First of all, I would like to express my deepest gratitude to my supervisor, Professor Romána Zelkó for her endless support, encouragement and guidance.
Special thanks should be given to Diána Balogh for her professional advice, selfless help and much encouragement. I am endlessly grateful for the fate, that we have met, and I wish to thank her for everything I could learn from her.
I am extremely grateful to SpinSplit Ltd. for ensuring the use of its electrospinning device during my second project.
I am also indebted to the following people, co-authors who helped me during my work:
Dr. Péter Szabó, Dr. Attila Domján, Dr. Károly Süvegh, Dr. Attila Farkas, Dr. Tamás Bozó, Dr. Miklós Kellermayer, Dr. András Darcsi, Dr. Szabolcs Béni, Dr. Tamás Vörös, Dr. János Madarász, Attila Balázs, Balázs Pinke and Dr. Lajos Szente.
Furthermore, I am grateful to all colleagues at the University Pharmacy Department of Pharmacy Administration.
Last, but not least, I would like to express my deepest gratitude to my parents and my twin brother for everything that I have been receiving throughout the course of my study.
Journal ofPharmaceuticaland BiomedicalAnalysis
jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a
Predictionofthehydroxypropylcellulose—poly(vinylalcohol)ratio in aqueoussolutioncontainingpapaverinehydrochlorideinterms of drugloadedelectrospun fiberformation
AdriennKazsoki,PéterSzabó,RománaZelkó∗
UniversityPharmacyDepartmentofPharmacyAdministration,SemmelweisUniversity,H ˝ogyesEndreSt.7-9,H-1092Budapest,Hungary
a r t i c l e i n f o
Articlehistory:
Received31December2016
Receivedinrevisedform14February2017 Accepted15February2017
Papaverinehydrochlorideloadedelectrospunfiberswerepreparedforbuccaldrugdeliverywiththeaim ofimprovingtheoralbioavailabilityofthecrystallinedrug,whichcanbeachievedbytheincreased solu-bilityandbythecircumventionoftheintensivefirstpassmetabolism.Thewatersolublehydroxypropyl cellulose(HPC)waschosenasamucoadhesivepolymer.Inordertoimprovetheelectrospinnabilityof HPC,thealsomucoadhesivepoly(vinylalcohol)(PVA)wasused.Duringtheexperiments,thetotal poly-merconcentrationwaskeptconstantlyat15%(w/w),andonlytheratioofthetwopolymerswaschanged.
FivedifferentHPC:PVAratios(5:5,6:4,7:3,8:2,9:1)wereexamined.Combinationofrheological mea-surementsandmolarreflectancecharacterizationwithscanningelectronmicroscopywasappliedforthe determinationoftheoptimumcompositionofthegelsforfiberformation.Thecrystalline-amorphous transitionofpapaverinehydrochloridewasalsotrackedbyFouriertransforminfraredspectroscopy.A correlationwasfoundbetweenthemacrostructuralpropertiesofthepolymersolutionsandtheir elec-trospinnabilityandtheconsequentmorphologyoftheresultantsamples.Alongwiththechangesofthe polymerratio,thecorrespondingmorphologyoftheelectrospunsamplesalsovaried.Withdecreasing HPCratioofthesystem,atransitionfromthespray-driedfilm-likestructurethroughfibrousfilmto fiberswasobserved.Polymersolutionsofthelowestelasticityandsmallestintermolecularinteractions contributedtothebestfibercharacteristicsofthesamples.Theresultsenablethedeterminationofthe polymerratiofortheformationofapplicablequalityofelectrospunfibers.Accordingtotheresults5:5 and6:4polymerratiosenabledthebestfiberperformance.
©2017ElsevierB.V.Allrightsreserved.
1. Introduction
Cardiovasculardiseasesareamongtheleadingcausesofdeath intheworld.Cerebralischaemiarepresentsoneofthemajor cardio-vascularhealthconcernsbecauseofnotonlythemorbiditybutits roleinthedevelopmentofdementiaandotherdisabilities. Consid-eringtheeffectofcerebralischaemiaonoverallpublichealth,itis obviousthatpropermanagementofsuchconditionsisessentialto achievethebestpossibleoutcome,thereforepromptandtargeted treatmentcanenormouslycontributetobothpublicandindividual health[1].However,pharmacodynamicpropertiesofpapaverine arebeneficial forthetreatmentofstroke; itsunfavourable bio-pharmaceutical behavior confines the clinicalapplicability. The
Abbreviations:HPC,hydroxypropylcellulose;PVA,poly(vinylalcohol).
∗Correspondingauthor.
E-mailaddress:zelko.romana@pharma.semmelweis-univ.hu(R.Zelkó).
benzylisoquinoline-typeopiumalkaloidpapaverine(Fig.1)hasa non-specificdirectrelaxanteffectonsmoothmuscle.The vasodi-lationisresultedbytheinhibitionofphosphodiesteraseenzymes andthedirectactionsoncalciumchannels[2].
Orallyadministered papaverine hasbeen shown toincrease regional cerebralblood flow in humans.Papaverine hydrochlo-rideisslightlysolubleinwater,anditspoorperoralbioavailability isassociatedwithhighintra-andinterindividualvariability.This variationcanbeexplainedbytheextensivefirstpassmetabolism andtheinadequateabsorptiondependingonincompleteinvivo drugrelease[3].Furthermore,theutilizationofalternative admin-istrationroutesishastenedbythafactthatpapaverinehasbeen reportedtocarrytheriskofpotentialliverdamage[4,5].Asaresult oftheseproperties,papaverinecanonlybeadministered parenter-allyinemergencycases,suchascerebralischemiainordertoensure thetherapeuticdruglevelsinthebrainandtherapidonset.Since theadministrationofinvasiveparenteraldosageformshas sev-eralunfavourablefeatures,theseproblemscanbeaddressedby
Fig.1.Chemicalstructureofpapaverinehydrochloride.
usingalternativedosageforms,suchasinhalationorbuccal,rectal ortransdermaladministrationroutes[6].
Nanofibershavebeensuccessfullyappliedinvariousfieldsofthe medicine,suchastissueengineeringscaffolds,inwoundhealing, vasculargraftimplantsanddrugdelivery[7–9]becauseoftheir pharmaceuticallyluringadvantages,suchashighsurfaceareato volumeratioandveryhighporosity[10,11].
Electrospinningrepresentsthemostcommonlyusedfiber form-ingtechnique,whichutilizeselectricalforcestoproducenanofibers frompolymers[12].Widevarietyofpolymerscanbeusedto con-trolthedrugreleasekineticsortomodifythedissolution[13].The incorporationofpoorlywater-solubledrugsintopolymer-based nanofibersisofspecialinterestbecauseoftheincreasedsolubility andtheconsequentimprovedoralbioavailability.Such improve-ment in the solid state properties can beenabled by thehigh amorphization efficacyofthe fiberformingprocess and bythe uniquecharacteristicsoftheformedfibroussystem[14–16].
Theoralcavityisapossiblesiteforthedeliveryofdrugs.With themucosaldeliverylocalpharmacologicaleffectscanbeachieved.
Forthisapplication,theresidencetimeandthelocaldrug concen-trationarethemostimportantfactors,influencingtheamountof absorbeddrug[17].Systemiceffectscanalsobeachievedvia trans-mucosalroutes,whichisduetothehighlyvascularized,richblood suppliedandrelativelypermeablepropertiesofthemucosa. Per-meationandconsequentlythebioavailabilitycanincreasebyusing enhancers(forexamplepolysorbate,sodiumlaurylsulfate)[18,19].
Becauseoftheadvantagesprovidedbybuccalformulations,such asrapidonsetofeffect,goodbioavailability,eliminationof hep-aticfirst-passmetabolismandconsequentlythereducedamount ofdrug,thebuccaldeliverycouldbeanalternativeadministration oftheparenteralroutes[20–26].
Chemically-modifiedcellulosederivatives(ethers andesters) arecommonlyusedforbuccaldrugdelivery[20].Thefirst genera-tionofmucoadhesivepolymerssuchasthehydroxypropylcellulose (HPC)andpoly(vinyl-alcohol)(PVA)areabletoformnon-covalent bondswiththemucosa,thusensuringabetteroralresidenceand drugreleaseattheabsorptionsite.HPCpresentshigh mucoadhe-sivepotentialanditsfurtheradvantageistheaqueoussolubility, thus avoiding the need of the use of organic solvents during production[27–29].Mostofthenaturalandsemisynthetic poly-mersexhibit poor spinnabilitythus the application of polymer ofgoodspinnabilitycouldimprovefiberformingcapability.The combinationoftwo polymersenablesadequatecompositefiber performance.
Thefiberformingmechanism isvery complexand the elec-trospinnability of polymer gels is widely studied, but still not completelyunderstood.Thesurfacetension,conductivity, entan-glementconcentration,viscosityandthedynamicmoduli:storage (G’) and loss (G”) modulus are the most commonly examined
are thesame, the conductivityand the surface tensionremain unchanged[7,30–35].
Molar refraction was successfully used to examine the intermolecularforcesbetweenpolymerandplasticizer[36]. Deter-mination of molarrefractionscould beappliedto examinethe interactions between thepolymers. Best of ourknowledge the molarrefractioneffectonthefiberformingprocesshasnotbeen publishedyet.
Theprimaryaimofthepresentstudywastopreparepapaverine hydrochloride loaded buccal nanofibrous films, and to deter-minetheoptimumcompositionofHPC-PVAaqueoussolutionsfor electrospunfiberformationbased ontherheologicalandmolar reflectancecharacterizationofthesystem.Withthisformulation intendedfortransmucosaldrugadministration,theunique prop-erties of thenanofibrous sheets and thebenefits of thebuccal formulationcouldbecombinedpreferably.
2. Materialsandmethods 2.1. Materials
Hydroxypropyl cellulose (Klucel EXF Pharm, Ashland, USA;
Mw∼80000;themolesofsubstitution=3.8)andpoly(vinyl alco-hol) (18-88 Ph. Eur., Merck, Darmstadt, Germany) polymers, polysorbate20(Ph.Eur.,Molarchemicals,Hungary)anddistilled waterwereusedforthepreparationofgels.Themodeldrugwas papaverinehydrochloride(Ph.Eur.).
2.2. Preparationofpapaverine-HClgels
Papaverine-HClstocksolutionwaspreparedbydissolving0.6g drugin18.8gofdistilledwaterinthepresenceof0.6gpolysorbate 20(3%w/w)applyingafewminutesofheating.Theclearsolution wascooledtoroomtemperatureandthenitwasdilutedto20.0g withdistilledwater.Thegelswerepreparedbytheadditionofthe necessaryamountofHPC,PVAand30mg/gpapaverine-HCl aque-ousstocksolution.Thetotalpolymerconcentrationofthegelswas 15%(w/w).TheHPC:PVAmassratioswere5:5,6:4,7:3,8:2,9:1 whichcorrespondedto7.5,12.2,10.5,9and13.5HPC concentra-tion(%w/w),respectivelywiththefinalpolymerconcentrationof 15%(w/w).TherequiredamountofPVAandstocksolutionwas measuredintoabeaker,thanitwasheatedon80–90◦Candstirred onmagneticstirplateuntilahomogenousgelwasachieved.The gelswerecooledtotheroomtemperatureandfinallythe neces-saryamountoftheHPCwasaddedanditwasalsostirreduntil homogenousgelwasachieved.Thesampleswerestoredatroom temperature(25±2◦C).
2.3. Calculationofthemolarrefraction
Themolar refractionvalues werecalculated by the Lorentz-Lorenzequation[36]:
R =Vm−r (1)
whereVmisthemolarvolume(dm3mol−1)andristhespace-filling factor.Vmwascalculatedas
Vm = M/ (2)
whereMisthemolecularweight(gmol−1)andisthevolumetric massdensityofthegels(gcm−3).
Densityvaluesweredeterminedwithcalibratedflaskmethod.
as
r =(n2−1)/(n2 +2) (3) where n is the refractive index, which was determined at 22.6±0.1◦CbyKrüssRefractometer(Germany).
2.4. Rheologicalpropertiesofthegels
Therheologicalproperties ofthegelswerecarried out with KinexusProrheometer(MalvernInstrumentsLtd,UK).Measured datawere registeredwithrSpace forKinexus Pro1.3 software.
Samplesweremeasuredusingparallelplates geometry(50mm diameter)inconstant 25±0.1◦Ctemperature.Thegapbetween theplateswas0.0300mm.
Storageandlossmoduliweredeterminedbyoscillatorytests.
Theelasticorstoragemodulus(G’),whichrepresentstheabilityof thematerialstostoreenergy,wascalculatedas
G’= (a/a)∗cosı
andtheviscousorlossmodulus(G”),whichcanbedefinedas theabilityofthematerialtodissipateenergy,wascalculatedas G = (a/a)∗sinı,
whereaistheshearstress,aisthedeformationandıisthephase shiftangle[37].
Theoscillatoryshearmeasurementswereperformedat ampli-tudewithinthelinearregionwhichwaschosento40%withinthe viscoelasticregionandthefrequencywasintherangeof0.1–10s−1. Threeparallelsweremeasuredofeachgelinbothtests.
2.5. Electrospinning
Thepreparedgelsweretransferredintoplastic syringesand thenthesyringeswerevented. Afterall,a metallicneedle with 1.2mminnerdiameterwasattachedthroughasilicontube.The syringewasputintoasyringepumpwhichprovidedthecontrolled polymergelflow.
Theappliedvoltagewasexaminedinthreelevels:20,25and 30kV.Theneedletocollectordistancewas5,10and15cm.The flowratewas0.2or0.3ml/h.Inthecourseofthe10mincontinuous apparatusoperation,thefiberswerecollectedonaluminumfoil.
2.6. Fouriertransforminfrared(FT-IR)spectroscopy
Physicochemicalpropertiesofthefiberswereexaminedusing Jasco FT/IR-4200 spectrophotometer which was equipped with JascoATRPRO470-Hsinglereflectionaccessory.Themeasurements wereperformedinabsorbancemode.Thespectrawerecollected overawavenumberrangeof4000and1800cm−1.After50scans, themeasurementswereevaluatedwiththeFT-IRsoftware(Spectra Manager-II,Jasco).
2.7. Preparationofphysicalmixtures
Physical mixtures of the components of the drug loaded
(r)andtheLorentz-Lorenzplot(Rm-Rc)/Rmvalues(%)incaseofthedifferentHPC:PVA massratio.
Fig.2.Lorentz-Lorenzplotvaluesofpapaverine-HClloadedgels(Rmisa measured-andRcisacalculatedmolarrefractionindex).
2.8. Scanningelectronmicroscopicanalysis(SEM)
Detailedmorphologyofthefiberswascharacterizedby scan-ningelectronmicroscope(SEM).SEMstudieswereperformedbya HitachiS-4300instrumentequippedwithaBrukerenergy disper-siveX-rayspectroscope(HitachiScienceSystems,Ltd.,Japan).The surfacesofsampleswerecoveredbyasputteredgoldconductive layer,and5–10kVacceleratingvoltagewasusedfortakinghigh resolutionelectronmicrographs.
3. Results
3.1. TheLorentz-Lorenzanalysis
Densityandrefractiveindexweredeterminedincaseofeach gels.Rmvaluesandspace-fillingfactorswerecalculatedusingEqs.
(1and 3),respectively. Rm values wereplottedasa functionof PVAconcentrations(%w/w)andalinearwasfitted.Theslopeof linearregression was2.4024and thelinearregressionaccuracy values(R2)was0.9999.Accordingtotheregressionlines,Rcvalues werecalculated.Table1summarizesthemeasured(Rm)andthe calculated(Rc)molarrefractions,thespace-fillingfactorsandthe Lorentz-Lorenzplot(Rm-Rc)/Rmvalues(%).Thenearlyunchanged space-fillingfactorvaluesrefertoalmostequalmolecular mobil-ity.Lorentz-Lorenzplotvaluesofthepapaverine-HClloadedgels asfunctionofHPC:PVAmassratioareshowninFig.2.According totheresults,alongwithincreasingHPCratio,RmandRc values arecontinuouslydecreasing,whichindicatesthelackofspecific
Fig.3.Ratioofstorage(G’)andloss(G”)moduliofpapaverine-HClloadedgelsasa functionofHPC:PVAmassratio(dynamicmodulimeasuredatafrequency1.995Hz).
3.2. Rheologicalmeasurements
Theviscoelasticityofgelsinfluencesthejetformationandthe jetstability, soit is oneof themostcriticalparameters forthe successfulelectrospinning process. Oscillatorytest was usedto determinethestorageandlossmoduli.Thetestswerecomparedat differentfrequencyvalues.TheresultsofG”/G’valuesdisplayedthe sametendencyatthedifferentfrequencyvalues,thereforeonlythe resultscorrespondingto1.998s−1valuewerepresentedinFig.3.
Alocalminimumwasobservedinthedynamicmodulusratio ofHPC:PVA=8:2sample.Themodulusratiodecreases monoton-icallyreachingaminimumvalueandfollowingtheincrease.The smallestratioreferstothemostelasticbehaviorofthecomposite gelcomparetotheotherHPC:PVAratios.Thelattersuggeststhat thepreponderantviscoelasticityofthesamplecouldbe disadvanta-geousfromthepointoffiberformation,sincetheelectricalforces cannotbeeffectivelytransferredtotheviscoelasticelongationof thepolymerjetsinthecourseofthefiberformation.
3.3. Morphologystudyofthesamples
Theelectrospinningofpapaverine-HClcontaininggelsresulted inawell-defined,moreorlessroundshapedsamplesonthe alu-minumfoil.Mostofthepreparedsamplescouldbeeasilypeeledoff fromthealuminumfoil,whichisbeneficialforthefurtherstudies andalsoforthefinalapplications.OnlythesampleofHPC:PVA9:1 massratiowastheexception,sincethefirmadhesioninhibitedits removal.
ThemorphologyofthepreparedsampleswastrackedbySEM measurements.TheresultsindicatethatwithincreasingHPCratio toPVAthemorphologywaschanged;transitionfromspray-dried film-likestructuresthroughfibrousfilmstofiberscanbeobserved whichisillustratedbyFig.4.
Partiallyspray-driedfilmsand beadedfiberswereformedin caseofHPC:PVA9:1massratio,whichcanbeobservedinFig.4E.
Thesimultaneouselectrostaticsprayingandelectrospinningofthe viscouspolymersolutionresultedintheappearanceoffilmsand beadedfibers.
Incaseof8:2ratio,afibrousfilmmorphologycanbeobserved (Fig. 4D), while at 7:3 ratio also the fibrous film formation is dominant(Fig. 4C), but in the latter case thefibrous structure
Fig.4.SEMphotosofthespray-driedfilm-to-fiber transitionasafunctionof HPC:PVAmassratio5:5(A),6:4(B),7:3(C),8:2(D),9:1(E).
(Thetotalpolymerconcentrationwas15%(w/w).Duringthefiberforming pro-cess30kVvoltagewasapplied.Theflowratewas0.2ml/handneedletocollector distancewas15cm.)
With the decreasingHPC ratio the fibrous morphology will become thedominant feature. Thetransition from film forma-tiontothe mainlyfibroussystemis initiated at8:2 massratio andcompletedby6:4ratio.AlongwiththedecreaseofHPCratio theproportion of thefibrous parts in the electrospunsamples increased.
3.4. FT-IRanalysis
The FT-IR spectra of various samples,physical mixture and thecomponentsarepresentedinFig.5.Thecharacteristic peak of themodeldrug aroundat 2300–2700cm−1 can beobserved clearly in thephysical mixture.Another characteristicpeaks of thepapaverine-HClwereoverlappedwithpeaksfromfunctional groupofthepolymers,sothattoverifytheamorphoustransitionof thedrugthe2300–2700cm−1wavenumberrangewasevaluated.
Incontrasttocrystallinematerials,wherethelimited rotational-vibrationaltransitionsareallowed,inamorphousmaterialsseveral transitionscantakeplace.Sincetheabsorbedenergyisdissipated amongthenumeroustransitions,broadeningandmergingofthe spectrum is a common phenomenon as a result of crystalline-amorphoustransition.
Thelackofthehighintensitycharacteristicpeaksofvariousfiber samplesindicatesthatthepapaverine-HClwasincorporatedinto thenanofibersinanamorphousstateasaresultofthe electrospin-ningprocess.
4. Discussion
Thereisadesiredchainentanglementdensity,whichis nec-essary to produce electrospun fibers [30]. The optimalchange entanglement,where overlappingofpolymericchainsrendersa flexiblesupramolecularstructuretothegels,isdevelopedatagiven concentrationrange resulting in good fiber formingproperties.
Ifthechainentanglementbecometooextensive,inotherwords unfavourableoverlappingofpolymericchainstakesplace,instead oftheformationofdefect-freeindividualfibersthefibrousfilm for-mationisdominant.Alterationofpolymericchainentanglement, thusthechangeofpolymericchainoverlappingcanbeexplained bytheobtainedresults,since molarreflectanceand rheological parametersalsodependontheinter-andintrachaininteraction ofpolymers.Themolarreflectancecharacterizationresults indi-catedthattheincreaseinHPCcontentresultedintheenhanced entanglementsofpolymerchains,whichisdisadvantageousinthe
Fig.5.FTIR spectraofpapaverine-HCl(a),hydroxypropylcellulose (HPC)(b), poly(vinylalcohol) (PVA)(c), polysorbate20 (d), physicalmixture (e),fibers
elasticbehaviorofthesamplesresultedinmorefibrousstructure.
Theobservedphenomenonisingoodagreementwiththeauthors’
previousstudies,wheretheadhesivenessandtheviscosityvalues hadalocalminimum,whichcouldleadtohigherproductivityof thefiberformationprocessandoptimummicromorphologyofthe emittedfibers[38].
5. Conclusion
Papaverine-HCl loadedHPC-PVA composite nanofibrous sys-temswerepreparedsuccessfullybyelectrospinningprocess.The optimumcomposition ofHPC-PVAfiberswasdeterminedbased ontherheologicalandmolarreflectancecharacterizationoftheir solution.
This formulation could enable the transmucosal amorphous drugdelivery,thusimprovingtheoralbioavailabilityofthepoorly soluble crystallinepapaverine-HCl, meanwhile avoiding itsfirst passmetabolism.
This formulation could enable the transmucosal amorphous drugdelivery,thusimprovingtheoralbioavailabilityofthepoorly soluble crystallinepapaverine-HCl, meanwhile avoiding itsfirst passmetabolism.