Enhancement of pharmacokinetic and pharmacological behavior of ocular dorzolamide after

16 Pages·2011·3.61 MB·English

RESEARCHARTICLE
Enhancement of pharmacokinetic and
pharmacological behavior of ocular
dorzolamide after factorial optimization of
self-assembled nanostructures
EnasA.M.R.Afify1,IbrahimElsayed2,3,MaryK.Gad1,MagdyI.Mohamed2,AbdEl-
MoneimM.R.Afify4*
1 NationalOrganizationforDrugControlandResearch(NODCAR),Giza,Egypt,2 Departmentof
PharmaceuticsandIndustrialPharmacy,FacultyofPharmacy,CairoUniversity,Cairo,Egypt,3 Department
ofPharmaceuticalSciences,CollegeofPharmacy,GulfMedicalUniversity,Ajman,UnitedArabEmirates,
a1111111111 4 BiochemistryDepartment,FacultyofAgriculture,CairoUniversity,Cairo,Egypt
a1111111111
a1111111111 *[email protected]
a1111111111
a1111111111
Abstract
Dorzolamidehydrochlorideisfrequentlyadministeredforthecontroloftheintra-ocularpres-
sureassociatedwithglaucoma.Theaimofthisstudyistodevelopandoptimizeself-assem-
OPENACCESS
blednanostructuresofdorzolamidehydrochlorideandL-α-Phosphatidylcholinetoimprove
Citation:AfifyEAMR,ElsayedI,GadMK, thepharmacokineticparametersandextendthedrugpharmacologicalaction.Self-assem-
MohamedMI,AfifyAE-MMR(2018)Enhancement
blednanostructureswerepreparedusingamodifiedthin-filmhydrationtechnique.Thefor-
ofpharmacokineticandpharmacologicalbehavior
mulaecompositionsweredesignedbasedonresponsesurfacestatisticaldesign.The
ofoculardorzolamideafterfactorialoptimizationof
self-assemblednanostructures.PLoSONE13(2): preparedself-assemblednanostructureswerecharacterizedbytestingtheirdrugcontent,
e0191415.https://doi.org/10.1371/journal. particlesize,polydispersityindex,zetapotential,partitioncoefficient,releasehalf-lifeand
pone.0191415
extent.Theoptimizedformulaehavingthehighestdrugcontent,zetapotential,partition
Editor:AamirAhmad,UniversityofSouthAlabama coefficient,releasehalf-lifeandextentwiththelowestparticlesizeandpolydispersityindex
MitchellCancerInstitute,UNITEDSTATES
weresubjectedtofurtherinvestigationsincludinginvestigationoftheirphysicochemical,
Received:November11,2017 morphologicalcharacteristics,invivopharmacokineticandpharmacodynamicprofiles.The
Accepted:January4,2018 optimizedformulaewerepreparedatpH8.7(F5andF6)andcomposedofL-α-Phosphati-
dylcholineanddrugmixedinaratioof1:1and2:1w/w,respectively.Theyshowedsignifi-
Published:February5,2018
cantlyhigherC ,AUC24andAUC1attheaqueoushumorwithextendedcontroloverthe
Copyright:©2018Afifyetal.Thisisanopen max 0 0
accessarticledistributedunderthetermsofthe intra-ocularpressure,whencomparedtothemarketedproduct;Trusopt®.Thestudyintro-
CreativeCommonsAttributionLicense,which ducednovelandpromisingself-assembledformulaeabletopermeatehigherdrugamount
permitsunrestricteduse,distribution,and
throughthecorneaandachievesustainedpharmacologicaleffectatthesiteofaction.
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
availablewithinthepaperanditsSupporting
Informationfile. Introduction
Funding:Theauthorsreceivednospecificfunding Glaucomaisanophthalmicdiseaseassociatedwithanincreaseintheintra-ocularpressure
forthiswork. (IOP).PersistenthighIOPaccompaniedwithglaucomacandamagetheopticnervewithtime
[1].Untreatedglaucomamayleadtoblindnesswithinfewyears[2].Theanti-glaucomather-
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. apyrequirescontinuedadministrationofmedicinesforlongtimeandso,sustainedrelease
PLOSONE|https://doi.org/10.1371/journal.pone.0191415 February5,2018 1/16
Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
systemsareneededtohelpinkeepingconstantdruglevelsatthesiteofactionandenhancing
patientcompliancethroughdecreasingthedosingfrequency[3].
Dorzolamidehydrochloride(DH)isacarbonicanhydraseinhibitorusedintreatmentof
glaucomathroughdecreasingtheproductionofaqueoushumor[4].DHis20timesmore
activethanacetazolamideinloweringtheIOPwhenappliedtopically[5].Thedrugisavailable
inthemarketaseyedrops;Trusopt1(Merck,N.J.,USA)containing2%DHintheformof
aqueoussolutionbufferedatpH5.6[6].Trusopt1isadministeredthreetimesadaytoachieve
asufficientcontrolovertheIOPbutthiscanexaggerateanypossiblesideeffectandaffectthe
patientcompliance[7].ReformulationofDHintheformofsustainednanoparticlesornano-
vesiclesmayenhancepatientcompliance,pharmacokineticandpharmacologicalbehavior[8].
Self-assemblydrugnanostructures(SADN)isawell-definedstablestructuremolecules
formedunderthermodynamicandkineticconditionsthroughnoncovalentinteractions,elec-
trostaticinteractions,hydrophobicinteractions,hydrogenbonding,-πstacking,andVander
Waalsforces,etc.,tomaintainmoleculesatastablelow-energystate[9].Recently,manyself-
assemblydrugnanostructureshavebeensynthesizedfrombiomaterialsincludingcarbohy-
drates,nucleicacids,andpeptidestoachieveabetterunderstandingoftheself-assemblymech-
anismandutilizethemforseveralbiomedicalapplicationssuchastissueregeneration,drug
delivery,andbiosensors[10].Self-assemblydrugnanostructurescreatenewmaterialswith
suitablemorphologiesanddesiredfunctionsthroughsingle-moleculedesignandthisresults
incontrollingthebulkpropertiesoftheresultantmaterialbymodulatingindividualmono-
mericbuildingblocks[11].SADNareconsideredsuperioroverotherencapsulatingnanopar-
ticlesand/ornanovesiclesinhavinghigherdrugloadingcapacity,lowerdrugleakageand
betterpermeationcapabilitiesthroughdifferentbiologicalmembranes[12].
TheaimofthisstudyistodevelopSADNoftwoamphiphiliccompounds;DHandL-α-
Phosphatidylcholine(PC).ThepreparedSADNisproposedtohavesustainedinvitrorelease
profile,superiorpermeationpowerandextendedinvivodrugactionwhencomparedtothe
conventionaldrugsolutionavailableinthemarket.
Materialsandmethods
Materials
DorzolamideHydrochloride(DH)waskindlygiftedfromJamjoomPharma,Jeddah,Saudi
Arabia.L-α-Phosphatidylcholine(PC)waspurchasedfromSigma–Aldrich,St.Louis,USA.All
otherchemicalsandsolventswereofanalyticalgrade,andutilizedwithoutfurtherpurification
orprocessing.AllrabbitsusedintheexperimentwereobtainedfromFacultyofAgriculture,
CairoUniversity,Cairo,Egypt,andwerebredforresearchpurposes.
Preparationtechnique
Theself-assembleddrugnanostructures(SADN)werepreparedusingamodifiedthin-film
hydrationtechnique[13].Briefly,DH(200mg)aloneorinamixturewithPCinratio1:1and
1:2w/wwasdissolvedin10mLdichloromethaneasshowninTableAinS1File.Theyielded
solutionwasevaporatedunderreducedpressureinarotaryevaporator(Rotavapor,Heidolph
VV2000,Burladingen,Germany)equippedwitha50mLrounded-bottomflaskrotatingat
100rpmINthermostaticwaterbath(40oC).Theformedresidueswerehydratedfor1husing
10mLofeithercitratebuffer(pH5.4),Tris-HClbuffer(pH7.05)orboratebuffer(pH8.7)to
yieldafinaldrugconcentrationof2%w/v.Theprepareddispersionsweresonicatedfor1min,
filteredonfilterpapertoremoveanyaggregatesandkeptinrefrigeratorforcharacterization.
PLOSONE|https://doi.org/10.1371/journal.pone.0191415 February5,2018 2/16
Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
Statisticaldesignofthestudy
Responsesurfacedesignwasutilizedtoinvestigatetheinfluenceofformulationvariableson
physicochemicalcharacteristicsofthepreparedSADNformulaeusingDesign-Expert17Soft-
ware(Stat-EaseInc.,Minneapolis,MN,USA).AsdisplayedinTable1,theeffectsofpH(X )
1
anddrugtopolymerratio(P/D:X )onthedrugcontent(DC:Y ),particlesize(PS:Y ),poly-
2 1 2
dispersityindex(PDI:Y ),zetapotential(ZP:Y ),partitioncoefficient(K:Y ),releasehalf-life
3 4 5
(T :Y )andreleaseextent(RE:Y ).Basedonthat,thecompositionof6formulaewere
50% 6 7
obtainedtobepreparedandcharacterized.Consequently,theselectedcomposition(s)was/
weredeterminedbasedonthedesirabilityfunctionforsimultaneousoptimizationofthe
tracedresponses.
Invitrocharacterizationofthepreparedself-assembleddrug
nanostructures
Drugcontent. Samplesweretakenfromeachpreparedformulaanddilutedwithmetha-
nol(100times).Thediluteddispersionswerekeptovernightinathermostaticshakingwater
bath(Gesellschaftfu¨rLabortechnik,Burgwedel,Germany)runningataspeedof100rpm[14].
Then,clearaliquotsweretakenafterfiltrationusing100nmmembranefilters(Bu¨chiLabor-
technikAG,Flawil,Switzerland).Theclearsolutionswereanalyzedforitsdrugcontentusing
UVspectrophotometer(SPD-10A,Shimadzu,Tokyo,Japan)atλ of254nm.Drugcontent
max
ofeachformulawascalculatedusingthefollowingequation:
A
Drug content %¼ PSx100 ð1Þ
A
TS
where,A andA arethepracticalandtheoreticaldrugamountintheemployedsamplevol-
PS TS
ume,respectively.
Particlesize,particlesizedistributionandzetapotential. Particlesize,particlesizedis-
tributionandzetapotentialoftheoptimizedformulaeweremeasuredusingdynamiclight
scatteringtechnique(NanoZS-90Zetasizer,MalvernInstruments,Malvern,UK)afterdilution
ofeachnanodispersionwithbi-distilledwaterinratio1:10v/v.
Partitioncoefficient. EffectsofthepHandD/Pratioonthedrugpartitioncoefficient
weredeterminedbasedonshake-flasktechnique[15].Samplesofthepreparedformulaewere
shakenwithequalvolumesofn-octanolfor24hinathermostaticshakingwaterbath.The
mixtureswereshakenat100rpmandtemperature32oCtosimulatethesurfacetemperature
Table1. CompositionofthepreparedSADNformulaebasedontheresponsesurfacedesignandthemeasuredresponses.
Formulae Factors Responses
pH P/Dratio DC(%) PS(nm) PDI ZP(mV) K T (min) RE(%)
50%
F1 Low Low 69.22 604.1 0.575 -39.3 0.188 605 88.34
61.10 594.8 0.603 -41.7 0.179 627 84.61
F2 Low High 83.63 385.8 0.273 -40.4 0.125 840 76.26
81.09 372.9 0.312 -41.5 0.121 859 75.81
F3 Medium Low 23.97 386.3 0.499 -51.3 1.914 1332 78.32
F4 Medium High 30.77 190.4 0.475 -46.0 2.821 1569 68.20
F5 High Low 92.64 237.4 0.256 -55.5 0.852 243 82.84
97.81 236.8 0.240 -60.7 0.864 274 86.67
F6 High High 98.28 103.9 0.714 -54.5 2.330 355 85.28
99.33 100.1 0.670 -57.8 2.180 316 81.74
https://doi.org/10.1371/journal.pone.0191415.t001
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Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
oftheeye.Then,theaqueousphasewasseparatedandthedrugconcentrationwasdetermined
usingUVspectrophotometeratthepredetermineddrugλ .Thepartitioncoefficient(K )
max o/w
wascalculatedbasedonthefollowingequation:
C C (cid:0) C
K ¼ oct: ¼ wi wf ð2Þ
o=w C C
wf wf
where,C andC arethedrugconcentrationsinoctanolandwateratequilibrium,respec-
oct. wf
tively,while,C istheinitialdrugconcentrationinwater.Thepercentofsolubilityinboth
wi
layershowninTableBinS1File.
Invitrorelease. Thedrugreleaserateandextentfromeachformulawerestudiedand
comparedtotheequivalentdrugsolutionusingmodifiedFranzdiffusioncell.Capacityofthe
acceptorcellwas10mLwitha0.5cmaperturefacingthedonorcell.Overnightsoakeddialysis
tubingcellulosemembrane(Typicalmolecularweightcut-off=14,000Da,Sigma–Aldrich,
St.Louis,USA)werefixedbetweenthetwocellafterfillingoftheacceptoronewithsimulated
tearfluid(pH7.4)rotatingatspeed100rpmusingTefloncoatedmagneticbead[16].Thetem-
peratureofthewaterjacketsurroundingtheacceptorcellwasadjustedat32±0.5˚C.Samples
(0.2mL,each)weretakenatdifferenttimeintervals(15,30min.,1,2,3,4,5,6,7,8and24h),
filtered,dilutedandthenspectrophotometricallyanalyzedthepredeterminedDHλ .Each
max
formulawastestedthriceandtheaverageDHreleasepercentageswerecalculatedandplotted
againsttimetodemonstratethedrugreleaseprofilesfromdifferentformulae.
Thereleasekineticsweredeterminedbasedonsubstitutioninthedifferentorderequations
(zero,1standHiguchidiffusion)tocomparethecorrelationcoefficientanddeterminethe
mostrepresentativeorder[17].Basedontheselectedorder,thereleasehalf-life(T )wascal-
50%
culatedandutilizedtocomparethereleaserateofDHfromdifferentformulae.
InvitrocharacterizationoftheoptimizedSADNformulae
DifferentialscanningcalorimetryandX-raydiffraction. SamplesweretakenfromDH,
PCandtheoptimizedformulae;andplacedinsealedaluminumpans.Temperaturewasraised
upto300˚Cunderacoverofnitrogengaswithheatingrateof10˚C/minusingdifferential
scanningcalorimeter(Mettler-ToledoInternationalInc.,OH,USA).Thesamplesthermo-
gramswereconstructedandtheDHpeak(s)was/weretracedineachthermogramforpresence
ofanyshiftordisappearance.
PatternsofX-raydiffractionweredeterminedusingMD-10mini-diffractometer(MTICor-
poration,CA,USA)operatingatavoltage25kVandacurrentof30mA[18].Thetestwasper-
formedforDH,PCandtheoptimizedformulae.
Transmissionelectronmicroscopy. Thesurfacesoftheoptimizednano-complexeswere
visualizedusingtransmissionelectronmicroscope(JEM-2100,JEOL,Tokyo,Japan)operated
atavoltageof100kV.Thiswasdoneafterspreadingthetakensamplesoveracoppergrid,
stainingthemwith1%phosphotungisticacidandair-dryingofthestainedsamples.
InvivocharacterizationoftheoptimizedSADNformulae
Animals. Theanimalexperimentsweredoneinfullcompliancewithregulatoryprinciples
andafterapprovalofethicscommitteeofFacultyPharmacy,CairoUniversity(ApprovalNo.
PI1181).MaleNewZealandalbinorabbitsweighting2–3kgwerehousedatcontrolledtem-
perature(25±2˚C),andhumidity(60±5%),witha12/12hlight-darkcycles.Nineanimals
wereemployedinthepharmacologicalstudyandsixinthebioavailabilityevaluation.
Bioavailabilityenhancement. Onedrop(50μl)fromeachoptimizedformulawasapplied
torighteyeoftheemployedanimalsafterbeingrandomlydividedintotwogroups,onegroup
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Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
performula.Trusopt1eyedropwasappliedtothelefteyesofallanimalsasareferencetoact
asareferenceandevaluatetheimprovementoftheDHpharmacokineticsabsorbedfromthe
optimizedformulae.Therabbits’eyelidswerekindlykeptclosedforfewsecondstoprevent
dosedrainage.Afteranesthetizingtheanimalsusingintramuscularlyinjectedketaminehydro-
chloride(50mg/kg)andxylazine(10mg/kg),samplesoftheaqueoushumor(50μl)werewith-
drawnatthefollowingtimeintervals:1,2,4,6,8,24h,usinginsulinsyringeequippedwith29
gauge[19].Sampleswerefrozenat-20˚Ctillthetimeofanalysis.
Beforeanalysis,theaqueoushumorsamplescontainingDHweremixedwiththeinternal
standardsolution(Mepivacainehydrochloride,50μg/mL)inratio5:1v/v,andthenextracted
usingacetonitrile(HPLCgrade)[19].Theorganicphasewasseparated,driedundervacuum
usingvacuumconcentrator(Eppendorf5301;Hamburg,Germany)andreconstitutedbya
mobilephasecomposedofacetonitrile:potassiumdihydrogenphosphate(30mMol)contain-
ing0.1%triethanolamine,pH3.5,mixedinratio20:80v/vasshowninFigureAinS1File.
Sampleswereinjectedataflowrateof1mL/minintoHPLC(Shimadzu,Kyoto,Japan)
equippedwithUV-Visibledetector,HypersilC18columnand20μLinjector.
DHconcentrationateachtimeintervalwascalculatedbasedonapre-establishedcalibra-
tioncurve.Theobtaineddataweresubjectedtonon-compartmentalpharmacokineticsanaly-
sisusingPKsolveradd-inprogram[20].Foreachcurve,themaximumplasmaconcentration
(C ),itsequivalenttime(T ),areasunderthecurves(AUC24andAUC1)andmeanresi-
max max 0 0
dencetime(MRT)werecalculated.
Pharmacologicalreinforcement. Animalswererandomizedinthreegroups,eachcom-
posedofthreerabbits.Experimentalglaucomawasinducedbyfourdailyintraocularinjections
of2%w/vmethylcellulose[21].Asingledose(50μl)oftheoptimizedformulaewasappliedto
thecorneaofthefirsttwogroups.Inaddition,equalvolumeofTrusopt1eyedropwasadmin-
isteredtothethirdgroupasareferenceforthecomparisonpurposes.Theintraocularpressure
(IOP)oftherabbitsweremeasuredusinganindentationtonometer(RudolfRiesterGmbH
Co.,Jungingen,Germany)atdifferenttimeintervals(0.5,1,2,3,4,5,6,7,8and24h)[22].
ThenormalizedIOPpressurewascalculatedandtheobtaineddatawererecordedasthemean
values±standarddeviations.
Invitro–invivocorrelation(IVIVC). MultiplelevelsCcorrelationwasadoptedtocorre-
latethepercentagesoftheinvitroreleaseateachtimeintervalwitheachoftheareaunderthe
curveofthepharmacokineticstudyandthenormalizedIOPmeasuredduringthepharmaco-
logicalstudy[23].Datawereplottedandthemostfittingregressionmodelwillbeconsidered
todeterminethecorrelationcoefficients.
Resultsanddiscussion
FormulationandpreparationofSADNformulae
pHvalueswereselectedcarefullytoprovidesuitablemolecularenvironmentforelectrostatic
interactionbetweenDHandPC,asillustratedinFig1.Thedrughastwoisoelectricpointsat
6.4and8.5[24].AtpH5.4,DHcarriedpositivechargeonthequaternaryammoniumgroup
andhadanegativechargeloadedonthesulfonamidenitrogenatpHat8.7whileitisneutralat
theintermediatepH(7.05)[25].Ontheotherhand,PCremainednegativelychargedneverthe-
lessthesurroundingpHbecauseallselectedpHvalueswerehigherthantheisoelectricpoint
ofPC(4)[26].InteractionbetweenthecationicDHandtheanionicPCcouldhappenatpH
5.4whilethechanceofelectrostaticmigrationwasexpectedtobeminimalatpH7.04dueto
theabsenceofanychargeatthedrugstructure.Ontheotherhand,thenegativelycharged
drugatpH8.7wasproposedtoreplacethehydroxylgroupsneutralizingthepositivelycharged
quaternaryammoniumgroupofPC[27].
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Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
Fig1. Theelectrostaticinteractionsbetweenthedrugandphosphatidylcholineatlow(A),medium(B)andhigh(C)pHvalues.
https://doi.org/10.1371/journal.pone.0191415.g001
Invitrocharacterizationofthepreparedself-assembleddrug
nanostructures
Drugcontent. Highdrugcontentwasdesiredtoreducethevolume,equivalenttothe
drugdose,instilledineacheye[28].Thedeterminedvalueswererangedbetween23.97%and
99.33%throughoutthepreparedformulae.Quadraticmodelwasutilizedtoanalyzethedata
andvalidatedbasedonitsnon-significantlackoffit(p-value=0.5561),highadequatepreci-
sion(32.29)andthereasonableagreementbetweentheadjustedandpredictedr2(0.9854and
0.9691,respectively)[29].Resultsanalysisyieldedthefollowingpolynomialequation:
DC¼27:37þ11:62X þ4:83X (cid:0) 3:40X X þ58:02 ð3Þ
1 2 1 2
FactorialanalysisofvarianceshowedthepresenceofsignificanteffectsofpHandP/Dratio
onthedrugcontent(p-values=0.0002and0.0058,respectively).Thelowestdrugcontentval-
ueswereatthemediumpH(7.05),asshowninTable1andFig2A.Thiscouldbeexplainedin
termsofthelowdrugsolubilityatneutralmediaasperthestudyperformedbySigurdsson
etal[25].Thelowsolubilitymightleadtothepresenceofexcessundissolveddruginthefor-
mulawhichcouldberemovedbyfiltrationafterpreparation.Ontheotherhand,increasing
thePCratioleadtoasignificantincreaseinthedrugcontent.Thiscouldindicatetheefficiency
of2:1w/wP/Dratio(nearerto1:1Mol/Mol)toformSADNwiththedrugwhencomparedto
thelowerratio.ThiscouldsupporttheequimolarinteractionbetweenPCandthecandidate
drugsaspreviouslystatedinseveralresearches[13,14and30].
Particlesize,particlesizedistributionandzetapotential. Particlesizewaspreservedin
thenanorangewherevaluesvariedbetween100.1and604.1nmthroughouttheprepared
SADNformulae,asdemonstratedinTable1andFig2B.Quadraticmodelwasthemostfitting
onefortheanalysisoftheobtaineddatawithnon-significantlackoffit(p-value=0.1162),
highsignaltonoiseratio(adequateprecision=96.87)andgoodmatchingbetweenthe
adjustedandpredictedR2values(lessthan0.2)[31].Thefollowingequationrepresentsthe
effectofthetracedfactorsontheparticlesize:
PS¼288:35(cid:0) 159:35X (cid:0) 90:64X þ21:24X X þ41:13X2 ð4Þ
1 2 1 2 1
SignificantdecreaseintheparticlesizewasobservedwithincreasingpHandP/Dratio(p-
value<0.0001)withsignificantinteractionbetweenthetwofactors(p-value=0.0004).The
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Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
Fig2. ResponsesurfaceplotsfortheeffectsofpHandP/Dratioonthedrugcontent(A),particlesize(B),polydispersityindex(C),zetapotential(D),partition
coefficient(E),releaseT (F)andreleaseextent(G)ofDorzolamideself-assemblednanostructures.
50%
https://doi.org/10.1371/journal.pone.0191415.g002
correlationbetweenpHandinterfacialtensionofPCwaspreviouslystudiedbyPetelskaetal
[26]wheretheinterfacialtensionwasmaximalatthePCisoelectricpoint((cid:25)4)andgradually
declinedbyincreasingpH[26].Reducingtheinterfacialtensioncouldfacilitatetheformation
ofsmallernanostructures[32].Ontheotherhand,thedecreaseintheparticlesizewithincreas-
ingP/Dratiomightbereferredtothepreviouslydiscussedefficientcomplexationbetweenthe
drugandPCatratio2:1w/wwhichcouldleadtoformationofnanostructureswithhigheryield
andsmallersize.Thistheorycouldbeopposedbythedirectrelationbetweenparticlesizeand
polymerconcentrationobservedincaseofconventionalnanoparticles,physicallyencapsulating
thedrug[33].
Polydispersityindexvalueswereextendedfrom0.240and0.714.Itwastakenasaparameter
forevaluatingthedegreeofparticlesizevariation.Datawereanalyzedusingtwo-factorinter-
actionmodel.Adjustedandpredictedr2wereclosetoeachother(0.9604and0.9388,respec-
tively)andadequateprecisionwashigherthan4(18.77)indicatingtheabilityoftheselected
modeltonavigatethedesignspacewithefficientpredictivecapability[34].Therepresenting
equationwasasfollows:
PDI ¼0:46þ0:01X þ0:03X þ0:03X þ0:18X X ð5Þ
1 2 2 1 2
pHandP/Dratiohavenosignificanteffectsonpolydispersityindexasp-valueswere0.2913
and0.0535,respectively,althoughtherewasaninteractionbetweenthetwofactors(p-
value<0.0001),asdisplayedinTable1andFig2C.ElevationofpHreducedpolydispersity
indexatP/Dratio1:1w/wwhilepolydispersityindexwereraisedwithincreasingpHatP/D
ratio2:1w/w,butgenerally,changeswereinsignificant.
Zetapotentialvaluesliedbetween-39.3and-60.7,asillustratedinTable1andFig2D
impartinghighphysicalstabilityonthenanostructuressurfacesthroughhinderingtheiraggre-
gationduringstorage[35].Linearmodelwasthemostsuitableforthefactorialanalysisofzeta
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Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
potentialvalueswithnon-significantlackoffit(p-value=0.6135)whencomparedtothepure
errorindicatingthatthemodelfitswell[36].Moreover,r2(adjusted:0.9250,predicted:0.8793)
andadequateprecision(15.02)valuesconfirmedthevalidityandsuitabilityofthelinearmodel
toanalyzethezetapotentialdata.Theoutputtedequationwasasfollows:
ZP¼(cid:0) 48:87(cid:0) 8:2X þ0:83X ð6Þ
1 2
pHhadasignificanteffectonzetapotential(p-value<0.0001)whileP/Dratiohadnosig-
nificanteffect(p-value=0.2708).ZetapotentialincreasedbyelevatingpHfrom5.4to8.7pass-
ingthroughpH7.05.Thiscouldbeexplainedinthelightoftheelectrostaticinteractions
occurredateachpHvalue.ThepositivedrugmoietiesatpH5.4couldneutralizethenegative
chargesonPCsurfacesminimizingthezetapotentialwhilethenegativelychargeddrugmole-
culesformedatpH8.7couldaddanegativepotentialonthedoublebilayerofPCraisingthe
zetapotentialupto-60[27].
Partitioncoefficient. Partitioncoefficientvaluesrangedbetween0.121and2.821,as
showninTable1andFig2E,andstatisticallyanalyzedusingresponsesurfacequadratic
model.Themodelfittingandprecisionwereconfirmedthroughdeterminingthelackoffit
significanceandadequateprecision(p-value=0.1051and105.97,respectively).Theoutput
quadraticequationwasasfollows:
K ¼0:36þ0:48X þ0:07X þ0:15X X (cid:0) 0:70X2 ð7Þ
1 2 1 2 1
BothpHandP/Dratiohadsignificanteffectonthepartitioncoefficientvalues(p-
value<0.0001).ThemaximumpartitioncoefficientswereobservedattheneutralpHwhile
thelowestatpH5.4withintermediatevaluesatpH8.7.Therelationbetweenpartitioncoeffi-
cientandcornealpermeabilitywaspreviouslystudiedinmanyresearchesanditwasfound
thattheoptimumcornealpermeationwasatpartitioncoefficientrangeof2.5–3[37].More-
over,themaximumcorneal/conjunctivalpermeabilityratiowasobservedatpartitioncoeffi-
cienthigherthantwo[38].Asthemaximummeasuredvalueinourstudywas2.821which
couldbeoptimumforthecornealpermeation,theoptimizationtargetwassettomaximizethe
partitioncoefficient.
Invitrorelease. Invitroreleasethroughdialysismembraneswastakenasapredictivetool
fortheinvivobehaviorafterinstillationintoeyes.Twooptimizationtargetsweresetforthein
vitroreleasetesting;maximizingthereleaseT andextent.AteachpHvalue,theSADNfor-
50%
mulaeshowedslowerreleaseratesthanthemarketproduct,asshowninFig3.Furthermore,
thereleaseextentvalueswereminimumattheneutralpH.ReleaseT valuesrangedbetween
50%
243and1569minwhilereleaseextentvaluesliedbetween68.20%and88.34%,asdemon-
stratedinTable1,Fig2Fand2G.Tostatisticallyevaluatethedata,quadraticmodelwas
selectedforbothresponses;T andreleaseextent.Theselectedmodelwasauthenticatedfor
50%
theitscapabilitytofitandnavigatethedesignspacethroughensuringthatthelackoffitwas
non-significantlydifferentfromthepureerror(p-values=0.0643and0.2997forT and
50%
releaseextent,respectively),inadditiontoadequateprecisionexceeding4inbothresponses
(61.75and10.16forT andreleaseextent,respectively).Factorialequationsforthetwo
50%
responseswereasfollows:
T ¼1450:50(cid:0) 217:87X þ85:80X (cid:0) 39:12X X (cid:0) 935:62X2 ð8Þ
50% 1 2 1 2 1
RE¼73:6þ1:44X (cid:0) 3:35X þ2:30X X þ9:43X2 ð9Þ
1 2 1 2 1
PLOSONE|https://doi.org/10.1371/journal.pone.0191415 February5,2018 8/16
Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
PLOSONE|https://doi.org/10.1371/journal.pone.0191415 February5,2018 9/16
Self-assembleddorzolamidenanostructuresforhighintra-ocularpressure
Fig3. Releaseprofilesofthedrugfromtheformulaepreparedatlow(A),medium(B)andhigh(C)pHvalues.
https://doi.org/10.1371/journal.pone.0191415.g003
ChangingpHhadsignificantinfluenceontheT (p-value<0.0001)wherethelowest
50%
releaseratewasobservedattheneutralpH.Thiscouldbecorrelatedtothehighpartitioncoef-
ficientandhence,lipophilicityofDHattheseconditionsthatmighthinderthedrugreleaseto
theaqueousmedium[39].Ontheotherhand,bothreleaserateandextentweresignificantly
affectedbytheP/Dratio(p-values=0.0003and0.0065,respectively).Thismightbeattributed
tothelipophilicnatureofPCanditsabilitytoefficientlyassociatedrugmoleculesatratio2:1
w/w,asdiscussedbeforewithinthedrugcontentdata[40].
SelectionoftheoptimizedSADNformulae
Desirabilitywasconsideredasastatisticaltooltoselecttheoptimumlevelforeachfactorthat
couldsimultaneouslyachievethedesiredresponses[41].Optimizationprocesswasbuilton
maximizingdrugcontent,zetapotential,partitioncoefficient,releaseT andextent,inaddi-
50%
tiontominimizingparticlesizeandpolydispersityindex.TheformulaepreparedatpH8.7(F5
andF6)hadthehighestdesirabilityvalues(0.64and0.55,respectively).Consequently,both
formulaewereselectedforfollowingfurtherinvestigation.
InvitrocharacterizationoftheoptimizedSADNformulae
Differentialscanningcalorimetryandx-raydiffraction. Thedrughadacharacteristic
endothermicpeakat131.25oC,asdemonstratedinFig4A,whilePChadahigherendothermic
peakappearedat181.54oC.Theoptimizedformulae(F5andF6)showeddown-shiftingofthe
drugandPCpeaks.ThesefindingsindicatedthepresenceofinteractionbetweenDHandPC
thatcouldbephysicalofchemical[42,43].X-raydiffractionwasperformedtoinvestigate
natureofthedetectedinteractionandtheobtaineddiffractogramsweredisplayedinFig4B.
Thedrughadcharacteristicspeaksat12.7,16.9,20.8,24.8,32.1and45.8(2θ),whilePClacked
thepresenceofanypeakthroughoutitsdiffractogram.Thesedatacouldpointoutthecrystal-
linenatureofDHandtheamorphousnatureofPC.IncaseofF5formula,thepeakat16.9
(2θ)disappearedwhileanewpeakarousedat30.0(2θ).Moreover,twopeaksappearedwithin
F6diffractogramat21.3and30.0(2θ).Changesinthedrugcharacteristicpeaks,especially
appearanceofnewpeaks,despitetheamorphousPCnature,couldelucidatethechemical
interactionbetweenDHandPCratherthanthedrugphysicalinclusionwithintheconven-
tionalPCliposomes[44,42].
Transmissionelectronmicroscopy. Imagingbytransmissionelectronmicroscopewas
employedtoinvestigatethemorphologicalcharacteristicsoftheoptimizedSADNformulae.
Fig4Cand4Dshowedtheexistenceofuniformnanoparticleswithasizerangematchingthe
dataobtainedbytheZetasizeranalysis.Moreover,almostallparticleshadsphericaloutline
withsmoothsurfaces.
InvivocharacterizationoftheoptimizedSADNformulae
Bioavailabilityenhancement. Thepharmacokineticbehavioroftheoptimizedformulae
wascomparedtothemarketproduct(Trusopt1)todeterminethedegreeofenhancementin
therateand/orextentofdrugpermeationtotheaqueoushumorasshowninTableCinS1
File.Theoptimizedformulaedidnotshowsustainedinvivoprofilesrelativetothemarket
productastheintermsoftheT andMRTvalues,asdisplayedinTable2.Ontheother
max
hand,FormulaeF5andF6hadsignificantlyhigherC ,AUC24andAUC1thanthatofTru-
max 0 0
sopt1(p-value<0.001)indicatingthecapabilityoftheformulatedSADNtoenhancethe
PLOSONE|https://doi.org/10.1371/journal.pone.0191415 February5,2018 10/16