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
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Articlehistory:
Received21October2014
Receivedinrevisedform9February2015 Accepted10February2015
Availableonline17February2015 Keywords: Electrospinning Nanofibers Hydroxypropylcellulose Cyclodextrin Sulfisoxazole Molecularmodeling
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Herein,hydroxypropyl-beta-cyclodextrin(HPCD)inclusioncomplex(IC)ofahydrophobicdrug, sul-fisoxazole(SFS)wasincorporatedinhydroxypropylcellulose(HPC)nanofibers(HPC/SFS/HPCD-IC-NF) viaelectrospinning.SFS/HPCD-ICwascharacterizedbyDSCtoinvestigatetheformationofinclusion complexandthestoichiometryofthecomplexwasdeterminedbyJob’splot.Modelingstudieswere alsoperformedonSFS/HPCD-ICusingabinitiotechnique.SEMimagesdepictedthedefectfreeuniform fibersandconfirmedtheincorporationofSFS/HPCD-ICinnanofibersdidnotalterthefiber morphol-ogy.XRDanalysesshowedamorphousdistributionofSFS/HPCD-ICinthefibermat.Releasestudies wereperformedinphosphatebufferedsaline(PBS).TheresultssuggesthigheramountofSFSreleased from HPC/SFS/HPCD-IC-NF when comparedtofree SFS containing HPC nanofibers(HPC/SFS-NF). ThiswasattributedtotheincreasedsolubilityofSFSbyinclusioncomplexation.Sandwich configu-rationswerepreparedbyplacingHPC/SFS/HPCD-IC-NFbetweenelectrospunPCLnanofibrousmat (PCL-HPC/SFS/HPCD-IC-NF).Consequently,PCL-HPC/SFS/HPCD-IC-NFexhibitedslowerreleaseofSFS ascomparedwithHPC/SFS/HPCD-IC-NF.Thisstudymayprovidemoreefficientfuturestrategiesfor developingdeliverysystemsofhydrophobicdrugs.
©2015ElsevierB.V.Allrightsreserved.
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, nanoporousstructureanddesignflexibilityforfurther functional-ization[1,2].Additionally,bygiventhemorphologicalsimilarities betweenelectrospunnanofibersandextracellularmatrix, bioma-terialsforwoundhealing,drugdeliverysystemsandscaffoldsfor tissueengineeringcouldbedevelopedthroughtheelectrospinning [1,2].
Certain hydroxyl groups of cellulose are substituted with anotherfunctionalgroupyieldsitsderivates.Forexample, hydrox-ypropylcellulose(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].HPCcouldbeusedinthefieldofbiomedical engineer-ingfor drugdeliveryapplications[4,5].Hence,electrospunHPC nanofibersalsocouldbequiteapplicableintheareaof biotech-nologyduetotheirdistinctivecharacteristicsasmentionedearlier. However,asfaraswecanascertain,intheliteraturethereareonly fewstudiesrelatedtoelectrospinningofHPC[6–8].Shuklaetal. haveproducedelectrospunHPCnanofibersfromethanoland 2-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 (HPCD)inwhichsomeofthehydroxylgroupsaresubstitutedwith hydroxypropylgroups.CDsarewater-solublemoleculeswithrigid andwelldefinedmolecularstructures.CDstaketheshapeofa trun-catedconeasshowninFig.1c[9].Asoutstandingsupramolecules http://dx.doi.org/10.1016/j.colsurfb.2015.02.019
Fig.1. Chemicalstructureof(a)sulfisoxazole,(b)HPCD;schematicrepresentationof(c)HPCD,(d)SFS/HPCD-IC,(e)electrospinningofnanofibersfromHPC/SFS/HPCD-IC solution,(f)thereleaseofSFSfromHPC/SFS/HPCD-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 modifiedCDlikeHPCDismoresuitableforthesolubilizationof hydrophobicdrugs,becauseofitsbetteraqueoussolubility com-paredtonativeCDs[11].IthasbeenshownthatCDsandCD-ICs canbeusedinfunctionalizationofelectrospunnanofibersfor dif-ferentapplicationslikebiomedical[12–17],filtration[18–20]and foodpackaging[21–26].
Thesulfonamidedrugshaveabasicchemicalstructure compris-ingasulfanilamidegroupandfiveorsix-memberedheterocyclic ring.Sulfisoxazole(SFS)(Fig.1a)is asulfonamidedrugwithan oxazolesubstituent.Itisaweaklyacidicinnaturewithantibiotic activityandpoorlysolubleinwater[27].ThelowsolubilityofSFS preventsitsfastdissolutionand leadstopooravailabilityatthe targetsite.Henceinthisstudy,SFSwasselectedasamodeldrug forreleaseexperiments.Therehavebeenreportsintheliterature regardingCD-ICofSFStoevaluatechemicalstability,thermal sta-bilityandsolubilitybeforeandaftercomplexation[28].Another studydealswiththecharacterizationofICviasolubility,thermal anddissolutionstudies[29].Ontheotherhand,absorptionand flu-orescencespectraofsulphadrugswereanalyzedinthepresence ofCD-ICtodeterminethestoichiometryofsulphadrugsandCD [27].
In this study, HPC nanofibers incorporating SFS (HPC/SFS-NF)and ICofSFSwithHPCD(Fig.1d)(HPC/SFS/HPCD-IC-NF) wereobtainedvia electrospinningfor ourdrugdeliverysystem (Fig.1e).SinceHPCnanofibersarewatersoluble,HPC/SFS-NFand
HPC/SFS/HPCD-IC-NF matsweresandwiched between electro-spunpolycaprolactone(PCL)nanofibrousmats (PCL-HPC/SFS-NF and PCL-HPC/SFS/HPCD-IC-NF). PCL is a semi crystalline and hydrophobicpolymerthatiscommonlyusedforbiomedical appli-cationssuchastissue engineeringscaffold [30]wounddressing [31] and drug delivery system [32], thanks to its biocompati-bilityand biodegradability. SFS/HPCD-ICwascharacterizedby DSCand continuousvariation technique(Job’s plot)in orderto investigatethestoichiometryoftheIC.Inaddition,inclusion com-plexationhasbeeninvestigatedbymolecularmodelingusingab initio techniques. HPC/SFS-NF and HPC/SFS/HPCD-IC-NF were characterizedbySEM,XRDandUV-Visspectroscopy.Release stud-ieswereperformedinphosphatebufferedsaline(PBS)(Fig.1f)and quantifiedthroughHPLC.Inaddition,SFS(powder),HPC/SFSfilm, HPC/SFS/HPCD-ICfilm;HPC/SFS-NFand HPC/SFS/HPCD-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(HPCD,average sub-stitution degree per anhydroglucose unit 0.6, Wacker Chemie AG,Germany),ethanol (99.8%,Sigma Aldrich),dichloromethane (DCM,extrapure, SigmaAldrich), N,N-dimethylformamide pes-tanal(DMF,Riedel),acetonitrile(ACN,chromasol,SigmaAldrich), methanol (extra pure, Sigma Aldrich), potassium phosphate monobasic(RiedeldeHaen),disodiumhydrogenphosphate dodec-ahydrate(RiedeldeHaen),sodiumchloride(SigmaAldrich)were purchasedandusedasreceivedwithoutanyfurtherpurification. Thewaterusedinexperimentswasdistilled–deionizedfroma MilliporeMilli-Qultrapurewatersystem.
2.2. Preparationofsolutions
TwotypesofSFScontainingHPCnanofibers(HPC/SFS-NFand HPC/SFS/HPCD-IC-NF) wereprepared by incorporating 9% SFS (w/w,withrespecttopolymer).For producingHPC/SFS/HP CD-IC-NF,theamountofSFSwasdeterminedas1:1molarratiowith HPCD,and thesame amountof SFSwasusedforHPC/SFS-NF. In order to prepare HPC/SFS-NF, SFS and 3% (w/v) HPC were
solution),SFS wasdissolvedinethanol atRT.Then50% HPCD (w/w,withrespecttopolymer)wasaddedintothesolutionand stirredovernightatRT.Finally,3%(w/v)HPCwasaddedintothe system,and dissolved for the electrospinning. For comparison, wehavealsoelectrospunHPCinethanol(3%,w/v)withoutSFSor SFS/HPCD-IC.Table1summarizesthecompositionsofthe solu-tionsusedfortheelectrospinning ofthenanofibers.Toproduce PCLnanofibers,10%(w/v)PCLwasdissolvedinthebinarysolvent systemcontainingDMF:DCM(v/v)(3:1).
2.3. Electrospinningofnanofibersandpreparationoffilms
HPC,HPC/SFS,HPC/SFS/HPCD-ICandPCLsolutionswere indi-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,HPCandHPCD.Notethattheearlierdescribed procedurewasemployedtopreparethevarioussolutionsfor cast-ing.
2.4. Characterizationandmeasurements
InordertoinvestigatethethermalpropertiesofSFS-HPCD-IC, powderofSFS-HPCD-ICwasobtainedbyevaporatingthesolvent. Inaddition,physicalmixtureofSFSandHPCD(SFS-HPCD-PM) wasalsoprepared ascontrol.ThepowdersofSFS,HPCD, SFS-HPCD-PM and SFS-HPCD-IC were analyzed with differential scanningcalorimetry(DSC)(Netzsch,DSC204FI).ForDSC measure-ment,SFS,HPCD,SFS-HPCD-PMwerepreparedinanaluminum pan,heldisothermallyat25◦Cfor3minandheatedfrom25◦Cto 250◦Catarateof20◦C/minundernitrogenpurge.SFS-HPCD-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 ofSFSandHPCDpreparedinethanolweremixedtoastandard volumevaryingthemolarratio(r,[SFS]/[SFS]+[HPCD])from0to 1whilekeepingthetotalconcentrationofeachsolutionconstant. Afterstirring thesolutions for1h atRT,theabsorbance ofthe
theviscosityofHPC,HPC/SFS,HPC/SFS/HPCD-ICandPCLsolutions atRT.Therheometerwasequippedwitha cone/plateaccessory (spindletypeCP40-2)ataconstantshearrateof100sec−1.The con-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 HPCD;HPCNF,HPC/SFS-NFandHPC/SFS-HPCD-ICNFmatswere recordedusingaPANalyticalX’Pertpowderdiffractometer apply-ingCuK␣radiationinthe2rangeof5–30◦.
Since HPC is water-soluble,thesandwich configuration was prepared by placing the HPC/SFS/HPCD-IC-NF between PCL nanofibrousmatforachievingthecontrollablereleaserate (PCL-HPC/SFS/HPCD-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/HPCD-ICfilm,HPC/SFS-NFand HPC/SFS/HPCD-IC-NFwerealsotested.Atotalimmersionmethod was used tostudy the cumulative release profiles of SFS from thecomposite matsandcontrolsampleswherethethicknessof composite mats is ∼400m (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 3mpacking(ACE, C8)and thedetection wasaccomplishedat 270nm.Mobilephase,flowrate,injectionvolumeandthetotalrun timewere100%ACN,0.1mL/min,20Land4min,respectively.The calibrationsampleswerepreparedinethanol.Accordingto pre-determinedcalibrationcurveforSFS,thedatawerecalculatedto determinethecumulativeamountofSFSreleasedfromthesamples foreachspecifiedimmersionperiod.Theexperimentswerecarried outintriplicateandtheresultswerereportedasaverage±standard deviation.
Inordertodeterminetheactualloading(%)ofSFSinnanofibers, aknownweightofthesamplewasdissolvedinethanolandthe amountof SFS inthe sample wasmeasured byHPLC in tripli-cate.Accordingtocalibrationcurvepreparedinethanol,thetotal amountofSFSinthesampleandactualloading(%)wascalculated.
Fig.2. DSCthermogramsoffirstheatingscan ofSFS,HPCD,SFS/HPCD-PM, SFS/HPCD-ICandsecondheatingscanofSFS/HPCD-IC.
Theexperimentswerecarriedoutintriplicateandtheresultswere reportedasaverage±standarddeviation.
Todeterminethesolubilityof SFSineach fiber,certain con-centrationofSFS(powder) (0.094mM)andthesameamountof SFScontainingHPC/SFS/HPCD-IC-NFweredissolvedinwater.For comparison,HPC/SFS-NFandHPC/SFS/HPCD-IC-NFwerescaledto thesameweight.Lastly,UVabsorbanceofSFSinthesampleswas measuredat270nmviaUV–Vis–NIRspectroscopy(Varian,Cary 5000).
2.5. Computationalmethod
Thestructures of SFS, pristine -CD [34], HPCD, and their ICswereoptimizedbyusingabinitiomethodsbasedondensity functionaltheory(DFT)[35,36]implementedintheViennaAb ini-tiosimulationpackage[37,38].Theinitialgeometryof-CDwas obtainedfromCambridgeStructuralDatabase[39].HPCDis con-structedmanuallybyaddingfour2-hydroxypropylgroupsonthe primarygroupsof -CDcorrespondingtoa substitutiondegree peranhydroglucoseunit of0.6 which wascompatiblewiththe experiments[40].The exchange-correlation wastreated within Perdew-Burke-Ernzerhofparametrizationofthegeneralized gradi-entapproximation(GGA-PBE)[41]withinclusionofVanderWaals correction[42].Theelementpotentialsweredescribedbyprojector augmented-wavemethod(PAW)[43]usingaplane-wavebasisset withakineticenergycutoffof400eV.TheBrillouinzoneintegration wasperformedatthe-point.Allstructureswereconsideredas isolatedmoleculesinvacuumandwererelaxedusingthe blocked-Davidsonalgorithmwithsimultaneousminimizationofthetotal energyandinteratomicforces.Theconvergenceonthetotalenergy andforcewastestedandthensetto10−5eVand10−2eV/Å, respec-tively.
3. Resultsanddiscussion
3.1. ThermalpropertiesofSFS/HPˇCD-IC
DSCthermogramsofthepowderofSFS,HPCD,SFS-HPCD-PM andSFS-HPCD-ICaredisplayedinFig.2.TheDSCcurveofpure SFSexhibitedasharpendothermicpeakat203◦C(H=115.4J/g) corresponding to the melting point of SFS [29]. The thermal transitionofHPCDrangedfrom40◦Cto170◦Ccorrespondingto dehydrationofHPCD-ringcavity,andappearsasanendothermic transitionwithanenthalpyof184.1J/g.Theendothermictransition ofSFSwasretainedinSFS-HPCD-PM(between170◦Cand205◦C), andabroadendothermictransitioncorrespondingtodehydration ofHPCDappearedintherangeof40◦Cto160◦C.Theenthalpies
Fig.3. Continuousvariationplot(Job’splot)forthecomplexationofSFSwithHPCD obtainedfromabsorptionmeasurements.
oftheabove-mentionedtransitionswere12.97J/gand 169.2J/g, respectively. These transitions observed in DSC thermogram of SFS-HPCD-PM might be attributed to the presence of less or nointeractionbetweenSFSand HPCD.Whencrystallineguest molecules formIC with CDs,the melting point either shifts or disappearsintheDSCthermogram[29].Notably,inDSC thermo-gramofSFS/HPCD-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/HPCD-IC.Inthefirst heatingscanofSFS/HPCD-IC,theenthalpiesoftheendothermic transitionswere1.91J/gand58.78J/g,respectively.Therefore,the enthalpiesoftransitionsofSFS/HPCD-ICweremuchsmallerthan thoseofSFS/HPCD-PM.Theenthalpyreductionalsoshowsthe interactionbetweenSFSandCDinSFS/HPCD-IC[44].
3.2. Stoichiometrydeterminationbythecontinuousvariation method(Job’splot)
Thecontinuousvariationmethod(Job’splot)wasusedto deter-minethestoichiometryofSFS/HPCD-IC.Accordingtothismethod, maximumpointofthemolarratio(r)correspondstothe complexa-tionstoichiometry.TheplotinFig.3showsthemaximumatamolar ratioofabout0.5,indicatingthatthecomplexeswereformedwith 1:1stoichiometry.
3.3. MolecularmodelingofSFS/HPˇCD-IC
ThestabilityofCD-ICswhenSFSincludedinHPCDis exam-ined by first-principles modeling techniques. Firstly, HPCD 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].The opti-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,singleSFSmoleculeisintroducedintoHPCD at various positions. In addition, two different orientationsare
Fig.4.SideandtopviewofoptimizedstructuresofSFS/HPCD-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(Ecomp)iscalculatedby
Ecomp=EHPCD+ESFS−EHPCD+SFS
whereEHPCD,ESFS,EHPCD+SFSisthetotalenergy(includingvander
Waalsinteraction)ofHPCD,SFS,andSFS/HPCD-IC,respectively. Ecompiscalculatedas35.05and32.49kcal/molforopenandclose
HParms,respectively. WhenHP armsgetclosernarrowingthe rim,Ecompslightlyreduces.Ourresultsareinagreementwiththe
Job’splotandsupporttheformationofstableICofSFSandHPCD 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 setata1:1ratiowithHPCDinSFS/HPCD-IC.Fig.5displaysSEM imagesandAFD along withfiberdiameterdistributions ofHPC NF, HPC/SFS-NF and HPC/SFS-HPCD-IC NF. By optimizing the electrospinningparameters,wewereabletoobtainbead-freeand uniformnanofibers from HPC, HPC/SFSand HPC/SFS-HPCD-IC systems.TheAFDofHPCNFwas125±50nm,whereasAFDof elec-trospunHPC/SFS-NFandHPC/SFS/HPCD-IC-NFwere90±40nm and60±25nm,respectively.TheslightdecreaseinAFDcouldbe explainedbydifferencesinconductivityandviscosityofHPC/SFS and HPC/SFS/HPCD-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/HPCD-ICsolutionshavemuchhigherconductivity and slightlyhigher viscositythanHPCsolution;thereby AFDof HPC/SFS-NF and HPC/SFS/HPCD-IC-NF were lower than HPC nanofibers. Moreover, HPC/SFS/HPCD-IC-NF was thinner than HPC/SFS-NFpossiblyduetothehigherconductivityandslightly lowerviscosityoftheparentsolution.
Furthermore,weinvestigatedtheeffectofSFSincorporationin theformoffreeSFSandSFS/HPCD-IConnanofibermorphology. IncorporationofSFSinHPCnanofibersdidnotdeterioratetheshape anduniformity.Moreoverrelativelythinnerfiberswereobtained duetothehigherconductivityofHPC/SFSandHPC/SFS/HPCD-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-like compos-itematswerepreparedinwhichtheinnerlayerwasHPC/SFS-NF orHPC/SFS/HPCD-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 HPCD; HPC NF, HPC/SFS-NF and HPC/SFS-HPCD-IC NF mats.XRDpatternofasreceivedSFSindicated thatSFS isa
Fig.5.SEMimagesandfiberdiameterdistributionswithaveragefiberdiameter(AFD)oftheelectrospunnanofibersobtainedfromsolutionsof(a)HPC,(b)HPC/SFS,(c) HPC/SFS/HPCD-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 sharppeakinthepatternofHPCDrevealsthatitpossessesan 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.Allsamplesdepicted sus-tainedreleasebehaviorofSFSoveraperiodof720min(Fig.7a). SFS followeda typicaldual-stagerelease profilefromallofthe samples,aninitialrelativelyfastreleasefollowedbya constant release.HPC/SFS/HPCD-IC-NFreleasedmuchmoreamountofSFS intotalthanSFS(powder)andHPC/SFS/HPCD-ICfilm.Thiscould beduetothehighsurfaceareaofHPC/SFS/HPCD-IC-NFcompared toSFS(powder)andHPC/SFS/HPCD-ICfilm.Theactualloading (%)ofSFSinHPC/SFS-NFandHPC/SFS/HPCD-IC-NFwere deter-minedas76±1%and73±1%,respectively.However,totalamount ofreleasedSFSwasmuchhigherfromHPC/SFS/HPCD-IC-NFthan HPC/SFS-NF.Possiblereasonforthiscouldbethehighersurface areaofHPC/SFS/HPCD-IC-NFduetothelowerfiberdiameteras
Fig.6. XRDpatternsofSFS,HPCD,HPCNFandHPCnanofiberscontainingSFS.
Fig. 7.The cumulative release of SFS from SFS (powder), HPC/SFS film, HPC/SFS/HPCD-ICfilm,HPC/SFS-NFwithoutPCL,HPC/SFS/HPCD-IC-NFwithout PCL,PCL-HPC/SFS-NFandPCL-HPC/SFS/HPCD-IC-NFfor(a)720min,(b)120min (n=3,theerrorbarsinthefigurerepresentthestandarddeviation(SD)),and(c)the solubilityofSFSaspowder,inHPC/SFS-NFandHPC/SFS/HPCD-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/HPCD-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/HPCD-IC-NFreleased SFSslowerthanHPC/SFS/HPCD-IC-NFwithoutPCLbecauseofthe presenceofanextralayerforreleaseofSFS.Asaconsequence, PCL-HPC/SFS/HPCD-IC-NFexhibitedarapidreleaseattheinitialstage andthensustainedrelease atthefinalstage.Thismightbe cru-cialformanyapplicationsthatarerelatedwiththepreventionof bacteriaproliferation.Bringingtothecontext,higherreleaseatthe initialstageisofgreatimportancewhichlimitstheproliferationof bacteriainthebeginningwhilesustainedreleaseinhibitsthefew bacteriawhichmanagedtoproliferate[50].
HPC/SFS/HPCD-IC-NFreleasedhigheramountofSFSinthe ini-tialstageand ineach time period givenonthegraph;and the maximumamountofreleasedSFSwasalmost14%higheras com-paredtoHPC/SFS-NF.Aswementionedbefore,thissituationmight berelatedwiththeexistenceofHPCDinthematrix[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/HPCD-IC-NFwasverifiedbysolubilitytest.TheUV–Vis spectroscopyofSFS(powder),HPC/SFS-NFandHPC/SFS/HP CD-IC-NFdissolvedinwaterisshowninFig.7c.HPC/SFS-NFallowsthe SFSdrugtobedispersedinthemediumwhichfacilitatesits disso-lution.AlthoughsameamountofSFSwasusedforeachsamplethe solubilityofSFShasincreasedmuchmoreinHPC/SFS/HP CD-IC-NF.Additionally,thepresenceofCDmaylowertherequireddose ofanactivemoleculebyimprovingitssolubility.Consequently, HPC/SFS/HPCD-IC-NFcouldbeusedasanefficientdrugdelivery systemforwounddressingpurpose.
4. Conclusion
The concept of employing electrospunnanofibers as matrix andimprovingthesolubilityofhydrophobiccompoundsby CD-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/HPCD-IC. ThestoichiometryofthecomplexbetweenSFSand HPCDwas determinedtobe1:1byJob’splot.Furthermore,modeling stud-ies performedusing ab initiotechniques alsorevealed that the
stoichiometryoftheSFS/HPCD-ICwas1:1(SFS:HPCD). There-fore,theresultsofmolecularmodelingareingoodagreementwith theexperimentaldata.SFSreleasefromPCL-HPC/SFS/HP CD-IC-NFthatwaspreparedbyplacingHPC/SFS/HPCD-IC-NFbetween PCLnanofibrousmatswasslowercomparedtoHPC/SFS/HP CD-IC-NFwithoutPCL.Controlledrelease ofSFS wasattainedfrom HPC/SFS-NFandHPC/SFS-HPCD-ICNFforaperiodof720min,yet weobservedthathigheramountofSFSwasreleasedfrom HPC/SFS-HPCD-IC-NFwhencomparedtoHPC/SFS-NF.Thisispossiblydue tothepropertyofCD-ICtoenhancethesolubilityofhydrophobic SFSmoleculesviainclusioncomplexationwithHPCDandhigher surfaceareaofHPC/SFS/HPCD-IC-NFcomparedtoHPC/SFS-NF. Asaresult,PCL-HPC/SFS/HPCD-IC-NFdepictedslowreleaseand highestamountoftotalSFSrelease.Thisstudycontributestothe existingliteratureforimprovingCD-ICfunctionalizedelectrospun nanofibersthatmightbeusedaswounddressingwith character-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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