New routes to the preparation of silver-soft liner nanocomposites as an antibacterial agent
ChulJae Leea,Ki YoungNamb,Dong Yeub Kima,Hee JinKimc,Won Hui Dohd, HyunKukChoie,Maeng JoonJunge,*
aSchoolofChemicalIndustry,YeungnamCollegeofScience&Technology,Daegu705-703,RepublicofKorea
bDepartmentofDentistry,CollegeofMedicine,KeimyungUniversity,Daegu700-712,RepublicofKorea
cDepartmentofChemicalEngineering,PohangUniversityofScienceandTechnology(POSTECH)San31,Hyoja-Dong,NamGu,Pohang,Gyungbuk790-784, RepublicofKorea
dLaboratoireLMSPCUMR7515,CNRS,UniversityofStrasbourg,F-67087Strasbourgcedex2,France
eSchoolofNano&MaterialsScienceEngineering,KyungpookNationalUniversity,Sangju742-711,RepublicofKorea
1. Introduction
Newtechnologiesrequirenewmaterialswithspecialcombi- nations of properties. Recently, the surface modification of polymer materials has been a major challenge in medical applications such as implants or other devices in the human body[1].Particularlyintheoralcavity,acrylicsoftdentureliners havebeencommonlyusedtoenhancetherecoveryofdenture- bearing tissues from trauma and damage, which are usually caused by ill-fitting dentures. However, lining materials have complicated molecular structures and are easily degradable, alongwithbeingsusceptibletobacterialadhesion.Theadhered bacteria can be released from denture plaque into salivary secretionsandthen aspiratedinto the lower respiratorytract, causingpneumonia, especiallyin elderlypeopleor thosewith disabilities;suchindividualshaveadiminishedgeneralimmune responseandpharyngealreflex[2–6].
Systemic or local antibiotic agents have been prescribed to eliminate the bacterial population for treatment in cases of infection;however,asmicrobialresistanceand healthcarecosts are both increasing, the development of antimicrobial denture liningmaterialcouldbenecessaryforthepreventionandcareof increasedbacterialpopulations[7,8].Althoughseveralinvitroand invivostudieshaveshownthebeneficialeffectsofantimicrobial agents combined in polymeric soft denture liners [9–11], no effective,commercialantimicrobialagenttobecombinedhasyet beendeveloped.
Silver nanoparticles are emerging as a new generation of antibacterial agent[12]; theyhave beenused forhygiene [13], medicalapplications[14–16],andantibacterialwaterfilters[17].
Recently,silver nanoparticleshaveshown superiorantibacterial activitycomparedtothatofothersilvercompounds,aswellas bulk silver. Silver nanoparticles do not only possess strong antibacterial activity, but can also inhibit a broad spectrum of bacteriaandfungi[13,18].Althoughsilvernanoparticleshavebeen integratedwithvariousmaterials,noneoftheseworkswascarried outusingsoftliners (SLs).Therefore,thepurposesofthis study weretogenerateanewsynthesisofsilver-softlinernanocompo- sites(Ag-SLN)andtoassessitsantimicrobialeffects.
JournalofIndustrialandEngineeringChemistry20(2014)1276–1279
ARTICLE INFO
Articlehistory:
Received21January2013 Accepted2July2013 Availableonline9July2013
Keywords:
Silver Nanoparticles Soft-liner Antibacterial E.coli
ABSTRACT
Thisstudyevaluatedtheantimicrobialandphysicalpropertiesofsilver-softlinernanocomposites(Ag- SLN).Polymerizedacrylic soft dentureliner diskspecimens containing0 (control),1500,3000, or 6000mg/Lofsilvernanoparticleswereplacedontheflatbottomoftheseparate12-wellcellculture platedishand100mLsamplesofmicrobialsuspensionsofEscherichiacolistrainwereinoculatedoneach specimen and incubated at 378C, for24 and or 72h. Theantimicrobial effects weredetermined according to the number of viable cells in the retrieved suspension. Characterization of silver nanoparticleswas carriedoutbasedonUV–visspectroscopyandtransmissionelectronmicroscopy (TEM)analysis.Thespecimenswerecharacterizedbythermalgravimetricanalysis(TGA),fieldemission scanning electron microscope and energy dispersive X-ray analysis (FE-SEM/EDAX), and atomic absorptionspectroscopy(AAS).WesuccessfullypreparedAg-SLNandidentifiedthegreat excellent antimicrobialactivityforEcoli.
ß2013PublishedbyElsevierB.V.onbehalfofTheKoreanSocietyofIndustrialandEngineering Chemistry.
*Correspondingauthor.Tel.:+82545301336;fax:+82545365330.
E-mailaddress:[email protected](M.J.Jung).
ContentslistsavailableatSciVerseScienceDirect
Journal of Industrial and Engineering Chemistry
j o urna l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / j i e c
1226-086X/$–seefrontmatterß2013PublishedbyElsevierB.V.onbehalfofTheKoreanSocietyofIndustrialandEngineeringChemistry.
http://dx.doi.org/10.1016/j.jiec.2013.07.004
2. Experimental
Silver nitrate (AgNO3, 99.9%) was purchased from Kojima ChemicalsCo.Ltd.(Japan).PVP(MW.avg.=10,000)wasobtained fromSigma–AldrichCo.GCSoft-LinerwaspurchasedfromGCCo.
Ltd.(Japan). All chemical reagents were used withoutfurther purification. Colloidalsilvernanoparticles werepreparedfrom thesolutionsof0.3MofAgNO3and5wt.%PVPindistilledwater.
Thewholepreparationprocedurecan beexplainedasfollows:
12.50g of PVP was dissolved in 250mL of distilled water;
followingthis,12.74gofsilvernitratewasaddedtothesolution.
Then, the solution changed in color from yellow to dark olive green. The chemical process continued for 2h at room temperature.
The size of the silver nanoparticles was measured using transmission electron microscopy (TEM; Hitachi H-7100). The UV–visspectraofthesilvernanoparticleswererecordedwitha SCINOUVS-2100UV-Visspectrophotometer.
DeterminationofeachAg-SLNsample(about1.0g)wasdried underavacuumuntilitmaintainedaconstantweightandthen placedinaflask.Distilledwater(50mL)wasaddedtotheflaskand themixturewasshakenforabout24,48,or72h.Theconcentration ofsilvernanoparticlesintheobtainedsolutionwasdetermined usingatomicabsorptionspectroscopy(AAs;PerkinElmerAAnalyst 100,USA).Asilverhollowcathodelamp(Perkin-Elmer,USA)witha 328.1nmline and0.7nmspectralbandpassoperatedat12mA wastheradiationsource.
GCSoft-Liner(GCcooperation,Tokyo,Japan)wasselectedasa soft denture liner in this study. Doses of Ag8 added to the conditioner liquid are shown in Table 1. Colloidal Ag8 was preliminarily combinedand homogenized withtheconditioner liquidinasterileglassbeakerataconcentrationrangingfrom0 (control) to 1500,3000, and 6000mg/L (vol/vol; colloidal Ag8/
conditionerliquid).Acrylicpowderwasaddedandmixedfor30s atadesignatedpowder/liquidratiofollowingthemanufacturer’s instructions.Togenerateauniformshapewitharegularsurfacefor samples,mixedconditionerpastewaspouredontoacustom-made brassmoldwithahole(20mmdiameter3.0mmheight),which sandwiched the paste between glass slides until the SL was solidified.Sixtyspecimens werepreparedand dividedintofour groups (N=15) according to the concentration of Ag8. Before microbialassay,allsamplesweresterilizedwithethyleneoxide gas for 24h to ensure the initial sterility of samples. The microstructureofobtainedAg-SLNwasstudiedbyfieldemission scanning electron microscope (FE-SEM; Philips XL30S FEG) equipped with EDAX (Sapphire detector with LEAP+Crystals).
TheAg-SLN wasgold sputtered underhighvacuum before the analysis.EDAXstudieswerealsoperformedforAg-SLNtoidentify thesilvernanoparticlesofthesurfaces.
Thermalgravimetricanalyses(TGA)werecarriedoutsimulta- neouslyusingaTG-DTA92instrument(SETARAM,France)witha heatingrateof108Cmin 1from308Cto6008C.Eachsamplewas analyzedintriplicateandmeanvalueswereestimated.
Microbial suspensions were obtained from a single colony (Escherichia coli; ATCC 6538) isolated on agar plates and then inoculatedin appropriatebrothforovernight cultures.Bacterial strainsweregrowninbrain–heartinfusion(BHI;Difco,Franklin
Lakes,NJ,USA)brothandontoMuellerHintonagarplatesat378C.
After incubating microbial cells at 378Covernight, the optical density(OD)ofthesuspensionat600nmwasadjustedto1.0using aspectrophotometer(MiltonRoyspectrophotometer20D+,Milton Roy,Ivyland,USA).Thesuspensionwasdilutedwithphosphate- buffered saline (pH 7.4) to 1:100 and suspended to final concentrationof1.0107cells/mL[19,20].
SamplesofAg-SLNandcontrolwereplacedontheflatbottom of separate12 well cell culture plates (Costa1, Corning, New York,USA),andthen100mLoftheinitialmicrobialsuspension in 1.0mLofSabouraudbroth wasinoculatedin eachwelland incubated at 378C. After incubation for 24hand 72h for an extended contact period, the suspension (100mL) was with- drawn through serial dilution; viable cells (colony forming units[CFU])inthesuspensionweredeterminedusingthespread plate methodat alevelof detectionwithin500CFUperplate.
Assays were independently performed with three repetitive testsanddatawererecordedasmeansandstandarddeviations.
According to conventional standards [21,22], the borderline of antimicrobialeffectwasdeterminedtobe1500mg/L viable cells (99.9% reduction of CFU) as the minimum bactericidal concentration(MBC)ofantibiotics.AsimplecomparisonofCFU means was carried out (t-test, p<0.05indicated a significant correlation).
3. Resultsanddiscussion
Fig.1givesaTEMimageofsilvernanoparticlespreparedusing the in situ reduction method. The average size of the silver nanoparticleswasabout408.0nm.TheUV–visspectrumofthe silvernanoparticlesispresentedinFig.2,whereweidentifiedthatthe peak at 410nm was the surface plasmon band of the silver nanoparticlessynthesizedbytheinsitureduction.
Itiswellknownthatasurfaceplasmonbandofsphericalsilver nanoparticles appears at around the 400nm region. The color
Table1
PreparationrecipeandmixingpatternofAg-SLN.
Group N SolAg8/liquid(vol/vol,%) Ag8dose(mg/L) Mixingpattern Strainstested
Control 15 0 0 GCSoft-Liner(GCSoft-Liner,GCcooperation)powder/
(solAg8+conditionerliquid)=2.2/1.8
E.coli
I 15 0.5 1500
II 15 1.0 3000
III 15 2.0 6000
Fig.1.TEMimageofsilvernanoparticlesprepared by theinsitureduction method.
C.J.Leeetal./JournalofIndustrialandEngineeringChemistry20(2014)1276–1279 1277
propertiesofsilvernanoparticlesarequitesensitivetothesizeof nanoparticlesat a constant number density; at constant mass loading,theyappeartobeinsensitivetosmallchangesinthesizeof silvernanoparticlesbecauseoftheinversecubiccouplingbetween thesizeofparticlesandnumberdensityatconstantmassloading [23].Therefore,thecharacteristicsofaggregated silverparticles areingoodagreementwithmanypreviousstudies[24].
Inordertoinvestigatethepresenceofsilvernanoparticleson thesurfaceofAg-SLN,theFE-SEMandEDAXelementalanalysis.
The SEM photographs in Fig. 3 were taken at 250
magnification to observe the surface morphology of Ag-SLN.
Fig.3showstheAg-SLNsurfacecoveredwithsilverparticles;the diameter of a silver particle was about 5–50mm. The EDAX spectrumforAg-SLNisillustratedinFig.4.Onlycarbon,oxygen, andsilverpeakscanbeobserved.Thesehavebeenidentifiedas theprincipalelementsofAg-SLN.Inthiscase,carbonandoxygen atomswerethemaincomponentsoftheSL.Inaddition,thepeak of the silver atoms showed that silver nanoparticles were immobilizedontotheSLsurface.Therefore, weconfirmedthat thesilverpeakisclearlyshowninFig.4,whichindicatesthatthe silvernanoparticles weresuccessfullyimmobilizedontotheSL surface.
SLsareusedforshortperiodstoimprovethecomfortandfitof anolddentureuntilitcanberemadeorpermanentlyrelined.SLs are composed of powder containing poly ethyl methacrylate (PEMA)and a liquid containinganaromaticester-ethylalcohol mixture.SLsareverysoftelastomerswithahardnessoffrom13to 49ShoreAhardnessunits24haftermixing.Theroleofliquidisto speedupthepolymerizationofthemonomeratroomtemperature.
Fig.5givesacomparativethermaldecompositionpatternfor (a) SL, (b) 1500mg/L Ag-SLN, (c) 3000mg/L Ag-SLN, and (d) 6000mg/L Ag-SLN. SL gave two thermal degradation patterns correspondingtothedegradationoftheethylmethacrylateand aromaticester-ethylalcohol.Fig.5showssimilarthermogramsfor (b)1500mg/LAg-SLN,(c)3000mg/LAg-SLNand (d)6000mg/L Ag-SLNfrom308Cto6008C.Asharpweightreductionisobserved between2508Cand3508C.
Despitetheuseofsilvernanoparticles,theonsetofdegrada- tion for all samples appeared to start at about the same temperature.Attheendofthedegradationprocess,theamounts of residues were proportional to the concentrations of silver nanoparticlesinSL,sincethesilvernanoparticleswerestableat theexperimentaltemperature.Allthesamples,includingbothSL Fig.2.UV–visspectrumofsilvernanoparticlespreparedbytheinsitureduction
method.
Fig.3.SEMimageofAg-SLN.
Fig.4.Energy-dispersiveX-rayanalysisofAg-SLN.
Fig.5.TGAcurvesof(a)SL,(b)1500mg/LAg-SLN,(c)3000mg/LAg-SLN,and(d) 6000mg/LAg-SLN.
C.J.Leeetal./JournalofIndustrialandEngineeringChemistry20(2014)1276–1279 1278
andAg-SLN,exhibitedarathersimilarpatternofdegradation.This suggeststhatsilvernanoparticlesareuniformlyimmobilizedwith SL. In addition, there was a slight sign of thermal stability improvement obtained from the immobilization of the silver nanoparticles.
Fig.6showsthereleaseofsilvernanoparticlesasafunctionof immersiontimeinwater.ItcanbeseenthatforAg-SLN,asmall amountofsilvernanoparticleswasreleased.When(a)1500mg/L Ag-SLN, (b) 3000mg/L Ag-SLN, or (c) 6000mg/L Ag-SLN was shakenfor3days,theremainingsilvernanoparticlesontheSLwas reducedto0.08mg/L,0.21mg/L,and0.49mg/L,respectively.In addition,theaverage releaserates of(a)1500mg/LAg-SLN,(b) 3000mg/LAg-SLN,and(c)6000mg/LAg-SLNwere0.0017,0.0035, and0.0088mg/Lfor1h,respectively.
The silver nanoparticle release tests were combined with antimicrobialefficacytestsagainstE.coliinordertodetermine whethertherewasadirectcorrelation.Theinitialconcentrationof E.coliwas1.7107CFUmL 1.After24hand72h,theconcentra- tionoflivingE.coliinthesuspensionwasmeasured.Theresultsare summarizedin Table2. Whencompared totheCFUat0h, the controlgroup(0mg/LAg8)didnotshowanymicrobialinhibitory effectagainstthestrains.FortheE.colibacterialstrains,theMBCof the Ag8-incorporated samples was identified at doses above 1500mg/L;noviable cellsweredetected(noCFU)atconditions of3000mg/Labove.
TheaboveresultsshowthattheE.coli werekilled bysilver nanoparticlesreleasedfromtheAg-SLN(bactericidaleffect).TheE.
coli survived, but couldnot grow into colonies on the Ag-SLN surfaces, because in the suspensions taken from the Ag-SLN surfacessilver nanoparticleswerepresent. Therefore,we found thatthesilvernanoparticlesinhibitedthegrowthofE.colicells.
4. Conclusion
ThispaperreportedanewrouteforthepreparationofAg-ASDL compositesasanantimicrobialagent. Ourresultsillustratedthe characteristicsofAg-SLNthroughUV–Visspectroscopy,TEM,FE- SEM/EDAX,XRD,AAs,andantimicrobialefficacytests.TheAg-SLN surfacewascoveredwithsilverparticlesandthediameterofthe silverparticleswasabout5–50mm.Asaresultofthermalanalysis, wefoundthatthesilvernanoparticleswereuniformlyimmobilized withSLandtheAg-SLNshowedaslightsignofthermalstability improvement obtained from the immobilization of the silver nanoparticles.Thesilvernanoparticlereleasetestswerecombined withantimicrobialefficacytestsagainstE.coli.Asaresult,wefound thattherewasadirectcorrelationbetweenthereleaseamountsof silver nanoparticles and the antimicrobial effect on E. coli.
Suspensionsof3000mg/LAg-SLNand6000mg/LAg-SLNshowed greater antimicrobial effects(99.9%) on the E.coli (ATCC6538).
Therefore,wedemonstratedanewrouteforthepreparationofAg- SLN,andthiscouldbeproposedasanantimicrobialdentalmaterial.
Acknowledgement
ThisresearchwassupportedbyKyungpookNationalUniversity ResearchFund,2011.
References
[1]J.I.Kroschwitz,Polymers:BiomaterialsandMedicalApplications, Wiley-Inter- science,JohnWiley&Sons,NewYork,1989.
[2]Y.Sumi,H.Miura,Y.Michiwaki,S.Nagaosa,M.Nagaya,ArchivesofGerontology andGeriatrics44(2007)119.
[3]F.A.Scannapieco,JournaloftheAmericanDentalAssociation10(2006)25.
[4]P.S.Wright,JournalofDentistry12(3)(1984)19.
[5]S.Qudah,A.Harrison,R.Hugget,InternationalJournalofProsthodontics3(1990)477.
[6]T.Hamada,H.Murata,DentureLining,DentalDiamondCo,Tokyo,2001,pp.68–
76(inJapanese).
[7]L.M.DeVisschere,L.Grooten,G.Theuniers,J.N.Vanobbergen,Gerodontology23 (2006)195.
[8]L.A.Casemiro,C.H.GomesMartins,C.Pires-de-SouzaFde,H.Panzeri,Gerodon- tology25(2008)187.
[9]D.M.Quinn,JournalofOralRehabilitation12(1985)177.
[10]M.R.Truhlar,K.Shay,P.Sohnle,JournalofProstheticDentistry71(1994)517.
[11]C.K.W.Chow,D.W.Matear,H.P.Lawrence,Gerodontology16(1999)110.
[12]M.Rai,A.Yadav,A.Gade,BiotechnologyAdvances27(2009)76.
[13]N.H.Chau,L.A.Bang,N.Q.Buu,T.T.N.Dung,H.T.Ha,D.V.Quang,Advancesin NaturalSciences9(2008)241.
[14]T.W.Chang,L.Weinstein,AntimicrobialAgentsandChemotherapy8(1975)677.
[15]B.S.Atyeh,M.Costagliola,S.N.Hayek,S.A.Dibo,Burns33(2007)139.
[16]D.Roe,B.Karandikar,N.Bonn-Savage,B.Gibbins,J.B.Roullet,JournalofAntimi- crobialChemotherapy61(2008)869.
[17]P.Jain,T.Pradeep,BiotechnologyandBioengineering90(2005)59.
[18]T.T.N.Dung,N.Q.Buu,D.V.Quang,H.T.Ha,L.A.Bang,N.H.Chau,N.T.Ly,N.V.Trung, JournalofPhysics:ConferenceSeries187(2009)012054.
[19]E.Budtz-Jørgensen,E.Theilade,J.Theilade,H.A.Zander,ScandinavianJournalof DentalResearch89(1981)149.
[20]J.Chandra,P.K.Mukherjee,S.D.Leidich,F.F.Faddoul,L.L.Hoyer,L.J.Douglas,M.A.
Ghannoum,JournalofDentalResearch80(3)(2001)903.
[21]W.Bruns,H.Keppeler,R.Bancks,AntimicrobialAgentsandChemotherapy27 (1985)632.
[22]A.G.Gristina,R.A.Jennings,P.T.Naylor,Q.N.Myrvik,L.X.Webb,Antimicrobial AgentsandChemotherapy33(1989)813.
[23]C.Advait,S.Praveen,S.Suvarna,T.Rochish,M.Anurag,ColloidsandSurfacesA:
PhysicochemicalandEngineeringAspects404(2012)83.
[24]L.Suber,I.Sondi,E.Matijevi,D.V.J.Goia,JournalofColloidandInterfaceScience 288(2005)489.
Table2
ResultsforantimicrobialtestofAg-SLN.
Strain(CFUat0h) Ag8dose(mg/L)
Incubatedtime(h) MeanCFUvalues(s.d)
0(Control) 1500(I) 3000(II) 6000(III)
E.coli(107) 24 2.41081.4107 30102 0 0
72 1.71085.8107 80103 0 0
Fig.6.Releaseratesofsilverinto(a)1500mg/LAg-SLN,(b)1500mg/LAg-SLNand (c)1500mg/LAg-SLN.
C.J.Leeetal./JournalofIndustrialandEngineeringChemistry20(2014)1276–1279 1279
