structure and Collagen-chitosan scaffold with Au modiﬁed and Ag nanoparticles:Synthesis Applied Surface Science

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ContentslistsavailableatScienceDirect

Applied Surface Science

j o ur na l ho me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / a p s u s c

Collagen-chitosan scaffold modiﬁed with Au and Ag nanoparticles:

Synthesis and structure

M.S. Rubina

^a

, E.E. Kamitov

^a

, Ya. V. Zubavichus

^b

, G.S. Peters

^b

, A.V. Naumkin

^a

, S. Suzer

^c

, A.Yu. Vasil’kov

^a,∗

aA.N.NesmeyanovInstituteofOrganoelementCompounds,RussianAcademyofSciences,Moscow,119991RussianFederation

bNationalResearchcenter«KurchatovInstitute»,Moscow,123182RussianFederation

cDepartmentofChemistry,BilkentUniversity,Ankara,06800Turkey

a r t i c l e i n f o

Articlehistory:

Received7May2015 Receivedinrevisedform 28December2015 Accepted13January2016 Availableonline14January2016

Keywords:

Collagen-chitosanscaffolds Metal-vaporsynthesis Nanoparticles XPS XRD SAXS XANES/EXAFS

a b s t r a c t

Nowadays,thedermalbiomimeticscaffoldsarewidelyusedinregenerativemedicine.Collagen-chitosan scaffoldoneofthesematerialspossessesantibacterialactivity,goodcompatibilitywithlivingtissuesand hasbeenalreadyusedasawound-healingmaterial.Inthisarticle,collagen-chitosanscaffoldsmodiﬁed withAgandAunanoparticleshavebeensynthesizedusingnovelmethod-themetal-vaporsynthesis.

ThenanocompositematerialsarecharacterizedbyXPS,TEM,SEMandsynchrotronradiation-basedX-ray techniques.AccordingtoXRDdata,themeansizeofthenanoparticles(NPs)is10.5nmand20.2nmin Au-Collagen-Chitosan(Au-CollCh)andAg-Collagen-Chitosan(Ag-CollCh)scaffolds,respectivelyinfair agreementwiththeTEMdata.SAXSanalysisofthecompositesrevealsanasymmetricsizedistribution peakedat10nmforAu-CollChand25nmforAg-CollChindicativeofparticle’saggregation.According toSEMdata,themetal-carryingscaffoldshavelayeredstructureandthenanoparticlesareratheruni- formlydistributedonthesurfacematerial.XPSdataindicatethatthemetallicnanoparticlesareintheir unoxidized/neutralstatesanddominantlystabilizedwithinthechitosan-richdomains.

1. Introduction

Naturallyoccurringpolymersarewidelyappliedinmedicine duetotheirpronouncedbiocompatibility,availability ofrenew- ablesources,easiness ofchemical modiﬁcation,etc.[1].Among them,chitosan is ofspecial importance,which isessentiallyan N-deacetylatedchitinderivative,alinearpolymercomposedofD- glucosamineandN-acetylglucosamineresidues.Chitosanisknown topromoteskinregeneration,woundsandburnshealing.Further- more,itmanifestshemostaticandimmunomodulatoryproperties [2–4],aswellasantibacterialandantifungalactivity[5,6].Chitosan isabiocompatiblepolymerandpossessesahighsorptioncapac- ity.Apartfrompristinechitosan,chitosan-basedhybridmaterials, especiallyoneswithcollagen[7],cellulose[8],polyethyleneglycol [9],polyvinylpyrrolidone[10],gelatin[11],polyvinylalcohol[12], etc.,arepromisingmaterialsforbiomedicalapplications[13].

Collagenisastructuralproteinactivelyusedinmedicine[14].

Availabilityofhydroxylicgroupsandaminoacidresiduesonitssur- facemakesitpossibletoadjustitssurfacechargetoeithernegative

∗ Correspondingauthor.Tel.:+74991359380.

E-mailaddress:alexandervasilkov@yandex.ru(A.Yu.Vasil’kov).

or positive values by changing pH[15].Collagen ﬁbrils(cross- linkedornot,nativeordenaturated)stronglyaffectmorphology andphysiologyofcells[16].Collagenisnearlyasbiodegradable andbiocompatibleaschitosan,thatiswhyitiswidelyusedintis- sueengineeringaswoundandburndressingandsponges.Collagen andchitosandonotoccurinnaturetogether,butspeciﬁcproper- tiesofbothpolymerscanbeutilizedtodesignahybridmaterial withuniquestructuralandmechanicalproperties[17].

Numerousexamples of similarmaterialsused for thetissue engineering, drugdelivery matrices, bandage dressing, etc.,are availablein literature[18,19].Incorporation ofnoble metalNPs withantibacterialactivityfurtherextendsapprovedﬁeldsofappli- cationsofcollagen-chitosanscaffolds.Themajorityofmethodsfor theincorporationofmetalNPsinpolymermatricesrequireschem- icalreductionofmetalsaltsimpregnatedintothepolymermatrix (e.g.,borohydrideorcitrate methods)[20,21].These techniques aretypicallymulti-stepandusepotentiallybiologicallyhazardous orenvironmentally unfriendlyreactants,stabilizers, orreducing agents[22],whichpreventorstronglylimittheuseoftheresultant compositesinmedicine[23].

Metal-vapor synthesis (MVS) is an efﬁcient route to pro- ducebiologicallyactivemetalnanoparticlesand thecomposites derivedfromthemwithbiocompatiblematerialsforbiomedical http://dx.doi.org/10.1016/j.apsusc.2016.01.107

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applications[24,25].Thetechniquehasbeensuccessfullyapplied tomodifycommonmedicalmaterials,includingsurgicalsuture anddressingmaterialsorimplants,withmetalnanoparticlespos- sessingantibacterialandantifungalproperty[26,27].Incontrastto themajorityofmethodsforpreparationofnanoparticles,theMVS methodisfullyenvironmentallysafeandcaneasilybeintegrated intodiversetechnologicalcycles.TheMVSmethodaffordscolloidal suspensions of NPsin commonmedical solvents, which makes anyfurtherpuriﬁcationunnecessary[28].Thetargetcomposites arepreparedviathemodiﬁcationofabiopolymerwithorganosols containingmetalnanoparticlesfollowedbysolventremoval.

Here, we report for the ﬁrst time on the synthesis of metal-bearinghybridsystems based ontheintrinsicallyporous collagen-chitosanscaffolds.Thestructureandchemicalstatesof metalsinthecompositenanomaterialsareaddressedbymodern physicochemicaltechniques.

2. Experimental 2.1. Materials

Collagen-chitosanscaffold, denoted as CollCh,was prepared accordingtothemethoddescribed elsewhere[29].Et3N(Sigma Aldrich,purity≥99.5%)andi-PrOH(Fluka,purity99.8%)wereused asorganicsolvents.Allotherreactantsusedfortheexperiments wereofanalyticalgrade.Priortouseinthesynthesis,allsolvents weredried,distilledinanatmosphereofpuriﬁedAranddegassed byseveralconsecutivepump-freeze-thawcyclesat10⁻¹Pa and RTfor1h.Theresultantcollagen-chitosanscaffoldwasdegassed invacuo.

2.2. Metal-vaporsynthesisofhybridmaterials

Theoriginal metal-vapor synthesis (MVS)method wasused in this work to produce metal-modiﬁed composites based on thecollagen-chitosanscaffold.The (MVS)methodis efﬁcientin preparationofhighlyreactivemetalnanoparticlesandtheirincor- poration into various matrices to induce practically important magnetic[30],antibacterial[26],catalytic[31]andtribological[32]

properties.

NanocompositesﬁlledwithgoldandsilverNPswereprepared byimpregnationofthemetal-containingorganosolsfromMVSinto thecollagen-chitosanscaffoldasdescribedelsewhere[26].Metals wereevaporated at a basepressure of 10⁻²Pa by a resistively heatedevaporatorintheformofeithertungstenrodorsmalltan- talumvesselforAu(99.99%)orAg(99.99%),respectively.Specially preconditionedorganicsolventsEt₃Nandi-PrOHwereusedtosta- bilize,respectively,AuandAgnanoparticlesassols.Metalvapor wascondensed simultaneouslywiththesolventvaporontoliq- uidnitrogen-cooledwallsofaglassreactorwithavolumeof5L.A typicalsolvent-to-metalmolarratiointhesynthesiswas300:1.

Afterthesynthesis,theco-condensate washeatedtothemelt- ingpoint andtheresultantorganosolwasimpregnatedintothe collagen-chitosanscaffoldplacedinaSchlenkﬂaskundervacuum.

Theexcessiveamountoftheorganosolwasremovedandthetarget productwasdriedinvacuumat60^◦C.

2.3. Characterizationmethods

2.3.1. Synchrotronradiation-basedtechniques

X-raystructuralstudiesofthecompositesbasedoncollagen- chitosanscaffoldsmodiﬁedwithAuandAgnanoparticles,including suchtechniquesaspowderX-raydiffraction,small-angleX-ray scattering,andX-rayabsorptionspectroscopyXANES/EXAFSwere performedat theKurchatov synchrotronradiation source(NRC

“KurchatovInstitute”,Moscow).

X-rayabsorptionspectraandpowderdiffractionpatternswere measuredattheStructuralMaterialsSciencebeamline [33]. Au L₃-edgeXANES/EXAFSforthegold-containingsampleAu-CollCh aswellasfortheAufoilreferenceweremeasuredinthetrans- missionmodeusingionchambersfilledwithappropriateN2-Ar mixtures.ForsimilarAg-containingcomposites,thesilverconcen- trationappearedinsufficientfortransmissionmeasurementsand thusthespectraweremeasuredintheX-rayfluorescenceyield modeusingaSiavalanchephotodiode.SpectrafortheAgfoilrefer- enceweremeasuredinthetransmissionmode.Experimentaldata processingandanalysiswereperformedusingtheIFEFFITsoftware package[34].

PowderX-raydiffractionpatternsforthesamesampleswere measuredin thetransmission (Debye-Sherrer)modeusing Fuji FilmImagingplatesasa2Ddetector.Diffractionmeasurements wereperformedatanX-raywavelength=0.06889nm.Themean nanoparticlesizewasestimatedbyproﬁleanalysisassumingthe Pseudo-Voightlineshapeofthediffractionpeakswithinstrumen- talfunctionresponsiblefortheGaussianpartofbroadeningand Lorentzian-typesample-drivenphysicalbroadening.

Additionally, small-angle X-rayscattering curves were mea- suredforthepristineandAg-,Au-ﬁlledcollagen-chitosanscaffold to reﬁne the diffraction data on NP sizing. The measurements wereperformedattheDICSIbeamlineofthesamesynchrotron sourceanda2DMAR165CCDdetectorwasused.Thesample-to- detectordistancewas2400mmandtheX-raywavelengthwasset to=0.162nm.Toimprovesamplingandsignal-to-noisestatis- tics,severaldatasetsatdifferentspotsonasampleweremeasured and averaged.The sizedistributionof Agand Au nanoparticles wasretrievedfromthecorrespondingSAXS“Ag-CollCh-CollCh”

and“Au-CollCh-CollCh”differencecurves,respectively.Theindi- rectFouriertransformationapproachasimplementedintheGNOM code under assumption of a polydisperse distribution of non- interactinghardspheres[35]hasbeenused.

2.3.2. X-rayphotoelectronspectroscopy(XPS)

X-rayphotoelectronspectrawereacquiredwithanAxisUltra DLD(Kratos,UK)spectrometerusingAlK_␣radiationatanoperat- ingpowerof150WoftheX-raytube.Surveyandhigh-resolution spectraofappropriatecorelevelswererecordedatpassenergiesof 160eVand40eVandwithscanningstepsof1eVand0.1eV,respectively.Sampleareaof300␮m×700␮mcontributedtothespectra.

Thesamplesweremountedonasampleholderwithatwo-sided adhesivetape,andthespectrawerecollectedatroomtemperature.

ThebasepressureintheanalyticalUHVchamberofthespectrom- eterduringmeasurementsdidnotexceed10⁻⁸Torr.Thebinding energyscaleofthespectrometerwascalibratedtoprovidethefol- lowing valuesfor referencesamples(i.e., metalsurfacesfreshly cleanedbyionsputtered):Au4f_7/2–83.96eV,Cu2p_3/2–932.62eV, Ag3d_5/2–368.21eV.Theelectrostaticchargingeffectswerecom- pensatedbyusinganelectronneutralizer.For eachsample,the spectrawererecalibratedagainsttheadventitiouscarbonsignalat 285.0eV.Backgroundwithinelasticlosseswassubtractedfromthe high-resolutionspectraaccordingtotheShirleyprescription.The AgMNNAugerspectrawerecorrectedusingalinearbackground.

Thesurfacechemicalcompositionwascalculatedusingstandard elementsensitivityfactorscodedinthespectrometercontrolsoft- warecorrectedforthetransferfunctionoftheinstrument.

2.3.3. TEM

Micrographsofthesamplesweremadeusingatransmission electronmicroscopeLEO912ABOMEGA,Zeiss(Germany).

2.3.4. SEM

SEManalysiswasperformedwithascanningelectronmicro- scopeTescanMiraLMU(CzechRepublic).Thesampleswereﬁxed

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Fig.1.XRDpatternsforthenanocompositesstudiedAu-CollCh,Ag-CollCh,and pristineCollCh(=0.06889nm).

onaconductivetapeandexaminedunderhighvacuumwiththe Everhart-Thornleystandardsecondaryelectrondetector.

3. Resultsanddiscussion

Theexperimental X-raydiffraction patternsof thecollagen- chitosanscaffoldsmodiﬁed bygoldandsilvernanoparticlesare showninFig.1.Thecollagen-chitosanscaffoldappearstobeamor- phoussothatalldiffractionpeakspresentintherespectivepatterns canbeattributedtofccatomicpackingoftheAgandAunanoparti- cles[PDF#040783and#040784,respectively].Thecrystallitesizes estimatedfromthelinebroadeninganalysisassumingthatmicros- trainsmakenegligiblecontributionyield10.5nmand20.2nmfor Au-CollChandAg-CollCh,respectively,thenominalaccuracyofthe estimateis10–15%.

0.1 0.1

1 10 100 1000 10000

0 25 50 75 100

Scattering Intensity, a.u.

q, nm^-1

Volume fraction, a.u.

d, nm

Fig.2.ExperimentalSAXScurvesforAu-CollCh(ﬁlledsquares),Ag-CollCh(open circles),andpristineCollCh(line).Theinsetshowsvolumedistributionofmetal nanoparticlediametersreconstructedfromthedifferencecurves.

Forthecomposites,anindependentestimateforthenanopar- ticlessizewasobtainedfromtheSAXSdata.Thecorresponding experimentaldataareshowninFig.2.

The experimental curve for the metal-containing composite (correctedfortheincidentintensityandthesampleX-rayabsorption) is characterizedby a higher scattering intensity than the pristineCollChscaffoldovertheentirerangeofscatteringwave vectorsmeasured, which makes it possibleto reliably separate thecontributionofthescatteringby thenanoparticlesviasim- plenumericalsubtractionprocedure.Ananalysisofthedifference curveusingtheindirectFouriertransformationwiththeTikhonov’s regularizationunderassumptionofnone-interactinghardspheres yieldsarathernarrowandslightlyasymmetricsizedistribution peakedat10nmforAu-CollChandmuchbroaderasymmetricdis- tributioncurvewithamaximumat25nmextendinguptosizes

Fig.3.TEMmicrographs(scalebaris100nm),correspondingSAEDpatternsandparticlesizedistributionfortheAu-CollCh(a–d)andAg-CollCh(e–h)composites.

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Fig.4.SEMmicrographsofthecross-sectionoftheAu-CollCh(a,b)andAg-CollCh(c,d)composites.

of 120–150nm for Ag-CollCh. Apparently, this means that the nanoparticlesareagglomeratedintoaggregatesfromfewtoseveral thousandindividualnanoparticleswithinthecomposite.

Fig.3showsmicrographs,SAEDpatternsofmetalnanoparticles andsizedistributionhistogramfortheAu-CollChandAg-CollCh.

AccordingtoTEM,theparticlesarespherical,polydisperseandhave fairlyuniformdistributioninthescaffold.Theaveragesizeofthe AuandAgparticlesis4.6nmand6.6nm,respectively.Itisdemon- stratedinFig.3(candg)thatAg-CollChcompositehavethebroader particlesizedistributionthanAu-CollChone.

The presence of larger Ag nanoparticles with size of about 60–70nminthematerialshowninFig.3(eandf)canbeexplained byaggregationofsmallernanoparticlesduringthemodiﬁcationof polymermatrixwithorganosol,aswellassurfacediversityofthe pristinescaffold.

Fig.4showstheinternalcross-sectionalmicrostructureofthe Ag-CollChandAu-CollChcomposites. Collagen-chitosanscaffold hasalayeredstructure,consistingoflamellasandmicrocavaties formedbypolymernetworkofﬁbrils(Fig.4a–c).

Theaveragediameterofmicrocavitiesisabout40–80microns andthethicknessoftheﬁbrilscaffoldis2–5microns.Itisexhibited thattheaggregates,withsizesfromafewtensofnanometersto severalmicrons,haveslightlyuniformdistributionsonthesurfaces oftheﬁbrilsandlamellas(Fig.4bandd).

Supplementaryinformation ontheelectronic stateandlocal environmentofAuandAgatomsinthecompositeswasobtainedby X-rayabsorptionspectroscopyXANES/EXAFS.AuL3-andAgK-edge XANESspectraforthemetal-modiﬁedCollCh-basedcompositesare comparedwithreferencespectraofmetalfoilsinFig.5.

ThesimilarshapeandenergypositionofEXAFSspectralfeatures indicatethatthechemicalstateofthemetalatomsinthecompos- itesissimilartothatintheirbulkmetals,whichmeansthatthe fractionofsurfaceatomsstronglyinteractingwiththematrixis ratherlow.

FortheXANESspectrumoftheAg-CollChcomposite,anincrease intheperiodofoscillationsisapparent,whichcorrespondstoan Ag-Agbondlengthcontractionwithrespecttothebulkmetal.This ﬁndingisfurthersupportedbyaquantitativeanalysisoftheEXAFS

Fig.5. XANESspectraofcollagen-chitosanscaffoldmodiﬁedwithAu(left)andAg(right).

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Fig.6.FTsofEXAFSspectraforcollagen-chitosanscaffoldsmodifiedwithAu(left)andAg(right):experimental(line)andbest-fittheoretical(opencircles)curves.Thelocal environmentparameterscorrespondingtothefitsaresummarizedinTable1.

Fig.7. SurveyXPSspectraofAu-CollCh(a)andAg-CollCh(b)composites;high-resolutionAu4f,Ag3d,AgMNNandN1score-levelspectraofAu-CollCh(c),Ag-CollCh(d, e)and(f1,f2),respectively.

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Table1

Parametersofthelocalenvironmentofthemetalatomsinthechitosan-basedcom- positesaccordingtoEXAFS:interatomicdistances(R),coordinationnumbers(n), andDebye-Wallerfactors(²).

Sample Path n R[Å] ²[Å²]

Au(foil) Au-Au 12.0 2.85 0.0079

Au-CollCh Au-Au 11.9 2.84 0.0095

Ag(foil) Ag-Ag 12.0 2.88 0.0095

Ag-CollCh Ag-Ag 9.7 2.85 0.0119

data.FourierTransforms(FTs)ofEXAFSspectraforthecompos- itesunderstudyareshowninFig.6.Theyareessentiallysimilar tothoseofthereferencemetalfoils.Best-ﬁtcoordinationnumbers andinteratomicdistancesfortheﬁrstmetal-metalcoordination spheresarelistedinTable1.

Thecompositesarecharacterizedbysomewhatdecreasedcoor- dination numbers when compared with the data the for bulk metals,whichisverycommonfornanometer-sizedmetalparti- cles.Theminimummetal-metalcoordinationnumberisobserved fortheAg-CollChcompositedespiteitsratherlargercrystallitesize asestimatedfromdiffractiondata.Furthermore,theAg–Aginter- atomicdistancederivedfromtheEXAFSforthatcompositeisby 0.03 ˚AshorterthanthatforAgfoil.Thebondlengthcontractionis quitetypicalofsmallmetalnanoparticles.Butinthecaseofsil- ver,bondlengthelongationissometimesobservedduetopartial oxidationandsuboxideformation[36].Nevertheless,aprominent changein thecoordinationnumberand bondlengthfortheAg- CollChdespitenominallylargesizefromdiffractionmayindicatea distortedlocalstructureofsilvernanoparticleswithfrequentpoint defects.

Inordertoevaluatethesurfaceconcentrationsand chemical statesofmetalatomsinthecomposites(withinalayerof5–8nm), X-rayphotoelectronspectroscopywasapplied(Fig.7).Surveyspec- tra of Au-CollCh (Fig. 7a) and Ag-CollCh (Fig. 7b) reveal peaks attributabletoelementsofthenominalchemicalcomposition,i.e., C,O,N,Au/Ag,aswellasadmixtureelementsSiandF.Quantifi- cationdatabasedonatomicsensitivityfactorsarepresented in Table2.AsitcanbejudgedfromC/NandC/Oatomicratios,the incorporationofmetals intothepristine collagen-chitosanscaf- foldsgivesrisetosomeenrichmentofthecompositesurfaceswith carbonspecies(seeTable2).Partly,thiscanbeduetoco-sorption oftheorganicsolventusedinthesynthesis.Theabsenceofdra- maticchangesinelementconcentrationsratherconfirmsthatthe CollChscaffoldsdonotdegradeuponmodificationwithnoblemetal nanoparticles.

TheAu4fbindingenergyobservedforthecomposite(Fig.7c)is characteristicoftheAu⁰state.Aminuteshiftby0.05eVtoahigher energywithrespecttofoilandpronouncedasymmetryofthepeak shapeareduetothesizeeffects[37,38]andthustheyalsoconﬁrm thepresenceofnm-sizedgoldnanoparticlestherein.

High-resolutionAg3dcore-levelsandMNNAugerlinesforthe Ag-containingcompositeareshowninFig.7dande.Theexperi- mentalvaluesfortheAg3dbindingenergy,AgMNNAugerkinetic energy,andthecorrespondingAugerparameterallconﬁrm[39,40]

theAg⁰stateofsilveratomsinthecomposite.TheN1sspectrain metal-containingcompositesandpristinescaffoldsarecharacter- izedbydifferentbindingenergiesandfull-widthsathalf-maxima

Table2

SurfacechemicalcompositionsofthecompositesfromXPSdata.

Sample Relativeconcentration,at.%

C N O Au Ag C/N C/O O/N

CollCh 74.2 8.5 17.3 – – 8.7 4.3 2.0

Au-CollCh 74.0 7.8 15.8 2.4 – 9.5 4.7 2.0

Ag-CollCh 75.2 8.1 15.8 – 1.0 9.3 4.8 2.0

(Fig.7f),whichcanbeexplainedbyanalterationofbalancebetween thecollagenandchitosanN-functionalgroupsuponthemodiﬁca- tion.TypicalN1score-levelbindingenergiesinchitosan(C-NH₂) andcollagen(C(O)N)are399.67eV[41]and399.77–399.96[42], respectively.

4. Conclusions

Thenovelmethodforthesynthesisofhybridmaterialsbasedon thecollagen-chitosanscaffoldmodiﬁedwithAgandAunanopar- ticles is reported. XRD and SAXS data show that the Au- and Ag-bearingcompositesarecharacterizedbymeanparticlesizeof 10nmand25nm,respectively.Itisrevealedthatnanoparticlesin thebulkofmaterialshaveaslightlyuniformdistributionwiththe meansizeis4.6nmand6.6nmforAuandAg,respectively.Onthe surfaceitwasobservedthelargeraggregates,withsizesfromafew tensofnanometerstoseveralmicrons,consistingofsmallerparti- cles.Thewholestructureoftheobtainednanocompositesissimilar.

However,theAg-CollChcompositehasmuchbroaderasymmetric sizedistributionthanAu-CollCh.

AccordingtoXPS,metalatoms areintheunoxidized/neutral statesandpreferablystabilizedinthechitosanmatrix.Similarly, XANES/EXAFSdataindicatethechemicalstateandlocalstructure ofmetalatomsinthecompositesissimilartothatofbulkmetals apartfromasmalldecreaseinmetal-metalcoordinationnumber inbothcomposites,andmetal-metalbond-lengthcontractionin Ag-CollCh.

Wesuggestthatcollagen-chitosanscaffold,containingAuand Agnanoparticles,canbepromising antibacterialwound-healing materialformedicine.

Acknowledgements

ThisworkwaspartiallysupportedbytheRussianFoundation forBasicResearch(grantsnos.14-03-01074and15-53-61030).

References

[1]A.Aravamudhan,D.M.Ramos,A.A.Nada,S.G.Kumbar,Naturalpolymers:

polysaccharidesandtheirderivativesforbiomedicalapplications,in:S.

Kumbar,C.Laurencin,M.Deng(Eds.),NaturalandSyntheticBiomedical Polymers,ﬁrsted.,Elsevier,USA,2014,pp.67–89.

[2]R.Jayakumar,M.Prabahran,P.T.SudheeshKumar,S.V.Nair,H.Tamura, Biomaterialsbasedonchitinandchitosaninwounddressingapplications, Biotechnol.Adv.29(2011)322–337.

[3]S.-Y.Ong,J.Wu,S.M.Moochhala,M.-H.Tan,J.Lu,Developmentofa chitosan-basedwounddressingwithimprovedhemostaticandantimicrobial properties,Biomaterials29(2008)4323–4332.

[4]A.Pattani,V.B.Patravale,L.Panicker,P.D.Potdar,Immunologicaleffectsand membraneinteractionsofchitosannanoparticles,Mol.Pharm.6(2009) 345–352.

[5]I.Younes,S.Sellimi,M.Rinaudo,K.Jellouli,M.Nasri,Inﬂuenceofacetylation degreeandmolecularweightofhomogeneouschitosansonantibacterialand antifungalactivities,Int.J.FoodMicrobiol.185(2014)57–63.

[6]J.Xu,X.Zhao,X.Han,Y.Du,Antifungalacivityogoligochitosanagainst Phytophtoracapsiciandotherplantpathogenicfungiinvitro,Pest.Biochem.

Physiol.87(2007)220–228.

[7]J.-P.Chen,G.-Y.Chang,J.-K.Chen,Electrospuncollagen/chitosannanoﬁbrous membraneaswounddressing,Coll.Surf.A:Physicochem.Eng.Asp.313–314 (2008)183–188.

[8]M.I.NiyasAhamed,S.Sankar,P.MohammedKashif,S.K.HayathBasha,T.P.

Sastry,Evaluationofbiomaterialcontainingregeneratedcelluloseand chitosanincorporatedwithsilvernanoparticles,Int.J.Biol.Macromol.72 (2015)680–686.

[9]C.Prego,D.Torres,E.Fernandez-Megia,R.Novoa-Carballal,E.Qui ˜noá,M.J.

Alonso,Chitosan–PEGnanocapsulesasnewcarriersfororalpeptidedelivery:

effectofchitosanpegylationdegree,J.ControlledRelease111(2006)299–308.

[10]T.C¸aykara,A.Alaslan,M.S.Ero˘glu,O.Güven,Surfaceenergeticsof

poly(N-vinyl-2-pyrrolidone)/chitosanblendﬁlms,Appl.Surf.Sci.252(2006) 7430–7435.

[11]C.-M.Deng,L.-Z.He,M.Zhao,D.Yang,Y.Liu,Biologicalpropertiesofthe chitosan-gelatinspongewounddressing,Carbohydr.Polym.69(2007) 583–589.

(7)

[12]E.Salehi,S.S.Madaeni,Inﬂuenceofpoly(ethyleneglycol)aspore-generator onmorphologyandperformanceofchitosan/poly(vinylalcohol)membrane adsorbents,Appl.Surf.Sci.288(2014)537–541.

[13]M.Rinaudo,ChitinandChitosan:propertiesandapplications,Prog.Polym.

Sci.31(2006)603–632.

[14]M.Chvapil,R.L.Kronenthal,W.V.WinkleJr.,Medicalandsurgicalapplications ofcollagen,Int.Rev.ConnectiveTissueRes.6(1973)1–61.

[15]T.Nezu,F.M.Winnik,Interactionofwater-solublecollagenwithpoly(acrylic acid),Biomaterials21(2000)415–419.

[16]K.Yoshizato,A.Makino,K.Nagayoshi,Regulationofmorphologyand physiologyofepithelialcellsbycollagenﬁbrils,Biomed.Res.9(1988) 33–45.

[17]T.Garg,O.Singh,S.Arora,R.S.R.Murthy,Scaffold.Anovelcarrierforcelland drugdelivery,Crit.Rev.Therap.DrugCarrierSyst.29(2012)1–63.

[18]X.H.Wang,D.P.Li,W.J.Wang,Q.L.Feng,F.Z.Cui,Y.X.Xu,X.H.Song,M.vander Werf,Crosslinkedcollagen/chitosanmatrixforartiﬁciallivers,Biomaterials 24(2003)3213–3220.

[19]L.Ma,C.Gao,Z.Mao,J.Zhou,J.Shen,X.Hu,C.Han,Collagen/chitosanporous scaffoldswithimprovedbiostabilityforskintissueengineering,Biomaterials 24(2003)4833–4841.

[20]L.-PingDing,Y.Fang,Aninvestigationofthesurface-enhancedRaman scattering(SERS)effectfromlaserirradiationofAgnanoparticlespreparedby trisodiumcitratereductionmethod,Appl.Surf.Sci.253(2007)4450–4455.

[21]W.Zhanga,X.Qiao,Q.Chen,Y.Cai,H.Chen,Theinﬂuenceofsynthesis conditionandagingprocessofsilvernanocrystalsontheformationofsilver nanorods,Appl.Surf.Sci.258(2012)5909–5913.

[22]D.Wei,W.Sun,W.Qian,Y.Ye,X.Mac,Thesynthesisofchitosan-basedsilver nanoparticlesandtheirantibacterialactivity,Carbohydr.Res.344(2009) 2375–2382.

[23]Y.-K.Twu,Y.-W.Chen,C.-M.Shih,Preparationofsilvernanoparticlesusing chitosansuspensions,PowderTechnol.185(2008)251–257.

[24]L.N.Nikitin,Yu.A.Vasil’kov,M.Banchero,L.Manna,A.V.Naumkin,V.L.

Podshibikhin,S.S.Abramchuk,M.I.Buzin,A.A.Korlyukov,A.R.Khokhlov, Compositematerialsformedicalpurposesbasedonpolyvinylpyrrolidone modiﬁedwithketoprofenandsilvernanoparticles,Russ.J.Phys.Chem.A85 (2011)1190–1195.

[25]O.A.Belyakova,A.V.Shulenina,Ya.V.Zubavichus,A.A.Veligzhanin,A.V.

Naumkin,Yu.A.Vasil’kov,Diagnosticsofgold-containingsurgical-dressing materialswithX-rayandsynchrotronradiation,J.Surf.Invest.X-ray SynchrotronNeutronTech.7(2013)509–514.

[26]G.Cárdenaz,J.DíazVisurraga,M.F.Meléndrez,C.Cruzat,A.GarcíaCancino, ColloidalCunanoparticles/chitosancompositeﬁlmobtainedbymicrowave heatingforfoodpackageapplications,Pollym.Bull.62(2009)511–514.

[27]S.I.Stoeva,A.B.Smetana,C.M.Sorensen,K.J.Klabunde,Gram-scalesynthesis ofaqueousgoldcolloidsstabilizedbyvariousligands,J.ColloidInterfaceSci.

309(2007)94–98.

[28]C.Zhu,D.Fan,X.Ma,W.Xue,Y.Yu,Y.Luo,B.Liu,L.Chen,Effectsofchitosan onpropertiesofnovelhuman-likecollagen/chitosanhybridvascularscaffold, J.BioactiveCompatiblePolym.76(2009)560–576.

[29]PatentRU2108114.

[30]I.P.Suzdalev,Yu.V.Maksimov,Yu.A.Vasil’kov,A.V.Naumkin,V.L.

Podshibikhin,I.O.Volkov,ElectronicandmagneticpropertiesofAu-Fecluster nanocompositespreparedbyasequentialsolvatedmetalatomdispersion process,Nanotechnol.Russia3(2008)72–78.

[31]Yu.A.Vasil’kov,A.V.Naumkin,I.O.Volkov,V.L.Podshibikhin,G.V.Lisichkin, A.R.Khokhlov,XPS/TEMcharacterisationofPt–Au/Ccathodeelectrocatalysts preparedbymetalvapoursynthesis,Surf.InterfaceAnal.42(2010)559–563.

[32]A.P.Krasnov,V.N.Aderikha,O.V.Afonicheva,V.A.Mit’,N.N.Tikhonov,Yu.A.

Vasil’kov,E.E.Said-Galiev,A.V.Naumkin,Yu.A.Nikolaev,Categorization systemofnanoﬁllerstopolymercomposites,J.Frict.Wear31(2010)68–80.

[33]A.A.Chernyshev,A.A.Veligzhanin,Y.V.Zubavichus,Structuralmaterials scienceend-stationatthekurchatovsynchrotronradiationsource:recent instrumentationupgradesandexperimentalresults,Nucl.Instrum.Methods Phys.Res.A603(2009)95–98.

[34]B.Ravel,M.Newville,ATHENA,ARTEMIS,HEPHAESTUS:dataanalysisfor X-rayabsorptionspectroscopyusingIFEFFIT,J.Synchrotr.Rad.12(2005) 537–541.

[35]D.I.Svergun,Determinationoftheregularizationparameterin

indirect-transformmethodsusingperceptualcriteria,J.Appl.Crystallogr.25 (1992)495–503.

[36]A.I.Frenkel,A.Yevick,C.Cooper,R.Vasic,Modelingthestructureand compositionofnanoparticlesbyextendedX-rayabsorptionﬁne-structure spectroscopy,Annu.Rev.Anal.Chem.4(2011)23–39.

[37]C.R.Henry,Surfacestudiesofsupportedmodelcatalysts,Surf.Sci.Rep.31 (1998)235–325.

[38]F.Karadas,G.Ertas,E.Ozkaraoglu,S.Suzer,X-rayinducedproductionofgold nanoparticlesonSiO2/SisystemandinPMMA,Langmuir21(2005)437–442.

[39]R.Romand,M.Roubin,J.P.Deloume,ESCAstudiesofsomecopperandsilver selenides,J.ElectronSpectrosc.Relat.Phenom.13(1978)229–242.

[40]G.B.Hoﬂund,J.F.Weaver,W.S.Epling,AgFoilbyXPS,Surf.Sci.Spectra3 (1994)151–156.

[41]N.Li,R.Bai,C.Liu,Enhancedandselectiveadsorptionofmercuryionson chitosanbeadsgraftedwithpolyacrylamideviasurface-initiatedatom transferradicalpolymerization,Langmuir21(2005)11780–11787.

[42]G.Beamson,D.Briggs,HighResolutionXPSofOrganicPolymers:TheScienta ESCA300Database,Wiley,Chichester,1992.