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Availableonlinewww.
jocpr.
comJournalofChemicalandPharmaceuticalResearchISSNNo:0975-7384CODEN(USA):JCPRC5J.
Chem.
Pharm.
Res.
,2011,3(4):410-421410ThesystemSLN-Dextranhydrogel:AnapplicationforthetopicaldeliveryofKetoconazolePatriziaPaolicellia,FedericaCorrentea,DanielaSerricchioa,FeliceCerretoa,StefaniaCesaa,BeatriceTitab,FedericaVitalib,FeliciaDiodataD'Auriac,GiovannaSimonettic,MariaAntoniettaCasadei*aaDipartimentodiChimicaeTecnologiedelFarmaco,"Sapienza"UniversitàdiRoma,PiazzaleAldoMoro5,00185Rome,ItalybDipartimentodiFisiologiaeFarmacologia,"Sapienza"UniversitàdiRoma,PiazzaleAldoMoro5,00185Rome,ItalycDipartimentodiSanitàPubblicaeMalattieInfettive"Sapienza"UniversitàdiRoma,PiazzaleAldoMoro5,00185Rome,ItalyABSTRACTKetoconazole(KTZ)isawidelyusedantifungaldrug.
Despiteitsbroadspectrumasantifungalagent,itsuffersfromtwocharacteristicsthatmakedifficultitsutilization.
Itispoorlywatersolubleandundergoesveryeasilychemicaldegradation,suchasoxidationandhydrolysis.
Thepaperreportsanewsystembasedonsolidlipidnanoparticles(SLN)entrappedintopolysaccharidichydrogelstobeemployedasmodifieddeliverysystemofketoconazoleintopicalformulations.
Threedifferentlipidphases(compritol,precirolandamixtureprecirol/almondoil)wereemployedtoprepareSLNandallthenanoparticleswerecharacterizedfortheirmeanparticlediameter,polidispersionindexandzetapotential.
Allthesystemswereabletoefficientlyencapsulatethedrug(entrapmentefficiency>90%).
SLNformulationswereabletoprotectthedrugfromUVdegradation,evenifpartially.
TheincorporationofKTZ-loadedSLNintodextranhydrogelsallowedthepreparationofsystemshavingrheologicalcharacteristicssuitablefordifferenttopicalapplications.
TheefficacyofthesystemhasbeenexploitedtestingtheantifungalactivityagainstCandidaalbicans,whereastheskintolerabilityofallthepreparationcomponentswastestedonrabbits.
Keywords:Solidlipidnanoparticles;Dextranmethacrylatehydrogels;Ketoconazole;Modifiedrelease;Candidaalbicans.
INTRODUCTIONInrecentyearsaremarkableincreaseoffungalinfectionshastakenplace.
Opportunisticinfections,suchasaspergillosis,candidiasis,andcryptococcosis,haveemergedasmajorproblemsincancerpatients,transplantrecipients,andotherimmunocompromisedindividuals,MariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421411includingthosewithAIDS[1-3].
Vulvovaginalcandidiasisisacommonmucosalinfectionaffectingmanywomen,withseveralcaseofrecurrenceandtherapeuticfailure,upto75%ofallwomenexperienceatleastoneepisodeduringtheirlife[4].
Candidaalbicansistheprincipalresponsibleaccountingforover80%oftotalvulvovaginalcandidiasis.
TheantifungalpharmacotherapywasrevolutionizedsincetheNinetiesbytheintroductionofazolicdrugs.
Ketoconazole(KTZ),cis-1-acety-l-4-[4-2-(2,4-dichlorophenyl)-2-2-(1H-imidazole-1yl-methyl)-1,3-dioxolan-4-yl]methoxypiperazine,isabroadspectrumagentusedinthetreatmentofsystemicandsuperficialfungalinfections[5].
Recently,newformulationsofKTZhavebeenbroughttothemarketforthetreatmentofseborrheicdermatitis,givinganewlifetoitstopicaluse[6].
KTZshowslimitedwatersolubility;moreoverthiscompoundisnotverystablebecauseitundergoeschemicaldegradation,suchasoxidationandhydrolysis,easily[7].
Solidlipidnanoparticles(SLN)representaninterestingapproachtoaddressbothsolubilityandstabilityproblemsofKTZ.
SLNareanefficientcolloidalcarriersystemforthemodifieddeliveryoflipophilicdrugs[8-10].
Theyarecomposedofphysiologicallipidsandfortheirhighbiocompatibilityfindapplicationincosmeticanddermatologicalpreparations[11]aswellasinparenteralandoraldrugformulations[12-16].
Furthermorethistypeofsystemscanoffereffectiveprotectiontolight-and/orwater-sensitivedrugs.
RecentlysomeattemptsweremadetoloadKTZintoSLNorNLCinordertoimproveitsstability[17,18].
Wehavealreadyproposedanewsystemconstitutedbysolidlipidnanoparticles(SLN)entrappedintoachemicaldextran-basedhydrogelforthecontrolleddeliveryoflipophilicdrugsinoralformulations[19]aswellasintopicalones[20].
Themodellipophilicdrug(S)-(+)-2-(4-isobutylphenyl)-propionicacid(ibuprofen)wasusedtotestthesystem.
WenowplannedtoinvestigatethepossibilityofemployingtheSLN-hydrogelsystemtoformulateKTZ.
Twolipidswithdifferentmeltingpoints,addedornotwithalmondoilwereusedtopreparetheSLNandtheobtainednanoparticleswerecharacterizedfortheirphysical-chemicalproperties.
ThenSLNwereentrappedintochemicalpolysaccharidichydrogelsandthereleaseprofilesofketoconazolewerestudied.
MoreovertheSLN-loadedhydrogelsweresubmittedtorheologicalmeasurementsinordertoassesstheirpossibleemploymentastopicalformulations,whereastheirantifungalactivitywasevaluatedinvitroagainstCandidaalbicans.
Finallyprimaryskinirritationstudiesofallthecomponentsoftheformulationwereperformedonrabbits.
EXPERIMENTALSECTION2.
1.
MaterialsAlltheusedreagentswereofanalyticalgrade.
Dextran(DEX)fromLeuconostocssp.
(Mr40000),dimethylsulfoxide(DMSO),glycidylmethacrylate(GMA),4-dimethylaminopyridine(DMAP),ammoniumperoxydisulphate(APS),N,N,N',N'-tetramethylethylendiamine(TEMED)andcholicacidsodiumsaltwerepurchasedfromFluka(Switzerland),Sephadex75-GfromSigma(USA),D2OfromAldrichChemical(USA),LutrolF68(poloxamer188)fromBASF(Germany).
Ketoconazole(KTZ)wasakindgiftofJansenn(Italy).
PrecirolATO5andCompritol888ATOwerekindlyprovidedasagiftbyGattefossè(France).
PrecirolATO5consistsofamixtureof40%tri-,45%di-and14%monoglyceridesofpalmiticandstearicacids.
Compritol888ATOisamixtureof13-21%mono-,40-60%di-and21-35%triglyceridesofpalmitic,stearic,arachidic,behenic,erucicandlignocericacids,withbehenicacidasprincipalcomponentaccountingformorethan83%ofthetotalfattyacids.
PurealmondoilwaspurchasedfromAgrar(Italy).
Dialysismembranes(cut-off12,000-14,000)werefromMedicellInternational(U.
K.
).
CandidaalbicansATCC24433wasobtainedfromAmericantypecultureMariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421412collection(ATCC,Rockville,MD,USA).
RPMI1640,MOPSandsabourauddextroseagarwerepurchasedfromSigma-Aldrich(USA).
2.
2.
Synthesisofdextranmethacrylate(DEX-MA)Dextranmethacrylatewassynthesizedasalreadyreported[21].
4-DMAP(2.
0g)andGMA(1.
5g,2mol/6molofrepetitiveunit)wereaddedtoasolutionofdextran(5.
0g)inDMSO(40ml).
Thesolutionwasmaintainedunderstirringatroomtemperaturefor24h.
ThepolymerwasprecipitatedwithEtOH(200ml),recoveredbyfiltration,dissolvedinwater(15ml)andthesolution,adjustedtopH8with1.
0MHCl,wassubmittedtoexhaustivedialysis.
Afterfreeze-dried,thepolymerwascharacterizedbyFT-IR,13C-and1H-NMR.
FT-IRspectrawererecordedwithaPerkinElmerParagon1000spectrophotometerintherange4000-400cm-1usingKBrpellets(resolutionof1cm-1).
1H-NMRspectrawereobtainedwithaBrukerAC-400instrument.
Thedegreeofderivatization(amountofmethacryloylgroupsfor100glucopyranosylresidues,DD),calculatedonthebasisofthe1H-NMRspectrum,asreportedintheliterature[21,22],was40±1.
DEX-MAhavingDD=20±1wassynthesizedwiththesameprocedure,using1.
0gof4-DMAPand0.
73gofGMAfor5.
0gofstartingpolymer.
2.
3.
Preparationofsolidlipidnanoparticles(SLN)Solidlipidnanoparticleswerepreparedwiththehothomogenizationtechnique[23].
Fivegoflipid[PrecirolATO5,PrecirolATO5/almondoil85:15(w/w)orCompritol888ATO]wereheatedatatemperatureatleast10°Cabovetheirmeltingpoint(55.
2,54.
5and73.
2°Crespectively).
Whencompletelymelted,thelipidphasewasaddedwithanaqueoussolution(45ml)ofthesurfactants(sodiumcholate,0.
50gandpoloxamer,1.
25g),heatedatthesametemperature[24].
ThemixturewashomogenizedwithanUltra-TurraxT18Basic(IKA-WERK,Germany)for10minutesat24000rpmandthenlefttocooldowntoroomtemperature.
DrugloadedSLNwerepreparedbyaddingKTZ(0.
20g)tothelipidphaseandfollowingthepreviouslydescribedprocedure.
SampleswerelabeledasSLNP,NLCPO,SLNC,SLNPK,NLCPOKandSLNCK,whereP,O,CandKstandforPrecirolATO5,almondoil,Compritol888ATOandketoconazolerespectively.
2.
4.
ParticlesizeanalysisandzetapotentialmeasurementsTheparticlesizeanalysisofallSLNsampleswasperformedbyphotoncorrelationspectroscopy(PCS)immediatelyafterthepreparation.
ThePCSanalysisyieldedthemeandiameteroftheparticles(Z-average)andthepolidispersityindex(PDI),asameasureofthewidthoftheparticlesizedistribution.
ThesampleswereproperlydilutedwithtwicedistilledwaterandanalyzedwithaMalvernZetasizernanoZS90(MalvernInstruments,UK).
Thesurfacechargeofallthesampleswasdeterminedbymeasurementsofthezetapotentialcarriedoutwiththesameinstrument.
2.
5.
Differentialscanningcalorimetry(DSC)measurementsDSCmeasurementswerecarriedoutwithaSETARAMDSC131(France),equippedwithSETSOFT2000.
Thesampleswerequicklyfrozeninliquidnitrogenandfreeze-driedatroomtemperatureandpressureof0.
4-0.
5Hgmm.
Allfreeze-driedsampleswereleftundervacuumonP2O5for3daysbeforetheDSCinvestigationwascarriedout,inordertoensurethecompleteeliminationofthemoisture.
Exactlyweighedsamples(10mg)weresealedinstandardaluminiumpans(30l)andsubmittedtocalorimetricanalysisatascanrateof10°C/mininthetemperaturerange15-160°C,usinganemptypanasreference.
Analyseswereperformedundernitrogenpurge.
MariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-4214132.
6.
DeterminationofencapsulatedketoconazoleTheamountofKTZencapsulatedintothethreeSLNsystemswasdeterminedbyextractingthedrugfromthenanoparticlesafterseparationoftheun-encapsulatedKTZ.
Tothisend,aliquotsofeachSLNpreparationwerequicklypassedthroughacolumnofSephadex75-G,employingdistilledwateraseluant.
Therecoverednanoparticleswerelyophilizedandtheresiduesextractedwithmethanol.
TheamountofthedrugwasdeterminedbymeansofHPLCanalysis,monitoringat240nm.
HPLCapparatusconsistedofaPerkinElmerSeries200LCpump,equippedwitha235DiodeArrayDetector.
HPLCanalyseswerecarriedoutusingaMerckHibarLiChrocart(250-4,5m)RP-18column,withaflowof1ml/min.
Amixtureofaqueousdiethylamine(1%w/w)andmethanol(20:80)wasusedaseluantfortheanalyses.
Alltheexperimentswereperformedintriplicateandtheresultsreportedasmeanvalues±S.
D.
2.
7.
KetoconazolestabilitytoUVirradiationKTZ(20.
0mg)wasdissolvedinMeOH(5ml)andsubmittedtoUVirradiationperformedwithaHeliosItalquartzPhotochemicalMultiraysReactor(Italy)equippedwithten,14wattmediumpressure,mercurylamps(λmaximum310nm)for4h.
Atpre-establishedtimeintervals(1,2,3and4h),aliquotsoftheKTZsolutionwerewithdrawnandtheamountofresidualdrugwasdeterminedbymeansofHPLCanalysis,aspreviouslydescribed.
SameexperimentswereperformedonSLNPK,NLCPOKandSLNCKsamples.
Aftertheirradiation,thenanoparticlessampleswerefreeze-dried,theresidueswereextractedwithMeOHandanalyzedasalreadydescribed.
2.
8.
HydrogelspreparationDEX-MA(DD=20and40,1.
25g,2.
5%w/w)wasaddedtoalltheSLNsamples.
Thecross-linkingreactionofDEX-MAwascarriedoutaddingappropriateamountsofAPS(0.
77mmolAPS/mmolmethacrylicgroups)andTEMED(0.
2mmol)to10mlofeachpreparation.
Inthesameway,DEX-MA20hydrogelswithapolymercontentof3.
75and5%w/vwerealsopreparedbyfreeradicalpolymerization.
2.
9.
DrugreleasestudiesDrugreleaseexperimentswereperformedonfreshSLN-dextranhydrogels.
Samplesoftheformulations(0.
05g)wereplacedin50mlofacetatebuffer(pH=4.
0)andmaintainedunderstirringat37.
0±0.
1°Cfor8hours.
Atappropriatetimes,0.
5mlofreleasemediumwerewithdrawnandreplacedwiththesameamountoffreshmedium.
ThereleaseofthedrugwasfollowedbymeansofHPLCanalysis,asdescribedbefore.
Alltheexperimentswerecarriedoutintriplicateandtheresultswerereportedasmeanvalues±SD.
2.
10.
RheologicalmeasurementsRheologicalexperimentswereperformedwithaHaakeRheoStress300RotationalRheometer(Germany)equippedwithaHaakeDC10thermostat.
OscillatoryexperimentswereperformedonalltheSLNsuspensions,preparedwiththedifferentlipidphases.
MechanicalspectrawerealsorecordedafteradditionofDEX-MAandcross-linkingreaction.
Enoughquantityofeachsamplewascarefullypouredtocompletelycoverthe6cmcone-plategeometry(angleof1°).
Frequencysweepexperimentswereperformedonallthesamplesat25.
0±0.
2°Cintherange0.
01–10Hz,inthelinearviscoelasticregion,assessedbypreliminarystresssweepexperiments.
Themechanicalspectrawerethenrecordedapplyingaconstantdeformationinthelinearregion:usuallya1%maximumdeformationwasused.
MariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-4214142.
11.
MicrobiologicalstudiesTheactivityofsamplesofdextranhydrogelentrappingSLNPKwastestedagainstC.
albicansATCC24433,recommendedasreferencestraininCLSIstandardprotocol[25]andcomparedwithacommercialcream(Nizoral2%,J&J)andaketoconazolesolution.
Experimentswereperformedaspreviouslydescribedwithsomemodifications[26].
ExperimentswerecarriedoutinRPMI1640mediumattwodifferentpHvalues(7.
0and5.
0).
RPMI1640waspreparedaccordingtoCLSIstandard[25]andmediumwasbufferedtopH7.
0or5.
0withMOPS.
Forketoconazolesolution,thedrugwasfirstdissolvedinDMSOataconcentration100timeshigherthanthetestconcentration,asrecommendedbyCLSI[27]andthendilutedinbufferedmediumataconcentrationof50g/ml.
Atuseddrugconcentrationthesolventdidnotinterferewithcellviability.
SamplesofSLNPKDEX-MAhydrogel,commercialcreamandketoconazolesolution,containing50g/mlofdrug,wereinoculatedwithC.
albicanscellssuspensionadjustedtoattainafinalconcentrationof1x106cells/mlandincubatedat37°Cfor4,6,24and48h.
Suspensionswerethenappropriatelydilutedinsaline,spreadbyinclusioninsabourauddextroseagarandincubatedat37°Cfor24h;Colony-FormingUnitswerethenquantified.
Theexperimentswereperformedintriplicateonthreeconsecutivetestdays.
2.
12.
In-vivoskintolerabilitystudiesThreehealthymaleNewZealandWhiterabbitswerepurchasedfromCharlesRiver(Calco,Lecco,Italy)andacclimatedinthelaboratoryforaweek.
Therabbitswereindividuallyhousedandreceivedstandarddietandtapwateradlibitum.
Principlesoflaboratoryanimalcare(EECDirectiveof1986;86/609/EEC)guidelineswerefollowed.
Thebackoftheanimalswasclippedfreeoffurwithanelectricclipper24hbeforeapplicationofthesamples.
Theclippedareasoftheskinweredividedintofourpartsofsamearea(30*30mm).
DEX-MAhydrogelandthesystemsDEX-MAhydrogel/SLNP(loadedornotwithKTZ)wereappliedtothreesites(approximately500mg/site);whereasthelastpartwasusedasacontrol.
Thetreatedandthecontrolsiteswerecoveredbygauzeandthebackoftherabbitwaswrappedwithanon-occlusivebandage,thereaftertheanimalswerereturnedtotheircages.
After4h,thebandageandthetestmaterialwereremovedand1hlaterthesiteswereexaminedforskinirritation.
Observationofthesiteswithmaterialandcontrolwasrepeatedafter24,48and72h.
Thereaction,definedaserythema(Er)oredema(Ed),wasevaluatedaccordingtothescoreoftheskinreactionsalreadyreported[28].
TheScoreofPrimaryIrritation(SPI)wascalculatedforeachrabbitasthedifferencebetweenthesumofthescoresforerythema(Er)andedema(Ed),at24,48and72hdividedbythenumberoftheobservationsforthetreatedsitesandthesumofthescoresforerythema(Er)andedema(Ed),at24,48and72hdividedbythenumberoftheobservationsforthecontrolsites,accordingtotheformula:withT,treated;C,control;Er,erythema;Ed,edema.
ThePrimaryIrritationIndex(PII)wascalculatedasthearithmeticalmeanoftheSPIvaluesofthethreeanimals.
TheevaluationofPIIwasperformedaccordingtothecategoriesreportedintheliterature[28].
MariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421415RESULTSANDDISCUSSION3.
1.
Preparationandcharacterizationofsolidlipidnanoparticles(SLN)samplesSolidlipidnanoparticleswerepreparedwiththehothomogenizationprocedurealreadyreported[23].
Allthesampleswereanalyzedfortheirmeanparticlesize,sizedistributionandzetapotentialvalueandtheresultsarereportedinTable1.
Table1.
Physico-chemicalcharacteristicsofSLN.
Meandiameter(Z-average),polidispersityindex(PDI),zetapotentialandentrapmentefficiencyvaluesoftheSLNformulationsobtainedwithdifferentlipidphases(meanvalue±SD,n=3),containingornotketoconazole.
SampleMeandiameter(nm)PDIZetapotential(mV)EntrapmentEfficiency(%)SLNP148±30.
281±0.
008-26.
8±1.
0------SLNPK132±20.
270±0.
030-33.
6±1.
989±4SLNC202±10.
212±0.
010-31.
7±0.
7------SLNCK266±30.
234±0.
012-23.
3±1.
494±3NLCPO131±30.
104±0.
007-36,4±1.
1------NLCPOK156±20.
177±0.
006-30.
4±1.
795±2Ingeneral,theaverageparticlediameteroftheSLNdispersionsincreaseswiththemeltingpointofthelipids,becauseofthehigherviscosityofthedispersedphase[23].
InfacttheaveragesizeofSLNPwasfoundtobesignificantlysmaller(148±3nm)thanthesizeofSLNC(202±4nm).
InaccordancewiththishypothesistheadditionofanoiltoPrecirolATO5gaverisetoparticleswithanaveragediameterof131±3nm.
Inallthecasesthevaluesofpolidispersityindex(PDI),ameasureofthewidthoftheparticlesizedistribution,werelowerthan0.
3soindicatingagoodhomogeneityofthedispersions.
Furthermorethesurfacechargeoftheparticleswasalwaysnegativeandthevalueswerehighenoughtoassureagoodstabilityofthenano-suspensions.
DSCexperimentswerealsocarriedoutonthecomponentsofthepreparations(Table2).
Table2.
Calorimetricstudies.
DSCmeltingpeakdataofSLNC,SLNPandSLNPOsamples.
Meltingpeak(°C)BulklipidSLNSLNKCompritolATO88873.
272.
971.
4PrecirolATO555.
254.
654.
1PrecirolATO5/almondoil54.
552.
351.
5ThemeltingpointofbulkCompritol888ATOis73.
2°C.
WhenthislipidisformulatedasSLN,thevalueofthemeltingpointdecreasesto72.
9°C,probablyforthepresenceofthesurfactants.
InthesampleofSLNcontainingKTZ(SLNCK),themeltingpointdecreasestill71.
4°Cshowingthataninteractionbetweenthelipidphaseandthedrug(havingmeltingpoint153.
5°C)hastakenplaceandthatthelatterisatleastinpartloadedintotheparticles.
Thisbehaviorisalreadydescribedintheliteratureforsimilarsystems[29,30].
AnalogousresultswereobtainedfortheformulationsSLNPandNLCPO.
EveninthesecasestheformulationofthelipidsasnanoparticlesMariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421416causedashiftofthemeltingpeak,whichwasfurthermodifiedbytheincorporationofthedrug,thusindicatingtheoccurrenceofaninteractionbetweenthelipidmatrixandKTZ.
TheamountofKTZencapsulatedintoeachSLNsamplewasdeterminedbyextractingthedrugfromthenanoparticles,afterseparationoftheun-entrappedKTZbygelfiltrationchromatography.
Theresultsshowveryhighentrapmentefficiencyforallthesystemsandaverylittleinfluenceofthelipidnature(Table1).
3.
2.
KetoconazolestabilitytoUVirradiationInourpreviouspapers[19,20]dextranhydrogelswereobtainedbyUV-inducedcross-linkingofDEX-MA.
UnlikelyKTZisaphotosensitivedrug[7].
ThereforeasetofpreliminaryexperimentswereperformedinordertoassessifdrugencapsulationintoSLNmayprotectitagainstUVinduceddegradation.
Tothisend,KTZloadedSLNpreparedwiththethreelipidphasesweresubmittedtoirradiationat310nmfor4hourstogetherwithsolutionsofthedruginmethanol,usedasacontrol.
AllthesampleswereanalyzedbyHPLCandtheresultsarereportedinFigure1.
0204060801001234%recoveredKTZtime(h)MeOHSLNckSLNpkNLCpokFigure1.
UVstabilityofketoconazole.
Percentagesofketoconazole(%w/w)recoveredafterUVirradiation(310nm).
ItcouldbeobservedthatKTZmethanolicsolutionsunderwentaprogressivedegradationandturnedbrown-colored,withlessthan6%ofdrugrecoveredafter4hofUVirradiation.
ThepercentagesofresidualdrugweremuchhigherintheSLNsamples,suggestingthatthedegradationmainlyoccurredontheparticlessurface,withatrenddependentonthemeltingpointofthelipid.
InfactCompritol,thehighermeltinglipid,seemedtobeabletoprotectKTZmoreefficientlythanPrecirol,addedornotwithalmondoil.
ItislikelythatthemobilityofthedrugwithinSLNCwasrestricted,thereforeitmovedtowardsthenanoparticlessurfaceinlesserextentduringtheirradiationtime.
Howevertheencapsulationreduceddeeplybutnotcompletelythedrugdegradation;therefore,inordertoavoidthelossofnotnegligibleamountofactiveprinciple,thehydrogelswereproducedbychemicalreagents,accordingtoaprocedurereportedintheliterature[22].
3.
3.
HydrogelspreparationandreleasestudiesThehydrogelscontainingthethreeSLNformulations(SLNPK,NLCPOK,SLNCK)weresubmittedtoreleasestudies.
ThereleaseprofilesarereportedinFigure2.
MariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421417Figure2.
Effectofthelipidphaseonthereleaserateofketoconazole.
Releaseprofiles[(Mt/M∞)x100]ofketoconazolefromthesystems--SLNPK/DEX-MA40,--SLNCK/DEX-MA40and--NLCPOK/DEX-MA40,maintainedat37.
0±0.
1°Cinaceticbuffersolution(pH4.
0)for6h.
Thelipidnatureinfluencesthedrugreleaseprofile,infactSLNPKreleaseKTZatafasterratethanSLNCKandNLCPOKevenif,inanycase,alltheloadedketoconazoleisreleasedwithin6hours.
Nosignificantdifferencescouldbeevidencedwhendextranswithdifferentderivatizationdegreewereemployed(Figure3).
Figure3.
Effectofthecross-linkingdegreeofthehydrogelonthereleaserateofketoconazole.
Releaseprofiles[(Mt/M∞)x100]ofketoconazolefromthesystems--SLNPK/DEX-MA20and--SLNPK/DEX-MA40maintainedat37.
0±0.
1°Cinaceticbuffersolution(pH4.
0)for8h.
3.
4.
RheologicalmeasurementsRheologicalbehaviourisacrucialcharacteristicfortheapplicationofthehydrogelsintopicalformulations[31].
Atopicaldosageformneedstohaveasemisolidconsistency,tobeeasilyspreadableontheskinorabletoremainintheapplicationsitewhenemployedforvaginalapplications.
Inourpreviousstudy,theeffectofthelipidphaseontherheologicalbehaviourofdextranhydrogelsloadedwithdifferentSLN,wasinvestigated[20].
SLNsamplesshowedmechanicalspectra,recordedintherange0.
01–10Hz,typicalofaviscoelasticliquidwiththedissipativemodulusG''biggerthantheelasticoneG'[16].
AftertheadditionofDEX-MAandphotocross-linkingreaction,themechanicalspectrachangeddeeplyandthesystemsshowedweakgelbehaviour.
Inparticular,thegelstrengthwasaffectedbythelipidphaseaswellasbythederivatizationdegreeofDEX-MA.
Takingintoaccountthesepreviousresults,inordertoobtainformulationswithasuitabletexture[31,32],DEX-MA20wasemployedforSLNsamplesMariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421418preparedwithlipidshavinghighermeltingpoint,whereasDEX-MA40wasusedfortheotherones.
Therefore,DEX-MA40(2.
5%w/w)wasaddedtothenano-suspensionsofSLNPandNLCPOandafterchemicalreticulationthemechanicalspectrareportedinFigure4wereobtained.
Figure4.
Rheologicalstudies.
MechanicalspectraofthehydrogelsSLNPK/DEX-MA40(G';G")andNLCPOK/DEX-MA40(G';G")at25.
0±0.
2°C.
G'andG''valueswereinfluencedbythelipidnatureandforSLNPresultedcomparablewiththoseofthehydrogelsobtainedbyphoto-crosslinking[20].
DEX-MA20at2.
5%w/wwasemployedforSLNCsamplesandtherelativemechanicalspectrumisreportedinFigure5andcomparedwiththesystemcontainingSLNP.
Figure5.
Rheologicalstudies.
MechanicalspectraofthehydrogelsSLNCK/DEX-MA20(G';G")andSLNPK/DEX-MA20(G';G")at25.
0±0.
2°C.
Figure6.
Rheologicalstudies.
MechanicalspectraofthehydrogelsSLNPK/DEX-MA20obtainedwithdifferentamountofthepolymer:5%w/v(G';G"),3.
75%w/v(G';G")and2.
5%w/v(G';G")at25.
0±0.
2°C.
MariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421419Itisevidentthatthenatureofthelipidphaseinfluencesthegelstrength.
Allthesystemsshowedaweakgelbehaviour,withtheelasticmodulusG'biggerthanthedissipativemodulusG'',aG'/G''ratio<10,andvaluesofbothmodulusquiteindependentfromtheappliedfrequency.
Theinfluenceofthepolymerconcentrationwasalsoevaluated.
ThemechanicalspectraobtainedonsamplesofSLNPafteradditionofDEX-MA20atdifferentconcentrationsandcross-linkingreactionarereportedinFigure6.
Theconsistenceoftheformulationcanbeopportunelymodulatedvaryingtheconcentrationofthepolymer,becausethevaluesofG'andG''arerelatedtoDEX-MAconcentrationandincreasewiththislater.
Inallthesamplestheviscosityisdependentfromtheshearstress;inparticularηdecreaseswhenγincreases,accordingtoabehaviourcharacteristicofpseudo-plasticsystems(Figure7).
Figure7.
Rheologicalstudies.
FlowcurvesofthehydrogelsSLNCK/DEX-MA40(--);SLNPK/DEX-MA40(--)andNLCPOK/DEX-MA40(--)at25.
0±0.
2°C.
3.
5.
MicrobiologicalstudiesWeinvestigatedtheinvitroantifungalactivityofSLNPKDEX-MAhydrogel,freeketoconazoleandatopicalskinformulationavailableonthemarket(Nizoral).
Forthepurposeofthisstudywehavechosentoincubatethesamplesfordifferenttimewithaninoculumof1x106aspreviouslydescribedwithsomemodifications[26].
AsitcanbeobservedinFigure8,thesamplesofSLNPKDEX-MAhydrogelinducedthegrowthinhibitionofCandidaalbicansATCC24433withareductionofcellsgrowthatpH5after4hofincubation,respecttountreatedcontrol.
Aremarkablereductionofcellsviabilitywasobservedat48hofincubationatbothpH5and7.
Finally,noappreciabledifferencesbetweenourpreparationandthemarketedonewereobserved.
WealsoevaluatedtheeffectofthepHofthemediumonantifungalactivity,becauseisknownthesusceptibilityofketoconazoleactivitytoenvironmentalpH[33].
AsseeninFigure8differentpHdidnotinterferewithsampleantifungalactivity.
3.
6.
In-vivostudies:primaryskinirritationexperimentsPrimaryskinirritationexperimentswerecarriedoutonrabbits,inordertoverifytheuseofthesystemSLN/dextranhydrogelastopicalformulation.
InparticularDEX-MAhydrogelscontainingornotSLNPandSLNPKweretestedaccordingtotheproceduredescribedinsection2.
12.
Individualresultsofskinirritationscoreswerealwaysequalto0.
00.
Intheanimalserythemaoredemawereabsentafter24,48and72hofapplicationofthethreesamples(DEX-MAhydrogel,SLNP/DEX-MAhydrogel,SLNPK/DEX-MAhydrogel)aswellasinthecontrol.
ThePIIofallthecomponentswasnegligible,indicatingtheabsenceofskinirritationand,asa.
MariaAntoniettaCasadeietalJ.
Chem.
Pharm.
Res.
,2011,3(4):410-421420consequence,thepossibilitytoemploythesystemSLNPK/DEX-MAhydrogelintopicalformulations.
Figure8.
Microbiologicalstudies.
InvitroactivityofSLNPK/DEX-MAhydrogel,acommercialcream(Nizoral)andaketoconazolesolutionagainstCandidaalbicansATCC24433.
Reportedresultsrefertoaninoculumof1x106cells/mlandtoadrugconcentrationof50g/ml.
ThepHofmediawas5.
0and7.
0.
Datarepresentmeans±standarddeviationfromthreeindependentexperiments,eachperformedintriplicate.
CONCLUSIONKetoconazolecanbeefficientlyencapsulatedintosolidlipidnanoparticlesthatareabletodeliverthedruginasustainedmannerwithoutcompromisingitsactivity.
TheincorporationofthesenanoparticlesintoDEX-MAhydrogelsgaverisetoabiocompatiblesystem,whoserheologicalpropertiescanbeconvenientlyandeasilyadjustedchangingthederivatizationdegreeand/ortheconcentrationofthepolymer,inordertoassureasemisolidconsistency,agoodspread-abilityandapleasanttexture,ortoremainintheapplicationsitewhennecessary.
OveralltheseresultssuggestagoodpotentialofthesystemSLN/dextranhydrogelforthetopicalmodifieddeliveryofketoconazole.
AcknowledgementsThisworkwassupportedbytheEnricoandEnricaSovenaFondation.
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Chem.
Pharm.
Res.
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