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International Journal of Pharmaceutics
j o u r n al ho m e p age :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
Nanostructured lipid carrier versus solid lipid nanoparticles of simvastatin:
Comparative analysis of characteristics, pharmacokinetics and tissue uptake
Radheshyam Tiwari, Kamla Pathak
∗DepartmentofPharmaceutics,RajivAcademyforPharmacy,Mathura,UttarPradesh281001,India
a r t i c l e i n f o
Articlehistory:
Received12February2011
Receivedinrevisedform16May2011 Accepted18May2011
Available online 26 May 2011
Keywords:
Simvastatin
Nanostructuredlipidcarriers Factorialdesign
Invitrodrugrelease Invitro–invivocorrelation
a b s t r a c t
Nanostructuredlipidcarrier(NLC)systemofsimvastatinwasinvestigatedforimprovementinrelease, pharmacokineticsandbiodistributionoveritssolidlipidnanoparticles(SLN).TheNLCformulationspre- paredbysolventinjectiontechniquewereoptimizedby23 fullfactorialdesign.OptimizedNLCwas deducedonthebasisofdependentvariablesthatwereanalyzedusingDesignexpert8.0.2®software(Stat Ease,Inc.,USA).Paretochartsandresponsesurfaceplotswereutilizedtostudytheeffectofvariableson theresponseparameters.TheoptimizedNLCwasasuspensionofnanosizedhomogeneousparticleswith significantlyhigherentrapmentefficiency(>90%)andlowerrecrystallizationproperties(p<0.01)than SLNs.ThepharmacokineticparametersofTc99labeledoptimizedNLCinmice,obtainedusingQuick- calsoftware(Plexus,India)revealed4.8foldsincreaseinbioavailabilityascomparedtosimvastatin suspensionand2.29foldsascomparedtoSLNs.Biodistributionstudyrevealedpreferentialaccumula- tionofNLCintheliverandthisisadvantageousbecauseliveristhetargetorganforsimvastatin.IVIVC studiesdemonstratedlevelAcorrelationbetweeninvitroreleaseandpercentdrugabsorbed.Thisinves- tigationdemonstratedthesuperiorityofNLCoverSLNforimprovedoraldeliveryanditwasdeduced thattheliquidlipid,oleicacidwastheprincipalformulationfactorresponsiblefortheimprovementin characteristics,pharmacokineticsandbiodistributionofNLCs.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Attention is being focused on nanotechnology-based drug delivery systems including biodegradable polymeric nanopar- ticles, smart polymeric micelles, nanocrystals, nanosuspension, nanoemulsionsandlipidnanoparticleswiththeaimtoimprovelow aqueoussolubilityororalbioavailabilitytobringaboutsatisfactory therapeuticefficacy.Overthepastfewyears lipidnanoparticles have been especially considered, because these are composed of natural or synthetic lipids, show good biocompatibility and have potential to exhibit controlled release of drugs (Li et al., 2009).Lipid-basednanoparticleformulations mayalsoenhance drugabsorptionviaimprovementindissolutionandsolubilization withintheintestinalmilieu,areductioningastricemptyingrate andincreaseinmucosalpermeability.Lipidsareknowntoenhance lymph formationand simultaneously promote lymphflow rate (Sureshetal.,2007).Therefore,lipidnanoparticleshavethepoten- tialtoenhancetheoverallextentofabsorptionaswellasincrease theproportionofwhatisabsorbedandtransportedtothesystemic
∗Correspondingauthorat:RajivAcademyforPharmacy,Delhi-MathuraBypass, P.O.Chattikara,Mathura,UttarPradesh281001,India.Tel.:+915652913018;
fax:+915652825050.
E-mailaddress:kamla [email protected](K.Pathak).
circulationviaintestinallymph(HumberstoneandCharman,1997;
Aungst,2000).
Simvastatin(SV),alipophilicactiveconstituentderived from fungi, is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitorwithbeneficialeffectsoncoronarydiseasesandmortal- ityrateinpatientswithhypercholesterolemia.Itisadministered as aninactive lactoneprodrug and hastwoseparate metabolic pathways.Oxidativebiotransformationisoneofthepathwaymedi- atedprimarilyby cytochromeP3A (Christains etal.,1998), and hydrolyzationofsimvastatinacidbycarboxylesteraseisanother pathwayleadingtonon-enzymaticmetabolismintoanactivecom- petitivestatin.TheSVthenreducestheamountofmevalonicacid, aprecursorofcholesterol,therebyinhibitingdenovosynthesisof cholesterol.Consequently,thesynthesisoflowdensitylipoproteins receptorsincreaseswhilecholesterolsynthesisdecreases;result- ingintheincreasedclearanceoflowdensitylipoproteinsfromthe bloodstream(Liljaetal.,2004).
Mostoftheavailablestatins,includingsimvastatin,havebeen developed asimmediate-releaseformulations.However,SVisa poorly water solubledrug(aqueous solubility≈0.03g/L)witha short half-life time of about 2h, and is cleared by extensive metabolismin theintestinal gutand liverby cytochrome P3A (MahleyandBersot,2006).Duetotheslowdissolutionrateinthe intestinaltractandsignificantfirst-passeffect,theoralbioavail- ability of SV in humans is as low as 5%. Recently number of 0378-5173/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.05.044
strategies havebeenemployed toaddressthe issuesrelated to lowbioavailabilityandpooraqueoussolubilityofSV.Theseinclude selfmicroemulsifyingdrugdeliverysystem(Kangetal.,2004;Patil etal.,2007),selfnanoemulsifyinggranules(DixitandNagarsenker, 2008),soliddispersion(Silvaetal.,2010),cubicnanoparticles(Lai etal.,2009),andPolyringdevice(Vishwanathan,2008).
Inaseriesofinvestigationscarriedoutinourlab,SVloadedSLN formulationshavebeenworkedoutasapotentialoraltherapeu- ticcarriersystem.SLNswereformulatedandoptimizedusingtwo lipids,glycerylmonostearate(GMS)andCompitrol888ATO.The optimizedSLNformulationmadewithGMSexhibitedaparticlesize of258.5nm,%entrapmentefficiencyof75.81%,with82.67%cumu- lativedrugreleaseafter55handitsrecrystallizationindexwas foundtobe65.51%(ShahandPathak,2010).A1.87foldsincrease inbioavailabilitywasrecordedwithSVSLNmadeusingglyceryl monostearatewithrespecttoSVsuspensionin.Ontheotherhand, theoptimizedformulationofCompitrolbasedSLNwithamean particlesizeof271.18nm,%EEof68.16%and%CDRof76.23%after 55hdocumentedarelativebioavailabilityof220%,substantiating theprotectiveactionofSLNsagainstlivermetabolism.Oncom- parison,itwasconcludedthattheSLNsmadewithCompitrol888 ATOdemonstratedhigherbioavailabilitythanSLNsmadewithGMS becauseofhigherlipophilicnatureoftheformerthatwasrespon- sibleformoresustainedreleaseofdrug(Shahetal.,2010).Though theseformulationsprovidedsustainedreleaseofSVwithpotential forimprovedtherapeuticefficacy,SLNssufferfromtwomajorlim- itationsthatarelimiteddrugloadingandexpulsionofdrugduring storage.
Amongthelipid-basedformulations,thenanostructuredlipid carriers(NLCs),regardedasthesecond-generationoflipidnanopar- ticles,areattractingforemostconsiderationasalternativecolloidal drugcarriers. NLCs have evolvedfrom solid lipidnanoparticles (SLN)andarecomposedof amixtureofspatiallydifferentlipid molecules,i.e.,solidlipidisblendedwithliquidlipidtoovercome thedisadvantagesofSLNssuchaslimiteddrugloading,largerpar- ticlesize,riskofgelationanddrugleakageduringstoragecaused bylipidpolymorphism. Inviewof thisNLCmayberegardedas analternativetoSLNand canbeexploitedasa novelapproach forimprovingthedeliveryofSV.Lipidformulationsloadedwith poorlywater-solubledrugsfororalroutehavebeeninvestigated andreportedtoimprovetheoralbioavailabilitybymanyresearch teams(Mohamedetal.,1998;Paliwaletal.,2009)butthereare fewreportsonNLCsystemfororaladministration.Inthisstudywe havemadeeffortstoinvestigatethefeasibilityofimprovingoral bioavailabilityofSVthroughNLC.
Thustheobjectiveofpresentstudywastodevelopandopti- mize NLC suspension of SV using solid lipid and liquid lipid agents,inorder toincrease thedrugentrapmentefficiencyand tofurtherreducetheparticlesize;themajorlimitationsofSLNs and thereby increase bioavailability bypreventing hepatic first passmetabolismuptosomeextentalongwithcomparisonwith the SLN and drug suspension. Thus, the SV-loaded NLCs were developedandscreenedandthephysicochemicalcharacteristics, invitrodrugreleaseproperties,pharmacokineticsandbiodistribu- tionwereinvestigatedindetail.ThenalltheparametersofNLCs werecompared withthe SLNs. Finally, the possibleabsorption mechanismofNLCformulationisdiscussed.
2. Materialsandmethods
2.1. Materials
SimvastatinwasakindgiftfromRanbaxyLaboratories,India.
Glycerolmonostearate(M.P.52–54◦C;molecularweight358.63) waspurchasedfromCDH,India.Poloxamer407(molecularweight
12.5)waspurchasedfromBASF, USA.Oleicacidwaspurchased fromRanbaxyfinechemicalslimited,India.Dialysisbag(molec- ularweightcutoff12–14kDa;poresize2.4nm)wassuppliedby HiMedia,Mumbai,India.Otherchemicalswereofanalyticalgrade.
2.2. Experimentaldesign
Inthis study,a23 full-factorialdesign wasused tooptimize NLCs.In ordertooptimize,theamountofGMS(X1),amountof OA(X2)andconcentrationofpoloxamer407(X3)wereselectedas independentvariables.Eachfactorwassetatahighlevelandlow level.Theactualvaluesandcodedvaluesofdifferentvariablesare giveninTable1.EightformulationsofsimvastatinNLCs(F1–F8) werepreparedaccordingtothefactorialdesign.Theparticlesize,
%entrapmentefficiencyand%cumulativedrugreleaseat55hwere takenasresponseparameters.Thestatisticalanalysisofresponses wasmadebyDesignexpert8.0.2.(Stat-EaseInc.,USA)thathasbeen detailedinSection2.7.
2.3. PreparationofsimvastatinloadedNLCs
Nanostructuredlipidcarrierswerepreparedbysolventinjec- tiontechnique.Simvastatin(SV)(15mg)andspecifiedamountof GMSand OAasgiven inTable 1weredissolvedin 2mlof iso- propylalcohol(boilingpoint81–83◦C)withheatingatthemelting temperature of GMS.Though GMS is soluble in IPA it requires someheatfor easeofsolubilization.Theresulting solutionwas rapidlyinjectedintothe10mlofaqueousphasecontainingspec- ified amount of poloxamer 407 as given in Table 1 that was continuouslystirredat400rpmfor30minonamagneticstirrer.
0.1NHCl(4ml)wasaddedtothedispersiontodecreasethepH around1.5–2forcausingaggregationofNLCsfortheeaseofsep- aration.Thereafter,thedispersionwascentrifugedat10,000rpm (5590×g)for30minat10◦CinREMIcoolingcentrifuge(ModelC- 24BL,VACO-779,Vasai,India),andaggregateswerere-suspended in10mldoubledistilledwatercontaining4%poloxamer407(by weight)asstabilizerwithstirringat1000rpmfor10min.
2.4. PurificationofSV-loadedNLCs
PurificationofSV-loadedNLCswasdonebydialysistechnique.
Re-suspendedsuspensionwastakeninthedialysisbagandsealed atbothends.Thedialysisbagthenimmersedinto100mlofdou- bledistilledwatercontaining0.2%(w/v)sodiumlaurylsulphate andstirred at100rpmfor20min.Fivemilliliter ofsample was withdrawnattimeintervalsof5,10,15,and20min,dilutedappro- priatelyandanalyzedforamountofdrugbyHPLC.Pairedt-testwas usedtocheckanysignificantdifferencebetweenthepercentfree drugremovedat15andat20min.
2.5. HPLCassayforSV
TheSVcontentwasassayedusingADEPTHPLCsystems–Cecil CE4201,equippedwithaRheodyne injectorHamilton 702NR, Adept4100dualpistonpumpanddetectorCE4200setat239nm, withamicrosorbC18analyticalcolumn(RP-18,250mm×4.6mm, 5m). The mobile phase was a mixture of acetonitrile:0.01M ammoniumacetate buffer(35:65,v/v), eluted at a flow rateof 1.0ml/min andasampleinjectionvolumeof20l.Thelimitof detectionandthelimitofquantificationofthisanalyticalmethod were0.163g/mland0.493g/mlrespectively.Linearcalibration curvewasobtainedforSVwithr2=0.9998intherange5–25g/ml.
Table1
23Full-factorialdesignofsimvastatin-loadedNLCs,andformulationofG-SLNandC-SLNandevaluatedresponseparameters(n=3).
Formulationcode GMSX1 OAX2 Poloxamer407X3 Particlesize(nm) %EE Zetapotentiala(mV) PDIa %CDRafter55h
F1 −1 −1 −1 209.9 83.91±2.25 −31.1 0.355 73.65±2.54
F2 −1 −1 +1 206.7 82.92±1.13 −32.1 0.351 81.23±2.76
F3 −1 +1 −1 194.4 93.33±3.26 −35.8 0.242 88.34±3.23
F4 −1 +1 +1 180.9 86.49±2.83 −39.8 0.250 91.49±3.78
F5 +1 −1 −1 260.0 88.64±1.61 −29.1 0.191 57.95±3.18
F6 +1 −1 +1 254.9 81.74±3.45 −35.4 0.359 59.13±4.11
F7 +1 +1 −1 239.4 91.46±1.02 −39.1 0.360 65.59±3.12
F8 +1 +1 +1 230.6 90.54±2.91 −30.2 0.371 69.98±3.76
F9 0 0 0 211.54 83.97±3.46 −34.5 0.344 77.81±4.20
G-SLN −1 – 0.38 258.5 75.81±4.54 −22.34 0.245 82.67±3.46
C-SLN −1 – 0.57 271.18 68.16±3.88 −25.94 0.361 76.23±3.33
Actualvalues:X1,+1=200mg,−1=100mg,0=150mg;X2,+1=30mg,−1=15mg,0=22.5mg;X3(%,w/w),+1=0.8%,−1=1.2%,0=1.0%,0.38=1.1%,0.57=1.1%.
aNotselectedfortheoptimization.
2.6. CharacterizationofNLC 2.6.1. Particlesizeandzetapotential
Theparticlesizeandzetapotentialoftheformulationswere determined bylaser scatteringtechnique using Malvern Hydro 2000SM(MalvernInstruments,UK)afterappropriatedilutionwith doubledistilledwater.Lightscatteringwasmeasuredatanangle of90◦.Theaqueousnanoparticulatedispersionwasaddedtothe sampledispersionunitcontainingstirrerandstirredtominimize theinterparticleinteractions,whilethelaserobscurationrangewas maintainedbetween10%and20%.
2.6.2. Entrapmentefficiency
Inordertodeterminetheentrapmentefficiency,theequation reportedbyShahand Pathak(2010)wasutilized, and%EEwas calculatedbythefollowingequation:
%EE=
Wa−(Ws+Wp) Wa
×100 (1)
whereWaistheamountofdrugaddedinsystem,Wsistheamount of drug in supernatant after the centrifugation, and Wp is the amountofdruginthepurificationmedium.
2.6.3. Invitrodrugrelease
Invitrodrugrelease studyof NLCswasperformed bydialy- sisbagdiffusiontechnique.NLC-suspensionequivalentto5mgSV wasfilledindialysisbagandimmersedinareceptorcompartment containing100mlofphosphatebuffer,pH7.4,stirredat100rpm andmaintainedatatemperatureof37±0.5◦C.Thereceptorcom- partmentwascoveredtopreventevaporationofthemedium.Five milliliterofaliquotswerewithdrawnatvarioustimeintervals(1, 2,3,4,5,6,7,8,9,10,11,23,24,25,26,27,28,29,30,31,32,33, 34,35,48,49,50,51,52,53,54and55h),andreplacedwithfresh volumeofdissolutionmedium,dilutedappropriately,andconcen- trationwasmeasuredbyHPLCat239nm.Theexperimentswere performedintriplicate.
2.7. Statisticalanalysisofresponsesbydesignexpert
DesignExpert8.0.2.(Stat-EaseInc.,USA)wasusedfortheanal- ysisofeffectofeachvariableonthedesignatedresponse.Pareto chartsweremadefortheanalysisofeachresponsecoefficientfor itsstatisticalsignificance.Quantitativeandqualitativecontribution ofeachvariableoneachoftheresponsewasanalyzed.Thesignif- icantpolynomialequationsgeneratedbyDesignExpertwereused tovalidatethestatisticaldesign(BoltonandBon,2004).Validation ofdesignofSV-NLCwasdonebypreparingextradesigncheckpoint formulation(F9),andforthatcenterpointofeachvariablelevel wasselected.Thiscodedlevelwastransformedtoactualvalueof
eachvariableusingprincipleoftransformation.Experimentalvalue and predictedvalueofeach response weredeterminedbyper- formingexperimentandusingdesignexpertsoftwarerespectively.
Responsesurfaceplotsweregeneratedtovisualizesimultaneous effectofeachvariableoneachresponseparameter.
2.8. Selectionofoptimizedformulation
Fortheselectionofoptimizedformulation,DesignExpertsoft- warewasutilized.Itisanadvantageoustool,whichvarieseach variablesimultaneouslyandgivesallpossibleoptimumselections basedonwhichtheoptimizedformulationisselected.Thus,opti- mizedformulationwasselectedonthebasisofsmallparticlesize, higherentrapmentefficiency,andhigherinvitrocumulativedrug releaseafter55h.
2.9. Characterizationoftheoptimizedformulation
Besides particle size, zeta potential, entrapment efficiency and invitrorelease, additionalinvestigationsincludingelectron microscopy,differentialthermalanalysis,radiolabelingandphar- macokineticandtissueuptakestudieswereperformedonselected optimizedformulation(F4).
2.9.1. Transmissionelectronmicroscopy
ThemorphologyofSV-NLCsuspensionwasobservedbytrans- missionelectronmicroscopy[Hitachi(H-7500)80kV,Japan].The samplewaspreparedbyplacingadropofSV-NLCthatwasprevi- ouslydiluted50-foldswithdouble-distilledwaterontoa400-mesh coppergridcoatedwithcarbonfilmandfollowedbynegativestain- ingwith1%phosphotungsticacid.Thesamplewasdriedintheair beforeTEMobservation.
2.9.2. Differentialthermalanalysis
In order to evaluate the recrystallization index, differential scanningcalorimetrywasperformedusingUniversalV4.5ATAdif- ferentialscanningcalorimeter.Accuratelyweighedsample(s)was placedinaluminumpanandsealedwitha lid.Aluminumoxide wasusedasthereference.Inthescanningprocess,aheatingrate of10◦C/minwasappliedinthetemperaturerangefrom−50◦Cto 200◦Cwithanitrogenpurgeof0.2ml/min.Therecrystallization index(RI)wascomputedbythefollowingequation(Soutoetal., 2006):
%RI= HNLC
Hbulklipid× fractionoflipidphase×100 (2) where HNLC is enthalpy of fusion of NLC, and Hbulklipid is enthalpyoffusionofGMS.
2.9.3. Radiolabelingofformulations
Radiolabelingofsimvastatinsuspensionandoptimizedsimvas- tatinNLCswasdonebydirectmethodusingstannouschlorideas reducingagent.Briefly,1mlofsimvastatinsuspensionandopti- mized simvastatinNLCs was mixedwith stannouschloride. To adjustthepHofthismixture,10lofsodiumhydrogencarbonate solutionwasadded.Then,0.1mloffreshlyelutedTc-99m(Regional CenterforRadiopharmaceuticals,BRIT,NewDelhi,India)(1.5mCi) wasaddedtoeachpreparation,mixedwell,andincubatedatroom temperature.Finalradioactivity present in thepreparation was checkedusinggammaraycounter(Capintech,CAPRAC-R,USA).
2.9.3.1. Optimizationof radiolabeling efficiency. The effectof the amountofstannouschloride,thefinalpHofthepreparation,and theincubationtimeonlabelingefficiencywasoptimizedbychang- ingoneparameteratatime.Foroptimizingamountofstannous chloride,a range of 25–400g of stannous chloridewas used.
Similarly,pHofthereactionmixturewasvariedfrom4to7and incubationtimewasvariedbetween5and30min.
2.9.3.2. Determinationoflabelingefficiency. Thelabelingefficiency ofsimvastatinsuspension,and optimizedsimvastatinNLCswas determinedbyascendingthinlayerchromatography(TLC)using instantthin-layerchromatography (ITLC)stripscoatedwithsil- icagel(GelmanScienceInc.,AnnArbor,MI).TheITLCstripswere spottedwith1–2loflabeledcomplexat1cmabovethebottom.
Thesestripsweredevelopedusingsolventsystem(pyridine:acetic acid:waterionratioof3:5:1.5)todeterminefreeTc-99m.Thesol- ventfrontwasallowedtoreachtoaheightofapproximately6–8cm fromtheorigin,and,thestripwascuthorizontallyintotwohalves.
Radioactivityineach halfwasdeterminedbyusing gammaray counter.
2.9.3.3. Stabilitystudyoflabeledcomplex. StabilityoftheTc-99m labeledsimvastatinsuspensionand optimizedsimvastatinNLCs wasdeterminedinvitroinmiceserumandnormalsalinebyascend- ingTLCtechnique. Thelabeledcomplex (0.1ml)wasincubated separately,with0.9mlfreshlycollectedmiceserumand0.9mlnor- malsalineseparatelyatroomtemperature.Aliquotssampleswere withdrawnattimeintervalsof1,2,4,24and48handITLCwas performed.Thesestripswerecountedforradioactivity.Theprepa- rationswerestableinmiceserumandnormalsalinefor48h.Atall timepoints99Tcmlabeledpreparationsdemonstratedradiolabel- ingefficiencyofmorethan96%.
2.9.4. Pharmacokineticsandtissueuptakestudy
Allanimalexperimentswerecarriedoutaccordingtotheprin- ciplesofCommitteeforthePurposeofControland Supervision ofExperimentsonAnimals(CPCSEA)Chennai,India.Theproto- cols for the animal studies were approved by theInstitutional AnimalEthicalCommittee(IAEC)RefNo-IAEC/RAP/2908/2010of RajivAcademyforPharmacyMathura,India.Balb/Cmiceweigh- ing25–30gwereobtainedfromAnimalExperimentalCenterof InstituteofNuclearMedicinesandAlliedSciences,NewDelhiand housedatatemperatureof22±2◦Cand45–50%relativehumidity.
Themiceweredividedrandomlyintotwogroups(group1and group2)comprisingfifteenanimalseachandfastedovernight,but allowedtofreeaccesstowaterbeforeexperiment.Theradiolabeled SV-suspensionandoptimizedSV-NLCswithafinalradioactivityof 1.5mCi/ml(equivalenttothedoseof5mg/kgofsimvastatin)was administeredorallytogroup1andgroup2animalsrespectively.
Forpharmacokineticevaluation,themicewereanaesthetizedusing chloroformat1,2,4,24,48hpostadministrationandbloodwas collectedviacardiacpuncture.Fortissueuptakestudyateachtime point,threemiceweresacrificedandorgansofinterestsuchasliver, lungs,spleen,kidney,heart,intestineandstomachwerequickly
removed.Theorgansamplesincludingblood wereanalyzedfor radioactivitybygammaraycounter.Radioactivityinthesample wasdeterminedintheunitofcounts/minuteand%uptakepergram organweightwascalculatedbythefollowingequation:
% uptake/gramorganweight= [counts/weight]×100
totaladministeredcounts (3) Thepharmacokineticparameterswereacquiredwiththehelpof pharmacokineticprogramQuickcalsoftware(Plexus,Ahmadabad, India).TherelativebioavailabilityofNLCformulationswascalcu- latedusingthefollowingequation:
Fr (%)= AUCSV-NLC
AUCSV-suspension×100 (4)
whereFr(%)istherelativebioavailability,AUCistheareaunder theplasma concentration–time curve.Various pharmacokinetic parameterssuchasCmax,Tmax,eliminationhalflife,absorptioncon- stant,eliminationconstantvolumeofdistributionandclearance werealsocalculated.
2.9.5. Invitroinvivocorrelation
ThepercentageofSVabsorbedatspecifictimeswascalculated fromthebloodconcentrationdatausingtheWagnerandNelson method.The methodwasbased onutilization of thefollowing equation:
AT
Vd =CT+Kel(AUC)t=0−T (5)
whereATistheamountofdrugabsorbedattimet,Vdistheapparent volumeofdistribution,CTisthebloodconcentrationattimet=T, KelistheeliminationrateconstantandAUCistheareaunderthe curvebetweenthelimitst=0tot=T.AvalueofKel=0.134h−1was calculatedfromtheslopeobtainedbyaleast-squaresregression ofthelinearregionofthelogblood%A/Gversustimeprofilefor freeSVsuspensionafteroraldosing.Usingthismethod,successive valuesofAT/Vdwerecalculatedtoobtainthemaximum(AT/Vd)∞, whichwasthendividedintoeachvalueofAT/Vdtodeterminethe percentageabsorptiondataasafunctionoftime.
3. Results
3.1. Formulationconsiderations
NLCswerepreparedbysolventinjectiontechniquethatrelies onrapiddiffusionofthesolventacrossthesolvent–lipidinterfaced withtheaqueousphaseand this physicalphenomenonis criti- calforthenanosizedlipidparticleprecipitation.Owingtotheir smallsizecoupledwithlowdensityoflipids,NLCspresentdiffi- cultyinsettlinguponcentrifugation.Toovercomethislimitation, inthepresentstudy,thepHofthedispersionwasreducedto1.5–2 toadjustthezetapotentialtoalevelthatpromotesaggregation ofnanosizedparticles,facilitatescentrifugationandconsequently separation. Anothersignificantaspectin preparationof NLCs is purityoftheproductobtained. Apossibilityofpresence offree simvastatinparticlesinthesedimentofsimvastatin-loadedNLCs cannotbedeniedandhencewasexplored.Freedrugparticlescan affectboth,theinvitroandinvivoreleasebehaviorofsimvastatin.
Therefore,freedrugparticleswereremovedfromthesedimentof NLCsbydialysistechnique.Thistechniquewasconsideredsuitable, assimvastatinwithalowmolecularweightof418.57Dacouldbe efficientlyremovedusingdialysisbagmadeofthemembranewith cutoff 12–14kDa.Consequently,purificationwasaccomplished bymonitoringpercentfree drugremovedwithrespecttotime.
Initially,thepercentfree drugremoved increasedlinearlywith timeandreachedplateaulevelswithin20min.Pairedt-testwas usedtocheckanysignificantdifferencebetweenthepercentfree
Fig.1.Invitroreleaseprofilesofsuspension(F0)andNLCsofsimvastain(F1–F8)in phosphatebuffer,pH7.4.
Fig. 2.Comparative invitro release profilesof NLCs, G-SLN, C-SLN and SV- suspensioninphosphatebuffer,pH7.4.
drugremovedat15and20minandnosignificantdifferencewas observed(p>0.01).Thus,freeSVfromsedimentofNLCscouldbe efficientlyremovedbydialysisfor15minandwasusedthroughout theexperiment.
3.2. Analysisofdependentvariables 3.2.1. Particlesizeandzetapotential
Values of theparticle size and surface charge of thedevel- opedNLCsaredocumentedinTable1.Theresultsshowedthatthe
amountsofGMSandOAwerecriticalparametersingoverningthe particlesize.Afterproduction,themeandiameteroftheparticles rangedbetween180.9and260nmandpolydispersitywasbetween 0.191and0.371.ThemeanparticlesizewasleastwhenGMSwas atlowlevelandOAandpoloxamerwereathighlevels(F4)andthe particlesizewaslargestwhenGMSwasathighlevelandbothOA andpoloxamerwereatlowlevels(F5).Thus,uponincreasingthe OApercentageinthelipidmatrixfrom15%to30%,themeanparti- clesizedecreased.HenceF4wasthesmallestsizednanostructured lipidnanoparticlewithazetapotentialof−39.8mV.Asdepicted inTable1,thezetapotentialvaluescloselyrangedbetween−29.1 and−39.8mV.Leastvalueofzetapotentialwasassociatedwith F5andthehighestwithF4andrestoftheformulationsshowed intermediatevalues.
3.2.2. Drugentrapmentefficiency
Animportant issuewithrespect totheuseof nanoparticles as drug carriers is their capacity for drug loading and conse- quently the effects of OA concentration on drug entrapment efficiency were investigated. It is clear that the entrapment efficiency of nanoparticles increased from 81.74% to 93.33%
respectively upon increasing the percentage of OA from 15 to 30wt%. These values are considerably higher than the encap- sulation efficiency of SLN made with SV in our lab. The entrapment efficiency of optimized SLNs made with glyceryl monostearate (Shah and Pathak,2010)and Compitrol888ATO (Shahetal.,2010)was75.81%and68.16%respectively(Table1).
Thus,incorporationofOAinlipidmatrixresultedinhigherentrap- mentefficiencyascomparedtoSLN.
3.2.3. Invitrodrugrelease
In ordertodevelopa prolongedrelease system,it isvital to understandtherelease mechanismandkinetics. TheinvitroSV releaseprofilesofF1–F8andSVsuspension(F0)areshowninFig.1.
Theinvitroreleaseprofilesofthetestformulations(F1–F8)were differentfromthereferenceformulationF0 (f2rangedbetween 92and96).ThecumulativeamountofSVreleasedfromeachfor- mulationwasplottedasafunctionoftimeandasevidenttheSV suspensionshowedquickreleaseofsimvastatin,withacumulative releaseofmorethan90%within6h.Ontheotherhandtherelease ofSVfromtheNLCs(F1–F8)sloweddownconsiderablyanditvar- iedfrom57.95% to91.49%in 55hdependingupontheGMS/OA levels.Asisevident,formulationF4formulatedusinglowlevels ofGMSandhighlevelsofbothOAandpoloxamer407displayed highest%CDR(88.3%±3.23)thatwasquiteincontrasttoformula- tionF5formulatedwithhighlevelsofGMSandlowlevelsofboth OAandpoloxamer407exhibitedleast%CDR(57.95%±3.18)after 55h.Thereleaseprofileswerenotsmoothcurvesbutwereidenti- fiedasbiphasicwithinitialfasterreleasephasetill10hfollowedby aslowerreleasephasethereaftertill55h.Oncomparingtherelease
Fig.3. Paretochartsforanalysisofresponsecoefficientsignificanceonthedependentvariables(a)particlesize,(b)%EE,and(c)%CDR.
Fig.4.ResponsesurfaceplotsshowingsimultaneousinfluenceofindependentvariablesonresponseparametersofSVloadedNLCformulatedwithintheexperimental design.
trendsofoptimizedSLNs(G-SLN(glycerylmonostearateSLN)and C-SLN(Compritol888ATOSLN)withNLC(Fig.2),evidentlythe releasepatternsweresimilarbutthequantumofreleasewashigher incaseofSVNLCthanSVSLNs.TheSLNsexhibited<80%releasein 55hwhereasNLCrelease>90%ofSVin55h.Consequently,F4that displayedmaximum%CDRwithin55handbasedontheevaluation ofthethreedependentvariables,theoptimizedformulationF4was selectedastheoptimizedformulation.
3.3. Validationofexperimentaldesign
Theselectedindependentvariables liketheamountofGMS, amountofOAandconcentrationofpoloxamer407influencedthe particlesize,%EE,and%CDRthatisquiteevidentfromtheresults inTable1andhasbeendocumentedinprecedingsections.Con- sequently,foreachdependentvariable,theresponsepolynomial coefficientsweredeterminedinordertoevaluatetheeffectofeach response.Eachresponsecoefficientwasstudiedforitsstatistical significancebyParetochartsasshowninFig.3.Paretochartsestab- lishtvalueofeffectthatisstudiedbytwolimitlinesnamelythe Bonferronilimitline(tvalueofeffect=3.082)andtlimit line(t valueofeffect=2.1199).Coefficientswithtvalueofeffectabovethe Bonferronilinearedesignatedascertainlysignificantcoefficient;
coefficientswitht valueofeffectbetweenBonferronilineand t limitlinearetermedascoefficientslikelytobesignificant,while tvalueofeffectbelowthetlimitlineisstatisticallyinsignificant coefficientandareremovedfromtheanalysis.Thus,non-significant
responsecoefficientsweredeletedandfollowingsignificantpoly- nomialresponseequation(s)forparticlesize,%EE,and%CDRwere generated.
Particlesize (nm)=222.10+24.11X1−0.78X2−3.84X3
−1.76X2X3+0.83X1X2X3 (6)
%EE=87.27+0.83X1+3.19X2−1.84X3+1.59X1X2X3 (7)
%CDR=73.41−10.26X1+5.43X2+2.04X3−0.80X1X2
+0.95X1X2X3 (8) Theseequationswereutilizedforvalidationoftheexperimental designbyformulatinganextradesigncheckpointformulationF9.
Utilizingtheaboveequations,particlesizeof222.10nm,entrap- ment efficiency of 87.27% and %CDR of 73.41% at 55h were predicted.ExperimentalrunofF9yieldedvaluesclosetothepre- dictedindicatingvalidityofthegeneratedmodel.Tofurtheranalyze theeffect ofvariables on theresponses,response surface plots (Fig.4)weregeneratedthatrepresentthesimultaneouseffectof anytwo variablesonresponseparametertakingonevariableat constantlevel.Oncarefullyobservingtheseplots,thequalitative andinteractiveeffect(s)ofindependentvariablesoneachresponse parametercanbevisualizedandtheresultsaresummarized in Table2.
Fig.5.DifferentialscanningcalorimetricprofilesofSV,GMS,SV-GMSphysicalmix- tureandtheformulationF4.
3.4. Recrystallizationindex
ExaminationbyDSCpatterns(Fig.5)revealedsinglepeaksin theendothermsofSV(m.pt=139.6◦C;H=71.19J/g)andglyceryl monostearate(m.pt=57.65◦C;H=79.19J/g).Theendothermof F4recordedadistinctivebroad,slightlyasymmetricsinglepeakat 54.95◦CwithHof29.1J/gandthepeakforSVwasnotevident.
Themeltingpointofthedevelopedformulationwaslowerthan thatofbulkGMS;however,therewasadetectablemeltingevent over50◦Cconfirmingsolidstateofthelipidmatrixatroomtem- perature.TheshiftinmeltingpointofNLCmayattributedeither totheinteractionofsolidlipidwiththeliquidlipidandthesur- factants,orduetothecolloidalsizerange ofthelipidparticles.
ThedisappearanceofmeltingpeakintheendothermofF4canbe explainedbyKelvineffect,accordingtowhichsmall,isolatedpar- ticles(SV)wouldmeltatatemperaturelowerthanthemelting temperatureofbulkmaterial(glycerylmonostearate).Asthedif- ferentialscanninganalysiswasaimedatevaluatingchangesupon storage,theenthalpyvalueswereutilizedforcalculatingrecrystal- lizationindex(RI).TheRIofF4wascalculatedas36.71%againstthe referencevalueof100%forGMSthatwasmuchlowerthantheRI of91.1%forphysicalmixtureofSVandGMS(1:10).Itisclearthat therecrystallizationofoptimizedNLCformulationwasdepressed bytheadditionoftheOAthatdecreasedcrystallinity,promoted disorderedarrangementandconsequentlyreducedthetendency ofsolidlipidtorecrystallize.
3.5. Pharmacokineticstudy
Theblood%A/G–timeplots,inmiceafteroraladministrationof testformulations(SVsuspension,SV-SLNandSV-NLC)areshown in Fig. 6 and the pharmacokineticparameters are tabulated in Table3.TheTmaxwas2handtheCmaxwas0.09%A/Gafteroral administrationof SVsuspension. However,thetime to achieve
Fig.6.%A/GinbloodversustimeprofileofSVsuspension,SLNandNLCafteroral administrationoftheradiolabeledformulationsinmice.
Fig.7. AbsorptionprofilesofSVloadedNLCandSLNgeneratedfromdeconvolu- tionofthecorrespondingbloodconcentrationprofilesusingtheWagner–Nelson method.
maximumconcentrationofSVwasdelayedby2hinbothcaseof NLCandSLN.TheCmaxofSV-NLCwas0.16%A/Gwhichwassignifi- cantly(p>0.01)higherthanthatobtainedwiththeSVsuspension (0.09%A/G)butlowerthanSLN(1.24%A/G).Meanwhile,atalltime points,the%A/GinbloodofSV-NLCwereremarkablyhigherthan thoseadministeredwithSVsuspension.Twenty-fourhoursafter oral administrationof SV-NLCs,theblood %A/G wasstill0.08%, whereastheactivitywasundetectable24hafteradministrationof SVsuspensionandwasmuchlowerincaseofSLN.TheAUC(0–∞)of SV-NLCwas3.92%A/G/h,thatwas4.87foldshigherthanAUC(0–∞) of0.804%A/G/hforSVsuspensionand2.74timesmorethanSLN formulationofSV,clearlydefiningperformancesuperiorityofNLC overSLNandsuspensions.LowervaluesofVdforNLCincomparison toSLNandsuspensionareindicativeoflargerfractionofformula- tionbeingretainedinthecentralcompartmentandhencemore bioavailable.
Table2
Acompilationofinteractivestatusandsimultaneousqualitativeeffectofindependentvariablesoneachresponseparameter.
Parameter Assessmentof Interactingvariables
X1X2 X1X3 X2X3
Particlesize Interaction − − +
Effectondependentvariable Increase Increase Decrease
Percententrapment efficiency
Interaction + − −
Effectondependentvariable Increase Increase Increase
PercentCDRin55h Interaction + + −
Effectondependentvariable Decrease Decrease Increase
(−)antagonisticinteraction,(+)synergisticinteraction.
Table3
PharmacokineticparametersofradiolabeledSV-suspension,NLCandSLNafteroraladministrationofthetestformulations.
Evaluationparameters SV-suspension NLC SLN
Cmax(%A/G) 0.09±0.02* 0.16±0.04 1.24±0.076*
Tmax(h) 2* 4 4
Eliminationhalflife(h) 5.20±1.2* 10.86±1.74 6.72±2.69*
Absorptionconstant(h−1) 0.32±0.094 0.31±0.082 0.13±0.011*
Eliminationconstant(h−1) 0.134±0.09* 0.06±0.092 0.12±0.08*
Volumeofdistribution(L) 9.3±2.3* 4.0±1.3 6.89±2.21*
Clearance(L/h) 1.24±1.3* 0.26±0.9 0.713±0.97
AUC(0–∞)(%A/G/h) 0.804±0.65* 3.92±1.1 1.43±1.43*
Relativebioavailability(%) 100* 488 186*
Eachvaluerepresentsthemean±SD(n=3);*p<0.01,comparisonwithNLC.
ThetableshowsthemeanandSD(N=3)ofpharmacokineticparameterscalculatedfollowingoraladministrationofSVsuspension,NLCandSLNinBalb/Cmice.Thepvalues foreachparametersisanunpairedt-testcomparisonamongSVsuspension,NLC,andSLNafteroraldelivery[<0.01(*)].
3.6. Invitro–invivocorrelation
CorrelationbetweeninvitroandinvivodataforSV-NLCand SLNwasinvestigated.ThreelevelsofIVIVCareclassifiedaccording totheUSFoodandDrugAdministration(DressmanandReppas, 2000). The best correlation Level A, is point-to-point relation- ship betweeninvitro dissolution and invivo absorption.Using Wagnor–Nelsonmethod,thepercentageabsorptiondataasafunc- tionoftimetogettheplotsshowninFig.7.Theabsorptionprofiles ofboththeSVNLCformulationandSLNweresimilarbutvaried inextentofabsorption.TheNLCsexhibitedhigherpercentabsorp- tionofSVthanSLNintheinitialphasethatgraduallyincreased withtime(4h)andthedifferencewasmaintainedtill24h.There- afterboththeprofilesmergedintoasinglepointin48h.Onplotting thefractionSVabsorbedagainstthefractionSVreleasedfromthe lipidnanoparticulatesystem,linearcorrelationwithR2valuesof 0.940and0.941wereobtainedforSV-NLCandSV-SLNrespectively (Fig.8)indicatingthattheinvitroreleasetestcanbeusedtopredict theinvivoperformanceofthedevelopedformulations.
3.7. Tissueuptakestudy
Thetissue uptakestudyof99mTclabeledformulations(max- imum labeling efficiency of 99.36% observed with 200mcg/ml SnCl2,pH6–6.5and anincubationtime of25min) wasinvesti- gatedinthevitalorgansheart,lungs,liver,spleen,kidney,stomach andintestine and it wasobserved thatthedistribution pattern varieddepending onthe dosageform (Table 4).On comparing thedistributionofdrugfromSVsuspensionandSVloadedNLC, higherradioactivitywasdemonstrableinstomachwithin1hoforal administrationfromboththeformulationsthatstartedtodeclinein thesecondhouranddiminishedbytheendof24h.Fastdeclineof radioactivityinstomachisattributabletoshortgastricresidence time of SVsuspension and theSVloaded NLCadministered as suspension.Declineofradioactivityinstomachcorrespondedto increaseinradioactivityinintestineduetogastrointestinaltransit ofsuspension.Incaseofintestine,theradioactivitywasmaximum insecondhourforSVsuspensionbutforSVNLCmaximumactivity wasseenupto4hafteradministrationindicativeofhigherres- idencetimeofNLCinintestinethatcanprovideanopportunity forprolongedabsorptionofSVfromNLCs.Thisresultisconsistent withthesharpincreaseinthepercentdrugabsorbedwithin4has showninFig.7.
Asthedruggotabsorbedanddistributedtovariousorgans,the uptakepattern ofradiolabeled formulationsinvital organswas analyzed.TheaccumulationofSVNLCwashigherinliverthanSV suspensionatalltimepointsthanfromsuspension.IncaseofSV suspensionmaximumactivitywasobservedwithin2hofadminis- trationthatwasmaintainedtill24handdeclinedthereafter.Thisis quitedifferentfromthedistributionkineticsofSV-NLCs.The%A/G
thatwasinitiallyhigherthanSVsuspensionkeptonincreasingup to24hsuggestingaccumulationofNLCsinliver.Thisisadvanta- geousbecausetheliveristhetargetorgan,toreducethecholesterol levelinblood.
Inrestoftheorgansanalyzedforbiodistribution,the%A/Gwas notverysignificantlydifferentexceptforfewtestpointsmarked inthetable.However,theradioactivityfromSVNLCwasgrossly higherascomparedtosuspensioninlungs,heart,spleenandkid- ney.
4. Discussion
NLCs of SV wereformulated using a combination of highly ordered and less ordered lipids. As documented in literature, thelessorderedlipidenhancestheentrapmentefficiencyofthe nanoparticulatesystembecauseentropyofthelipidprovidesspace for entrapment of drug molecules. In present investigationOA wasusedasthelessorderedlipidanditsconcentrationmarkedly affecteddrugloadingandalsothesizeofNLC.Thus,athigherlevels ofOA (30wt%)small,sphericalnanoparticleswithhighentrap- mentefficiencywereobtained.OAisconsideredasthemainfactor responsibleforthereductioninparticlesizeanddecreasedpoly- dispersity indexbecause itsinclusion inthe formulationledto reductioninviscosityandsurfacetensioninsideNLCsthathelped intheformationofnanosizedhomogenouslydistributednanopar- ticles.Thenanoparticlesarethermodynamicallyunstablesystems and for the stability of colloidaldrug carriers, a zetapotential valueofequaltoormorethan30mVisdesirable.Inthepresent studythezetapotentialofNLCswithinthedesigncloselyranged between−29.1and−39.8mVsuggestingthermodynamicallysta- blesystems.Buthighzetapotentialvaluecoupledwithnanosized particlespresentsdifficultyinseparationbycentrifugation,so0.1M hydrochloricacidwasaddedtothesystemtoreducezetapotential andfacilitatecentrifugation.
AsOAaffects particlesize sodoesitaffects theentrapment efficiency.Researchershaveexplainedthattheliquidlipidscause numerouscrystaldefectsinsolidlipidandprovideimperfections inhighlyorderedcrystal.Inthisspacemoredrugmoleculesget entrapped. Asboth particlesize and entrapment efficiency are determinants of drug release from a given carrier system,the invitrodrugreleaseprofileswereexpectedtovaryaccordingly.
Thus, the formulation F4with smallestparticle size of 180nm (Fig. 9) and highest entrapmentefficiency displayed maximum
%CDRin asustained manner.Itcan berealizedthatOA played keyroleininfluencingthe%CDRversustimeprofile.Thus,athigh levelofOAtheparticlesizereducedleadingtoincreasedspecific surfaceareaand consequently%CDR.The%CDRprofiletypically consistedoftwoparts,theinitialfasterphasefollowedbygrad- ualslowrelease.Thisbehaviorissimilartotheonedocumented byZhuangetal.(2010).Accordingtotheauthors,whensolvent
Fig.8. Invitroinvivocorrelationof(a)SVloadedNLCand(b)SVloadedSLN.
injectionmethodisutilizedforpreparingNLCs,thedifferencein meltingpointsofsolidandliquidlipid,leadstothecrystallization ofsolidlipid,formingaliquidlipidfreecoreoracorewithlittle lipid.MostoftheliquidlipidislocatedattheperipheryofNLCspar- ticle.InourcasethisphenomenonledtoformationofOA-enriched peripherallayer(s)thatpossessessubstantiallyhighersolubilityfor thelipophilicdrug.Thus,simvastatinwasreadilyloadedinhigher amountsintheperipheralregionandwaseasilyreleasedbythe drugdiffusioncontributingtoinitialfasterdrugreleasephasefol- lowedby slowreleasefromtherelativelysolid core.Thus,it is possibletomodifythereleaseprofilesasa functionofthelipid matrixandthisisdemonstratedinFig.6wheretheinvitrorelease profiles ofSV-SLNs made upof complete solid coreof glyceryl monostearateandCompitrol888ATOshowedslowerreleaserate.
Itcanberealizedthatthemobilityofdrugisdrasticallyreduced insolidifiedorcrystallizednanoparticles,resultinginslowdrug release.AmongtheNLCformulationsevaluatedforinvitrorelease, F4displayedmaximum%CDRwithin55handbasedontheevalu- ationofthethreedependentvariables,theoptimizedformulation F4wasselectedforfurtherinvestigation.
Inadditiontoaffectingtheparticlesize,entrapmentefficiency andinvitrodrugreleasecharacteristics,theliquidlipidsintegrated intothe solidlipids inNLCs cause reduction ofordered matrix structureandallowadequatespacestolodgethedrugmolecule thatisfirmlyembracedinthedisorderedstructureduringlong- termstorage(Mulleretal.,2000).Thus,NLCscanoffersolutionsto thechangesthatmayoccuruponaging,crystallinityandpolymor- phismofSLNs.Therecrystallizationindexwasusedasaparameter toevaluatetheabilityofnanostructuredlipidcarrierinretarding
Fig.9. TransmissionelectronmicroscopicimageoftheoptimizedSVloadedNLC (F4).
anypotentialchangesthatcanoccuruponlongtermstorage.Sig- nificantly(p<0.001)lowvalueoftherecrystallizationindexthan theSLNssignified therole ofliquid lipidinretardingrecrystal- lization.Asawelldocumentedfactcrystallizationoccursdueto supersaturationandthepresenceofliquidlipidprobablytendsto maintainsubsaturationstateofthesolidlipidretardingcrystalliza- tion.Hence,theSLNsmadewithGMS(ShahandPathak,2010)and Compitrol888ATO(Shahetal.,2010)hadhigherrecrystallization tendencythanNLCsofsimvastatin.
NLCs of SV had superior pharmacotechnicalproperties than SLNs and hence were assessed for their pharmacokinetics and biodistributioninmiceusingSVsuspensionasreferenceformu- lation.ComparedtoSV-suspension,the%A/Gversustimeprofile ofSV-NLCdisplayedasmoother%A/Gversustimeprofile,witha delayedTmax,andprolongedt1/2.Thisoffersachanceforreduction indosingfrequency.ThevisibledifferencebetweenTmaxvalueof SV-NLCandSVsuspensionmanifestedthattheratesofabsorption fortheformulationswerenotsame.SVfromsuspensiondissolved intheintestinaltractfluidsandwasthenabsorbedintothesystemic circulationincontrasttoSV-NLCthatreleasedSVverygradually intothegastrointestinaltract,asindicatedbytheinvitrorelease studiesconsequently leadingtoslow absorption.The sustained releaseofdrugfromtheNLC’snotonlysloweddowntheabsorption italsoaffectedtheeliminationhalf-lifeofSV.Consequentlythet1/2 ofSVfromNLCsalmostdoubledthanthet1/2ofSVfromsuspension andevenhigherthanthet1/2ofSVfromSLN.
Poorwater-solubility,extensivehepaticfirstpassmetabolism andtheseparationfromthebulkaqueousphaseofintestinalcon- tentsbyunstirredwaterlayerofthebrushborderedmembrane of theenterocytesliningthesmallintestine, aremajor barriers to absorption of poorly water soluble lipid digestion products.
Thesearealsothepossiblereasonsoflowbioavailabilityofsimvas- tatin.ConsideringthestructureofSV,aweaklyalkalinecompound (pKa=4.7),thatshowshighersolubilityatlowpHvalues,thesolu- bilityofSVthroughoutthegastrointestinaltractcanbeanalyzed.
Afteroraladministration,thedrugwouldfirstdissolveinthegas- tricfluidandpresentitselfforabsorption.Whenthedrugreaches theintestinaltract,increaseinpHwillresultinprecipitationof the drug, that will affect the absorption. Additionally, SV that getsdirectlyabsorbedfromtheintestine intothebloodcircula- tionwould meet extensivemetabolismin theliverresultingin poorbioavailability.In thecase ofSV-NLCand SLN,thepresys- temichepaticeffectcanbelargelyavoidedbecausethatthedrugis encapsulatedinnanoparticlesandowingtotheircolloidalnature thesewereavailableforthelymphaticabsorbingpathway.Thiscan enhancethebioavailabilityofthedrugandthesustainedrelease propertyofSV-NLCandSLNcouldalsoachievealongerretention timeofdruginvivo.
AnalysisofthecompositionofNLCscanalsoexplainenhance- mentinthebioavailability.GMSandOA,whichissimilartofatrich
Fig.10.DiagrammaticrepresentationoftheproposedabsorptionmechanismsofradiolabeledSVNLC.
dietandinducethebilesecretioninsmallintestine,andmixedwith thebiletoformmixedmicelles.Thesemicellescanenhancethe luminalsolubilityofthelipiddigestionproducts,andalsofacilitate theirpassageacrosstheunstirredwaterlayertherebyproviding a concentration gradient for absorption (Tso, 1994; Porter and Charman,2001).Additionally,thepresenceofaregionoflowpH, adjacenttotheenterocytesurfacemayprovokeachangetothecol- loidalstructureofthemicellarspecies,presentingtheintactNLCs availableforlymphaticuptake(Jacobsetal.,2001).Theuptakeand lymphatictransportofintactcolloidalnanosizedNLCssupposedly playedadominantroleinpromotingabsorption.Adiagrammatic representationoftheproposedabsorptionmechanismofSVNLCis presentedinFig.10.
Predominantly,threemechanismshavebeendocumentedfor theabsorptionoftheNLCfromtheintestinethatinclude direct uptakethroughtheGItract,increaseinpermeabilitybysurfactants anddecreaseddegradationandclearance.Theparticlesizeofthe NLCformulationswasalmostlessthan200nm,andthisreduced particlesizeimprovedthesurfaceareaoftheNLCs.Thefundamen- talequationusedtosupportthedesignofnanoparticlessystemsis theequationofOstwald–Freundlichwhichestablishestheincrease insolubilityofagivensubstanceonbasisoftheincreaseofinterfa- cialenergyathighcurvaturesofsmallsizedparticle(Kipp,2004).
Thissmallsizeallowstheefficientuptakeintheintestineparticu- larlyinthelymphoidalsectionofthetissuethusbypassingthefirst passmetabolism.AnotherfactorthatsupportsabsorptionofNLCs intheintestinalmilieu,isthehighdispersibilityofNLCs.Besides this,theNLCscanalsoadhereontothegutwallprolongingthe residencetime,andconsequentlytheabsorption.Thepresenceof poloxamer407usedassurfactantinformulationalsosupportsthe absorptionofNLCs,duetoitseffectonintestinalepithelialperme- ability.Poloxamerisdocumentedtodeformthecellmembraneand
openthetightjunctionoftheintestinalepithelialcellfacilitating paracellulartransportofNLCs(WuandLee,2009).Poloxamer407 alsorestrainsp-glycoproteineffluxpumpandincreasesNLCtrans- portacrosstheintestinalmucosa(Luoetal.,2006).Recently,ithas beenreportedthatenzymeactivityofCYP3Acouldbeinhibitedby oleicacid(Mountfieldetal.,2000).Thusitcanbeconcludedthat significantdifferencebetweenthebioavailabilitiesofSV-NLCand SLNswasmainlyduetothesmallparticlesizeofNLCandpresence ofoleicacidinNLC.
Biodistribution studiesindicated longer duration of radioac- tivity in the intestine that is attributable to higher amount of villipresentintheintestine,inwhichnanoparticlescanbeeas- ilyentrappedforlongperiodoftime.Biodistributionofparticulate drugcarriersisgreatlyinfluencedbytheirsize,surfacecharacter- isticsandopsonizationprocess.Asawellknownfactopsoninsget adsorbedonthenanoparticlesurfaceandpromoteparticlerecogni- tionbyRESorgans.InourstudyNLCsshowedhigheraccumulation inliverthansuspensionbuttheextentandpatternofbiodistribu- tionwassimilarforboththeformulations,inspleen.Preferential uptakeofNLCsbylivercanbecorrelatedtosmallersizeofparticles andlymphaticuptakeoflipidnanoparticleswhereasthepresence ofchargeonthesurfaceofNLCsdidnotfacilitateefficientcoat- ingwiththeopsonizingcomplementsystemresultinginreduced uptakebyspleen(ManjunathandVenkateswarlu,2005).
InterestinglythetransportofNLCsthroughtheintestinallym- phaticviathethoraciclymphducttothesystemiccirculationat thejunctionofthejugularandleftsubclavianvein,bypassesthe liver.SV is consideredto bea reasonable substrate for intesti- nallymphatictransportbecauseofitshighlogpvalue.Asevident frombiodistributionstudiesthatSV-NLCscontinuouslyenteredto livereven after24hof administration.Thisisattributedtothe longcirculatory time of SV-NLCsin blood. The longcirculatory
Table4 Percentuptakepergramorganweight(%A/G)followingoraladministrationofradiolabeledSVsuspensionandSV-NLCindifferentorgansofBalb/Cmice. Organs%A/Gatdifferentorgansatdifferenttimepoints SVsuspensionSV-NLC 1h2h4h24h48h1h2h4h24h48h Heart0.001±0.007*0.2±0.016*0.01±0.02*0.009±0.0010.004±0.0010.01±0.0010.02±0.10.006±0.0010.005±0.0010.002±0.001 Lungs0.013±0.0010.11±0.03*0.04±0.03*0.007±0.0010.003±0.0010.03±0.0030.06±0.0170.007±0.0020.004±0.0010.002±0.001 Liver0.009±0.002*0.06±0.01*0.06±0.01*0.04±0.008*0.032±0.05*0.04±0.0050.09±0.010.36±0.080.73±0.140.11±0.02 Spleen0.039±0.0050.03±0.0060.04±0.010.001±0.0020.001±0.0010.02±0.010.03±0.0070.01±0.0010.004±0.0010.002±0.001 Kidney0.003±0.001*0.06±0.010.05±0.0150.04±0.010.02±0.0130.06±0.0150.07±0.020.04±0.010.04±0.0120.02±0.01 Stomach16.51±1.3214.75±1.813.6±1.86*1.1±0.02*0.09±0.02716.96±1.5111.5±2.039.08±1.90.8±0.130.014±0.06 Intestine3.3±0.62*1.35±0.72*0.9±0.25*0.04±0.006*0.02±0.00511.9±1.8618.36±1.4819.3±3.123.6±0.730.012±0.01 Blood0.01±0.0070.09±0.020.05±0.010.005±0.0010.0002±0.0010.03±0.0060.07±0.0160.16±0.040.08±0.0160.01±0.001 Thepvalueforeachorganisanunpairedt-testcomparisonbetweenSVsuspensionandSV-NLCsuspensionoraldelivery[*p<0.05].
time ofSV-NLCinblood iscreditedtothestabilizationofNLCs suspension,thatwasprobablyachievedbytheadditionofasur- factantpoloxamer407.DuringformationofNLCs,poloxamergets adsorbedonthehydrophobicsurfaceofNLCsviatheirhydrophobic polyoxypropylenecenter-block,andhydrophilicpoloyoxyethylene sidechainextendtowardsoutsidefromNLCssurface.Thistype of arrangementprovidesa thermodynamically stablesystemin suspensionbyrepulsioneffectthroughastericstabilizationmech- anism.Hydrophobic center blockof poloxameradheres oncell membraneandiscapableofalteringthebiodistributionprofileof NLCs.Thehydrophilicpoloyoxyethylenetailsofpoloxamerpre- ventstheinteractionbetweenanapproachingparticlesand the cells,andstickingoflipidnanoparticlestothebloodvesselendothe- lium.Thisalsosuggeststheirphagocyticresistanceandexplainsthe longcirculatorytimeofNLCsintheblood(Mohigimietal.,2001).
Longcirculatorytimeofdruginthebloodofferedmorepartitioning ofdrugtothetissuesresultinginhigherorganuptake.
5. Conclusion
SimvasatinNLCsuspensionsuccessfullyoptimizedtoachieve higherandsustainedreleaseaswellasbioavailabilityofsimvas- tatin.TheobtainedNLCwassuspensionofnanosizedhomogeneous particleswithhighentrapmentefficiencyand lowrecrystalliza- tionproperties.Thepharmacokineticstudydemonstratedahigher relativebioavailabilitythanbothSVsuspensionandtheSLNfor- mulationthatwasdirectlyafunctionofthepresenceofoilylipid intheNLC.TheNLCscouldimprovethegastrointestinalabsorption ofSVandlevelAcorrelationcouldbeestablishedbetweeninvitro dissolutionandinvivoabsorption.Thisworkfacilitatestheuseof nanostructuredlipidcarrierasanoveldeliverysystemtoenhance oralbioavailabilityofsimvastatin.
Acknowledgments
FinancialsupportwasprovidedbyAllIndiaCouncilforTechnical Education(AICTE),NewDelhi,India.AuthorsaregratefultoPunjab University,Chandigarh,India,forprovidingTEMfacility;IIT,Delhi, India,forprovidingassistanceinDSCscan;CDRI,Lucknow,India, forprovidingzetasizingfacility.
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