JPP2009,61:3113212009TheAuthorsReceivedJuly28,2008AcceptedDecember01,2008DOI10.1211/jpp/61.03.0005ISSN0022-3573Correspondence:DrSarasijaSuresh,Prof.&HeadofPharmaceutics,Al-AmeenCollegeofPharmacy,HosurRoad,Bangalore-560027,India.E-mail:sarasija_s@hotmail.comResearchPaperNovelformulationofsolidlipidmicroparticlesofcurcuminforanti-angiogenicandanti-inflammatoryactivityforoptimizationoftherapyofinflammatoryboweldiseaseVivekRamshankarYadava,SarasijaSuresha,KshamaDevibandSeemaYadavcaSRF,DepartmentofPharmaceutics;bDepartmentofPharmacology,Al-AmeenCollegeofPharmacy,HosurRoad,Bangalore-560027andcDepartmentofPharmacology,NootanDentalCollege&Hospital,Visnagar,Mehsana,Gujarat,IndiaAbstractObjectives This project was undertaken with a view to optimize the treatmentof inflammatory bowel disease through a novel drug delivery approach for localizedtreatment in the colon. Curcumin has poor aqueous solubility, poor stability in thegastrointestinal tract and poor bioavailability. The purpose of the study was to prepare andevaluatetheanti-inflammatoryactivityofsolidlipidmicroparticles(SLMs)ofcurcuminfor the treatment of inflammatory bowel disease in a colitis-induced rat model by a colon-specificdeliveryapproach.Methods Wehavedevelopedanovel formulationapproachfor treatingexperimentalcolitis intherat model. SLMs of curcuminwerepreparedwithvarious lipids, suchaspalmiticacid, stearicacidandsoyalecithin, withanoptimizedpercentageofpoloxamer188. The SLMs of curcumin were characterized for particle size, drug content, drugentrapment, in-vitro release, surface morphology and infrared, differential scanningcalorimetry and X-ray studies. The colonic delivery systemof SLMformulations ofcurcumin were further investigated for their anti-angiogenic and anti-inflammatory activityusingchickembryoandratcolitismodels.Keyfindings Particlesize, drugcontent, drugentrapment andin-vitroreleasestudiesshowed that formulationF4 containing one part stearic acid and0.5%surfactant hadthe smallest diameter of 108 mm, 79.24%entrapment and exhibited excellent in-vitroreleasecharacteristicswhencomparedwithotherformulationsandpurecurcumin. SLMsof curcumin(F4)provedtobeapotent angio-inhibitorycompound, asdemonstratedbyinhibition of angiogenesis in the chorioallantoic membrane assay. Rats treated withcurcumin and its SLM complex showed a faster weight gain compared with dextran sulfatesolution (DSS) control rats. The increase in whole colon length appeared to be significantlygreaterinSLM-treatedratswhencomparedwithpurecurcuminandDSScontrolrats.AnadditionalfindingintheDSS-treatedratswaschroniccellinfiltrationwithpredominanceof eosinophils. Decreasedmast cell numbers inthe mucosa of the colonof SLMs ofcurcuminandpurecurcumin-treatedratswasobserved.Conclusions Thedegreeof colitis causedbyadministrationof DSSwas significantlyattenuatedbycolonicdeliveryof SLMs of curcumin. Beinganontoxicnatural dietaryproduct, curcumincouldbe useful inthe therapeutic strategyfor inflammatoryboweldiseasepatients.Keywords angiogenesis; colonic delivery; curcumin; inflammatorybowel disease; solid lipidmicroparticles (SLMs)IntroductionInflammatory bowel disease (IBD), such as ulcerative colitis and Crohns disease, is associatedwith chronic relapsing inflammation of the intestinal tract of unknown aetiology.[1]Histologically, mucosal accumulationofleucocytesisacharacteristicfeatureofIBDandactivationofTcellsandmonocytes/macrophageshasbeenregardedasacrucial factorinits pathogenesis. Treatment of IBDdepends on drugs such as 5-aminosalicyclic acid,corticosteroids, azathioprine, mercaptopurines andciclosporin.[1,2]However, useof these311drugs is sometimes limited by drug-inducedtoxicity.Thereisincreasingneedforalternativeagentsthatmaybeequally,ormore, effective but toxicity free, as well as less expensive.Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a low-molecular-weight polyphenolderived fromthe rhizomes of turmeric (Curcuma longaLinn.),isayellowpigment,widelyusedasacoloringagentand spice in many foods and has been used in Ayurvedic andChinesemedicineforcenturies.Turmericcontainsbioactivesubstances known as curcuminoids, such as curcumin,demethoxycurcumin, bisdemethoxycurcuminanddimethox-ycurcumin.[3]Interest in this dietary polyphenol has grown inrecent years due toits vast arrayof beneficial pharmaco-logical effects, including antioxidant, anti-inflammatory, anti-carcinogenic,[46]hypocholesterolaemic,[7]antibacterial,[8]woundhealing, antispasmodic, anticoagulant, antitumour[9]and hepatoprotective[10]activity. It has also been reported thatcurcumin has a protective effect on 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice; curcumin reducedsignificantly the degree of both neutrophil infiltration andlipid peroxidation, as well serine protease activity.[11]It is alsoa potent free radical scavenger, having superoxide anion,singlet oxygen, hydroxyl radical scavenging and lipidperoxidationinhibitoryactivity.[12]Despitethepromisingbiological effectsofcurcumin, itspoor oral bioavailability in both rodents and humans, asreported by several workers,[13,14]has restricted its use in themanagement of human ailments. The main drawbacks for theclinical application of curcumin are its low solubility in waterat acidic and physiological pH[9]and its rapid hydrolysisunderalkalineconditions.[15,16]Extensivepresystemicmeta-bolism ofcurcumin mayberesponsible fortheunfavourablepharmacokineticsof this molecule. Theseproblemscanbeaddressed by incorporation of curcumin into a colloidalcarriersystemandothermethods, suchasadministrationofcurcuminalongwithpiperine, useof liposomal curcumin,nanoparticles of curcumin and synthetic analogues ofcurcumin. Amongmoderndrugdeliverycarrierssolidlipidmicroparticles (SLMs) seemto be a promising colloidalcarriersystem. SLMsmadefrombiodegradablesolidlipidsexist inthesubmicronsizerangeandcanbepreparedbyseveral methods. The advantages of SLMs are as follows: thepossibility of controlled drug release and drug targeting;protection of incorporated compound against chemicaldegradation; no biotoxicity of the carrier; avoidance oforganicsolvent; controlledreleasecharacteristics; chemicalandphysicalstoragestability(forbothdrugandcarrier)andfeasibilityofscalingupproduction.[1719]Angiogenesis is a strictly controlled process in the normalhumanbodyandis regulatedbya varietyof endogenousangiogenic and angiostatic factors.[20]However, at shortnotice the microvascular system appearscapable ofrespond-ing with rapid capillary growth to physiological demandssuch as ovulation, as well as to pathologic conditions such aswounds, chronic inflammation, certain immune reactions andtumours.In this study we have developed a novel formulationapproach for treating experimental colitis in a rat model. WehavepreparedSLMsof curcuminwithvariouslipids, suchas palmitic acid, stearic acid and soya lecithin, with anoptimizedpercentageofpoloxamer188andevaluatedthemfor increasing the solubility of curcumin. The effect onangiogenesis was determined by the chick embryo chor-ioallantoic membrane (CAM) system. Further, a modifiedpulsatile capsule has been designed to deliver the drug to theterminal ileumandcolon. Modifiedpulsatilecapsulesweredesigned by filling the optimized SLMformulation ofcurcuminintoa hardgelatincapsule followedbycoatingthecapsulewithacrylicpolymers(pH-dependentpolymers).Thedevelopeddeliverysystemwasevaluatedinadextransulfate (DSS)-induced experimental colitis model for itspotential as a new therapeutic approach for treatment of IBD.MaterialsandMethodsMaterialsCurcumin was a gift from Natural Remedies Pvt Ltd(Bangalore, India). Soyalecithin, stearicacidandpalmiticacid were purchased from Hi-Media (Mumbai, India).Methanol and Tween-80 were purchased from ThomasBaker (Mumbai, India). Poloxamer 188 was a gift fromBPRL (Bangalore, India). Dialysis membrane, diameter76mm, withanumber 150waspurchasedfromHi-Media(Mumbai, India). Eudragit S100, Eudragit L100, EudragitRL100 and Eudragit RS100 were obtained fromRohmGmbH(Degussa India Pvt. Ltd, Mumbai, India). EmptyporcinehardgelatincapsuleNo. 09wasobtainedasagiftsamplefromTORPACInc. (NJ, US). All other chemicalsusedwereofanalyticalgrade.PreparationofcurcuminloadedsolidlipidmicroparticlesCurcumin loaded SLMs were prepared by a modifiedmicroemulsion technique according to the method developedby Gasco.[21]The oil phase, consisting of 110% (w/w) lipidand 1% (w/w) curcumin, was heated to 45C in a china dish.The aqueous phase, consisting of 0.5% (w/w) poloxamer 188andde-ionizedwater,washeatedto35C.Themeltedmassofdrugandlipidwasaddeddrop-wiseandstirredat8000g(Remi Sales, Bangalore, India). The microemulsion obtainedwas sonicated using a Probesonicator (12T-probe; RoopTELSONICUltrasonixLtd, Mumbai, India) for 5minandimmediatelycooledto8Cinarefrigerator for oneweek.Due to the small particle size and the presence of emulsifiers,lipidcrystallizationmaybehighlyretardedandthesamplemayremainasasupercooledmeltforseveralmonths.[22]InitialcharacterizationofsolidlipidmicroparticlesofcurcuminThesamplesofpurecurcuminandSLMsofcurcuminweresubjectedtoinitial characterizationimmediatelyafter theirpreparationasfollows.VesiclesizeandsizedistributionSLMformulationsofcurcuminwerecentrifugedat14000gat 10Cfor 40min. Thesupernatant wasremovedandtheresidue was washedtwicewithdistilledwater andfurthercentrifugedat thesamespeedfor 30min. Theresiduewasredispersed inwater, and the mean particle size and size312 JournalofPharmacyandPharmacology 2009;61:311321distributionwere determinedusinga MalvernMastersizer(Hydro 2000S units). The sample was placed in anautomated dispersion unit and subjected to particle sizeanalysis (Table 1). A collimated laser beamwas madeincident to the suspended sample particles. The intensitysignals of the scattered light were processed into particle sizedistribution.DrugcontentThe amount of drug present in a 10-mg equivalent amount ofSLMs was determined by dissolving the SLMin TritonX-100 (0.1% in methanol) and the dispersion was centrifugedat 14000gfor30min. Further, it wassuitablydilutedwithmethanolandthevisiblespectrumwasmeasuredat425nm.Drugconcentrationwas determinedspectrophotometricallyfromastandardgraphofdrugdevelopedinmethanolusinga Shimadzu-1700 UV-Visible spectrophotometer. DummySLMsofthesamelipidwereusedastheblank. ResultsareshowninTable1.DrugentrapmentefficiencyAll formulations were centrifugedat 14000gat 10Cfor40min. Thesupernatant wasremovedandtheresiduewaswashedtwicewithwaterandfurthercentrifugedatthesamespeedfor 30min. TheresiduewasredispersedwithTritonX-100 (0.1% in methanol) solution to obtain a clear solution.Further, dilutions were made with methanol and theabsorbance was measuredat 425nm. Blankwas preparedby using dummy SLMs of the same lipid and the entrapmentefficiency(showninTable1)wascalculatedasfollows.%Entrapmentefficiency=(DrugpresentinSLM/Totaldrugcontent) 100(1)LyophilizationMicroemulsionscontainingSLMsof curcuminwerefreezedriedusinga 1:1ratio(onweight basis) of sucrose andlipid. Non-encapsulateddrugwasremovedbycentrifugationat 14000g at 10C for 40min. The supernatant was removedandthe residue was washedtwice withwater andfurthercentrifuged at the same speed for 30min. The dispersion wasfrozen to -35C and freeze drying was performed in a freezedrier(Operon, Japan)untiladrypowderwasobtained. Thewhole drying process took 24h with primary drying at-40Cfor16handsecondarydryingat -20Cfor8h.In-vitroreleasestudyIn-vitro drug release studies were performed for purecurcumin and the SLMs of curcumin to select the appropriateformulationfor further analysis. In-vitrodrugreleasefromthe formulations was determined by using dialysis bagsNo. 150 (Hi-Media, Mumbai, India). SLMs of curcuminequivalent to 25mg pure curcumin was transferred to adialysis bag. The bagwas suspendedin a 250-ml beakercontaining 100ml simulated intestinal solution consistingof a pH6.8phosphate buffer andPEG400. The beakerswereplacedonamagneticstirrer(Remi, Bangalore, India),the temperature was maintained at 37 1C throughouttheexperiment, androtatedat 50g. Samplesof dissolutionmedium were collected periodically and replaced with a freshdissolutionmedium. Asampleof 5ml waswithdrawnandfilteredthroughWhatmanfilterpaperNo.1and1mlofthefiltrate was made up to 10ml with methanol in a 10-mlvolumetricflask.Suitabledilutionswerefurthermadewhenrequired. The samples were analysed by a spectophotometricmethod. CumulativepercentagedrugreleasewascalculatedusingPCPDissoVersion2.08software(PoonaCollegeofPharmacy,Pune,India);thetimerequiredfor50%and85%drug release was calculated based on the Korsmeyer andPeppasmodel.[23]EmptySLMswithout thedrugwerealsosubjectedtoanin-vitroreleasestudy. Thesampleobtainedservedasablankforanalysis. In-vitroreleasestudieswerecarriedoutforpurecurcumin.KineticmodellingofdrugreleaseThedissolutionprofileofallthebatcheswasfittedtozero-order, first-order, Higuchi, Hixon-Crowell, Korsmeyer andPeppas and Weibull models to ascertain the kineticmodelling of drug release by using PCP Disso Version2.08software, andthe model withthe highest correlationcoefficientwasconsideredtobethebestmodel.InfraredstudiesThe instrument usedwas a ShimadzuFTIR-8700spectro-photometer. In this study, the potassium bromide discmethodwasemployed. BothpuredrugandSLMformula-tions were subjected to infrared (IR) studies. Apinch ofpowderedSLMsamplewaswell mixedwith20mgof dryTable1 Drugcontent,entrapmentefficiencyandparticlesizeanalysisofsolidlipidmicroparticlesNo. Code Ratio Lipidused Drugcontent(%) Entrapmentefficiency(%) Size of SLMs (mm)1 F1 1:1 Soyalecithin 87.45 1.22 68.12 0.12 290 2.122 F2 1:2 Soyalecithin 82.74 1.11 62.35 0.06 315 1.663 F3 1:3 Soyalecithin 76.45 1.66 58.4 0.18 342 1.784 F4 1:1 Stearicacid 94.67 1.22 79.24 0.21 108 0.255 F5 1:2 Stearicacid 91.34 1.18 73.83 0.28 268 1.236 F6 1:3 Stearicacid 84.56 1.02 67.29 0.11 298 1.667 F7 1:1 Palmiticacid 92.93 1.11 74.58 0.03 131 2.228 F8 1:2 Palmiticacid 87.35 1.33 72.56 0.06 275 1.449 F9 1:3 Palmiticacid 83.26 1.66 66.29 0.11 312 1.54Valesaremeans SD,n=6.SLMs,solidlipidmicroparticles.SolidlipidmicroparticlesofcurcuminforIBD VivekRamshankarYadavetal. 313powdered potassium bromide. The mixture was thencompressedintoatransparent discunder highpressure.[24]The disc was placed in an IRspectrophotometer using asample holder and the spectrum was scanned over awavenumberrangeof4000400cm-1.ScanningelectronmicroscopestudiesThe surface morphology of the layered sample was examinedby using a scanning electron microscope (SEM) (AkashiSX-40, Japan). Asmall amount of powder was manuallydispersedontoacarbontab(doubleadhesivecarboncoatedtape) adhered to an aluminium stub. These sample stubs werecoated with a thin layer (30 A) of gold by employing aPolaron-E3000sputtercoater. Thesampleswereexaminedby SEM and photographed under various magnifications withdirectdatacaptureoftheimagesontoacomputer.DifferentialscanningcalorimetrystudiesDifferential scanning calorimetry (DSC) studies of pure drugand all the selected SLMformulation was performed byusingdifferential scanningcalorimeter(MettlerToledoStarsystem). Samples were weighed (5.008.00mg 0.5mg)and placed in sealed aluminium pans. The coolant was liquidnitrogen.Thesampleswerescannedat10C/minfrom20Cto300C. DSCthermogramsof puredrugandSLMswererecorded.X-raydiffractionstudiesX-raydiffractionpatternsof theselectedSLMformulationwasdeterminedusingadiffractometer(SMART&SAINT,Bruker, 2000) equipped with a rotating target X-ray tube anda wide-angle goniometer. The X-ray source was Ka radiationfroma copper target with graphite monochromator. TheX-ray tube was operated at a potential of 50 kV and a currentof 150 mA. The range (2q) of scans was from 0 to 70 and thescanspeedwas2/minatincrementsof0.02,respectively.FillingandcapsulecoatingforcolonspecificdeliverySelectedSLMformulations of curcuminbasedonparticlesize, drug entrapment, drug content and in-vitro release werechosenfor colon-specific deliverybydevelopingpulsatilecapsules. Around 80mg of formulation was filled intocapsules manually. Capsules were coated with EudragitRS100,RL100,S100andL100andweresoakedfor20minin acetone containing the specified quantities of talc and PEG6000. The various parameters of coating conditions werestandardized, such as dipping time (5s), coating solutionconcentration, temperature(25 1C), dryingtime(5min)in 50Cair. The coating process was carried out until adesiredweight wasgained. Thecapsuleswerecoatedwithtwo coats, the first was with pH-independent polymer(Eudragit RS100andEudragit RL100ina ratioof 6:2)andthesecondwithpH-dependent polymer(Eudragit S100and Eudragit L100 in a ratio of 60:40). PEG 6000 was usedasplasticizerinboththecoatings. Capsuleswereplacedinbetweenthearmsoftheforcepsanddippedintothecoatingsolution for 5s and dried artificially for 5min. This alternatedipping and drying was repeated until the desired weight wasgainedbythe capsule. The filledcapsules were subjectedtofurtherstudyforsite-specificdeliveryofcurcuminintheterminalileum.AngiostaticactivityinthechickembryochorioallantoicmembraneTheangiostaticactivityoftheselectedSLMformulationofcurcumin was demonstrated using chick chorioallantoicmembrane (CAM). Fertilized, domestic five-day-oldchickembryos were purchased from a poultry farm (GKVKVeterinary Sciences, Hebbal, Bangalore, India) and wereincubatedat 37 1C under 60%relative humidity.A small(0.50.6cm2) opening was made with a 4.0-mmscalpelblade, throughtheshell at theendoftheeggdirectlyoverthe embryonic bloodvessels, as previouslydeterminedbycandling, to observe the growth, development and location oftheembryo. Theshell membranewasremovedslowlyandcarefully. SelectedSLMsofcurcuminweighingabout 15 0.2mg containing curcumin (5mg) and pure curcumin5mg/ml (ethanolPBS1:9v/v), respectively, weretrans-ferred directly on to the CAM surface and the shell was closedusingparafilm.After48hofincubationthevasculaturewasobservedusingadissectingmicroscopeat amagnificationof 10andphotographsweretakenusinga4.2megapixelcamera (Cannon cybershot Model V3X). Six eggs were testedin each group and the experiment was performed in triplicateto ensure reproducibility.In-vivostudyAllexperimentalprocedureswereinaccordancewithguide-lines for animal welfare set out bythe Medical ResearchCouncil of India. Animal Ethics Committee approval wasobtained.MaleSpragueDawleyrats, aged 67 weeks(175205g), obtained fromthe Indian Institute of Science(Bangalore, India), wereusedfor in-vivoevaluation. Theyallreceivedthestandardlaboratorydietandwerenotfastedbefore the experiment; they were allowed free access todrinkingwater except duringtheinductionof colitis. Ratswere housed (two per cage) and kept in a regulatedenvironment (temperature, 22 1C; humidity, 50 5%;lightdarkcycle, 12h). Theratswererandomizedintofourgroups (n = 6). Rats in groups 1, 2 and 3 received 5% dextransulfatesodium(DSS)solution(MW40000;SigmaChemi-cals, Gujarat, India) in drinking water for 9 days. Control ratsreceivedplaindrinkingwater.Afterninedays,ratsingroup1receivedSLMsofcurcumin, 100mg/kg(bodyweight)inpulsatile capsules,[25]rats in group 2 receivedpure curcumin(100mg/kg in pulsatile capsules) and rats in group 3 served asaDSScontrol. Theclinical resultsrecordedinDSS-treatedandcontrolratswerebodyweight, stoolconsistency, faecalbleeding, and diarrhoea.Theexperimentwasterminated atthe endof90 daysandall rats were killedbycervical dislocationafter overnightfasting. The colon was slit open longitudinally and cleaned offaecal material with a gentle jet of water. Then the colon wasstored in formalin (10%) solution and subjected to histo-pathologicalexamination.314 JournalofPharmacyandPharmacology 2009;61:311321Assessmentofinflammation:symptomsandinflammatoryscoreClinical assessment of inflammation included weeklymonitoringof bodyweight andgeneral health. Themacro-scopicappearanceofthecolon(inflammatoryscore), basedonthedegreeofinflammationandthepresenceofoedemaorulceration,stoolconsistency(diarrhoeascore)andvisiblefaecalblood,werescoredseparatelyonascaleof03.[26]Thedistalcolon,proximalcolonandileumwerestudied.Each specimen was sliced down the antimesenteric border togive two symmetrical halves, stretched andfixed in 10%bufferedformalinfor 4handthenrinsedin70%alcohol.The formalin-fixed sections were embedded in paraffinblocks and sections were cut, dewaxed and stained withhaematoxylineosin. The severity of inflammation wasassessed first macroscopically. The freshly opened colonwasexaminedunderamagnifyingglassbytwoindependentobservers blindedtotheprocedure. Theextent of mucosaldamagewas assessedusingthemacroscopicinflammatoryscoring(MIS)system.[27]HistologyTissue sections (5 mm) of distal andproximal colonwerestained with haematoxylineosin to address the degree ofinflammationandwithMassonstrichrometovisualizetheconnectivetissue. Thestainedtissuewasanalysedindepen-dentlyandinablindedfashionwithastandardmicroscope(NikonChargecoupledevice(CCD)withdigital displayat40and100magnification). Thepathophysiologyof thetissue was characterized and the lesions were scored asdescribed by Vilaseca et al.[26]By the presence of ulceration,inflammatorycells(neutrophils, macrophages, lymphocytesand plasma cells), signs of oedema, crypt loss, surfaceepithelialcellhyperplasia,gobletcellreductionandsignsofepithelialregenerationwereexamined.[28]StatisticalanalysisAll values are expressed as the mean SD. Statisticalanalysis was performed using analysis of variance fordependentvariables. P