Development of a polystyrene sulfonate/silver nanocomposite with self-healing properties for biomaterial applications

Development

of

a

polystyrene

sulfonate/silver

nanocomposite

with

self-healing

properties

for

biomaterial

applications

Je´roˆme

Girard

a

,

Priscilla

S.

Brunetto

a

,

Olivier

Braissant

b

,

Zarko

Rajacic

c

,

Nina

Khanna

c

,

Regine

Landmann

c

,

Alma

U.

Daniels

b

,

Katharina

M.

Fromm

a,

*

a

DepartmentofChemistry,UniversityofFribourg,9,cheminduMuse´e,1700Fribourg,Switzerland

b_{Biomechanics&CalorimetryCenterBasel/Pharmazentrum,UniversityofBasel,Klingelbergstrasse50-70,4056Basel,Switzerland}

c_{InfectionBiology,DepartmentofBiomedicine,UniversityHospital,4056Basel,Switzerland}

1. Introduction

Nowadays, more and more biomaterials, especially polymeric ones, are used for insertion into the body. Despite sterilization procedures, bacterial infection remains a major problem of medical implants, suchas catheters,prostheticjointimplants.In2004,intheUnited States, over 3.6 million implants were inserted in the humanbodyandmorethan4%ofthembecameinfected [1].Onepossiblewaytopreventtheseinfectionsistheuse ofsilver-basedcompoundse.g.incoatingsorasingredient in thebiomaterial itself, as silver is wellknown for its antimicrobialeffectandthissinceantiquity[2].Thereare twomaindifferentwaystoassemblesilverandpolymer: thefirstoneisthesynthesisofmetallopolymerscontaining silver ions (orcoordination polymers), a methodwhich

waspreviouslystudiedinourgroup[3].Thesecondoneis the use of silver nanoparticles, which are directly generatedinthepolymermatrixorbymixingapolymer with already prepared silver nanoparticles [4]. Silver nanoparticlescanbeeasilysynthesizedfromsilversalts, generally silver nitrate, by using a reducing agent, like ascorbicacid,citricacid, sodiumborohydride,hydrazine hydrate and, undercertain conditions,theycan alsobe obtainedbylightreduction[5].Onemajorchallengewith nanoparticlesistheir stability,astheynaturallytendto aggregate to form larger particles, a reason why often surfacestabilizingagentsareaddedduringthesynthesisof nanoparticles[6].Thesenanoparticlescaneasilybemixed witha polymerduringtheextrusionprocessinorderto haveagoodhomogeneity ofthefinalcompositesample [7].Onedrawbackofthismethodisthatonlynanoparticles near the surface can release silver ions to act as antimicrobial agent,while mostof them remain in the bulkof thepolymerand neverreact.Thisrendersthese materialsratherexpensive.Onesolutioncanbetodesign

Asilverpolystyrenesulfonatepolyelectrolytewassynthesized,showingaspontaneous

reductionofsilverionstosilvernanoparticlesoveraperiodofapproximately1month.A

follow-upofthenanoparticleformationviascanningelectronmicroscopy(SEM)reveals

migrationofthenanoparticlestowardsthecracksofthepolymerovertime,leadingtoa

self-healingprocessofthenanocomposite.Antibacterialtestsshowexcellentantibacterial

activityofourcompound,whichallowsustousethiscompounde.g.forexternalmedical

applications.

* Correspondingauthor.

E-mailaddress:katharina.fromm@unifr.ch(K.M.Fromm).

Published in "&RPSWHV5HQGXV&KLPLH±

which should be cited to refer to this work.

polymersinwhichnanoparticlescanmigratetoreache.g. thesurfaceofthepolymeror,inviewofself-healing,the cracksofthepolymer[8].

Inthiscontext,wedecidedtostudypolyelectrolytes,in particular polystyrene sulfonate. A polyelectrolyte is a polymer inwhich a substantial portionof the constitu-tionalunitscontainsionicorionizablegroups,orboth[9]. These groups can be acids, bases, or salts that will dissociate in solution to form a charged polymer. The use of a negatively charged polyelectrolyte has the advantagetobeabletoformasaltwithsilverions,and that,comparedtocoordinationpolymersbasedonneutral ligands,theanionoftheinitialsilversaltcanbeeliminated. Indeed, the counter ion can have important effects regarding the biocompatibility and the antimicrobial activity itself of a compound [10]. Polystyrene sulfonic acid(PSS)isapolymermadebythedirectsulfonationof polystyrene[11].Recently,anewmethodtosynthetizePSS useslivingpolymerizationofprotectedstyrenesulfonates [12].ThesodiumsaltsofPSSareusedassuperplastifiersin cements, dye-improvingagents, proton exchange mem-branesinfuelcells,andalsoinionexchangeresinswhen PSSiscrosslinked.Itisalsousedassodiumorcalciumsalt, asadrugforhyperkalemia[13].

Inordertoeasilyeliminatetheanioninthepreparation ofsilverPSScomposites,weusedsilveroxideasstarting silver salt andammonia todeprotonatethePSS. Inthis work, wepresent a newPSS/silvernanocompositewith self-healing properties due to the migration of the nanoparticles towards the cracks of the polymer. This nanocompositealsoshowsgood antibacterialproperties evenatlowconcentrations.

2. Resultsanddiscussion

2.1. ComplexationofPSSwithsilveroxide

Inordertogeneratecomplexesof PSSwithsilver,a solution of poly(4-styrene sulfonic) acid in water was mixedwithasolutionofsilveroxideindilutedammonia. After 1day of mixing, water was evaporated under vacuumtoyieldaviscoussolution,whichwastransferred inaPetridishatroomtemperatureinthedarkuntilasolid, pale yellow film of PSS–Ag is formed. X-ray powder diffraction(XRPD)analysiswasperformedafterthefilm formation (Supplementary data Fig. S1). The diffracto-gram showed only the contribution of the pure PSS polymer with a broad peak at 188, indicating an amorphousstructure.Wedidnotobserveanypeakfrom thesilveroxidealoneorfromapolymersilvercomplex. Thus,theformedcomplexislikelytobeamorphousorhas thesamestructureasPSSalone.Theformedcompositeis paleyellow just after thefilm formation, but overthe periodof1month,itshowscolorchangesfromyellowto paleorange(SupplementarydataFig.S2).Thisrangeof colorsindicatestypicallythesilvernanoparticle forma-tion stages,from smalltolarger nanoparticles[14]. To confirmthis,anXRPDwasperformed.Thediffractogram after1monthwasdifferentfromthefirstone(Fig.1).The peakat188isstillpresent,butnewpeaksappeared.All newpeakscanbeattributedtometallicsilverAg0_.The

average size of the crystallite was determined by the Scherrerequation[15]:

d¼

_b

K

l

cos

u

wheredisthemeansizeofthecrystallite,Kistheshape factor,

l

istheX-raywavelength,

b

isthefullpeakwidthat halfmaximumand

u

istheBraggangleofthe correspond-ingpeak.

Assumingthatwehavesphericalnanoparticles,which arethemostcommonformfor thesilvernanoparticles, thisequationgivesavalueofca17nmforthesizeofthe silver crystallites. For confirmation and in order to determinetherealparticle sizeandshape, transmission electronmicroscopy(TEM)imagesofthesilver nanopar-ticleswerecarriedout.Todoso,thesamplewasdissolved inwaterandcentrifugedtoremovemostofthepolymer. Fig. 2 shows that the polymer could not be removed completely even after high centrifugation speed (15,000rpm), as it actsvery likely as surface stabilizer forthenanoparticlesthemselves.Thisisconfirmedbythe absence of agglomerates of nanoparticles in the TEM

Fig.1.X-ray powderdiffractionofthenanocomposite after1month

(blue),silvertheoreticalpattern(purple).

Fig.2.Transmissionelectronmicroscopyimageofthenanocomposite

aftercentrifugation.

picture.Theanalysisof67differentparticlesontheimage givesanaveragediameterof2513nm,whichisinthe rangeofthesizeofthecrystallitesizefoundbypowderX-ray analysis. Itcan thusbe concludedthat thecrystallitesize correspondstotheparticlesize.

Wetriedtoelucidatetheprocessofsilverreductionto nanoparticles,butcouldnotfindanychangestothePSSby NMRorotheranalyticalmeans,whichcouldgivehintsto anoxidationprocessinthepolymer.Possiblepathwaysof thesilverreductioncouldthusbe:

autocatalyticlightreduction[16](althoughwetriedto workinthedarkasmuchaspossible);

reductionincontactwithair/water;

incompletesulfonationofthepolymerwithremaining polystyrenegroups,thelatterofwhichcanbeoxidized [17].ThechemicalreductionofthesilverionsinthePSS– Agnanocompositebyaddingascorbicacidyieldssilver nanoparticlesof16038nmonaverage.

2.2. Kineticsofnanoparticleformation

In order to study the kinetics of the nanoparticle formation,twodifferentmethodsweretested:ICPtitration and isothermal microcalorimetry. The first one was realized by dissolving the PSS-Ag composite in water andsubsequentseparationofthesilverionsby centrifuga-tionfromthepolymerandthenanoparticles,followedby theanalysisoftheremainingconcentrationofsilverionsin the solution.The resultsobtained bythis method were however not coherent, exhibitinghuge variances in the concentrationofremainingsilverions.Oneexplanationfor thiseffectcanbethecontentofwater(relativehumidity) of the polymer,which wasshown tovary dramatically from 1day to the next, resulting in a variation of the concentrationoftheremainingsilver.Thesecondreason wastheincompleteseparation.Acompleteseparationof thepolymerandthenanoparticlesevenatultra-highspeed (>20,000rpm)couldnotbeachieved.TEMimagesofthe supernatant solutionalwaysshowremaining nanoparti-cles in the solution, surrounded by polymer, hence, falsifyingtheICP-measurementevenafterfiltration.The second method tested to follow the kinetics of silver nanoparticleformationwasisothermalmicrocalorimetry. Mostchemicalreactionsreleaseoradsorbheat,whichcan be followedwitha microcalorimeter over many daysif thesereactions areslow.Thismethodwasthus usedto follow the reduction of silver in our sample. A freshly preparedpolymerfilm wasintroducedina microcalori-metervessel,andthetemperaturewaskeptat37_8Cduring theexperiment.Atthebeginningoftheexperiment,the sampleshowedasmallreleaseofheat(104Jfor1.2gof sample,which means6.5J/mol)which decreasedslowly withtimeto7.107J(4.102permol)after20days.Ifwe assumethatallthereleasedenergycomesfromthesilver reduction,thenanoparticleformationagainsttimecanbe plotted(Fig.3).Thecurvehasalogarithmicbehaviorwitha fast formation atthebeginning (around50% in 2days), then,theformationbecomesslowerandslower.Withthis method, we couldonly seetherateof formationof the nanoparticles,andwedidnotgaininformationifthesilver

reductionwastotalorifsilverionswerestillpresentinthe sample.

2.3. Follow-upbySEM

A freshly prepared sample was placed on a sample holderandsputteredwithathinlayercarbontoimprove thedissipationoftheelectrons.Allimageswererecorded withaback-scatteredelectrondetector[18](Fig.4).The firstSEMimageatlowmagnificationwasrecorded2days afterthesynthesis.Thesurfaceisregularwithfewwhite spotsofasizeofca.100nm,atthismagnificationonlythe biggeronesarevisible,correspondingtosilver nanopar-ticles.Thepolymeritselfappearsalsoquitebrightdueto the highsilver ion concentration remaining in it. After 5days,manycracksbecomevisibleatthesurfaceofthe sample. This crack formation could be due to thehigh vacuum required for the SEM, but even without any vacuum,wealsoobservedtheformationofmacro-cracks on the sample due to drying. More nanoparticles are present with different sizes, most of them have a size between 20nm to 50nm and few of them are around 200nm.Particlessmallerthan20nmwerenotvisiblewith thismagnification.Athighmagnificationafter9days,the nanoparticles are homogeneously distributed in the polymer and their size is in thesame range as before. Thebordersofthecracksarebrighter thantheaverage: thiscanbeexplainedbytwophenomena,thefirstoneis the difference of height between the border and the polymeritselfandthesecondoneisahigherconcentration ofsilverattheborderduetoe.g.aphaseseparation.After 24days, many more nanoparticles are present, still homogeneously distributed. After 1month, the borders ofthecracksaremuchbrighterthantherestofthepolymer andmostofthelargernanoparticlesarenearsuchaborder. These two observations clearly indicate a separation processbetweenthesilvernanoparticlesandthepolymer. Thisisconfirmedbytheimagetakenafter2months,where the brighter cracksseem to befilled by silver. As seen previously,thecontentofwaterisvaryingalot,depending onthestorageconditionsandrelativeairhumidity.This watercontentcouldhelpsilverionstomigratenearacrack wheretheywillbereducedagainincontactwithairand

Fig.3.Heatflowofthenanocompositeasafunctionoftime(redcurve–

decreasing)andcorrespondingformationpercentageofnanoparticles

(bluecurve–increasing).

water.AnSEMimageatlowmagnificationrevealsthatthe cracksarepartiallyfilledandalsoshowsagglomerationof nanoparticles in parts of the composite sample. This migrationofthenanoparticlesthroughthecracksofthe polymercanbeconsideredasaself-healingprocess[19]as the nanoparticles, which are available in the sample, migrate to the cracks in order to fill them [20]. This phenomenon may be explained by the stretching and extensionofthepolymerchainsclosetothenanoparticles, whicharedrivenbythetendencytominimizeinteraction betweenthepolymerandthenanoparticles[21].Inmost cases,the compositemust beheated in ordertogivea sufficientmobilitytothepolymerchainstohaveamobility ofthenanoparticlesbutinourcase thehighcontentof waterinthepolymerseemstobesufficient tohavethe self-healing process without any heating. After almost 6months,mostofthenanoparticlesarenownearorinthe cracks.Allthecracksarefilledwithsilverandsomeofthe nanoparticles are just next to them, with only few nanoparticlesstillinthemiddleofthebulkofthepolymer. The silver nanoparticles accumulating in and near the crackdonothavearegularshapeattheborderandthe TEMimagenevershowsrod-shapedparticlesnorwires,so itseemsthatwehaveratheragglomerationof nanopar-ticlesinthecracks.Thefollow-upbySEMimagesshows twodifferentstepsinthenanoparticleformationprocess. Thefirstoneistheformationofthenanoparticlesduring approximately 1month, which is consistent with the microcalorimetry experiment. Energy-dispensive X-ray

spectroscopywasnotpossibleduetothedegradationof thesample.Withthemicrocalorimeter,weneed20daysto formthenanoparticlesat378Cwhereasthesamplesfor theSEMwerestoredatroomtemperature.Duringthisfirst month,cracksappearonthesurfaceofthepolymer.After 1month, the nanoparticles start to migrate along the cracksand after 6monthsall the cracksare filled with silvernanoparticles.

2.4. AntibacterialandcellviabilityassaysofPSS–Ag nanocomposite

SinceourPSS–Agcompositeshowedgoodmobilityof silvernanoparticles,andasthiscanbeinterpretedasan advantage over other polymers in which only the nanoparticlesclosetothesurfacecanactasantimicrobial agents, we tested our composite in this context. Anti-bacterialtests(Kirby–Bauertests)andcellviabilityassays were performed on our nanocomposite todetermine a possibleuseasbiomaterial.Forallthetestsdoneatleastin triplicata, we used a 4months old sample in order to ensurethatallsilvernanoparticleshavebeenformed.It should be said that PSS alone does not show any antimicrobial activity for all tested concentrations and against all bacterial strains. In order to observe an antimicrobialeffectforournanocomposite,a concentra-tionof at least 500

m

g/mL (11.6

m

g of silver per mLof polymersolution) nanocompositeisrequired tocombat a bacterial concentration of 104 _{and 10}6_{CFU/mL of}

Fig.4.Scanningelectronmicroscopyimagesofthenanocompositewithmigrationofsilvertowardsthecracks(topleft:2days,topright:31days,bottom

left:61days,bottomright:165days).

Staphylococcus epidermidis,and 1000

m

g/mL (23.2

m

g of silver per mL of polymer solution) for the bacterial concentration of 107_{CFU/mL (Fig. 5). At 2000}

m

g/mL (46.3

_m

gofsilverpermLofpolymersolution)a plateau fortheinhibitionzoneofallthebacteriaconcentrations wasreached.ForStaphylococcusaureus,aconcentrationof atleast500

_m

g/mL(11.6

_m

gofsilver permLofpolymer solution) for a concentration of 104_{CFU/mL isrequired,}

1000

m

g/mL(23.2

m

gofsilverpermLofpolymersolution) for a concentration of 106_{CFU/mL and 2000}

m

g/mL (46.3

m

g of silver per mL of polymer solution) for a concentrationof107CFU/mLtoobserveanantimicrobial activity.AsforS.epidermidisat2000

_m

g/mL,aplateaufor the inhibition zone of all bacterial concentrations is reached(Fig.6).Theinhibitionzoneatdifferent concen-trations of the composite at differentconcentrations of bacteria shows a plateau for all concentrations in the inhibitionzone,after2000

m

g/mL(46.3

m

gofsilverpermL of polymersolution),thevalueoftheinhibition zoneis stable,probablyindicatingthemaximumdiffusionspeed ofsilverwithintheobservedtimespan.Belowthisvalue, the maximum reached value of the inhibition zone dependson boththeconcentrationofinoculumand the concentration ofthecompound.Theinhibitionzoneand

thustheantimicrobialactivityareinoculumand concen-tration dependent for both bacteria, from 25mm for 104_{CFU/mLofS.epidermidisto13mmfor10}7_CFU/mLofS.

aureus. In parallel, we determined the basic minimum inhibitoryconcentrations(MIC)andminimumbactericidal concentrations(MBC)values.TheMICvaluesare78

_m

g/mL (1.81

m

gof silver per mLof polymer solution) forboth types of bacteria,while theMBC values are 313

_m

g/mL (7.25g of silver per mL of polymer solution) for S. epidermidis and 156

m

g/mL (3.61g of silver per mL of polymersolution)forS.aureus.ThisMICvalueisverylow comparedtoMICofsilvernitrateof10

_m

g/mL(6.30

_m

gof silverpermLofpolymersolution)forS.epidermidis.These valuesareconsistentwiththeonesfoundfortheinhibition zones. S. aureus thus seems to be as sensitive to our compoundasS.epidermidis.Withsuchgoodantimicrobial properties,weproceededtoevaluatethebiocompatibility. ThemostsimplecellviabilitytestinvitroistheMTT assay, based on a colorimetric test for measuring the percentageoflivecells comparedtoareferencesample, carriedoutonfibroblastcellsL-929.Thefirsttestwasdone onPSSalone,withacontrolexperimentat0

_m

g/mL.The results show cell viability around 90% for all tested concentrations after 48, 72 or 96h, confirming the biocompatibilityofPSS.These resultsare notsurprising becausesodiumcomplexesofPSSareusedasa drugto treathyperkalemia[22].Thesecondtestwasdoneonthe nanocomposite,thesamplebeingoldenoughtobesure thatallthenanoparticleswereformed.Thecellviabilityis goodforconcentrationsbetween5and40

m

g/mL(0.93

m

g ofsilverpermLofpolymersolution),forconcentrations between50

_m

g/mL(1.16

_m

gofsilverpermLofpolymer solution) and 70

m

g/mL (1.62

m

g of silver per mL of polymer solution). Thecell viabilityis around 75%, and drops to 40% for concentrations between 80

_m

g/mL (1.85

m

g of silver per mL of polymer solution) and 100

m

g/mL(2.31

m

gofsilverpermLofpolymersolution) ofournanocomposite.For150

_m

g/mL(3.47gofsilverper mLofpolymersolution),thecellviabilityisonly15%after 4days(Fig.7).Thus,whilecytotoxicityassaysshowagood biocompatibilitybut no antimicrobialproperties forthe PSSalone,ourcompoundisalreadyhighlyantimicrobial

Fig.5.Inhibitionzoneatthreedifferentbacterialconcentrations104

,106

and107_{CFU/mLofStaphylococcusepidermidis.Forinterpretationofcolors,}

seetheonlineversionofthisarticle.

Fig.6.Inhibitionzoneatthreedifferentbacterialconcentrations104_,106

and107

CFU/mLofStaphylococcusaureus.Forinterpretationofcolors,see

theonlineversionofthisarticle.

Fig.7.MTTassaysonthenanocompositeafterthreedifferenttimesof

incubation48h,72hand96h.Forinterpretationofcolors,seetheonline

versionofthisarticle.

forbothS.aureusandS.epidermidis,butalsocytotoxicat theconcentrationsrequiredfortheMBC.Thisdifferenceof toxicitybetweenthepolymeraloneandthe nanocompo-sitecanbeexplainedbythepresenceofnanoparticles.The cytotoxicityofsilverparticlesincreasesdramaticallywith their size reduction [23].It hasbeen shown that small silvernanoparticlescanenterintothecell,leadingtoan increaseofoxidativeprocessesleadingtocelldeath[24]. Basedontheseresults,ourcompoundcannotbeusedasan implantablematerialduetothecytotoxicity,butitcanbe used for external applications, such as antimicrobial clothesorwounddressings.

3. Conclusions

Weshowed thatthesilver ions of a polyelectrolyte complexof PSSwith silvercould beslowly reducedto give silver nanoparticles of around 25nm in size. The mechanismofthisreductionhasnotyetbeenprovenbut the complexation between the silver atoms and the sulfonategroupseemstobemandatory.Thisreductionis aslowprocess,whichtakesca.1monthunderstandard conditions for a loading with 2.5wt% of silver oxide. ChemicalreductionofthesilverPSScomplexgiveslarger particles than the spontaneous, slow reduction. SEM follow-up of the nanoparticles formation shows two different steps, the first one is the formation of the nanoparticles themselves during the first month followed by a phase separation process between the nanoparticlesandthepolymer,resultinginan accumu-lationofsilverinthecracksofthepolymer. Antimicro-bial tests show a good antimicrobial activity of our compoundevenatlowconcentrations>1mg/mL. Unfor-tunately,theproductiscytotoxicattheseconcentrations dueto the presenceof silver nanoparticles in solution when the polymer dissolves in the medium. This restrains the possible use of our nanocomposite to external applications, such as antimicrobial clothes or wounddressings.

4. Experimental

Allthechemicalswerestandardreagentgradeandwere used without further purification. 1_{H NMR spectrawas}

recorded with 360MHz spectrometer with residual solventsignalusedasreference.PowderX-ray diffracto-gram were collected on a Stoe StadiP using Cu K_a1

radiation (1.5406A˚). The morphology and the size of particles were determined by scanning electron micro-scopy(SEM,PhilipsXL30), transmissionelectron micro-scopy(TEM).

4.1. Preparationofthenanocomposite

In a one-neck flask, polystyrene sulfonic acid [Mw: 75,000g/mol(200mg)]and200mLofwaterweremixed thenasolutionofsilveroxide([5mgfora],[20mgforb] and[50mgforc])in3mLofconcentratedammoniawas added.Theresultingsolutionwasstirredfor24hthenthe solventwasevaporatedtoformaviscousliquidwithapH of 6.5. Slow evaporation of theremaining solvent in 1

or 2days leads to the nanocomposite film with a thickness<1mm.Thefilmsarenotcompletelydry,and thecontentofwaterisbetween13and18%afterthefilm formation.1_{HNMR(360MHz,D} 2O)

d

1.2–1.9(3H)6.2–6.8 (2H)7.3–7.8(2H).IR

n

=3489(mb),3209(mb),3055(wb), 2364(w),2368(w),1600(w),1415(m),1166(s),1126(s), 1033(s),1006(s),835(s),777(s),671(s). 4.2. Antimicrobialtests

For the antimicrobial tests, the nanocomposite was alwaysdissolvedinthemediumusedforthegrowthofthe bacteriaorcells.

4.2.1. Bacterialstrainsandgrowthconditions

S. epidermidis1457 and S. aureus 113 bacteria were freshlygrownintryptic soybroth (TSB)for 7hat378C withoutshakingandthendiluted1:100foranovernight (ON) culture, which was used for the experiments. Bacterial numbers were estimated by determining the opticaldensityat600nmand assessedbyplatingserial dilutionsonMu¨llerHintonAgar(MHA)[25].

4.2.2. Invitroantimicrobialsusceptibility

Astandardinoculumof1105_to5105_CFU/mLofS.

epidermidis1457orS.aureus113wasused.MICandMBC, respectively of silver compounds for logarithmically growingbacteriaweredeterminedusingamacrodilution methodaccordingtotheClinicalandLaboratoryStandards Institute(CLSI) guidelines[26]. TheMICwasthelowest concentrationofsilver compoundsthatinhibitedvisible bacterial growth. The MBC was defined as the lowest concentrationofsilvercompoundswhichkilled99.9%of theinitialbacterialcounts(i.e.,3log10[CFU/mL])in24h.

4.2.3. Agarinhibitionassays

Agarinhibitionassayswereperformedindisheswitha sizeof2424cmcontaining160mLofagar.S.epidermidis 1457orS.aureus113wasdilutedto104_,106_and107_CFU/

mL in the agar. Solutions of silver compounds were pipettedintothewholeseachof1cmdiameterintheagar. Theagarplateswereincubatedfor18hat378C,andthe inhibition zones around the solutions of the silver compoundsweremeasured.

4.3. Fibroblastcellculture

L-929fibroblastmurinecelllines(ATCCnumber: CCL-1)wereusedasacellmodeltoinvestigatetheeffectsof materialvariationsonsofttissueresponse.Thefibroblast culturesweremaintainedinRPMIsupplementedwith0.25 mM HEPES, 10% foetal bovine serum, 1XNEAA, 1 mM sodiumpyruvateand1%penicillin/streptomycinat378C inhumidifiedairand5%CO2.Culturesweresubdividedby

trypsination using Trypsin–EDTA solution. The culture mediumwaschangedevery3days.

4.4. MTTtestsoffibroblastcells

A quantitative colorimetricMTT test was performed after2, 3 and 4days of culture to characterize cellular

metabolism (vitality)and,byimplication,proliferation. Cells were seeded at the right concentration ontothe 96-well plates, grew for 24hbefore adding the silver compounds. As a control substrate forcell attachment andgrowth,fibroblastswereplateddirectlyontotissue culturepolystyrene plastic.Atday2, 3and4,10

_m

Lof MTT solution [5mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide in PBS] was added to eachwell,andthe cellswere incubatedat 378C for 4h. The reaction was stopped at 48C for hours. The mediumwasthenremovedand100

m

Lof dimethylsulf-oxide was added to each well, followed by 30min incubationatroomtemperatureonashaker.Theoptical density (OD) was measured at 540nm with an ELISA Reader.TheODisdirectlyproportionaltothenumberof cells alive [27]. The mean absorbance values were corrected for a blank (medium only) and results were reportedasOD.

4.5. Microcalorimetricanalysis

The IMC instrument(TAM48,TA instruments, New CastleDEUSA)wassetat378C.Thenanocompositewas placed in air, in microcalorimetric ampoules. The ampoules were closed under aerobic conditions and eachwasplaced intheequilibrationpositionin oneof the 48 chambers of the IMC instrument for 15min to allow initialequilibration.Afterthis,thesampleswere lowered to the measuring position and measurement started after45min.Duringall themeasurementtime, theinstrumentmaintainedthesettemperatureof378C andheatflowversuselapsedtimewasrecordedforeach ampoule.

Acknowledgements

TheauthorsthanktheSwissNationalScience Founda-tion,theUniversityofFribourgandtheFribourgCenterfor Nanomaterials(FriMat)forgeneroussupport.

AppendixA. Supplementarydata

Supplementarydata associatedwiththisarticlecanbe found, in the online version

References

[1](a)E.M.Hetrick,M.H.Schoenfisch,Chem.Soc.Rev.35(2006)780; (b)R.O.Darouiche,N.Engl.J.Med.350(2004)1422.

[2]K.M.Fromm,NatureChem.3(2011)178.

[3](a)P.S.Brunetto,T.V.Slenters,K.M.Fromm,Materials4(2011)355; (b)O.Gordon,T.VigSlenters,P.S.Brunetto,A.E.Villaruz,D.E. Sturde-vant,M.Otto,R.Landmann,K.M.Fromm,Antimicrob.Agents Che-mother.54(2010)4208;

(c)P.S.Brunetto,K.M.Fromm,Chimia62(2008)249;

(d)K.M.Fromm,J.L.Sague,L.Mirolo,Macromol.Symp.291–292(2010)75.

[4](a)A.C.Balazs,T.Emrick,T.P.Russell,Science314(2006)1107; (b)W.J.E.Beek,M.M.Wienk,R.A.J.Janssen,Adv.Mater.16(2004)1009.

[5]N.Kallay,S.Zˇalac,J.ColloidInterfaceSci.253(2002)70.

[6](a)K.Belser,T.VigSlenters,C.Pfumbidzai,G.Upert,L.Mirolo,K.M. Fromm,H.Wennemers,Angew.Chem.Int.Ed.48(2009)3661; (b)H.H.Huang,X.P.Ni,G.L.Loy,C.H.Chew,K.L.Tan,F.C.Loh,J.F.Deng, G.Q.Xu,Langmuir12(1996)909.

[7](a)U.Chatterjee,S.K.Jewrajka,S.Guha,Polym.Compos.30(2009)827; (b)J.Liu,Y.Gao,D.Cao,L.Zhang,Z.Guo,Langmuir27(2011)7926.

[8]S.Gupta,Q.Zhang,T.Emrick,A.C.Balazs,T.P.Russell,Nat.Mater.5 (2006)229.

[9]M.Nicˇ,J.Jira´t,B.Kosˇata,A.Jenkins,A.McNaught(Eds.),Polyelectrolyte, IUPAC,ResearchTriaglePark,NC,2009.

[10]S.Dutta,A.Shome,T.Kar,P.K.Das,Langmuir27(2011)5000.

[11](a)H.W.Gibson,F.C.Bailey,Macromolecules13(1980)34; (b)H.Asthana,B.Erickson,L.Drzal,J.Adhes.Sci.Technol.11(1997)1269.

[12](a)K.Lienkamp,I.Schnell,F.Groehn,G.Wegner,Macromol.Chem. Phys.207(2006)2066;

(b)M.A.Mannan,K.Fukuda,Y.Miura,Polym.J.39(2007)500.

[13]I.Meyer,Nephrol.Nurs.J.20(1993)93.

[14]Y.Sun,Y.Xia,Analyst128(2003)686.

[15](a)A.Patterson,Phys.Rev.56(1939)978;

(b)P.Scherrer,Go¨ttingerNachrichtenGesell.2(1918)98.

[16]K.T.Nam,Y.J.Lee,E.M.Krauland,S.T.Kottmann,A.M.Belcher,ACSNano 2(2008)1480.

[17]N.Ktari,C.Combellas,F.Kanoufi,J.Phys.Chem.C115(2011)17891.

[18]J.I.Goldstein,D.E.Newbury,P.Echlin,D.C.Joy,c.Fiori,E.Lifshin, ScanningElectronMicroscopyandx-ray Microanalysis:ATextfor Biologists,MaterialsScientistsandGeologists, PlenumPress,New York,1984.

[19]D.Y.Wu,S.Meure,D.Solomon,Prog.Polym.Sci.33(2008)479.

[20](a)J.Y.Lee,G.A.Buxton,A.C.Balazs,J.Chem.Phys.121(2004)5531; (b)S. Tyagi,J.Y.Lee, G.A.Buxton,A.C.Balazs,Macromolecules 37 (2004)9160.

[21]J.Y.Lee,Q.Zhang,T.Emrick,A.J.Crosby,Macromolecules39(2006)7392.

[22]M.Watson,K.C.Abbott,C.M.Yuan,Clin.J.Am.Soc.Nephro.5(2010)1723.

[23]L.Braydich-Stolle,Toxicol.Sci.88(2005)412.

[24]W.Liu,Y.Wu,C.Wang,H.C.Li,T.Wang,C.Y.Liao,L.Cui,Q.F.Zhou,B. Yan,G.B.Jiang,Nanotoxicology4(2010)319.

[25](a)R.M.Atlas,HandbookofMicrobiologicalMedia,3rded.,CRCPress, BocaRaton,Fla,2004;

(b)M.T. Madigan, J.M. Martinko, T.D. Brock, Brock Biology of Microorganisms, 11thed., PearsonPrenticeHall,UpperSaddleRiver, NJ,2006.

[26]P.R.Murray,E.J.Baron,J.H.Jorgensen,M.A.Pfaller,R.H.Yolken(Eds.), ManualofClinicalMicrobiology,7thed.,AmericanSocietyfor Micro-biology,Washington,DC,2003.

[27]T.Mosmann,J.Immunol.Methods65(1983)55.