Core-shell nanofibers of curcumin/cyclodextrin inclusion complex and polylactic acid: enhanced water solubility and slow release of curcumin

Core-shell

nano

fibers

of

curcumin/cyclodextrin

inclusion

complex

and

polylactic

acid:

Enhanced

water

solubility

and

slow

release

of

curcumin

Zeynep

Aytac

a,b

,

Tamer

Uyar

a,b,

*

a

InstituteofMaterialsScience&Nanotechnology,BilkentUniversity,Ankara06800,Turkey

b

UNAM-NationalNanotechnologyResearchCenter,BilkentUniversity,Ankara06800,Turkey

ARTICLE INFO Articlehistory:

Received30November2016

Receivedinrevisedform24December2016 Accepted31December2016

Availableonline3January2017 Keywords: Electrospinning Core-shell Curcumin Hydroxypropyl-b-cyclodextrin Slowrelease Antioxidantactivity ABSTRACT

Core-shell nanofibers were designed via electrospinning using inclusion complex (IC) of model hydrophobicdrug(curcumin,CUR)withcyclodextrin(CD)inthecoreandpolymer(polylacticacid,PLA) intheshell(cCUR/HPbCD-IC-sPLA-NF).CD-ICofCURandHPbCDwasformedat1:2molarratio.The successfulformationofcore-shellnanofiberswasrevealedbyTEMandCLSMimages.cCUR/HPb CD-IC-sPLA-NFreleasedCURslowlybutmuchmoreintotalthanPLA-CUR-NFatpH1andpH7.4duetothe restrictionofCURinthecore ofnanofibers andsolubilityimprovement showninphase solubility diagram, respectively.ImprovedantioxidantactivityofcCUR/HPbCD-IC-sPLA-NFin methanol:water (1:1)isrelatedwiththesolubilityenhancementachievedinwaterbasedsystem.Theslowreactionof cCUR/HPbCD-IC-sPLA-NFinmethanolisassociatedwiththeshellinhibitingthequickreleaseofCUR.On theotherhand,cCUR/HPbCD-IC-sPLA-NFexhibitedslightlyhigherrateofantioxidantactivitythan PLA-CUR-NFinmethanol:water(1:1)owingtotheenhancedsolubility.Toconclude,slowreleaseofCURwas achievedbycore-shellnanofiberstructureandinclusioncomplexationofCURwithHPbCDprovideshigh solubility.Briefly,electrospinningofcore-shellnanofiberswithCD-ICcorecouldofferslowreleaseof drugsaswellassolubilityenhancementforhydrophobicdrugs.

©2017ElsevierB.V.Allrightsreserved.

1.Introduction

Curcumin(CUR) (Fig.1a) isa polyphenoland apartfromits usageasatherapeuticagent,itiswidelyemployedasaspice,food preservative,flavoringandcoloringagent(Aggarwaletal.,2003). Its common application for various diseases including cancer, cardiovascularandAlzheimer’sdisease,inflammatoryand neuro-logicaldisordersisowingtotheoutstandingbiologicalfunctions suchasantioxidant,anti-tumor,andanti-inflammatoryactivities ofCUR(Yallapuetal.,2015).But,italsoexhibitsdrawbackslikelow bioavailability,instabilitydependingonpH,insolubilityinwater, slow uptake by the cells and rapid metabolism inside thecell (Sivieroetal.,2015).Severalstrategiesweredevelopedpreviously toimprovepharmacokinetics,systemicbioavailability,and biolog-icalactivityofCUR(Sivieroetal.,2015).Amongthesestrategies, cyclodextrin(CD)inclusioncomplexes(ICs)isacommonlyapplied methodtoovercomethelimitationsofCUR.CDsarenontoxicand biodegradable cyclic oligosaccharides which are capable of

forming ICswith avariety ofmolecules toenhance solubility, bioavailability, andthermal stabilityof hydrophobicguest com-pounds; reduce the volatility of molecules with low thermal stability,mask off malodors/bittertastes,and controlrelease of activeagents (DelValle,2004;Hedges 1998;Szejtli,1998).The most commonCDsare

a

-CD,

b

-CD and

g

-CDwith6, 7, and 8 glucoseunits,respectively.Inaddition,chemicallymodifiedCDs including hydroxypropyl-beta-cyclodextrin(HP

b

CD) (Fig.1b) in which someof thehydroxyl groups in the

b

-CD structure are substituted with hydroxypropyl groups were also synthesized. HP

b

CDismoresuitableforthesolubilizationofhydrophobicdrugs duetoitsbetteraqueoussolubilitycomparedtonative

b

-CD(Del Valle,2004;Hedges1998;Szejtli, 1998).ICofCURandHP

b

CDwere studiedbeforeforseveralaimssuchasenhancingthesolubility andfluorescence(Bagloleetal.,2005),oralbioavailability(Bansal et al., 2011)of CUR; treating melanoma(Sunet al.,2014), and inflammatoryboweldisease(Yadavetal.,2009).

Nanofibers are quite appropriate to carry active agents including drugs, antioxidant, and antibacterial agents owingto highsurfacetovolumeratioandporousstructure(Agarwaletal., 2008).Furthermore,owing tothemorphological similarities of nanofibers with extracellular matrix, biomaterials for wound healingandscaffoldsfortissueengineeringcouldbedevelopedby

* Correspondingauthorat:InstituteofMaterialsScience&Nanotechnology, BilkentUniversity,Ankara06800,Turkey.

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

http://dx.doi.org/10.1016/j.ijpharm.2016.12.061

0378-5173/©2017ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

International

Journal

of

Pharmaceutics

usingnanofibers(Greinerand Wendorff, 2007; Wendorffet al., 2012). Recently, there has been significant interest on electro-spinningwhichisasimpleandcommontechniqueforproducing nanofibers(GreinerandWendorff,2007; Wendorffetal.,2012). Designflexibilityofelectrospunnanofibersfacilitatesthe encap-sulationofactiveagentsforbiomedicalapplications(Greinerand Wendorff,2007;Wendorffetal.,2012).CURloadedelectrospun nanofiberswerereportedpreviouslyintheliterature(Guoetal., 2011; Sampath et al., 2014; Suwantong et al., 2007).However, ratherthanloadingfreeactiveagentsintoelectrospunnanofibers, incorporatingtheir CD-ICs is advantageousin many aspects as previously reported in the studies of our group. For instance, volatilemoleculeswerehighlypreserved(Aytacetal.,2014;Kayaci etal.,2013a,2014;KayaciandUyar,2012;Uyaretal.,2009a,2009b, 2011)andthesolubilityofhydrophobicmoleculeswereimproved (Aytacetal.,2015,2016a,2016b;AytacandUyar,2016;Kayacietal., 2013b) by CD-IC incorporated nanofibers. Sun et al. (2013) published a study concerning CUR/CD-IC loaded electrospun nanofibers(Sunetal.,2013).FasterreleasewasseenfromCUR/ HP

b

CD-ICincorporatedpolyvinylalcohol(PVA)nanofibersthan CUR incorporated PVA nanofibers owing to the solubility enhancementanditisexpectedforCUR/HP

b

CD-ICincorporated PVA nanofibers to exhibit higher systemic bioavailability and enhanced in vivo efficacy. On the other hand, it is of great importanceforsomecompoundstobeprotectedagainstorganic solvents,encapsulatedin largeamountand releasedin a more controlledmanner.Duetotheflexibilityoftheset-up,nanofibers withdifferentmorphologiessuchascore-shell,alignedandhollow nanofiberscanbeobtainedviaelectrospinning(Ramakrishnaetal., 2005).Particularly, electrospinning of core-shellnanofibers has severaladvantagessuchaspossibilitytoelectrospunnanofibers from non-spinnable solutions (Sun et al., 2003), protecting sensitive active agents against harsh environment of organic solvents(Jiangetal.,2014),controllingthereleaseofactiveagents inamoreefficientwayduetothepresenceofshellactingasan additionallayer(Jiangetal.,2005),encapsulatingmorethanone drugatthesametime(Llorensetal.,2015),designingactiveagent containingnano_fibersfortargetedrelease(Wangetal.,2015).In thestudyofLlorensetal.(2015),triclosanloadedpoly(ethylene glycol)andCURloadedpoly(butylenesuccinate)solutions were used as core or shell solutions at different compositions. The releaseof triclosanandCURwereinvestigatedin PBSand PBS/ ethanol(30:70, v/v).CUR couldnotbereleasedinPBSfromall

compositionsbecauseofitshighhydrophobicityandinteraction withpoly(butylenesuccinate);whereasitwascompletelyreleased in PBS/ethanol (30:70,v/v) (Llorens et al., 2015). Kumar et al. (2014)producedcore-shellnanofibersbyencapsulatingCURand 5-fluorouracilinthecoreandthen,bothcoreandshellpolymers was crosslinked in type I nanofibers; whereas only shell was crosslinkedintypeIInanofibers.But,crosslinkingofcoreandshell oronlyshelldidnotaffectthereleaserateandamountofCURin contrast to 5-_{fluorouracil}(Kumar et al., 2014).In the studyof SedghiandShaabani(2016)core-shellpolymer-freecorestructure nanofiberswasproducedbyusingCURsolutionin thecoreand PVAandchitosanintheshell.AlthoughtheburstreleaseofCUR was prevented by core-shell nanofibers compared to blend nanofibers, core-shell nanofibers released less amount of CUR thanblendnanofibersduetothelowsolubilityofCURinaqueous solutions(SedghiandShaabani,2016).

Inthisstudy,core-shellnanofibersofCUR/HP

b

CD-IC(asacore) (Fig.1c)andpolylacticacid(PLA)(asashell)whichisanaliphatic polyester and widely used in biological applications due to its biodegradabilityand biocompatibilitywasproducedvia electro-spinning(cCUR/HP

b

CD-IC-sPLA-NF)(Fig.1d).Asacontrolsample, CURblendedwithPLAwasalsoelectrospunintonanofibers (PLA-CUR-NF).Themolarratioof theCUR:HP

b

CDinclusion complex was1:2andthephasesolubilitytestconfirmedthewatersolubility increase of CUR with the inclusion complexation. Core-shell morphologyofcCUR/HP

b

CD-IC-sPLA-NFwasconfirmedbyTEM andCLSMimaging.InvitroreleaseofCURfromPLA-CUR-NFand cCUR/HP

b

CD-IC-sPLA-NFwastestedin0.1NHCl(pH1),PBS(pH 7.4),methanol,andmethanol:water(1:1).Theantioxidantactivity of nanofibers wasinvestigatedby 2,2-diphenyl-1-picrylhydrazyl (DPPH)radicalscavengingassaywithrespecttoconcentrationand time.

2.Experimental 2.1.Materials

Polylacticacid(PLA)(Natureworks,productcode6252D)and hydroxypropyl-beta-cyclodextrin (HP

b

CD) (Wacker Chemie AG, Germany) was donated to our research group for laboratory studies.Curcumin(CUR,95%,AlfaAesar),zincacetatedehydrate (SigmaAldrich),fluoresceinisothiocyanate(FITC,SigmaAldrich), potassium phosphate monobasic (Sigma Aldrich), sodium

Fig.1.(a)ChemicalstructureofCUR,(b)chemicalstructureandschematicrepresentationofHPbCD;schematicrepresentationof(c)formationofCUR/HPbCD-IC,and(d) electrospinningofcore-shellnanofibersfromcCUR/HPbCD-IC-sPLAsolution.

phosphatedibasicheptahydrate(SigmaAldrich),sodiumchloride (SigmaAldrich),methanol(extrapure,SigmaAldrich),chloroform (CHCl3,extrapure,SigmaAldrich),deuterateddimethylsulfoxide

(DMSO-d6,deuterationdegreemin99.8%forNMRspectroscopy, Merck), hydrochloric acid (HCl, 36.5–38%, Sigma-Riedel), 2,2-diphenyl-1-picrylhydrazyl(DPPH,SigmaAldrich)werepurchased andusedas-receivedwithoutanyfurtherpurification. Distilled-deionized water was supplied from Milliporemilli-Q ultrapure watersystem.

2.2.Preparationofsolutionsforelectrospinning

Core-shellnanofibersofCUR/HP

b

CD-inclusioncomplex(CUR/ HP

b

CD-IC) as a core and PLA as a shell were produced via electrospinning (cCUR/HP

b

CD-IC-sPLA-NF). As control samples, pristinePLAnanofibers(PLA-NF)andCURblendedwithPLAwas also electrospun (PLA-CUR-NF). PLA solution was prepared by dissolvingPLA(15%,w/v)inCHCl3:Methanol(2:1)for3h.Inorder

to produce PLA-CUR-NF, PLA-CUR solution was prepared by dissolving3.33%CUR (w/w,withrespecttopolymer)in CHCl3:

Methanol(2:1),thenPLA(15%,w/v)wasaddedintothesolution. Thesolutionwasstirredatroomtemperature(RT)for3hpriorto electrospinning. For core-shell nanofibers, core solution was prepared by dissolving HP

b

CD in water and then adding CUR (CUR:HP

b

CD,1:2molarratio)and thecoresolutionwasstirred overnightatRT.Inaddition,PLA(15%,w/v)wasdissolvedinCHCl3:

Methanol (2:1) for 3hat RT to beused as shell solution.The compositions of the solutions used for the electrospinning of nanofibersaresummarizedinTableS1.

2.3.Electrospinning

PLA-NF and PLA-CUR-NF was produced by single-nozzle electrospinning. PLA and PLA-CURsolutions wereloaded sepa-ratelyinaplasticsyringe(innerdiameter:0.8mm)andmounted onasyringepump(WPI,SP101IZ).Then,thesolutionswerefedat arateof1mL/hand meanwhile15kVwas appliedfroma high voltage power supply (AU Series, Matsusada Precision Inc.). Nanofibers were deposited on a grounded cylindrical metal covered with aluminum foil at a distance of 10cm from the needle tip. In order to electrospun cCUR/HP

b

CD-IC-sPLA-NF, home-madecore-shellsetupwas used(Fig.1d).Core andshell solutionswereloadedinplasticsyringesmountedontwosyringe pumps.Thecoresolutionsentatarateof1mL/h,whereastheshell solutionwas sent at a rate of 3mL/h towardsto collector.The electrospinningofthenanofiberswas performedat25Cunder 18–20%relativehumidity.

2.4.Characterizationsandmeasurements

Phase solubility study was performed in aqueous solution according to the previously described method (Higuchi and Connors, 1965). Excess amount of CUR was added into the solutionscontainingvaryingamountofHP

b

CD(0_–20mM).The suspensionswerestirredovernightatRTandspectrophotometric determinationwas doneat425nm(Varian,Cary100)after the filtrationofthesolutions.Themeasurementswerecarriedoutin triplicateandthephasediagramwasdrawnbyplottingthemolar concentrationofCURagainstthemolarconcentrationofHP

b

CD accordingtothecalibrationcurve.

CUR/HP

b

CD-ICwas formedaccordingtotheco-precipitation methodat1:2molarratio(CUR:HP

b

CD)andthefinalmolarratio of CUR/HP

b

CD-IC was confirmed by proton nuclear magnetic resonance (1_{H NMR) measurement. First of all, HP}

_b

_{CD was}

dissolved in aqueous solution; then CUR was added and the solutionwasstirredfor12hatRT.Finally,thesolutionwasfiltrated

afterkeepingitinrefrigeratorfor6handdriedinhoodfor2days.

1

HNMRspectraofCUR,HP

b

CD,andCUR/HP

b

CD-ICdissolvedin DMSO-d6 were taken on Bruker DPX-400. The assignment of protonsofCURandHP

b

CDaredepictedinFig.S1.Then,themolar ratioofCURandHP

b

CDinCUR/HP

b

CD-ICwascalculatedbyusing theintegrationofthechemicalshifts(

d

)giveninpartspermillion (ppm)calculatedviaMestrenovasoftware.

Themorphological characterizationofcCUR/HP

b

CD-IC-sPLA-NF was performed by transmission electron microscopy (TEM, TecnaiG2F30),confocallaserscanningmicroscopy(CLSM,Zeiss LSM510),andscanningelectronmicroscopy(SEM,FEI–Quanta 200FEG).TheSEMimagingofPLA-CUR-NFasacontrolsamplewas alsoperformed.Thenanofibersamplesweresputteredwith5nm of Au/Pd (PECS-682) to avoid charging problem during SEM imaging. Thecalculationof averagefiberdiameter(AFD)ofthe nanofiberswasmadeonSEMimages(n100)andtheresultsare givenasaveragestandarddeviation.Fortheproofofcore-shell morphologyof cCUR/HP

b

CD-IC-sPLA-NF,zinc acetatedehydrate was added tothecoresolution(CUR/HP

b

CD-IC) and core-shell nanofiberswerecollectedonTEMgrids.ForCLSMimaging,FITC was addedtothecoresolution(CUR/HP

b

CD-IC) andnanofibers werecollectedonglassslides.

X-ray diffraction (XRD) was employed to investigate the crystallinestructureofCUR(powder),HP

b

CD(powder),PLA-NF, and cCUR/HP

b

CD-IC-sPLA-NF. XRD datawere recordedusing a PANalyticalX’PertpowderdiffractometerapplyingCuKradiation inthe2thetarangeof5–30_.

ThermalpropertiesofCUR(powder),HP

b

CD(powder),PLA-NF, PLA-CUR-NF, and cCUR/HP

b

CD-IC-sPLA-NF were examined by thermal gravimetricanalysis(TGA,TAQ500,USA).Thesamples wereheatedupto500Cataconstantheatingrateof20C/min undernitrogenatmosphereforTGAmeasurement.

NanofibershavingequivalentamountofCURwasimmersedin 25mLof0.1MHCl(pH1),PBS(pH7.4),methanolandmethanol: water(1:1)atRTfor120,480,60,and600min,respectively.The cumulativeamountofCURreleasedfromPLA-CUR-NFandcCUR/ HP

b

CD-IC-sPLA-NFwasinvestigatedviaUVspectroscopyfor0.1M HCl (pH 1) and PBS (pH 7.4) and high performance liquid chromatography (HPLC, Agilient, 1200 series) equipped with VWD UV detector (425nm) for methanol and methanol:water (1:1).0.5mLofsolutionwaswithdrawn atpredeterminedtime intervalsandanequalamountoffreshmediumwasrefilledfor HPLC measurement. The separation was accomplished by C18 column(Inertisil,columndimension:4.6mm50mm,particlesize: 5

m

m)operatingat 1mL/min usingmethanolas aneluent. The calibrationcurveswereobtainedtoconvertabsorbanceandarea valuesobtainedfromUVspectroscopyandHPLCtoconcentration (ppm). The experiments were performed in triplicate and the results were reported as averagestandard deviation. The morphologyofnanofiberswasalsoevaluatedafterimmersionof nano_fibersinto0.1MHCl(pH1)andPBS(pH7.4).

According to 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengingassay,antioxidantactivityofPLA-CUR-NFandcCUR/ HP

b

CD-IC-sPLA-NFweretesteddependingonconcentrationand time. Concentration dependent antioxidant activity tests were donebyimmersingnanofibershavingequivalentamountofCURin methanol(for60min)andmethanol:water(1:1)(for600min)as decidedfromreleasetests.Thedilutionofthesolutionsweredone inmethanolandmethanol:water(1:1),respectively.Then,1mLof thosesolutionsweremixedwith2mLof10 4DPPHpreparedin methanol.AfterincubationofthesolutionsindarkatRTfor60min, absorbanceofthesolutionswasdeterminedbyUVspectroscopy (Varian, Cary 100) at 517nm. In order tocalculate antioxidant activity(%),theabsorbanceofDPPHwasdefinedas100%andthe antioxidant activity (%) was calculated based on the following equation:

Antioxidantactivity(%)=(Acontrol Asample)/Acontrol100 (1)

where Acontrol and Asample represent the absorbance values of

control DPPH solution and DPPH solution with nano_fibers, respectively. Efficient concentration (EC50) was defined as the amount of antioxidant molecule necessary to decrease DPPH concentration by 50% (Brand-Williams et al., 1995). For time dependenttests,nanofiberswithequivalentamountofCURwere immersedinmethanol(for60min)andmethanol:water(1:1)(for 600min)and1mLofthosesolutionsweremixedwith2mLof10 4 DPPHpreparedinmethanol.Then,themixtureswereincubatedin dark at RT for 60min. The absorbance of the solutions was measuredbyUVspectroscopyat517nm.

3.Resultsanddiscussion 3.1.Phasesolubilitystudies

PhasesolubilitydiagramforCUR:HP

b

CDsystem isshownin Fig.2.TheinsetphotographsgiveninFig.2presentedthechangeof transparentcolorofthesolutiontoyellowastheconcentrationof dissolved CUR increased. Solubility study was performed with increasingamountofCDinaqueoussolutionatRT.Thesolubilityof CURenhancedlinearlyupto16mMofHP

b

CD,beyondthatpoint thecurvedeviates ina positivedirectionfromlinearity.So,the solubilitycurve of CUR:HP

b

CD system is classified as Ap type

(BrewsterandLoftsson,2007;Takahashietal.,2012).Aptypephase

solubility diagram suggests the formation of higher order complexeswithrespecttoCDathigherconcentrationofHP

b

CD (CUR:HP

b

CD, 1:>1) as well (Brewster and Loftsson, 2007; Takahashietal.,2012).Therefore,thisresultisinagreementwith theinitiallyusedmolarratiowhichis1:2(CUR:HP

b

CD)andthisis alsoconfirmedwiththeproton nuclearmagneticresonance(1H NMR)resultasdiscussedindetailbelow.

3.2.Themolarratioofinclusioncomplex

The molar ratio of 1:2 (CUR:HP

b

CD) was used for the preparation of inclusion complex between the guest molecule (CUR)andthehostmolecule(HP

b

CD).OncetheCUR/HP

b

CD-ICis formed,wecheckedthetruemolarratio ofCUR:HP

b

CD inthe sampleby1_{HNMR(Fig.S1inSupplementarymaterial).Forthe}

analyses, CUR, HP

b

CD, and CUR/HP

b

CD-IC was dissolved in DMSO-d6andthen1_{HNMR spectrawererecorded.The}

assign-mentof protons of CUR and HP

b

CD aredepicted in Fig.S1 in Supplementarymaterial.ThemolarratioofCURtoHP

b

CD was calculatedas1:2bytakingtheintegrationofCURpeakat6ppm andHP

b

CDpeakat1ppm.Therefore,itwasconcludedthatthe

initialamountofCURandHP

b

CD(1:2)waspreservedperfectly aftertheinclusioncomplexationprocess.

3.3.Morphologyanalysesofnanofibers

Transmission electron microscopy (TEM) and confocal laser scanningmicroscopy(CLSM)imagesofcCUR/HP

b

CD-IC-sPLA-NF areshowninFig.3aand3b.Bothoftheimagesconfirmedthe core-shellstructureofnanofibers.ThemorphologyofPLA-CUR-NFand cCUR/HP

b

CD-IC-sPLA-NF was further examined by scanning electron microscopy (SEM) Fig. 4a-b. AFD of nanofibers were determinedas 780_375nm and 695_280nm,respectively. As seen from the photographs given in Fig. 4c–d, both of the nanofibershaveyellowcolorbuttheshadeof thenanofibersis obviouslydifferentfromeachother.Thereasonofthepaleyellow colorofcCUR/HP

b

CD-IC-sPLA-NFisbecausetheCUR/HP

b

CD-ICis coveredwiththePLAlayerasashellinthissample.

3.4.Crystallinestructureofthenanofibers

Thecrystallinestructure ofCUR, HP

b

CD, PLA-NF, andcCUR/ HP

b

CD-IC-sPLA-NFwereinvestigatedviaX-raydiffraction(XRD) (Fig. S2). CUR is a crystalline molecule, whereas HP

b

CD is amorphous moleculeas seen from the diffraction patterns. As seenfromthediffractionpattern,cCUR/HP

b

CD-IC-sPLA-NFdidnot showcrystallinepeaksofCUR.TheCUR/HP

b

CD-ICisloadedinthe fibermatrixasacoreanditisexpectedthatCURwouldpreserveits crystallinephaseifthereisnotrueinclusioncomplexationwith theHP

b

CD.Itiswellknownthatonceinclusioncomplexisformed, guestmoleculesareseparatedfromeachotherbythecavityofthe CDmoleculeandthereforecannotformcrystals(Giordanoetal., 2001).Here, theabsence ofcrystalline peaksof CUR forcCUR/ HP

b

CD-IC-sPLA-NF sample suggested that the true inclusion complexationbetweenCURandHP

b

CDwaspreservedevenafter theelectrospinningprocess.

3.5.Thermalanalysesofnanofibers

Thermalgravimetric analysis(TGA) ofCUR, HP

b

CD, PLA-NF, PLA-CUR-NF,andcCUR/HP

b

CD-IC-sPLA-NFaregiveninFig.S3.The thermal degradation of CUR started at around 200C. Native HP

b

CDexhibiteditsmaindegradationwithaweightlossinthe temperaturerangefrom290Cto425C.Theweightlossof PLA-NFisobservedbetween200Cand375Candcorrespondstothe degradationofPLA.TheweightlossinTGAthermogramsof PLA-CUR-NFandcCUR/HP

b

CD-IC-sPLA-NFisseenbetween215–375_C and 215–425_{C, respectively. So, the thermal stability of CUR} slightly improved in PLA-CUR-NFand cCUR/HP

b

CD-IC-sPLA-NF. BecauseoftheoverlappinginthethermaldegradationofCUR,PLA, and HP

b

CD, the amount of CUR in nanofibers could not be determinedfromTGAdata.

Fig.2.PhasesolubilitydiagramofCUR/HPbCDsysteminwater.Insetphotographs showthechangeofthesolutioncolorwithincreasingHPbCDconcentration(0–

3.6.invitroreleasestudy

ThepHdependentreleaseofCURfromPLA-CUR-NFandcCUR/ HP

b

CD-IC-sPLA-NF were investigated in 0.1M HCl (pH 1, simulated gastric fluid) and PBS (pH 7.4, simulated intestinal fluid)atRT(Fig.5a–b).HigheramountofCURreleasedfrombothof thenanofibersatpH1comparedtopH7.4.SinceCURisknownto existincationic,neutraloranionicformsdependingonpHand theseformsinfluencethesolubilityof CUR.Hence,whenpHis acidic,CURreleasesreadilyfromthenanofibersduetoincreased solubility when it is in cationic form (Massaro et al., 2016). Moreover,therateofreleasefromcCUR/HP

b

CD-IC-sPLA-NFwas slowercomparedtoPLA-CUR-NFattheinitialstepatbothpH1and pH7.4owingtoshellstructureincCUR/HP

b

CD-IC-sPLA-NF.Onthe other hand, CUR released from cCUR/HP

b

CD-IC-sPLA-NF was muchmore intotalthan PLA-CUR-NFat pH1 andpH7.4 most probablydue to thesolubilityenhancement of CUR byHP

b

CD inclusioncomplexationasshowninFig.2.Afterinvitrorelease test,possibilityoffibermatrixdegradationwasinvestigatedbythe morphology change in SEM images (Fig. S4 in Supplementary material).If nanofiber matrix undergoes degradation overtime, erosionbasedrelease mechanism is anticipated. However,SEM images clearly showed that PLA-CUR-NF and cCUR/HP

b

CD-IC-sPLA-NF samples preserved their fibrous structure during the releaseperiod,andtherefore,inourcasethereleasemechanismis diffusionbased.

ThereleaseofCURwasevaluatedinmethanolandmethanol: water(1:1)aswell(Fig.6a–b).PLA-CUR-NFreleasedmuchmore CUR in methanol; whereas cCUR/HP

b

CD-IC-sPLA-NF released slightlymoreamountofCURinmethanol:water(1:1)comparative toitscounterpart.ThisislikelyduetothehighsolubilityofCURin methanolwhenitisinfreeformandenhancedwatersolubilityof CURinmethanol:water(1:1)whenitisformedanICwithHP

b

CD.

Fig.4.SEMimageofelectrospunnanofibersobtainedfromthesolutionsof(a)PLA-CURand(b)cCUR/HPbCD-IC-sPLA;thephotographsof(c)PLA-CUR-NFand(d)cCUR/ HPbCD-IC-sPLA-NF.

Fig.5. ThecumulativereleaseofCURfromPLA-CUR-NFandcCUR/HPb CD-IC-sPLA-NFinto(a)pH1and(b)pH7.4(n=3).Theerrorbarsinthefigurerepresentthe standarddeviation(SD).

3.7.Antioxidantactivity

Thereductionofchronicdiseases,DNAdamage,mutagenesis, carcinogenesis, and inhibition pathogenic bacterial growth is mostly related with the free radical scavenging ability of antioxidant compounds (Ak and Gulcin, 2008). Antioxidant activityofmoleculesisoftenduetothepresenceofthephenolic hydrogenin theirstructure, butsince CUR’s phenolic hydrogen atoms are intramolecularly hydrogen-bonded to the methoxy groups,hydrogenabstractionfromphenolicringofCURisdifficult (Gulcin,2012).However,abstractionofhydrogenfromthecarbon atom which is in the heptadienone linkage between the two methoxyphenolringsisrelativelyeasierandthisabstractionisthe mainreasonofantioxidantactivityofCUR(Gulcin,2012).

AntioxidantactivityofPLA-CUR-NFandcCUR/HP

b

CD-IC-sPLA-NF was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) with respecttoconcentrationandtimefirstlybyextractingCURfrom nanofibersinmethanolormethanol:water(1:1)(Figs.7and8).For methanol,PLA-CUR-NFandcCUR/HP

b

CD-IC-sPLA-NFhas442– 950%and341–943%antioxidantactivityinthe concentra-tion range of 5–160ppm, respectively (Fig. 7a). According to concentrationdependenttestmadeinmethanol,efficient concen-tration50(EC50)wasdeterminedbetween5and10ppmand20– 40ppmforPLA-CUR-NFandcCUR/HP

b

CD-IC-sPLA-NF, respective-ly.LowerEC50showshigherfreeradicalscavengingcapabilityof PLA-CUR-NF.Thisresultisalsoconsistentwiththereleasestudy madeinmethanolinwhichPLA-CUR-NFreleasedhigheramountof CURcomparedtocCUR/HP

b

CD-IC-sPLA-NF.Thevisual investiga-tionoftheresultingsolutionsalsoshowsthecoherencewiththe calculated antioxidant activities (Fig. 7b). For instance, cCUR/ HP

b

CD-IC-sPLA-NFexhibited341%ofantioxidantactivityand thecolorofthesolutionwaspurple;when943%ofantioxidant activitywasseen,thecolorofthesolutionbecameyellow.

Timedependentantioxidantactivityofnanofibersfromwhich methanol was used to extract CUR was evaluated for 60min (Fig. 7c). PLA-CUR-NF has reached its maximum antioxidant activity in 2min (931%); whereas cCUR/HP

b

CD-IC-sPLA-NF showsitsmaximumantioxidantactivity(843%)in15min.The slowantioxidantactivityofcCUR/HP

b

CD-IC-sPLA-NFascompared

toPLA-CUR-NFisduetothepresenceofanadditionalhydrophobic barrier(shell)delayingtheaccessofwateranddissolutionofCUR. TheantioxidantactivityofPLA-CUR-NFandcCUR/HP

b

CD-IC-sPLA-NFextractedusingmethanol:water(1:1)wascalculatedas 443–891% and 391–923% in the range of 5–160ppm,

Fig.6. ThecumulativereleaseofCURfromPLA-CUR-NFandcCUR/HPbCD-IC-sPLA-NFinto(a)methanoland(b)methanol:water(1:1)(n=3).Theerrorbarsinthefigure representthestandarddeviation(SD).

Fig.7.(a)ConcentrationdependentantioxidantactivityofPLA-CUR-NFandcCUR/ HPbCD-IC-sPLA-NF(methanol),(b)thephotographsofthesolutionswithrespectto concentration;(c)timedependentantioxidantactivityofPLA-CUR-NFandcCUR/ HPbCD-IC-sPLA-NF(methanol).

respectively (Fig. 8a). EC50 for both PLA-CUR-NF and cCUR/ HP

b

CD-IC-sPLA-NFwasdecidedtobeslightlyhigherthan20ppm. So, cCUR/HP

b

CD-IC-sPLA-NFexhibitedquite similarantioxidant capability withPLA-CUR-NF. Thisresult is compatiblewith the releasestudy;thus,whenthesolubilityofCURwasenhancedby complexation, cCUR/HP

b

CD-IC-sPLA-NF exhibited antioxidant activityquite well.Thephotographs of each solutionshow the changeofpurpletoyellowcolorwiththeincreasingconcentration ofCUR(Fig.8b).

TimedependentantioxidantactivityofPLA-CUR-NFandcCUR/ HP

b

CD-IC-sPLA-NF was measured for 60min and antioxidant activitiesweredecidedas78_2%and87_2%forPLA-CUR-NFand whereas cCUR/HP

b

CD-IC-sPLA-NF, respectively (Fig. 8c). The maximumantioxidantactivityofPLA-CUR-NFandcCUR/HP

b

CD-IC-sPLA-NFwereobtainedin15min(742%)and8min(814%), respectively. This result indicated relatively quick antioxidant activityof cCUR/HP

b

CD-IC-sPLA-NFin methanol:water (1:1) in comparisonwithmethanolowingtothegreatersolubilityofCUR inwaterbasedsystem.

4.Conclusions

Core-shell nanofibers were produced using cyclodextrin-inclusioncomplex(CD-IC)ofamodelhydrophobicdrug(curcumin, CUR)in the coreand polylactic acid(PLA) in the shell (cCUR/ HP

b

CD-IC-sPLA-NF)byelectrospinning.CURreleasedfromcCUR/ HP

b

CD-IC-sPLA-NFwasmuchmorethanPLA-CUR-NFatpH1and pH7.4mostprobablyduetothesolubilityenhancementasshown inphasesolubilitydiagram.Inaddition,duetothepresenceofa shelllayerCURreleasedslowerfromcCUR/HP

b

CD-IC-sPLA-NFas comparedtoPLA-CUR-NF.Efficientconcentration50(EC50)was

lower for cCUR/HP

b

CD-IC-sPLA-NF in methanol:water than in methanol since inclusion complexation of CUR and HP

b

CD improvesthesolubilityofCURinaqueoussolution.Therelatively slowantioxidantactivityofcCUR/HP

b

CD-IC-sPLA-NFinmethanol islikelyduetotheadditionalpolymericbarrier(shell)delayingthe access of water; whereas slightly quick antioxidant activity is associatedwiththehighsolubilityofCUR/HP

b

CD-ICinmethanol: water(1:1).In short,drugdeliverysystemsbased oncore-shell nanofiberstructureinwhichhydrophobicdrugsareplacedinthe corestructureintheformofinclusioncomplexwithcyclodextrins couldprovideslowreleaseaswellashighwatersolubilityforsuch hydrophobicdrugs.

Acknowledgements

ThisworkwassupportedbyTheScientificandTechnological ResearchCouncilofTurkey(TUBITAK)-Turkey(Project#111M459). Dr. Uyar also acknowledgesThe Turkish Academy of Sciences-Outstanding Young Scientists Award Program (TUBA-GEBIP)-Turkeyforthepartialsupport.Z.AytacthankstoTUBITAK-BIDEB (2211-C)andTUBITAK(project#111M459)forthePhD scholar-ship.WeexpressourspecialthankstoDr.AnithaSenthamizhanfor herhelpintheconfocallaserscanningmicroscopyimaging. AppendixA.Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2016.12.061.

References

Agarwal,S.,Wendorff,J.H.,Greiner,A.,2008.Useofelectrospinningtechniquefor biomedicalapplications.Polymer49(26),5603–5621.

Aggarwal,B.B.,Kumar,A.,Bharti,A.C.,2003.Anticancerpotentialofcurcumin: preclinicalandclinicalstudies.AnticancerRes.23(1A),363–398.

Ak,T.,Gulcin,I.,2008.Antioxidantandradicalscavengingpropertiesofcurcumin. Chem.Biol.Interact.174(1),27–37.

Aytac,Z.,Uyar,T.,2016.Antioxidantactivityandphotostabilityofa-tocopherol/

b-cyclodextrininclusioncomplexencapsulatedelectrospunpolycaprolactone nanofibers.Eur.Polym.J.79,140–149.

Aytac,Z.,Dogan,S.Y.,Tekinay,T.,Uyar,T.,2014.Releaseandantibacterialactivityof allylisothiocyanate/b-cyclodextrincomplexencapsulatedinelectrospun nanofibers.ColloidsSurf.B:Biointerfaces120,125–131.

Aytac,Z.,Sen,H.S.,Durgun,E.,Uyar,T.,2015.Sulfisoxazole/cyclodextrininclusion complexincorporatedinelectrospunhydroxypropylcellulosenanofibersas drugdeliverysystem.ColloidsSurf.B:Biointerfaces128,331–338.

Aytac,Z.,Kusku,S.I.,Durgun,E.,Uyar,T.,2016a.Encapsulationofgallicacid/ cyclodextrininclusioncomplexinelectrospunpolylacticacidnanofibers: releasebehaviorandantioxidantactivityofgallicacid.Mater.Sci.Eng.C63, 231–239.

Aytac,Z.,Kusku,S.I.,Durgun,E.,Uyar,T.,2016b.Quercetin/b-cyclodextrininclusion complexembeddednanofibres:slowreleaseandhighsolubility.FoodChem. 197,864–871.

Baglole,K.N.,Boland,P.G.,Wagner,B.D.,2005.Fluorescenceenhancementof curcuminuponinclusionintoparentandmodifiedcyclodextrins.J.Photochem. Photobiol.A:Chem.173(3),230–237.

Bansal,S.S.,Kausar,H.,Aqil,F.,Jeyabalan,J.,Vadhanam,M.V.,Gupta,R.C.,Ravoori,S., 2011.Curcuminimplantsforcontinuoussystemicdelivery:safetyand biocompatibility.DrugDeliv.Transl.Res.1(4),332–341.

Brand-Williams,W.,Cuvelier,M.E.,Berset,C.L.W.T.,1995.Useofafreeradical methodtoevaluateantioxidantactivity.LWT-FoodSci.Technol.28(1),25–30.

Brewster,M.E.,Loftsson,T.,2007.Cyclodextrinsaspharmaceuticalsolubilizers.Adv. DrugDeliv.Rev.59(7),645–666.

DelValle,E.M.,2004.Cyclodextrinsandtheiruses:areview.ProcessBiochem.39 (9),1033–1046.

Giordano,F.,Novak,C.,Moyano,J.R.,2001.Thermalanalysisofcyclodextrinsand theirinclusioncompounds.Thermochim.Acta380(2),123–151.

Greiner,A.,Wendorff,J.H.,2007.Electrospinning:afascinatingmethodforthe preparationofultrathinfibers.Angew.Chem.Int.Ed.46(30),5670–5703.

Gulcin,I.,2012.Antioxidantactivityoffoodconstituents:anoverview.Arch.Toxicol. 86(3),345–391.

Guo,G.,Fu,S.,Zhou,L.,Liang,H.,Fan,M.,Luo,F.,Qian,Z.,Wei,Y.,2011.Preparationof curcuminloadedpoly(e-caprolactone)-poly(ethyleneglycol)-poly (e-caprolactone)nanofibersandtheirinvitroantitumoractivityagainstGlioma 9Lcells.Nanoscale3(9),3825–3832.

Fig.8. (a)ConcentrationdependentantioxidantactivityofPLA-CUR-NFandcCUR/ HPbCD-IC-sPLA-NF(methanol:water,1:1),(b)thephotographsofthesolutions withrespecttoconcentration;(c)timedependentantioxidantactivityof PLA-CUR-NFandcCUR/HPbCD-IC-sPLA-NF(methanol:water,1:1).

Hedges,A.R., 1998.Industrialapplicationsofcyclodextrins.Chem.Rev.98(5),2035– 2044.

Higuchi,T.K.,Connors,A.,1965.Phase-solubilitytechniques.Adv.Anal.Chem. Instrum.4,117–212.

Jiang,H.,Hu,Y.,Li,Y.,Zhao,P.,Zhu,K.,Chen,W.,2005.Afaciletechniquetoprepare biodegradablecoaxialelectrospunnanofibersforcontrolledreleaseofbioactive agents.J.Control.Release108(2),237–243.

Jiang,H.,Wang,L.,Zhu,K.,2014.Coaxialelectrospinningforencapsulationand controlledreleaseoffragilewater-solublebioactiveagents.J.Control.Release 193,296–303.

Kayaci,F.,Uyar,T.,2012.Encapsulationofvanillin/cyclodextrininclusioncomplexin electrospunpolyvinylalcohol(PVA)nanowebs:prolongedshelf-lifeandhigh temperaturestabilityofvanillin.FoodChem.133(3),641–649.

Kayaci,F.,Ertas,Y.,Uyar,T.,2013a.Enhancedthermalstabilityofeugenolby cyclodextrininclusioncomplexencapsulatedinelectrospunpolymeric nanofibers.J.Agric.FoodChem.61(34),8156–8165.

Kayaci,F.,Umu,O.C.,Tekinay,T.,Uyar,T.,2013b.Antibacterialelectrospunpoly (lacticacid)(PLA)nanofibrouswebsincorporatingtriclosan/cyclodextrin inclusioncomplexes.J.Agric.FoodChem.61(16),3901–3908.

Kayaci,F.,Sen,H.S.,Durgun,E.,Uyar,T.,2014.Functionalelectrospunpolymeric nanofibersincorporatinggeraniol-cyclodextrininclusioncomplexes:high thermalstabilityandenhanceddurabilityofgeraniol.FoodRes.Int.62,424–431.

Kumar,S.U.,Matai,I.,Dubey,P.,Bhushan,B.,Sachdev,A.,Gopinath,P.,2014. Differentiallycross-linkablecore-shellnanofibersfortunabledeliveryof anticancerdrugs:synthesis,characterizationandtheiranticancerefficacy.RSC Adv.4(72),38263–38272.

Llorens,E.,Ibañez,H.,DelValle,L.J.,Puiggalí,J.,2015.Biocompatibilityanddrug releasebehaviorofscaffoldspreparedbycoaxialelectrospinningofpoly (butylenesuccinate)andpolyethyleneglycol.Mater.Sci.Eng.C49,472–484.

Massaro,M.,Amorati,R.,Cavallaro,G.,Guernelli,S.,Lazzara,G.,Milioto,S.,Noto,R., Poma,P.,Riela,S.,2016.Directchemicalgraftedcurcuminonhalloysite nanotubesasdual-responsiveprodrugforpharmacologicalapplications. ColloidsSurf.B:Biointerfaces140,505–513.

Ramakrishna,S.,Fujihara,K.,Teo,W.E.,Lim,T.C.,Ma,Z.,2005.AnIntroductionto ElectrospinningandNanofibers,vol.90.WorldScientific,Singapore.

Sampath,M.,Lakra,R.,Korrapati,P.,Sengottuvelan,B.,2014.Curcuminloadedpoly (lactic-co-glycolic)acidnanofiberforthetreatmentofcarcinoma.ColloidsSurf. B:Biointerfaces117,128–134.

Sedghi,R.,Shaabani,A.,2016.Electrospunbiocompatiblecore/shellpolymer-free corestructurenanofiberswithsuperiorantimicrobialpotencyagainstmulti drugresistanceorganisms.Polymer101,151–157.

Siviero,A.,Gallo,E.,Maggini,V.,Gori,L.,Mugelli,A.,Firenzuoli,F.,Vannacci,A.,2015. Curcumin,agoldenspicewithalowbioavailability.J.Herb.Med.5(2),57–70.

Sun,Z.,Zussman,E.,Yarin,A.L.,Wendorff,J.H.,Greiner,A.,2003.Compound core-shellpolymernanofibersbyco-electrospinning.Adv.Mater.15(22),1929–1932.

Sun,X.Z.,Williams,G.R.,Hou,X.X.,Zhu,L.M.,2013.Electrospuncurcumin-loaded fiberswithpotentialbiomedicalapplications.Carbohydr.Polym.94(1),147– 153.

Sun,Y.,Du,L.,Liu,Y.,Li,X.,Li,M.,Jin,Y.,Qian,X.,2014.Transdermaldeliveryofthein situhydrogelsofcurcuminanditsinclusioncomplexesof

hydroxypropyl-b-cyclodextrinformelanomatreatment.Int.J.Pharm.469(1),31–39.

Suwantong,O.,Opanasopit,P.,Ruktanonchai,U.,Supaphol,P.,2007.Electrospun celluloseacetatefibermatscontainingcurcuminandreleasecharacteristicof theherbalsubstance.Polymer48(26),7546–7557.

Szejtli,J.,1998.Introductionandgeneraloverviewofcyclodextrinchemistry.Chem. Rev.98(5),1743–1754.

Takahashi,A.I.,Veiga,F.J.B.,Ferraz,H.G.,2012.Literaturereviewofcyclodextrins inclusioncomplexescharacterization–PartI:Phasesolubilitydiagram, dissolutionandscanningelectronmicroscopy.Int.J.Pharm.Sci.Rev.Res.12(1), 1–6.

Uyar,T.,Hacaloglu,J.,Besenbacher,F.,2009a.Electrospunpolystyrenefibers containinghightemperaturestablevolatilefragrance/flavorfacilitatedby cyclodextrininclusioncomplexes.React.Funct.Polym.69(3),145–150.

Uyar,T.,Nur,Y.,Hacaloglu,J.,Besenbacher,F.,2009b.Electrospinningoffunctional poly(methylmethacrylate)nanofiberscontainingcyclodextrin-menthol inclusioncomplexes.Nanotechnology20(12),125703.

Uyar,T.,Hacaloglu,J.,Besenbacher,F.,2011.Electrospunpolyethyleneoxide(PEO) nanofiberscontainingcyclodextrininclusioncomplex.J.Nanosci.Nanotechnol. 11(5),3949–3958.

Wang,X.,Yu,D.G.,Li,X.Y.,Bligh,S.A.,Williams,G.R.,2015.Electrospunmedicated shellacnanofibersforcolon-targeteddrugdelivery.Int.J.Pharm.490(1),384– 390.

Wendorff,J.H.,Agarwal,S.,Greiner,A.,2012.Electrospinning:Materials,Processing, andApplications.JohnWiley&Sons,Weinheim.

Yadav,V.R.,Suresh,S.,Devi,K.,Yadav,S.,2009.Effectofcyclodextrincomplexationof curcuminonitssolubilityandantiangiogenicandanti-inflammatoryactivityin ratcolitismodel.AAPSPharmSciTech.10(3),752–762.

Yallapu,M.M.,Nagesh,P.K.B.,Jaggi,M.,Chauhan,S.C.,2015.Therapeuticapplications ofcurcuminnanoformulations.AAPSJ.17(6),1341–1356.