pcl/peg electrospun fibers as drug carriers for the...

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Journal of In Silico & In Vitro Pharmacology2015

Vol. 1 No. 1:2

1© Copyright iMedPub This article is available in http://pharmacology.imedpub.com/

Repanas A1,Wolkers WF1, Gryshkov O1, Müller M1 Glasmacher B1

Institute for Multiphase Processes,Leibniz Universität Hannover, 30167Hannover,Germany

Corresponding author: Repanas A

[email protected]

Dipl.-Pharm., MSc, Leibniz Universität Hannover, Institute for Multiphase Processes, Callinstrasse 36, 30167 Hannover, Germany

Tel: +49(0)5117623824Fax: +49(0)5117623031

Pcl/Peg Electrospun Fibers as Drug Carriers for the Controlled

Delivery of Dipyridamole

AbstractElectrospinningisaversatileanddiversetechnologyfortheproductionofnano-andmicrofibersthatcanbeusedasadrugdeliverysystem(DDS).The aim of this studywas to create fibrous scaffolds from amixture ofpolycaprolactone(PCL)andpolyethyleneglycol(PEG)andtoevaluatethesuitability of the fibers as DDS. Dipyridamole (DPA), an anti-thromboticand anti-proliferative pharmaceutical agent, was used as amodel drug.Two typesof PEGwithdifferent chain lengthwereused. The structural,mechanical and physicochemical characteristics of the fibers with andwithoutDPA,weredetermined,andthereleasekineticsofDPAwerestudied.TheobtainedfibersloadedwithDPAandwiththehighermolecularweightPEGwere smooth andhad an averagediameterof 586.75± 204.79nmandanaverageYoung’smodulusof57.14±4.35MPa,whereasthetensilestrainatbreakwas0.61±0.05mm/mmandtheultimatetensilestrength(UTS)was16.79±3.08MPa.ThecumulativereleaseofDPArevealedtwostages:an initialburstphenomenon followedbyslowerFickiandiffusion(releaseexponentn=0.432).

Keywords: Anti-platelet treatment; Biomaterials; Electrospinning;Polycaprolactone;Polyethyleneglycol;Dipyridamole

Abbreviations:DPA:Dipyridamole;PCL:Polycaprolactone;PEG:PolyethyleneGlycol;DDS:DrugDeliverySystem;TFE:2,2,2-Trifluroethanol;PBS:PhosphateBufferSolution;SEM:ScanningElectronMicroscopy;FTIR:FourierTransformInfraredSpectroscopy;UTS:UltimateTensileStrength

IntroductionElectrospinning is a promising technology that can be used tocreate fibrous scaffolds in the nanoscale range for biomedicalapplications[1].Thebasicprincipleofthissimple,versatileandcost-efficient technique is the application of a strong electricalfieldtoejectapolymersolutionfromareservoir toacollectorproducing non-woven fiber networks. Solution properties suchas the concentration and molecular weight of solvents andpolymers,aswellasprocessingparameters,suchasthespinningdistance and the solution flow rate affect the morphological,physical and biomechanical properties of the generated fibers[2].A typical setupof anelectrospinning apparatus consists ofthree basic elements: a high voltage power supply, a solutionreservoirandagroundedcollector[3].Duetothewiderangeofavailablematerials, electrospinning technologyhas turned into

apowerfultoolforthefabricationofmicro-ornano-structuressuitable for applications in thefieldsoftissueengineering anddrugdelivery[4-7].Polycaprolactone(PCL) isasemi-crystalline,highly hydrophobic polymer that is easily soluble in mostorganicsolvents,andhasbeenapprovedbytheFoodandDrugAdministration (FDA) in the USA for biomedical uses [8]. PCLhas good miscibility properties, possesses mechanical stabilityanddisplaysprolongeddegradation.Moreover, it canbeeasilyelectrospun and can be used for long-term sustained deliveryofpharmaceuticalagentsinthefieldofdrugdeliveryandtissueengineering[9-13].Ontheotherhand,polyethyleneglycol(PEG)is a water soluble polyether with a wide range of molecularweightsthathasbeenfoundtobebiocompatibleandhasbeenused in hydrogels, for surface modification of other polymerstoobtainco-polymers, controlofproteinadsorptionandasanadhesionmolecule[14].Therefore,theuseofbothpolymersas

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2015Vol. 1 No. 1:2

Journal of In Silico & In Vitro Pharmacology

© Copyright iMedPub This article is available in http://pharmacology.imedpub.com/

asingleblendcouldleadtoproductsthatexhibitacombinationofproperties,suitableforapplicationsindrugdelivery.Theabilitytodiffuseintothesurroundingmediumaswellastherelease kineticsof a drug that hasbeen loaded in a polymericcarrier depend on various factors, including the solubilityand the possibility of swelling of the polymeric matrix in themedium according to Tungprapa et al.[15]. Moreover, whena pharmaceutical agent is loaded inside a biodegradablepolymeric carrier, two distinct major mechanisms are knownto facilitate the drug release,mainly polymer degradation andsmallmoleculediffusion [16]. PCLdisplays littledegradation inaqueousenvironmentduringthefirstmonths[8],whereasPEGis a water soluble polymer that can be immediately dissolvedin the surroundingmedium.PEGalsoenhances the stabilityofbiomoleculesandaffectsreleasecharacteristics [17].Inaddition,ifPCListhepredominantpolymerinthefabricatedelectrospunfibers,diffusionthroughthepolymericmatrixisexpectedtobethemajormechanism[18]. TheaimofthisworkwastofabricatefibrousscaffoldsfromapolymericblendofPCLandPEGwithanincorporatedmodeldrug,inordertocreateadrugdeliverysystem(DDS),whichwouldenableencapsulationandsustaineddeliveryofthepharmaceuticalagent.Dipyridamole(DPA)wasusedasamodeldrug.Thereleasekineticsfromelectrospunscaffoldsandtheeffectofitspresenceonthepropertiesofthefabricatedfiberswerestudied.DPAisusedasanantiplateletagent,eitheraloneorincombinationwithotherpharmaceuticalagentsinordertoreducetheriskofstrokeincidentsafteraseverecardiovascularevent,suchasmyocardial infarction[19,20].Additionally, itcanincreasegapjunctioncouplinginaorticendothelialandvascularsmooth muscle cells [21,22]. One of the conclusions of thesestudieswasthatDPA isbestutilized insustainedrelease formsof administration likemicrospheres.ADDS enabling controlledreleaseofDPAcanbeusedindrugdeliveryapplications[23,24].PEGwithdifferentmolecularweightwasusedtoinvestigatetheinfluenceofthepolymericchainlengthonthepropertiesofthescaffolds.ElectrospunPCLfiberswithoutPEGservedascontrol.The structural,morphological,mechanical andphysicochemicalproperties of the electrospun scaffolds were evaluated. Thecumulative in vitroreleaseofDPAwasmonitoredandanalyzedthroughoutaperiodofninetysixdaystoinvestigatethereleasekinetics of DPA by fitting the obtained data according to themodel describedbyRitger andPeppas (1987) [25]. The choiceofPCLasthemainpolymerwasbasedonthefactthatinordertocreateaproperDDSsystemforthesustainedreleaseofDPA,aslowdegradingpolymer(recognizedasasuitablematerialforthefabricationofscaffolds,asitwaspreviouslydiscussed),whichwouldbealsoinexpensiveandeasytoprocess,wasneeded.Inaddition,asitwaspreviouslymentioned,PEGwasusedtoenhancethestabilityoftheencapsulatedpharmaceuticalmoleculesandtoaugmentthemiscibilityofthepolymericsolution.Furthermore,sincePEGtypeswithvariousmolecularweightsareavailable,itenablestoadjustthefibers’characteristicswithregardtoreleasekineticsoftheencapsulateddrug,viaitspolymericchainlength.It is also inexpensiveandpresents goodmiscibilitywithPCL, afeaturethathasbeenpreviouslydescribed[26].Therefore, thecombination of the two polymers was considered suitable tocreateaDDSforDPAencapsulation.Inanutshell,aDDSsystembased on PCL/PEG fibers can be considered as a suitable drug

carriercandidateofDPAinordertobefurthertestedasanovelsolution for an alternative anti-coagulant therapeutic strategyforpatientsthatareathighriskofstroke,myocardialinfraction,thrombotic incidents and other life threatening situations. Theuseofelectrospinningandfiber technology for the creationofa proper DDS system for DPA has only been used once in thepastforthecreationofpoly-urethanetubularscaffolds[23]andbyourgroupforthecreationofPCLbasedDDS[24].Therefore,furtherresearchneedstobeconductedinordertohaveabetterunderstandingoftheuseoffibersforthisapplication.However,theideaofcontrollingthecharacteristicsofthefibersandDIP’srelease kinetics via the polymeric chain length of PEG is novelandverypromisingforthementionedDDS,anditwasoneofthebasic goalsof thiswork. Finally, theoutcomeof this study canbeused as an initial step topromote future researchonDDSsbased on PCL/PEG fibers made by electrospinning for similarpharmaceuticalagents.

Materials and MethodsMaterialsPolycaprolactone (PCL) (Mn 70000-90000) and dipyridamole(DPA)(≥98.0%)werepurchasedfromSigma-Aldrich.Polyethyleneglycol (PEG) (Mr3500-4500&Mr~35000)waspurchased fromFluka. 2,2,2-trifluroethanol (TFE) was purchased from abcrGmbH&Co.KG.Allmaterialsandreagentswereusedasreceivedwithoutanyfurtherpurification.

Preparation of the polymeric solutionsBoth the polymers and DPA were dissolved in TFE. The totalpolymer concentration was 150 mg/mL and the mass ratiobetweenPCLandPEGwas3:1.TheconcentrationofDPAinthesolution was 10 wt% of the mass of PEG. The concentrationsof PCL and PEG were selected based on previous preliminaryexperiments taking into account the ease of electrospinningofthesolutionsandthesolubilityofthematerialsused inTFE.Forexample,a50:50polymerratioofPCLandPEGledtofiberswith beads, wider diameter distributions and to an unstableelectrospinning process (Figure 1). In addition, a solutionwiththe same polymer concentration and mass ratio but withoutDPA was also prepared to serve as a control. Furthermore,blendsolutionsofPCLwithandwithoutDPAwerepreparedasadditionalcontrolgroups.Inthatcase,theconcentrationofPCLwas 150mg/mLwhile the concentration of DPA remained thesameasthatof thesolutionswithPEG.Allpolymericsolutionswereconstantlystirredfor18hatroomtemperatureinordertoachievehomogenousmixtures.

Electrospinning processDisposable blunt-tipped needles (21G, Nordson EFD) with aninnerdiameterof0.6mmand5mLsyringes(Omnifix,B.Braun)were used as the polymeric solution reservoirs. The solutionflowratewaskeptat4mL/hwithanappliedvoltageof25kVin order to achieve continuous flow with no jet breaks. Thedistancebetweenthetipoftheneedleandthegrounded,squarealuminumcollector (15ˣ15cm2)waskeptconstantat25cm. Inaddition,analuminumfoilcoveringthesurfaceofthecollector

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was used to retrieve the electrospun fiber mats. Moreover,electrospinning was performed under stable conditions oftemperature and relative humidity (T=21 ± 1.5, Rel. Humidity=30±5%)for30minpersample.Afterthepress,thesampleswereremovedfromthecollectorandlefttodryundervacuumfor24h.ThesamplegroupwithDPAandPEG(Mr35000,PEG35k)aredesignatedasF1andtheonewithoutDPAasF0forbettercomprehension.Furthermore,theelectrospunfiberscomposedofPEG(Mr4000,PEG4k)butwithoutDPAandaredesignatedasF2andtheoneswithincorporatedDPAasF3.Finally,thesamplegroupswithoutDPAandonlyPCLaredesignatedasF4andtheones with incorporated DPA as F5. The concentrations of allmaterialsandtheshortcodenamesaresummarizedinTable 1.

Characterization of the electrospun fiber matsElectrical conductivity: A conductometer (SevenMulti, MettlerToledoAG)wasusedtomeasuretheelectricalconductivityofthepolymericsolutions.2mLofeachsolutionweremeasuredat25.Allmeasurementswerecarriedoutinquintuplicate.

Scanning electron microscopy (SEM): Square strips of 5ˣ5mm2 were carefully punched out from all sample groups andweresputtercoatedwithAu/Pd for60sbeforebeingmountedona steel stage inside theSEM (S3400N,Hitachi), (15kV,highvacuum). In order to determine the average fiber diameter,pictures obtained at a magnification of 4000x were pressedusing the image analysis software ImageJ (National Institutesof Health), takingmeasurements of 75 different fibers from 5differentsamplesfromallgroups.

Fourier transform infrared spectroscopy (FTIR): InfraredabsorptionmeasurementswerecarriedoutwithaPerkinElmer100 Fourier transform infrared (FTIR) spectrometer (PerkinElmer,Norwalk,CT,USA),equippedwithatriglycinesulfate(TGS)detectorandanattenuatedtotalreflection(ATR)accessorywitha diamond/ZnSe crystal. The acquisition parameters were: 4cm-1resolution,8co-addedinterferograms,and4000-650cm-1 wavenumberrange.SpectraanalysisanddisplaywerecarriedoutusingPerkinElmersoftware(PerkinElmer,Norwalk,CT,USA).

Mechanical tensile testing: Rectangular stripsof15mmgaugelength and 10mmwidth were carefully punched out from allgroups.Thethicknessofeachindividualspecimenwasmeasuredwith a thickness gauge (Quickmini,Mituotoyo). Double-edgedduct tape was carefully placed at both edges of the strips tofacilitate their mounting on the metallic grips of the testingdevice.Uniaxial tensile testswereconductedwithan INSTRON5967DualColumntensiletestinginstrumentwitha100Nloadcell (Instron, USA). The ultimate tensile strength (UTS), thetensilestrainatmaximumforceandtheYoung’smodulusvalueswere analyzed. All the experiments were conducted at roomtemperature.Anelongationrateof1mm/swasapplied to thesamples.Sixsamplesweretestedpergroup.

In vitro drug release study: For the drug release experiments,squarestripsof10ˣ10mm2werepunchedoutfromtheelectrospunspecimens,subsequentlyimmersedin1mLofphosphatebuffersolution (PBS) and kept in 2 mL tubes (Eppendorf) inside awaterbathat37.Atpredefinedtimepoints,thetotalvolumeofthe PBS medium was removed for measuring the absorbance

andfreshmediumwasaddedbacktothetube.Theabsorbanceof DPAwasmeasured at 284 nm using a UV-Vis spectrometer(LIBRAS22,Bihrom)andplastic,disposableUV-cuvettes(1.5mL,12.5ˣ12.5ˣ45mm,Brand).AcalibrationplotofabsorbanceversusconcentrationwascreatedusingstandardsolutionsofDPAinPBSwithconcentrationsrangingfrom1μg/mLupto50μg/mL.ThecumulativereleaseofDPAwasmeasuredforatotalperiodof96days.Allexperimentswerecarriedoutintriplicates.

Release kinetics mechanism: The release kineticsofDPAwereanalyzedusingtheequationdescribedbyRitgerPLandPeppasNA(1987)[25].

Mt/M∞=ktn Equation1

where Mt is the drug released at the time point t, M∞ is thetotalamountofdrugreleased,kisaconstantdependingonthestructuralandgeometricalcharacteristicsofthedrugcarrierandnisthereleaseexponentwhichindicatesthereleasemechanism.

Statistical analysis: All data are expressed asmean± standarddeviation unless otherwise mentioned. One-way analysis ofvariance (ANOVA) with post h Tukey means comparison testswereconductedandpvalue<0.05wasconsideredsignificant.The distribution of values in terms of normality was assessedbeforeone-wayANOVAanalysisinordertoensurethequalityoftheobtainedresults.

ResultsStructural and morphological analysisTheobtainedSEMpicturesofthefibersareshowninFigure 2. SEM images of the investigated specimens revealed no drugaggregates and beads, indicating adequate miscibility of allthe materials and confirming a stable and repeatable press.In all cases, electrospinning resulted in cylindrical fibers withsmooth surface and diameters in the sub-micron/nano- scaleforming dense networks. Thatwasmade possible by selectingtheappropriatePCL/PEGratioduringpreliminaryresults(Figure 1). The averagefiber diameter of specimens F0, F1, F2, F3, F4and F5fiberswere found tobe661.33± 196.94nm,586.75±204.79nm,617±208.96nm,558±190.08nm,1005.33±319.44nmand771.33±247.9nm,respectively.Figure3captures the

Samplename

PCLconcentration

mg/mL

PEGconcentration

mg/mL

DPAconcentration

mg/mL

F0 112.5 37.5(PEG35k)a -

F1 112.5 37.5(PEG35k)a 3.75

F2 112.5 37.5(PEG4k)b -

F3 112.5 37.5(PEG4k)b 3.75

F4 150.0 - -

F5 150.0 - 3.75a PEGMr~35000,b PEGMr3500-4500

Table 1 Short codenamesandmaterials’ concentrations (mg/mL) forelectrospunfiberspreparedusingPolycaprolactone(PCL),Polyethyleneglycol (PEG, two different average molecular weight types) andDipyridamole(DPA).

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Figure 1 SEM pictures of electrospun fibers with a 50:50mass ratio between PCL and PEG (A) and fiberswitha75:25massratiobetweenthetwopolymers(B); 15 kV accelerating voltage, high vacuum, 500xmagnification,scalebars=20μm.

differencesbetweenallthedifferentelectrospunfiberspecimens.No significant differences were observed between fibers F0andF1,aswellasbetweenfibersF2andF3.Theaveragefiberdiameter of specimen F5 differed significantly from fibers F1,F2,F3(p<0.001).Finally,fibersF5alsodifferedsignificantlywithfibers F4 (p<0.05). The histograms of the calculated diametersof the electrospun fibers are also depicted in Figure 2. Thefiber diameter distributionof the specimens F0, F1, F2 and F3werefoundtobesimilar,whereasF4displayedabroaderfiberdiameterdistribution.IncaseofspecimenF5,theadditionofDPAresultedinanarrowerfiberdiameterdistribution(Figure2I).Inaddition,noparticularalignmentwasobserved(Figure2).

Physichemical characterizationThe addition of DPA to the polymeric solutions significantlyaffected the electrical conductivity (Figure 4), which increasedfrom1.556±0.09μS/cm for F0fibers to6.272±0.152μS/cmforF1fibers; from1.912±0.03μS/cm forF2fibers to6.716±0.12 μS/cm for F3 fibers and from 0.521 ± 0.023 μS/cm forF4 fibers to 18.81 ± 0.058 μS/cm for F5 fibers, presentinga statistically significant difference (p <0.001). In order toinvestigatephysichemical interactionsbetweentheelectrospunfibers loadedwith DPA and the bulkmaterials, FTIRwas used

as a means of chemical characterization. Figure 5 depicts theFTIR spectraof F1andF0fibers comparedwith theones frombulkPCLandPEGpelletsaswellasDPAcrystallinepowder.ThespectrumofthePCLpelletsexhibitedcharacteristicpeaksat2943cm-1(-CH3,asymmetricstretching),2869cm-1(-CH3,symmetricstretching) and1725 cm-1 (-C=O, stretching) in agreementwithprevious studies [26]. Furthermore, the FTIR spectrum of bulkPEGexhibitedcharacteristicpeaksat2882cm-1(-CH3,symmetricstretching), 1342 cm-1 (-CH3, scissoringandbending) and1095cm-1(-C-O-C-(ether),stretching).Moreover,thespectrumofDPAinpowderformexhibitedthefollowingcharacteristicpeaksamongother: at 2921 cm-1 (-CH3, asymmetric stretching) and at 1531cm-1 (-C=N, stretching). Additionally, F0 specimens exhibitedcharacteristicpeaksat2946cm-1(-CH3,asymmetricstretching),1723 cm-1 (-C=O, stretching), 1342 cm-1 (-CH3, scissoring andbending),1103cm-1(-C-O-C-(ether),stretching).Ontheotherhand,thespectrumoftheF1fibersexhibitedamixtureofcharacteristicpeaksfromthespectraofthebulkmaterials.Morespecifically,F1 fibers exhibited characteristic peaks at 2946 cm-1 (-CH3,asymmetric stretching),1726cm-1 (-C=O, stretching),1342cm-1 (-CH3,scissoringandbending),1100cm-1(-C-O-C-(ether),stretching)andalowabsorbancepeakat1536cm-1,correspondingto–C=Nstretching fromDPA.Thedifferences inpeaksof theF0andF1specimensaswellasfromthebulkmaterialsaresummarizedinTable 2.

Mechanical characterizationTheobtaineddatafromthemechanicaltensiletestsarepresentedinFigure 6.TheaverageUTSincreasedfrom14.32±1.51MPafortheF0fibersto16.79±3.08MPafortheF1electrospunfibers(Figure 6A).Moreover,F3specimensexhibitedhigherUTS(13.94±1.87MPa)comparedtoF2specimens (11.32±1.93MPa). Inaddition,theaverageUTSvaluesofspecimensF4andF5,whichwere58.15±4.75MPaand60.27±6.48MParespectively,weresignificantly higher than the rest of the specimens (p <0.001).However,theadditionofDPAinspecimensF1,F3andF5didnotresult in significant increaseof theUTSvalues (p>0.05) (Figure 6A).Additionally,thetensilestrainatbreakwas0.62±0.06mm/mmand0.61±0.05mm/mmforfibersF0andF1,respectively(Figure 6B), indicating that DPA has little effect onmechanicalproperties. A minor decrease in the tensile strain values wasobservedbetween specimensF2andF3.More specifically, thetensilestrainatbreakdroppedfrom0.38±0.05mm/mmto0.30±0.02mm/mm,butnotsignificantly(p>0.05).TheuseofPEGwithdifferentmolecularweightwith andwithout incorporatedDPAledtoastatisticaldifferenceintermsoftensilestrain(p<0.001).Moreover, the specimens fabricatedonlybyPCL (F4) exhibitedthehighest tensilestrainatbreak,namely0.8±0.09mm/mm,and were statistically different from all the other specimens(p<0.001).Finally,theaveragetensilestrainatbreakoffibersF5was0.45±0.05mm/mm.Thedifferencesbetween the Young’smodulusvaluesaredepictedinFigure 6C.ThepresenceofDPAinspecimensF1,F3andF5ledtoanincreaseintheaverageYoung’smodulusvaluesfrom48.69±6.46MPato57.14±4.35MPaforfibersF0andF1;from65.87±6.66MPato107.05±9.29MPaforfibersF2andF3andfrom98.9±16.35MPato199.98±33.78MPaforfibersF4andF5,respectively.FibersF5exhibitedthehighestaverageYoung’smodulus,whichwasstatisticallydifferent from

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Figure 2 SEMpicturesofelectrospunF0fibers(PCL&PEG35k)withnoDipyridamole(DPA)encapsulated(A);F1

fibers(PCL&PEG35k)withDPAencapsulated(B);HistogramofthefiberdiameterdistributionsoffibersF0andF1(C);F2fibers(PCL&PEG4k)withnoDipyridamole(DPA)encapsulated(D);F3fibers(PCL&PEG4k)withDPAencapsulated(E);HistogramofthefiberdiameterdistributionsoffibersF2andF3(F);F4fibers(PCL)withnoDipyridamole(DPA)encapsulated(G);F5fibers(PCL)withDPAencapsulated(H);HistogramofthefiberdiameterdistributionsoffibersF4andF5(I);15kVacceleratingvoltage,highvacuum,4000xmagnification,scalebars=1μm.

Figure 3 Average fiber diameter values from electrospun fibers

F0-F5obtainedafterimageanalysisusingImageJ;(mean±SD,n=75);One-wayANOVAwithposthocTukey (*p<0.05,**p<0.001,***p<0.001,NS=notsignificant.

Figure 4 Electrical conductivity values from the polymeric

solutionsusedtofabricateelectrospunspecimensF0-F5,(mean±SD,n=5);One-wayANOVAwithposthocTukey(*p<0.05,**p<0.001,***p<0.001,NS=notsignificant).

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Figure 5 FTIR spectra of PCL pellets, bulk PEG, powderDPA, F0and F1 electrospun fibers from 4000 to 650 cm-1; thedotted rectangular sites indicate thedifferences in thespectrabetweenF0,F1fibersandbulkmaterials.

all the other samples (p<0.001). In addition, Young’s modulusvaluesfromfibersF2andF3werestatisticallydifferent(p<0.01)whiletherewasnosignificantdifferencebetweenfibersF0andF1 (p>0.05). Finally, the use of PEG with different molecularweightledtoasignificantdifferenceinYoung’smodulusvaluesbetweenfibersF1andF3(p<0.001).

Cumulative drug release experimentsThe cumulative amount of DPA released from F1, F3 and F5specimenswascalculatedasthefractionofthetotalDPAcontentinside a PBS solution (pH 7.4,T=37 ). The drug release studieswereperformed in an incubatoroveraperiodof96days. ThecumulativereleaseprofileofDPAfromtheelectrospunfibersisshown inFigure 7,while the insetdepicts thefirstdayofDPArelease.Twodistinctreleasestagescouldbeidentifiedforallthedifferentelectrospunspecimens:arapidburstreleasefollowedbyarelativelyslowreleasephase.Duringtheinitial24h,F1fibersshowedaburstreleaseofDPA,where52.4±1.1%ofDPAreleasedinto the PBS medium. In comparison with this, F3 and F5showed60.32±1.82%and63.14±1.07%releaseofDPAintothemedium,respectively.ThefirststagewheremorethanhalfofDPAmasswasalreadyreleasedforalldifferentelectrospunspecimens was followed by a successive slow second stage.DuringthisstagethecumulativereleaseofDPAwaslowerandmore gradual, reaching 84.74 ± 1.06% until the 18th day forfibersF1.Inaddition,thecumulativereleaseofDPAfromfibersF3andF5was93.79±2.02%and95.89±1.43%,respectively.Moreover,thereleaserateofDPAwasgraduallydecelerating,ultimatelyreachingaplateau.ThecumulativereleaseofDPAreached 89.07 ± 1.29% for F1 fibers, 97.55 ± 1.71% for F3fibersand98.17±1.59%forF5fibersonthe96thday.In order to comprehend the mechanism that regulates thereleaseofDPA,thedataobtainedfromthecumulativereleasestudieswerefittedaccordingusingEquation1[27].InFigure 7 (inset)thereleaseexponentnforF1,F3andF5fibersisdepictedtogetherwithanattempttofittheobtainedexperimentaldata.

Figure 6 Mechanical properties of the F0-F5 electrospunfibers.Ultimatetensilestrength(UTS)(A);tensilestrainatbreak(B);andYoung’smodulus(C);elongationrate=1mm/sec;(mean±SD,n=6);One-wayANOVAwithposthocTukey(*p<0.05,**p<0.001,***p<0.001,NS=notsignificant).

Thereleaseexponentsn forspecimensF1,F2andF3were0.432,0.454and0.426respectively,correspondingtoFickiandiffusion(R2>0.93forallcases).

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DiscussionElectrospun fibers loaded with DPA and without DPA werefabricatedusingdifferentmolecularweightofPEG.Theobtainedfibershadanaveragediameterbetween600nmand1000nm(Figure 3).TheadditionofDPAinthepolymericsolutionsresultedinasignificant increaseof thesolution’selectricalconductivity,while at the sametimedecreasing the averagefiber diameter.Onetheotherhand,additionofPEGledtoasignificantincreasein the average values of electrical conductivity and a decreaseintheaveragefiberdiametercomparedtothefiberscontainingonlyPCL.Additionally,highermolecularweightofPEGresultedin polymeric solutions with lower average values of electricalconductivityandconsequentlytothickerfibers.Similarfindingsconcerning the influence of molecular weight of PEG on fiberdiameterandelectricalconductivityweredescribedbySarafetal [28].AccordingtoLeeetal.,decreaseintheaveragefiberdiametercouldbetheresultoftheincreaseinthesolutionsconductivityduetoaugmentedtransferofelectricalchargesintothepolymericjet [29], resulting in greater bending instability of the solutioninsidetheelectricalfield.Consequently,furtherelongationofthejetcurred,leadingtothinnerfibers.Furthermore,thepresenceof chemical groups (pH dependant) in DPA (hydroxyl groups),could have further increased the electrical conductivity [30].FTIR spectroscopy was used to study the possible differencesbetween theelectrospunfibersand thebulkmaterials,aswellasbetweenthepolymersandDPA(Figure 5).Shiftsinthepeakscouldbeindicativeofdifferentintermolecularinteractionsinthefibers.Morespecifically,therearevariouschangesinthepeaksoftheF1fiberscomparedwiththespectraoffibersF0,bulkPCLandPEG(Table 2).TheresultingpeaksoftheF1spectrumcouldbeanaftermathofapossibleinteractionbetweenPCLandPEG.On theother hand, thepeaksofDPAwere verydifficult to be

Figure 7 CumulativereleaseofDPAfromF1,F3andF5electrospunfibersduringatotal96daysperiod;cumulativereleaseof DPA from F1, F3 and F5 fibers during the initial 24hours(inset);(mean±SD,n=3);T=37oC;Experimentalandfitteddata(usingEquation1describedbyPeppasetal.,Mt/M∞=kt

n)fromF1,F3andF5fibersduringthefirst60%ofDPAreleased;(mean±SD,n=3).

identified in the fibers due to themass ratio of thematerialsused. Nevertheless, a low intensity peak at 1536 cm-1 (-C=N,stretching)wasdetectedintheF1spectrum,whichwasshiftedcomparedwiththepeakintheDPAspectrum,whereasnopeakwas observed in the spectrum of F0 fibers. Furthermore, thedifferencesbetweenthespectraofbulkPCLandF1fiberscouldbetheresultofdifferentdegreeofPCLcrystallinitybeforeandafter itwas dissolved in TFE and electrospun [31]. In addition,formationofhydrogenbondsbetweentheestergroupsofPCL(in a semi-crystalline state) and the hydroxyl groups of TFE (inan amorphous state)molecules could explain the shifts in theabsorption bands [32,33]. The addition of DPA in the blendsolutions increased the average values of UTS and Young’smoduluswheretheadditionofDPAresultedinstifferfibers.TheinfluenceofDPAandPEGonthesolution’selectricalconductivityas well as on the average fiber diameter can also affect themechanicalproperties.Thedecreaseintheaveragefiberdiameterandminorchangeinthegeneralalignmentandstructureofthefibersaffectmechanicalproperties[34].Moreover,differencesinintermolecularinteractionsbetweenthepolymersmightexplaindifferencesinmechanicalproperties.TheFTIRdataendorsethishypothesissincetherewereshiftsincharacteristicpeaksofthefiberspectracomparedwiththatofthebulkmaterials,butalsobetweenF0andF1.Theuseof lowmolecularweightPEGwithlower average polymeric chain length (F2& F3) led toweakerand less flexible but stiffer fibers indicating that the length ofthe polymeric chain determines mechanical properties of thefabricatedscaffolds.Moreover,thefibrousscaffold’sarchitectureinthenano-/submicronlevelisconsideredveryimportantforthereleaseoftheencapsulatedmolecule.Thestressesthatascaffoldwould possibly confront from the surrounding environment atthe site of implantation affect the structure of the carrier andthusthereleasekinetics[35].TwodistinctstagesofDPAreleasewereobservedinallcases:astrongburstreleasephenomenonfollowedbyaslowerreleasephase.Theinitialburstreleasestagethatwasparticularlyintenseduringthefirst12hours(Figure 7,inset), has been previously described by other groups [36-38].AccordingtoZamanietal., itispossiblethataconsiderableamountof the drug could havemigrated to the surface of the ejectedpolymer solutionduring electrospinning [11].Additionally,DPAmighthavebeenincorporatedintotheamorphousregionsofthesemi-crystalline PCL. Kim et al. proposed that drug aggregatescan be forced to migrate to the surface of the fibers duringelectrospinning because of the semi-crystalline nature of PCL[33].AnothermajorreasonforthereleaseofDPAduringthefirststageistherapiddissolutionofPEGinthemedium.TherehavebeenpreviousreportsonfasterosionofPEGorsimilarpolymersin polymeric blends that led to burst release phenomena andchangesinthestructureanddiameterofthefibers[33,35].Kimetal.alsodescribedthepossibilityofphaseseparationbetweenthesemi-crystallinePCLandthewatersolublepolyethyleneoxide(PEO)thatmighthaveledtoacoreskeletonmainlycomposingofPCLanda surrounding coatingofPEO [33]. The latter couldcur during electrospinning, when the blend solution is quicklyejected to the collector, while fast solvent evaporation couldenable phase separation between PCL and PEG. The use of alowmolecularweightPEGforfibersF3ledtoafasterreleaseofDPAand toamore intenseburstphenomenonduring thefirst

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hours.Theaveragemolecularweightandthusthelengthofthepolymeric chains affects the degradation rate of the polymer[8].ThereleaseratefromF5fiberswasfoundtobethefastest,whichispossiblyduetotheamorphousregionsofPCLandthetendencyofthemoleculestotravelnearthesurfaceofthefibers.Moreover, the absence of PEG might have resulted in lowerhomogeneity of the blend solution, leading to amore intensephase separation during electrospinning and consequently ahigherpercentageofDPApresenton the surfaceof thefibers.F1fiberswerethemostappropriateforsustainedreleaseofDPAexhibitingthelowestburstreleasesuggestingthatPEGcouldbebeneficial in a polymeric blend rendering it suitable as a DDS.Ontheotherhand,duringthesecondstage,thereleaseofDPAwas slower andmore gradual, mainly depending on diffusion.Thecumulativereleaseofthedrugwasmainlyattributedtothediffusion of DPA through the polymericmatrix combined withtheinitialerosionofPEG(onlyforspecimensF1andF3).DuringthesecondstageofthereleasethereisnoPCLdegradation[8],while the vast amountofPEGhadbeenalreadydissolved intothemedium.Therefore,thereleaseofDPAfromtheelectrospunfibersispredominantlycontrolledbyFickiandiffusion,whiletheadditionofPEGtotheblendsolutionanditsmolecularweightdidnotaffecttherelease.

Sample

Peaks(cm-1)

-CH3,asymmetricstretching

-C=O,stretching

–C=Nstretching

-CH3,

scissoringandbending

-C-O-C-(ether),stretching

DPAbulk 2921 1531

PCLbulk 2943 1725

PEG35kbulk 2882 1342 1095

F0 2946 1723 1342 1103

F1 2946 1726 1536 1342 1100

Table 2 Characteristicpeaks(cm-1)intheFTIRspectraofthebulkmaterials(PCL,PEGandDPA)andintheFTIRspectraofF0andF1electrospunfibers.

ConclusionBlend electrospinning can be used to fabricate PCL/PEG fibersloadedwithDPA,whichcanbeusedasaDDS.TheadditionofDPAtothepolymericblendusedforelectrospinningresultedinthinner,strongerandstifferfibers.Moreover,thepolymerchainlengthofthePEGcanbeusedasatooltoadjustfiberpropertiesifnecessary.TheelectrospunfibersshowsustainedreleaseofDPA,mainlythroughFickiandiffusion,indicatingthattheyaresuitableasdrugdeliverycarriers,aslongastheinitialburstphenomenoniscontrolled.

AcknowledgementsThisresearchwasgrantedbytheGermanResearchFoundation(Deutsche Forschungsgemeinschaft, DFG) by the Cluster ofExcellenceREBIRTH(FromRegenerativeBiologytoReconstructiveTherapy, DFG EXC 62/1). The authors express their sinceregratitude to fellow researcher Poulami Basu MSc from theBiomedicalEngineeringDivision(VelloreInstituteofTechnology,Vellore, India), for editing themanuscript, as well as Dipl.-Ing.Panagiotis Kalozoumis from the Department of Cardiothoracic,TransplantationandVascularSurgery(HannoverMedicalSchool,Hannover, Germany), for his contribution to the mechanicaltensileteststhatwereperformed.

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