeelandrespondtothe

stiffnessoftheirsubstrate,Science310(2005)1139–1143,http://dx.doi.org/

10.1126/science.1116995.

[34]Q.S.Li,G.Y.H.Lee,C.N.Ong,C.T.Lim,AFMindentationstudyofbreastcancer cells,Biochem.Biophys.Res.Commun.374(2008)609–613,http://dx.doi.

org/10.1016/j.bbrc.2008.07.078.

[35]M.S.Yousafzai,F.Ndoye,G.Coceano,J.Niemela,S.Bonin,G.Scoles,etal., Substrate-dependentcellelasticitymeasuredbyopticaltweezers indentation,Opt.LasersEng.76(2016)27–33,http://dx.doi.org/10.1016/j.

optlaseng.2015.02.008.

[36]M.G.Haugh,C.M.Murphy,R.C.McKiernan,C.Altenbuchner,F.J.O’Brien, Crosslinkingandmechanicalpropertiessignificantlyinfluencecell attachment,proliferation,andmigrationwithincollagenglycosaminoglycan scaffolds,TissueEng.PartA.17(2011)1201–1208,http://dx.doi.org/10.

1089/ten.TEA.2010.0590.

[37]C.M.Lo,H.B.Wang,M.Dembo,Y.L.Wang,Cellmovementisguidedbythe rigidityofthesubstrate,Biophys.J.79(2000)144–152,http://dx.doi.org/10.

1016/S0006-3495(00)76279-5.

[38]S.Kidoaki,T.Matsuda,Microelasticgradientgelatinousgelstoinduce cellularmechanotaxis,J.Biotechnol.133(2008)225–230,http://dx.doi.org/

10.1016/j.jbiotec.2007.08.015.

[39]H.-W.Liu,C.-P.Lin,Y.-J.Liou,K.-W.Hsu,J.-Y.Yang,C.-H.Lin,NBT-IIcell locomotionismodulatedbyrestrictingthesizeoffocalcontactsandis improvedthroughEGFandROCKsignaling,Int.J.Biochem.CellBiol.51 (2014)131–141,http://dx.doi.org/10.1016/j.biocel.2014.04.009.

[40]F.G.M.Russel,J.M.Bindels,C.H.Van,Celllocomotionandfocaladhesionsare regulatedbysubstrateflexibility,Proc.Natl.Acad.Sci.U.S.A.94(1997) 13661–13665,http://dx.doi.org/10.1073/pnas.95.20.13661.

[41]L.Bacakova,E.Filova,D.Kubies,L.Machova,V.Proks,V.Malinova,etal., Adhesionandgrowthofvascularsmoothmusclecellsincultureson bioactiveRGDpeptide-carryingpolylactides,J.Mater.Sci.Mater.Med.18 (2007)1317–1323,http://dx.doi.org/10.1007/s10856-006-0074-1. [42]Y.Zhu,C.Gao,X.Liu,T.He,J.Shen,Immobilizationofbiomacromolecules

ontoaminolyzedpoly(L-lacticacid)towardaccelerationofendothelium regeneration,TissueEng.10(2004)53–61,http://dx.doi.org/10.1089/

107632704322791691.

[43]R.D.Abbott,D.L.Kaplan,Strategiesforimprovingthephysiological relevanceofhumanengineeredtissues,TrendsBiotechnol.33(2015) 401–407,http://dx.doi.org/10.1016/j.tibtech.2015.04.003.

[44]C.Xue,B.-C.Choi,S.Choi,P.V.Braun,D.E.Leckband,Proteinadsorption modesdeterminereversiblecellattachmentonpoly(N-isopropyl

acrylamide)brushes,Adv.Funct.Mater.22(2012)2394–2401,http://dx.doi.

org/10.1002/adfm.201103056.

[45]M.A.C.Stuart,W.T.S.Huck,J.Genzer,M.Müller,C.Ober,M.Stamm,etal., Emergingapplicationsofstimuli-responsivepolymermaterials,Nat.Mater. 9(2010)101–113,http://dx.doi.org/10.1038/nmat2614.

[46]J.Ran,L.Wu,Z.Zhang,T.Xu,Atomtransferradicalpolymerization(ATRP):A versatileandforcefultoolforfunctionalmembranes,Prog.Polym.Sci.39 (2014)124–144,http://dx.doi.org/10.1016/j.progpolymsci.2013.09.001. [47]S.Edmondson,V.L.Osborne,W.T.S.Huck,Polymerbrushesvia

surface-initiatedpolymerizations,Chem.Soc.Rev.33(2004)14–22,http://

dx.doi.org/10.1039/b210143m.

[48]J.Draper,I.Luzinov,S.Minko,I.Tokarev,M.Stamm,Mixedpolymerbrushes bysequentialpolymeraddition:anchoringlayereffect,Langmuir20(2004) 4064–4075,http://dx.doi.org/10.1021/la0361316.

[49]B.Zdyrko,I.Luzinov,Polymerbrushesbythegraftingtomethod,Macromol. RapidCommun.32(2011)859–869,http://dx.doi.org/10.1002/marc.

201100162.

[50]K.S.Iyer,B.Zdyrko,H.Malz,J.Pionteck,I.Luzinov,Polystyrenelayersgrafted tomacromolecularanchoringlayer,Macromolecules36(2003)6519–6526,

http://dx.doi.org/10.1021/ma034460z.

[51]M.Husseman,E.E.Malmström,M.McNamara,M.Mate,D.Mecerreyes,D.G. Benoit,etal.,Controlledsynthesisofpolymerbrushesbylivingfreeradical polymerizationtechniques,Macromolecules32(1999)1424–1431,http://

dx.doi.org/10.1021/ma981290v.

[52]N.Ayres,C.D.Cyrus,W.J.Brittain,Stimuli-responsivesurfacesusing polyampholytepolymerbrushespreparedviaatomtransferradical polymerization,Langmuir23(2007)3744–3749,http://dx.doi.org/10.1021/

la062417+.

[53]U.Mansfeld,C.Pietsch,R.Hoogenboom,C.R.Becer,U.S.Schubert,Clickable initiators,monomersandpolymersincontrolledradicalpolymerizations–a prospectivecombinationinpolymerscience,Polym.Chem.1(2010)1560,

http://dx.doi.org/10.1039/c0py00168f.

[54]P.Liu,Modificationofpolymericmaterialsviasurface-Initiated Controlled/Livingradicalpolymerization,EPolymers7(2007)1–3,http://

dx.doi.org/10.1515/epoly.2007.7.1.725.

[55]P.Król,P.Chmielarz,RecentadvancesinATRPmethodsinrelationtothe synthesisofcopolymercoatingmaterials,Prog.Org.Coat.77(2014) 913–948,http://dx.doi.org/10.1016/j.porgcoat.2014.01.027.

[56]C.-Y.Hsiao,H.-A.Han,G.-H.Lee,C.-H.Peng,AGETandSARAATRPofstyrene andmethylmethacrylatemediatedbypyridyl-iminebasedcopper complexes,Eur.Polym.J.51(2014)12–20,http://dx.doi.org/10.1016/j.

eurpolymj.2013.11.013.

[57]N.Singh,X.Cui,T.Boland,S.M.Husson,Theroleofindependentlyvariable graftingdensityandlayerthicknessofpolymernanolayersonpeptide adsorptionandcelladhesion,Biomaterials28(2007)763–771,http://dx.doi.

org/10.1016/j.biomaterials.2006.09.036.

[58]W.Feng,J.Brash,S.Zhu,Non-biofoulingmaterialspreparedbyatom transferradicalpolymerizationgraftingof2-methacryloloxyethyl

phosphorylcholine:separateeffectsofgraftdensityandchainlengthon proteinrepulsion,Biomaterials27(2006)847–855,http://dx.doi.org/10.

1016/j.biomaterials.2005.07.006.

[59]J.Pyun,T.Kowalewski,K.Matyjaszewski,Synthesisofpolymerbrushes usingatomtransferradicalpolymerization,Macromol.RapidCommun.24 (2003)1043–1059,http://dx.doi.org/10.1002/marc.200300078.

[60]J.Qin,Z.Cheng,L.Zhang,Z.Zhang,J.Zhu,X.Zhu,Ahighlyefficient iron-mediatedAGETATRPofmethylmethacrylateusingFe(0)powderas thereducingagent,Macromol.Chem.Phys.212(2011)999–1006,http://dx.

doi.org/10.1002/macp.201000737.

[61]R.Gong,S.Maclaughlin,S.Zhu,Surfacemodificationofactivemetals throughatomtransferradicalpolymerizationgraftingofacrylics,Appl.Surf. Sci.254(2008)6802–6809,http://dx.doi.org/10.1016/j.apsusc.2008.04.101. [62]C.Hou,R.Qu,C.Sun,C.Ji,C.Wang,L.Ying,etal.,Novelionicliquidsas

reactionmediumforATRPofacrylonitrileintheabsenceofanyligand, Polymer(Guildf)49(2008)3424–3427,http://dx.doi.org/10.1016/j.polymer.

2008.06.013.

[63]Y.Wang,Y.Zhang,B.Parker,K.Matyjaszewski,ATRPofMMAwithppm levelsofironcatalyst,Macromolecules44(2011)4022–4025,http://dx.doi.

org/10.1021/ma200771r.

[64]J.Cao,L.Zhang,X.Jiang,C.Tian,X.Zhao,Q.Ke,etal.,Facileiron-mediated dispersant-freesuspensionpolymerizationofmethylmethacrylatevia reverseATRPinwater,Macromol.RapidCommun.34(2013)1747–1754,

http://dx.doi.org/10.1002/marc.201300513.

[65]L.Zhang,Z.Cheng,Z.Zhang,D.Xu,X.Zhu,Fe(III)-catalyzedAGETATRPof styreneusingtriphenylphosphineasligand,Polym.Bull.64(2009) 233–244,http://dx.doi.org/10.1007/s00289-009-0139-7.

[66]C.Bolm,J.Legros,J.LePaih,L.Zani,Iron-catalyzedreactionsinorganic synthesis,Chem.Rev.104(2004)6217–6254,http://dx.doi.org/10.1021/

cr040664h.

[67]R.Poli,L.E.N.Allan,M.P.Shaver,Iron-mediatedreversibledeactivation controlledradicalpolymerization,Prog.Polym.Sci.39(2014)1827–1845,

http://dx.doi.org/10.1016/j.progpolymsci.2014.06.003.

[68]S.-I.Nakanishi,M.Kawamura,H.Kai,R.-H.Jin,Y.Sunada,H.Nagashima, Well-definedironcomplexesasefficientcatalystsforgreenatom-transfer radicalpolymerizationofstyrene,methylmethacrylate,andbutylacrylate withlowcatalystloadingsandcatalystrecycling,Chemistry20(2014) 5802–5814,http://dx.doi.org/10.1002/chem.201304593.

[69]M.KleinGunnewiek,A.DiLuca,X.Sui,C.A.vanBlitterswijk,L.Moroni,G.J. Vancso,Controlledsurfaceinitiatedpolymerizationof

N-isopropylacrylamidefrompolycaprolactonesubstratesforregulatingcell attachmentanddetachment,Isr.J.Chem.52(2012)339–346,http://dx.doi.

org/10.1002/ijch.201100118.

[70]J.Liu,W.He,L.Zhang,Z.Zhang,J.Zhu,L.Yuan,etal.,Bifunctional nanoparticleswithfluorescenceandmagnetismviasurface-initiatedAGET ATRPmediatedbyanironcatalyst,Langmuir27(2011)12684–12692,

http://dx.doi.org/10.1021/la202749v.

[71]K.Matyjaszewski,W.Jakubowski,K.Min,W.Tang,J.Huang,W.A. Braunecker,etal.,Diminishingcatalystconcentrationinatomtransfer radicalpolymerizationwithreducingagents,Proc.Natl.Acad.Sci.U.S.A. 103(2006)15309–15314,http://dx.doi.org/10.1073/pnas.0602675103. [72]W.Jakubowski,K.Matyjaszewski,Activatorgeneratedbyelectrontransfer

foratomtransferradicalpolymerization,Macromolecules38(2005) 4139–4146,http://dx.doi.org/10.1021/ma047389l.

[73]K.Min,H.Gao,K.Matyjaszewski,Useofascorbicacidasreducingagentfor synthesisofwell-definedpolymersbyARGETATRP,Macromolecules40 (2007)1789–1791,http://dx.doi.org/10.1021/ma0702041.

[74]B.V.Bhut,K.a.Conrad,S.M.Husson,Preparationofhigh-performance membraneadsorbersbysurface-initiatedAGETATRPinthepresenceof dissolvedoxygenandlowcatalystconcentration,J.Memb.Sci.390–391 (2012)43–47,http://dx.doi.org/10.1016/j.memsci.2011.10.057.

[75]P.Shivapooja,L.K.Ista,H.E.Canavan,G.P.Lopez,ARGET–ATRPsynthesisand characterizationofPNIPAAmbrushesforquantitativecelldetachment studies,Biointerphases7(2012)1–9,

http://dx.doi.org/10.1007/s13758-012-0032-z.

[76]Y.Zhang,Y.Wang,C.Peng,M.Zhong,W.Zhu,D.Konkolewicz,etal., Copper-MediatedCRPofmethylacrylateinthepresenceofmetalliccopper: effectofligandstructureonreactionkinetics,Macromolecules45(2012) 78–86,http://dx.doi.org/10.1021/ma201963c.

[77]C.H.Worthley,K.T.Constantopoulos,M.Ginic-Markovic,R.J.Pillar,J.G. Matisons,S.Clarke,Surfacemodificationofcommercialcelluloseacetate membranesusingsurface-initiatedpolymerizationof2-hydroxyethyl methacrylatetoimprovemembranesurfacebiofoulingresistance,J.Membr. Sci.385–386(2011)30–39,http://dx.doi.org/10.1016/j.memsci.2011.09.017. [78]X.Qiu,X.Ren,S.Hu,Fabricationofdual-responsivecellulose-based

membraneviasimplifiedsurface-initiatedATRP,Carbohydr.Polym.92 (2013)1887–1895,http://dx.doi.org/10.1016/j.carbpol.2012.11.080. [79]K.Pan,X.Zhang,R.Ren,B.Cao,Doublestimuli-responsivemembranes

graftedwithblockcopolymerbyATRPmethod,J.Memb.Sci.356(2010) 133–137,http://dx.doi.org/10.1016/j.memsci.2010.03.044.

[80]C.Zhang,P.T.Vernier,Y.H.Wu,W.Yang,Surfacechemicalimmobilizationof paryleneCwiththermosensitiveblockcopolymerbrushesbasedon N-isopropylacrylamideandN-tert-butylacrylamide:synthesis,

characterization,andcelladhesion/detachment,J.Biomed.Mater.Res.—Part BAppl.Biomater.100B(2012)217–229,http://dx.doi.org/10.1002/jbm.b.

31941.

[81]W.Zhen,C.Lu,Surfacemodificationofthermoplasticpoly(vinyl alcohol)/saponitenanocompositesviasurface-initiatedatomtransfer radicalpolymerizationenhancedbyairdielectricdischargesbarrierplasma treatment,Appl.Surf.Sci.258(2012)6969–6976,http://dx.doi.org/10.1016/

j.apsusc.2012.03.145.

[82]J.-K.Chen,C.-Y.Hsieh,C.-F.Huang,P.Li,Characterizationofpatterned poly(methylmethacrylate)brushesundervariousstructuresuponsolvent immersion,J.ColloidInterfaceSci.338(2009)428–434,http://dx.doi.org/10.

1016/j.jcis.2009.06.040.

[83]Y.Liu,V.Klep,B.Zdyrko,I.Luzinov,Synthesisofhigh-densitygrafted polymerlayerswiththicknessandgraftingdensitygradients,Langmuir21 (2005)11806–11813,http://dx.doi.org/10.1021/la051695q.

[84]S.J.Eichhorn,A.Dufresne,M.Aranguren,N.E.Marcovich,J.R.Capadona,S.J. Rowan,etal.,Review:currentinternationalresearchintocellulose nanofibresandnanocomposites,J.Mater.Sci.45(2009)1–33,http://dx.doi.

org/10.1007/s10853-009-3874-0.

[85]J.Lindqvist,E.Malmström,Surfacemodificationofnaturalsubstratesby atomtransferradicalpolymerization,J.Appl.Polym.Sci.100(2006) 4155–4162,http://dx.doi.org/10.1002/app.23457.

[86]C.Kang,R.M.Crockett,N.D.Spencer,Molecular-weightdeterminationof polymerbrushesgeneratedbySI-ATRPonflatsurfaces,Macromolecules47 (2014)269–275,http://dx.doi.org/10.1021/ma401951w.

[87]M.Barsbay,O.Güven,Ashortreviewofradiation-inducedraft-mediated graftcopolymerization:apowerfulcombinationformodifyingthesurface propertiesofpolymersinacontrolledmanner,Radiat.Phys.Chem.78 (2009)1054–1059,http://dx.doi.org/10.1016/j.radphyschem.2009.06.022. [88]C.Barner-Kowollik,T.P.Davis,J.P.a.Heuts,M.H.Stenzel,P.Vana,M.

Whittaker,RAFTingdownunder:talesofmissingradicals,fancy architectures,andmysteriousholes,J.Polym.Sci.PartAPolym.Chem.41 (2003)365–375,http://dx.doi.org/10.1002/pola.10567.

[89]K.Kusolkamabot,P.Sae-ung,N.Niamnont,K.Wongravee,M.

Sukwattanasinitt,V.P.Hoven,Poly(N-isopropylacrylamide)-Stabilizedgold nanoparticlesincombinationwithtricationicbranched

phenylene-Ethynylenefluorophoreforproteinidentification,Langmuir29 (2013)12317–12327,http://dx.doi.org/10.1021/la402139g.

[90]H.Takahashi,M.Nakayama,M.Yamato,T.Okano,Controlledchainlength andgraftdensityofthermoresponsivepolymerbrushesforoptimizingcell sheetharvest,Biomacromolecules11(2010)1991–1999,http://dx.doi.org/

10.1021/bm100342e.

[91]J.Bigot,D.Fournier,J.Lyskawa,T.Marmin,F.Cazaux,G.Cooke,etal., Synthesisofthermoresponsivephenyl-andnaphthyl-terminated poly(NIPAM)derivativesusingRAFTandtheircomplexationwith cyclobis(paraquat-p-phenylene)derivativesinwater,Polym.Chem.1 (2010)1024–1029,http://dx.doi.org/10.1039/c0py00085j.

[92]HandbookofRAFTPolymerization,in:C.Barner-Kowollik(Ed.),Wiley-VCH VerlagGmbH&Co.KGaA,Weinheim,Germany,2008,http://dx.doi.org/10.

1002/9783527622757.

[93]F.Audouin,A.Heise,Surface-initiatedRAFTpolymerizationofNIPAMfrom monolithicmacroporouspolyHIPE,Eur.Polym.J.49(2013)1073–1079,

http://dx.doi.org/10.1016/j.eurpolymj.2013.01.013.

[94]H.Alem,A.-S.Duwez,P.Lussis,P.Lipnik,A.M.Jonas,S.

Demoustier-Champagne,Microstructureandthermo-responsivebehavior ofpoly(N-isopropylacrylamide)brushesgraftedinnanoporesof track-etchedmembranes,J.Membr.Sci.308(2008)75–86,http://dx.doi.

org/10.1016/j.memsci.2007.09.036.

[95]I.Lokuge,X.Wang,P.W.Bohn,Temperature-Controlledflowswitchingin nanocapillaryarraymembranesmediatedbyPoly(N-isopropylacrylamide) polymerbrushesgraftedbyatomtransferradicalpolymerization†,

Langmuir23(2007)305–311,http://dx.doi.org/10.1021/la060813m. [96]L.Ren,S.Huang,C.Zhang,R.Wang,W.W.Tjiu,T.Liu,Functionalizationof

grapheneandgraftingoftemperature-responsivesurfacesfromgraphene byATRPonwater,J.NanoparticleRes.14(2012),http://dx.doi.org/10.1007/

s11051-012-0940-3.

[97]P.Zhuang,A.Dirani,K.Glinel,A.M.Jonas,Temperaturedependenceofthe surfaceandvolumehydrophilicityofhydrophilicpolymerbrushes, Langmuir32(2016)3433–3444,http://dx.doi.org/10.1021/acs.langmuir.

6b00448.

[98]W.Wu,J.Li,W.Zhu,Y.Jing,H.Dai,Thermo-responsivecellulosepapervia ARGETATRP,FibersPolym.17(2016)495–501,http://dx.doi.org/10.1007/

s12221-016-5877-1.

[99]L.L.Yang,J.M.Zhang,J.S.He,J.Zhang,Z.H.Gan,Synthesisand characterizationoftemperature-sensitive

cellulose-graft-poly(N-isopropylacrylamide)copolymers,Chin.J.PolymSci. (EnglishEd.)33(2015)1640–1649,

http://dx.doi.org/10.1007/s10118-015-1703-2.

[100] R.B.Vasani,S.J.P.McInnes,M.A.Cole,A.M.M.Jani,A.V.Ellis,N.H.Voelcker, Stimulus-responsivenessanddrugreleasefromporoussiliconfilms ATRP-graftedwithpoly(N-isopropylacrylamide),Langmuir27(2011) 7843–7853,http://dx.doi.org/10.1021/la200551g.

[101] P.-W.Chung,R.Kumar,M.Pruski,V.S.-Y.Lin,Temperatureresponsive solutionpartitionoforganic-Inorganichybrid

poly(N-isopropylacrylamide)-coatedmesoporoussilicananospheres,Adv. Funct.Mater.18(2008)1390–1398,http://dx.doi.org/10.1002/adfm.

200701116.

[102] A.Hufendiek,V.Trouillet,M.A.R.Meier,C.Barner-Kowollik,Temperature responsivecellulose-graft-copolymersviacellulosefunctionalizationinan