Prolonged Antibacterial Effects and Bacterial Interaction Mechanisms for Wound Dressings

19 Pages·2017·6.75 MB·English

nanomaterials
Article
Nanosilver–Silica Composite: Prolonged
Antibacterial Effects and Bacterial Interaction
Mechanisms for Wound Dressings
DinaA.Mosselhy1,2,* ID,HenrikaGranbohm1 ID,UllaHynönen3 ID,YanlingGe1,AiriPalva3,
KatrinaNordström4andSimo-PekkaHannula1 ID
1 DepartmentofChemistryandMaterialsScience,SchoolofChemicalEngineering,AaltoUniversity,
02150Espoo,Finland;henrika.granbohm@aalto.fi(H.G.);yanling.ge@aalto.fi(Y.G.);
simo-pekka.hannula@aalto.fi(S.-P.H.)
2 MicrobiologicalUnit,FishDiseasesDepartment,AnimalHealthResearchInstitute,Dokki,Giza12618,Egypt
3 DepartmentofVeterinaryBiosciences,DivisionofVeterinaryMicrobiologyandEpidemiology,
UniversityofHelsinki,P.O.Box66,00014Helsinki,Finland;ulla.hynonen@helsinki.fi(U.H.);
airi.palva@helsinki.fi(A.P.)
4 DepartmentofBioproductsandBiosystems,SchoolofChemicalEngineering,AaltoUniversity,
02150Espoo,Finland;katrina.nordstrom@aalto.fi
* Correspondence:dina.mosselhy@aalto.fi;Tel.:[phone]
Received:24July2017;Accepted:3September2017;Published:6September2017
Abstract: Infectedsuperficialwoundsweretraditionallycontrolledbytopicalantibioticsuntilthe
emergenceofantibiotic-resistantbacteria. Silver(Ag)isakernelforalternativeantibacterialagents
to fight this resistance quandary. The present study demonstrates a method for immobilizing
small-sized (~5 nm) silver nanoparticles on silica matrix to form a nanosilver–silica (Ag–SiO )
2
compositeandshowstheprolongedantibacterialeffectsofthecompositeinvitro. Thecomposite
exhibited a rapid initial Ag release after 24 h and a slower leaching after 48 and 72 h and was
effectiveagainstbothmethicillin-resistantStaphylococcusaureus(MRSA)andEscherichiacoli(E.coli).
Ultraviolet(UV)-irradiationwassuperiortofilter-sterilizationinretainingtheantibacterialeffects
of the composite, through the higher remaining Ag concentration. A gauze, impregnated with
the Ag–SiO composite, showed higher antibacterial effects against MRSA and E. coli than a
2
commercialAg-containingdressing,indicatingapotentialforthemanagementandinfectioncontrol
of superficial wounds. Transmission and scanning transmission electron microscope analyses of
thecomposite-treatedMRSArevealedaninteractionofthereleasedsilverionswiththebacterial
cytoplasmicconstituents,causingultimatelythelossofbacterialmembranes. Thepresentresults
indicatethattheAg–SiO composite,withprolongedantibacterialeffects,isapromisingcandidate
2
forwounddressingapplications.
Keywords: silvernanoparticles; silica; composite; prolongedsilverleaching; antibacterialeffects;
mechanismsofaction;wounddressings
1. Introduction
Theskinisthelargestbodyorgan,formingaprotectivebarrieragainstharmfulbacteria. Skin
damageallowsforbacterialpenetration,enablinglocalwoundinfectionsorsystemicsepticemia[1].
Healingofacutewoundsisanorderlyandtimelyregenerativeprocess. Therefore,themanagement
ofacutewoundsessentiallymeanspreventingcomplications,suchaswoundinfections,whichcan
halttheregenerationprocessoftissuesandconvertacutewoundstochronicwounds[2]. Asclassified
intermsofthedepthoftheskininjury,superficialwoundscompriseinjuriesoftheepidermisand
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Nanomaterials2017,7,261 2of19
papillarydermisonlyandarehealedwithintendays,providedthatinfectionshavebeenprevented[1].
Wounddressingsvaryaccordingtothetypeofwound[3]andplayavitalpartinwoundhealing[4–7]
by acting as physical barriers and by preventing wound contamination and infection [1]. Topical
antibiotics are administered for the initial treatment of infected superficial wounds [8]. However,
theunbridleduseofantibioticshasresultedintheemergenceofbacterialantibioticresistance[9–11].
Thereisagrowingconcernthatthesehighlyresistantbacterialpopulationsmaybeopeningupanera
ofnon-treatableinfections[12]. Mostnotably,theincreaseofseriousinfectionscausedbyMRSAis
alarming,conventionallyinhospitalenvironmentsandwoundcare[13,14],andmayleadtothedeath
ofpatients[4]. Inaddition, MRSAisnowapredominantpathogeninthecommunityreservoiras
well[15,16],alsocausingfatalinfections[17]. Thiswillincreasetheadministrationofvancomycin[15],
aglycopeptideantibioticthatisconsideredasanultimatearsenalfortreatingMRSA[18],whichmay
provoke the tenacity of antibiotic-resistant Gram-positive bacteria [15]. To further complicate the
resistancequandary,vancomycinresistancehasalreadybeenidentifiedinMRSA[19]. Accordingly,
the emergence of antibiotic-resistant bacteria calls for the rapid formulation of new therapeutic
modalitiesthatarelesslikelytopromotethedevelopmentofbacterialresistance[10,14,20].
Silver (Ag) has received resurgent interest for use in medicine, particularly in wound
management[4,21–23]. Notably,Aginwounddressingshasshownpromisingantibacterialeffects[24]
and it has been shown that silver nanoparticles (Ag NPs) are highly antibacterial agents [25–27].
The weaker tendency of Ag to elicit bacterial resistance is due to the complex interference of Ag
NPsandreleasedsilverions(Ag+)withbacterialcells[24]. Forinstance,theinteractionofAgNPs
withthebacterialcellmembranesleadstotheformationof“pits”anddamagetothemembranes,
whichincreasethepermeabilityofmembranes,resultinginbacterialdeath[28]. Moreover,AgNPs
can form free radicals that cause damage to the membranes, leading to an antibacterial effect [29].
Furthermore, Ag+ can interact with phosphorus moieties in DNA, hindering bacterial replication,
as well as interfere with sulfur-containing proteins in the bacterial cell walls and thiol groups of
bacterialenzymes,resultingintheirdamageandinactivation[30]. Consequently,Ag-baseddressings
aregenerallypreferredinthetopicalmanagementofwoundinfections,diabeticwounds[31,32],and
particularlyintheprophylaxisandcontrolofinfectionscausedbyantibiotic-resistantbacteria[4,21,33].
Ontheotherhand,AgNPsaresusceptibletoaggregation,whichresultsinlossoftheirantibacterial
properties[34,35].
Silica (SiO ) particles can be efficiently utilized as a stabilizing matrix for preventing the
2
aggregationofAgNPs[7,27,36–38].Moreover,SiO particleshavehighchemicalandthermalstabilities,
2
are inert and biocompatible, which propose them as an excellent system to deliver antibacterial
agents[11]. ImmobilizationofAgNPscanalsoprovideprolongedantibacterialeffects,asAg+have
beenshowntoexhibitsustainedreleasefromtheimmobilizedAgNPsonsubstrates[39].Oneapproach
toimmobilizeAgNPsisbyutilizingthecore-shellsystems. Themainchallengesinthisapproachare
theaggregationoftheAgcoreswhendecreasingthethicknessofSiO shellsandtheslowdissolution
2
rate of the Ag cores when increasing the shell thickness [40]. Such characteristics can prevent the
fullutilizationofAgNPsinthecore-shellsystems. Therefore,inthisstudy,tomaximizethebenefits
ofimmobilizationandtheprolongedreleaseofAgtosafeguardtheantibacterialeffectsinwound
dressingapplications,wehavedevelopedacompositebyimmobilizingAgNPsonSiO matrix.
2
Previousstudieshavedescribedbroad-spectrumantibacterialeffectsforAg–SiO composites,
2
with more efficacy against Gram-negative bacteria [41–43]. The present study, in turn, aims to
investigate the prolonged antibacterial performance of the composite against both Gram-positive
andGram-negativebacteria(MRSAandE.coli,respectively). AsMRSAandE.coliarecommonwound
pathogens[13,44],therefore,theexaminationoftheirsensitivitytothecompositeisofparticularinterest
consideringtheproposedwounddressingapplications. WhilsttheadministrationofAg-containing
dressingsisincreasing,debatecontinuesconcerningtheirefficacy[4]. Atpresent,thereisonlylittle
publisheddataontheantibacterialefficacyofthedressingsthathaverecentlyreachedthemarket.
IthasevenbeendemonstratedthatthereisnodirectrelationbetweentheAgcontent,Agreleaseand
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Nanomaterials 2017, 7, 261 3 of 20
theantibacterialeffectsoftheAg-containingdressings,andthatahighreleaserateofAgfromthe
dresshinavges riescnenottlya rgeuacahraedn ttehee fmorartkheeti.r Ita nhtaisb aevcteenr ibaeleenf fidceamcoyn[s4tr5a]t.edT htheraet ftohreer,e wise nhoa dviereaclts orelcaotimonp ared
between the Ag content, Ag release and the antibacterial effects of the Ag-containing dressings, and
theantibacterialeffectsofacurrentlyavailablecommercialAg-containingdressing(CSD)withthe
that a high release rate of Ag from the dressings is not a guarantee for their antibacterial efficacy
Ag–SiO composite-impregnatedgauze(Ag–SiO -G)invitro. Thespecificobjectivesofthisstudyare
2 2
[45]. Therefore, we have also compared the antibacterial effects of a currently available commercial
(i)thepreparationandcharacterizationofaAg–SiO composite;(ii)thedeterminationoftheleaching
Ag-containing dressing (CSD) with the Ag–SiO2 2composite-impregnated gauze (Ag–SiO2-G) in
profileandtheprolongedantibacterialeffectsofthecompositeagainstMRSAandE.coli,incomparison
vitro. The specific objectives of this study are (i) the preparation and characterization of a Ag–SiO2
withacoCmSpDo,siwtei;t h(iit)h tehea idmetoefrmaciunatetiowno oufn tdhem laeancahgienmg epnrotfailned anindf etchtei opnrocloonntgreodl; aanntdib(aicitie)rtihale eifdfeecnttsi fiocf ation
ofthethaen ctoibmapctoesritiea lamgaeinchsta MniRsmSAs oanfdth Ee. ccoolmi, pino scoitme.parison with a CSD, with the aim of acute wound
management and infection control; and (iii) the identification of the antibacterial mechanisms of the
2. Recsoumltpsoasnitde. Discussion
2.1. C2h. aRreascutelrtsiz aantido nDoisfcAugss–iSoinO 2CompositeandSiO2Particles
The prepared Ag–SiO composite and SiO particles were characterized utilizing a range of
2.1. Characterization of Ag–2SiO2 Composite and SiO22 Particles
instrumental techniques, such as X-ray diffraction (XRD), scanning electron microscope (SEM),
transmissTiohne perleepctarroend mAgic–rSoiOsc2 ocpoem(pToEsiMte )a,nhdig ShiO-r2e spoalrutitciloens wTEerMe c(hHarRaTctEerMiz)e,de nuetirligzyindg ias prearnsgiev eofX -ray
instrumental techniques, such as X-ray diffraction (XRD), scanning electron microscope (SEM),
spectroscopy(EDX)ofthescanningtransmissionelectronmicroscope(STEM),andZetasizer. TheXRD
transmission electron microscope (TEM), high-resolution TEM (HRTEM), energy dispersive X-ray
patternsoftheAg–SiO compositeandSiO particlesaredisplayedinFigureS1. Thehumpsaround
spectroscopy (EDX)2 of the scanning trans2mission electron microscope (STEM), and Zetasizer. The
25◦ (2θ) in both patterns are attributed to the amorphous structure of the SiO . The XRD pattern
XRD patterns of the Ag–SiO2 composite and SiO2 particles are displayed in Figure2 S1. The humps
ofthearcooumndp o2s5i°t e(2dθo) eins nbootthr epvaettaelrndsi fafrrea cattitorinbupteeadk tso ftohre tahmeocrrpyhsotaulsl isnteruActgu.reT ohfe tahbe sSeinOc2e. Tohfed XifRfrDa ction
peakspaftotrertnh eofA tgheN cPomscpaonsitbee daotetsr inboutt erdevteoalt hdeiffsrmacatilolns ipzeeakosf tfhore tAheg cNryPsstaollbintea iAnegd. Tahtet haebsleonwce hoefa ting
tempdeirfafrtaucrteio(n3 0p0ea◦kCs )foorf tthhee Acogm NpPos sciaten. bTeh aitstriisbucotends itsot ethnet wsmitahll asipzer eovf itohues Arge sNeaPrsc hob[t4a6in]etdh aatt hthaes also
reporltoewd htheaetianbgs etenmcepeorfadtuirfefr (a3c0t0io °nC)p oefa kthsef coormthpeosiimtem. Tohbisil iisz ecdonAsigsteonnt SwiOith aa tp4re0v0io◦uCs hreesaetatrrceha [t4m6]e ntin
2
airanthdadt ehtaesc atelsdo Aregpodritfefdr atchtei oanbspeneacek sofo dnilfyfrawcthioenn ptheaekms feoarn thseiz iemomfoAbiglizNePd sAign corne aSsiOed2 awt 4it0h0 °thCe hienactr ease
treatment in air and detected Ag diffraction peaks only when the mean size of Ag NPs increased
oftheheatingtemperature. Thisrelationshipbetweentheabsenceofdiffractionpeaksandthesmall
with the increase of the heating temperature. This relationship between the absence of diffraction
sizeofAgNPs,7to9nm[47],and2to3nm[48],hasfurtherbeenreportedintheliterature.
peaks and the small size of Ag NPs, 7 to 9 nm [47], and 2 to 3 nm [48], has further been reported in
TheSEMimagesshowthesurfacemorphologyofthesphericalpristineSiO particles(Figure1A)
the literature. 2
withmedianandaveragesizesof673nmand674±22nm,respectively(Table1),andtheraspberry-like
The SEM images show the surface morphology of the spherical pristine SiO2 particles (Figure
Ag–S1iAO)2 wcoitmh pmoesdiitaenw aintdh athveerasguer fsaiczees-i mofm 67o3b inlimze adndA g67N4 P± s22e xnpmo,s eredsp(eFcitgivuerley 1(BTa).blTeh 1e), TaEnMd thime ages
(Figurraesp2bAer,Bry)-lriekve eAagl–tShieO2s cpohmepriocsailt,e rweiltaht itvheel ysudrfaarcke-iAmgmoNbPilsizeimd mAgo bNiPlisz eedxpaolsledo v(Feirguthree 1SBi)O. T2hme atrix
formTinEgMa irmaasgpebse (rFriyg-ulirkee 2cAo,Bm) proevseitael. tTheh espmheerdiciaaln, raenladtiavveleyr adgaerks iAzge sNoPfst himemAogbNiliPzesdo afltl hoevecro mthpe osite
are5SniOm2 manadtri5x f±or2mninmg ,ar reassppebcetrirvye-lliyk,ew coitmhpaossiitzee. Tdhies tmriebduiatino annrda anvgeirnaggef rsoizmes 2oft oth2e 0Angm NP(Ts aobf ltehe1 and
FigurceomS2p)o.sTithei sarsem 5a lnlmsi zaendo f5A ±g 2N nPms, hreasspaenctiivmelpyl,i cwaittiho na csoiznes iddisetrriinbguttiohne sraizneg-idnegp feronmde 2n ttoa n2t0i bnamct erial
(Table 1 and Figure S2). This small size of Ag NPs has an implication considering the
effectsofAgNPs:smallerAgNPs,preferablyintherangeof1to10nm,haveshownbetterantibacterial
size-dependent antibacterial effects of Ag NPs: smaller Ag NPs, preferably in the range of 1 to 10
effectsthanlargerones[49,50]. Furthermore,theTEMimagesdisplaytheAgNPswithauniform
nm, have shown better antibacterial effects than larger ones [49,50]. Furthermore, the TEM images
distributionthroughouttheSiO matrixwithoutaggregation. Thisuniformdistributionisfavorable,
display the Ag NPs with a un2iform distribution throughout the SiO2 matrix without aggregation.
asaggregationreducestheactivesurfacesofAgNPs,andthusresultsinlossoftheirantibacterial
This uniform distribution is favorable, as aggregation reduces the active surfaces of Ag NPs, and
effecttshu[3s4 r,e4s2u]l.ts in loss of their antibacterial effects [34,42].

Figure1. SEMimagesshowing(A)thesphericalpristineSiO particles;and(B)theraspberry-like
2
Ag–SiO compositewithsurface-immobilizedAgNPs.
2
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Figure 1. SEM images showing (A) the spherical pristine SiO2 particles; and (B) the raspberry-like
NanomAatge–riSalisO220 c1o7,m7,p2o6s1ite with surface-immobilized Ag NPs. 4of19
Table 1. The sizes of SiO2 particles and Ag NPs on the composite. The number of measured particles
Table1.ThesizesofSiO particlesandAgNPsonthecomposite.Thenumberofmeasuredparticlesis
is 50 at a minimum for e2ach sample. standard deviation (SD).
50ataminimumforeachsample.standarddeviation(SD).
Ag Nanoparticles (NPs) of
Materials SiO2 Particles
the Composite
Materials SiO Particles AgNanoparticles(NPs)oftheComposite
2
Median size (nm) 673 5
Mediansize(nm) 673 5
Mean size (nm) 674 5
Meansize(nm) 674 5
SD (nm) 22 2
SD(nm) 22 2
Minimum particle size (nm) 616 2
Minimumparticlesize(nm) 616 2
MaMximaxuimmupmar ptiacrleticsilze esi(znem ()nm) 724 724 20 20
The selected-area electron diffraction (SAED) pattern of the composite (Figure 2C) shows the
Theselected-areaelectrondiffraction(SAED)patternofthecomposite(Figure2C)showsthering
ring pattern with the d values calculated, corresponding to plane spacing of the {111}, {200}, {220},
patternwiththedvaluescalculated,correspondingtoplanespacingofthe{111},{200},{220},and{311}
and {311} planes of the face-centered cubic (fcc) crystal structure of Ag reported in the international
planesoftheface-centeredcubic(fcc)crystalstructureofAgreportedintheinternationalcentrefor
centre for diffraction data (ICDD, reference code: 04-016-6676). The HRTEM images of the
diffractiondata(ICDD,referencecode:04-016-6676).TheHRTEMimagesofthecompositedemonstrate
composite demonstrate (i) single-crystal Ag NPs as indicated by the one-directional lattice fringes
(i) single-crystal Ag NPs as indicated by the one-directional lattice fringes in Figure 2D showing
in Figure 2D showing d-spacing of 0.241 nm, which matches the {111} plane spacing of the fcc Ag
d-spacingof0.241nm,whichmatchesthe{111}planespacingofthefccAgcrystal;and(ii)twinned
crystal; and (ii) twinned and multi-grain Ag NPs (Figure S3). The multi-grain Ag NPs may be
andmulti-grainAgNPs(FigureS3). Themulti-grainAgNPsmaybeattributedtothegrowthofthe
attributed to the growth of the small single Ag crystals into larger Ag NPs [51]. Together these
smallsingleAgcrystalsintolargerAgNPs[51]. Togethertheseresultsprovideimportantinformation
results provide important information on the crystalline structure of Ag NPs of the composite that
onthecrystallinestructureofAgNPsofthecompositethathasnotbeenrevealedbyXRD.TheEDX
has not been revealed by XRD. The EDX results (Figure 2E) show peaks of Ag, Si, and O, which
results(Figure2E)showpeaksofAg,Si,andO,whichfurtherconfirmthepresenceofAgwithinthe
further confirm the presence of Ag within the SiO2 matrix. The detected peaks of copper (Cu) are
SiO matrix.Thedetectedpeaksofcopper(Cu)areoriginatingfromthecoppergrid.Thezetapotential
orig2inating from the copper grid. The zeta potential values of the Ag–SiO2 composite and SiO2
valuesoftheAg–SiO compositeandSiO particlesare−68.3±1mVand−66.9±0.7mV,respectively,
particles are −68.3 ± 21 mV and −66.9 ± 02.7 mV, respectively, indicating the negative surface charge
indicatingthenegativesurfacechargeandtheelectrostaticstabilityofthepreparedmaterials.
and the electrostatic stability of the prepared materials.

Figure2.Cont.
Nanomaterials2017,7,261 5of19
Nanomaterials 2017, 7, 261 5 of 20
Nanomaterials 2017, 7, 261 5 of 20

FFiigguurree2 2..( A(A,B,B))T TEEMMi mimaaggeesss shhoowwininggs spphheerricicaallA AggN NPPssi mimmmoobbiliilzizeeddt h thrroouugghhoouuttt htheeS SiOiO22 mmaattrriixx inin
tthhee rraassppbbeerrrryy--lliikkee ccoommppoossiittee aatt ddiiffffeerreenntt mmaaggnniififcicaattiioonnss;; ((CC)) TThhee sseelelecctteedd--aarreeaa eeleleccttrroonn ddififfrfraaccttioionn
Figure 2. (A,B) TEM images showing spherical Ag NPs immobilized throughout the SiO2 matrix in
((SSAAEEDD)) rriinngg ppaatttteerrnn ooff ththeec crryyssttaalllilninee AAgg NNPPss oofft htheec coommppoossitiete;; aanndd (D(D))t htheeh higighh-r-reessooluluttioionn TTEEMM
the raspberry-like composite at different magnifications; (C) The selected-area electron diffraction
((HHRRTTE(SEMAME))D imi)m raiagngege opofaftt thtehrenel aolabf betehleleed dcrs ysusurtarffallacicneee--i miAmgmm NooPbbsiil lioizzfe etdhde A Acgogm NNpPoPs sishtheo;o wawniidnn gg(D tth)h eteh lela athtttiigicchee- rffrerisinonglgueetsiso (n(d d-T-ssEppMaac ciinngg))
aanndd tth(hHeeRc cTooErrrMrees)s pipmoonandgdeiin noggf ftfahasest tl FaFbooeuluerrdiie ersru trtrrfaaancness-fifomorrmmmo (b(FFilFiFzTTe)d) p pAaatgtt teNerrnPn (s(ihinnoswseetitn));g; ( (EtEh))e T Tlhahteeti eceenn eferrrigngygyed sdi si(sdpp-eseprrsasicivivneeg X)X --rraayy
ssppeeccttraronosdscc otohppey yc( o(ErEDrDeXsXp))oe neledleminmgeen fntaatsalt laF anonuaalrylieysrsi sitsroa ofnfst hftoheremA A g(gF––FSSTiOi)O p22ac tcotoemmrnpp (ooinssisiteteet.).; (E) The energy dispersive X-ray
spectroscopy (EDX) elemental analysis of the Ag–SiO2 composite.
22.2.2.. AAgg LLeeaacchhiinngg PPrroofifillee
2.2. Ag Leaching Profile
IInndduuccttiviveelylyc ocouuppleldedp lpalsamsma-ao-potpictiaclaelm emissiisosniosnp sepctercotmroemteert(eIrC (PIC-OPE-OS)EwS)a swuatsi liuzteidlizteodi dteon itdifeyntthifey
Inductively coupled plasma-optical emission spectrometer (ICP-OES) was utilized to identify
pstntrhoooecnl ko-pfntnoihrgolfoetnee lA-dorpfeingrlAdot–ge lgSeorseidnrtOdoe g 2lceAesdktcago os cAoemkrfge fp rolAeorofea gmslAsi–eteaSgte sh–iefO(Ser1 io2fOA mrmoc2g omg –cm/toS hmmitpOehpo eL2o sA)isActigioetsg–em – 5S(S(7p1i1iO.O o 8mm2s2 ±itggcceo/o/1.mmmm0TL.pLph4)o) oe sµissiitgtsi oet 5/e.t 57am. T.7l8T.hLc 8eo±h ( ne±1t1 c o00ett10.ao4n0%l tt. a4μr)cla .go tμ/nTcimogcohenL/nenm coti(erLn1fan0 Atvt0i(ro%i1gatn0rt)i o0i.n oo%Tflnt ehh) aA.ee oc gTfhni nhioiAn nenvg g-itifit hpnrlioe rtn eov rfiitethlrdeoe
olefaAcghliefnragoch mpinrtgoh fepilrfieo lftoielfer eAodfg AA fggr o–fmSroiO mth2 tech oefm ilftipletoreersedidt e AAagsg––tSShiieOOf22u ccnoocmmtipoponosisotieft eta isma steh teih sfeus hnfuoctwniocnnti oionnf Ftoiimgf uet irmies 3es. hiAosw tsnthh oienw snta ritn
oFfigthuerFeei gx3up. reAer 3ti m.t Aheetn tstht(ea0 rstht ao)r,ft ttohhfe eth fieelx teprxaeptrieiomrinmeonefnt tt( h0(0e hhs))t,,o ttchhkee sffiuilltstrrpaatetionionsn ioo ofn ftsh tehh aestd osctroke csskuu lsstpueedsnpsieinonn7ssi.5 ohn±asd h1r.ea2sduµ lrtgee/sdum ilntL edA ign
c7o.n5 c±e7 n.15t.r 2a± t μi1og.2n/ m,μwgL/ mhAiLcg h Acogren cpcorenencseternnattrtiasotin~o,1n w3, %whhicoichfh tr rheepeprrseetssoeecnnkttssA ~~1g133%c%o on ofc fteh tneht esrta osttciookc nAk.g A Acgofn tcecorenn2ct4reanhtit,ornaA.t igAofnwte. arA s2f4qt ehur,i c2k4 lhy,
lAeagc hweAdags fwrqoaumsi cqktulhyiec klcleyoa mclehapecodhs eifdtre ofmrwom itthh teha ec cocoomnmcppeonosstiirtteae t iwwoniitthho afa 2cco2on.1ncec±nentr2tar.t3aiotµino gno/f mo2f2L .21(2 ~±.13 28±..32 2%μ.g3)./ mμTLgh /m(e~n3L,8 .a2(~%s3l)8o. .w2%er).
sTuhsetaniT,n haee dnsl, loeawa scelhorw inseugrs stoaufisnAtaeigdn ewlde aalsecahdcihenitgne gco tfeo dfA ,Agag s wwtahasse ddceeottneecccteteenddt, r,a aast sito htneh sec oocnfocn2e7nce.t1rnatt±rioant2is.o4 nofµs 2go7/f. 1m2 7±L .12( .~4± 4 μ62g..9/4m% μL)g a/mndL
2(8~.446±.9(~%24.6)2 .9aµ%ngd)/ am2n8dL. 42( 8~±.44 92±..12 %2 μ.2)g wμ/mge/rLme L(d~ e(4~t94e.9c1.t1%e%d) ) awwfteeerrree 4dd8eeatteenccdtteed7d 2a afhtfe,trer er4 s84p 8ae ncadtni vd7e2 l 7yh2., Arhe,s pproeesscpstiivebceltleyiv.e exAlpy pl.a oAnssa iptbioloesn sifbolre
explanation for the subsequent slower sustained release of Ag is the depletion of the immobilized
tehxepsluanbasetiqoune fnotrs tlohwe esurbsusesqtauiennetd srleolweaesre soufsAtaginiesdt hreeldeaespel eotifo Ango fist htheeim dmepolebtiiloizne dofA tgheN iPmsmfroobmilitzheed
sAurgf aNcAPegso NffrSPoismO fr 2tohpmea trshtuiecr lsfeuascr.fea coef oSfi OSi2O p2 apratritcilceless. .

Figure3.InvitroleachingprofileofAgfromthefilteredaqueoussuspensionsoftheAg–SiO composite
2
(1mg/mL),shakenatregulartimeintervals,shownastheaveragevaluesoftriplicatemeasurements.

T hebarsrepresentthestandarderrorsoftheaverages.
Nanomaterials2017,7,261 6of19
Overall,thepresentresultshavethreeimportantimplications. First,theinitialquickleachingof
Agisdesirable,asarapidantibacterialactionisapropertyofanidealwounddressing[52]. Secondly,
thesustainedleachingofAgallowsforaprolongedantibacterialactionofAg. Thirdly,theremaining
AgconcentrationoftheembeddedAgNPsthroughouttheSiO matrixshouldbeinterpretedwith
2
somecaution,asifsub-lethalconcentrationsofAgarereleased,Ag-resistancemightevolve[22,24].
Ag-resistancegeneshavepreviouslybeendocumentedinaplasmidofaSalmonellastrainisolated
from a hospital burn unit [53], and homologs of these genes have also been identified in E. coli
chromosomes[54]. WhiletheincidenceofAgresistanceremainsrare,cliniciansandscientistsshould,
however, be aware of the Ag concentrations needed to be administered for achieving the desired
antibacterialeffectsofAg,butsimultaneouslystrivetoavoidtheemergenceofresistance. Ithasbeen
recommendedthatprolongeduseofAg-dressingsshouldbeavoidedifwoundsshownoresponseto
Agafter3to5timesofchangingdressingswithin10to15days[32].
2.3. AntibacterialEffectsofAg–SiO CompositeandDressings
2
ThesusceptibilityofMRSAandE.colitotheAg–SiO andSiO powderswastestedinthefirst
2 2
setofagardiffusionassays. Noinhibitionzones(IZs)aredetectedonplatesofMRSAandE.coliwith
SiO particles(Figure4A,B,respectively),whichdemonstratesthattheSiO particleshavenorolein
2 2
theantibacterialeffectsofthecomposite. Incontrast,theAg–SiO compositeproducesIZsofboth
2
MRSAandE.coli. TheantibacterialeffectsoftheAg–SiO compositearemostlikelycontributedtothe
2
smallsize(5nm)oftheAgNPs. Thesesmallsized-AgNPspossesslargesurfaceareas,enablingthem
tohavelargecontactareaswiththebacterialcells[26,50,55]andtoreleasehighamountsofAg+[27,56].
Moreover,theaerobicenvironmentsoftheantibacterialtestsallowforthepartialsurfaceoxidationof
theAgNPs. PartiallyoxidizedAgNPspossessinghighlevelsofAg+mayfacilitatetheantibacterial
effects, aspreviouslyreportedbyLoketal.[34]. Thepresentfindingsareconsistentwiththoseof
Agnihotrietal.[57],whohavesuggestedthatthehighantibacterialefficacyoftheimmobilizedAg
NPsispartlyattributedtotheirsmallsize,whichenhancesthefasterdissolutionandthemorerelease
ofAg+. Furthermore,immobilizationallowsforthecontact-modeinteractionofAgNPswithalarge
numberofbacterialcells,astheAgNPsdonotbecomesequesteredinsidethebacterialcells.
TherewasnodifferencebetweenthegrowthinhibitionofMRSAandE.colibythecomposite
in the agar diffusion assay (Figure 4C), which emphasizes two major aspects. First, the present
compositeexertsantibacterialeffectsagainstbothGram-positiveandGram-negativebacterialspecies
tested,whichiscrucialinthecontextofwounddressingapplications. Secondly,thecompositeshows
antibacterialeffectsagainstthebacterialspeciesmostofteninvolvedinwoundinfections,especially
theantibiotic-resistantbacterium,MRSA,posingaseverethreattowoundmanagement[14,33]. Ithas
beensuggestedbyCuttingetal.[32]thatAgdressingsdonotleadtoacureofinfections,butrather
theycanefficientlyinhibitbacterialpenetrationintowounds,duetotheirbroad-spectrumofaction.
Accordingly,thepresentresultssuggestthattheAg–SiO compositecanbeusedinwounddressing
2
applicationsfortheprophylaxisandcontrolofantibiotic-resistantbacterialinfections.
Themostsuitabledecontaminationmethodthatretainstheantibacterialeffectsofthecomposite
wasassessedbytheparallelagardiffusionassaysofthefilter-sterilizedAg–SiO composite. Figure4
2
showsthattheUV-treatedAg–SiO compositeproduceslargerIZs(11.5±0.7mm)ofbothstrains
2
testedthanthefilter-sterilizedcomposite(8±1.4mmand10±1.4mmagainstMRSAandE.coli,
respectively). ThisisclearlyduetothehigherAgconcentrationoftheUV-treatedcompositecompared
to that of the filter-sterilized composite, as detected by ICP-OES. According to the present data,
UV-irradiationisarobustmethodfordecontaminatingthecompositeandcanbeusedtoavoidthe
problemofcloggingoftenobservedwhenmembranefiltersareused.
Nanomaterials 2017, 7, 261 7 of 20
Nthaen opmraetesreianlst2 d01a7t,a7,, 2U61V-irradiation is a robust method for decontaminating the composite and ca7no fb1e9
used to avoid the problem of clogging often observed when membrane filters are used.

FFiigguurree 44.. AAnnttiibbaacctteerriiaall eeffffeeccttss ooff UUVV--ttrreeaatteedd aanndd fifilltteerr--sstteerriilliizzeedd AAgg––SSiiOO22 ccoommppoossiitteess ddeetteecctteedd bbyy tthhee
ddiiaammeetteerrss ooff iinnhhiibbiittiioonn zzoonneess( I(ZIZs)s)o nonp lpaltaetsesw withith(A ()AM) RMSRASAan dan(dB )(BE). cEo.l i.co(lCi.) (TCh)e Tdhiea mdeiatmerestoefrsI Zosf.
TIZhse. aTvheer aagveesraagneds satnadn dsatarnddearrrdor esrorofrtsw oof itnwdoe pinednedpeenntdaegnatr adgifafru dsiioffnuasisosnay assasareyss haorew snh.own.
TThhee eefffificcaaccyy ooff tthhee ccoommppoossiittee iinn wwoouunndd ddrreessssiinngg aapppplliiccaattiioonnss wwaass iiddeennttiiffiieedd iinn tthhee sseeccoonndd sseett
ooff aaggaarr ddiiffffuussiioonn aassssaayyss.. TThhee AAgg––SSiiOO2--GG iiss eeffffeeccttiivvee aaggaaiinnsstt bbootthh MMRRSSAA aanndd EE.. ccoollii,, aass cclleeaarr IIZZss
2
((FFiigguurree 55AA,,BB,, rreessppeecctitviveelyly))a arereo bosbesrevrevdeda fateftretrh tehgea guazuezhea hsabse ebneesno askoeadkeind ainq uaeqouuesosuuss psuesnpsieonnssioonfst hoef
cthoem pcoomsitpeofsoitreo nfolyr 1o5nlmy in1,5h mighinli, ghhitginhgligthhetirnagp itdhea nrdapeifdfe catnivde aefnfteicbtaivctee raianltiabcatciotenrioafl thaceticoonm pofo stihtee.
Icnosmtepaods,inteo. IIZnsitseaodb,s enrov eIZd iws iothbstehrevpedri swtiinthe cthoen tprorilsgtianuez ceo(nFtirgoul rgea5uAz)e, (inFidgiucartei n5gAt)h, iantdthiceaatinntgib tahcatte rtihael
eafnfteicbtascotefrtihael eAffge–cStsiO of- Gthea rAego–nSlyiOa2t-tGri baurete odntloy tahtetrciboumtpedo stiote t.hTeh ceoCmSpDoswitaes. Thhyde rCaSteDd wbeafso rheytdersatitnedg
2
tboefmorime tiecstthinegm tooi smtiwmoiuc nthdee nmvoiirsotn wmoeunntda nednvwiraosnpmlaecnetd awndit hwiatss gprlaaycemd ewshitshi dites ignrcaoyn mtaecsthw siitdhet hine
icnoonctualcatt weditphl athtees itnooaclulolawtetdh eprlealteeass teoo aflAlogw+ tinhteo rtehleeaasgea orfp Alagte+ si.nHtoo twheev aegr,atrh pelCatSeDs. dHooewsnevoetrin, thhibe iCttShDe
gdrooews tnhoot fiMnhRibSiAt t(hFeig gurroew5Ath) aonf dMoRnSlyAs l(iFgihgtulyrei n5hAib) iatsntdh eongrlyo wsltihghotflyE .icnohliib(Fitisg uthree 5gBr)o;wthteh porfo dEu. cceodli
I(ZFigisufraer 5sBm);a tllheer pthraonduthceadt pIrZo disu fcaerd sbmyatllheer Athga–nS itOhat- Gp.roFdiguucreed5 bCys thhoew Asgt–hSeiOre2m-Ga.r kFaigbulered 5ifCfe rsehnocwess
2
btheetw reemenarthkeabcloer rdeicffteerdenincheisb biteiotwnezeonn ethse( CcoIZrrse)cotfedth ienAhigb–itSiioOn z-Gon(e4s.5 (CanIZds4) .2o5f tmhem Aagg–aSiniOst2-MGR (S4.A5 aanndd
2
E4..2c5o lmi,mre sapgeacitnisvte lMy)RaSnAd aCnSdD E(. 1comli,m reosnpleyctaivgealiyn)s tanEd. cColSi)D. I(t1 hmasmb eoennlys uaggagiensstte dE. tchoalti).h Iytd hraasti obneeins
rseuqgugiersetdedf otrhaatn heyffidcraietniotnl eias crheiqnugiroefdA fogr+ afnro emffiAcige-ncto lnetaacihniinngg odfr eAsgsi+n fgrsomto Aacgh-cieovnetaainninang tdibraecstseinrigasl
to achieve an antibacterial effect [44,52]. Liang et al. [58] have shown that Ag NPs on the
effect[44,52]. Liangetal.[58]haveshownthatAgNPsonthehydrophilicsurfaceofanasymmetric
hydrophilic surface of an asymmetric wettable AgNPs/chitosan composite dressing inhibit bacterial
wettable AgNPs/chitosan composite dressing inhibit bacterial growth. In the present study, such
growth. In the present study, such favorable hydration conditions were maintained by soaking the
favorablehydrationconditionsweremaintainedbysoakingthegauzeinanaqueoussuspensionof
t hecompositewithknownconcentration(1mg/mL)for15min. However,theexactAgconcentration
Nanomaterials 2017, 7, 261 8 of 20
Nanomaterials2017,7,261 8of19
gauze in an aqueous suspension of the composite with known concentration (1 mg/mL) for 15 min.
However, the exact Ag concentration within the gauze after impregnation has not been determined.
withinthegauzeafterimpregnationhasnotbeendetermined. Moreover, theconcentrationofAg
Moreover, the concentration of Ag within the CSD has not been elucidated. Therefore, further
withintheCSDhasnotbeenelucidated. Therefore,furtherstudiesarenecessarytodeterminethe
studies are necessary to determine the concentrations of Ag–SiO2 composites that are needed to
concentrationsofAg–SiO compositesthatareneededtoimpregnatethedressingsinamannerthat
2
impregnate the dressings in a manner that allows a sustained release and an effective antibacterial
allowsasustainedreleaseandaneffectiveantibacterialactionofAg.Collectively,thefollowingaspects
action of Ag. Collectively, the following aspects of our results are of importance: first, antibacterial
ofourresultsareofimportance: first,antibacterialeffectsareobservedagainstboththeGram-positive
effects are observed against both the Gram-positive MRSA and the Gram-negative E. coli. Secondly,
MRSAandtheGram-negativeE.coli. Secondly,theantimicrobialagarsusceptibilitytestresemblesthe
the antimicrobial agar susceptibility test resembles the administration of dressings in the clinical
administrationofdressingsintheclinicalsettingsandsuggeststhatthisbacterialgrowthinhibition
settings and suggests that this bacterial growth inhibition can also occur at the wound-dressing
canalsooccuratthewound-dressinginterface. Thirdly,thehydrationconditionsthatpermittedthe
interface. Thirdly, the hydration conditions that permitted the leaching of Ag+ can provide a basis
leachingofAg+canprovideabasisfortheuseofAg–SiO -Gasatopicalwounddressing.
for the use of Ag–SiO2-G as a topical wound dressing. 2

FFigiguurere5 .5.( A(A,B,B))A Anntitbibaaccteterriaialle efffefecctstso offA Agg––SSiOiO22 ccoommppoossiittee--iimmpprreeggnnaatteedd ggaauuzzee ((AAgg––SSiiOO22--GG) )aaggaaininsst t
MMRRSSAA( A(A))a annddE E..c coolili( B(B)) inin ththee aaggaarr ddiiffffuussiioonn aassssaayy.. TThhee bbllaacckk aanndd wwhhiittee nnuummbbeerrss rreepprreesseennt tththee
siszizeesso offt htheed drreessssininggssa annddt thhee pprroodduucceedd IIZZss iinn mmmm,, rreessppeeccttiivveellyy.. TThhee wwhhiittee aarrrrooww ppooiinnttss ttoo tthhee ssmmaalll l
IZIZp prorodduucceeddb byyt htheec coommmmeerrcciaiall AAgg--ccoonnttaaiinniinngg ddrreessssiinngg ((CCSSDD)) ((HHaannssaappllaasstt)) aaggaaiinnsstt EE. .ccoollii. .(C(C) )TThhee
cocorrrercetceteddi ninhhibibitiitoionnz zoonneess( C(CIIZZss))o offA Agg––SSiiOO22--GG aanndd CCSSDD..
TThhee mmiinniimmuumm iinnhhiibbiittoorryy ccoonncceenntrtartaitoionns s(M(MICIsC) so)f othfet hAeg–ASgiO–S2 icOomcpoomsipteo asigtaeinasgt aMinRstSAM aRnSdA
2
aEn.d coEl.i acoreli daerteerdmetienremd,i naes d2,50a san2d50 50a0n dμg5/0m0Lµ, gre/smpeLc,tirveeslpye. cFtuivrethlye.rmFourreth, tehrem SoirOe2, pthaertiScilOes sphaorwti cnloes
2
sh ownoinhibitionofbacterialgrowthevenatthehighestconcentration(1mg/mL)tested,which
Nanomaterials2017,7,261 9of19
further indicates that only the Ag in the Ag–SiO composite inhibits the bacterial growth. The
2
present results advocate previous findings that SiO particles have no antibacterial effect [37,59].
2
ThecorrelationbetweentheMICsandtheinvitroleachingprofileofAgfromthecompositeshows
promisingantibacterialeffectsfor thecompositebecauseaconcentrationof1mg/mLofAg–SiO
2
suspension has released 22.1 ± 2.4 µg/mL Ag after 24 h. It is evident that the MICs of 250 and
500µg/mLofAg–SiO compositehavereleased~5.5and11.1µg/mLAg,respectively,afterovernight
2
incubationinthebrothmicrodilutiontest. Hence,itcanbearguedthat~5.5and11.1µg/mLarethe
elementalAgconcentrationsthatshouldbeleachedfromtheAg–SiO compositeattheirprolonged
2
antibacterialadministrationinwounddressingstoinhibitthegrowthofMRSAandE.coli,respectively.
The present MICs are encouraging, as based on their elemental Ag concentrations, they are less
than the previously reported MICs of Ag–SiO composites with pure Ag concentration of 50 and
2
12.5µg/mLagainstS.aureusandE.coli,respectively[43],and6.72to13.44µg/mLagainstS.aureus[27].
ContrarytoexpectationsthatGram-positivebacteriaarelesssusceptibletoAg–SiO compositesthan
2
Gram-negative bacteria [35,41,42], owing to the thicker cell wall of Gram-positive bacteria [27,43].
ThepresentstudydidnotfindremarkabledifferencesinthesusceptibilityofMRSAandE.colitothe
compositeintheagardiffusionassays. Moreover,basedontheMICs,theGram-positiveMRSAis
evenmoresusceptibletothecompositethantheGram-negativeE.coli. Alloftheresultsdescribed
sofarinthepresentstudyindicatethattheAg–SiO compositedisplayseminentantibacterialeffects
2
against representatives of both Gram-positive and Gram-negative bacteria. Dong et al. [7] have
demonstrated that Ag–SiO /poly-ε-caprolactone nanofibrous membranes promote good and fast
2
woundhealing,withlessinflammationandepithelialshrinkageofwoundsinducedinWistarrats,
whichwasattributedtotheantibacterialeffectsofthereleasedAg–SiO . Theaforementionedstudy
2
utilizedapreviouslysynthesizedAg–SiO compositewithsmall-sizedAgNPs(2to10nm)andaMIC
2
of6.72to13.44µg/mLelementalAgagainstS.aureus[27]. Thecompositesynthesizedinourstudy
iscomposedofsmall-sizedAgNPs(5nm)andhasalowMICof~5.5µg/mLelementalAgagainst
MRSA.InlightofthefindingsofDongetal.[7],arolefortheAg–SiO compositeinwoundhealing
2
invivoseemsplausibleandfurtherstudiesarewarranted.
TheprolongedantibacterialeffectsofAg–SiO -Gareshownintheturbidityassays(Figure6);
2
the quantitative results are shown in Figure 7 and Table S1. When comparing the growth of the
bacterial cultures containing different dressings to that of the positive controls, it is clear that the
Ag–SiO -GinhibitsthegrowthofMRSAandE.coliafter24h,andpowerfullyreducestheirproliferation
2
after48h,indicatingprolongedantibacterialeffectsoftheAg–SiO -G.Thisprolongedantibacterial
2
effectisrequiredinpracticalapplications[35]anddesiredfeatureinAg-containingdressings[44],
decreasingthefrequencyofdressingchanges[60]. TheCSDonlyslightlydelayedthebacterialgrowth
after24and48h,indicatingafarlesslastingantibacterialeffectwhencomparedtotheAg–SiO -G.
2
Thelackofanyantibacterialeffectofthepristinegauzewasfurtherconfirmedbythatthebacterial
cultures containing pristine gauze reached almost the same turbidity and bacterial growth as the
positivecontrols. ThepresentfindingspointtotheprolongedantibacterialeffectsoftheAg–SiO -G
2
against both the Gram-positive MRSA and Gram-negative E. coli that are promising for wound
dressingapplications.
Nanomaterials2017,7,261 10of19
Nanomaterials 2017, 7, 261 10 of 20
Nanomaterials 2017, 7, 261 10 of 20

FFFiiggiguuurreere 66 .6. .TT Thhheee pp prroorolloolonnngggeeeddd aa annnttiitbbibaaaccctteeterriiraaiall lee efffffeefecccttsst soo off fAA Aggg–––SSSiiOOiO22-2-G-GG a aagggaaaiininnsstst t( (A(AA)) )MM MRRRSSSAAA aa annnddd (( BB(B)) )EE E.. .cc oocloliil iii nnin tt hhtheee
MMuuelellelre–rH–Hinitnotnonb rborthot(hM (HMBH)Btu)r btuidrbitiydaitsys aaysssaoybss eorvbesderavfetder a2f4t,e4r8 2,4a,n d487,2 ahndof 7in2c uhb aotfi oinn.cuGb,aptrioisnti.n Ge,
Mueller–Hinton broth (MHB) turbidity assays observed after 24, 48, and 72 h of incubation. G,
gpaurizseti.n+eC g,abuazcet.e +riCa,l bsuacspteerniasli osnusspweinthsioountsd wreitshsoinugts d.r–eCs,siMngHsB. –wCi, tMhoHuBtb wacittheoriuat. bacteria.
pristine gauze. +C, bacterial suspensions without dressings. –C, MHB without bacteria.

Figure7.Cont.