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: DOI: 10.1016/j.colsurfb.2016.12.007
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: 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
a,
S.
Cavalie
a,
C.
Combes
b,
J.
Torrisani
c,
N.
Carrere
c,d,
A.
Tourrette
a,∗aCIRIMAT,UniversityofToulouse,CNRS,INPT,UPS,UniversitéPaulSabatier,FacultédePharmacie,35ChemindesMaraichers,31062Toulousecedex9, France
bCIRIMAT,UniversitédeToulouse,CNRS,INPT,UPS,ENSIACET,4alléeEmileMonso,CS44362,31030Toulousecedex4,France
cUniversitéFédéraleMidi-Pyrénées,UniversitédeToulouseIIIPaulSabatier,InsermU1037,CRCTdeToulouse,2avenueHubertCurienCS5371731037 ToulouseCedex1,France
dGastrointestinalSurgeryDepartment,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 newbiomaterialsinapplicationsinvolvinginnovativestrategies
∗ Correspondingauthor.
E-mailaddress:audrey.tourrette@univ-tlse3.fr(A.Tourrette).
for disease treatment. These biomaterialshave to fulfilvarious requirementsdependingontheapplicationbutinallcases 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 in thefunction 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.
Plasmatreatmentis probablythemostcommonprocess 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 the case 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]. Theintroductionof reactivegroups alsopermits compoundsor chemical functionsto begrafted onto thematerial surfaces. In thepresence of oxygen, surfacesactivated viatheintroduction 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 lowlevel ofsurfacestructuring 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, ATRPandRAFTpolymerizationmethodswillbereviewed along withthewaysinwhichtheyhavebeencharacterized.Finally, engi-neeringoftunablesurfacesusingPNIPAMwillbepresentedwith itsmainandmostrecentapplications.
2. Bioadhesion
2.1. Bioadhesionmechanisms
Withasizefrom1to100mm,cellsarecomposedofvarious enti-ties.Somearededicatedtoitsstructure:aphospholipidbilayer, thecellmembrane,maintainstheseparationbetweenthe cyto-sol(internalliquidphase)andtheextra-cellularmatrix(ECM)and thecytoskeleton,composedmainlyofmicrotubulesandactin fil-amentnetworks, control therigidity of the structure [13]. The ECMcompositionvariesdependingonthetissueconcerned.It con-tainsanumberofproteins:fibronectin,collagen,laminin,butalso growthfactorsandalltheproteinsneededfor cellsupportand 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 areresponsiblefor cell adhesiontosurfaces(intercellularcohesionisregulatedbyother mechanisms,e.g. cadherin-mediated homotypic 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.Thissecondstepleadstoa cascadeofactionsincluding 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 human foreskin 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.
Amodel involvinga 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].Thisgroupshowedthatwater/surfaceinteractionsare 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)surfacewitha contactanglearound70◦.Additionally,it isnowacceptedthatsurfaceswhicharetoohydrophobicleadto thedenaturationofproteins[1,28].Moreprecisely,hydrophobic andhydrophilic aminoacids,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 the geometrical 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 surroundingmediumnotonlyviachemicalstimulationbutalso 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 stiffnesscontrolloop is setup:cytoskeletonand adhesionwilladaptdepending onthe feedback.Asaconsequence,stiffermatricesleadtoanincreasein theelasticityofthecells,and canbemeasuredbyatomicforce 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,dependingon theirtype, 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.3Darchitecturesarecommonlymodulated inthefield of 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)
Asisgenerallythecase 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, respectively asshown inFig. 4.Atthe presenttime, due toits highcatalyticactivitycopperisusedmost[61,62].Iron catalyzed-ATRPcanbeperformedusinglowamountsofcatalyst,reducing thetoxicologicalrisksasiron isconsideredlesstoxicand 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,thistechniqueincreasestheairtolerance andcan 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 copper catalyzed 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. ATRPcanbeachievedincombination withplasma 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 initiatorssuch as azobisisobutyronitrile(AIBN)or4,4′ -azobis(4-cyanovalericacid),whichhastheadvantageofbeingcarboxylic acidend-functionalized.Achaintransferagent(CTA)(alsocalled RAFTagent)ensuresthisequilibriumduringthepropagationsteps, asshowninFig.5[87,88].Thereductionofactivechain concen-trationresultsinanarrowdistributionofthechainlength,witha polydispersityindex(PDI),forPNIPAM,abletoreachvaluesaround 1.20,but PDIbelow1.10canbeobtainedinoptimalconditions
[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 stabilizingthe adductradical, Risaleaving group.A completediscussion aboutthechoiceof theRAFTagentisavailableintheliterature[92].Thesurface initi-atedRAFT(SI-RAFT)polymerizationofPNIPAMcanallowtoobtain polydispersitiesbelow1.3[93].Thankstothesulfurylgroupsof theCTA,elementalanalysiscan,insomecases,beusedto deter-minethequantityofgraftedRAFTagent.Thetheoreticalmolecular massofthepolymer,Mnth,canbeestimatedthroughthefollowing equation:
Mnth= [Mono]0 [CTA]0
×MMono×con
v
.+MCTAwhereMMonocorrespondstothemolecularweightofthemonomer, [Mono]0toitsinitialconcentration,conv.istheconversionrateof themonomer,MCTAisthemolecularweightoftheCTAand[CTA]0 itsinitialconcentration[91,93].
InordertoperformSI-RAFTpolymerization,theinitiator[90]
ortheCTA[93]havetobepreviouslygraftedtothesurface.Inthe firstcasethehomolyticcleavageoftheinitiatorwillleadtogrowth eitheronthesurfaceorinthemedium,bothwiththefreeCTA.In thecaseofCTAgraftedsurfaces,theinitiatorandanotheramount oftheRAFTagentareintroducedtopermitthepolymerizationof freechainsandtheircharacterization.FewexamplesofSI-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.From opticalwaveguide lightmodespectroscopy(OWLS)themassofadepositedpolymer canalsobeobtained[23].
Moreover,themorphology,graft densityand 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:
s= hrNA Mn
withsthegraftdensity,hthedrythickness,NAAvogadro’s num-berandMnthemolecularweight.ThedensityofdryPNIPAM,r,is sometimesarbitrarilytakenequalto0.95g/cm3bycertainauthors
[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(afewnmforXPSagainstupto1mm 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 affinityaccording tothetemperatureof thesurrounding medium,turningfrom hydrophilictohydrophobic.Thischange occurs at around32◦C, whose temperature is calledthe lower criticalsolutiontemperature(LCST),andleadstoachangeinits conformation.Abovethistemperature,PNIPAMcollapsesin solu-tion.Thiscoiltoglobuletransitionisendothermicandisrelated towater/polymerand polymerinterand intra-molecule hydro-genbonding.In otherwords,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−andCH 3COO− 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, occurs for small particles, 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)facilitates primaryandternaryadsorbed 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].It isobviousthatinter-chainattractive interactionswouldplayaroleonproteininclusion.
VariouscellsadheretoheatedPNIPAMsurfacesandarereleased during cooling [122]. Typically, cell detachment is 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 hastriedtodeterminethecompositionofthis 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,noadhesionisobserved fortemperatures belowLCST 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 andmolecular weightof 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 PNIPAMstronglyreducedplatelet 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/detach to/from breast cancer cells, through specific and selectiveinteractions[134].ComparedtoflatSi-PNIPAMsurfaces, theintroduction ofnanopillar architecturewidenedthe overall potentialcontactsurfacebutlimitedtheavailablespacefor inter-actions between cells and surfaceswhen adhered. As a result, the3Darchitectureofthesesurfacesenhancedcellcapture, 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.
Whilebrushes dopresent someinterestingproperties,other non-brushsystemshavebeenusedtodevelopthesamekindof functionalities. Ignacio et al. made a wound dressing using UV grafted PNIPAMpolyurethane membranes [139]. New subcuta-neousconnectivetissuegrewbutnotoxicitywasobserved.The detachmentonmiceskinwoundswastriggeredbythereduction of temperaturebelow theLCST. We canalsomentionthe easy removalofretinalimplantsachievedwithPNIPAMsurfaces[140]. Inthisstudy,bioadhesion,measuredbyapull-offtest,appearedone minuteafterpassingthroughtheLCST.Thecorrelationbetweenthe cellculturebehaviorandthermo-sensitivetissueadhesionclearly indicates thatbioadhesionontissuesisrelated 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.It isknownthatthegraftingmethodcan leadtodifferentproperties(e.g.cross-linking),andimpactthe sur-faceinteractionwithcells.PNIPAM,asathermo-sensitivepolymer, is widelystudied and is a promising polymerin the 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).
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