In situ reduction of antibacterial silver ions to metallic silver nanoparticles on bioactive glasses functionalized with polyphenols

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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

Full

length

article

In

situ

reduction

of

antibacterial

silver

ions

to

metallic

silver

nanoparticles

on

bioactive

glasses

functionalized

with

polyphenols

S. Ferraris

a,∗

_,

_M.

_Miola

a,b

_,

_A.

_Cochis

b

_,

_B.

_Azzimonti

b

_,

_L.

_Rimondini

b,1

_,

_E.

_Prenesti

c,1

_,

E. Vernè

a,1

a_Department_of_Applied_Science_and_Technology,_Politecnico_di_Torino,_C.so_Duca_degli_Abruzzi_24,_10129,_Torino,_Italy

b_Department_of_Health_Sciences,_Università_del_Piemonte_Orientale_UPO,_Via_Solaroli_17,_28100,_Novara,_Italy

c_Department_of_Chemistry,_Università_degli_Studi_di_Torino,_Via_Pietro_Giuria_7,_Torino,_10125,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28June2016

Receivedinrevisedform24October2016

Accepted27October2016

Availableonline3November2016

Keywords: Bioactiveglasses Polyphenols Silvernanoparticles Insitureduction Antibacterialactivity

a

b

s

t

r

a

c

t

Therealizationofsurfaceswithantibacterialpropertiesduetosilvernanoparticlesloadedthrougha greenapproachisapromisingresearchchallengeofthebiomaterialﬁeld.

Inthisresearchwork,twobioactiveglasseshavebeendoublysurfacefunctionalizedwith polyphe-nols(gallicacidornaturalpolyphenolsextractedfromredgrapeskinsandgreentealeaves)andsilver nanoparticlesdepositedbyinsitureductionfromasilvernitrateaqueoussolution.Thepresenceof biomolecules–showingreducingabilitytodirectlyobtaininsitumetallicsilver–andsilver nanopar-ticleswasinvestigatedbymeansofUV–visspectroscopy,X-RayPhotoelectronSpectroscopy(XPS)and FieldEmissionScanningElectronMicroscopy(FESEM).Theantibacterialactivityofthemodiﬁedsurfaces wastestedagainstamultidrugresistantStaphylococcusaureusbacterialstrain.

1. Introduction

Silverisknownfromancienttimesforitsbroadspectrumof

antibacterialactivityandiswidelyinvestigatedasmulti-purpose

antibacterialagent.Theraisingdiffusionofbacterialresistanceto

commonantibiotics,recentlydeﬁnedasglobalthreat[1],increases

theinterestinalternativeantibacterialsubstancesandinparticular

inorganicones,suchassilver.Thedevelopmentof

nanotechnolo-giesfocuses the attention onsilver nanoparticles as promising

antibacterial agents [2,3] and numerous silver

nanoparticles-loadedproductscomeintothemarketinvariousapplicationﬁelds

(e.g. drugs, personal care/cosmetics, textiles/shoes, electronics,

householdproducts, ﬁltration/sanitization, medical devices)[4].

Despitethewidediffusionofsilvernanoparticles,itspotential

tox-icityforhealthandenvironmentisnotcompletelyknownupto

now[4].Theantibacterialmechanismofsilvernanoparticlesisnot

yetfullyunderstood,howeveramultipleactionwasproposed:i)

interactionwiththebacterialcellwallanditsconsequentdamage,

ii)inductionofoxidativestressbyReactiveOxygenSpecies(ROS)

productionandiii)sustainedreleaseofsilverions[5,6].Thanksto

∗ Correspondingauthor.

E-mailaddress:sara.ferraris@polito.it(S.Ferraris).

1 _Co-shared_authorship.

thehighsurfacetovolumeratioandtheprobablemultiplemodeof

action,silvernanoparticlespresentsuperiorantibacterialactivity

comparedtobulkmetallicsilver.

Silvernanoparticlescanbeproducedbytopdownapproaches,

whichforeseethedimensionalreductionoflargerstructures(e.g.

mechanical, ball milling, chemical etching, thermal/laser

abla-tion,sputtering)orbottomupapproaches,basedonaggregation

processes(suchaschemical/electrochemicalprecipitation,vapor

deposition,atomic/molecularcondensation,sol-gel,spray

pyroly-sis,laserpyrolysisoraerosolpyrolysis)[7,8].Themaindrawbacksof

commonsynthesisroutesforsilvernanoparticlesarethe

employ-ment of toxic chemicals and high temperature, pressure and

energy,sothatnewgreenandenvironmentallyfriendlystrategies

areneeded.Variousgreensynthesisapproacheshavebeen

investi-gatedsuchasthoseusingmicroorganisms(bacteria,fungi,yeasts)

ratherthanalgaeorpolysaccharidesand plantsextracts[8–11].

Amongthem,theuseofnaturalsubstancessuchasplantextracts

seemsmorepromisingfortheindustrialapplicationbecauseitis

easilyscaledupanddoesnotrequirecomplexsystemsfor

microor-ganisms’ cultureand related biohazardconcerns [8]. Moreover,

reducing agents canbeobtainedfromvegetal productscoming

fromthewastesoffoodandwineproductionchains,openingthe

opportunityforasustainableuseofresourcesobtainedfrom

vege-talresiduesthatwouldbecomewastes.

http://dx.doi.org/10.1016/j.apsusc.2016.10.177

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Table1

Glass composition (molar percentages), melting temperatures and annealing

conditions.

Glasscomposition(mol%) Tmelt[◦C] Annealing SiO2 Na2O CaO Al2O3

SCNA 57.0 6.0 34.0 3.0 1550 10h@600◦_C

SCN1 57.0 9.0 34.0 0.0 1500 12h@550◦_C

Numerousexperimentalstrategieshavebeenreportedinthe

recentscientiﬁcliteratureforthegreensynthesisofcolloidal

sil-vernanoparticles[8–21]althoughfewpapersconsiderspeciﬁcally

insitureductionofsilvernanoparticlesonsubstrates[22–24].

In the present paper, for the ﬁrst time, silver nanoparticles

(Ag-NPs)have been obtainedby in situ reduction onbioactive

glassesfunctionalized withgallic acid(usedas a simple model

moleculeto represent thepolyphenols reactivity) or with

nat-ural polyphenols extracted from red grape skin and green tea

leaves.Theeffectofglasssurfacereactivityontheabilitytograft

biomolecules and, subsequently, to induce in situ reduction of

silverionswasinvestigatedbymeansofUV–visphotometry,

X-RayPhotoelectronSpectroscopy(XPS)andFieldEmissionScanning

ElectronMicroscopy(FESEM).TheantibacterialactivityofAg-NPs

dopedbioactiveglasseswasevaluatedagainstamultidrug

resis-tantstrainofStaphylococcusaureusbymeansofbioactivecoating

derivedmetabolicreductionevaluation.

Thisresearchworkpresentsapromisingstrategytoobtain

inno-vativesmartpolyfunctionalbiomaterialscombiningthepeculiar

featuresofi)bioactiveglasses(bioactivity,ionreleaseability),ii)

polyphenols(antioxidant,antibacterial,vascularprotective,bone

stimulatingactivities)andiii)silver(antibacterial).Inthisroute,

Ag-NPsresultsembeddedontheglasssurfacereducingrisk

con-cernsrelatedtothefreemetallicnanoparticles.

2. Materialsandmethods

2.1. Glasssynthesis

Twobioactive glasses,designed in theauthors’laboratories,

wereconsidered:SCNAandSCN1.Themolarcompositionand

melt-ing/annealingconditionsarereportedinTable1.Thereactivityof

bioactiveglassesstronglydependsontheircomposition.In

par-ticular,itwasevidencedthatthesilicacontent(formeltderived

bioactiveglasses)shouldnotexceed60%(mol),inordertoobtain

abioactivebehavior[25]andthattheadditionofaluminainthe

glasscompositionreducetheglassbioactivitybecauseitinhibits

theionexchangeimprovingthematerialstability[25,26].

More-oversodiumisinvolvedintheionexchangeprocess,whichisthe

ﬁrststepinthebioactivitymechanism[25–27].Thetwobioactive

glassesconsideredinthisresearchworkpresentthesamesilica

contentanddiffersforthepresence/absenceofaluminaandforthe

sodiumcontent.SCNAisahighlystableglassduetothepresence

ofaluminaamongitsconstituentoxides.SCN1presentsalarger

reactivitybecauseofahigherNacontentandtheabsenceof

alu-mina.Theseglasseswerechosenfortheirsimplecompositionsand

controllablereactivityinordertoinvestigatetheirabilitytograft

polyphenolsandinduceinsitureductionofsilvernanoparticles.

2.2. Surfacefunctionalizationwithpolyphenols

Gallicacid(GA),usedassimplemodelmolecule,orpolyphenols

extractedfromredgrapeskin(GPH)andgreentealeaves(TPH)

wereconsideredfortheorganicsurfacefunctionalization.

GallicacidwaspurchasedfromSigmaAldrich(GA97.5–102.5%

titration,G7384,SigmaAldrich,Milan,Italy)whilenatural

polyphe-Table2

Samplesnames,treatmentsandsurfacefeatures.

Samplename Treatment Surfacefeature

SCNA-wash Acetoneandwater

washing

OHgroups

SCNA+GA Acetoneandwater

washing+GAgrafting

GAmolecules

SCNA+GPH Acetoneandwater

washing+GPHgrafting

GPHmolecules

SCNA+TPH Acetoneandwater

washing+TPHgrafting

TPHmolecules

SCN1-wash Acetoneandwater

washing

OHgroups

SCN1+GA Acetoneandwater

washing+GAgrafting

GAmolecules

SCN1+GPH Acetoneandwater

washing+GPHgrafting

GPHmolecules

SCN1+TPH Acetoneandwater

washing+TPHgrafting

TPHmolecules SCNA-wash+Ag Acetoneandwater

washing+insitu reductionAgnps

OHgroups/AgNPs

SCNA+GA+Ag Acetoneandwater

wash-ing+GAgrafting+in situreductionAgNPs

GAmolecules/AgNPs

SCNA+GPH+Ag Acetoneandwater

wash-ing+GPHgrafting+in situreductionAgNPs

GPHmolecules/AgNPs

SCNA+TPH+Ag Acetoneandwater

wash-ing+TPHgrafting+in situreductionAgNPs

TPHmolecules/AgNPs

SCN1-wash+Ag Acetoneandwater washing+insitu reductionAgnps

OHgroups/Agnps

SCN1+GA+Ag Acetoneandwater

wash-ing+GAgrafting+in situreductionAgNPs

GAmolecules/AgNPs

SCN1+GPH+Ag Acetoneandwater

wash-ing+GPHgrafting+in situreductionAgNPs

GPHmolecules/AgNPs

SCN1+TPH+Ag Acetoneandwater

wash-ing+TPHgrafting+in situreductionAgNPs

TPHmolecules/AgNPs

nolswereextractedbyconventionalsolventextractionmethod,as

previouslydescribedbytheauthorsin[28,29].

Theabovecitedbiomoleculesweredirectlygraftedonthe

sur-faceof SCNA and SCN1 afterhydroxylsexposition ontheglass

surface.ReactiveOHgroupswereexposedbymeansofacetoneand

waterwashingsinultrasonicbath,asdescribedin[28–32].Washed

sampleswerenamedSCNA-washandSCN1-wash(Table2).The

graftingofbiomoleculeswasperformedbysoakingwashedglasses

inasolutionofpolyphenols(1mg/mlforGAandTPHand5mg/ml

forGPH)for3hat37◦C[29,33].Attheendofthesoakingperiod

samplesweregentlywashedtwotimesinultrapurewaterandlet

dryunderalaminarﬂowcabinet(FASTERCYTOSAFE)indark

condi-tions.FunctionalizedsampleswerenamedSCNA+GA,SCNA+GPH,

SCNA+TPH,SCN1+GA,SCN1+GPHandSCN1+TPH(Table2).

2.3. Insitureductionofsilvernanoparticles

Functionalized biomaterials with the mentioned organic

molecules were soaked1h at 37◦C in a 0.005MAgNO3

aque-ous solution in order to obtain the in situ reduction of silver

nanoparticles(AgNPs)ontheglasssurface,exploitingthereducing

actionofpreviouslygraftedpolyphenols.Attheendofthe

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dryunderalaminarﬂowcabinetindarkconditions.Polyphenols

graftedandsilvermodiﬁedsampleswerenamedSCNA+GA+Ag,

SCNA+GPH+Ag,SCNA+TPH+Ag,SCN1+GA+Ag,SCN1+GPH+Ag

and SCN1+TPH+Ag(Table2).Washedsamples weresubjected

tothesametreatmentforcomparisonpurposesandwerenamed

SCNA-wash+AgandSCN1-wash+Ag(Table2).

2.4. Physico-chemicalcharacterization

PhotometricmeasurementsintheUV–visspectralregion(CARY

500Varianspectrophotometer)wereperformedtoquantifythe

amountofactivepolyphenols ontheglass surfacebymeansof

theFolin&Ciocalteumethod[34],aspreviouslydescribedbythe

authors[29,33].Astandardcalibrationcurvewasobtainedwith

GAsolutionsofknownconcentrationandusedforGA

quantiﬁca-tiononthesamples[30,28,29,33].Asfarasnaturalpolyphenols

(acomplexblendofvariousmono-andpolyciclictypeof

phenol-basedmolecules)areconcerned,theirconcentrationwascalculated

ingallicacidequivalentsunits,employingtothesamecalibration

curve[28,29].

Surfacechemicalcompositionandchemicalstateofelements

wereanalyzedbymeansofX-rayPhotoelectronSpectroscopy(XPS,

PHI5000VERSAPROBE,PHYSICALELECTRONICS)inorderto

deter-mine the presence of biomolecules after functionalization and

silvernanoparticlesafterinsitureduction.

Field Emission Scanning Electron Microscopy (FESEM-EDS

SUPRATM40,Zeissand MerlinGeminiZeiss)wasemployedfor

theinvestigationofsilvernanoparticlesprecipitationonsamples

surface.SamplesweresputtercoatedwithathinCrlayer(<5nm)

beforeanalyses.

2.5. Antibacterialactivityevaluation

SCNA and SCN1 samples functionalized with polyphenols

extractedfromgreentealeavesandthesameafterinsitureduction

ofAgNPswereconsideredfortheantibacterialtests,becausethey

showedthebestresultsintermsoffunctionalizationamongthe

moleculesofnaturalorigin.Justwashedsampleswerealsotested

forcontrolpurposes.

2.5.1. Bacterialstrainsandgrowthconditions

The exponentially-growing bioﬁlm pathogen Staphylococcus

aureus(clinicalisolatefromtheHospitalMaggioreofNovara)strain

wasusedtoevaluatetheantibacterialactivityofsamples.Bacteria

werecultivatedonblood-agarplates(SintakS.r.l.,Corsico,Milan,

Italy) at 37◦C in aerobicconditions for 48huntil roundsingle

colonieswereobtained.Plateswerethenstoredat4◦Cuntiluse.

2.5.2. Bioﬁlmformation

Specimenswereplacedintothewellsofa12multiwellplate

(NuncDelta, Nunclone). Then,500mLof freshbacterialculture

were prepared by inoculating about 4–5 single colonies into

LuriaBertanibroth(LB,Sigma-Aldrich,Milan,Italy);cultureswere

incubatedat 37◦C in a Gallenkamp orbitalshaker incubator at

200rpm for 16h. Exponentially-growing bacterial suspensions

werethen diluted in freshLB medium ata ﬁnal concentration

of 1_×107_cells_mL−1 _according _to_McFarland _standard _1.0 _[35]_.

1mLofthebrothculturewascollectedandusedtocontaminate

specimens;platewasincubatedat37◦Cinrotation(90rpm)for

90min(adhesionphase).Thesupernatant containingplanktonic

cellswasthenremoved,whilebioﬁlmformercells,attachedtothe

specimens’surfaces,wererinsedwith1mLof freshLBmedium

(separationphase)[36,37].Platewasincubated24hat37◦Cina

humidatmospheretoallowmaturebioﬁlmgrowth.

Table3

Polyphenolsamountonthesurfaceofglasssamples.

Glass Polyphenolcontent–GA-equivalents[mg/ml](mean±stdev)

GA GPH TPH

SCNA 0.0000±7.2832·10−8 _0.0000_±_7.071·10−6 _0.0000_±_7.071·10−6 SCN1 0.0004±0.0002 0.0000±0.0005 0.0025±0.0006

2.5.3. Bacterialcellviability

To assess the growth capacity of the bacterial after 24h

of direct contact to specimens’ surface, compared to that

of untreated controls, bacterial viability was evaluated by

the validated quantitative colorimetric metabolic 2,3-bis

(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenyl amino)

carbonyl]-2H-tetrazoliumhydroxide assay(XTT,Sigma). Brieﬂy, 100␮Lof

XTTsolution(3mgmL-1inacetonecontaining0.1Mmenadione)

wereaddedtoeachwellandplateswereincubatedat37◦Cfor

5hinthedark;100␮Lwerethencollectedfromeachwell,

cen-trifugedfor2minat1200rpmtoremoveanydebris,andtheoptical

density(o.d.)wasevaluatedusingaspectrophotometer

(Spectra-Count,IBM,NY,USA)at490nm[38].Experimentswereperformed

intriplicate.

2.6. Statisticalanalysis

Alltestswereperformedintriplicate.TheKruskal–Wallis

anal-ysisfollowedbyConovertestaspost-hocwereusedtodetermine

signiﬁcancethatwassetatp<0.05

3. Resultsanddiscussion

3.1. Photometricdeterminationofthepolyphenolcontentonthe

glasssurface

The resultsof theFolin&Ciocalteu test onglass samplesare

reportedinTable3.Itwasimpossibletodetectasigniﬁcantamount

ofpolyphenols(GA,GPHandTPH)onSCNAsurfacebythis

tech-nique,whileacertainamountofGAandTPHwasregisteredon

SCN1 one. These resultsare in accordance with previous ones

obtainedby the authorsonthe same glass[30,28] and can be

attributedtothelow reactivityof SCNAand themoderate

sur-faceareaofglassslices.Theincreaseinreactivityobtainedwith

SCN1compositionallowsamoreeffectivegraftingofbiomolecules,

exceptforGPH.

3.2. XPSanalyses

Atomic percentages of elements from survey spectra are

reportedinTable4.

NosigniﬁcantdifferencesinthesurfaceamountofSiandCacan

behighlightedbetweenSCNAandSCN-1, inaccordancetotheir

theoreticalbulkcomposition.Thelowamountofsodiumrecorded

onSCN-1anditsabsenceonSCNA(comparedtothetheoretical

bulkcomposition)canbeascribedtobothionreleaseinthe

wash-ingsolutionandalsotoasortofhidingduetocarbonpresence

atthesurface.XPSissensitivetotheoutermostsurfacelayer(few

nanometers)andasigniﬁcantamountofcarbonhasbeen

regis-teredalsoonwashedsamplesduetohydrocarbonabsorptionfrom

theatmosphere,aswidelydocumentedintheliteraturefor

reac-tivesurfaces[39–42].EDSanalyses(notreported),thatinteresta

higherpenetrationdepth(around1␮m)detectasodiumamount

closertothetheoreticalone.ThisphenomenonmakesXPSsurvey

analysesnotcompletelysuitableandexhaustiveforthe

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Table4

AtomicpercentagesofelementsfromXPSsurveyspectra.

SCNA

Element wash wash+Ag GA GA+Ag GPH GPH+Ag TPH TPH+Ag

O 56.8 55.1 59.6 55.6 57.0 49.9 53.6 54.6 C 21.7 18.0 15.1 15.2 20.6 26.3 22.3 20.5 Si 15.4 20.1 22.9 23.4 21.3 19.3 19.6 19.2 Ca 3.5 0.8 0.3 1.3 0.5 Al 2.6 2.6 1.6 1.2 2.7 N 0.4 Ag 4.2 5.9 4.0 5.2 Cl 0.2 0.1 SCN1 O 53.4 48.7 53.0 49.3 52.6 50.7 50.0 50.9 C 25.3 29.3 27.1 29.5 30.0 27.4 31.2 26.6 Si 17.1 16.8 17.9 16.4 14.3 16.4 17.5 16.4 Ca 2.8 2.1 1.1 0.2 1.9 0.3 1.0 1.1 Na 1.3 1.0 1.0 0.3 0.9 N 1.0 0.9 0.5 0.2 0.1 0.1 Ag 1.1 4.1 5.0 0.2 3.9

spectraoftheoxygenregion(notreported)hasbeenrecordedand

conﬁrmedhydroxylsexpositiononbothglassesafterwashing.

Nosigniﬁcanttrendscanberegisteredforcarbonandoxygen

ascharacteristicelementsof graftedbiomolecules.The detailed

analysisofcarbonregionisnecessaryinordertoinvestigatethe

effectivenessof the surfacefunctionalizationprocedure, as

dis-cussedinthefollowing.

Ontheotherhand,asigniﬁcantamountofsilvercanbedetected

onAg-treatedsamples,conﬁrmingthesurface abilitytoinduce

thereductionof Ag(I)andthesubsequentuptake. Ahigher

sil-vercontentcanbeobservedonpolyphenolgraftedSCN1,whileno

signiﬁcantdifferencesbetweenfunctionalizedandwashed

sam-plescanbenotedonSCNAsamples.Thenatureofdepositedsilver

(metallic/ionic)was investigatedby the detailed analysisof Ag

regioninthefollowing.Asmallamountofsilverhasbeendetected

onSCN1+TPH,theamountcanbeconsiderednegligiblebecauseit

fallsintheinstrumentalerror(0.1–0.2%at.)andcanbeattributed

tocontamination.

Thedetailedanalysisofcarbonregionforwashedand

function-alizedsamplesisreportedinFig.1.

On thewashed samples (Fig. 1a and e) two main

contribu-tions,atabout284.8eVand289eV,canbedetectedandattributed

to C C/C H from hydrocarbon contaminants and carbonates

respectively [39–42]. Surface contaminations by atmospheric

hydrocarbonsarealwayspresentontoreactivesurfaces[39–41],

asalreadyobserved,togetherwithcarbonates,onbioactiveglasses

bytheauthors[28–30,43].Asmallsignalatabout287eVcanbealso

detectedonSCN1-wash;C Obondscancorrespondtothisenergy,

butitcanalsocomefromcontaminantsonthissample.

Thesignalofcarbonatesdisappearsaftersurface

functionaliza-tion,aspreviouslyobservedbytheauthors[28–30].Asigniﬁcant

signalataround 286eVappearsonall thepolyphenols grafted

glasses(Fig.1b,c, d, f–h) and can beattributed toC Obonds

in polyphenols molecules [44–46]. This signal was previously

observedonpolyphenolsgraftedbioactiveglassesbytheauthors

[28–30].A moderate contributionin the 288eV around canbe

observedonSCN1samplesafterpolyphenolsgrafting. Itcanbe

attributed to the C O bonds [44–46] in carboxylic groups of

polyphenols or in hydroxyl groups of polyphenols oxidized to

quinones, as previously observed by the authors [28–30]. The

appearanceoftheC Osignalonlyonthemostreactiveglasscan

beassociatedwitha higherion release inthefunctionalization

medium withconsequent higher alkalinization and polyphenol

oxidationduringgraftingforthismaterial.

Thesignalat284.8eVpersistsafterthefunctionalizationbut

itsrelativeintensityislowerthanonthewashedsurfaces,

espe-ciallyfor SCNA. Itmust beunderlinedthattheC C/C Hsignal

onthewashedsamplecanbeattributedtosurfacehydrocarbon

contaminantsfromtheatmosphere.Inabsenceofsurface

function-alization,andforastableglassforwhichcarbonatationislow,itis

themostsigniﬁcantsignalinthecarbonregion.After

functionaliza-tionthesignalrelatedtothecharacteristicfunctionalgroupsofthe

biomoleculesbecomemorerelevantcomparedtothiscontribution

anditsrelativeintensitydecreases.

ThedetailedanalysisofthesilverregionforAg-treatedsamples

isreportedinFig.2.Forallthesamples,themaincontributionis

givenbythesignalsatabout368.2eVand374.3eV,attributableto

metallicsilver(Ag0₎_[47,48]_._A_second_contribution_at_lower_binding

energies(about367.7eVand373.1eV)canbeobserved,exceptfor

SCN1-wash+Agsample,andattributedtosilveroxides(AgO,Ag2O)

[47,48].ThiscontributionisalmostnegligibleforSCNAsamples,

whileit becomes signiﬁcantforSCN1+GA+Ag,SCN1+GPH+Ag

andSCN1+TPH+Ag.

Thepresenceofmetallicsilveronthesurfacesconﬁrmstheir

abilitytoreducesilverionsfromtheAgNO3solutionofthe

treat-ment.Thisreducingabilitycanbeascribedtograftedpolyphenols

forfunctionalizedsurfaces.Inthecase ofwashedglassesa

cer-tainreducingabilitycancomefromthesurfaceexposedhydroxyl

groups.Thisresultisinaccordancewiththeantioxidantabilityof

bioactiveglassespreviouslyobservedbytheauthors[29].

Thepresenceofsilveroxidescanberelatedtotheabsorption

ofsilverionsinthesurfacereactionlayerduringthemodiﬁcation

process.ThisphenomenonismoreevidentonfunctionalizedSCN1

glassbecauseofahigherreactivityandamorepronouncedreaction

layer,asconﬁrmedbyFESEMobservations(paragraph3.3).

3.3. FESEMobservations

FESEMimageshavebeenrecordedbothinsecondaryelectrons

andinback-scatteredonesmodesinordertoinvestigatesurface

morphologyanddiscriminatethepresenceofsilvernanoparticles.

InfactAgisheavierthantheglassconstituentsandsilver

nanopar-ticlescanbedetectedasbrightspotsonback-scatteredelectrons

images.

FESEM images of samples (secondary electrons and

back-scatteredones)arereportedinFig.3.

Numerousparticles,withabrightappearanceinback-scattered

images, can be observed on all the samples except of

SCN1-wash+Ag. Particles form aggregates of at about 200nm on

SCNA-wash+Ag while particles with dimensions lower than

100nmandsmallaggregatescanbeobservedonSCNA+GA+Ag,

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Fig.1. XPShighresolutionspectraofcarbonregion.

dimensionscanbeevidencedonSCN1+GA+Ag,SCN1+GPH+Ag

andSCN1+TPH+Ag.Ontheotherhandonlyprecipitateswith

big-gerdimensions(few␮m)andcaltrop shapecanbedetectedon

SCN1-wash+Ag(Fig.4).

AmoderatereactionlayercanbeobservedonSCNAsamples

afterthevarious modiﬁcationprocesses asan irregulartexture

(Fig.3).Ontheotherhandasigniﬁcantreactionlayer,moreevident

aftergraftingofbiomolecules,canbeobservedonSCN1samples,

conﬁrmingthehigherreactivityofthisglass(Fig.3).Considering

bothFESEMandXPSresultsitcanbehypothesizedthatsilver

pre-cipitatesonlyasmetallicnanoparticlesonSCNAsubstratesandon

SCN1-washones,whileitisalsoadsorbedinionicforminthe

reac-tionlayeronSCN1+GA+Ag,SCN1+GPH+AgandSCN1+TPH+Ag.

EDSanalysesconﬁrmthepresenceofsilverinalltheobserved

precipitates.

3.4. Antibacterialactivity

SinceTPHgraftedandTPHgrafted/Agtreatedsamplesshowed

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Fig.2. XPShighresolutionspectraofthesilverregion.

ofnaturalorigin,theywerebothconsideredfortheantibacterial

teststogetherwithwashedcontrolsinordertoevaluatetheeffect

ofbothnaturalpolyphenolsandsilvernanoparticlesonbacterial

viability.Themainadvantagesintheuseofnaturalextractsinstead

ofsyntheticchemicalsarethepossibilityofrecoveryfromnatural

sources,witheconomicandenvironmentalbeneﬁts,andreduced

toxicologicalconcerns.

TheresultsofantibacterialtestsarereportedinFig.5.

XTTanalysis(Fig.5a)revealed a statisticalsigniﬁcant

differ-encebetween washed control specimens (wash) and

polyphe-nols+silver doped (TPH+Ag) ones for both SCNA (p<0.05,

indicatedby*)andSCN-1(p<0.05,indicatedby#).Nosigniﬁcant

differenceswerenoticedbetweencontrolsandpolyphenols(TPH)

graftedspecimens(p>0.05forbothSCNAandSCN-1bioglasses).

Finally,bacteriaviabilityafter24hisreportedinFig.5bin

func-tionofcontrols.

No signiﬁcant differences have been observed between the

behaviorofthetwoglassesdespiteofthedifferentreactivityand

the differentchemical stateof silver on theirsurfaces (mainly

metalliconSCNAand metallicandioniconfunctionalized

SCN-1).Theantibacterialactionofsilverionsandnanoparticlesiswidely

discussed in the literature and not completely understood yet

[49–51]aswellastheoptimaleffectivetherapeuticdosage[52].As

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Fig.4.FESEMimage(secondaryandback-scatteredelectrons)ofSCN1-wash+Ag(5000xmagniﬁcation).

Fig.5.S.aureusviabilityonthetestedsamples.XTTassay(a)showedsigniﬁcantdifferencesbetweencontrol(washed)andTPH+Agspecimens(p<0.05,indicatedby*and

#forSCNAandSCN-1,respectively).In(b)the24hsbioﬁlmviabilitynormalizedtowardscontrolsarereported.Barsrepresentmeansandstandarddeviations.

themechanismoftheantibacterialactionshouldbeinvestigated

inmoredetailtogetherwiththesilverreleaseandthe

biocompat-ibility,butthisisoutofthescopeofthepresentpreliminarystudy

andwillbediscussedinafutureresearchpaper.

4. Conclusions

Gallicacidandnaturalpolyphenolsextractedfromredgrape

skinandgreentealeaveshavebeensuccessfullygraftedontwo

bioactiveglasseswithdifferentsurfacereactivity. Theabilityto

graft biomolecules increases with the glass surface reactivity.

Polyphenolicbiomolecules(asgallicacidornaturalonesextracted

byspeciﬁcplantsources)graftedonthesurfaceofbioactiveglasses

and,inacertainmeasure,alsothesurfacehydroxylgroupsofthe

glass,makeitpossiblethereductionofthesilverions(comingfrom

asilvernitrateaqueoussolution)tometallicsilvernanoparticleson

theglasssurface.Thespeciﬁcnoveltyofthispaperconsistsinthe

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onthesurfaceofeach bioactiveglassunderexamination

previ-ouslygraftedwithnaturalorganicreducingmolecules(gallicacid

orblendsofpolyphenolsextractedfromplants)alsoabletoact

asprotective agents (antioxidant,antibacterial, vascular

protec-tive,bonestimulatingactivities).Bioactiveglassesfunctionalized

withteapolyphenolspresentastatisticallysigniﬁcant

antibacte-rialactivityagainstStaphylococcus aureusafterinsitu reduction

ofsilvernanoparticles.Finally,thestrategyofpreparationofthe

functionalizedbiomaterialsdevelopedopenstheopportunityfor

asustainableuseofresourcesobtainedfromvegetalresiduesthat

wouldbecomewastes.Thevegetablescrapsofdifferent

machin-ingoperationscanbecomevaluablesourcesofbiomoleculeswhose

activitiesareusefulinvarioussectorsofimpacttothehealthand

totheeconomy.

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