Electrospun zein nanofibers incorporating cyclodextrins

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ContentslistsavailableatSciVerseScienceDirect

Carbohydrate

Polymers

jo u r n al h om ep a ge : w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Electrospun

zein

nanoﬁbers

incorporating

cyclodextrins

Fatma

Kayaci, Tamer

Uyar

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UNAM-InstituteofMaterialsScience&Nanotechnology,BilkentUniversity,Ankara,06800,Turkey

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Articlehistory:

Received26December2011

Receivedinrevisedform16March2012 Accepted22May2012

Available online 30 May 2012 Keywords: Cyclodextrin Electrospinning Zein Nanoﬁbers

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Zeinnanofiberscontainingcyclodextrins (zein/CD)wereproducedvia electrospinning.Three types ofCDs(␣-CD,␤-CDand␥-CD)having10%,25%and50%(w/w)wereindividuallyincorporatedinto zeinnanofibers.SEMimagingelucidatedthatthemorphologiesoftheelectrospunzein/CDnanofibers dependedontheCDtypeandweightpercentage.TheincorporationofCDsinzeinimprovedthe electro-spinnabilityandbead-freenanofiberswereobtainedatlowerzeinconcentrations.Zein/CDnanofibers havingfiberdiameters∼100–400nmwereobtaineddependingonthezeinconcentrations,typesand weightpercentagesofCD.XRDstudiesrevealedthatCDsweremostly distributedwithoutforming crystallineaggregatesforzein/CDnanofiberscontaininglowerweightpercentageofCDs.Thesurface analysesofzein/CDnanofibersbyATR-FTIRandXPSindicatedthatsomeoftheCDswerepresentonthe fibersurface.Thermalanalysesshowedthatzein/␤-CDnanofibershaveshownhigherglasstransition temperaturesandhigherdegradationtemperaturewithincreasingCDcontent.

1. Introduction

Recently,electrospinningtechniquehasgainedagreat inter-est since this technique is quite versatile for fabricating nanofibers/nanowebs from various synthetic or natural poly-mers,polymerblends,sol–gels,ceramics,etc.(Agarwal,Greiner, &Wendorff,2009;Bhardwaj&Kundu,2010;Ramakrishnaetal., 2006; Teo&Ramakrishna, 2009).Moreover, functional electro-spunnanofibrouscompositestructurescanalsobeproducedby incorporating functional additives and/or nanoparticles in the fiber matrix or on the fiber surface (Andrew & Clarke, 2008; Anitha,Brabu,Thiruvadigal,Gopalakrishnan,&Natarajan,2012; Dong, Wang, Sun, & Hinestroza, 2008; He, Hu, Yao, Wang, & Yu, 2009; Roso, Sundarrajan, Pliszka, Ramakrishna, & Modesti, 2008; Zhang, Shao, et al., 2011).Unique properties of electro-spun nanofibers/nanowebs including a relatively large surface area tovolume ratio and pore sizes within the nanoscale and multi-functionality due to the presence of functional additives andnanoparticlesmakethemfavorablecandidatesinavarietyof applicationareassuchasmembranes/nanofilters,wounddressing, tissue engineering,drugdelivery, nanotextiles,nanocomposites, energy,environment,etc.(Agarwaletal.,2009;Bhardwaj&Kundu, 2010; Chigome,Darko, &Torto, 2011;Lu, Wang,&Wei, 2009; Ramakrishnaetal.,2006;Teo&Ramakrishna,2009;Thavasi,Singh, &Ramakrishna,2008;Xie,Li,&Xia,2008;Yoon,Hsiao,&Chu,2008).

∗ Correspondingauthor.Tel.:+903122903571;fax:+903122664365. E-mailaddresses:[email protected],[email protected](T.Uyar).

Inrecentyears,biopolymersfromrenewableresourcessuchas zeinhavegainedattentionforeconomicalandenvironmental rea-sons(Paraman, &Lamsal,2011;Selling&Woods,2008;Selling, Woods,Sessa,&Biswas,2008).Zein,themajorproteinofcornand aby-productofthebioethanolindustry,isanon-toxic, biocompat-ible,biodegradablepolymerandthispolymercanformfilms.Zein filmsandzeinmicro/nanoparticlescanbeusedforencapsulation ofessentialoils,aromasandflavors, controlledreleaseofactive additives and as an activefood packagingmaterial, etc. (Alkan etal.,2011;Parris,Cooke,&Hicks,2005;Patel,Heussen,Hazekamp, Drost,&Velikov,2012;Sanchez-Garcia,Hilliou,&Lagaron,2010; Shi,Kokini,&Huang,2009;Zhong,Jin,Davidson,&Zivanovic,2009). Intherecentyears,electrospinningofzeinnanofibershavereceived muchattentionaswell(Jiang,Reddy,&Yang,2010;Jiang&Yang, 2011;Jiang,Zhao,&Zhu,2007;Miyoshi,Toyohara,&Minematsu, 2005;Sellingetal.,2007,2008;Torres-Giner,Gimenez,&Lagaron, 2008;Yao,Li,&Song,2009).Thesestudiesaremostlyrelatedtothe optimizationoftheelectrospinningparametersofzeinnanofibers (Miyoshietal.,2005;Sellingetal.,2007;Torres-Gineretal.,2008), crosslinkingof zeinnanofibers(Jianget al.,2010;Jiang&Yang, 2011;Sellingetal.,2008)andblendingofzeinwithsomeother typeofbiopolymers(Jiangetal.,2007;Yaoetal.,2009a;Yao,Li, Song,Li,&Pu,2007).Inaddition,␤-carotenewhichisabioactive antioxidant(Li,Lim,&Kakuda,2009)and(−)-epigallocatechin gal-letethatisaplantpolyphenol(Fernandez,Torres-Giner,&Lagaron, 2009)wereincorporatedintoelectrospunzeinnanofibermatrixfor thestabilizationoftheseactiveadditives.

Ourparticularinterestisthefunctionalizationofelectrospun nanoﬁbers with cyclodextrins (CDs). CDs are cyclic oligosac-charides having a toroid-shapedmolecular structure. The most 0144-8617/$–seefrontmatter © 2012 Elsevier Ltd. All rights reserved.

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Fig.1. (a)Chemicalstructuresof␣-CD,␤-CDand␥-CDand(b)schematicrepresentationofCD.

commonCDs are named as ␣-CD, ␤-CD and ␥-CD having 6, 7 and8glucopyranoseunits,respectively(Fig.1).Hydrophobic cav-ityof CD actsas a host forthe variousmolecules, and CD can formnon-covalenthost–guestinclusioncomplexes.Thephysical andchemicalpropertiesoftheguestmoleculesaretailoredand becomemorestablewhencomplexedwithCDs(DelValle,2004; Hedges,1998; Szejtli, 1998), therefore, CDsand their inclusion complexesarequiteapplicableinmanyfieldsincluding pharma-ceuticals,functionalfoods,cosmeticsandhome/personalcareand textiles(DelValle,2004;Hedges,1998;Szejtli,1998,2003).Up todate,severalstudieshavebeencarriedoutdealingwith incor-porationofCDsinelectrospunnanofibersfordifferentpurposes suchascrosslinking offibermatrix(Li&Hsieh,2005), molecu-larfiltration(Uyar,Havelund,Hacaloglu,Besenbacher,&Kingshott, 2010;Uyar,Havelund,Nur,etal.,2010,2009;Zhang,Chen,&Diao, 2011)andCDwasalsousedasareducingandstabilizingagentfor gold(Baietal.,2008)andsilver(Chae,Kim,Yang,&Rhee,2011) nanoparticlesformation.Inourrecentstudies,CDsandCD inclu-sioncomplexes(CD-ICs)ofvolatilefragrancesweresuccessfully incorporatedintoelectrospunnanofibers,andtheseCD function-alizedelectrospunnanofiberswereusedasmolecularfilters(Uyar, Havelund,Hacaloglu,etal.,2010;Uyar,Havelund,Nur,etal.,2010, 2009), and CD-ICshaveprovided longershelf-lifeand stabiliza-tionofvolatilefragrancesathighertemperature(Kayaci&Uyar, 2012;Uyar,Hacaloglu,&Besenbacher,2009;Uyar,Hacaloglu,& Besenbacher,2011;Uyar,Nur,Hacaloglu,&Besenbacher,2009).

Inthisstudy,wereportontheelectrospinningofzeinnanofibers incorporatingCDs.Electrospunzein/CDnanofiberswereobtained byusingthreetypesofCDs;␣-CD,␤-CDand␥-CDandtheweight loadingsoftheseCDswerevariedfrom10%upto50%(w/w)with respecttozein.WefoundthattheadditionofCDinthepolymer solutionsimprovetheelectrospinnabilityofthezeinnanofibers at lower polymer concentration. The morphological, structural, surfaceand thermal characteristicsof theresultingelectrospun zein/CDnanofibers werecharacterizedby SEM, XRD,ATR-FTIR, XPS,DSCandTGA.Thisstudymainlydealswiththeoptimization ofelectrospinningofzein/CDnanofibersandtheirmorphological, structural,surfaceandthermalcharacterizations.

2. Experimentalpart

2.1. Materials

Zeinfrommaize(Sigma–Aldrich)andN,N-dimethylformamide (DMF, Pestanal, Riedel) were purchased. The alpha-, beta- and gamma-cyclodextrins(␣-CD,␤-CDand␥-CD)werepurchasedfrom WackerChemieAG(Germany).Allmaterialswereusedas-received withoutanypuriﬁcation.

2.2. Preparationofthesolutions

First,40%,50%and60%(w/v)zeinweredissolvedinDMFand electrospinningofzeinsolutionswithoutCDswasperformed.For theelectrospinningofzein/CDsolutions,10%,25%and50%(w/w, withrespecttozein)CDs(␣-CD,␤-CDand␥-CD)weredissolved inDMFandthen,40%,50%and60%zein(w/v,withrespectto sol-ventofDMF)wereaddedtoeachCDsolutionseparatelyandstirred for1hat roomtemperature.Thecompositionsof thesolutions weresummarizedin Table1.Homogeneous andclearsolutions wereobtainedforallofthezein/_␤-CDcompositions.Ontheother hand,thezeinsolutionscontaining25%(w/w)␣-CDwasslightly turbidandalsothesolutionscontaining50%(w/w)␣-CDand␥-CD werehighlyturbid.Theresultingzeinandzein/CDsolutionswere electrospun.

2.3. Electrospinning

Thesolutionswereplacedina3mLsyringefittedwitha metal-licneedlehavinginnerdiameterof0.8mm.Thesyringewasfixed horizontallyonthesyringepump(Model:SP101IZ,WPI).Several parameterswereappliedinordertooptimizetheelectrospinning ofthesolutionsandtheoptimalparameterswerechosenasfollows. Voltageof15kVwasappliedtothemetalneedletipbyusinghigh voltagepowersupply (AUSeries,MatsusadaPrecision Inc.).The polymersolutionwaspumpedwithflowrateof0.5mL/hduring electrospinningandthetip-to-collectordistancewassetto12cm. Thegroundedstationarycylindricalmetalcollector(height:15cm, diameter:9cm)coveredwithaluminumfoilwasusedforthe depo-sitionoftheelectrospunnanofibers.Theelectrospinningprocess wascarriedoutat24◦Cand30%relativehumidityinanenclosed Plexiglasbox.

2.4. Measurementsandcharacterization

TheviscosityofthesolutionswasmeasuredbyusingAntonPaar PhysicaMCR301Rheometerequippedwithcone/plateaccessory usingthespindletypeCP40-2at22◦Candaconstantshearrateof 100s−1.Theconductivitymeasurementofthesolutionswas per-formedbyusingMultiparametermeterInoLab®_Multi₇₂₀_(WTW)

atroomtemperature.

Themorphologyandthediameterofthenanofiberswere exam-ined by usingscanning electron microscope (SEM) (FEI-Quanta 200FEG).Thenanofiberswerecoatedwith5nmAu/Pdpriorto SEMimaging.Around100fiberdiametersweremeasuredfromthe SEMimagestodeterminetheaveragefiberdiameter(AFD)ofthe nanofibers.

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Table1

Propertiesofzeinandzein/CDsolutionsandtheresultingzeinandzein/CDnanoﬁbers. Solutions %zein (w/v)a TypeofCD(%) (w/w)b Viscosity (Pas) Conductivity (␮S/cm)

Fibermorphology Averageﬁberdiameter (AFD)(nm)

zein40 40 – 0.0332 435 Nanoﬁberswithmanybeads –

zein50 50 – 0.0859 344 Nanoﬁberswithfewbeads 80±35

zein60 60 – 0.206 264 Bead-freenanoﬁbers 170±30

zein40/␣-CD10 40 ␣-CD,10 0.0421 359 Nanoﬁberswithmanybeads –

zein40/␤-CD10 40 ␤-CD,10 0.0428 357 Nanoﬁberswithmanybeads –

zein40/␥-CD10 40 ␥-CD,10 0.0439 333 Nanofiberswithfewbeads 60±10 zein40/␣-CD25 40 ␣-CD,25 0.0522 270 Nanofiberswithfewbeads 60±20 zein40/␤-CD25 40 ␤-CD,25 0.0562 283 Nanofiberswithfewbeads 70±20 zein40/␥-CD25 40 ␥-CD,25 0.0732 267 Nanofiberswithfewbeads 60±10 zein40/␣-CD50 40 ␣-CD,50 0.0849 96.8 NanofiberswithbeadsandCDaggregates – zein40/␤-CD50 40 ␤-CD,50 0.0727 78.8 NanofiberswithbeadsandCDaggregates – zein40/␥-CD50 40 ␥-CD,50 0.101 115.6 NanofiberswithbeadsandCDaggregates –

zein50/␣-CD10 50 ␣-CD,10 0.125 286 Bead-freenanoﬁbers 90±20

zein50/␤-CD10 50 ␤-CD,10 0.171 278 Bead-freenanoﬁbers 100±25

zein50/␥-CD10 50 ␥-CD,10 0.212 268 Bead-freenanoﬁbers 110±30

zein50/␣-CD25 50 ␣-CD,25 0.212 138 NanoﬁberswithCDaggregates 185±45

zein50/␥-CD25 50 ␥-CD,25 0.239 161 Bead-freenanoﬁbers 155±35

zein50/␣-CD50 50 ␣-CD,50 0.39 74.3 NanofiberswithCDaggregates 240±85 zein50/␤-CD50 50 ␤-CD,50 0.381 97.8 NanofiberswithCDaggregates 360±140 zein50/␥-CD50 50 ␥-CD,50 0.354 126.5 NanofiberswithCDaggregates 265±110

zein60/␣-CD10 60 ␣-CD,10 0.329 211 Bead-freenanoﬁbers 225±30

zein60/␥-CD10 60 ␥-CD,10 0.218 189.4 Bead-freenanoﬁbers 170±40

zein60/␣-CD25 60 ␣-CD,25 0.69 89.8 NanoﬁberswithCDaggregates 375±80

zein60/␥-CD25 60 ␥-CD,25 0.664 109.6 Bead-freenanoﬁbers 380±240

zein60/␣-CD50 60 ␣-CD,50 1.56 41.6 Noﬁberformation –

zein60/␤-CD50 60 ␤-CD,50 1.02 85.6 Noﬁberformation –

zein60/␥-CD50 60 ␥-CD,50 0.752 85.8 Noﬁberformation –

a_With_respect_to_solvent_(DMF). b_With_respect_to_polymer_(zein).

X-raydiffraction(XRD)dataofthenanoﬁberswerecollectedby usingPANalyticalX’PertPowderdiffractometerwithCuK␣ radia-tioninarange2=5–30◦.

Surface characterizations of the nanofibers were performed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) (Bruker, VERTEX 70) and K-Alpha-monochromated high-performance X-ray photoelectron spec-trometer (XPS) (Thermo Scientific). The ATR-FTIR spectrawere recordedfrom700to4000cm−1witharesolutionof4cm−1by tak-ing64scansforeachsample,andthesespectrawereobtainedwith FTIRspectrometerequippedwithaliquidnitrogencooledmercury cadmiumtelluride(MCT) detectorbyusingATRsetup contain-ingagermaniumcrystal.XPSwasusedbymeansofafloodgun chargeneutralizersystemequippedwithamonochromatedAl K-␣X-raysource(h=1486.6eV).Wideenergysurveyscans(WESS) wereobtainedoverthe0–1360eVbindingenergy(BE)rangeata detectorpassenergyof150eVinordertodeterminethesurface ele-mentalcompositionofthenanofibers.Thehighresolutionspectra wererecordedforC1sregionatpassenergyof50eV.

Thethermalpropertiesofthenanoﬁberswereinvestigatedby usingdifferentialscanningcalorimetry(DSC)(TAQ2000)and ther-mal gravimetric analyzer (TGA) (TA Q500). DSC analyseswere

carriedoutwithabout5mgofsamplesundertheN2 asapurge

gas.Initially,thesampleswereequilibratedat25◦Cthentheywere heatedto200◦Cat10◦C/min.TGAwasperformedfromroom tem-peratureto500◦Cataheatingrateof20◦C/minunderthenitrogen atmosphere.

3. Resultsanddiscussion

3.1. Electrospinningofzeinnanoﬁbers

In the literature,the electrospinning of zeinnanofibers was mostlycarriedoutbyusingethanol/watermixturesolventsystem whichresultedinribbon-likefibermorphologyduetotherapid skinformationandcollapseofthefibercorebecauseoftheveryfast evaporationofthesolvent(Miyoshietal.,2005;Sellingetal.,2007; Torres-Gineretal.,2008).However,round-shapedzeinnanofibers canbeobtainedbyusingsolventsystemshavinghighboilingpoints suchasDMF(Jiangetal.,2007).Inourstudy,theelectrospinningof zeinnanofiberswascarriedoutbyusingDMFasasolventsystem. ThereasonofchoosingDMFisbecausethinnerandmoreuniform zeinfiberscanbeobtained(Jiangetal.,2007)whencomparedto ethanol/watersystem(Miyoshietal.,2005;Sellingetal.,2007;

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Torres-Gineretal.,2008),andothermoreimportantreasonisthat DMFisaverygoodsolventforCDs,andthereforewewereableto preparezein/CDhomogeneoussolutionsinmostcases.

Thecharacteristics (composition, viscosityand conductivity) ofthe zeinand zein/CD solutions and themorphologiesof the electrospunnanofibersandtheiraveragefiberdiameter(AFD)are summarizedin Table1. Zeinsolutionshavingdifferent concen-trationswereelectrospuninorder tofindtheoptimalpolymer concentrationforobtainingbead-freeuniformnanofibers.The rep-resentativeSEMimagesofzeinnanofiberselectrospunfrom40%, 50%and60%(w/v)zeinsolutionsinDMFaredepictedinFig.2.At lowerzeinconcentration(40%,w/v),micronsizeirregular spher-icalbeadedstructureswereobtainedduetothelowviscosityof thepolymersolution.Astheconcentrationofzeinsolutionwas increasedto50%(w/v),thenumberofbeadswasdecreased sig-nificantly and theshape of beads became more elongated and nanofibershavingAFDof80±35nmwereobtained.Uniformand bead-freezeinnanofibershavingAFDof170±30nmwereobtained when60%(w/v)zeinsolutionwaselectrospunindicatingthat60% (w/v)istheoptimalzeinconcentrationforproducinguniformzein nanofibersattheappliedelectrospinningconditions.Ourresults correlatewiththeliteraturefindingswherethebead-freeuniform zeinnanofiberswereproducedabove50%(w/v)zein concentra-tionwhenDMFwasusedasasolventsystem(Jiangetal.,2007). Thisbehaviorisverytypicalfortheelectrospinningofpolymeric solutionswherethetransitionfrombeadedstructuretobead-free nanofibersisobservedbyincreasingthepolymerconcentration. Higherpolymerconcentrationresultedinhighersolution viscos-ityduetothepresenceofmorepolymerchainentanglementsand thereforethebeadedstructuresareeliminatedsincetheelectrified polymerjetcanbestretchedfullyyieldingbead-freenanofibers (Ramakrishna,Fujihara,Teo,Lim,&Ma,2005;Uyar&Besenbacher, 2008).

3.2. Electrospinningofzein/CDnanoﬁbers

Thezein/CDsolutionswereclearandhomogeneousexceptfor thesolutionscontaininghigherweightpercentageof␣-CDand ␥-CD.Thezein/␤-CDsolutionswereclearinallcompositionswhereas thezein/CDsolutionscontaining50%(w/w)␣-CDand␥-CDwere highlyturbid, andzein/CDsolutioncontaining25% (w/w)␣-CD wasslightlyturbid.Theturbiditywasobservedpossiblybecauseof theprecipitationofthe␣-CDand␥-CDathigher%loading,andit isanticipatedthattheelectrospinningofthesezein/CDsolutions wouldcontainCD aggregatesin thefibermatrix. Inthecase of clearzein/CDsolutions,thehomogeneousdistributionoftheCDsin thefibermatrixisexpectedfortheelectrospunzein/CDnanofibers containingloweramountofCDs.TheSEMimagingofthe electro-spunzein/CDnanofibersgavesomeinsightfulinformationforthe presenceofCDaggregatesinthefibermatrix.

The representative SEM images of the electrospun zein/CD nanoﬁbersaredepictedinFig.3.Itwasobservedthattheaddition ofCDstozeinsolutionsimprovedtheelectrospinnability,andless beadedstructuresand/orbead-freenanoﬁberswereobtainedat lowerzeinconcentrationswhencomparedtozeinsolutions with-outCDs.

The electrospinning of 40% (w/w) zein solutions containing CDs resulted in nanoﬁbershaving much less beaded structure whencomparedto40%(w/w)pristinezeinsolution.Fig.3ashows theSEMimagesofelectrospunzein/CDnanoﬁbersobtainedfrom 40%(w/v)zeinsolutioncontaining10%,25%and50%(w/w,with respecttozein)CDs(␣-CD,␤-CDand␥-CD).Theelectrospinning of40%(w/w)zeinsolutions containing10%(w/w)CDsresulted inreduction of beadstosomeextent(Fig.3a1–a3). Inthecase ofzein40/␥-CD10sample,theeliminationofbeadsismuchmore pronounced which is possibly because of the higher solution

viscositycompared tozein40/␣-CD10and zein40/␤-CD10 solu-tions.Furthermore,itwasclearlyobservedthattheadditionof25% (w/w)CDstothe40%(w/v)zeinsolutionsimprovedthe electro-spinnabilityofthezein/CDsolutions,andyieldednanofiberswith muchlessbeadshavingmoreelongatedstructures(Fig.3a4–a6). Thisispossiblybecauseofthehighersolutionviscosityofzein/CD systemswherethebeadedstructuresaremostlyeliminateddue tothemorestretchingofelectrifiedsolutionjet.However,even theadditionof50%(w/w)CDsresultedinmoreviscoussolutions, theelectrospinningofthesezein/CDsolutionsyieldednanofibers havingirregularstructures(Fig.3a7–a9)suggestingthatuniform zein/CDnanofiberscannotbeproducedwhenthehighweight per-centageofCDswasused.Theirregularstructuresconsistingofnot onlybeadsbut alsoCDaggregates weremuch moreprominent inzein40/␣-CD50andzein40/␥-CD50nanofiberswhencompared tozein40/␤-CD50,sincethezein40/␣-CD50and zein40/␥-CD50 solutions werehighlyturbidprior toelectrospinning indicating thatCDaggregateswerealreadypresentinthesolution,andthese CDaggregatespossiblycouldnotbestretchedoutalongthefiber matrixduringtheelectrospinningprocess.Inthecaseofzein40/ ␤-CD50,thebeads wereless in number since thezein40/␤-CD50 solutionwasclear,andsomeCDaggregateswerepossiblyformed duringtheelectrospinningprocesswhenthesolventevaporation tookplace.

The electrospinning of 50% (w/v) zein solution yielded nanofiberswithfew beads as mentioned above(Fig.2b), how-ever, bead-free nanofibers were obtained from 50% (w/v) zein solutionwiththeadditionof10%and25%(w/w)CDs(␣-CD, ␤-CDand␥-CD)exceptforzein50/␣-CD25system(Fig.3b).Inthe caseofzein50/␣-CD25nanofibers,someirregularstructureswere observedwhichisbecauseofthepresenceof␣-CDaggregatesas discussedlaterintheXRDsection.Similarto40%(w/v)zein sys-tem,theadditionof 50% (w/w)CDsin 50% (w/v)zeinsolution yieldednanofibershavingirregularstructuresduetothe aggre-gationof CD crystalsasconfirmed byXRD results.It wasclear thattheadditionofCDsincertainratios(10%and25%,w/w)to the50%(w/v)zeinsolutionsassistedtoeliminatethebead forma-tionandprovidedbead-freezein/CDnanofiberswithoutincreasing polymerconcentration.We observedsimilareffectonthe mor-phologyoftheelectrospunPS(Uyar,Havelund,Hacaloglu,etal., 2009),PMMA(Uyar,Balan,Toppare,&Besenbacher,2009)andPEO (Uyar&Besenbacher,2009)nanofiberscontainingCDsinourrecent studies.

The electrospinning of 60% zein (w/v) solutions contain-ing 10% and 25% (w/w) CDs resulted in bead-free nanoﬁber morphology except for zein60/␣-CD25 system (Fig. 3c). The zein60/␣-CD25nanoﬁbershavesomebead-likestructuressimilar tozein50/␣-CD25systemwhichispossiblyduetoCDaggregates. Furthermore,60%zein(w/v)solutioncontaining50%(w/w)CDs couldnot be electrospundue tothe very highviscosity ofthe solutions.

In brief, the addition of CDs to zein solutions significantly affected the electrospinning, and bead-free nanofibers were obtainedfromlowerzeinconcentrationsforzein/CDsystemswhen comparedtopurezeinsolution.Thisismostlyduetothehigher viscosityofthe zein/CDsolutions,and highersolutionviscosity resultedinmoreuniformfiberswhenelectrospun(Ramakrishna etal.,2005;Uyar&Besenbacher,2008).Inaddition,the morpholo-giesof thezein/CDnanofibers containingdifferentkindof CDs (␣-CD,␤-CDand␥-CD)haveshownslightvariationsamongeach otherbecauseofthedifferencesinviscosityandconductivityof thesesolutions.ItwasobservedinTable1thattheAFDincreasesas thecontentoftheCDsincreasessincethepresenceofCDscausesa viscosityincreaseofthesolutionswhileitreducestheconductivity ofthesolutions.Therefore,zein/CDsolutionshavinghigher viscos-ityandlowerconductivityvaluesyieldedthickerfibersduetothe

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Fig.3.RepresentativeSEMimagesofelectrospunnanoﬁbersof(a1)zein40/␣-CD10,(a2)zein40/␤-CD10,(a3)zein40/␥-CD10,(a4)zein40/␣-CD25,(a5)zein40/␤-CD25,(a6) zein40/␥-CD25,(a7)zein40/␣-CD50,(a8)zein40/␤-CD50and(a9)zein40/␥-CD50;(b1)zein50/␣-CD10,(b2)zein50/␤-CD10,(b3)zein50/␥-CD10,(b4)zein50/␣-CD25,(b5) zein50/␤-CD25,(b6)zein50/␥-CD25,(b7)zein50/␣-CD50,(b8)zein50/␤-CD50and(b9)zein50/␥-CD50;(c1)zein60/␣-CD10,(c2)zein60/␤-CD10,(c3)zein60/␥-CD10,(c4) zein60/␣-CD25,(c5)zein60/␤-CD25and(c6)zein60/␥-CD25.

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Fig.4.XRDpatternsof(a)(i)zein50,(ii)zein50/␣-CD10,(iii)zein50/␣-CD25,(iv) zein50/␣-CD50and(v)␣-CD;(b)(i)zein50,(ii)zein50/␤-CD10,(iii)zein50/␤-CD25, (iv)zein50/␤-CD50and(v)␤-CD;(c)(i)zein50,(ii)zein50/␥-CD10,(iii) zein50/␥-CD25,(iv)zein50/␥-CD50and(v)␥-CD.

lessstretchingoftheelectriﬁedjet(Ramakrishnaetal.,2005;Uyar &Besenbacher,2008).

3.3. Structuralcharacterizationofzein/CDnanoﬁbers

TheXRDpatternsofelectrospunzeinnanofibersandzein/CD nanofibers are shown in Fig. 4 and the XRD patterns of as-receivedCDswerealsoshownforcomparison.Zeinnanofibershave showntwobroadpeakshavingmaximaat2=8.99◦(9.8 ˚A)andat

2=19.38◦(4.58 ˚A).Itisreportedthatthelargerd-spacingaround 10 ˚Aisassociatedwiththemeandistanceofapproachof neighbor-inghelices(thespacingoftheinter-helixpackingofzeinchains) whereas theshorter d-spacingat around4.5 ˚Ais relatedtothe averagebackbonedistancewithin␣-helixstructureofzein(Yao, Li,Song,Li,&Pu,2009).

CDs(␣-CD,␤-CDand␥-CD)arecrystallinematerialshaving dis-tinctdiffractionpatternsat2=5–30◦(Fig.4).CDsgenerallyhave twotypesofcrystalstructures;in‘cage-type’thecavityofeach CD moleculeis blockedby theadjacentCD molecules whereas theCDmolecules arealignedand stackedontopof eachother inthe‘channel-type’structure.TheXRDofas-receivedCDshave showndiffractionpatternsfor‘cage-type’crystallinestructuresas reportedintheliterature(Harata,1998;Rusaetal.,2002;Saenger etal.,1998).

Some structural changes were observed for the zein/CD nanofibersdependingontheweightpercentagesandtypesofCDs. TheXRDofzein50/␣-CD10nanofibershaveshowntwobroadhalo diffractionpatternscenteredat2=8.94◦(9.9 ˚A)andat2=20.18◦ (4.4 ˚A)whichisverysimilartothezeinnanofibers.Thediffraction peaks for the _␣-CD crystals were absent in this sample indi-cating that ␣-CD molecules were distributed in the zein fiber matrixwithoutforminganyphase separatedcrystalaggregates. Forzein50/␣-CD25andzein50/␣-CD50nanofibers,thedecreaseof thepeakat2=8.99◦suggestedthatthespacingoftheinter-helix packingofzeinchainswasdisturbedandthezeinmolecular aggre-gatesweresomehowdestroyedwiththepresenceof␣-CDathigher weightpercentages.Moreover,slightly intensediffractionpeaks wereobservedforzein50/␣-CD25nanofiberssuggestingthatsome aggregationof␣-CDcrystalswaspresentinthissample.The␣-CD crystallinepeaksweremuchmorepronouncedforthezein50/ ␣-CD50sample.ThisfindingcorrelateswiththeSEMimageswhere thebead-likestructuresforzein50/␣-CD25andirregularstructures forzein50/␣-CD50wereobservedfor thesesamplesdue tothe presenceofsome␣-CDaggregates.TheXRDpatternscorrespond tochannel-typepackingof␣-CDsincethesalientdiffractionpeak 2_∼_{= 20}◦ischaracteristicforthe_␣-CDchannel-type(Harata,1998; Rusaetal.,2002;Saengeretal.,1998).Ingeneral,the ‘channel-type’packingofCDisassociatedwiththeinclusioncomplexstate. For instance, Tonelliet al. reportedthat protein based polymer suchasBombyxmorisilkfibroincanformaninclusioncomplex with␥-CD(Cristian,Bridges,Ha,&Tonelli,2005).However,here wedidnotanticipatetheinclusioncomplexationofzeinwith ␣-CD due tothe small size cavity of ␣-CD. As mentioned in the experimentalpart,thezein50/␣-CD25and zein50/␣-CD50 solu-tionswereturbidanditismostlikelythat␣-CDprecipitatedas ‘channel-type’crystalsinthezein/DMFsolutionsystem.Wehave alsoobservedsimilarsituationsforelectrospunPScontaining ␣-CDwherethe␣-CDprecipitatedas‘channel-type’crystalswithout forminginclusioncomplexation(Uyar,Havelund,Hacaloglu,etal., 2009).Additionally,theXRDofzein50/␤-CD50andzein50/␥-CD50 havealsoshown somediffractionpeaksdue tothepresenceof someCDcrystallineaggregatesbutthesediffractionsdidnot corre-spondtochannel-typepackingsuggestingthatCDswerenotinthe complexstatewithzeinchainsinzein50/CD50nanofibers.InXRD, thetypicalchannel-type␤-CDhastwomajorpeaksat2 ∼= 11.5◦

and 18◦ (Harada,Okada, Li,&Kamachi, 1995), and the charac-teristicdiffractionforchannel-type␥-CDhasonemajorpeakat 2 ∼= 7.5◦withminorreﬂectionsat2 ∼= 14◦,15◦,16◦,16.8◦and22◦ (Uyar,Hunt,Gracz,&Tonelli,2006).However,forzein50/␤-CD50 andzein50/␥-CD50nanoﬁbers,thediffractionpeaksweredifferent thanthechannel-typepackingasdiscussedbelow.

InXRD,itwasobservedthatthediffractionpatternsofzein50/ ␤-CD10 and zein50/␤-CD25 nanofiberswere very similar to that of purezeinnanofibers (Fig.4b).In thecase of zein50/␤-CD50 nanofibers, the intensity of first peak at around 2=9.0◦ was

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loweredsignificantlyindicatingthattheinter-helixpackingofzein molecularaggregates were substantially disturbed. In addition, veryweakdiffractionpeaksataround2=6.9◦,13.6◦ and24.4◦ wereobservedforthissamplepossiblybecauseofthepresenceof some␤-CDaggregatesinthefibermatrix.However,thesepeaksdid notcorrespondtoeithercage-typepackingorchannel-type pack-ingindicatingthattheregularpackingof␤-CDwasdisturbedbythe zeinchains.Forzein50/␤-CD10andzein50/␤-CD25nanofibers,no crystallinepeakswereobservedsuggestingthat_␤-CDmolecules weredistributedinthefibermatrixwithoutforminganycrystal aggregates.

Moreovertheincorporationof␥-CDintozeinfibermatrixhasa verysimilarstructuraleffectasseenforzein/␣-CDandzein/␤-CD. TheXRDpatternofzein50/␥-CD10nanofiberswassimilartopure zeinnanofibersshowingtwodistinctbroadhaloataround2=9◦ and2=20◦(Fig.4c).Theincorporationof25%and50%(w/w) ␥-CDtofibermatrixresultedindisruptionofinter-helixpackingof zeinchainsasdeducedfromtheXRDpatternsofzein50/␥-CD25 andzein50/␥-CD50sincethepeakataround2=9◦ was consid-erablyweakened.Inaddition,certaindiffractionpeaksataround 2=6◦,8.4◦ and17.4◦ wereobservedfor zein50/␥-CD50sample suggestingthatsomecrystalline_␥-CDaggregateswerepresentin thissampleasobservedintheSEMimageofthissample.Yet,these peakssomewhatdifferentthanthecage-typepackingor channel-typepackingsuggestingthat␥-CDpackingwasdisturbedbythe zeinchainswhichwassimilarlyobservedforthezein50/␤-CD50.

Inbrief,itwasobservedthattheshorterd-spacingca4.5 ˚A corre-latedtotheaveragebackbonedistancein␣-helixstructureofzein didnotchangewiththeadditionofCDs,whiletheintensityofthe largerd-spacingaround9 ˚Aassociatedwiththemeandistanceof approachofneighboringhelicesdecreasedsignificantlyasthe con-tentofCDincreasedfrom10%to50%(w/w)inzein/CDnanofibers. Thisresultimpliedthestructuralchangesdependingonthe side-chainpackingwereobservedforzein withtheadditionofCDs. Moreover,XRDdatasuggestedthatCDsweremostlydistributed inthezeinfibermatrixwithoutformingcrystallineaggregatesat lowerweightpercentages(10%of␣-CD,and10%and25%of ␤-CDand␥-CD),but,incorporationof25%␣-CDand50%ofallthree typesofCDsyieldedsomecrystallineCDaggregatesinthezeinfiber matrix.

3.4. Surfacecharacterizationofzein/CDnanoﬁbers

The surface analyses of zein/CD nanofiberswere performed byusingsurfacesensitivetechniques,ATR-FTIRandXPSinorder to corroborate the presence of CDs on the surface of the zein nanofibers.TheATR-FTIRspectraoftheelectrospunzeinnanofibers andzein/CDnanofibersareshowninFig.5a.Zeinprotein back-bonehastwocharacteristicvibrationalbands;amideIandamide II.ThecharacteristicabsorptionbandofamideIcorrespondstothe C Ostretching,whilethatofamideIIcorrespondsN Hbending andC Nstretching(Fernandezetal.,2009;Yaoetal.,2009b).The characteristicabsorptionbandsat1653and1540cm−1indicated thepresenceofamideIandamideII,respectivelyforpurezein nanofibers(Fig.5a1–a3).

ThecharacteristicpeakofcoupledC–C/C–Ostretching vibra-tionsand the antisymmetric stretchingvibration of the C–O–C glycosidicbridgeofCDwereobservedforzein/CDnanofibersat 1028,1080and1150cm−1,respectively(Uyar,Balan,etal.,2009). ItwasalsoclearthattheintensityofCDrelatedpeakswasincreased astheCDcontentincreasedfrom10%to50%(w/w)inthezein/CD nanofibers.TheATR-FTIRdataconfirmedthesuccessful incorpora-tionofCDsinthezeinnanofibersandsomeCDswerepresenton thesurfaceofthezein/CDnanofibers.

InATR-FTIRstudy,itwasobservedthattheamideIandamide IIpeakswereslightlyshiftedtolowerwavenumberforzein/CD

Fig.5.ATR-FTIRspectraofelectrospunnanoﬁbersof(a1)(i)zein50,(ii) zein50/␣-CD10,(iii)zein50/␣-CD25and(iv)zein50/␣-CD50;(a2)(i)zein50,(ii) zein50/␤-CD10,(iii)zein50/␤-CD25and(iv)zein50/␤-CD50;(a3)(i)zein50,(ii) zein50/␥-CD10,(iii)zein50/␥-CD25and(iv)zein50/␥-CD50and(b)overlayofXPSC1sspectra ofthezeinandzein/␤-CDnanoﬁbersandpure␤-CDpowder.

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Fig.6.(a1)DSCthermograms;(a2)enlargedregionofDSCthermogramsbetween140and190◦_C_of_electrospun_nanoﬁbers_of_(i)_zein50,_(ii)_{zein50/␤-CD10,}_(iii)_{zein50/␤-CD25}

and(iv)zein50/␤-CD50and(b)TGAthermogramsofzein50andzein50/␤-CDnanoﬁbersandpure␤-CDpowder.

nanofiberswhencomparedtopurezeinnanofibers.Forinstance, theamideIpeakwasobservedat1651,1650and1648cm−1 for zein50/␣-CD10,zein50/␣-CD25andzein50/␣-CD50,respectively. Similarly,theamideIIpeakwasshiftedtolowerwavenumberas theweightpercentageofthe␣-CDwasincreasedfrom10%to50%. Thatis,absorptionpeakofamideIIwasobservedat1536,1535 and1520cm−1forzein50/␣-CD10,zein50/␣-CD25andzein50/ ␣-CD50,respectively.ThepeakshiftofamideIandamideIItolower wavenumbersfor zein/CDnanofiberssuggested thepresence of interactionbetweenzeinand␣-CD,andtheinteractionbecame morepronouncedfornanofibersampleshavinghigherloadingof ␣-CD.Inthecaseofzein/␤-CDandzein/␥-CDnanofibersamples, theshiftintheamideIwasnotsignificantbuttheamideIIpeakwas shiftedtoaround1535cm−1suggestingthepresenceofinteraction betweenCDmoleculesandzeinchainsforthesesamplesaswell. But,thepeakshiftforamideIandamideIIwasmuchmore signif-icantinthecaseofzein/␣-CDnanofiberscomparedtozein/␤-CD andzein/␥-CDnanofiberspossiblebecause␣-CDhassmallersize whichcaninteractmorewiththezeinchains.

AsobservedintheSEMimaging,theuniformnanofiberswere obtainedinthecaseof␤-CD,therefore,moredetailedsurface anal-ysesandthermalcharacterizationswerecarriedoutforzein/␤-CD nanofibers.Thein-depthsurface chemistryanalysesfor zein/ ␤-CDnanofiberswereperformedbyXPSinordertodetermineto whatextent␤-CDmoleculesarepresentonthesurfaceofthezein nanofibers.Table2showselementarycompositionsbasedonwide energysurvey spectraof the␤-CD,zeinnanofibersand zein/ ␤-CDnanofibers.Oxygencontentoutersurfaceofthesampleswas increasedwiththeincreasingtheamountof␤-CD(from10%to

50%,w/w)usedintheelectrospinningofnanofibers.Highenergy resolutionC1sspectrawerealsorecordedtogetmoredetailed informationaboutsurfacechemistryofthezein/␤-CDnanofibers. Theoverlay of normalized C 1s spectraof thezein nanofibers, zein/␤-CDnanofibersand␤-CDaregiveninFig.5b.Therearethree differentcomponentsforalloftheC1shigh-resolutionspectra.The positionofoneisataround284.5eV,C1,isassignedtoaliphatic car-bons,C–Cand/orC–H(Shietal.,2009;Uyar,Havelund,Hacaloglu, etal.,2010,2009;Uyar,Havelund,Nur,et al.,2010,2009).It is aprominentpeakforpurezeinnanofibers.ThecomponentC2at about285.7eVisarisenfromeitherC–O–CorC–OH;andC3 (cor-relatedtoO–C–O)locatedataround287.7werefoundinbothzein nanofibersand␤-CD(Shietal.,2009;Uyar,Havelund,Hacaloglu, etal.,2010;Uyar,Havelund,Nur,etal.,2010,2009).Thepeaks aremore distinctivefor _␤-CD,therefore therelative concentra-tionsofC2andC3increasedwithincreasingamountofthe␤-CD usedinthepreparationofzein/␤-CDnanofibers.Itisfoundthatthe increaseinoxygencontentisduetothepresenceofC–O–C/C–OH and/orO–C–Oonthesurfaceofzeinnanofibers.Thepresenceof

Table2

AtomicconcentrationsgeneratedfromXPSwideenergysurveyscans.

Samples C(%) O(%) N(%) ␤-CD 36.61 63.39 – zein50 78.19 13.04 8.77 zein50/␤-CD10 77.06 15.14 7.8 zein50/␤-CD25 75.43 16.25 8.32 zein50/␤-CD50 72.62 18.64 8.74

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the␤-CDonthesurfaceofthezein/␤-CDnanoﬁbersisconﬁrmed

withthese results. On the otherhand, the␤-CD concentration

oftheprobedvolumeiscalculatedasapproximately4%,7%and

13%forzein/␤-CD10,zein/␤-CD25andzein/␤-CD50,respectively

fromtheelementary compositionsin Table2. Thesurface

con-tentofCDforallthreezein/␤-CDsamplesislowerthantheCD contentofthesolutionstheywerepreparedfrom.Thisindicates thatthesomeof theCDmoleculeslocatedonthefibersurface whereassomeCD moleculesareburied inthebulkof thefiber matrix.Zeinisausefulfoodpackagingmaterial(Alkanetal.,2011; Shietal.,2009),andCDshaveinclusioncomplexationcapability withvarietyofmoleculesincludingaromas,colors,antioxidants, antibacterials,odors,andotherfunctionalingredients(DelValle, 2004;Hedges,1998;Szejtli,1998),therefore,zein/CDnanofibers mayhavethepotentialstobeusedasactivefoodpackaging( López-de-Dicastillo,Gallur, Catalá,Gavara,&Hernandez-Mu ñoz,2010; López-de-Dicastillo,Jordá-Beneyto,Catalá,Gavara,& Hernandez-Mu ñoz,2011)materialsowingtosurfaceassociatedCDmolecules andtheirhighsurfaceareas.

3.5. Thermalcharacterizationofzein/CDnanoﬁbers

Thethermalcharacteristicsofthezein/␤-CDnanofiberswere studiedbyDSCandTGA.Fig.6adisplaystheDSCthermograms ofzeinnanofibersandzein/␤-CDnanofiberscontaining10%,25% and50%(w/w)␤-CD.Zeinnanofibersandzein/␤-CDnanofibers haveshownabroadendothermicpeakhavingapeakmaximumat around100◦CintheDSCthermogramindicatingthatthesamples containsomeamountofwater.Theglasstransitiontemperature (Tg)ofthesampleswasalsodetectedfromtheDSCthermograms.

TheTgofthepurezeinnanoﬁberswasobservedataround154◦C

which is in closeagreement withtheTg value reportedin the

literatureforzein(Torres-Giner,Gimenez,&Lagaron,2008; Torres-Giner,Ocio,&Lagaron, 2009).TheTg values forzein50/␤-CD10,

zein50/␤-CD25andzein50/␤-CD50nanoﬁberswereobservedat around158◦C,165◦C and172◦C,respectively. Itwasclearthat thehigherTgvalueswereobservedastheweightloadingsof

␤-CD wereincreasedfrom10% to50%.Theaddition ofCD inthe zeinnanoﬁberscausedanincreaseintheTgvalueswhichis

pos-sibly due tothe less chain mobility of zein in the presence of CD.

Fig. 6b shows the TGA thermograms of pure _␤-CD, zein nanofibersandzein/␤-CDnanofibers.TheTGAofpure␤-CDhas aninitialweightloss(∼12%)below100◦Candmajorweightloss between325and350◦Cowingtowaterlossandmain degrada-tion of ␤-CD, respectively (Anitha et al., 2012).Similar tozein nanofibers,waterlossforzein/␤-CDnanofiberswerealsoobserved butthewaterweightpercentagewasaround3–5%(w/w)indicating thatlessamountofwaterwaspresentinthenanofibers.In addi-tion,anotherminorweightlossregimebetween125and200◦C wasobservedforzeinnanofibersandzein/␤-CDnanofibers.This weightlossispossiblyduetothepresenceofremainingsolvent (DMF)inthenanofibersamples.

Themajorweightlossforthezeinnanofiberswasrecordedat around275–350◦Cwhichisconsistentwiththemaindegradation temperaturereportedfortheelectrospunzeinnanofibers( Torres-Giner&Lagaron,2010;Woods,Selling,&Cooke,2009).Sincethe degradationtemperatureforzeinand_␤-CDwasoverlapped,we observedasinglebutbroaderweightlossforzein/␤-CDnanofibers. Moreover,weobservedthatthethermaldegradationofzein/CD nanofiberswasshiftedslightlytohighertemperaturewith increas-ing␤-CDcontent.Hence,zein/␤-CDnanofibershaveshownslightly higherdegradationtemperaturecomparedtopurezeinnanofibers indicatingthattheincorporationoftheCDmoleculesinthezein fibermatrixresultedinhigherthermalstability.

4. Conclusion

Zein/CD nanofibers were obtained from electrospinning of zein/CDsolutions inDMF.Threetypes ofCDs(␣-CD,␤-CD and ␥-CD)usingdifferentweightloadings(10%,25%and 50%,w/w) wereincorporatedinzeinsolutionshavingvariousconcentrations (40%,50%and 60%,w/v),and thesezein/CDsolutionswere suc-cessfullyelectrospun.WefoundthattheadditionofCDinthezein solutions causedanincrease in solutionviscosityandtherefore resultedinimprovementoftheelectrospinnability,andlessbeaded structuresand/orbead-freezein/CDnanofiberswereobtainedat lowerzeinconcentrationswhencomparedtopristinezein solu-tions.Dependingonthezeinconcentration,CDweightpercentage andCDtype,bead-freezein/CDnanofibershavingfiberdiameters in the range of ∼100–400nm were obtained. The morphologi-cal,structural, surfaceand thermalcharacterizationsof zein/CD nanofiberswerestudiedbySEM,XRD,ATR-FTIR,XPS,DSCandTGA. SEMimagingrevealedthatthemorphologiesoftheelectrospun zein/CDnanofibersweresignificantlyaffectedbytheCD weight percentageandCDtypeaddedinzein/CDsolutions.XRDstudy sug-gestedstructuralchangesforzeinchainpackingwherethespacing oftheinter-helixpackingofzeinchainswasdisturbedwiththe additionofCDs,inaddition,itwasfoundthatCDsweremostly dis-tributedinthefibermatrixwithoutformingcrystallineaggregates whenlowerweightpercentagesofCDswereused(10%and25%of ␤-CDand␥-CDand10%of␣-CD),however,incorporationof50% (w/w)ofallthreetypesofCDsand25%of␣-CDyieldedcrystalline aggregatesinthezeinfibermatrix.Thethermalanalysescarriedout byDSCandTGAindicatedtheimprovementofthermalproperties forzein/␤-CDnanofibers,thatis,zein/␤-CDnanofibershaveshown higherglasstransitiontemperatureandhigherdegradation tem-peraturewithincreasing␤-CDcontentwhencomparedtopristine zeinnanofibers.ThesurfaceanalysesbyATR-FTIRandXPSshowed thatsomeCDmoleculeswerepresentonthesurfaceofzein/ ␤-CDnanofibers.Theseelectrospunzein/CDnanofibersmayhavethe potentialtobeusedasactivefoodpackagingmaterialsowingto veryhighsurfaceareaofzeinnanofibersandsurfaceassociatedCD moleculessinceCDmoleculeshaveinclusioncomplexation capa-bilitywithvariousmoleculesand thereforefunctionaladditives suchasantioxidants,flavors,aromas,antibacterialagentscanbe complexedwithCDsorremovalofunpleasantodorsfromthe sur-roundingscanbeachievedbyCDs.

Acknowledgements

StatePlanningOrganization(DPT)ofTurkeyisacknowledged forthesupportofUNAM-InstituteofMaterialsScienceand Nano-technology. Dr. Uyar acknowledges Marie Curie International ReintegrationGrant(IRG) NANOWEB(PIRG06-GA-2009-256428) and The Scientiﬁc &Technological Research Council of Turkey (TUBITAK)(project #111M459)for funding. F. Kayaci thanksto TUBITAK-BIDEBfornationalPhDstudyscholarship.

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