system cellulose hydroxypropyl as drugdelivery nanoﬁbers Sulﬁsoxazole/cyclodextrin inclusion incorporated complex inelectrospun Colloids and Surfaces B: Biointerfaces

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delivery system

Zeynep Aytac

^a,b

, Huseyin Sener Sen

^b

, Engin Durgun

^a,b

, Tamer Uyar

^a,b,∗

aInstituteofMaterialsScience&Nanotechnology,BilkentUniversity,Ankara06800,Turkey

bUNAM-NationalNanotechnologyResearchCenter,BilkentUniversity,Ankara06800,Turkey

a r t i c l e i n f o

Articlehistory:

Received21October2014

Receivedinrevisedform9February2015 Accepted10February2015

Availableonline17February2015

Keywords:

Electrospinning Nanoﬁbers

Hydroxypropylcellulose Cyclodextrin

Sulﬁsoxazole Molecularmodeling

a b s t r a c t

Herein,hydroxypropyl-beta-cyclodextrin(HP␤CD)inclusioncomplex(IC)ofahydrophobicdrug,sulfisoxazole(SFS)wasincorporatedinhydroxypropylcellulose(HPC)nanofibers(HPC/SFS/HP␤CD-IC-NF) viaelectrospinning.SFS/HP␤CD-ICwascharacterizedbyDSCtoinvestigatetheformationofinclusion complexandthestoichiometryofthecomplexwasdeterminedbyJob’splot.Modelingstudieswere alsoperformedonSFS/HP␤CD-ICusingabinitiotechnique.SEMimagesdepictedthedefectfreeuniform fibersandconfirmedtheincorporationofSFS/HP␤CD-ICinnanofibersdidnotalterthefibermorphol- ogy.XRDanalysesshowedamorphousdistributionofSFS/HP␤CD-ICinthefibermat.Releasestudies wereperformedinphosphatebufferedsaline(PBS).TheresultssuggesthigheramountofSFSreleased from HPC/SFS/HP␤CD-IC-NF when comparedtofree SFS containing HPC nanofibers(HPC/SFS-NF).

ThiswasattributedtotheincreasedsolubilityofSFSbyinclusioncomplexation.Sandwichconfigu- rationswerepreparedbyplacingHPC/SFS/HP␤CD-IC-NFbetweenelectrospunPCLnanofibrousmat (PCL-HPC/SFS/HP␤CD-IC-NF).Consequently,PCL-HPC/SFS/HP␤CD-IC-NFexhibitedslowerreleaseofSFS ascomparedwithHPC/SFS/HP␤CD-IC-NF.Thisstudymayprovidemoreefficientfuturestrategiesfor developingdeliverysystemsofhydrophobicdrugs.

1. Introduction

Electrospinningisawellrecognizedandversatiletechniquefor producing nanofibersfrompolymersolutions or polymermelts with the help of a very strong electric field [1]. Electrospun nanofibersandtheirnanofibrousmatsareverygoodcandidatesfor useinbiotechnology,textiles,membranes/filters,scaffolds,com- posites,sensorsduetotheirveryhighsurfaceareatovolumeratio, nanoporousstructureanddesignflexibilityforfurtherfunctional- ization[1,2].Additionally,bygiventhemorphologicalsimilarities betweenelectrospunnanofibersandextracellularmatrix,bioma- terialsforwoundhealing,drugdeliverysystemsandscaffoldsfor tissueengineeringcouldbedevelopedthroughtheelectrospinning [1,2].

Certain hydroxyl groups of cellulose are substituted with anotherfunctionalgroupyieldsitsderivates.Forexample,hydroxypropylcellulose(HPC)isanon-ioniccellulosederivativewhich

∗ Correspondingauthor.Tel.:+903122903571;fax:+903122664365.

E-mailaddresses:tamer@unam.bilkent.edu.tr,tameruyar@gmail.com(T.Uyar).

can be synthesized by substituting with hydroxypropyl ether groups[3].HPCcouldbeusedintheﬁeldofbiomedicalengineer- ingfor drugdeliveryapplications[4,5].Hence,electrospunHPC nanoﬁbersalsocouldbequiteapplicableintheareaofbiotech- nologyduetotheirdistinctivecharacteristicsasmentionedearlier.

However,asfaraswecanascertain,intheliteraturethereareonly fewstudiesrelatedtoelectrospinningofHPC[6–8].Shuklaetal.

haveproducedelectrospunHPCnanofibersfromethanoland2- propanolsolventsystem[6].Inanotherstudy,Francisetal.have obtainedHPCnanofibersinaqueoussolutionusingpolyethylene oxideascarriermatrix [7].InthestudyofPeriasamyetal.two typesofenzymeswereimmobilizedinHPCnanofibers[8].

Cyclodextrins(CDs)areaclassofcyclicoligosaccharides(Fig.1b) withseverald-glucopyranoseslinkedby ␣-1,4-glycosidicbonds [9].ThemostcommonnativeCDshave6,7,or8glucoseunitswhich arecalledas␣-CD,␤-CDand␥-CD,respectively[10].Therearealso chemically modiﬁed CDs like hydroxypropyl-beta-cyclodextrin (HP␤CD)inwhichsomeofthehydroxylgroupsaresubstitutedwith hydroxypropylgroups.CDsarewater-solublemoleculeswithrigid andwelldeﬁnedmolecularstructures.CDstaketheshapeofatrun- catedconeasshowninFig.1c[9].Asoutstandingsupramolecules http://dx.doi.org/10.1016/j.colsurfb.2015.02.019

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Fig.1. Chemicalstructureof(a)sulﬁsoxazole,(b)HP␤CD;schematicrepresentationof(c)HP␤CD,(d)SFS/HP␤CD-IC,(e)electrospinningofnanoﬁbersfromHPC/SFS/HP␤CD-IC solution,(f)thereleaseofSFSfromHPC/SFS/HP␤CD-IC-NF.

withhydrophobiccavity,CDsarecapableofformingnon-covalent complexeswithvariouscompoundsincludingpoorlysolubledrugs, antibiotics,andvolatilecompounds[9,10].Theinclusioncomplex (IC)formation in theCD systems is favored by substitution of thehigh-enthalpywatermoleculeswithintheCDcavity,withan appropriateguestmoleculeoflowpolarity[9].Here,eachguest moleculeisenclosedwithinthehydrophobiccavityoftheCD.CDs ﬁndapplicationsinvariousﬁeldssuchaspharmaceutical,cosmetic, food,chemicalindustriesandagriculture[10].Forexample,infood industrytheycansuppressunpleasantodorsand tastes.Onthe otherhandtheycanimprovethesolubilityandprovideprotection againstlightoroxygenforguestcompounds.FurthermoreCDscan delaythedegradationorevaporationofguestmoleculesthatare highlyvolatile[10].Inthecontextofpharmaceuticalindustry,the solubilityrelatedproblemsofsomedrugsremainamongthemost challengingissuesofthedrugformulation,whereCDscanenhance solubility,dissolutionrate,chemicalstability andbioavailability.

Interestinglycontrolledreleaseofpoorlysolubledrugsbymeans ofCD-IC[9,10] isworth mentioning.In particular,a chemically modifiedCDlikeHP␤CDismoresuitableforthesolubilizationof hydrophobicdrugs,becauseofitsbetteraqueoussolubilitycom- paredtonativeCDs[11].IthasbeenshownthatCDsandCD-ICs canbeusedinfunctionalizationofelectrospunnanofibersfordif- ferentapplicationslikebiomedical[12–17],filtration[18–20]and foodpackaging[21–26].

Thesulfonamidedrugshaveabasicchemicalstructurecompris- ingasulfanilamidegroupandfiveorsix-memberedheterocyclic ring.Sulfisoxazole(SFS)(Fig.1a)is asulfonamidedrugwithan oxazolesubstituent.Itisaweaklyacidicinnaturewithantibiotic activityandpoorlysolubleinwater[27].ThelowsolubilityofSFS preventsitsfastdissolutionand leadstopooravailabilityatthe targetsite.Henceinthisstudy,SFSwasselectedasamodeldrug forreleaseexperiments.Therehavebeenreportsintheliterature regardingCD-ICofSFStoevaluatechemicalstability,thermalsta- bilityandsolubilitybeforeandaftercomplexation[28].Another studydealswiththecharacterizationofICviasolubility,thermal anddissolutionstudies[29].Ontheotherhand,absorptionandflu- orescencespectraofsulphadrugswereanalyzedinthepresence ofCD-ICtodeterminethestoichiometryofsulphadrugsand␤CD [27].

In this study, HPC nanoﬁbers incorporating SFS (HPC/SFS- NF)and ICofSFSwithHP␤CD(Fig.1d)(HPC/SFS/HP␤CD-IC-NF) wereobtainedvia electrospinningfor ourdrugdeliverysystem (Fig.1e).SinceHPCnanoﬁbersarewatersoluble,HPC/SFS-NFand

HPC/SFS/HP␤CD-IC-NF matsweresandwiched betweenelectro- spunpolycaprolactone(PCL)nanofibrousmats (PCL-HPC/SFS-NF and PCL-HPC/SFS/HP␤CD-IC-NF). PCL is a semi crystalline and hydrophobicpolymerthatiscommonlyusedforbiomedicalappli- cationssuchastissue engineeringscaffold [30]wounddressing [31] and drug delivery system [32], thanks to its biocompati- bilityand biodegradability. SFS/HP␤CD-ICwascharacterizedby DSCand continuousvariation technique(Job’s plot)in orderto investigatethestoichiometryoftheIC.Inaddition,inclusioncom- plexationhasbeeninvestigatedbymolecularmodelingusingab initio techniques. HPC/SFS-NF and HPC/SFS/HP␤CD-IC-NF were characterizedbySEM,XRDandUV-Visspectroscopy.Releasestud- ieswereperformedinphosphatebufferedsaline(PBS)(Fig.1f)and quantifiedthroughHPLC.Inaddition,SFS(powder),HPC/SFSfilm, HPC/SFS/HP␤CD-ICfilm;HPC/SFS-NFand HPC/SFS/HP␤CD-IC-NF werealsousedascontrolsamplesforreleasestudies.

2. Materialsandmethods

2.1. Materials

Hydroxypropyl cellulose (HPC, Mw∼300,000g/mol, Scien- tiﬁc Polymer Products), polycaprolactone (PCL, Mn∼70,000–

90000g/mol,SigmaAldrich),sulﬁsoxazole(SFS,min.99%,Sigma Aldrich),hydroxypropyl-beta-cyclodextrin(HP␤CD,averagesub- stitution degree per anhydroglucose unit 0.6, Wacker Chemie AG,Germany),ethanol (99.8%,Sigma Aldrich),dichloromethane (DCM,extrapure, SigmaAldrich), N,N-dimethylformamidepes- tanal(DMF,Riedel),acetonitrile(ACN,chromasol,SigmaAldrich), methanol (extra pure, Sigma Aldrich), potassium phosphate monobasic(RiedeldeHaen),disodiumhydrogenphosphatedodec- ahydrate(RiedeldeHaen),sodiumchloride(SigmaAldrich)were purchasedandusedasreceivedwithoutanyfurtherpuriﬁcation.

Thewaterusedinexperimentswasdistilled–deionizedfroma MilliporeMilli-Qultrapurewatersystem.

2.2. Preparationofsolutions

TwotypesofSFScontainingHPCnanoﬁbers(HPC/SFS-NFand HPC/SFS/HP␤CD-IC-NF) wereprepared by incorporating 9% SFS (w/w,withrespecttopolymer).For producingHPC/SFS/HP␤CD- IC-NF,theamountofSFSwasdeterminedas1:1molarratiowith HP␤CD,and thesame amountof SFSwasusedforHPC/SFS-NF.

In order to prepare HPC/SFS-NF, SFS and 3% (w/v) HPC were

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solution),SFS wasdissolvedinethanol atRT.Then50% HP␤CD (w/w,withrespecttopolymer)wasaddedintothesolutionand stirredovernightatRT.Finally,3%(w/v)HPCwasaddedintothe system,and dissolved for the electrospinning. For comparison, wehavealsoelectrospunHPCinethanol(3%,w/v)withoutSFSor SFS/HP␤CD-IC.Table1summarizesthecompositionsofthesolu- tionsusedfortheelectrospinning ofthenanoﬁbers.Toproduce PCLnanoﬁbers,10%(w/v)PCLwasdissolvedinthebinarysolvent systemcontainingDMF:DCM(v/v)(3:1).

2.3. Electrospinningofnanoﬁbersandpreparationofﬁlms

HPC,HPC/SFS,HPC/SFS/HP␤CD-ICandPCLsolutionswereindi- viduallyloadedintoa3mLplasticsyringewithneedlediameter of0.8mmplacedhorizontallyonthesyringepump(KDScientific, KDS101).Theflowratesofthepolymersolutionswerecontrolled bysyringepumptoensurehomogeneousflowandfixedat1mL/h.

Thecylindricalmetalcollectorwasplacedatadistanceof11cm fromtheneedletipandcoveredbyaluminumfoil.Thegroundand thepositiveelectrodesofthehighvoltagepowersupply(AUSeries, MatsusadaPrecisionInc.)wereclampedtothecollectorandthe needlerespectively.Theelectrospinningapparatuswasenclosed inaPlexiglasbox,andelectrospinningwascarriedoutat16kV, 22^◦Cand20%relativehumidity.

SFScontainingHPCfilms(HPC/SFSfilmandHPC/SFS/HP␤CD- ICfilm)werepreparedbysolutioncastingmethodusingthesame amountsofSFS,HPCandHP␤CD.Notethattheearlierdescribed procedurewasemployedtopreparethevarioussolutionsforcast- ing.

2.4. Characterizationandmeasurements

InordertoinvestigatethethermalpropertiesofSFS-HP␤CD-IC, powderofSFS-HP␤CD-ICwasobtainedbyevaporatingthesolvent.

Inaddition,physicalmixtureofSFSandHP␤CD(SFS-HP␤CD-PM) wasalsoprepared ascontrol.ThepowdersofSFS,HP␤CD,SFS- HP␤CD-PM and SFS-HP␤CD-IC were analyzed with differential scanningcalorimetry(DSC)(Netzsch,DSC204FI).ForDSCmeasure- ment,SFS,HP␤CD,SFS-HP␤CD-PMwerepreparedinanaluminum pan,heldisothermallyat25^◦Cfor3minandheatedfrom25^◦Cto 250^◦Catarateof20^◦C/minundernitrogenpurge.SFS-HP␤CD-IC wassubjectedtoheatingand coolingcyclesconsistingof:hold- ingisothermallyat25^◦Cfor3min,rampingfrom25^◦Cto250^◦C at20^◦C/min,coolingatarateof20^◦C/mindownto25^◦C.Itwas subjectedtoasecondcycletoinvestigatethechangeinthermal behaviorfollowingtheﬁrstheatatarateof20^◦C/min.

The stoichiometryof IC was investigated by thecontinuous variationtechnique(Job’splot)[33].Equimolar(10⁻⁴M)solutions ofSFSandHP␤CDpreparedinethanolweremixedtoastandard volumevaryingthemolarratio(r,[SFS]/[SFS]+[HP␤CD])from0to 1whilekeepingthetotalconcentrationofeachsolutionconstant.

Afterstirring thesolutions for1h atRT,theabsorbance ofthe

theviscosityofHPC,HPC/SFS,HPC/SFS/HP␤CD-ICandPCLsolutions atRT.Therheometerwasequippedwitha cone/plateaccessory (spindletypeCP40-2)ataconstantshearrateof100sec⁻¹.Thecon- ductivityofsolutionwasdeterminedbyInolab^®Multi720-WTW atRT.

Scanningelectronmicroscopy(SEM,FEI–Quanta200FEG)was usedtoexaminethemorphologiesoftheelectrospunnanoﬁbers.

Thesamplesweremountedonmetalstubsusingadouble-sided adhesivetapeandcoatedwithAu/Pdlayer(∼6nm)(PECS-682)to minimizethecharging.Nearly100ﬁberswereanalyzedtocalculate averageﬁberdiameter(AFD).

Thecrystallinestructureofthematerialswasexaminedwith X-ray diffraction (XRD). XRD data for the powder of SFS and HP␤CD;HPCNF,HPC/SFS-NFandHPC/SFS-HP␤CD-ICNFmatswere recordedusingaPANalyticalX’Pertpowderdiffractometerapply- ingCuK␣radiationinthe2rangeof5–30^◦.

Since HPC is water-soluble,thesandwich configuration was prepared by placing the HPC/SFS/HP␤CD-IC-NF between PCL nanofibrousmatforachievingthecontrollablereleaserate(PCL- HPC/SFS/HP␤CD-IC-NF). The facile pressure is given by thumb forcepsatopenfaceofthemembranetowrapthestructure.The sameprocedurewasappliedtopreparesandwichconfiguration of HPC/SFS-NF (PCL-HPC/SFS-NF).Ascontrol, therelease of SFS (powder),HPC/SFSfilm,HPC/SFS/HP␤CD-ICfilm,HPC/SFS-NFand HPC/SFS/HP␤CD-IC-NFwerealsotested.Atotalimmersionmethod was used tostudy the cumulative release profiles of SFS from thecomposite matsandcontrolsampleswherethethicknessof composite mats is ∼400␮m (determined byZeiss AxioImager A2mopticalmicroscope).Inthistechnique,eachofthecomposite matsincluding24mgHPC/SFS-NFor 37mgHPC/SFS/HP␤CD-IC- NFand30mgPCLnanofibersandthecontrolsampleswithsame amountof SFS wereimmersed in 30mLof phosphate buffered saline(PBS)mediumat37^◦Cat 50rpm.Atpredeterminedtime intervalsbetween0and720min,0.5mLofthetestmediumwas withdrawnandanequalamountofthefreshmediumwasrefilled.

ThereleasedamountofSFSwasdeterminedbyhighperformance liquidchromatography(HPLC,Agilient,1200series)equippedwith VWD UVdetector.The columnwas2.1mm×50mm, contained 3␮mpacking(ACE, C8)and thedetection wasaccomplishedat 270nm.Mobilephase,ﬂowrate,injectionvolumeandthetotalrun timewere100%ACN,0.1mL/min,20␮Land4min,respectively.The calibrationsampleswerepreparedinethanol.Accordingtopre- determinedcalibrationcurveforSFS,thedatawerecalculatedto determinethecumulativeamountofSFSreleasedfromthesamples foreachspeciﬁedimmersionperiod.Theexperimentswerecarried outintriplicateandtheresultswerereportedasaverage±standard deviation.

Inordertodeterminetheactualloading(%)ofSFSinnanoﬁbers, aknownweightofthesamplewasdissolvedinethanolandthe amountof SFS inthe sample wasmeasured byHPLC in tripli- cate.Accordingtocalibrationcurvepreparedinethanol,thetotal amountofSFSinthesampleandactualloading(%)wascalculated.

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Fig.2. DSCthermogramsofﬁrstheatingscan ofSFS,HP␤CD,SFS/HP␤CD-PM, SFS/HP␤CD-ICandsecondheatingscanofSFS/HP␤CD-IC.

Theexperimentswerecarriedoutintriplicateandtheresultswere reportedasaverage±standarddeviation.

Todeterminethesolubilityof SFSineach ﬁber,certaincon- centrationofSFS(powder) (0.094mM)andthesameamountof SFScontainingHPC/SFS/HP␤CD-IC-NFweredissolvedinwater.For comparison,HPC/SFS-NFandHPC/SFS/HP␤CD-IC-NFwerescaledto thesameweight.Lastly,UVabsorbanceofSFSinthesampleswas measuredat270nmviaUV–Vis–NIRspectroscopy(Varian,Cary 5000).

2.5. Computationalmethod

Thestructures of SFS, pristine ␤-CD [34], HP␤CD, and their ICswereoptimizedbyusingabinitiomethodsbasedondensity functionaltheory(DFT)[35,36]implementedintheViennaAbini- tiosimulationpackage[37,38].Theinitialgeometryof␤-CDwas obtainedfromCambridgeStructuralDatabase[39].HP␤CDiscon- structedmanuallybyaddingfour2-hydroxypropylgroupsonthe primarygroupsof ␤-CDcorrespondingtoa substitutiondegree peranhydroglucoseunit of0.6 which wascompatiblewiththe experiments[40].The exchange-correlation wastreated within Perdew-Burke-Ernzerhofparametrizationofthegeneralizedgradi- entapproximation(GGA-PBE)[41]withinclusionofVanderWaals correction[42].Theelementpotentialsweredescribedbyprojector augmented-wavemethod(PAW)[43]usingaplane-wavebasisset withakineticenergycutoffof400eV.TheBrillouinzoneintegration wasperformedatthe-point.Allstructureswereconsideredas isolatedmoleculesinvacuumandwererelaxedusingtheblocked- Davidsonalgorithmwithsimultaneousminimizationofthetotal energyandinteratomicforces.Theconvergenceonthetotalenergy andforcewastestedandthensetto10⁻⁵eVand10⁻²eV/Å,respectively.

3. Resultsanddiscussion

3.1. ThermalpropertiesofSFS/HPˇCD-IC

DSCthermogramsofthepowderofSFS,HP␤CD,SFS-HP␤CD-PM andSFS-HP␤CD-ICaredisplayedinFig.2.TheDSCcurveofpure SFSexhibitedasharpendothermicpeakat203^◦C(H=115.4J/g) corresponding to the melting point of SFS [29]. The thermal transitionofHP␤CDrangedfrom40^◦Cto170^◦Ccorrespondingto dehydrationofHP␤CD-ringcavity,andappearsasanendothermic transitionwithanenthalpyof184.1J/g.Theendothermictransition ofSFSwasretainedinSFS-HP␤CD-PM(between170^◦Cand205^◦C), andabroadendothermictransitioncorrespondingtodehydration ofHP␤CDappearedintherangeof40^◦Cto160^◦C.Theenthalpies

Fig.3. Continuousvariationplot(Job’splot)forthecomplexationofSFSwithHP␤CD obtainedfromabsorptionmeasurements.

oftheabove-mentionedtransitionswere12.97J/gand 169.2J/g, respectively. These transitions observed in DSC thermogram of SFS-HP␤CD-PM might be attributed to the presence of less or nointeractionbetweenSFSand HP␤CD.Whencrystallineguest molecules formIC with CDs,the melting point either shifts or disappearsintheDSCthermogram[29].Notably,inDSCthermo- gramofSFS/HP␤CD-ICthemeltingofSFSisnotobserved,however endothermictransitions between 40–80^◦C and 80–190^◦C were observedduringtheﬁrstheatingscan.Thedisappearanceofan endothermicpeak of a guestmolecule can beattributed toan amorphousstate and/ortoaninclusion complexationin which guest molecules being completely included into the cavity of CD by replacing water molecules. Moreover,no transition was observedinthesecondheatingscanofSFS/HP␤CD-IC.Intheﬁrst heatingscanofSFS/HP␤CD-IC,theenthalpiesoftheendothermic transitionswere1.91J/gand58.78J/g,respectively.Therefore,the enthalpiesoftransitionsofSFS/HP␤CD-ICweremuchsmallerthan thoseofSFS/HP␤CD-PM.Theenthalpyreductionalsoshowsthe interactionbetweenSFSandCDinSFS/HP␤CD-IC[44].

3.2. Stoichiometrydeterminationbythecontinuousvariation method(Job’splot)

Thecontinuousvariationmethod(Job’splot)wasusedtodeter- minethestoichiometryofSFS/HP␤CD-IC.Accordingtothismethod, maximumpointofthemolarratio(r)correspondstothecomplexa- tionstoichiometry.TheplotinFig.3showsthemaximumatamolar ratioofabout0.5,indicatingthatthecomplexeswereformedwith 1:1stoichiometry.

3.3. MolecularmodelingofSFS/HPˇCD-IC

ThestabilityofCD-ICswhenSFSincludedinHP␤CDisexam- ined by ﬁrst-principles modeling techniques. Firstly, HP␤CD is manually built by adding four 2-hydroxypropyl groups onthe primary groups of pristine ␤-CD corresponding toa degree of substitutionof0.6.Variousdecorationpatternsofsubstituentsare considered(e.g.1-2-3-4,1-2-5-6,1-3-5-7wherenumbersindicate therelative positionofsubstitution glucoses)[40,45].Theopti- mizationresultsdonotindicateanysigniﬁcantenergydifferences onthesubstituentpattern.OntheothersidetheO-Hinteraction betweensubstituents reducestheenergytosomeextent(upto 10kcal/mol), thus hydroxypropyl (HP) arms may prefer to get closerasshowninFig.4b.Accordinglyweconsider1-3-5-7pattern asprototypeandexaminethepossibilityofICformation.Inorder toformacomplex,singleSFSmoleculeisintroducedintoHP␤CD at various positions. In addition, two different orientationsare

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Fig.4.SideandtopviewofoptimizedstructuresofSFS/HP␤CD-ICwhenHParmsare(a)openand(b)close.Gray,red,andyellowspheresrepresentcarbon,oxygen,and sulfuratoms,respectively.O HbondingbetweenHPgroupsareindicatedbydashedlines.(Forinterpretationofthereferencestocolorinthisﬁgurelegend,thereaderis referredtothewebversionofthisarticle.)

considered where NH₂- or CH₃-end points toward cavity. For eachcase thewholesystemisoptimized withoutimposingany constraints.ThelowestenergyconﬁgurationsareshowninFig.4.

Thecomplexationenergy(E_comp)iscalculatedby Ecomp=EHP␤CD+ESFS−EHP␤CD+SFS

whereE_HP␤CD,ESFS,E_HP␤CD₊_SFSisthetotalenergy(includingvander Waalsinteraction)ofHP␤CD,SFS,andSFS/HP␤CD-IC,respectively.

Ecompiscalculatedas35.05and32.49kcal/molforopenandclose HParms,respectively. WhenHP armsgetclosernarrowingthe rim,Ecompslightlyreduces.Ourresultsareinagreementwiththe Job’splotandsupporttheformationofstableICofSFSandHP␤CD with1:1molarratio.

3.4. Morphologyofthenanoﬁbers

Based on our preliminary experiments, ethanol was chosen asasolventfordissolvingboth HPCandSFS. Theconcentration ofHPCwasdeterminedas3%(w/v)inethanoltoyieldbead-free nanofibers.TheamountofSFS(9%,w/w,withrespecttoHPC)was setata1:1ratiowithHP␤CDinSFS/HP␤CD-IC.Fig.5displaysSEM imagesandAFD along withfiberdiameterdistributions ofHPC NF, HPC/SFS-NF and HPC/SFS-HP␤CD-IC NF. By optimizing the electrospinningparameters,wewereabletoobtainbead-freeand uniformnanofibers from HPC, HPC/SFSand HPC/SFS-HP␤CD-IC systems.TheAFDofHPCNFwas125±50nm,whereasAFDofelec- trospunHPC/SFS-NFandHPC/SFS/HP␤CD-IC-NFwere90±40nm and60±25nm,respectively.TheslightdecreaseinAFDcouldbe explainedbydifferencesinconductivityandviscosityofHPC/SFS and HPC/SFS/HP␤CD-IC solutions compared to HPC solution (Table 1). Stable jet formation usually depends on sufficiently high surface charge densities which are influenced by applied voltageandtheelectricalconductivityofthepolymersolution.As

theconductivityofasolutionincreases,thestretchingincreases due to the higher number of charges formed in the solution whichthinsthediameterofthefiber[46].Inaddition,viscosityis importantforthecontinuityofjetduringelectrospinningwhile lowerviscosityleadstolowerdiameternanofibers[47].HPC/SFS andHPC/SFS/HP␤CD-ICsolutionshavemuchhigherconductivity and slightlyhigher viscositythanHPCsolution;thereby AFDof HPC/SFS-NF and HPC/SFS/HP␤CD-IC-NF were lower than HPC nanofibers. Moreover, HPC/SFS/HP␤CD-IC-NF was thinner than HPC/SFS-NFpossiblyduetothehigherconductivityandslightly lowerviscosityoftheparentsolution.

Furthermore,weinvestigatedtheeffectofSFSincorporationin theformoffreeSFSandSFS/HP␤CD-IConnanoﬁbermorphology.

IncorporationofSFSinHPCnanofibersdidnotdeterioratetheshape anduniformity.Moreoverrelativelythinnerfiberswereobtained duetothehigherconductivityofHPC/SFSandHPC/SFS/HP␤CD-IC solutions.Ontheotherhand,neitherdrugcrystalsnoraggregates wereobservedonthesurfacesofnanofibers.Phaseseparationis not likely to occuras SFS is highly soluble in the HPC/ethanol system, while the solvent (ethanol) evaporates rapidly during electrospinning.Forreleaseexperiments,sandwich-likecompos- itematswerepreparedinwhichtheinnerlayerwasHPC/SFS-NF orHPC/SFS/HP␤CD-IC-NFmatandouterlayerswereelectrospun PCLnanofibrousmat.SEMimageandAFD(260±110nm)along with fiberdiameter distributions of bead-free and smooth PCL nanofibersisshowninFig.5d.

3.5. Crystallinestructureofnanoﬁbers

XRD was performed to investigate the crystallinity of the samples. Fig. 6 represents the XRD patterns of the powder of SFS and HP␤CD; HPC NF, HPC/SFS-NF and HPC/SFS-HP␤CD-IC NF mats.XRDpatternofasreceivedSFSindicated thatSFS isa

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Fig.5.SEMimagesandfiberdiameterdistributionswithaveragefiberdiameter(AFD)oftheelectrospunnanofibersobtainedfromsolutionsof(a)HPC,(b)HPC/SFS,(c) HPC/SFS/HP␤CD-ICand(d)PCL.

crystallinematerialwithcharacteristicdiffractionpeaks.TheHPC NF diffraction exhibits a diffused pattern with two diffraction haloswhichshowthatthepolymerisamorphous.InHPC/SFS-NF, thecharacteristicpeaksofSFSwereabsentandonlyahumpofthe amorphousformisnoticed.ThissuggeststhatSFSwasnolongerin crystallinestate;apparentlyitwasconvertedintoanamorphous state. In HPC/SFSethanol solution, SFS is well solubilized and homogeneouslydistributedwithinthepolymersolution,soduring the electrospinning process, drug mobility is restricted due to therapidevaporationofthesolventandtheSFSmoleculescould notformcrystallineaggregates.Theabsenceofanyintenseand sharppeakinthepatternofHP␤CDrevealsthatitpossessesan amorphous structure. In the XRD pattern of HPC/SFS/HP␤CD- IC-NF, the crystalline diffraction peak of SFS was also absent.

That means SFS was in amorphous state in the HPC/SFS/HP␤ CD-IC-NF[48].

3.6. Invitroreleasestudy

Thecumulativerelease(%)ofSFSfromdifferentsamplesisgiven inFig.7asafunctionofimmersiontime.Allsamplesdepictedsus- tainedreleasebehaviorofSFSoveraperiodof720min(Fig.7a).

SFS followeda typicaldual-stagerelease profilefromallofthe samples,aninitialrelativelyfastreleasefollowedbya constant release.HPC/SFS/HP␤CD-IC-NFreleasedmuchmoreamountofSFS intotalthanSFS(powder)andHPC/SFS/HP␤CD-ICfilm.Thiscould beduetothehighsurfaceareaofHPC/SFS/HP␤CD-IC-NFcompared toSFS(powder)andHPC/SFS/HP␤CD-ICfilm.Theactualloading (%)ofSFSinHPC/SFS-NFandHPC/SFS/HP␤CD-IC-NFweredeter- minedas76±1%and73±1%,respectively.However,totalamount ofreleasedSFSwasmuchhigherfromHPC/SFS/HP␤CD-IC-NFthan HPC/SFS-NF.Possiblereasonforthiscouldbethehighersurface areaofHPC/SFS/HP␤CD-IC-NFduetothelowerfiberdiameteras

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Fig.6. XRDpatternsofSFS,HP␤CD,HPCNFandHPCnanoﬁberscontainingSFS.

Fig. 7.The cumulative release of SFS from SFS (powder), HPC/SFS film, HPC/SFS/HP␤CD-ICfilm,HPC/SFS-NFwithoutPCL,HPC/SFS/HP␤CD-IC-NFwithout PCL,PCL-HPC/SFS-NFandPCL-HPC/SFS/HP␤CD-IC-NFfor(a)720min,(b)120min (n=3,theerrorbarsinthefigurerepresentthestandarddeviation(SD)),and(c)the solubilityofSFSaspowder,inHPC/SFS-NFandHPC/SFS/HP␤CD-IC-NFinaqueous solution.

mat can facilitate the dissolution of the drug in the medium and promote a rapid drugrelease [49]. In order toextend the releasetimeofSFSfromHPC/SFS-NFandHPC/SFS/HP␤CD-IC-NF, wehavepreparedPCL-HPC/SFS-NFand PCL-HPC/SFS/HP␤CD-IC- NF. SFS release from PCL-HPC/SFS-NFwas slowercompared to HPC/SFS-NFwithoutPCLandPCL-HPC/SFS/HP␤CD-IC-NFreleased SFSslowerthanHPC/SFS/HP␤CD-IC-NFwithoutPCLbecauseofthe presenceofanextralayerforreleaseofSFS.Asaconsequence,PCL- HPC/SFS/HP␤CD-IC-NFexhibitedarapidreleaseattheinitialstage andthensustainedrelease attheﬁnalstage.Thismightbecru- cialformanyapplicationsthatarerelatedwiththepreventionof bacteriaproliferation.Bringingtothecontext,higherreleaseatthe initialstageisofgreatimportancewhichlimitstheproliferationof bacteriainthebeginningwhilesustainedreleaseinhibitsthefew bacteriawhichmanagedtoproliferate[50].

HPC/SFS/HP␤CD-IC-NFreleasedhigheramountofSFSintheini- tialstageand ineach time period givenonthegraph;and the maximumamountofreleasedSFSwasalmost14%higherascom- paredtoHPC/SFS-NF.Aswementionedbefore,thissituationmight berelatedwiththeexistenceofHP␤CDinthematrix[51].CDs haveabilitytoenhancedrugreleasefrompolymericsystems,since theyincrease theconcentrationof diffusiblespecieswithin the matrix[52].WhenahydrophobicdrugmakescomplexwithCD itssolubilityincreasesconsiderablywhereCD-ICisusuallymore hydrophilicthanthefreedrug.ThenanofiberscontainingCD-IC weteasierandthestructuredisintegratesdissolvingthesubstance quickly [51,53]. The enhancement in thesolubility of SFS with HPC/SFS/HP␤CD-IC-NFwasverifiedbysolubilitytest.TheUV–Vis spectroscopyofSFS(powder),HPC/SFS-NFandHPC/SFS/HP␤CD- IC-NFdissolvedinwaterisshowninFig.7c.HPC/SFS-NFallowsthe SFSdrugtobedispersedinthemediumwhichfacilitatesitsdisso- lution.AlthoughsameamountofSFSwasusedforeachsamplethe solubilityofSFShasincreasedmuchmoreinHPC/SFS/HP␤CD-IC- NF.Additionally,thepresenceofCDmaylowertherequireddose ofanactivemoleculebyimprovingitssolubility.Consequently, HPC/SFS/HP␤CD-IC-NFcouldbeusedasanefficientdrugdelivery systemforwounddressingpurpose.

4. Conclusion

The concept of employing electrospunnanoﬁbers as matrix andimprovingthesolubilityofhydrophobiccompoundsbyCD- ICs are well-known approaches those have been investigated.

Therefore, we have anticipated that combination ofdrug/CD-IC andversatileelectrospinningprocesscouldenableHPCnanoﬁbers to be used for the deliveryof a variety of hydrophobic drugs.

Here, SFS wasusedasa model drugtostudyitsrelease kinet- icsfromelectrospunHPCnanoﬁbersincorporatingSFS/HP␤CD-IC.

ThestoichiometryofthecomplexbetweenSFSand HP␤CDwas determinedtobe1:1byJob’splot.Furthermore,modelingstud- ies performedusing ab initiotechniques alsorevealed that the

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stoichiometryoftheSFS/HP␤CD-ICwas1:1(SFS:HP␤CD).There- fore,theresultsofmolecularmodelingareingoodagreementwith theexperimentaldata.SFSreleasefromPCL-HPC/SFS/HP␤CD-IC- NFthatwaspreparedbyplacingHPC/SFS/HP␤CD-IC-NFbetween PCLnanoﬁbrousmatswasslowercomparedtoHPC/SFS/HP␤CD- IC-NFwithoutPCL.Controlledrelease ofSFS wasattainedfrom HPC/SFS-NFandHPC/SFS-HP␤CD-ICNFforaperiodof720min,yet weobservedthathigheramountofSFSwasreleasedfromHPC/SFS- HP␤CD-IC-NFwhencomparedtoHPC/SFS-NF.Thisispossiblydue tothepropertyofCD-ICtoenhancethesolubilityofhydrophobic SFSmoleculesviainclusioncomplexationwithHP␤CDandhigher surfaceareaofHPC/SFS/HP␤CD-IC-NFcomparedtoHPC/SFS-NF.

Asaresult,PCL-HPC/SFS/HP␤CD-IC-NFdepictedslowreleaseand highestamountoftotalSFSrelease.Thisstudycontributestothe existingliteratureforimprovingCD-ICfunctionalizedelectrospun nanoﬁbersthatmightbeusedaswounddressingwithcharacter- isticsofbothCDandelectrospunnanoﬁbersfordeliveryofpoorly solubledrugs.

Acknowledgments

Dr. Uyar acknowledges The Scientiﬁc and Technologi- cal Research Council of Turkey (TUBITAK) -Turkey (Project no. 111M459) and EU FP7-PEOPLE-2009-RG Marie Curie-IRG (NANOWEB,PIRG06-GA-2009-256428)andTheTurkishAcademy ofSciences–OutstandingYoungScientistsAwardProgram(TUBA- GEBIP)-Turkeyforpartialfundingoftheresearch.Z.Aytacthanks to TUBITAK (Project no. 111M459 and 213M185) for the PhD scholarship.

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