Int J Pharm

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ContentslistsavailableatScienceDirect

International

Journal

of

Pharmaceutics

j ou rn a l h om epa ge : w w w . e l s e v i e r . c o m / l o c a t e / i j p h a r m

Pharmaceutical

Nanotechnology

Design

of

cationic

lipid

nanoparticles

for

ocular

delivery:

Development,

characterization

and

cytotoxicity

Joana

F. Fangueiro

a

_,

_Tatiana

_Andreani

a,b,c

_,

_Maria

_A.

_Egea

d,e

_,

_Maria

_L.

_Garcia

d,e

_,

Selma

B. Souto

f

_,

_Amélia

_M.

_Silva

b,c

_,

_Eliana

_B.

_Souto

a,g,∗

a_Faculty_of_Health_Sciences,_Fernando_Pessoa_University_(UFP-FCS),_Rua_Carlos_da_Maia,_296,_4200-150_Porto,_Portugal b_Centre_for_Research_and_Technology_of_{Agro-Environmental}_and_Biological_Sciences_(CITAB),_Vila_Real,_Portugal c_Department_of_Biology_and_Environment,_University_of_{Trás-os-Montes}_e_Alto_Douro_(UTAD),_Vila_Real,_Portugal

d_Department_of_Physical_Chemistry,_Faculty_of_Pharmacy,_University_of_Barcelona,_Av._Joan_XXIII_s/n,₀₈₀₂₈_Barcelona,_Spain e_Institute_of_Nanoscience_and_{Nanotechnology,}_University_of_Barcelona,_Av._Joan_XXIII_s/n,₀₈₀₂₈_Barcelona,_Spain f_Division_of_{Endocrinology,}_Diabetes_and_Metabolism,_Hospital_de_Braga,_Braga,_Portugal

g_Institute_of_{Biotechnology}_and_{Bioengineering,}_Centre_of_Genetics_and_{Biotechnology,}_{Trás-os-Montes}_and_Alto_Douro_University_{(IBB/CGB-UTAD),}

VilaReal,Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11October2013 Receivedinrevisedform 10November2013 Accepted15November2013 Available online 23 November 2013

Keywords: Lipidnanoparticles Oculardelivery Multipleemulsion Cytotoxicity Turbiscan

Y-79humanretinoblastomacells

a

b

s

t

r

a

c

t

Inthepresentstudywehavedevelopedlipidnanoparticle(LN)dispersionsbasedonamultipleemulsion techniqueforencapsulationofhydrophilicdrugsor/andproteinsbyafullfactorialdesign.Inorderto increaseocularretentiontimeandmucoadhesionbyelectrostaticattraction,acationiclipid,namely cetyltrimethylammoniumbromide(CTAB),wasaddedinthelipidmatrixoftheoptimalLNdispersion obtainedfromthefactorialdesign.Therearealimitednumberofstudiesreportingtheidealconcentration ofcationicagentsinLNfordrugdelivery.Thispapersuggeststhatthechoiceoftheconcentrationofa cationicagentiscriticalwhenformulatingasafeandstableLN.CTABwasincludedinthelipidmatrix ofLN,testingfourdifferentconcentrations(0.25%,0.5%,0.75%,or1.0%wt)andhowcompositionaffects LNbehaviorregardingphysicalandchemicalparameters,lipidcrystallizationandpolymorphism,and stabilityofdispersionduringstorage.Inordertodevelopasafeandcompatiblesystemforoculardelivery, CTAB-LNdispersionswereexposedtoHumanretinoblastomacelllineY-79.Thetoxicitytestingofthe CTAB-LNdispersionswasafundamentaltooltofindthebestCTABconcentrationfordevelopmentof thesecationicLN,whichwasfoundtobe0.5wt%ofCTAB.

1. Introduction

Lipidnanoparticles(LN)havegainedinterest inrecent years asdrugcarriersfor oculardelivery,aiminga betterpermeation and/orprolongeddrugreleaseontotheocularmucosaandallowing drugsreachingthepostsegmentoftheeye(PignatelloandPuglisi, 2011).Oculardrugdeliveryisextremelyaffectedbyeyeanatomy and physiology that leads often to mechanisms that decrease bioavailabilityofapplieddrugs.Thesemechanismsincludereflex processes,suchaslacrimationandblinkingwhichreduces drasti-callythedrugresidencetime,anddifficultytodiffusethoughthe conjunctivaandnasolacrimalduct.Inaddition,thelowvolumeof theconjunctivalsacalsoleadstoapoorcornealorsclera pene-trationofdrugs(DieboldandCalonge,2010).Sinceoculardelivery

∗_{Corresponding}_author_at:_Faculty_of_Health_Sciences_of_Fernando_Pessoa

Univer-sity,RuaCarlosdaMaia,296,OfficeS.1,P-4200-150Porto,Portugal. Tel.:+351225074630x3056;fax:+351225504637.

E-mailaddresses:eliana@ufp.edu.pt,souto.eliana@gmail.com(E.B.Souto).

becameaproblemwhentheultimatetargetisintraoculardelivery, duetotheineffectivedrugconcentrationsandtimeresidencereach theinnertissues,alternativesystemsfordrugdeliveryarerequired (PignatelloandPuglisi,2011;Sultanaetal.,2011).Newdrug deliv-erysystemsbasedonlipids,namelyliposomes,andothermaterials suchaspolymers(poly-d-l-lacticacid(PLA)nanopsheres)were abletodeliveranantiviraldrug,acyclovir,intheinnertissuesof theeyecomprisingtheinnovationofthesesystems(Frestaetal., 1999;Giannavolaetal.,2003).

Oculardrugdeliverystrategiesmaybeclassifiedinto3groups: noninvasivetechniques,implants,andcolloidalcarriers.Colloidal drugdeliverysystems,suchasLN,canbeeasilyadministeredin aliquidformandhavetheabilitytodiffuserapidlyandare bet-terinternalizedinoculartissues.Inaddition,theinteractionand adhesionofLNocularsurfacewiththeendotheliummakesthese drugdeliverysystemsinterestingasnewtherapeutictoolsinocular delivery(delPozo-Rodriguezetal.,2013).

LN based onw/o/w emulsion are versatile colloidal carriers fortheadministrationofpeptides/proteinsandhydrophilicdrugs (Fangueiroetal.,2012).Dropletsfromtheinneraqueousphase,

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461 (2014) 64–73 65

wherethedrugisdissolvedor/andsolubilized,aresupportedby asolidlipidmatrixsurroundedbyanaqueoussurfactantphase. UsuallyLNarecomposedofphysiological solidlipids(mixtures ofmono-,di-ortriglycerides,fattyacidsorwaxes)stabilizedby surfactants. Inthecase of aw/o/w basedLN dispersion,a high hydrophilic–lipophilic balance (HLB) surfactant is added tothe externalaqueousphaseand,alowHLBsurfactantisaddedtothe lipidphase.Thetwosurfactantsareneededtostabilizethetwo existinginterfaces inthis typeofemulsion. Avarietyof surfac-tantscanbeapplied,suchasphospholipids,bilesalts,polysorbates, polyoxyethyleneethers (Gallarate etal., 2009; Fangueiroet al., 2012).MaterialsusedforLNproductionarelargelyusedin phar-maceuticalindustrywithprovedbiocompatibility(Severinoetal., 2012).

CationicLNhavebeenrecentlyinvestigatedfortargetingocular mucosa,namelytheposteriorsegmentoftheeye(e.g.retina).This isasmartstrategy thatcombinesthepositive surfacechargeof theparticlesandthenegativesurfacechargeofocularmucosaby meansofanelectrostaticattraction.Thisapproachcouldincrease thedrugsretentiontimeintheeyeaswellasimprovenanoparticles bioadhesion(Lallemandetal.,2012).

Intheoculardelivery, itis especiallyrelevant thecontrolof theparticlesizesinceitdirectlyinfluencethedrugrelease rate, bioavailability, and patient comfort and compliance (Shekunov etal.,2007;Soutoetal.,2010).Inaddition,itisknownthatthe smallertheparticlesize,thelongertheretentiontimeandeasier application(Araujoetal.,2009).

Physicochemicalcharacterizationandassessmentof nanotoxic-ityaremajorissuesfordevelopingandlarge-scalemanufacturingof nanocarriers.Furthermore,physicochemicalpropertiesofLNsuch asparticlesize,surfaceandcompositioncansignificantlyinfluence drugdeliveryonoculardelivery(Yingetal.,2013).

Inthepresentwork,thedevelopmentandcharacterizationof asystemofLNbasedonmultipleemulsionsusingablendof tri-acylglycerolsassolidlipid,wascarried out,in whichsonication methodwasemployed.Thefirstaimoftheworkwasthe appli-cation of a full factorial design todeterminewhich dependent variablescouldaffecttheLNdispersionproperties.Theanalyzed independentvariables,namelytheconcentrationofsolidlipidand bothhydrophilicandlipophilicsurfactants,werecheckedfortheir capacitytoinfluencethemeanparticlesize(Z-Ave),polydispersity index (PI) and zeta potential(ZP) of theproduced LNs disper-sions.Theoptimalformulationwasusedtoevaluatethetoxicity ofLN usingY-79 humanretinoblastoma cells employing differ-entcationiclipidconcentrationstoselectthebestformulationfor ocularinstillations.

2. Materialsandmethods

2.1. Materials

Softisan® ₁₀₀_(S100,_a_hydrogenated_{coco-glycerides}_C

10–C18

fattyacidtriacylglycerol)used assolid lipid wasa free sample fromSasolGermanyGmbH(Witten,Germany),Lipoid®_S75,_75%

soybeanphosphatidylcholine,usedassurfactant,waspurchased fromLipoidGmbH(Ludwigshafen,Germany),Lutrol®_F68_or

Polox-amer188 (P188)was a free sample fromBASF (Ludwigshafen, Germany).Cetyltrimethylammoniumbromide(CTAB)anduranyl acetatewereacquiredfromSigma–Aldrich(Sintra,Portugal). Anhy-drousglycerolwaspurchasedfromAcopharma(Barcelona,Spain). Ultra-purifiedwaterwasobtainedfromaMiliQPlussystem (Mili-pore,Germany).Allreagentswereusedwithoutfurthertreatment. TheY-79humanretinoblastomacelllinewaspurchasedfromCell LinesService(CLS,Eppelheim,Germany).Reagentsforcellculture werefromGibco(Alfagene,Invitrogene,Portugal).

Table1

Initial3-levelfullfactorialdesign,providingthelower(−1),medium(0)andupper (+1)levelvaluesforeachvariable.

Variables Levels Lowlevel (−1) Medium level(0) Highlevel (+1)

S100(wt%) 2.5 5.0 7.5

Lecithin(wt%) 0.25 0.5 0.75

P188(wt%) 0.5 1.0 1.0

S100:Softisan®_100;_P188:_Poloxamer_188.

2.2. Experimentalfactorialdesign

Afactorialdesignapproachusinga33_full_factorial_design

com-posedof3variableswhichweresetat3-levelseachwasapplied tomaximizetheexperimentalefficiencyrequiringaminimumof experiments.Forthispurposethreedifferentvariablesandtheir influenceonthephysicochemicalpropertiesoftheproducedLN wereanalyzed.Thedesignrequiredatotalof11experiments.The independentvariablesweretheconcentrationofsolidlipidS100, concentrationoflecithin(Lipoid® _S75)_and_the_{concentration}_of

hydrophilicsurfactantP188.Theestablisheddependentvariables werethemeanparticlesize(Z-Ave),polydispersityindex(PI)and zetapotential(ZP).Foreachfactor,thelower,mediumandhigher valuesofthelower,mediumandupperlevelswererepresentedby a(−1),a(0)anda(+1)sign,respectively(Table1).Thedatawere analyzedusingtheSTATISTICA7.0(Stafsoft,Inc.)software.

2.3. Lipidnanoparticlesproduction

LNdispersionswerepreparedusinganovelmultipleemulsion (w/o/w)technique(García-Fuentesetal.,2003).Briefly,aninner w/oemulsionwasinitiallyprepared.Avolumeof ultra-purified waterwasaddedtothelipidphase(5wt%)composedofglycerol, S100 and Lipoid® _S75_at _same_temperature _(5–10◦_C _above _the

meltingpointofthesolidlipidSoftisan®₁₀₀₍_T_≈₅₀◦_C)_and

homog-enized60swithasonicationprobe(6mmdiameter)bymeansofan UltrasonicprocessorVCX500(Sonics,Switzerland).Apower out-putwithamplitudeof40%wasapplied.AfewmillilitersofP188 solutionwasaddedandhomogenizedforadditional90s.This pre-emulsionwaspouredinthetotalvolumeofaP188cooledsolution undermagneticstirringfor15mintoallowtheformationoftheLN. TheobtainedLNdispersionswereusedforsubsequentstudies.The generalcompositionofLNdispersionsisdescribedinTable2.

2.4. Physicochemicalcharacterization

PhysicochemicalparameterssuchasZ-Ave,PIandZPwere ana-lyzedbydynamiclightscattering(DLS,ZetasizerNanoZS,Malvern Instruments,Malvern,UK).Allsamplesweredilutedwith ultra-purifiedwater andanalyzedintriplicate.ForanalysisoftheZP, ultra-purifiedwaterwithconductivityadjustedto−50␮S/cmwas

used.

Table2

CompositionofSLNdispersions(wt/wt%).

Components %(wt/wt)

Softisan®₁₀₀ _5.0

Glycerol 37.5

Lipoid®_S75 _0.5

Lutrol®_F68 _1.0

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66 461 (2014) 64–73

2.5. EvaluationoftheconcentrationofCTAB

Inordertoincreaseeyeretentiontimeandmucoadhesiononto theocularmucosa,acationiclipidwasaddedtothelipidmatrix.For thispurpose,CTABwasusedascationiclipidmaintainingthe5% oflipidmatrixvaryingtheproportionsofS100andCTAB.Thus,in theproductionstage,fourdifferentconcentrations(0.25,0.5,0.75 and1.0wt%)ofcationiclipid(CTAB)wasaddedinthelipidphase composedofS100andLipoid®_S75_of_the_optimal_formulation,

pre-viouslyobtainedfromthefactorialdesigntoevaluatetheinfluence inthephysicochemicalproperties.

2.6. StabilityanalysisofLNbyTurbiscanLab®

TheTurbiscanLab® _is_a _technique_used_to_observe_reversible

(creamingandsedimentationduetofluctuationonparticlesize andvolume)andirreversible(coalescenceandsegregationdueto particlesizevariation)destabilizationphenomenainthesample withouttheneed of dilution.TurbiscanLab® _is _useful_to_detect

destabilizationphenomena much earlier and also in a simpler waythanothermethods,sinceit isbasedonthemeasurement ofbackscattering(BS)andtransmission(T)signals(Araújoetal., 2009;Celiaetal.,2009;Marianeccietal.,2010;Liuetal.,2011).

ThephysicalstabilityofLNdispersionswasassessedwithan opticalanalyzerTurbiscanLab®_{(Formulaction,}_France)._The

disper-sionswereplacedinacylindricalglasscell,atroomtemperature (25◦_C)._The_equipment_is_composed_of_a_{near-infrared}_light_source

(=880nm),and2synchronoustransmission(T)and backscatter-ing(BS)detectors.TheTdetectorreceivesthelightcrossingthe sample,whereastheBSdetectorreceivesthelightscattered back-wardsbythesample(Araújoetal.,2009).Thedetectionheadscans theentireheightofthesamplecell(20mmlongitude),acquiringT andBSeach40␮m,3timesduring10minatdifferenttimesafter

production(7,15and30days).

2.7. Thermalanalysis

ThecrystallinityprofileofLNwasassessedbydifferential scan-ningcalorimetry(DSC).Thistechniqueis usefultoevaluatethe physicalstate,whichdirectlyaffectsthephysicochemical proper-tiesandthermodynamicstabilityofLNdispersions.Avolumeof LNdispersioncorrespondingto1–2mgoflipidwasscannedusing aMettlerDSC823eSystem(MettlerToledo,Spain).Heatingand coolingrunswereperformedfrom25◦_C_to₉₀◦_C_and_back_to₂₅◦_C

ataheatingrateof5◦_C/min,_in_sealed₄₀

␮Laluminumpans.An

emptypanwasusedasareference.Indium(purity>99.95%;Fluka, Buchs,Switzerland)wasemployedforcalibrationpurposes.DSC thermogramswererecordedforthefourdifferentCTAB concen-trationformulationsandforthebulklipids(CTABandS100).The DSCparametersincludingonset,meltingpointandenthalpywere evaluatedusingSTARe_Software_(Mettler_Toledo,_{Switzerland).}

2.8. X-Raystudies

X-raydiffractionpatternswereobtainedusingtheX-ray scat-tering(X’PertPRO,PANalytical)usingaX’Celeratorasadetector. Dataofthescatteredradiationweredetectedwithablend local-sensibledetectorusingananodevoltageof40kVandacurrentof 30mA.FortheanalysisofLNdispersionsandbulkmaterials,the samplesweremountedonastandardsampleholderbeingdriedat roomtemperaturewithoutanyprevioussampletreatment.

2.9. Alamarblueassayinhumanretinoblastomacellline

Y-79(Humanretinoblastomacellline)cellswereusedto per-formthecytotoxicityassay,inwhichfourLNdispersionscontaining

differentCTABconcentrationsweretested.Eachformulationwas testedat fourconcentrations (in␮gmL−1):10, 25,50 and 100.

Y-79 cells were maintained in RPMI-1640, supplemented with 10%(v/v)fetalbovineserum(FBS),2mMl-glutamine,and

antibi-otics(100UmL−1_penicillin_and₁₀₀

␮gmL−1ofstreptomycin)in

anatmosphereof5%CO2 inairat37◦Cellswerecentrifuged,

re-suspendedinFBS-freeculturemedia,countedandseeded,after appropriatedilution,at1×105_cell_mL−1_density_in_96-well_plates

(100␮L/well).Thedifferentformulationsweredilutedwith

FBS-freeculturemediatoachievethefinalconcentrations,andadded tocells24hafterseeding(100␮L/well).Cellviabilitywasassayed

withAlamarBlue(Alfagene,Invitrogene,Portugal)byadding10% (v/v)toeachwell,andtheabsorbanceat570nm(reducedform) and620nm(oxidativeform)wasread24,48and72hafter expo-suretotest compounds, datawereanalyzed by calculatingthe percentageofAlamarbluereduction(accordingtothe manufac-tures recommendation)and expressedas percentageof control (untreatedcells).

2.10. Transmissionelectronicmicroscopyanalysis

Transmissionelectronicmicroscopy(TEM)isatechniqueuseful toanalyzetheshape andsizeofLN dispersions.ThechosenLN dispersioncorrespondingtoCTAB-LNdispersionwith0.5wt%CTAB wasmountedonagridandnegativestainedwitha2%(v/v)uranyl acetatesolution.Afterdryingatroomtemperature,thesamplewas examinedusingaTEM(TecnaiSpiritTEM,FEI)at80kV.

2.11. Statisticalanalysis

Statistical evaluation of data was performed using one-way analysisofvariance(ANOVA).TheBonferronimultiplecomparison testwasusedtocomparethesignificanceofthedifferencebetween thegroups,ap-value<0.05wasacceptedassignificant.Datawere expressed as the mean value±standard deviation (Mean±SD) (n=3).

3. Resultsanddiscussion

TheproductionandoptimizationofLNbasedonmultiple emul-sionrequirespre-formulationstudiesandliteratureresearch.The stabilityandcompatibilityoftheemulsifiersandthelipidmatrix areessentialtoprovideastableandfunctionalsystem(Fangueiro etal.,2012).Sincethechoiceofthecomponentsarevitalfor dis-persions formation, Lipoid® _S75 _(soybean _{phosphatidylcholine)}

wastheselectedlipophilicemulsifierusedwitha HLBvalueof approximately7–9,andPoloxamer188wasselectedashydrophilic emulsifierwithaHLBvalueofapproximately22.

Physicochemicalpropertiesandstabilityofthenewdrug deliv-erysystemsaremajorissuestobeconsideredintheformulation stage, especially those intended for ocular administration. The useof dispersionswithappropriate physicochemical properties ensuresadequatebioavailabilityofadministereddrugs and bio-compatibilitywithocular mucosa.The LN dispersions obtained fromthefactorialdesign,revealedadequatephysicochemical sta-bilityduringtheanalyticaltesting.In addition,themacroscopic stability ofthe particles,monitoredbyvisual analysis,DLSand Turbiscan® _Lab_analysis,_did_not _suffer_any_changes. _Separation

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461 (2014) 64–73 67

Table3

Responsevalues(Z-Ave,PIandZP)ofthethreefactorsdepictedinTable1forthe11experimentformulations.

Run S100(wt%) Lecithin(wt%) P188(wt%) Z-Ave(nm)±SD PI±SD ZP(mV)

SLN1 2.5 0.25 0.5 256.40±1.20 0.181±0.02 −1.04

SLN2 7.5 0.25 0.5 268.50± 1.47 0.173± 0.04 −1.06

SLN3 2.5 0.75 0.5 165.85± 2.21 0.162± 0.01 −1.08

SLN4 7.5 0.75 0.5 171.24± 1.78 0.155± 0.03 −1.20

SLN5 2.5 0.25 1.5 241.30±2.54 0.182±0.05 −1.22

SLN6 7.5 0.25 1.5 235.40±1.87 0.192±0.02 −1.04

SLN7 2.5 0.75 1.5 189.75±1.99 0.163±0.08 −1.12

SLN8 7.5 0.75 1.5 164.50±1.14 0.158±0.03 −1.14

SLN9 5.0 0.5 1.0 165.90± 1.20 0.164± 0.04 −1.18

SLN10 5.0 0.5 1.0 164.50± 1.09 0.183± 0.05 −1.17

SLN11 5.0 0.5 1.0 164.70±1.01 0.177±0.02 −1.16

Table4

ANOVAstatisticalanalysisoftheZ-Ave.

Evaluatedfactorsandtheirinteractions Sumofsquares Degreesoffreedom Meansquare F-value p-Value

(1)S100concentration 23.32 1 23.32 0.01962 0.895380

(2)Lecithinconcentration 12032.66 1 12032.66 10.12031 0.033495

(3)P188concentration 120.44 1 120.44 0.10129 0.766206

1by2 84.89 1 84.89 0.07140 0.802522

1by3 295.73 1 295.73 0.24873 0.644149

2by3 533.99 1 533.99 0.44912 0.539455

Error 4755.85 4 1188.96

Total 17846.88 10

Thevaluesinboldarethestatiscallysignificantresults(p<0.5).

Table5

ANOVAstatisticalanalysisofthePI.

Evaluatedfactorsandtheirinteractions SumofSquares Degreesoffreedom Meansquare F-value p-Value

(1)S100concentration 0.000072 1 0.000072 1.02591 0.368411

(2)Lecithinconcentration 0.001352 1 0.001352 19.26425 0.011791

(3)P188concentration 0.000032 1 0.000032 0.45596 0.536541

1by2 0.000032 1 0.000032 0.45596 0.536541

1by3 0.000072 1 0.000072 1.02591 0.368411

2by3 0.000032 1 0.000032 0.45596 0.536541

Error 0.000281 4 0.000070

Total 0.001873 10

Thevaluesinboldarethestatiscallysignificantresults(p<0.5).

Theresultsforthe11producedformulationsisshowninTable3

andvariedfrom164.5±1.09nm(LN8orLN10)to268.50±1.47nm (LN2),whereasPIrangedfrom0.155±0.03(LN4)to0.192±0.02 (LN6).Theparticlesizedistributionwasverynarrowinallcases sincethePIwaslessthan0.2,correspondingtomonodispersed systems.Accordingtotheliterature(ZimmerandKreuter,1995,

Shekunovetal.,2007),theZ-aveforocularadministrationshould bebelow1␮mwithanassociatednarrowsizedistribution.Thus,all

formulationsrevealedaZ-AveandPIwithinacceptedrange(Fresta

etal.,1999;Giannavolaetal.,2003;Vegaetal.,2008;Soutoetal., 2010).Asexpected,theZPdidnotvary,since allusedreagents havenon-ionicnature.Foreachofthe3variables,analysisof vari-ance(ANOVA)wasperformed.FromTable4andFig.1,theonly factorthatwasshowntohaveasignificanteffect(p-value<0.05) onZ-Avewastheconcentrationoflecithin.Allotherevaluated fac-tors,werenotstatisticallysignificant(p-value>0.05),neitherthe interactionsbetweenthem.Thesameresultswereobservedforthe evaluationofthedependentvariablesonPI(Table5andFig.1).The

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68 461 (2014) 64–73

Fig.2.SurfaceresponsechartoftheeffectoftheconcentrationofS100andlecithinontheZ-Ave(A)andPI(C)andtheeffectoftheconcentrationoflecithinandpoloxamer 188ontheZ-Ave(B)andPI(D).

concentration of lecithin was the only independent variable affecting the PI. The lecithin used is composed of 75% of phosphatidylcholine, which is composed by phospholipids that resemblethe cellularmembranes. Theuse of this emulsifier is reportedassafeandbiocompatibleforseveralpurposes,suchas skinproducts(Fiume,2001)andalsoforophthalmic/oculardrug delivery(Bhattaetal.,2012).TheiruseinthedevelopmentofLN dis-persionsisessentialtodecreasetheinterfacialtensionbetweenthe oilphaseandtheinternalandexternalaqueousphase,andalsoto facilitatetheemulsificationofthelipidmatrix.Lecithinisuseddue toitshigherpowerofemulsificationabletoprovideaverygood sta-bilizationoftheoil-in-waterinterfacesandhasalsobeenreported todecreaseparticlesizeinemulsionsthatismainlyexplainedby itsamphiphiliccharacter(Trottaetal.,2002;Schubertetal.,2006; Kawaguchietal.,2008).Thelipophilicportionoflecithindissolves thelipidphase, i.e. lecithin likes tobeat theedge of thelipid phasebeingitslipophilictailsdirectedtothelipidphaseuntilthe hydrophilicportionisdirectedtothewaterphase. Thus,theoil phaseistotallyrecoveredbythelecithinpromotinglongtime stabi-lizationintheinterfaceoftheemulsions(Trottaetal.,2002).From theobtainedresults, acorrelationbetweentheconcentrationof lecithinandmeanparticlesizeoftheparticleswerefound,since highestconcentrationsleadstoadecreaseinZ-Aveand PI.This dependencyofZ-Aveonthetypeofemulsifierisduetheneedfor thecompletecoverageoftheinterface,whichisaffectedbythe selectedconcentration(Fig.2).

Theaimofthisfactorialdesignwastooptimizeaformulation withappropriatephysicochemicalparametersforthe incorpora-tionofhydrophilicdrugsforoculardelivery.Forthispurpose,the limitingfactorsweretheZ-AveandPIandfromtheobtainedresults anoptimalLNdispersionwasfound.ThisLNdispersionwasused forthefollowingstudies.

InordertoimproveLNadhesiontoocularsurfaceandalsoto improvestabilityofthedispersions,acationiclipidwasused.The

approachofusingacationiclipidisinterestingsinceocularmucosa depictsslightlynegativechargeaboveitsisoelectricpointandalso couldimprovesomelimitationsrelatedtoocularadministration, suchaspreventtearwashout(duetoteardynamics),increase ocu-larbioavailabilityandprolongtheresidencetimeofdrugsinthe cul-de-sac(Araujoetal.,2009).

Thestudyreportstheuseofdifferentconcentrationsofacationic lipid(CTAB)inthelipidmatrixofLN.TheresultsoftheCTAB con-centrationontheZ-Ave,PIandZPoftheLNdispersionsoptimized inthefactorialdesign aredepictedin Table6.Asexpected,the parametermostaffectedbythevariationonCTABconcentration istheZP.TheincreaseoftheZPwasdirectlyproportionaltothe increaseonCTABconcentration.Statisticalanalysisofthe physi-cochemicalpropertiesofCTAB-LNdispersionswasnotsignificant (p>0.05),indicatingthattheconcentrationofCTABdidnotaffect drasticallytheZ-Ave,PIandZPoftheformulations.All formula-tionspropertieswereinagreementwiththerequiredparameters foroculardelivery.However,nostatisticaldifferenceswerefound, theconcentrationofCTABimprovedtheZPfollowingaproportional relationship.Thisbehaviorwasexpectedbecauseofthecationic propertiesofCTABandisalsoexpectedthathigherZPvalues con-tributetohigherstabilityoftheparticlesduetoelectronicrepulsion betweenthemmaintaininglongertimeinsuspension.

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Table6

PhysicochemicalparametersfromCTAB-LNdispersionsattheproductiondayandalong-termstabilityafter7,15and30dayafterproductionat25◦

C(Mean±SD)(n=3).

Formulation Parameters Productionday Day7 Day15 Day30

0.25%CTAB-LN

Z-Ave(nm) 230.70±6.71 239.5±0.61 244.9±1.17 255.2±1.32

PI 0.308± 0.09 0.267± 0.01 0.261± 0.03 0.266± 0.01

ZP(mV) +24.80± 2.69 +17.4± 0.65 +16.7± 0.56 +16.4± 0.36

0.5%CTAB-LN

Z-Ave(nm) 194.40±0.43 199.25±0.30 201.45±0.08 213.7±0.74

PI 0.185± 0.02 0.186± 0.01 0.224± 0.02 0.225± 0.01

ZP(mV) +37.20± 1.27 +33.8± 0.45 +32.1± 0.53 +30.5± 0.03

0.75%CTAB-LN

Z-Ave(nm) 172.10±12.64 166.25±1.20 223±0.59 236.8±1.45

PI 0.182± 0.02 0.246± 0.01 0.204± 0.01 0.225± 0.04

ZP(mV) +41.70± 0.71 +40.15± 0.63 +38.6± 0.41 +37.4± 1.21

1.0%CTAB-LN

Z-Ave(nm) 169.10±2.51 144.7±1.61 188.3±1.52 200.7±1.65

PI 0.222± 0.022 0.211± 0.01 0.236± 0.02 0.245± 0.04

ZP(mV) +48.00± 0.31 +44.58± 0.50 +42.0± 0.05 +40.2± 0.63

stabilityovertime.Theotherconcentrationsseemtobemore sta-blesincevariationsarelowerthan10%duringthetimeofanalysis. Thisanalysiscouldpredictthegoodstabilityoftheformulations withCTABconcentrationrangingbetween0.5and1.0wt%.These resultsareinagreementwiththeZPvaluesrecordedforthefour formulations(Table6),sincehigherZPvaluescouldalsopredict ahigherstabilityofLNdispersionsasmentionedbefore.Inorder tosupportTurbiscan®_Lab_results_and_also_to_monitored_particles

overaperiodoftime,zetasizermeasurementsweremadeatthe samedaysafterCTAB-LNdispersionsproductionduringstorageat 25◦_C._All_dispersions_showed_a_milky_colloidal_appearance_where

noaggregationphenomenaandnophaseseparationweredetected duringtheperiodofanalysisandstorage.Fromtheresultsdepicted

inTable6ispossibletodetectaslightlyincreasebothintheZ-Ave andinthePIafter30daysofstorage.Duringtheperiodof analy-sis,CTAB-LNdispersionsdepictedparticlesizesbelow300nmand PI≤0.3,whichareacceptablevaluesforoculardelivery(Vegaetal., 2008;Gonzalez-Miraetal.,2010;Soutoetal.,2010;Gonzalez-Mira etal.,2011).Thelong-termstabilitystudiesconfirmedthat concen-trationsofCTABupto0.5wt%couldprovideabetterelectrostatic repulsionbetweenparticlesinsuspensionandfromthis sugges-tionprovidebetterstabilityavoidingparticleaggregationand/or flocculation.

Theanalysisofthecristallinity andpolymorphism oftheLN dispersionsobtainedwasanalyzedbyDSCandX-Ray.The thermo-dynamicstabilityofLNdependsmainlyonthelipidmodification

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70 461 (2014) 64–73

Fig.4. DSCthermogramsof(a)bulkCTAB,(b)bulkS100andCTAB-LNwithdifferent concentrations(c)0.25wt%,(d)0.50wt%,(e)0.75wt%and(f)1.0wt%.

thatoccursaftercrystallization.ThesolidlipidusedwasS100,a tri-acylglycerolblendofvegetablefattyacidswithC10–C18(Fangueiro

etal.,2013).Polymorphictransitionsaftercrystallizationof triacyl-glycerolbasedLNareslowerforlonger-chaintriacylglycerolsthan forshorter-chaintriacylglycerol(MetinandHartel,2005).Thetype ofemulsifierusedinLNdispersionsalsoaffectstheir thermody-namicbehavior,theirstoragetimeanddegradationvelocity(Han etal.,2008).

Triacylglycerols usually occur in three major polymorphic forms,namelya,ˇ′_and_ˇ_(in_order_of_increasing_{thermodynamic}

stability)whicharecharacterizedbydifferentsubcellpackingof thelipidchains.Theˇ′_modification_is_frequently_observed_in

com-plextriacylglycerolssuchasS100(BunjesandUnruh,2007). ThermalanalysisofthebulklipidS100(Fig.4)showsasingle endothermicpeakuponheatingwithaminimumof39.12◦_C_and

anenthalpyof−44.76Jg−1₍_Table₇_)._Our_results_are_in_agreement

withthose reportedbyotherauthors(Thomaand Serno,1983; SchubertandMüller-Goymann,2003)foraˇ′_modification_of

com-plextriacylglycerolsmixtures.ForCTABbulk,asmallmeltingevent wasobservedbetween40and50◦_C,_that_can_be_associated_to_the

meltingofacylchainsandamaintransitionataround106.34◦_C

correspondingtocompletemeltingofCTABandattributedtothe meltingofhead-groups(Doktorovovaetal.,2011).

With respect to CTAB-LN dispersions (Fig. 4), a very small endothermiceventwasreportedcomparedtotheother thermo-gramsofthebulklipidandbulkmatrix.Thistransitionisrelatively smallduetothelowestvaluesofenthalpypresentedbytheLN dis-persionscomparedtothebulklipid(Table7).Thepeaktemperature slightlydecreasedforallCTAB-LNdispersionsconfirmingthat poly-morphismofS100.InLNdispersions,aslightdecreaseofenthalpy isusuallyobserved,aswellasadecreaseonthepeaktemperature comparingtothebulkcounterpart,howeversincethislipidhasa

Fig.5. X-raydiffractionpatternsof(a)bulkCTAB,(b)bulkS100andCTAB-LNwith differentconcentrations(c)0.25wt%,(d)0.50wt%,(e)0.75wt%and(f)1.0wt%.

verylowmeltingpoint,itispossibletoformsupercooledmelts. ApossibleexplanationforthereductionofcrystallinityoftheLN dispersionisthecoexistenceoflipidbeingpresentinthea modifi-cationandalsoduetothecolloidalparticlesizeofferinginsufficient numberofdiffractionlevels.Thesedifferencescanbeattributedto thehighsurfacetovolumeratioofLNdispersions(Schubertand Müller-Goymann,2003).

TheX-rayresultsdepictedinFig.5 alsorevealthepresence oftwo signals oneat 0.42nm (2=21.1◦₎_and _other_at _0.38_nm

(2=23.2◦₎ _which _are _both _{characteristic} _of _the _orthorhombic

perpendicularsubcell,i.e.,ˇ′_modification₍_Schubert_and Müller-Goymann,2003).TheseresultsareinagreementwithDSCstudies, sinceallformulationsrevealedadecreasedcrystallinity compar-ingwithbulklipidsS100and CTAB.Also, themelting enthalpy ofCTAB-LNdispersionsis increasingwithincreasingCTAB con-tent, indicating higher crystallinity of the matrices containing 0.5–1.0wt%ofCTAB.Thus,theconcentrationofCTABinthelipid matrixisabletoprovidehighercrystallinitytothelipidmatrix; however,otherfeaturessuchasstability andtoxicitywere ana-lyzedtoprovide a full studyin order tochoosethe bestCTAB concentration.

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Table7

Differentialscanningcalorimetry(DSC)analysisoftheSLNformulationswithdifferentCTABconcentrations.

Formulation Onsettemperature(◦_C) _Melting_point₍◦_C) _Integral_(mJ) _Enthalpy_(J_g−1₎

Softisan100bulk 36.68 39.12 −3111.15 −44.76

CTABbulk 100.88 106.34 −677.30 −12.72

CTAB-LN0.25wt% 29.58 34.12 −17.50 −0.23

CTAB-LN0.5wt% 31.26 35.03 −116.43 −1.47

CTAB-LN0.75wt% 31.12 34.73 −110.44 −1.41

CTAB-LN1.0wt% 31.65 34.94 −129.98 −1.59

for furtherin vivo studies.Thisassay couldpredictif formula-tionscausecellulardamagewhichconsequentlyresultsinlossof themetaboliccellfunction.Alamarblueisasensitive fluoromet-ric/colorimetricgrowthindicatorusedtodetectmetabolicactivity of cells. Specifically, cells incorporate an oxidation-reduction (REDOX) indicator that, when in a reducing environment of o metabolicallyactivecell,isreduced.Whenreduced,Alamarblue becomes fluorescent and changes color from blueto pink.The reductionofAlamarblueisbelievedtobemediatedby mitochon-drialenzymes(Hamidetal.,2004).However,someauthorsalso suggestthatcytosolicandmicrosomalenzymesalsocontributeto thereductionofAlamarblue(GonzalezandTarloff,2001).

The evaluation of the Alamar blue assaywas based onthe percentviabilityoffourconcentrationsforeachCTAB concentra-tionin thedispersion.Thisassay isimportant topoint outthe importanceof theCTAB quantityin theformulation that could beadministeredforoculardelivery,avoiding celldamage.From

Fig. 6, we can observe that 10␮gmL−1 of all CTAB-LN

formu-lations is non-toxic to cells as cell viability is not statistically differentfromcontrol (untreatedcells)along the3 time-points of exposure. The concentrationof 50␮gmL−1 of CTAB-LNonly

reducedsignificantlycellviabilityintheCTAB-LNdispersions con-taining 0.75 and 1.0wt% of CTAB, along the 3 time-points of

exposure.Theconcentrationof100␮gmL−1ofCTAB-LNreduced

significantlycellviabilityinallCTABpercentages.CTAB-LN formu-lationcontaining0.25%(w/w)ofCTABisnon-toxicfortherangeof 10–50␮gmL−1(Fig.6a),butreducessignificantlycellviability,in

about50–75%ofcontrol(p<0.05).Theconcentrationrangewhere cellviabilityiskeptsimilartocontrolvaluesisreducedincreasing CTABconcentrationintheformulations.InFig.6b(0.5%ofCTAB, concentrationtwice that usedfor Fig.6a)we can observethat 50␮gmL−1 offormulationreducescellviabilityfrom40 to60%

ofthecontrolwhile100␮gmL−1practicallyabolishescell

viabil-ityforthe3timepointsofexposure.RaisingCTABconcentration intheformulationto0.75%(Fig.6c)andto1.0%(Fig.6d)reduces celltolerancetotheformulationasweobservethatwith increas-ing%ofCTABintheformulationreducestheconcentrationthat maintainscellviability.TheseresultssuggestthatahigherCTAB concentrationimplieshighercytotoxicitywhichisexpectedsince theeffectofcationic agentsonthehumanhealthis concentra-tiondependent.Inaddition,higherCTAB-LNconcentrationsalso implymorecytotoxicity.Fromourresults,asafeand biocompat-ibleLNsystemshouldbecomposedof,atmaximum,0.5wt%of CTAB.

TEManalysishasbeenperformedtoevaluatetheparticleshape andmorphologyofCTAB-LNdispersion.TEMimage(Fig.7)shows

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72 461 (2014) 64–73

Fig.7.TEMmicrographof0.5%CTAB-LN.

particlesmainlywithsphericalmorphology.From thisanalysis, theabsenceofaggregationphenomenaofCTAB-LNdispersionwas alsoconfirmed.TheseresultsareinagreementwithTurbiscan®_Lab

results.Itispossibletodetectaslightpolydispersityhoweverallthe particlesremainwithinthenanometerrange.Allparticlesshowed ameandiameterthatvariedbetween190and280nm,whichis alsoinagreementwiththezetasizermeasurements.TEManalysis suggestedthatimmediatelyafterproductionCTAB-LNdispersion with0.5wt%CTABcontainedparticlesnohigherthan1␮m,which

isusefulforoculardeliverypurposes.

4. Conclusions

ThedevelopmentofLNdispersionsforoculardeliveryshould becarriedoutregardingocularmorphologyandbiology.Afull fac-torialdesignwascarriedoutinordertofindoutwhichparameters couldinfluenceLNdispersionsbasedonmultipleemulsion tech-nique.ThesizeandPIofLNdispersionsarehighlydependenton thelecithinconcentrationmainlyduetoitshigheremulsification propertiesandamphiphiliccharacterabletodecreaseparticlesize inemulsions.Inordertoimproveocularmucoadhesion,acationic lipidwasaddedinthelipidmatrix.Theinsufficientinformationand lackofstudiesregardingnanotoxicityofcationicagentsforocular ordrugdeliveryleadsustostudydifferentCTABconcentrations onlipidmatrixanditseffectsonthephysicochemicalparameters andcelltoxicityofCTAB-LNdispersions.Thisstudydemonstrated thatthebetterCTABconcentrationforthedispersionpreviously optimizedbythefactorialdesignwas0.5%,providingbetter stabil-ityandbiocompatibility.Furtherstudiesencapsulatinghydrophilic drugsandevaluatingtheexvivoandinvivoperformanceofthe developedCTAB-LNdispersionarerequiredtoconfirmthese pre-liminaryresults.

Acknowledgements

Ms. Joana Fangueiro and Ms. Tatiana Andreani wish to acknowledge Fundac¸ão para a Ciência e Tecnologia do Min-istério da Ciência e Tecnologia (FCT, Portugal) under the refer-ences SFRH/BD/80335/2011 and SFRH/BD/60640/2009, respec-tively. FCT is also acknowledged under the research project PTDC/SAU-FAR/113100/2009andFCOMP-01-0124-FEDER-022696 (PEst-C/AGR/UI4033/2011).

References

Araujo,J.,Gonzalez,E.,Egea,M.A.,Garcia,M.L.,Souto,E.B.,2009.Nanomedicinesfor ocularNSAIDs:safetyondrugdelivery.Nanomedicine5,394–401.

Araújo,J.,Vega,E.,Lopes,C.,Egea,M.A.,Garcia,M.L.,Souto,E.B.,2009.Effectof polymerviscosityonphysicochemicalpropertiesandoculartoleranceof FB-loadedPLGAnanospheres.ColloidsSurf.B72,48–56.

Bhatta,R.S.,Chandasana,H.,Chhonker,Y.S.,Rathi,C.,Kumar,D.,Mitra,K.,Shukla,P.K., 2012.Mucoadhesivenanoparticlesforprolongedoculardeliveryofnatamycin: invitroandpharmacokineticsstudies.Int.J.Pharm.432,105–112.

Bunjes,H.,Unruh,T.,2007.Characterizationoflipidnanoparticlesbydifferential scanningcalorimetry,X-rayandneutronscattering.Adv.DrugDeliveryRev.59, 379–402.

Celia,C.,Trapasso,E.,Cosco,D.,Paolino,D.,Fresta,M.,2009.Turbiscanlabexpert analysisofthestabilityofethosomesandultradeformableliposomescontaining abilayerfluidizingagent.ColloidsSurf.B72,155–160.

delPozo-Rodriguez,A.,Delgado,D.,Gascon,A.R.,Solinis,M.A.,2013.Lipid nanopar-ticlesasdrug/genedeliverysystemstotheretina.J.Ocul.Pharmacol.Ther.29, 173–188.

Diebold,Y.,Calonge,M.,2010.Applicationsofnanoparticlesinophthalmology.Prog. Retin.EyeRes.29,596–609.

Doktorovova,S.,Shegokar,R.,Rakovsky,E.,Gonzalez-Mira,E.,Lopes,C.M.,Silva,A.M., Martins-Lopes,P.,Muller,R.H.,Souto,E.B.,2011.Cationicsolidlipid nanoparti-cles(cSLN):structure,stabilityandDNAbindingcapacitycorrelationstudies. Int.J.Pharm.420,341–349.

Fangueiro,J.F.,Andreani,T.,Egea,M.A.,Garcia,M.L.,Souto,S.B.,Souto,E.B.,2012. Experimentalfactorialdesignappliedtomucoadhesivelipidnanoparticlesvia multipleemulsionprocess.ColloidsSurf.B100,84–89.

Fangueiro, J.F., Gonzalez-Mira, E., Martins-Lopes, P., Egea, M.A., Garcia, M.L., Souto,S.B.,Souto,E.B.,2013.Anovellipidnanocarrierforinsulindelivery: production,characterization andtoxicitytesting.Pharm.Dev. Technol.18, 545–549.

Fiume,Z.,2001.Finalreportonthesafetyassessmentoflecithinandhydrogenated lecithin.Int.J.Toxicol.20,21–45.

Fresta,M.,Panico,A.M.,Bucolo,C.,Giannavola,C.,Puglisi,G.,1999. Characteriza-tionandin-vivoocularabsorptionofliposome-encapsulatedacyclovir.J.Pharm. Pharmacol.51,565–576.

Gallarate,M.,Trotta,M.,Battaglia,L.,Chirio,D.,2009.Preparationofsolidlipid nanoparticlesfromW/O/Wemulsions:preliminarystudiesoninsulin encap-sulation.J.Microencapsul.26,394–402.

García-Fuentes,M.,Torres,D.,Alonso,M.J.,2003.Designoflipidnanoparticlesfor theoraldeliveryofhydrophilicmacromolecules.ColloidsSurf.B27,159–168. Giannavola,C.,Bucolo,C.,Maltese,A.,Paolino,D.,Vandelli,M.A.,Puglisi,G.,Lee,

V.H.,Fresta,M.,2003.Influenceofpreparationconditionsonacyclovir-loaded poly-d,l-lactic acidnanospheres andeffectofPEGcoatingonoculardrug bioavailability.Pharm.Res.20,584–590.

Gonzalez-Mira,E.,Egea,M.A.,Garcia,M.L.,Souto,E.B.,2010.Designandocular tol-eranceofflurbiprofenloadedultrasound-engineeredNLC.ColloidsSurf.B81, 412–421.

Gonzalez-Mira,E.,Egea,M.A.,Souto,E.B.,Calpena,A.C.,Garcia,M.L.,2011. Opti-mizingflurbiprofen-loadedNLCbycentralcompositefactorialdesignforocular delivery.Nanotechnology22,045101.

Gonzalez,R.J.,Tarloff,J.B.,2001.Evaluationofhepaticsubcellularfractionsfor Ala-marblueandMTTreductaseactivity.Toxicol.InVitro15,257–259.

Hamid,R.,Rotshteyn,Y.,Rabadi,L.,Parikh,R.,Bullock,P.,2004.Comparisonof ala-marblueandMTTassaysforhighthrough-putscreening.Toxicol.InVitro18, 703–710.

Han,F.,Li,S.,Yin,R.,Liu,H.,Xu,L.,2008.Effectofsurfactantsontheformationand characterizationofanewtypeofcolloidaldrugdeliverysystem:nanostructured lipidcarriers.ColloidsSurf.A315,210–216.

Kawaguchi,E.,Shimokawa,K.-i.,Ishii,F.,2008.Physicochemicalpropertiesof struc-turedphosphatidylcholineindrugcarrierlipidemulsionsfordrugdelivery systems.ColloidsSurf.B62,130–135.

Lallemand,F.,Daull,P.,Benita,S.,Buggage,R.,Garrigue,J.S.,2012.Successfully improvingoculardrugdeliveryusingthecationicnanoemulsion,novasorb.J. DrugDeliv.,604204.

Liu,J.,Huang,X.-f.,Lu,L.-j.,Li,M.-x.,Xu,J.-c.,Deng,H.-p.,2011.TurbiscanLab®

Expertanalysisofthebiologicaldemulsificationofawater-in-oilemulsionby twobiodemulsifiers.J.Hazard.Mater.190,214–221.

Marianecci,C.,Paolino,D.,Celia,C.,Fresta,M.,Carafa,M.,Alhaique,F.,2010. Non-ionicsurfactantvesiclesinpulmonaryglucocorticoiddelivery:characterization andinteractionwithhumanlungfibroblasts.J.Control.Release147,127–135. Metin,S.,Hartel,R.W.,2005.Crystallizationoffatsandoils.In:Bailey’sIndustrialOil

andFatProducts.JohnWiley&Sons,Inc.,NewJersey,USA.

Pignatello,R.,Puglisi,G.,2011.Nanotechnologyinophthalmicdrugdelivery:a sur-veyofrecentdevelopmentsandpatentingactivity.RecentPatentsNanomed.1, 42–54.

Schubert,M.A.,Harms,M.,Müller-Goymann,C.C.,2006.Structuralinvestigations onlipidnanoparticlescontaininghighamountsoflecithin.Eur.J.Pharm.Sci.27, 226–236.

Schubert,M.A.,Müller-Goymann,C.C.,2003.Solventinjectionasanewapproach formanufacturinglipidnanoparticles–evaluationofthemethodandprocess parameters.Eur.J.Pharm.Sci.55,125–131.

(10)

461 (2014) 64–73 73

lipid nanoparticles (SLN and NLC) for oral drug delivery.J. Drug Deliv., http://dx.doi.org/10.1155/2012/750891.

Shekunov,B.Y.,Chattopadhyay,P.,Tong,H.Y.,Chow,A.H.L.,2007.Particlesize anal-ysisinpharmaceutics:principles,methodsandapplications.PharmRes.24, 203–227.

Souto,E.B.,Doktorovova,S.,Gonzalez-Mira,E.,Egea,M.A.,Garcia,M.L.,2010. Feasi-bilityoflipidnanoparticlesforoculardeliveryofanti-inflammatorydrugs.Curr. EyeRes.35,537–552.

Sultana,Y.,Maurya,D.P.,Iqbal,Z.,Aqil,M.,2011.Nanotechnologyinoculardelivery: currentandfuturedirections.DrugsToday(Barc)47,441–455.

Thoma,K.,Serno,P.,1983.ThermoanalytischerNachweisderPolymorphieder Sup-positoriengrundlageHartfett.Pharm.Ind.45,990–994.

Trotta, M., Pattarino, F., Ignoni, T., 2002. Stability of drug-carrier emulsions containing phosphatidylcholine mixtures. Eur. J. Pharm. Biopharm. 53, 203–208.

Vega,E.,Gamisans,F.,García,M.L.,Chauvet,A.,Lacoulonche,F.,Egea,M.A.,2008. PLGAnanospheresfortheoculardeliveryofflurbiprofen:drugreleaseand interactions.J.Pharm.Sci.97,5306–5317.

Ying,L.,Tahara,K.,Takeuchi,H.,2013.Drugdeliverytotheocularposteriorsegment usinglipidemulsionviaeyedropadministration:effectofemulsion formula-tionsandsurfacemodification.Int.J.Pharm.453,329–335.