ColloidsandSurfacesB:Biointerfaces120(2014)125–131
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Colloids and Surfaces B: Biointerfaces
jou rn a l h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b
Release and antibacterial activity of allyl
isothiocyanate/ -cyclodextrin complex encapsulated in electrospun nanofibers
Zeynep Aytac
a,b, Sema Y. Dogan
c, Turgay Tekinay
c,d, Tamer Uyar
a,b,∗aInstituteofMaterialsScience&Nanotechnology,BilkentUniversity,Ankara06800,Turkey
bUNAM-NationalNanotechnologyResearchCenter,BilkentUniversity,Ankara06800,Turkey
cGaziUniversity,LifeSciencesApplicationandResearchCenter,Ankara06830,Turkey
dGaziUniversity,PolatlıScienceandLiteratureFaculty,Ankara06900,Turkey
a r t i c l e i n f o
Articlehistory:
Received21December2013
Receivedinrevisedform19March2014 Accepted13April2014
Availableonline22May2014
Keywords:
Electrospinning Nanofibers Allylisothiocyanate
-Cyclodextrin Release
Antibacterialactivity
a b s t r a c t
Allylisothiocyanate(AITC)isknownasanefficientantibacterialagentbutithasaveryhighvolatil- ity.Herein,AITCandAITC/-cyclodextrin(CD)-inclusioncomplex(IC)incorporatedinpolyvinylalcohol (PVA)nanofiberswereproducedviaelectrospinning.SEMimageselucidatedthatincorporationofAITC andAITC/-CD-ICintopolymermatrixdidnotaffectthebead-freefibermorphologyofPVAnanofibers.
1H-NMRand headspaceGC-MSanalysesrevealedthatverylow amountof AITCwas remainedin PVA/AITC-NFbecauseoftherapidevaporationofAITCduringtheelectrospinningprocess.Neverthe- less,muchhigheramountofAITCwaspreservedinthePVA/AITC/-CD-IC-NFduetotheCDinclusion complexation.ThesustainedreleaseofAITCfromnanofiberswasevaluatedat30◦C,50◦Cand75◦Cvia headspaceGC–MS.WhencomparedtoPVA/AITC-NF,PVA/AITC/-CD-IC-NFhasshownhigherantibac- terialactivityagainstEscherichiacoliandStaphylococcusaureusduetothepresenceofhigheramountof AITCinthissamplewhichwaspreservedbyCD-IC.
©2014ElsevierB.V.Allrightsreserved.
1. Introduction
Electrospinningisawidelyusedmethodtoproducefunctional nanofibersfromvarietyofmaterialsincludingpolymers,inorganic materialsandcompositestructures[1,2].Generallyelectrospinning is a room temperature process in which the polymer solution isexposed toelectrostatic field and theelectrifiedjetis drawn towardtothegroundedcollectoranddepositedonthecollector asa fibrousweb. Asthesolventevaporates duringtheprocess, nanofibrousmaterialswithuniquepropertieshavingveryhighsur- faceareatovolumeratioandnanoporousstructuresareproduced [1,2].Theuniquepropertiesenableelectrospunnanofiberstobe usedinwounddressing,tissuescaffold,drugdelivery,foodpack- aging,filtration,energy,catalysis,sensors,etc.[1,2].Owingtothe roomtemperatureandambientprocessconditionsthatprovide protectionforthebioactivecompoundsinelectrospunnanofibers,
∗ Correspondingauthorat:InstituteofMaterialsScience&Nanotechnology (UNAM),UNAM,BilkentUniversity,06800Ankara,Turkey.Tel.:+903122903571;
fax:+903122664365.
E-mailaddresses:tamer@unam.bilkent.edu.tr,tameruyar@gmail.com(T.Uyar).
electrospinningisusedforproductionoffunctionalnanofiberscon- tainingactiveagentslikeantioxidants[3,4],flavors/fragrances[5,6]
andantibacterialagents[7].
Activeagentsloadedelectrospunnanofibersmaybeusefulin food packaging [3,4,7–11] and biomedical applications[12–14]
asreportedintheliterature.Vega-Lugoetal.madecomparison intermsofreleasecharacteristicsoffreeAITCandAITC/-CD-IC includingsoyproteinnanofiberswithchangingrelativehumidity andtheyalsostudiedtheeffectofAITCamountonthereleaseof AITC fromAITC includingpolylacticacidnanofibersin different relative humidity [8]. In another related study, Ge et al. pro- ducedmaterialsforfoodpackagingapplicationsbyimmobilizing glucoseoxidaseenzymeinpolyvinylalcohol/chitosan/teaextract electrospunnanofibrousmembrane[9].Inastudyconductedby ourresearchgroup,polylacticacidnanofibersincorporatedwith inclusioncomplexoftriclosanandcyclodextrinswereproduced viaelectrospinning[7].Ina studyofPérez-Masiá etal.,aphase changing material was encapsulated in electrospun zein fibers for the possible application as smart packaging materials [10].
Withregardstobiomedicalapplications,Mattanaveeetal.(2009) prepared different biomolecules immobilized electrospunpoly- caprolactonenanofiberswiththepurposeofusingthemintissue http://dx.doi.org/10.1016/j.colsurfb.2014.04.006
0927-7765/©2014ElsevierB.V.Allrightsreserved.
Fig.1.Chemicalstructureof(a)allylisothiocyanate,(b)-CD;schematicrepresentationof(c)-CD,(d)formationofAITC/-CD-ICand(e)electrospinningofnanofibers fromPVA/AITC/-CD-ICsolution.
engineering [12]. Merrell et al. obtained curcumin-antioxidant agent-loadedelectrospunpolycaprolactonenanofibersfordiabetic wounddressingapplications[13].InthestudyofUnnithanetal.,an antibacterialelectrospunnanofibersthatareintendedtobeused aswounddressingmaterialwereproducedbyelectrospinningof a drug– ciprofloxacinHCl –including solutionof dextran and polyurethane[14].
Cyclodextrins (CD) are cyclic oligosaccharides (Fig. 1b) and ableto form inclusion complex (IC)with variety of molecules.
CDhave␣-(1-4)linkagesformingatruncated-coneshapedstruc- ture(Fig.1c).␣-CD,-CDand␥-CDhaving6,7,8glucopyranose units are the most common CD types [15,16]. Owing to their hydrophobiccavity, CD are ableto formhost–guest complexes viaweakforces,suchasvanderWaalsinteractions,dipole–dipole interactions,andhydrogenbondingwithmoleculesinappropri- atepolarityand dimension.ICof CDwithaguestmoleculehas advantages likeproviding higher solubility,higherthermal sta- bility and bioavailability of hydrophobic guests; controlling of volatility,masking offunpleasant odors,and controllingrelease of drugs and flavors [15–17]. CD are widely used in pharma- ceutical,cosmetics, biotechnology, agriculture,textile,chemical, environmental protection industries [15–17]. There also exist studies concerning application of CD in active food packaging [18–20] and biomedical applications [21–23] in the literature.
Inthefoodpackagingstudies,CD wereincorporatedwithpoly- mersin free formtocaptureundesired molecules in thefoods like cholesterol [18], hexanal and cholesterol [19] or in guest molecule-CD-ICformtoreleaseanantimicrobialagent(trans-2- hexenal)[20].ICofCDand varioustypesofdrugsareprepared and theseCD-drug-ICcouldbeused in biomedicalapplications [21–23].
Allyl isothiocyanate (AITC) (Fig. 1a) is a major pungent, an antimicrobialcompoundfoundincabbage,horseradish,mustard, wasabiaandusedinfoodpackaging[24],andbiomedicalapplica- tions[25,26].ICofAITCwithCDwasinvestigatedintheliterature [27,28].InastudyofLietal.,releaserateofICofAITCand␣-CD andICofAITCand-CDwasinvestigatedagainstrelativehumidity [27].Inanotherstudy,Ohtaetal.studiedonthedecompositionand thermodynamicpropertiesofinclusioncomplexationofAITCand CD(␣-CDand-CD)[28].
AITC could be used in food packaging applications by incorporating it into polymeric films [29] or fibers [8]. Plack- ett et al. investigated the effect of CD type in the release behavior of AITC from AITC/CD-IC incorporated into polylactic acid–polycaprolactone films [29]. Vega-Lugo et al. studied the release ofAITC fromonlyAITC and AITC/-CD-ICincludingsoy proteinnanofibersatdifferentrelativehumidity[8].
Inthisstudy,AITCencapsulatedinelectrospunpolyvinylalco- hol(PVA)nanofibersweregeneratedbytwomethods.PVAasa nanofibermatrixwaschosensincePVAisasuitablepolymertype forfoodpackagingapplicationsshownintheliteraturebyincorpo- ratingdifferentmoleculesorbacteriaintoPVAfilms[30]andfibers [5,31]duetoitshightensilestrength,flexibility,highoxygenand barrierproperties.FreeAITCandinclusioncomplexofAITCwith
-CD(AITC/-CD-IC)(Fig.1d)preparedinaqueoussolutionwas incorporatedintoPVAsolution;thenPVA/AITCandPVA/AITC/- CD-ICnanofiberswereproducedviaelectrospinning(Fig.1e).The resultingnanofibrouswebswerecharacterizedbySEM,XRD,and
1H-NMR.Therelease of AITCfromnanofiberswasmeasuredat 30◦C,50◦Cand75◦CbyheadspaceGC–MS.Theantibacterialactiv- ities of nanofibers were tested against Escherichia coli (E. coli) andStaphylococcusaureus(S.aureus)accordingtocolonycounting method.
2. Materialsandmethods 2.1. Materials
Polyvinyl alcohol (PVA, Mw∼85.000–146.000g/mol, Sigma Aldrich,87–89%hydrolyzed),allylisothiocyanate(AITC,95%,Sigma Aldrich),beta-cyclodextrin(-CD,WackerChemieAG,Germany), deuterateddimethylsulfoxide(DMSO-d6,deuterationdegreemin 99.8% for NMR spectroscopy,Merck) werepurchased and used as-receivedwithoutanyfurtherpurification.Distilledwaterwas suppliedfromMilliporeMilli-Qultrapurewatersystem.
2.2. Preparationofsolutions
AITCcontainingPVAnanofiberswerepreparedbyincorporating AITCintoPVAsolutionsviatwomethods.Inthefirstmethod,free
Z.Aytacetal./ColloidsandSurfacesB:Biointerfaces120(2014)125–131 127
Table1
Thepropertiesofthesolutionsusedforelectrospinningandmorphologicalcharacteristicsoftheresultingelectrospunnanofibers(thefibersizeisreportedastheaver- age±standarddeviationand100fiberswereanalyzedforeachcase).
Solutions PVAa(g/kg,w/v) -CDb (g/kg,w/w)
AITCb (g/kg,w/w)
Viscosity (Pas)
Conductivity (S/cm)
Averagefiber diameter(nm)
Fiberdiameter range(nm)
Fibermorphology
PVA 75 – – 0.177 470 290±65 140–580 Bead-freenanofibers
PVA/AITC 75 – 40 0.267 487 210±45 120–330 Bead-freenanofibers
PVA/AITC/-CD-IC 75 227 40 0.194 523 235±90 90–540 Bead-freenanofibers
aWithrespecttosolvent(water).
bWithrespecttopolymer(PVA).
AITC(4%,w/w,withrespecttopolymer)wasdispersedinaque- oussolutionandthen7.5%PVA(w/v)wasputintothissolution.
Thesolution(PVA/AITCsolution)wasstirredatroomtemperature overnightandfinallyitwaselectrospun(PVA/AITC-NF).Inthesec- ondmethod,4%AITC(w/w,withrespecttopolymer)wasdispersed inaqueoussolution,then-CDwasaddedtogiveamolarratio of2:1(AITC:-CD)andresulting solutionwasstirred overnight at room temperature and white, turbidsolution was obtained.
Finally,7.5%PVA(w/v)wasadded(PVA/AITC/-CD-ICsolution), andPVA/AITC/-CD-ICsolutionwasstirred6hmoreatroomtem- peratureandelectrospun(PVA/AITC/-CD-IC-NF).Forcomparison, wehavealsoelectrospunPVAaqueoussolution(7.5%,w/v)without AITCorAITC/-CD-IC.Table1summarizesthecompositionsofthe solutionsusedfortheelectrospinningofthenanofibers.
2.3. Electrospinning
PVA,PVA/AITCandPVA/AITC/-CD-ICsolutionswereseparately loadedinto3mLplasticsyringe(metallicneedlewith0.8mminner diameter)andthenelectrospunbyusingthehorizontalelectro- spinning setup.The solutions were pumped through a syringe pump(WPI,SP101IZ)at1mL/hrate.Cylindricalmetalcovered withaluminumfoilwasusedasacollector.Theelectricfield(15kV) wasappliedfromahighvoltagepowersupply(AUSeries,Matsu- sadaPrecisionInc.)andthetip-to-collectordistancewas10cm.
Experimentswereperformedat24–25◦Cunder17–20%humidity.
2.4. Characterizationandmeasurements
The viscosityof PVA, PVA/AITC and PVA/AITC/-CD-ICsolu- tionsweredeterminedbyAntonPaarPhysicaMCR301rheometer equippedwithacone/plate accessory(spindletype CP40-2)at aconstantshearrateof100s−1,at25◦Candtheconductivityof solutionswasmeasuredbyInolab®Multi720-WTWatroomtem- perature.
The morphologies of the electrospun PVA-NF, PVA/AITC-NF and PVA/AITC/-CD-IC-NF wereexaminedby scanningelectron microscopy (SEM, FEI—Quanta 200 FEG). Prior to taking SEM images,nanofibersamplesmountedonmetalstubswithdouble- sidedadhesivetapewerecoatedwith5nmAu/Pd(PECS-682)to minimizecharging.Diametersofthenanofibersweremeasured directlyfromtheSEMimages(n≥100)andaveragefiberdiameter (AFD)wascalculated.
Thecrystalline structureof PVA-NF, -CD,PVA/AITC-NF and PVA/AITC/-CD-IC-NFwere recordedby X-raydiffraction (XRD, PANalyticalX’Pertpowderdiffractometer)applyingCuK␣radia- tionina2range7.5–30◦.
Theprotonnuclearmagneticresonance(1H-NMR)spectrawere recorded onBrukerDPX-400in DMSO-d6 at400MHzat 25◦C.
30mg/mLofAITC,PVA-NF,-CD,PVA/AITC-NFandPVA/AITC/- CD-IC-NFweredissolvedinDMSO-d6toexaminethepresenceof AITCinthesamplesandstoichiometryofPVA/AITC/-CD-IC-NF.
Integration of the chemical shifts (ı) given in parts per mil- lion(ppm)of thesampleswascalculatedbyusingMestReNova software.
The cumulative amount of AITC released from PVA/AITC- NF and PVA/AITC/-CD-IC-NF were determined for 240minby headspace gaschromatography–massspectrometry (GC–MS)of AgilentTechnologies7890AgaschromatographcoupledtoanAgi- lentTechnologies5975CinertMSDwithatriple-axisdetector.The capillarycolumnusedwasHP-5MS(Hewlett-Packard,Avondale, PA)(30m×0.25mmi.d.,0.25mfilmthickness).Theheadspace GC–MSexperiments wereperformedwitha CTCPAL autosam- pler.20mgofPVA/AITC-NFandPVA/AITC/-CD-IC-NFtakenfrom thealuminumfoilwereplacedin20mLheadspaceglassvials.The vialswereagitatedat500rpmat30◦C,50◦Cand75◦Cofincuba- tiontemperatureand20%relativehumidity.Heliumwasusedas carriergaswasataflowrateof1.2mL/min.500Lofvaporwas injectedtotheheadspaceGC–MSbyusingaheadspaceinjector (MSH02-00B,volume:2.5mL,scale:60mm).Thesyringetemper- aturewas50◦C.Oventemperaturewasheldat50◦Cfor1minand increasedto200◦Cattherateof20◦C/minandheldatthistem- peraturefor3min.Thermaldesorptionwasconductedinthesplit mode(20:1).HeadspaceGC–MSanalyseswerecarriedoutinthe completeselectedionmonitoringmode(SIM).Flavor2andNIST 0.5librarieswereusedtodecideAITCpeak.Thereleaseexperi- mentwasperformedintriplicateandtheresultswerereportedas average±standarddeviation.
The antibacterial activity of PVA/AITC-NF and PVA/AITC/- CD-IC-NF was assessed by colony counting method against Gram-negativeE.coliandGram-positiveS.aureusbacteria.200L oftheE.coliandS.aureusgrownovernightwereinoculatedinto 10mLofLuria–Bertani(LB)brothandnanofibersweresterilized byUVirradiationandputintotheflasks.About1mgofnanofiber samplesandcontrolswereincubatedat37◦Candstirred100rpm for24h.Samplesfromeachflaskandcontrolswereseriallydiluted, 100LofeachwasspreadontoLBagarandcolonieswerecounted andphotographedafter24h.Allexperimentswereperformedin triplicateandreportedasaverage±standarddeviation.
3. Resultsanddiscussion
3.1. Morphologyanalysisofnanofibers
As an aim toward producing PVA nanofibers (NF)encapsu- latingAITC,freeAITC andinclusioncomplexofAITC with-CD (AITC/-CD-IC)wasincorporatedintoPVAnanofibers(PVA/AITC- NFandPVA/AITC/-CD-IC-NF)byusingelectrospinningtechnique.
ThemorphologicalcharacterizationofPVA-NF,PVA/AITC-NFand PVA/AITC/-CD-IC-NFwascarriedoutviaSEM.SEMimagesand averagefiberdiameter(AFD)alongwithfiberdistributionsofthe nanofibersaredepictedin Fig.2.In thecase ofelectrospinning of pristinePVA solution,bead-free anduniformPVA-NF having AFDof290±65nmwasobtained.Then,weincorporatedAITCand AITC/-CD-ICintoPVAsolutionandthenelectrospinningwasper- formedinordertoobtainfunctional nanofibers.Asclearlyseen fromSEMimages,theincorporationofAITCorAITC/-CD-ICdid notcauseanynotablechangeinthemorphologyoftheelectrospun nanofibers.TheAFDofPVA/AITC-NFandPVA/AITC/-CD-IC-NFwas veryclosetoeachotherwhichwere210±45nmand235±90nm,
Fig.2. SEMimagesandfiberdiameterdistributionswithaveragefiberdiameter(AFD)oftheelectrospunnanofibersobtainedfromsolutionsof(a)PVA,(b)PVA/AITC,(c) PVA/AITC/-CD-IC.
respectively.ThesenanofiberswereslightlythinnerthanthePVA- NF. The slight variations of fiber diameters observed for the nanofibersampleswerepossiblyduetodifferencesinviscosityand conductivityofthesolutions[1,2,32].Theviscosity,conductivity andAFDvaluesofPVA,PVA/AITCandPVA/AITC/-CD-ICsolutions areshowninTable1.PVA/AITCandPVA/AITC/-CD-ICsolutions havehigherviscosityandhigher conductivitycompared toPVA solution.
3.2. Crystallinestructureofnanofibers
XRD was performed for PVA-NF, -CD, PVA/AITC-NF and PVA/AITC/-CD-IC-NFandthediffractionpatternsaredepictedin Fig.3.AITCisaliquidcompoundatroomtemperature,sowedid notrunXRDanalysisforpureAITC.PVAisasemi-crystallinepoly- merandtheXRDofPVA-NFhasshownabroaddiffractionataround 2∼19.6◦[33].XRDpatternofPVA/AITC-NFwassimilartoPVA-NF, thusPVAkeepsitssemi-crystallinestructureafterincorporationof AITC.Thecage-typepackingcrystalstructureobservedinCDgener- allytransformintochannel-typepackinginCD-IC.Theas-received
-CDhasacage-typepackinganditscharacteristicdiffractionpat- ternisgiveninFig.3.Thechannel-typepackingof-CDhassalient characteristicpeakat2∼12◦[6].Here,thepeakat2∼12◦inXRD patternofPVA/AITC/-CD-IC-NFwasobservedandweconcluded thattheICformationbetweenAITCand-CDinPVA/AITC/-CD-IC- NFwaspresent.Inaddition,thecage-typepackingof-CDpattern
wasabsentinthefibersampleandthismaysupporttheAITC/- CD-ICformationaswell.
3.3. AITCcontentinnanofibers
PVA/AITC-NF and PVA/AITC/-CD-IC-NF were tested by 1H- NMRtodeterminethestoichiometryofAITC/-CD-ICandwhether
Fig.3. XRDpatternsofPVAnanofibers,-CDandAITCcontainingPVAnanofibers.
Z.Aytacetal./ColloidsandSurfacesB:Biointerfaces120(2014)125–131 129
Fig.4. 1H-NMRspectraofAITC,PVA,-CDandAITCcontainingPVAnanofibers.
AITC was present in PVA/AITC-NF and PVA/AITC/-CD-IC-NF (Fig.4).ThepeaksforAITCwereobservedataround4.2,5.3,and 5.9ppmcorrespondingtotheprotonsofAITC[34].Thestoichiom- etryofAITCand-CD-ICinPVA/AITC/-CD-IC-NFwasdetermined
as0.68:1.00(AITC:-CD)byintegratingthepeakratioofthecharac- teristicchemicalshiftsofAITC(5.3ppm)and-CD(5.7ppm).Thus, initialmolarratioof2:1forAITC:-CDwhichwasusedtoobtain AITC/-CD-ICwaspreservedsubstantially.So,PVA/AITC/-CD-IC- NFstillcontainedsomeofAITCinthefibersamplewhereaswe couldnotobserveanypeakofAITCinPVA/AITC-NF.Therefore,we deducedthatextremelylowquantityofAITCexistinPVA/AITC-NF.
ThepossiblereasonforthissituationisevaporationofAITCdur- ingtheelectrospinningofsolutionsinceAITCisahighlyvolatile substance.Infact,wewereabletosmellAITCheavilyduringelec- trospinningofPVA/AITC-NF.Inbrief,1H-NMRstudyshowedthat
-CDpreventedevaporationofAITCtosomeextentduringelectro- spinningprocessandhighloadingofAITCinPVA/AITC/-CD-IC-NF wasachievedcomparedtoPVA/AITC-NF.
3.4. ThereleasestudyofAITCfromnanofibers
The application of AITC is somehow restricted because of its highly volatile nature, unpleasant odor and hydrophobic nature[27].Here, wehaveincorporatedAITC/-CD-ICintoPVA polymer solution, and PVA/AITC/-CD-IC-NF were obtainedvia electrospinning.Foracomparativestudy,wehavealsoproduced PVA/AITC-NFwithoutCD-IC.Therehavebeenreportsin thelit- eratureabouttheapplicationofCDinfoodpackagingmaterials tocontrolthedeliveryofactivecompounds[8,20,27,29].Here,we haveevaluatedthereleasebehaviorofAITCfromPVA/AITC-NFand PVA/AITC/-CD-IC-NFatthreedifferenttemperatures(30◦C,50◦C and75◦C)byusingheadspaceGC–MS(Fig.5).
ThecumulativeamountofAITCreleasedat30◦C,50◦Cand75◦C (for240min) fromPVA/AITC-NFwereminimalandsignificantly muchlowerthanthatofPVA/AITC/-CD-IC-NFsample.Itisevident thatverysmallamountofAITCwaspresentinthePVA/AITC-NF sampleasalsoconfirmedby1H-NMRstudieswhichwecouldnot even detectAITC inthis sample.In thecase ofPVA/AITC/-CD- IC-NF,theamountofAITCwassignificantandthereleaseofAITC fromthenanofibersamplewasincreasedgraduallywithtimeand finally becameconstant. Hence,we have successfully produced PVA/AITC/-CD-IC-NFwithasustainedreleasebehaviorofAITC.
Inaddition,asthetemperaturewasincreasedfrom30◦Cto75◦C, theamountofAITCreleasedwasalsoincreasedobviouslyforboth PVA/AITC-NFand PVA/AITC/-CD-IC-NFsamples.Thisis related withthehigherdiffusioncoefficientofmoleculesathighertem- peratures [35]. Athigher temperatures, the motionof polymer chainsincreases,sothemovementofactivemoleculethroughthe amorphous partsof thepolymeris favored [36]. Moreover,the incrementinthereleaseofAITCfromPVA/AITC/-CD-IC-NFwas muchclearerthanPVA/AITC-NF.Inshort,headspaceGC-MSresults clearlyconfirmedthattheevaporationofAITCwashinderedbyCD- ICandmuchhigheramountofAITCwasencapsulatedinthePVA nanofiberswhenAITCwascomplexedwith-CD.
3.5. Antibacterialactivityofnanofibers
AITC,themajorpungentcompoundinplants,hasbeenreported tohavestrongantimicrobialactivityinbothliquidandvaporforms.
Here,wehavetestedtheantibacterialactivityofPVA/AITC-NFand PVA/AITC/-CD-IC-NFbycolony countingmethodagainst Gram negativeE.coliandGrampositiveS.aureusbacteria.Fig.6ashows representativeimagesofbacteriacoloniestreatedbyPVA/AITC- NF and PVA/AITC/-CD-IC-NF; and Fig. 6b shows the effect of PVA/AITC-NFandPVA/AITC/-CD-IC-NFonthegrowthinhibition rateofE.coliandS.aureus.Thegrowthinhibitionrate(%)ofbacte- riawascalculatedbyassumingthatplateswithoutnanofibershave 100% growth.PVA/AITC-NFand PVA/AITC/-CD-IC-NFexhibited 31.98% and 94.41% antibacterialactivity againstE.coli whereas
Fig.5.CumulativereleaseofAITCfromPVA/AITC-NFandPVA/AITC/-CD-IC-NFat30◦C,50◦Cand75◦C(n=3).Theerrorbarsinthefigurerepresentthestandarddeviation (SD).
antibacterial activity against S. aureus was 53.55% and 99.82%, respectively.ItwasobviousPVA/AITC/-CD-IC-NFsample(includ- ingabout0.0107mg AITC accordingto NMR result)hasshown muchbetterantibacterialactivitywhencomparedtoPVA/AITC-NF.
ThisismostlikelyduetothepresenceofhigheramountofAITCin PVA/AITC/-CD-IC-NFwhichwaspreservedbyCD-IC.Inthecase ofPVA/AITC-NF,someantibacterialactivityagainstE.coliandS.
aureuswasobservedsinceverylowlevelofAITCwaspresentin thissampleasitwasdetectedbyheadspaceGC–MSanalysis.More- over,theelectrospinningprocessinwhichveryhighvoltageswere appliedhasnonegativeeffectontheantibacterialpropertyofAITC encapsulatedinPVA/AITC-NFandPVA/AITC/-CD-IC-NF.Inaddi- tion,thedifferenceintheantibacterialactivityofbothnanofiber samplesagainstE.coliandS.aureusmaybecausedbythedifference
Fig.6.(a)ExemplaryimagesofcoloniesofEscherichiacoli,Staphylococcusaureus;E.coliandS.aureuscoloniestreatedbyPVA/AITC-NF;E.coliandS.aureuscoloniestreated byPVA/AITC/-CD-IC-NF;(b)growthinhibitionrate(%)ofE.coliandS.aureusinPVA/AITC-NFandPVA/AITC/-CD-IC-NF(n=3).Theerrorbarsinthefigurerepresentthe standarddeviation(SD).
Z.Aytacetal./ColloidsandSurfacesB:Biointerfaces120(2014)125–131 131
betweencellwallcompositionoftwobacteria,whichareGram negativeandGrampositive,respectively.Gramnegativebacteria haveasemi-permeable barrierthatdeceleratespassingof com- pounds[37].
4. Conclusion
Inthisstudy,weperformedtheelectrospinningoffunctional PVAnanofibersincorporatingAITCandAITC/-CD-IC.SEMimages showedthatthebead-freefibermorphologyofPVAnanofibersdid notchangeaftertheincorporationofAITCandAITC/-CD-ICinto polymermatrix.Weobservedthattheextremelylowquantityof AITCexistinPVA/AITC-NFduetothequickevaporationofAITC duringtheelectrospinningofPVA/AITCsolutionsinceAITChasa highlyvolatilenature.On thecontrary,much higheramountof AITCwasencapsulatedinthePVA/AITC/-CD-IC-NFsampledue tothecyclodextrin inclusioncomplexation. Hence,PVA/AITC/- CD-IC-NFhasshownimprovedantibacterialactivityagainstE.coli and S. aureus when compared to PVA/AITC-NF without CD-IC.
Inbrief,newlydevelopedPVA/AITC/-CD-IC-NFwithhighload- ingefficiency,sustainedrelease behaviorandconsiderably high antibacterialactivitymayhavepotentialsforactivefoodpackaging andbiomedicalapplications.
Acknowledgments
Dr. Uyar acknowledges The Scientific and Technological Research Council of Turkey (TUBITAK) (Project no. 111M459) and EU FP7-PEOPLE-2009-RG Marie Curie-IRG (NANOWEB, PIRG06-GA-2009-256428) and The Turkish Academy of Sciences—Outstanding Young Scientists Award Program (TUBA- GEBIP) for funding the research. Z. Aytac thanks to TUBITAK (Projectno.111M459)forthePh.D.scholarship.Dr.Tekinaythanks GaziUniversityScientificResearchProjectUnitfortheirsupport.
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