Collagen-chitosan scaffold modified with Au and Ag nanoparticles: synthesis and structure

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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,∗

a_A.N._Nesmeyanov_Institute_of_{Organoelement}_Compounds,_Russian_Academy_of_Sciences,_Moscow,₁₁₉₉₉₁_Russian_Federation b_National_Research_center_«Kurchatov_Institute»,_Moscow,₁₂₃₁₈₂_Russian_Federation

c_Department_of_Chemistry,_Bilkent_University,_Ankara,₀₆₈₀₀_Turkey

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-carryingscaffoldshavelayeredstructureandthenanoparticlesarerather

uni-formlydistributedonthesurfacematerial.XPSdataindicatethatthemetallicnanoparticlesareintheir

unoxidized/neutralstatesanddominantlystabilizedwithinthechitosan-richdomains.

1. Introduction

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

Collagenisastructuralproteinactivelyusedinmedicine[14]. Availabilityofhydroxylicgroupsandaminoacidresiduesonits sur-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,thatiswhyitiswidelyusedin tis-sueengineeringaswoundandburndressingandsponges.Collagen andchitosandonotoccurinnaturetogether,butspeciﬁc proper-tiesofbothpolymerscanbeutilizedtodesignahybridmaterial withuniquestructuralandmechanicalproperties[17].

Numerousexamples of similarmaterialsused for thetissue engineering, drugdelivery matrices, bandage dressing, etc.,are availablein literature[18,19].Incorporation ofnoble metalNPs withantibacterialactivityfurtherextendsapprovedﬁeldsof appli-cationsofcollagen-chitosanscaffolds.Themajorityofmethodsfor theincorporationofmetalNPsinpolymermatricesrequires chem-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,withmetalnanoparticles pos-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 preparationofhighlyreactivemetalnanoparticlesandtheir incor-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 heatedevaporatorintheformofeithertungstenrodorsmall tan-talumvesselforAu(99.99%)orAg(99.99%),respectively.Specially preconditionedorganicsolventsEt3Nandi-PrOHwereusedto sta-bilize,respectively,AuandAgnanoparticlesassols.Metalvapor wascondensed simultaneouslywiththesolventvaporonto liq-uidnitrogen-cooledwallsofaglassreactorwithavolumeof5L.A typicalsolvent-to-metalmolarratiointhesynthesiswas300:1. Afterthesynthesis,theco-condensate washeatedtothe melt-ingpoint andtheresultantorganosolwasimpregnatedintothe collagen-chitosanscaffoldplacedinaSchlenkﬂaskundervacuum. Theexcessiveamountoftheorganosolwasremovedandthetarget productwasdriedinvacuumat60◦C.

2.3. Characterizationmethods

2.3.1. Synchrotronradiation-basedtechniques

X-raystructuralstudiesofthecompositesbasedon collagen-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 aswellasfortheAufoilreferenceweremeasuredinthe trans-missionmodeusingionchambersfilledwithappropriateN2-Ar mixtures.ForsimilarAg-containingcomposites,thesilver concen-trationappearedinsufficientfortransmissionmeasurementsand thusthespectraweremeasuredintheX-rayfluorescenceyield modeusingaSiavalanchephotodiode.SpectrafortheAgfoil refer-enceweremeasuredinthetransmissionmode.Experimentaldata processingandanalysiswereperformedusingtheIFEFFITsoftware package[34].

PowderX-raydiffractionpatternsforthesamesampleswere measuredin thetransmission (Debye-Sherrer)modeusing Fuji FilmImagingplatesasa2Ddetector.Diffractionmeasurements wereperformedatanX-raywavelength=0.06889nm.Themean nanoparticlesizewasestimatedbyproﬁleanalysisassumingthe Pseudo-Voightlineshapeofthediffractionpeakswith instrumen-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.The sample-to-detectordistancewas2400mmandtheX-raywavelengthwasset to=0.162nm.Toimprovesamplingandsignal-to-noise statis-tics,severaldatasetsatdifferentspotsonasampleweremeasured and averaged.The sizedistributionof Agand Au nanoparticles wasretrievedfromthecorrespondingSAXS“Ag-CollCh-CollCh” and“Au-CollCh-CollCh”differencecurves,respectively.The indi-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␣radiationatan operat-ingpowerof150WoftheX-raytube.Surveyandhigh-resolution spectraofappropriatecorelevelswererecordedatpassenergiesof 160eVand40eVandwithscanningstepsof1eVand0.1eV, respec-tively.Sampleareaof300␮m×700␮mcontributedtothespectra. Thesamplesweremountedonasampleholderwithatwo-sided adhesivetape,andthespectrawerecollectedatroomtemperature. ThebasepressureintheanalyticalUHVchamberofthe spectrom-eterduringmeasurementsdidnotexceed10−8Torr.Thebinding energyscaleofthespectrometerwascalibratedtoprovidethe fol-lowing valuesfor referencesamples(i.e., metalsurfacesfreshly cleanedbyionsputtered):Au4f7/2–83.96eV,Cu2p3/2–932.62eV, Ag3d5/2–368.21eV.Theelectrostaticchargingeffectswere com-pensatedbyusinganelectronneutralizer.For eachsample,the spectrawererecalibratedagainsttheadventitiouscarbonsignalat 285.0eV.Backgroundwithinelasticlosseswassubtractedfromthe high-resolutionspectraaccordingtotheShirleyprescription.The AgMNNAugerspectrawerecorrectedusingalinearbackground. Thesurfacechemicalcompositionwascalculatedusingstandard elementsensitivityfactorscodedinthespectrometercontrol soft-warecorrectedforthetransferfunctionoftheinstrument. 2.3.3. TEM

Micrographsofthesamplesweremadeusingatransmission electronmicroscopeLEO912ABOMEGA,Zeiss(Germany). 2.3.4. SEM

SEManalysiswasperformedwithascanningelectron micro-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 the collagen-chitosanscaffoldsmodiﬁed bygoldandsilvernanoparticlesare showninFig.1.Thecollagen-chitosanscaffoldappearstobe amor-phoussothatalldiffractionpeakspresentintherespectivepatterns canbeattributedtofccatomicpackingoftheAgandAu nanoparti-cles[PDF#040783and#040784,respectively].Thecrystallitesizes estimatedfromthelinebroadeninganalysisassumingthat micros-trainsmakenegligiblecontributionyield10.5nmand20.2nmfor Au-CollChandAg-CollCh,respectively,thenominalaccuracyofthe estimateis10–15%. 0.1 0.1 1 10 100 1000 10000 0 25 50 75 100 Sc at terin g Intens it y, a. u. q, nm-1 Volume fra ct ion , a. u . d, nm

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

Forthecomposites,anindependentestimateforthe nanopar-ticlessizewasobtainedfromtheSAXSdata.Thecorresponding experimentaldataareshowninFig.2.

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

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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.Itis demon-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 indicatethatthechemicalstateofthemetalatomsinthe compos-itesissimilartothatintheirbulkmetals,whichmeansthatthe fractionofsurfaceatomsstronglyinteractingwiththematrixis ratherlow.

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

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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-based com-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)ofEXAFSspectraforthe

compos-itesunderstudyareshowninFig.6.Theyareessentiallysimilar

tothoseofthereferencemetalfoils.Best-ﬁtcoordinationnumbers andinteratomicdistancesfortheﬁrstmetal-metalcoordination spheresarelistedinTable1.

Thecompositesarecharacterizedbysomewhatdecreased coor-dination numbers when compared with the data the for bulk metals,whichisverycommonfornanometer-sizedmetal parti-cles.Theminimummetal-metalcoordinationnumberisobserved fortheAg-CollChcompositedespiteitsratherlargercrystallitesize asestimatedfromdiffractiondata.Furthermore,theAg–Ag inter-atomicdistancederivedfromtheEXAFSforthatcompositeisby 0.03 ˚AshorterthanthatforAgfoil.Thebondlengthcontractionis quitetypicalofsmallmetalnanoparticles.Butinthecaseof sil-ver,bondlengthelongationissometimesobservedduetopartial oxidationandsuboxideformation[36].Nevertheless,aprominent changein thecoordinationnumberand bondlengthforthe Ag-CollChdespitenominallylargesizefromdiffractionmayindicatea distortedlocalstructureofsilvernanoparticleswithfrequentpoint defects.

Inordertoevaluatethesurfaceconcentrationsand chemical statesofmetalatomsinthecomposites(withinalayerof5–8nm), X-rayphotoelectronspectroscopywasapplied(Fig.7).Survey spec-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-chitosan scaf-foldsgivesrisetosomeenrichmentofthecompositesurfaceswith carbonspecies(seeTable2).Partly,thiscanbeduetoco-sorption oftheorganicsolventusedinthesynthesis.Theabsenceof dra-maticchangesinelementconcentrationsratherconfirmsthatthe CollChscaffoldsdonotdegradeuponmodificationwithnoblemetal nanoparticles.

TheAu4fbindingenergyobservedforthecomposite(Fig.7c)is characteristicoftheAu0_state._A_minute_shift_by_0.05_eV_to_a_higher energywithrespecttofoilandpronouncedasymmetryofthepeak shapeareduetothesizeeffects[37,38]andthustheyalsoconﬁrm thepresenceofnm-sizedgoldnanoparticlestherein.

High-resolutionAg3dcore-levelsandMNNAugerlinesforthe Ag-containingcompositeareshowninFig.7dande.The experi-mentalvaluesfortheAg3dbindingenergy,AgMNNAugerkinetic energy,andthecorrespondingAugerparameterallconﬁrm[39,40] theAg0_state_of_silver_atoms_in_the_composite._The_N_1s_spectra_in metal-containingcompositesandpristinescaffoldsare character-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-functionalgroupsuponthe modiﬁca-tion.TypicalN1score-levelbindingenergiesinchitosan(C-NH2) andcollagen(C(O)N)are399.67eV[41]and399.77–399.96[42], respectively.

4. Conclusions

Thenovelmethodforthesynthesisofhybridmaterialsbasedon thecollagen-chitosanscaffoldmodiﬁedwithAgandAu nanopar-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,consistingofsmaller parti-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

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