doi.org/10.26434/chemrxiv.11316455.v1

SilverSil: A New Class of Antibacterial Materials of Broad ScopeKeren Trabelsi, Rosaria Ciriminna, Yael Albo, Mario Pagliaro

Submitted date: 04/12/2019 • Posted date: 12/12/2019Licence: CC BY-NC-ND 4.0Citation information: Trabelsi, Keren; Ciriminna, Rosaria; Albo, Yael; Pagliaro, Mario (2019): SilverSil: A NewClass of Antibacterial Materials of Broad Scope. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.11316455.v1

Consisting of organically modified silica (ORMOSIL) physically doped with Ag nanoparticles, the SilverSil newclass of antibacterial materials of broad scope reported herein shows remarkably high and stable activityagainst representative Gram-positive and Gram-negative bacteria. The low cost, ease of application andexcellent health and environmental profile of SilverSil hybrid glassy coatings open the route to theirwidespread utilization across domestic, hospital, school, industrial and commercial environments and inconsumer products.

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COMMUNICATION

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SilverSil:ANewClassofAntibacterialMaterialsofBroadScopeKerenTrabelsi,aRosariaCiriminna,bYaelAlbo,a*MarioPagliarob*

Consisting of organically modified silica (ORMOSIL) physicallydoped with Ag nanoparticles, the SilverSil new class ofantibacterial materials of broad scope reported herein showsremarkablyhighandstableactivityagainstrepresentativeGram-positive and Gram-negative bacteria. The low cost, ease ofapplication and excellent health and environmental profile ofSilverSilhybridglassycoatingsopentheroutetotheirwidespreadutilization across domestic, hospital, school, industrial andcommercialenvironmentsandinconsumerproducts.Drivenbytheincreasingresistancetoconventionalantibacterial(AB) chemotherapy of many pathogens including meticillin-resistantStaphylococcusaureus,1thesearchofnewbroadscopeantibacterial agents is the aim of intense research activitiescarried out in numerous countries.2 One approach focuses ontheantimicrobialactivityofplantextractsandphytochemicals,often in combination with antibiotics.3 Another makes use ofinorganicspeciessuchassilverwhosetoxicitytohumancellsismuch lower than tobacteria.4 Knownandused in themedicalfield since ancient times, the antibacterial activity ofsilver ions (Ag+) and silver nanoparticles (NPs) is beingincreasinglyunderstood.5Silveractsinmultiplewaysonbacteria:Ag+andAgNPenterthecell membrane by porin proteins causing cell membranerupture, pore formation and cytoplasmic leakage.5 Within thecellmembrane,themetaldrivestheformationofseveralhighlyoxidizingspeciessuchashydroxylandsuperoxidefreeradicals,andH2O2whichquicklyoxidiseDNAanddenaturateproteins.

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Recently,Avnir and co-workersdemonstratedhowAg+ speciesare not deactivated by the killing mechanism, with the deadbacteria serving as an efficient sustained release reservoir forreleasing the lethal metallic cations for further action againstotherlivebacteria.7Starting in theearly2000s several companiesacross theworldstarted to embed Ag nanoparticles in antibacterial consumergoods including undergarments, T-shirts, towels, shirts andsocks,butalsoinwashingmachines,softtoysandevenpersonalcare products such as toothpaste.8 Washing fabricsfunctionalized with Ag NPs releases silver, but this does notaffecttheantimicrobialefficacyofthewashedfabricsasshownforexampleby>99.9% inhibitionofEscherichia coli growthontextilesretainingaslittleas2ppbAgafterprolongedwashing.9Knowledge of the impact of silver nanoparticles on humanhealth and on the environment is still limited, especiallyconcerning possible long-term toxic effects. For example,recently scholars inTaiwan reported the firstevidenceofbothcytotoxicityandimmunotoxicityofAgNPsontheleukocytesofcommonbottlenosedolphins.10Furthermore, it isalreadyknownthatmorethan90percentofsilver nanoparticles released in wastewater end up in thenutrient-rich biosolids residual of sewage treatment.11 Thesesolidsareoftenusedonlandasfertilizers,withplantstakingupsilverfromsoil.12Silveristhusconcentratedandintroducedintothefoodchain.To prevent environmental contamination with silvernanoparticles, scholars in North America lately suggested itsdirect treatment in the washingmachine by removing it fromwastewaterusinganion-exchangeresincartridge.13An alternative and even more desirable approach, since itreplacespollutioncontrolwithpollutionprevention,isbasedonimproving the microencapsulation technology so thatmicroencapsulatednanoparticlescanbeusedtoproduceleach-proofcoatingswithwhichtofunctionalizeconsumergoods.Surprisingly, the sol-gel encapsulation of Ag NPs has receivedlittleattention.Onlyrecently, for instance,thefirstexampleofantifoulingcoatingsbasedonsilvernanoparticlesembedded in

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an organosilica coating obtained from tetraethylorthosilicate(TEOS)andtriethyl(octyl)silanewasreported.14TheresultingcoatingshowedgoodinhibitionofbiocorrosionofanaluminumalloypromotedbychloridemediainoculatedwithPseudomonasaeruginosa.14Thereisawidespreadneedtodevelopnew,lowcostandbroadscopesilver-basedantibacterialsavoiding theunwantedhealthandenvironmentalcollateraleffectsofsilvernanoparticles,butstill showing the exceptionally broad scope and drug-resistantactivity of nanoparticulate silver against human pathogens,includingS.aureus.Now, we report the discovery that SilverSil, a new class ofantibacterialmaterials comprised of organicallymodified silica(ORMOSIL)physicallydopedwithAgnanoparticles,showssuchremarkably high and stable activity against representativeGram-positiveandGram-negativebacteria.Table1.CompositionofthedifferentSilverSilmaterialsSilverSil TEOS

(mol%)MTEOS(mol%)

Ag(0)load(mmolAg)

SpecificSurfacearea(m2/g)

Specificpore

volume(cm3/g)

90:10Ag

90 10 0.0325 394.036 0.9811

70:30Ag

70 30 0.0325 582.475 0.9918

50:50Ag

50 50 0.0325 506.046 0.9907

30:70Ag

30 70 0.0325 308.583 1.581

70:305Ag

70 30 0.1625 - -

70:3010Ag

70 30 0.325 - -

In detail, SilverSil materials of different composition (Table 1)were obtained by sol-gel hydrolytic co-polycondensation ofTEOS and methyltriethoxysilane (MTEOS) in the presence ofdissolvedsilvernitrate.Inatypicalpreparationofthe70:30SilverSil(70%TEOSand30%MTEOS, inmolar terms),6.0mLofaqueousHNO3 (0.2M)wasadded dropwise to a solution consisting of TEOS (8.8 mL),MTEOS(3.4mL)andethanol(13.3mL).Afterstirringfor10min,the mixture was added with 125 µl of (3-aminopropyl)triethoxysilane.An aliquot (5.0mL) of a 6.36mM aqueous solution of AgNO3was then added dropwise, after which water (5.0 mL) andaqueousammonia(0.01M,5.0mL)wereaddedtopromotethehydrolytic polycondensation. A wet gel was quickly obtained

which was dried at room temperature for approximately twoweeksuntilaconstantweightofthedrymatrixwasobtained.The drymatrix in amortarwas crushed into a powderwith apestle, after which the dried solid was treated with aqueousNaBH4(0.03M,100mL)topromotereductionoftheentrappedAg+ ions. The material obtained was air-dried again. Theresulting xerogel was crushed in a mortar and the resultingpowderusedassuchinantibacterialtrials.Thexerogelsaremesoporous,amorphousORMOSILglasses.Thepresenceof theAg(0)nanocrystalswasconfirmedbytheX-raypowderdiffraction (Figure1)usingaX’pertPROofPANalyticaldiffractometer,Cu-KαX-raysofwavelengthλ=1.54056Åwithdatatakeninthe40°-135°2θrangewithastepof0.02°.Figure1showsthepeaksatabout38.1°,44.3°,correspondingto(111)and(200)planesofcrystallineAgforboth0.5%and1%Agloadedxerogels.Thesepeaks,however,werenotobserved forthe 70:30 SilverSil matrix with 0.1% load due to the lowconcentrationofAg0NPs.

Figure1.XRDpatternsof70:30SilverSilwithdifferentsilverloading(1%,0.5%and0.1%).

The elements present on the surface of the xerogels, and theiroxidation states, were examined by X-ray photoelectronspectroscopy (XPS) using a Thermo Fisher Scientific, NEXSA XPSsystemwithmonochromatizedAlKα source(400microndiameter).The selected Pass Energy (“resolution”) of 200 eV for survey scansrevealed the general surface composition profile, whereas 50 eVhigh-resolutionscanswereusedforquantitativeanalysis.Therepresentativefull-scanXPSspectrumof70:30SilverSilinFigure2,showsthepresenceofSi,OandC.ThebindingenergyfortheC1speakat286.1eVwasusedasthereferenceforcalibration.TheXPSspectraofoxygen (O1s)at534.1eVand silicon (Si2p)at105.1eVoriginatefromtheorganosilicamatrices.

0

200

400

600

800

1000

1200

10 20 30 40 50 60 70 80

coun

ts

2θ

0.5%Agload

1%Agload

0.1%load

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Theweakpeakobtainedbyfocusedscanat368.9eV(Figure2B)dueto silver (Ag 3d), confirms the zero-valent metallic nature of AgpresentinSilverSil.Theveryweakintensityofthepeakindicatesthatmost Ag nanoparticles are locatedwithin the inner porosity of thesol-gelmatrix rather thanon thexerogel surface,awellknownandusefulfeatureofORMOSILsdopedwithmetalnanoparticles15orwithmolecularspecies.16

Figure2:XPSspectrumof70:30SilverSilxerogel; inset:FocusedscanforAg

peak.

TheenergydispersiveX-rayspectroscopy(EDXorEDS)chemicalmicroanalysisinconjunctionwithscanningelectronmicroscopycarriedoutwithanUltra-High-ResolutionMaia3FE-SEMusingan OXFORD EDS system equipped with a X-Max 80 detectorshowsthatSilverSilcompositionincludesonlySi,C,O,HandAgatoms.

Figure3.SEMimagesandEDXspectraof90:10SilverSil (a,bandc,respectively),andof70:30SilverSil(d,e,andf,respectively).

ThepresenceoftheAgnanocrystalsin90:10and70:30SilverSilxerogels is revealed as bright spots in the SEM photographs(Figures3aand3d).

TheantimicrobialactivityoftheSilverSilxerogelswasevaluatedby testing the materials against Staphylococcus aureus,representative of Gram-positive bacteria, and Escherichia coli,representativeofGram-negativebacteria.The general procedure formediumculturepreparationwas asfollows. Medium culture S. aureus from a stock culture wasinoculatedintobrainheartinfusionbroth(agrowthmediumformicroorganisms) until 0.3 optical density (OD) was obtained(measured with Thermo Genesis 10uv, UV-visspectrophotometerat600nm).The bacteria culture was then incubated at 37°C for 2 hours.MediumcultureE.coliwasgrownfollowingthesameprocedureusedforS.aureusgrowthexceptthatthegrowthmediumwasaLuriabrothmedium.BacteriaweregrownuntilobtainingOD=0.3,(logphaseofgrowth).Bythen,thebacterial inoculumwascentrifuged at 9,000 rpm for 10 minutes, washed with bufferphosphate (pH 7, 0.1M) and centrifuged again. The inoculumwaswashed2timeswithfreshbuffer.All antibacterial activity experimentswere performed inwaterbuffered at pH 7 using the aforementioned phosphate buffer(0.1 M). The inoculum was diluted to ~ 107 CFU/mL bacterialconcentrationand thendiluted toget final concentrationof ~103 CFU/mL. A flaskwas addedwith 0.5 g powdered SilverSil.Another flask was added with 0.5 g of blank material (anORMOSIL powder of identical composition but containing nosilver).Finally,athirdflaskwithnomaterialaddedwasusedascontrol. Allflasksweresterilizedinautoclaveat121°C.Asample(5mL)of buffer phosphate suspension of the bacteria was added toeach flask. The resulting suspensions (solutions, in the case ofno material added) were incubated at 37°C for 1 hour undergentle shaking. After 1 hour, a small sample (900 µL) of eachmixturewasretrievedandtransferredtoanEppendorftube.Thetubewascentrifugedat400rpmfor30safterwhich100μLof the upper liquid layerwere transferred to a Petri dish agarplate forbacterial lawn.Theplateswere incubatedat37°C for24 hours after which bacteria were counted by the colony-formingunits(CFU)technique.All SilverSil xerogels showed outstanding antibacterial activityagainstS.aureus(greenbarsinFigure4),andE.coli(bluebars)bacteria.The70%methyl-modifiedmatrixhadthehighestactivityagainstStaphylococcus aureus, hence it was selected as the optimalSilverSilantibacterialmaterial.

0,0E+00

2,0E+05

4,0E+05

6,0E+05

8,0E+05

1,0E+06

1,2E+06

0 200 400 600 800 1000 1200

coun

ts/s

bindingEnergy(eV)

O1s

C1s Si2p

7,6E+04

7,7E+04

7,8E+04

7,9E+04

8,0E+04

358 363 368 373 378

BindingEnergy(eV)

Ag3d

4

Figure 4. Antibacterial activity of different (10%, 30%, 50% and 70% methyl-modified) SilverSil xerogels againstS. aureus (greenbars), andE. coli (bluebars).Theactivityofdifferentblankmatricesandof thecontrol solution isalsoplotted(redbars).

LeachingofentrappedAgnanoparticlesinsolutionwasassessedbywashing the70:30 SiverSil xerogelwithwater anddifferentaqueous solutions in several consecutivewashingcycles (Table2).Table2.LeachingofAginsolutionfromthe70:30SiverSilxerogelmeasuredindifferentwashingcyclesviaICP-AESWashingcycle Agconcentration

(ppm)AmountofAgleached(mmol)

Agleaching(%)a

1 0.256 4.7510-5 0.15

2 0.486 9.0110-5 0.28

3 0.174 3.2310-5 0.10

4 0.468 8.6810-5 0.27

5 1.176 54.510-5 1.68

6 0.516 4.7810-5 0.15

7 0.386 3.5810-5 0.11aLeachingpercentagebasedonthetotalof3.25×10-2mmolsilverencapsulatedinthexerogelmatrix.

Thefirstfourcycles(entries1-4 inTable2)wereperformedbyimmersing the SilverSil matrix in 20 mL water for 30 min,followedbyfiltration.Inthetwosubsequentcycles(entries5-6),thematrixwasimmersedin50mLand10mLofbuffersolutionfor 30min and for 3 days, respectively. Finally, in cycle 7 thematrixwasimmersedin10mLwaterfor24h.Thesilverconcentrationinthewashingsolutionswasmeasuredeach time by inductively coupled plasma atomic emissionspectroscopy(ICP-AES)usingaSpectroArcos(Ametek,Berwyn,PA) instrument.Results inTable2showthattheamountofAgleached in solutionwasextremely low,amounting to88.510-5mmolAgafterthe7washingcycles.

Boththewashingsolutionsandthewashedmatrixweretestedfor antibacterial activity. Remarkably, regardless of modestleaching,bothshowedhighantibacterialactivity.Weascribethesurprisingly high AB activity of the washing solutions to thelethal effect of theAg+ present in ultralow concentration, in asimilar fashion to the 2 ppb Ag residual on textiles afterprolongedwashinginhibiting>99.9%ofEscherichiacoligrowth.9The antibacterial activity of SilverSil, furthermore, is due tocontactbetweenthebacteriaadsorbedandconcentratedattheORMOSILoutersurface,andthecagedAgnanoparticles.Accordingly, the accessibility of the ORMOSIL mesopores topolymeric molecules has been lately demonstrated with astructurally analogous ORMOSIL functionalized with anorganocatalystemployedintheoxidationofcellulose.17We remind thatceramicpot filters treatedwithcolloidal silverare since longused forwater sanitation inmany ruralareas inAfrica, SouthEastAsia and LatinAmerica.18Unfortunately, theceramic silver-impregnated pot filter is rapidly clogged bycolloidalparticlesreducingtherateofsafewaterproduction.19ThisworkprovidestheproofofconceptthatSilverSilisabroadscopeantibacterialsol-gelmaterialcapabletoquicklykillGram-positive and Gram-negative bacteria. Since ORMOSIL alcogelsrapidly and strongly bind virtually to any surface (includingceramic pots, but also metal, wood, cellulose or plasticobjects),20itwillbeenoughtomodifythesol-gelsyntheticroutevia procedures well known in sol-gel chemistry of silica-basedmaterials,21 tomakeSilverSilavailableasapaintwithwhich tofunctionalize the surface of any object needing prolongedantibacterialprotection.Considering the pronounced physical and chemical stability ofORMOSILs, but also their low cost, ease of preparation (andapplication) and excellent health and environmental profile,22these findings open the route to thewidespread utilization ofSilverSil coatings in life-savingapplications suchas theceramicpotfilter,aswellasinconsumerproductsandacrossdomestic,hospital,school,industrialandcommercialenvironments.

AcknowledgementsThis article is dedicated to the memory of Dr José FernandoMazariegos Anleu (1938-2018), inventor of the Ecofilter drinkingwater filter. K. T. thanksher colleaguesMichaelMeistelman, YannaGurianov, Dr. Olga Krichevski, Dr. Natalia Litvak, and Dr. FainaNakonechny.

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