Journal of Biomedical SciencesISSN 2254-609X

2017Vol. 6 No. 1:12

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Research Article

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DOI:10.21767/2254-609X.100056

Tarek A Ahmed1,2 and Bader M Aljaeid1

1 DepartmentofPharmaceutics,FacultyofPharmacy,KingAbdulazizUniversity,Jeddah,KSA

2 DepartmentofPharmaceutics&IndustrialPharmacy,FacultyofPharmacy,Al-AzharUniversity,Cairo,Egypt

Corresponding author: Dr.TarekAbdelnapyAhmed

dr_tarek_nour@yahoo.com

AssociateProfessorofPharmaceuticsandIndustrialPharmacy,DepartmentofPharmaceutics,FacultyofPharmacy,KingAbdulazizUniversity,Jeddah,KSA.

Tel: 96626400000/22250

Citation:AhmedTA,AljaeidBM.APotentialIn Situ GelFormulationLoadedwithNovelFabricatedPLGANanoparticlesforEnhancingandSustainingtheOphthalmicDeliveryofKetoconazole. JBiomedicalSci.2017,6:1.

A Potential In Situ Gel Formulation Loaded with Novel Fabricated PLGA Nanoparticles

for Enhancing and Sustaining the Ophthalmic Delivery of Ketoconazole

AbstractOralketoconazole therapy is commonlyassociatedwith serioushepatotoxicity.Improvingoculardrugdeliverycouldbesufficienttotreateyefungalinfections.The purpose was to develop an optimized ketoconazole poly lactide-co-glycolide (PLGA) nanoparticles (NPs) with subsequent loading into in situ gel(ISG) formulation forophthalmicdrugdelivery.Three formulation factorswereoptimized for their effect on particle size (Y1) and entrapment efficiency (Y2)utilizingcentralcompositeexperimentaldesign.Interactionamongcomponentswasstudiedusingdifferentialscanningcalorimetry (DSC)and fourier transforminfrared (FTIR). Ketoconazole crystalline state was studied using powder X-raydiffractometer(XRD).SixdifferentpolymericISGformulationswerepreparedandloadedeitherwithoptimizedNPsorpuredrug.Theprepared ISG formulationswerecharacterizedforin vitrogelation,drugreleaseandantifungalactivity.Thepermeation through human epithelial cell line was also investigated. ResultsrevealedthatallthestudiedformulationsparametersweresignificantlyaffectingY1andY2of thedevelopedNPs.DSCandFTIRstudies illustratedcompatibilityamongNPscomponentswhiletherewaschangefromthecrystallinetoamorphousstateintheNPs.Thein vitroreleasefromtheISGformulationsloadedwithdrugNPsshowedsustainedandenhanceddrugreleasewhencomparedtopuredrugformulations. Also, ISG loaded with NPs showed enhanced anti-fungal activitywhen compared to pure drug formulations. Alginate-chitosan ISG formulationloaded with optimized ketoconazole NPs illustrated higher drug permeationthrough epithelial cell lines and is considered as an effective ophthalmic drugdeliveryintreatmentoffungaleyeinfections.

Keywords: Ketoconazole;PLGA;Nanoparticles;ISG;Ophthalmicdelivery

Received: December07,2016; Accepted: December27,2016; Published: January03,2017

IntroductionKetoconazole is a synthetic imidazole drug used primarily totreatdifferenttypesofbodyfungalinfections.ThedrugworksbyinhibitingtheenzymecytochromeP45014α-demethylasewhichcontributesinthesynthesisofthefungalergosterol[1].Recently,the U.S. Food and Drug Administration (FDA) announced awarningthatoraltreatmentwithketoconazolecancausesevereliver damages and also adrenal glandproblems [2].Unlike theoral administration, topical drug formulations have not beenassociated with these adverse effects. When ketoconazole

is administered for treatment of fungal eye infection, it ischaracterized by a very short duration of action since itseliminationhalf-life intheaqueoushumorandcornea isabout19 and 43 minutes, respectively [3,4]. Although ketoconazolecharacterizedbyitshighlipidsolubility(logP=4.74),whichmaysupport permeation through biological membranes especiallythrough the corneal epithelium, yet the drug high molecularweight(531.44Da)hinderitstransport.Moreover,it isdifficultfor the drug to present in a solubilized form in the aqueouscornealsurfaceduetothelimitedaqueoussolubility[5].

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Ocular drug administration offers lower incidence of systemicdrug side effects and drug-drug interactions that are commonduringsystemictreatment.However,duetotheuniqueanatomyand physiology of the eye, ocular drug delivery represents amajor challenge during development of effective ophthalmicpreparations. The eye is supported with static and dynamicbarriers,andeffluxpumpsthatimpededrugdeliveryparticularlytotheposterioreyesegment[6].Nanoparticulatedrugdeliverysystems have been reported to deliver the administered drugsuccessfully across the different eye segments including theposterior part [5,7]. Polymeric nanoparticles (NPs) especiallythose that have been developed using biocompatible-biodegradable polymers, such as polycaprolactone, poly (alkylcyanocaprylate) and poly lactide-co-glycolide (PLGA), arepromisingoculardeliverysystemduetoenhancedbioavailabilityand reduced frequency of administration [8,9]. PLGA basedNPsarethemostsuitablepolymernanoparticulateoculardrugdeliveryowingtoeaseoffabricationandapprovalbytheFDAfordrugdelivery[10,11].

The aim of this work was to develop ketoconazole PLGANPs utilizing novel in vitro solvent exchange technique withsubsequentloadingintoISGformulationtoenhanceandsustainthe ocular drug delivery. The prepared ISG preparations werecharacterizedforin vitrogelation,drugrelease,antifungalactivityandpermeationthroughhumanepithelialcelllines.

Materials and MethodsMaterialsPoly (DL-lactide-co-glycolide), ester terminated, inherentviscosityrange:0.55-0.75dL/gfromLactelabsorbablepolymers,Duracet corporation (Birmingham, AL USA). Polyvinyl alcohol,cold water soluble, Mol. Wt: 140,000 was purchased fromHiMedia Laboratories Pvt. Ltd. (Mumbai, India). KetoconazolewasakindgiftfromDeefPharmaceuticalIndustriesCo.(Alqassim,KSA). Poloxamer 407 was supplied from Xi’an Lyphar BiotechCo., LTD (Shaanix Province, China). Chitosan of lowmolecularweight,sodiumalginateanddimethylsulphoxide(DMSO)werepurchasedfromSigma-Aldrich(St.Louis,MO).Carbopol940andhydroxypropyl methyl cellulose (HPMC) MW 86,000, viscosity4000cp(2%solution)wereprocuredfromAcrosOrganics(NewJersey,USA).

MethodsCentral composite experimental design: Three formulationfactorswereoptimizedfortheireffectsontheparticlesize(Y1)anddrugentrapmentefficiency(Y2)ofthedevelopedNPsutilizingStatGraphics Centurion XV version 15.2.05 software (StatPointTechnologies Inc,Warrenton, VA, USA).Table 1 illustrates theindependent variables, their levels and studied responses. Apreliminary study was conducted to identify the levels of theindependentvariables.

Preparation of the formulation: Based on the compositionof the sixteen formulations, obtained from the experimentaldesign,which is depicted inTable 2, DrugNPswere prepared

by dissolving the calculated amount of PLGA in the specifiedvolumeofDMSO toprepare3-6%organicpolymer solution.Aconstantconcentrationofketoconazole(0.1%w/v)basedonthetotalformulation,wasdissolvedinthisorganicsolution.AqueousPVAsolutions(1-3%)werepreparedbydissolvingthespecifiedamount of PVA in distilledwater undermagnetic stirring. Theorganic drug polymer solution was gradually added througha micropipette into the aqueous PVA solution on a magneticstirrer.Themilkycolloidaldispersionobtainedwaskeptstirringfor 30 minutes at 1200 rpm and then was probe sonicated(SonicsVibracell,VCX750,USA)for10minat60magnitudesforbettercolloidaldispersion.DrugloadedNPswereseparatedbycentrifugationat15000rpmfor1hat4°Cusing(SigmaLaboratorycentrifuge,3K30,Ostrode,Germany).TheprecipitatedNPsweresubjected to twocyclesofwashingwithdoubledistilledwaterandcentrifugationtoremovetheremainingofPVAandDMSO.Finally,theseparatedNPswerefreezedriedusing(alpha1-2LDplus,Christlyophilizer,Germany).

Characterization of the Prepared NPsMeasurement of NPs particle sizeKnownweightofthefreeze-driedNPswassuspendedindoubledistilledwaterbyvortexandtheparticlesizeofthesuspendedparticles was determined by dynamic light scattering using aZetatrac(MicrotracInc.,York,PA).

Entrapment efficiency Following centrifugation, the supernatantwas filtered through0.2µmcelluloseacetatemembranefilterandtheconcentrationofthedruginthefiltratewasdeterminedspectrophotometricallyat 231 nm using Jenway 6715 UV-Visible spectrophotometer(Jenway, Stone, UK). The entrapment efficiency (EE) wascalculatedusingthefollowingequation:

EE(%)=[(Dtotal–Dfreedrug)/Dtotal]×100

WhereDtotal is the total concentrationof thedrug loaded, andDfree drug represents the concentration of the free drug in thesupernatant.

Central Composite Experimental Design Statistical Analysis and Optimum DesirabilityTheobservedvalues forbothY1andY2, tabulated inTable 2,wereintroducedintotheresponsecolumnsoftheStatGraphicsoptimization software and analyzed to identify the statisticalsignificanceof the relationships between the studied variablesand obtained responses. A p-value˂0.05 was consideredsignificantfollowingstatisticalanalysisofthedata.Theoptimumdesirability was calculated, and an optimized formulation wasproposed. This formulation, containing the proposed optimumlevelsforX1,X2andX3,waspreparedandcharacterizedforY1and Y2 as previously described, and the differences betweenpredictedandobservedvalueswerecalculated.

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Physicochemical CharacterizationDifferential scanning calorimetry (DSC)Thethermalbehaviorofpureketoconazole,PLGA,PVAandtheoptimizedformulationwasstudiedusingShimadzuDSCTA-50ESIDSC apparatus (Shimadzu, Tokyo, Japan). Aluminium cruciblescontainingapproximately2mgofeachsampleunderadynamicnitrogenatmosphere(flowrate:50mL/min)andataheatingrateof10°C/mininatemperaturerangefrom25-950°Cwereused.

Fourier transform infrared spectroscopy (FTIR)Fouriertransforminfrared(FTIR)spectraofthesamplesusedintheDSCtechniquewererecordedusingNicoletiS10,ThermoScientific Inc. (Waltham,MA,USA) in the rangeof4000-400cm–1.

X-ray powder diffraction (XRPD) ThecrystallinestateoftheprepareddrugNPscomparedtothatof the pure ketoconazole powder was studied using powderX-ray diffractometer (D/max 2500, Rigaku, Tokyo, Japan). The

diffractionpatternsof the studied sampleswere recordedatascanspeedof0.5000degree/min.

Preparation of In Situ Gel (ISG) Formulations DifferentISGbasesolutionswerepreparedbysimpledispersionof the polymeric material, specifically sodium alginate (1%),chitosan (0.5%), poloxamer 407 (16%), or carbopol 940 (0.5%)indeionizedwaterwithcontinuousmixingonamagneticstirreruntil complete dissolving of the polymer. Polymeric solutionof chitosanwaspreparedbydispersion thepolymer in slightlyacidified solution.HPMC in a concentrationof 0.5% (w/v)wasadded to each solution as a viscosity enhancer. A quantityequivalentofNPs,containing0.3%ketoconazole,wasaddedtothe prepared homogenous polymeric dispersion solutions. Forcomparativestudy,thesamegelbasesolutionswerepreparedand loaded with 0.3% pure ketoconazole. ISG formulationscontainingmixtureofthestudiedpolymerswerealsoprepared.A total of twelve ISG preparations were prepared and thecompositionisillustratedinTable 3.

Independent VariablesLevel

Low Medium High%PLGAinorganicphase(X1) 3.0 4.5 6.0

%Organictoaqueousphase(X2) 4.0 5 6.0

%PVAinaqueousphase(X3) 1.0 2 3.0Dependent Variables GoalParticleSizeinnm(Y1) Minimize

%Entrapmentefficiency(Y2) Maximize

Tables 1KetoconazolePLGANPsindependentanddependentvariablesusedinthecentralcompositeexperimentaldesign.

Run X1(%)

X2(%)

X3(%)

Y1 (nm) Y2 (%)Observed Fitted Observed Fitted

1 3.0 6.0 3.0 457.0 ± 0.014 441.092 61.627±8.66 61.0401

2 4.5 5.0 3.682 729.0 ± 0.040 711.058 70.023±6.96 67.78793 3.0 4.0 3.0 417.0 ± 0.011 463.84 64.277 ± 0.47 66.78024 6.0 6.0 3.0 995.0 ± 0.024 1090.13 67.354±1.11 68.2275 4.5 3.318 2.0 483.0±0.019 526.614 83.453±3.08 78.03046 1.977 5.0 2.0 328.0±0.018 302.979 55.624 ± 0.44 54.90997 6.0 4.0 1.0 477.0 ± 0.012 520.366 69.122 ± 1.19 72.67548 3.0 4.0 1.0 446.0 ± 0.017 378.33 64.362±0.54 66.4575

9 7.023 5.0 2.0 982.0±0.083 968.19 69.666±0.78 66.182

10 4.5 6.682 2.0 945.0±0.043 862.554 67.524±3.06 68.748511 4.5 5.0 0.318 536.0±0.055 515.111 68.765±4.28 66.80212 6.0 6.0 1.0 962.0 ± 0.066 942.618 66.912 ± 0.51 67.377313 3.0 6.0 1.0 356.0±0.017 457.081 60.911 ± 0.99 60.50514 6.0 4.0 3.0 843.0±0.047 769.377 69.938±1.19 73.312615 4.5 5.0 2.0 885.0±0.183 893.331 67.847±3.10 67.569716 4.5 5.0 2.0 895.0±0.174 893.331 66.572 ± 0.96 67.5697

Table 2Experimentalrunsalongwiththeobservedandfittedvaluesforthestudiedresponses.

X1:%PLGAinorganicphase;X2:%Organictoaqueousphase;X3:%PVAinaqueousphase;Y1:Particlesizeinnm;Y2:%Entrapmentefficiency.

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Characterization of the Prepared ISG FormulationsIn vitro gelationThe prepared ISG formulationswere characterized for in vitro gelationbymixing25µLofeachformulationwith7µLsimulatedtear fluid (0.67 g sodium chloride, 0.2 g sodium bicarbonate,0.008gcalciumchloridedehydrateindeionizedwaterquantitysufficient to 100 ml and the pH was adjusted to 7.4 usinghydrochloricacid)at37°C.Aratioof25:7wasusedtomimicthein vivo ocular conditions since the volumeof tear in human isestimatedtobe7µL,andthecul-de-saccantransientlycontainaround30µLoffluid [12]. The in vitro gelationwasevaluatedas previously reported [13] by recording thetime required forgelation.

In vitro release studyThediffusionof ketoconazole in the formofNPsorpuredrugfrom the prepared ISG formulations was carried out usingautomated franz diffusion cell apparatus (MicroettePluss™,Hanson Research, Chatsworth, CA, USA). The apparatus isadapted with 1.76 cm2 of diffusion area. A quantity of ISGequivalentto400mgwasplacedinposition,donorchamber,andcoveredwithasyntheticnylonmembraneof0.45μmporesize.SimulatedtearfluidofpH7.4wasusedasareceivermediuminthereceptorchamber,thetemperaturewaskeptat32±0.5°Candthestirringratewasadjustedat400rpm.Samplesfromthereceptorchamberwereautomaticallywithdrawnat0.25,0.5,1,2,4,6and8hwithreplacementandanalyzedfordrugcontentspectrophotometrically. The release patterns of ketoconazolefromthestudiedISGformulationsweredeterminedbyplottingthecumulativeamountofthedrugpermeated(Q)perunitareaasafunctionoftimeandthedrugsteady-stateflux(JSS)foreachformulationwas determined from the slope. The permeabilitycoefficient (Pc)was estimatedbydividing thedrugfluxby theinitialdrug load (C0).Anotherplotof theQversus square rootof time was constructed and the diffusion coefficient (D) wasobtainedfromthefollowingequation:D=[slope/2Co]2×π

Anti-fungal activityThe antifungal activity of the prepared ISG formulations wastestedagainst standard strainofCandidaalbicansATCC76615that was obtained from the microbiology laboratory, KingAbdulazizUniversityHospital,Jeddah,KSA.Preliminaryscreeningof the antifungal activitieswas conducted using agar diffusiontechnique.Briefly,Petridishes(150mm)werefilledwith50mLMuller-Hintonagarcontaining1mLfungalculture(1×106CFU/mL)and the strainwas inoculated separately.Holesof12mmindiameterweremadeintheseededagarplatesandfilledwith200μLofeachformula.Disheswerethen incubatedfor4hat37°C. Inhibitory activity was defined as the absence of fungalgrowth in the area surrounding theholes, inhibition zone thatwasmeasuredusingacaliper.

Trans-corneal PermeationThehumancornealepithelium represents themainbarrier fortranscornealdrugpermeation.Todeterminetheimpactofocularbarriersontheophthalmicdrugdelivery,cellculturemodelscanbeused[14].Inthisstudy,evaluationofketoconazoletranscornealpermeabilityfromISGformulationloadedwitheitheroptimizeddrugNPsorpuredrug (F9andF10)wasperformedonhumanepithelialcellline.Cells(105)wereseededonT-25flasksfor24handthenweredividedintotwogroups.GroupIwasexposedtoF9(0.3mg/mlketoconazoleintheformofNPs)while,groupIIwas exposed to F10 (0.3mg/ml of pure ketoconazole). Cellswerecollectedafter2,4,6,12and24h(n=3)andwashedtwicewith icecoldphosphatebuffersaline.Thecollectedcellpelletswere ruptured by subjecting to two repeated cycles of freezeand thawing followed by ultrasonication for 10 minutes. Celllysates were centrifuged using Sigma Laboratory centrifuge(3K30,Ostrode,Germany)at15000rpmfor1hat4°Candtheconcentration of the drug in the supernatantwas determinedusing high-performance liquid chromatography (HPLC)methodpreviously reported [15] except for slight modifications. AnisocraticHPLCchromatographicmethodusingC18,4×250mm,5 μm, Thermo Fischer Scientific analytical column. Themobilephasecomposedofmethanolandwater80:20(v/v).Itsflowratewasadjustedat1.2ml/min.Thedetectionwavelengthwas240nm.Drug standards in the intracellular concentrate containingknownamountsofthedrugwerepreparedandanalyzedbeforedetermination of the unknown drug concentrations in thesamples.

Results and DiscussionPreparationofdrugnanoparticlesbasedonthein-vitrosolventexchange congealing method is a novel approach to developpolymeric drug delivery systemby easily achievable and faciletechnique. The term nanoparticle laden in situ gel has beenpreviously reported to describe the incorporation of PLGAnanoparticles, prepared by nanoprecipitation process, into in situ gel formulation suitable for ocular retention [16]. In thenanoprecipitationprocess, thedrugandpolymer inaspecifiedratio are dissolved in organic solvent at room temperatureand the resulting polymeric drug solution is slowly injected inaqueousPVAsolutionwhilestirringand,theorganicsolventandsomewaterareevaporated.

Inthisstudy,thein-vitrosolventexchangecongealingtechniquewhichdependsonsolventexchangemechanismwasemployedto develop PLGA NPs. PLGA is soluble in both water misciblesolventssuchasN-methylpyrrolidoneandDMSO,andinpartiallymiscibleones suchas triacetin,ethylacetate [17].WhenPLGAsolutionisinjectedintoanaqueousmedium,thesolventdissipateintotheaqueousenvironmentandthepolymersolidify[18-20].PVAwasaddedtotheaqueousphaseasastabilizertoproducesmall size and stable NPs [21,22]. The possible formulationparametersaffectingthepreparationoftheNPswereoptimizedto achieve the optimum concentration of PLGA in the organic

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solvent,organictoaqueousphaseratioandPVAconcentrationintheaqueousphase.

Effect on Particle SizeResults of the statistical analysis, using multiple regressionanalysis and two-way ANOVA, for the effect of the studiedindependent variables on particle size indicated that themaineffectsofX1,X2andX3alongwiththeirquadraticeffectsweresignificantly Y1 as illustrated inTable 4.When the sign of theestimatedeffectdenotesapositivevalue,thisisanindicationofasynergisticeffectofthisvariableontheselectedresponse,whileantagonisticeffectisexpectedwhenanegativesignisobtained.F-ratio is used to compare between the actual and expectedvariation of variable averages; an F-ratio greater than 1 is anindicationofa locationeffectandhencetheP-value isusedtoreportthesignificancelevel.AfactorisconsideredtosignificantlyinfluenceaselectedresponseiftheP-valuediffersfrom0andislessthan0.05.AstheconcentrationofPVAwasincreased,particlesizeofthepreparedparticleswasincreased.Thisbehaviorcouldbeattributedtotworeasons.First, the increase inparticlesizeis attributed to exceeding the PVA concentration required toprovidecompletecoverageofthenanoparticlessurfaceandstartformationofPVAmicellemolecules.Above this criticalmicelleconcentration,thereisalesssurfactantsurfaceadsorptionsincethemicellesmoleculesbegin tocompete for theadsorptionat

the solid particles, this effect results in destabilization of thepreparednanoparticlesandthereforecontributedtoincreaseintheparticle size [21-24].Second, increase in theconcentrationofPVA leads to increase in theviscosityof theaqueousphasewhich in turndecreases the rateofPLGAsolidificationand so,formationof large size particles. Results also revealed that, astheconcentrationofPLGAwasincreased,theparticlesizeofthepreparedNPswas increased.Thisfinding is ingoodagreementwithpreviousstudyindicatedanincreaseintheaverageparticlesize of PLA nanoparticles loaded with 5-Fluorouracil whenthe concentration of the polymer was increased. The authorsattributed this finding to high polymer load in the aqueousmedium during the solidification process [25]. High percentof the organic phase also increase the polymer load and soattributedtolargerparticlesize.ThestandardizedparetochartthatillustratestheeffectoftheX1,X2,X3,theirinteractionandquadraticeffectofY1confirmedtheabovefindingasdepictedinFigure 1.Averticalreferencelineatasignificantp-valueof0.05isrepresentedinthechartinwhichaneffectthatexceedsthislineis statistically significant. The polynomial equation that relatestheindependentvariablesandtheparticlesizeisasfollow:

Particle Size=-2009.41+155.604 X1+595.679 X2+458.835 X3-40.501X12+57.25X1X2+27.25X1X3-70.2679X22-25.375X2X3-99.0823X32.

ISGFormula

Composition Release parameters Zone diameter

(mm)

In vitro gelation

(min)Polymer Conc. (%) Drug form JSS (μg/cm2 min) P (cm/h) D

1 Na-Alginate 1 NPs 9.88x10-2 8.98x10-5 7.13x10-6 24 562 Na-Alginate 1 Puredrug 3.73x10-2 3.39x10-5 9.74x10-7 22 553 Carbopol 0.5 NPs 15.04x10-2 13.67x10-5 1.50x10-5 21 864 Carbopol 0.5 Puredrug 12.51x10-2 11.37x10-5 1.10x10-5 20 885 Poloxamer 16 NPs 39.45x10-2 35.86x10-5 10.9x10-5 20 656 Poloxamer 16 Puredrug 45.67x10-2 4.15x10-5 1.47x10-6 18 657 Chitosan 0.5 NPs 75.18x10-2 68.35x10-5 4x10-4 25 918 Chitosan 0.5 Puredrug 75.16x10-2 68.33x10-5 39.28x10-5 22 939 Chitosan-Alginatemixture 0.5-0.5 NPs 1.41 12.76x10-4 13.65x10-4 30 47

10 Chitosan-Alginatemixture 0.5-0.5 Puredrug 55.66x10-2 50.59x10-5 22.11x10-5 26 4811 Chitosan-Poloxamermixture 0.5-16 NPs 29.89x10-2 27.18x10-5 6.47x10-5 22 4612 Chitosan-Poloxamermixture 0.5-16 Puredrug 14.81x10-2 13.47x10-5 1.54x10-5 20 47

Table 3 CompositionofthepreparedISGformulations,in vitroreleaseparameters,antifungalactivitiesandgelationtime.

PF127:PoloxamerF127;NPs:nanoparticles;JSS:steadystateflux;P:permeabilitycoefficient;D:diffusioncoefficient.

FactorY1 Y2

Estimated effect F-ratio P-value Estimated effect F-ratio P-valueX1 395.537 72.32 0.0001 6.70238 10.27 0.0185X2 199.752 18.44 0.0051 -5.51903 6.96 0.0386X3 116.511 6.27 0.0462 0.58618 0.08 0.7887

X1X1 -182.254 10.42 0.0180 -4.96651 3.82 0.0983X1X2 171.75 7.99 0.0301 0.32725 0.01 0.9086X1X3 81.75 1.81 0.2272 0.15725 0.00 0.9560X2X2 -140.536 6.19 0.0473 4.11522 2.63 0.1563X2X3 -50.75 0.70 0.4356 0.10625 0.00 0.9702X3X3 -198.165 12.31 0.0127 -0.194251 0.01 0.9415

Table 4 Estimatedeffectsoffactors,F-ratio,andassociatedP-valuesfortheketoconazolePLGANPsparticlesize(Y1)andentrapmentefficiency(Y2).

X1:%PLGAinorganicphase;X2:%Organictoaqueousphase;X3:%PVAinaqueousphase;Y1:Particlesizeinnm;Y2:%Entrapmentefficiency.

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3D response surface plots were constructed to illustrate theeffect of changing two independent variables, when the thirdonewaskeptattheintermediatelevel,onY1wereconstructedandarerepresentedinFigure 2.

Effect on Entrapment EfficiencyStatisticalanalysisoftheobtaineddataforentrapmentefficiencyindicated that themaineffectsofX1andX2weresignificantlyaffectingY2astabulatedinTable 4andgraphicallyrepresentedin the pareto chart (Figure 1). As the concentration of theorganicphase(DMSO)wasincreased,theentrapmentefficiencywas decreased owing to the higher solubility of ketoconazoleinDMSOthat leads toescapeanddiffusionof thedrugwithinthe organic phase during the solvent exchange process. Thisbehaviorwasmorepronouncedwhentheconcentrationoftheorganicphasewasincreased.ThedrugentrapmentefficiencywasincreasedwhentheconcentrationofPLGAwasincreasedwhichis in agreement with previous work for PLA nanoparticles butwithwatersolubledrug[25].Thisbehaviorcouldbeattributedto rapid solidification and formation of the nanoparticles thatenclosehigher percentof thedrugwhen the concentrationofPLGAwas increased. Thepolynomial equation that relates theindependentvariablesandtheentrapmentefficiencyisasfollow:

Entrapment Efficiency=102.887+11.5169X1-23.9328X2+0.180091X3-1.10367 X12+0.109083 X1X2+0.0524167 X1X3+2.05761X22+0.053125X2X3-0.0971253X32.

To illustrate the effect of changing two independent variablesonY2,when the thirdonewaskeptat the intermediate level,3DresponsesurfaceplotswereconstructedandaregraphicallyrepresentedinFigure 2.

To prepare an optimized formulation characterized by smallerparticle size and high entrapment efficiency, the optimumdesirability that achieve an optimum combination of factorlevelswasidentifiedandtheoptimumlevelsfortheindependentvariablesalongwithpredicted,observedandresidualvaluesaretabulatedinTable 5.

Physicochemical CharacterizationTheDSC thermogram of ketoconazole showed a characteristicsharpendothermicpeakat151°Cas illustrated inFigure 3. No characteristicdrugpeakwasobservedinthedrugloadedPLGANPsformulationwhichisanevidencethattherewasnocrystallinedrug material in the optimized drug formulation. This is anindicationofthechangeinthedrugcrystallinityandhomogenousdispersion of the drug in the PLGA matrix. The same findinghas been previously reported for PLGA nanoparticles loadedcapecitabine[26]andmonensinloadedPLGAnanoparticles[27],andtheauthorsattributedthisbehaviortotheexistenceofthedrug inanamorphousordisordered-crystallinephasewhich isdistributedasamoleculardispersionorasolidsolutionstateinpolymericmatrix.

The FTIR spectroscopy of pure ketoconazole displayed acharacteristic drug peaks of carbonyl group C=O stretchingvibration at 1647.26 cm-1, C-O stretching of the drug aliphatic

ethergroupat1031.95cm-1andC-Ostretchingofcyclicetherat1244.13cm-1[28].ThespectraoftheoptimizedNPsloadedwithdrugexhibitedthesamecharacteristicpeaks,Figure 4,exceptforslightshiftordecreaseintheintensityofthepeaksduetoslightoverlappingbetween thedrug and thepolymers characteristicpeaksorduetothedrug-polymerratio.

XRPD patterns of pure ketoconazole and optimized NPsformulationareillustratedinFigure 5.Thediffractionspectrumofthepuredrugwascrystallineinnatureasindicatedbynumerousdistinct peaks. The spectrum of drug NPs was different fromthatofpureketoconazolewhichdemonstratedpeaksof lowerintensity,disappearance,andformationofsomenewpeaks.ThisfindingindicatescrystallinestatetransformationofketoconazoleintheNPsintotheamorphousformandabsenceofinteractionbetweenthedrugandthepolymersused.

Characterization of the Prepared ISG FormulationsPrevious studies have indicated that 1% sodium alginate, 16%poloxamerand0.5%carbopolweresuitableasin situvehiclesforophthalmic delivery [25,29,30]. Ionic gelation, thermosensitiveand pH induced transformation are the mechanisms involvedduring phase transition for alginate, poloxamer and carbopol,respectively.HPMCwasaddedtoallthepreparedformulationasviscosityenhancingagent.Chitosanisamuco-adhesivepolymerthat possesses viscosity enhancing properties and is soluble inaqueousacidicsolution.ThepHofthesimulatedtearfluid(STF),thepresenceofalkalinesubstances intheSTF,andadditionofHPMCtothechitosanbasedISGformulationcouldattributetoincreaseintheformulationviscosityuponcontactwiththeSTF.ItwasnoticedthatISGformulations7and8showedthelongestin vitro gelation time among all the studied ISG formulations.ISGformulationscontainingcombinationofchitosanandotherpolymers were also prepared except for carbopol which wasincompatiblewith chitosan as indicatedby formationofwhiteprecipitate during preparation. Mixing chitosan with alginateorpoloxamerleadstodecreaseinthetimeof in-vitrogelation.Whenall theprepared formulationsolutionwasaddedtoSTF,the viscosity was increased owing to gel formation by ionic,thermosensitiveorpHinteraction.Ifhighersheerrateisapplied,thehighshearrateofeye(4250-28,5001/s)duringblinking,theviscosityusuallydecreasedduetothepseudo-plasticbehavioroftheformulationaspreviouslydescribedbyNagarwaletalwhichsuggestssuitabilityoftheseformulationforoculardelivery[25].FastergelationofformulationISG9and10couldbeattributed

X1X2X3X3X1X1X1X2X3X2X3X1X3X2X3

X1X2X1X1X2X2X3X1X2X3X3X1X3X2X3

0 2 4 6 8 10

+-

+-

0 1 2 3 4

Standardized Pareto Chart for Particle Size Standardized Pareto Chart for Entrapment Efficiency

Standardized effect Standardized effect

StandardizedParetochartsfortheeffectoftheindependentvariablesonY1andY2.

Figure 1

2017Vol. 6 No. 1:10

Journal of Biomedical SciencesISSN 2254-609X

7© Under License of Creative Commons Attribution 3.0 License

tothepresenceofdoubleviscosityenhancingagent,HPMCandchitosan,andtotherapidionicgelationofalginatewithCa++intheSTF.

The in vitro release study revealed superior drug release fromNPs formulations when compared to the same formulationscontaining pure drug as indicated by the calculated releaseparameters illustrated in Table 3. Development of drug PLGANPs results in improvement in the drug permeation owingto a decrease in the drug particle size. The release from ISGformulation9and10wasthehighest, theeffectthatcouldbe

attributedtorapidgelationandeasydiffusionofthedrugfromthechitosan-alginatesystem.

Resultsfortheantifungalactivity,representedastheinhibitionzonediameter,wereindirectrelationtothereleasedata.Largeinhibition zone diameter was noticed with ISG formulation 9,containsdrugnanoparticlesinchitosan-alginatemixture,whichcouldberelatedtohighdrugpermeationfromthisformulationwhencomparedwithotherswithsubsequentinhibitioneffectonthestudiedfungalstrain.

520.0-600.0600.0-680.0680.0-760.0760.0-840.0840.0-920.0920.0-1000.01000.0-1080.01080.0-1160.01160.0-1240.01240.0-1320.01320.0-1400.0

1320

1120

920

720

520

75

7269

66

6360

69

67

65

63

61

74

72

70

68

66

3 3.5 4 4.5 5 5.5 6

3 3.5 4 4.5 5 5.5 6

4 4.4 4.8 5.2 5.6 6

Y1

Estimated Response Surface for the Efeect of X1 and X2 on Y1

Estimated Response Surface for the Efeect of X1 and X3 on Y1

Estimated Response Surface for the efeect of X2 and X3 on Y1 Estimated Response Surface for the Efeect of X2 and X3 on Y1

Estimated Response Surface for the Efeect of X1 and X3 on Y2

Estimated Response Surface for the feect of X1 and X2 on Y2

X3=2.0X3=2.0

Y1Y2

Y2

Y2

Y1

Y1

X2X2

X3

X3

X1

X1

X2X2

X1

X1

X1= 4.5

X3

X3

X2=5.0

X2

X1=4.0

X2=5.0

Y1

Y1

Y2

Y2

Y2

1080980880780680580480

940

840

740

640

540

3 3.5 4 4.5 5 5.5 6

4 4.4 4.8 5.2 5.6 6

3 3.5 4 4.5 5 5.5 6

1

1

1.4

1.4 1.4

4 4.44.8

5.25.66

1.8

1.8

2.2

2.2

2.6

2.6

3

3

11.82.2 2.6

3

1.411.82.2 2.6

3

4.444.85.2 5.6

6

60.0-61.561.5-63.063.0-64.564.5-66.066.0-67.567.5-69.069.0-70.570.5-72.072.0-73.573.5-75.075.0-76.5

60.0-61.561.5-63.063.0-64.564.5-66.066.0-67.567.5-69.069.0-70.570.5-72.072.0-73.573.5-75.075.0-76.5

60.0-61.561.5-63.063.0-64.564.5-66.066.0-67.567.5-69.069.0-70.570.5-72.072.0-73.573.5-75.075.0-76.5

520.0-600.0600.0-680.0680.0-760.0760.0-840.0840.0-920.0920.0-1000.01000.0-1080.01080.0-1160.01160.0-1240.01240.0-1320.01320.0-1400.0

520.0-600.0600.0-680.0680.0-760.0760.0-840.0840.0-920.0920.0-1000.01000.0-1080.01080.0-1160.01160.0-1240.01240.0-1320.01320.0-1400.0

3DResponsesurfaceplotsfortheeffectoftheindependentvariablesonY1(leftside)andY2(rightside).Figure 2

Factor Low level High level OptimumX1 1.977 7.023 5.494X2 3.318 6.682 3.318X3 0.3182 3.682 1.105

Response Predicted value Observed value ResidualY1 328.0 331.0 +3Y2 78.67 77.32 -1.35

Table 5 MultipleresponseoptimizationofketoconazolePLGANPs.

X1:%PLGAinorganicphase;X2:%Organictoaqueousphase;X3:%PVAinaqueousphase;Y1:Particlesizeinnm;Y2:%Entrapmentefficiency.

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DSCthermogramsofpureketoconazole,PLGA,PVAandoptimizedNPsloadedwithdrug.Figure 3

80

60

40

100

95

90100

908070

100

80

60

4000 3500 3000 2500 2000 1500 1000 500

Ketoconazole

% Tr

ansm

ittan

ce

% Tr

ansm

ittan

ce

% Tr

ansm

ittan

ce

% Tr

ansm

ittan

ce

PLGA

PVA

Optimized Formulation

Wavenumbers (cm-1)

FTIR-spectroscopyofpureketoconazole,PLGA,PVAandoptimizedNPsloadedwithdrug.Figure 4

Trans-Corneal Permeation Fungaleyeinfectionscanbeveryseriousifnottreated.Infectionof the clear, front layer of the eye (the cornea) is known askeratitiswhile, infectionintheinteriorpartoftheeyeiscalledendophthalmitis. Eye infectionsmay be attributed to differenttypesoffungithatresult inbothkeratitisandendophthalmitis.Candidaalbicans,atypeofyeastthatnormally livesonhumanskin and on the protective mucous membranes lining inside

the body, has been reported to be the most common causeof endogenous endophthalmitis. Mycotic keratitis is usuallyattributedtofilamentousfungisuchasLasiodiplodia theobromae although other species such as Fusarium, Alternaria, andAspergillushavebeenalsomentioned.Keratitisusuallyoccursinconjunctionwith trauma to the corneawith vegetablematter.KeratitisthatisattributedtoyeastssuchasCandidaismostlikelyto occur, and always happen in abnormal eyes, e.g., patientssufferingdryeye,chroniccornealulceration,orcornealscarring

2017Vol. 6 No. 1:10

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9© Under License of Creative Commons Attribution 3.0 License

[14,31]. In this study, the permeation of ketoconazole in theformofdrugNPsfromISGsystemwasstudiedtoevaluatetheusefulnessoftheformulationintreatmentofbothkeratitisandendophthalmitis. Effective permeation of the drug from thehumanepithelialcellscoupledwiththeantifungalactivitywillbeconsideredasasuccessfuldeliverysysteminmanagementoftheabovementionedeyeinflammationconditions.

Results of the trans-corneal permeation indicated higher andsustaineddrugpermeationfromformulationF9whencomparedtoF10asrepresentedinFigure 6.ThepreparedoptimizeddrugPLGANPsaugmentedthedrugpermeation,owingtothesmallerparticlesize,andsustainedthedrugreleaseduetoentrappingthe drug inside the polymericmatrix. It was previously statedthat drug release from PLGA polymericmatrix is attributed todiffusionfollowedbypolymererosion[32].LoadingthedrugorthepreparedoptimizedNPsintoISGvehiclerepresentsanotherfactor that could assist in sustaining the drug release but theeffectofthedrugreleasemechanismfromPLGA,diffusionanderosion,remainssuperior(Figure 7).

ConclusionIn this study, development of ketoconazole PLGA NPs basedonsolventexchange techniquewassuccessfullyachievedafteroptimizationtheformulationfactorsaffectingthedevelopmentprocess.CombinationofalginateandchitosanwasfoundtobeaneffectiveISGsystemasindicatedbyin vitrodrugpermeationand gelation, antifungal activity and trans-corneal permeation.Hence,ketoconazolePLGANPsloadedintoalginate-chitosanISGformulationcouldbeusedasaneffectiveantifungalmedicationintreatmentofbothkeratitisandendophthalmitis.Combinationofalginateandchitosanwasfoundtobeaneffectiveophthalmicdrug delivery system as indicated by in vitro drug permeationand gelation, antifungal activity and trans-corneal permeation.Hence, ketoconazole PLGA NPs loaded into alginate-chitosanISGformulationcouldbeconsideredasasuccessfulophthalmic

1400

1200

1000

800

600

400

200

0 10.0000 20.0000 30.0000 40.0000 50.0000 60.0000 10.0000 20.0000 30.0000 40.0000 50.0000 60.00002thela (deg.)

Ketoconazole Optimized NPs

2thela (deg.)

Intensity (cps)

D 3-60B1.raw 8 3-60B.raw

tensity (cps)600

400

200

0

X-raydiffractionpatternsofpureketoconazoleandoptimizedNPsloadedwithdrug.Figure 5

30

25

20

15

10

5

0

F1

F5 F6 F7 F8

F9 F10 F11 F12

F2 F3 F4

Cum

ubiti

ve %

drug

relea

sed

Cum

ubiti

ve %

drug

relea

sed

Cum

ubiti

ve %

drug

relea

sed

Time (hrs)

Time (hrs)

Time (hrs)

80

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60

50

40

30

20

10

0

90

8070

6050

403020

10

0

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8

DiffusionprofileofketoconazolefromthepreparedISGformulations.

Figure 6

2017Vol. 6 No. 1:10

Journal of Biomedical SciencesISSN 2254-609X

This Article is Available in: www.jbiomeds.com10

antifungal medication in treatment of both keratitis andendophthalmitis.

Conflict of Interest All authors of this work declare that there are no conflicts ofinterest.

AcknowledgmentThis paperwas funded by the Deanship of Scientific Research(DSR), King Abdulaziz University, Jeddah, under grant no. 166-822-D1435. The authors, therefore, acknowledge with thanksDSRfortechnicalandfinancialsupport.

350

300

250

200

150

100

50

0

Conc

(pg/

cell)

Time (h)0 4 8 12 16 20 24

F10

F9

Permeationofketoconazolethroughhumanepithelialcellline.

Figure 7

2017Vol. 6 No. 1:10

Journal of Biomedical SciencesISSN 2254-609X

11© Under License of Creative Commons Attribution 3.0 License

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