development of topical gel of capsaicin loaded solid lipid...

Development of Topical Gel of Capsaicin Loaded Solid Lipid Nanoparticles (SLNs):

Vol. 2, No. 1, January-June 2011 29

INDIAN JOURNAL OF PHARMACEUTICSVolume 2 • Number 1 • January-June 2011 • pp. 29-41I J P

Corresponding author: E-mail: [email protected]

Development of Topical Gel of Capsaicin Loaded Solid LipidNanoparticles (SLNs): in vitro and in vivo Evaluation

Arvind Sharma and Sandeep AroraChitkara College of Pharmacy, Chitkara University Punjab

Abstract: Purpose: The aim of this study was to evaluate and compare the in vitro and in vivo topical potential ofsolid lipid nanoparticles (SLNs) gel for capsaicin. Methods: SLNs were prepared by high shear homogenizationand ultrasonication using capsaicin. Then SLNs gel was formulated by hydration method using carbopol 940.Results: The mean particle size, width of distribution, zeta potential and entrapment efficiency of SLNs were100 nm, 0.374, -48.36 mV and 63.5 ± 2.9 respectively. The scanning electron microscopy (SEM) image revealedthat capsaicin in SLNs was in amorphous state. Capsaicin release was prolonged to 14 hr with encapsulation(94.3±3.8%). Drug loaded SLNs showed transdermal flux of 30.43±2.5 µg/cm2/h indicating synergistic effectsof encapsulation of capsaicin in SLNs. Permeation of optimized formulation was found to be about 1.6 foldtimes higher than plain drug. Drug loaded SLNs showed 51% remission in inflammation at the end of study(28th day). In the paw edema test, SLN-5 showed the best permeation and effectiveness. The results of thepresent study demonstrated SLN-5 gel formulation possesses great potential for enhanced skin accumulation,prolonging drug release and improving the site specificity of capsaicin Conclusion: SLNs containing capsaicincould be prepared successfully by using an ultrasonic technique, which will not only sustain the release of drugbut also increase permeation of drug to dermal layer of skin. The in vitro and in vivo studies showed that SLN-5 formulation prepared without organic solvent could be a new, alternative dosage form for effective therapy.Key words: Solid lipid nanoparticles (SLNs), capsaicin, topical gel

INTRODUCTIONSolid lipid nanoparticles (SLNs) have emerged asan alternative carrier system to traditional carriers,such as polymeric nanoparticles, emulsion,noisomes and liposomes, and they attract greatattention as a novel colloidal drug carrier for topicaluse [1]. The advantages of the carrier includenegligible skin irritation, controlled release andprotection of active substances [2]. Because they arecomposed of non-irritative and non-toxic lipids,SLNs seem to be well suited for use on inflammedand damaged skin. Moreover, SLNs have distinctocclusive properties due to the formation of anintact film on the skin surface upon drying, whichdecreases transepidermal water loss and favors thedrug penetrating through the stratum corneum [3-4]. Besides the nonspecific occlusion effect, the

enhanced drug penetration might be related withSLNs themselves, the highly specific surface areaof nanometer sized SLNs facilitate the contact ofencapsulated drug with stratum corneum [3]. Thenanometer sized particles can make close contactwith superficial junctions of corneocyte clusters andfurrows between corneocyte islands, which mayfavor accumulation for several hours allowing forsustained drug release [5-6]. Other advantages ofSLNs are reported to be as avoidance of organicsolvents, drug stability, high drug payload, andincorporation of liophilic and hydrophilic drugs [1].SLNs have been used to improve the skin/dermaluptake of several drugs such as triptolide,isotretinoin, podophyllotoxin and prednicarbate [7-11, 12] which supports that SLNs can be employedas the carrier for the topical delivery of capsaicin.

Capsaicin (8-methyl N-vanillyl-6 nonenamide),the active compound of hot peppers of the genus

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Arvind Sharma and Sandeep Arora

30 Indian Journal of Pharmaceutics

Capsicum, exhibits broad bioactivity [13] includingantinociception, antihypertensive and lipid-lowering activities [14-16]. Capsaicin is also usedtopically to treat various diseases such asrheumatoid arthritis, osteoarthritis, diabeticneuropathy and post therapeutic neuralgia [17]. Thepresent work is focused on the preparation,characterization, in vitro percutaneous permeationand skin targeting behaviors capsaicin-loadedSLNs. Topical capsaicin is well absorbed from theskin. Maximal cutaneous concentrations ofcapsaicin are rapidly achieved when capsaicin isapplied topically. These concentrations are greaterwith isopropyl preparations compared withpropylene glycol or mineral oil preparations. [18].In mice, capsaicin is distributed widely to the brain,spinal cord and liver after intravenousadministration. [19]. Capsaicin is a fat soluble,odorless, pungent tasting, off-white solid with amelting point of 62–65 ºC and a molecular weightof 305.4 kDa. As it is not water soluble, alcoholsand other organic solvents are used to solubilizecapsaicin in topical preparations and sprays [20].The drug had a poor water solubility, shortbiological half life and high lipophilicity that madeit an excellent candidate for SLNs encapsulation.Topical application circumvents the hepaticmetabolism and thus is suitable to develop deliverysystems to attain both systemic and local effects forcapsaicin. The present investigation was aimed atformulating topical gel of capsaicin loaded SLNsand explore its potential for topical delivery.

EXPERIMENTAL

MaterialsCapsaicin was obtained ex-gratis from AshianHerbex Ltd., Hyderabad. Glyceryl behnate, sodiumcollate, soyalecithin were bought from Sigmachemicals (USA). All other chemicals and solventswere of analytical grade.

Preparation of Solid Lipid NanoparticlesSLNs were prepared by high shear homogenizationand ultrasonication as in our previous studies.Capsaicin (80 mg) was added to Compritol 888 ATO(4 g) previously melted at 80 °C. This hot lipid phasefurther was dispersed in a surfactant solution (1.5%,w/w), at 8000 rpm, 80 °C for 1 min, using a high-

speed stirrer (Ultra Turrax T8, Alliance AnalyticalInc., California, USA). The surfactants used werepoloxamer and sodium cholate. The obtained pre-emulsion was ultrasonified using a probe sonicator(Vibra cell, Sonics, USA). In order to preventrecrystallization during homogenization,production temperature was kept at least 5 °C abovethe lipid melting point. The obtained nanoemulsion(O/W) was cooled down in an ice bath to form SLNsand finally diluted up to 200 ml with deionizedwater. Nanoparticle dispersions were stored at4 °C. Capsaicin was incorporated into SLNs, andniosomal formulations at saturating concentrationto obtain equal thermodynamic activities. Todetermine the maximum amount of drug that couldbe added, increasing amounts of drug were addedduring preparation of SLNs formulation. Sixformulations of SLNs (SLN-1 to SLN-6) wereprepared using drug concentration in the range of2-12%. Similarly 5 formulations of niosomes (Nio-1 to Nio-5) were prepared containing drug in therange of 2-10%. It was assumed that the presenceof capsaicin crystals would indicate that theformulation was saturated with drug. Therefore, alldrug loaded formulations were examined over aperiod of 14 days using phase contrast microscope(Leica, DMLB, Switzerland) for appearance of drugcrystals. From these results SLN- 5 (drug 10%) andNio-2 (8%) was optimized and used for furtherstudies.

Preparation of Carbopol GelCarbopol 940 (1% w/w) was dispersed in smallquantity of distilled water to prepare an aqueousdispersion which was allowed to hydrate for 4-5hours. Propylene glycol (10% w/w), glycerine (30%w/w) and 0.025% w/w of drug were addedsubsequently to the aqueous dispersion followedby neutralization with 0.3% triethanolamine toadjust pH 6. The final weight of the gel was adjustedto 100 g. The entrapped air bubbles were removedusing vacuum and left the gel overnight.

Preparation of Drug Loaded SLNs GelSLNs gel was prepared using the same formula. Forthis purpose equivalent amount of SLNs suspensioncontaining 0.025 g drug was centrifuged and thepellets obtained were incorporated instead of thedrug.



Preparation of Niosomal GelNiosomes were prepared by cast film method [21].Phosphatidylcholine, span80, cholesterol used forniosomes preparation were dissolved in minimumquantity of chloroform in a round bottom flask.The organic solvent was removed by rotaryevaporator under reduced pressure to form a thinfilm on the wall of flask. Final traces of solventwere removed under vacuum overnight. Thedeposited films were hydrated with water byrotation at 60rev/min for 1 h. To prepare smallervesicles, dispersion was bath sonicated for 15 min.Niosomal gel was prepared as described in abovesection.

Physicochemical Characterization of SLNsThe prepared SLNs were evaluated for size, zetapotential, Scanning Electron Microscopy (SEM),Transmission electron microscopy (TEM),differential scanning calorimetery (DSC),entrapment efficiency (EE%) and stability studiesas in our previous studies.

Measurement of Size and Zeta Potential ofSLNsThe size and zeta potential of SLNs were measuredby photon correlation spectroscopy using aZetasizer 3000 HSA (Malvern, UK). Samples werediluted appropriately with the aqueous phase of theformulation for the measurements, and the pH ofdiluted samples ranged from 6.9 to 7.2. Zetapotential measurements were done at 25ºC and thefield strength was 20 V/cm on a large boremeasuring cell (4 mm). Samples were diluted withbi-distilled water.

Transmission Electron Microscopy (TEM)TEM (JEM-100CXII, Japan) is a method of probingthe microstructure of rather delicate systems suchas micelles, liquid crystalline phases, vesicles,emulsions and also nanoparticles [13]. Withoutsurfactants the lyophilized SLNs were disperseddirectly into tristilled water. Then copper gridcoated with carbon film was put into the abovesolution several times. After being stained by 2%phosphotungstic acid (PTA) solution and driedunder room temperature, the sample was ready forthe TEM investigation.

Scanning Electron Microscopy (SEM)The morphology of the lyophilized empty andcapsiacin loaded SLNs were determined using SEM(Model LEO 1455VP, LEO, Cambridge, England).The lyophilized SLNs were evenly distributed ontoa conductive tab on a stud and then sputter coatedwith gold in a cathodic evaporator.

Stability StudiesThe prepared formulations were tested for stabilityon storing them in amber colored glass vials at 4 °Cand 25 °C for 30 days. On 7th,14th and 30th days, theywere evaluated for vesicle size, zeta potential, EE%and shape.

Entrapment Efficiency (EE %)2ml of nanosuspension solution of certainconcentration was prepared and added to SephadexG-25 column. Then methanol aqueous solution waspassed through the column. The collected washingout liquid was measured by the UV-detection(Unico, UV-2102PC, USA) at 310 nm. The amountof drug that was not incorporated into the SLNscould be obtained by the UV-detection absorptionpercent. EE% could be achieved by the followingequation.

% 100%initial drug freedrug

initial drug

W WEE

W−

= × (1)

Differential Scanning Colourimetry (DSC)Thermal behavior of capsaicin loaded SLNs wasanalyzed using DSC-7 (Perkin-Elmer, USA).Approximately 10mg of sample was placed inaluminum crimp cells and heated at the scanningrate of 10 °C/min from 30 to 400 °C in a nitrogenatmosphere. Aluminum oxide was used as thestandard reference material to calibrate thetemperature and energy scale of the DSCinstrument [22-23].

HPLC Analysis of CapsaicinThe drug content of capsaicin was analyzed by aHPLC system consisting of a Hitachi L-7100 HPLCpump, a Hitachi L-7200 sample processor and aHitachi L-7480 fluorescence detector. A 25 cm long,4 mm inner diameter C18 column (LichroCart 250-



4, Merck) was used. The mobile phase for capsaicinwas 55% pH 4 citrate–phosphate buffer and 45%acetonitrile at a flow rate of 1.0 ml/min. The columneffluent was passed through the fluorescencedetector set at an excitation wavelength of 280 nmand an emission wavelength of 310 nm. Thedetection limit of capsaicin was 20ng/ml.

In -Vitro Drug Release Studies using CellophaneMembraneThe in vitro release of capsaicin from differentcapsaicin formulations was studied using locallyfabricated Keshry Chein diffusion cell through thecellophane membrane (molecular weight cut off12000 to 140000). Pure capsaicin was dissolved in1:1 (v/v) ethanol-pH 7.4 citrate–phosphate buffersat a concentration of 20 mg/ml and used as control.The prepared formulation SLN-5 gel, SLN-5(suspension), NIO-2 gel, plain capsaicin gel,Zostrix®(marketed formulation) cream with 0.075%capsaicin (the same concentration of capsaicin as20 mg/ml pure drug solution) was transferred to adialysis bag (size cut off = 2.5 nm) immediately. Thedialysis bag was placed in a 50 ml-beaker containing1:1 (v/v) ethanol-pH 7.4 citrate–phosphate buffer.The outer phase was stirred continuously. Atpredetermined time intervals sample waswithdrawn and replenished with same amount ofreceptor fluid. The drug content in outer phase wasanalyzed by using HPLC as described in abovesection.

In Vitro Skin Permeation StudyThe in vitro permeation experiments weredetermined by using Franz diffusion cell. The dorsalskin of wistar rat (8–9 weeks old) was mounted onthe receptor compartment with the SC-side facingupwards into the donor compartment and thedermal side facing downwards into the receptor. A10 ml of 1:1 (v/v) ethanol-pH 7.4 citrate–phosphatebuffer was used as the receptor medium. The donorcompartment of the cell was filled with 2ml of drugsolution, SLN-5(suspension), 1g SLN-5 Gel, NIO-2Gel, Zostrix® (marketed formulation) with 0.075%capsaicin and plain capsaicin gel of formulationvehicle containing the test drug. The top of donorwas covered with paraffin paper. The availablediffusion area of cell was 0.785 cm2. The receptorphase of cell was sustained at 37°C and stirred by a

magnetic stirrer at 600 rpm. At appropriateintervals, 200 µl aliquots of the receptor mediumwere withdrawn and immediately replaced by anequal volume of fresh receptor solution. All studieswere performed in triplicate. The samplewas analyzed by HPLC method described earlier[24].

Determination of Drug Content in Epidermis andDermisThe drug amount uptaken in epidermis anddermis was determined at 2, 6, and 12 h aftertreating with the same method described in abovesection. The excessive SLN-5 gel, NIO-2, Zostrix®dispersion or cream was removed and the skinsamples with a penetration area of 3.8cm2 wererinsed with alcohol and water to eliminate thecapsaicin remained on the surface and then gentlydried with a cotton wool [25]. Heat-separatedmembrane separation was carried out based on thereported method [26]. The skin samples wereimmersed in distilled water at (60±1) °C for 2 minand then the epidermis was removed from thedermis using a dull scalpel blade [27-28]. Theepidermis sample or dermis cut into small pieceswas soaked in 5ml of 0.4% perchloric acid solutionfor 24 h and subjected to homogenization with aDiax 900 homogenizer (Heidolph Electro, Kelhaim,Germany) for 1 min and ultrasonication for 60minin an ultrasound bath (CX-250, Peking MedicalEquipments Ltd., Peking, China), followed bycentrifugal separation (0412-1 Shanghai SurgicalInstruments Co. Ltd., Shanghai, China). The drugextracted from epidermis or dermis was analyzedby HPLC [29-30].

LyophilizationThe SLNs were lyophilized using a programmablefreeze-dryer (Shin PVTFD10R, Shinil Lab, Korea).Cryoprotectant was added to the SLN dispersionbefore freezing. Trehalose, mannitol, sucrose andfructose were screened at the level of 5% and 10%w/v for their cryoprotectant efficiency. Slowfreezing was carried out on the shelves in the freezedryer (shelf temperature “40 °C). The samples werelyophilized for 24 h from”40 °C to 25 °C at anincreasing rate of 5 °C/h. Lyophilized productswere reconstituted by sonication (2 min, 500W,Power Sonic 510, Korea).



In Vivo Studies in Rats

Arthritis Induction in RatsArthritis was induced in the right knee joint of eachrat. On two occasions, 1 week apart, rats wereinjected subcutaneously with an emulsion of equalvolumes of methylated bovine serum albumin (m-BSA) and Freunds Complete Adjvant (FCA). Doseof this emulsion was 50 µl per rat per immunizationcycle. Fourteen days after the second immunization,the rats were given two intra-articular injection ofm-BSA (0.5 mg in 50 µl saline) into the right knee.One day after the induction dose, the rats developedinflammation in the right knee which peaked after3 days. The development of arthritis was assessedby histopathological results [31].

Treatment of Antigen-adjuvant Induced ArthritisThe inflammation was assumed to be 100% onseventh day. Thus seven days after the intra-articular dose of m-BSA; the rats were divided intothree groups (n=6). One group of rats was rubbedwith plain capsaicin gel. Second group of rats wasrubbed with capsaicin loaded SLNs gel onto theright knee (inflamed) and gel without capsaicin wasrubbed onto the left knee joint. The last group waskept as positive control in which only gel withoutcapsaicin was rubbed onto the right knee.

Measurement of Joint Knee DiameterThe development of arthritis was monitored atregular intervals by measuring the knee jointdiameter, with the knee flexed to the same extent,using digital micrometer. The percentage averageinflammation was measured by using formula.

% Average Inflammation =.

. 7 7Average joint dia of right knee Average joint diaof left

Avg joint dia of right kneeday Avg joint diaof left kneeday−

−

Histopathology Studies4 weeks after the m-BSA challenge, representativerats from first two groups were killed. The kneejoints were removed into and fixed in formalinbuffer (15%) one week before histologicalprocessing. The joints were decalcified by dippingin 7% nitric acid for 5 days and were then embeddedin paraffin wax (high m.p. 58ºC), then sectioned at5 µm and stained with haematoxylin/saline. The

sections were evaluated under light microscopy forproliferation of fibroblasts, cartilage damage, andpresence of inflammatory infiltrate and hyperplasiaof synovial membrane.

Anti-inflammatory Effect TestThe formulations of capsaicin were evaluated fortheir anti-inflammatory activity on a carrageenan-induced rat paw edema model [32-33] Inflammationwas produced in the rats (Male, Wistar, weighing200-250 g) using 100 µL of 1% carrageenan (wt/vol)in saline. This was injected into the plantar surfaceof the rats’ left hind paw. To evaluate the topicalanti-inflammatory activity of the formulations SLN-5 gel, plain capsaicin gel , NIO-2 gel, Zostrix® 4groups of animals (n = 3) with carrageenan-inducedpaw edema were examined. Thirty minutes later,100 µg of SLN-5gel, plain capsaicin gel, NIO-2gel,Zostrix® was applied topically on the edematouspaw. A fifth group of rats was used as a control(gel without capsaicin). The increase in pawthickness was measured with the help of digitalmicrometer before (time 0) and 1, 2, 3, 4, 5, and 6hours after carrageenan administration. Thepercentage of paw thickness increase from time 0was calculated.

Skin Irritation EvaluationIrritation evoked by experimental formulations onrat skin was microscopically judged after the endof experiments of in vivo percutaneous absorption.The site of application of each formula on the skinwas excised and fixed in 10% neutral carbonated-buffered formalin for at least 24 h before routineprocessing. Each section was rinsed with runningwater, dehydrated using a graded series of ethanolsolution and embedded in paraffin wax, and thenfrozen at –20°C prior to sectioning. The tissueswere cut into small sections (6 µm) and stainedwith hematoxylin and eosin for histologicalevaluation. Sections were examined by lightmicroscopy [34].

Statistical AnalysisResults were reported as arithmetic meanvalues±standard deviation (¯x ± S.D.). Statisticallysignificant differences were determined usingStudent’s t-test with P < 0.05 as a minimal level ofsignificance.



RESULTS AND DISCUSSION

Formulation OptimizationA sufficient high-energy input was necessary tobreak down the droplets into the nanometer range[35]. Besides the production parameters, lipidmatrix surfactant blend, viscosity of lipid andaqueous phase infiuenced the outcome of theprocedure [36]. In the formulation, glycerolbehenate was used as a lipid matrix as well assurfactant. It was suggested that this surface activepartial glyceride facilitated emulsification andformed more rigid surfactant films and thereforeimproved the long-term stability of SLNs. Foroptimization of drug amount the formulations wereprepared using varying concentrations of drug(2-12 w/w %) and these formulations wereevaluated for particle size, zeta potential,morphological characterization and EE%. Manydifferent drugs had already been incorporated inSLNs. According to professor M. Muller theprerequisite to obtain a sufficient loading capacitywas a sufficiently high solubility of the drug in thelipid melt [37]. Relative higher drug EE% was oneof the major advantages of SLNs. The maximumconcentration of capsaicin that could beincorporated into the SLN-5 and NIO-2formulations was found to be 10.0 mg and 8.0 mgwith EE% of 63.5 ± 2.9% and 54.5± 4.8% arespectively; on further increasing the amount ofdrug, drug crystals precipitated probably due to thesaturation of the formulation. It should be stressedthat these saturation concentrations are the totalconcentrations of capsaicin that could beincorporated in the formulation and not theentrapment value [38-39]. After one-monthrefrigerated storage the value only showed littledecrease. Therefore, it was revealed that SLNsproduced by this modified production methodcould achieve higher drug incorporation forlipophilic drugs like capsaicin. Drug loading wasfound to be 5.12%.The mean particle size and widthof distribution were 100 nm and 0.374 respectivelymeasured by LD (Mastersizer 2000) [40], shown in(Figure 1), thus the drug loading SLNs showed anarrow distribution width and considerable smallparticle size. An outstanding feature ofnanoparticles was the increase in saturationsolubility and consequently an increase in thedissolution velocity of the compounds. According

to the Kelvin and Ostwald-Freundlich [41]equation, for small particles especially thenanometer range, the saturation solubility could beincreased significantly. Both the increase ofsaturation solubility and the enlargement of surfacearea contributed to the improvement of dissolutionvelocity by the Noyes- whitney equation [42]. Drugsin the form of nanoparticles (nanocrystals) had beenreported to possess a full range of positive effectsand the data on increase in bioavailability was quiteimpressive [43]. One aim of this experiment was toimprove the bioavailability of the poorly solublelipophilic drug by transforming it intonanoparticles. From the above discussions, it shouldbe presumed that if the nanometer range particlescould be obtained, the increase in bioavailabilitywill became available. The zeta potential of drugloaded SLN-5 was -48.36 mV (Figure 2) Zetapotential can make a prediction about the stabilityof colloid dispersions. A high zeta potential (>|30|mV) can provide an electric repulsion to avoid theaggregation of particles [44]. The incorporation ofcapsaicin into SLNs found to have no influence onthe zeta potentials.

Figure 1: Average Particle Size and Particle Size Distributionof SLNs Loading Model Drug

Figure 2: Zeta Potential of the Ultrafiltrated Dispersions ofSLNs



Stability StudiesVesicular formulations were stored at 4 °C and at25 °C for 30 days. The stability testing data indicatedthat vesicular formulations stored at 4 °C were morestable than those stored at 25 °C. The zeta potentialof different vesicular formulations stored at 25 °Cwere very unstable whereas the zeta potentialvalues of formulations stored at 4 °C were morestable therefore the storage temperature of 4 °C isbetter than the room temperature in order tomaintain favorable zeta potentials.

TEM and SEM InvestigationThe TEM image (Figures 3a) shows the shape ofthe nanoparticles entrapping with the model drug.It was evident that the particles investigatedrevealed round and homogeneous shading, theparticle size ranging approximately from 100 to 110nm. SEM was used to examine the submicron sizeand morphology of the SLNs. The SEM imagerevealed that the SLNs were spherical in shape andhomogeneously distributed around 100–200nm indiameter, and that the incorporation of capsaicindid not seem to cause morphological changes.

DSC AnalysisDSC was a tool to investigate the melting andrecrystallization behavior of crystalline materialslike SLNs (Figure 4) shows an overview of themelting process of bulk matrix, lyophilized drug-free placebo and drug loading powder. The heatfiow is less than 1 mW. Thermogram of thelyophilized drug loaded SLNs did not show themelting peak of crystalline capsaicin, whichindicates that capsaicin in SLNs, was in amorphousstate. For this study, capsaicin and lipid were firstdissolved in ethyl acetate. Subsequently, ethylacetate was evaporated. Therefore capsaicin wasdispersed in the lipid homogeneously. There aresome similar results revealing that drug in SLNwere in amorphous state [45-46]. The bulk glycerylbehnate melted at 82.28°C with almost the samemelting point at 83°C.

Figure 3: (a) TEM of Capsaicin Loaded SLNs (b) SLN Imageswithout Drug (A) Obtained by Scanning ElectronMicroscopy

Figure 4: Differential Scanning Calorimetric Heating Curvesof (a) glyceryl behnate (b) blank SLN; (c) SLN withCapsaicin Added and Dissolved Inside at 80 °C

In-vitro Drug Release StudiesThe release rate from capsaicin loaded SLNs is animportant parameter since a sustained release isnecessary in order to decrease the dose dependentside effects of capsaicin and improve its therapeuticindex. The drug entrapment and release rates wereevaluated by dialysis method usingspectrophotometric estimation [47]. Capsaicinloaded SLNs were stable in1:1 (v/v) ethanol-pH 7.4citrate–phosphate buffer (used as the receptormedium) and a slow release of drug from thecomplex was found. (Figure 5) shows thecomparative % drug release from different drugloaded SLN-5 gel, SLN-5 suspension, and NIO-2gelformulations in comparison with plain capsaicin



gel, Cream Zostrix® and drug solution. Significantprolongation of capsaicin release across the dialysismembrane was achieved with the capsaicin loadedSLNs in comparison with plain drug. Thecumulative amount of drug released in 1 hr fromthe SLN-5 gel formulation was 6.5 ± 0.3% comparedwith 8.89±1.2%, 14.5±0.8%, 78.75±2.8%, 45.78±0.8%and 95.8 ± 4.1%, from SLN-5(suspension), NIO-2gel,plain capsaicin gel, Cream Zostrix® the control drugsolution respectively. Capsaicin release wasprolonged to 14 hr with encapsulation (94.3±3.8%).Drug release from the capsaicin was steady andslow and decreased as a function of time.

In Vitro Skin Permeation StudiesSkin permeation studies were carried out usingabdomen rat skin. (Figure 6) shows % amount ofcapsaicin permeated across rat skin as a function oftime from different formulations SLN-5gel, SLN-5

suspension and NIO-2gel, compared with marketedformulation (Zostrix®) and drug solution. The invitro skin permeation of drug is mainly assessed byits flux, Jss(µg/cm2/h). This was calculated from theslope of the linear steady portion of % cumulativeamount released versus time (h) plot as shown in(figure 6). Another important parameter is diffusionlag time (LT) for the drug to reach the receptorcompartment. Diffusional lag time is importantparameter, which is necessary for calculatingdiffusivity, partition coefficient and permeabilitycoefficient. All these skin permeation parametersfor capsaicin have been calculated and reported in(Table 1). The value of transdermal flux for differentformulations was observed between 22.52±0.6 to50.20±2.3 µg/cm2/h. This is about 1.2 to 2.5 timeshigher than that obtained from drug solution(18.15±0.6) and two-fold higher than Ceram(marketed formulation) and liposomal formulation.SLN-5 showed significant increase in transdermalflux to 30.43±2.5 µg/cm2/h indicating synergisticeffects of encapsulation of capsaicin in SLNs.

Figure 5: In Vitro Drug Release of Different Capsaicin LoadedFormulation in Comparison to Plain Capsaicin Gel,Marketed Cream (Zostrix®) and Drug SolutionAcross the Artificial Dialysis Membrane (MWCO12000-14000)

Figure 6: %Amount of Capsaicin Permeated Across Rat Skinfrom Different Capsaicin Loaded Formulations inComparison to Drug Formulation

Table 1Permeation Parameters of Different Capsaicin Formulations across Rat Skin

Formulation code Jssa(µg/cm2/h) LTb(h) Dc(cm2/h) Pd(cm/h) R2e Def (µg) ERD ERP

SLN-5(gel) 30.43±1.2 2.1 9.891 15.22 0.978 307.56±1.5 12.4 1.59SLN-5 (suspension) 28.19±2.1 1.71 .3891 14.09 0.972 198.37±2.7 4.34 1.47NIO-2 26.15±0.6 .82 .3773 9.57 0.951 45.74±1.1 2.23 1.21Cream(Zostrix®) 24.52±2.3 2.26 .2952 11.76 0.953 158.92±2.8 3.47 1.23Plain capsaicin gel 20.15±0.6 .82 .2673 9.57 0.951 45.74±1.1 - -Drug Solution 18.15±0.6 1.82 .3673 9.57 0.951 45.74±1.1 - -

Jssa = Transdermal flux; LTb = Lag time; Dc = Diffusion coefficient;Pd = Permeability coefficient; R2e = Correlation coefficient; Def = Amt. of drug deposited in skin; ERD = Enhancement ratio ofdrug deposition in skin.ERP = Enhancement ratio of drug permeation in skinSLN-5= Nanosuspension of solid lipid nanoparticles



Skin Uptake BehaviorsThere was no significant difference in cumulativeamounts of capsaicin in epidermis between thecream (Zostrix®) marketed formulation, NIO-2geland SLN-5gel as shown in Figure 7(a) (p > 0.05),while the SLN-5 gel significantly increased thecumulative uptake of capsaicin in dermis as shownin Figure 7(b) (p < 0.05). The amount of capsaicinpenetrated into dermis from SLN-5gel at 12 h wasnearly 2 fold that of commercial cream. The smallsize and close interaction between SLNs and thestratum corneum are the possible reasons whySLNs can increase the drug amount penetrating intothe viable skin. The increase amount of drug in thedermis is also related to the occlusion properties.Following the water evaporation from the SLNssuspension applied to the skin surface, an adhesivelayer occluding the skin surface is formed. Then thehydration of stratum corneum can increase, whichcan facilitate drug penetration into deeper skinstrata by reducing corneocyte packing andwidening the inter-coenocytes gaps [48-49]

In Vivo Studies in Antigen Adjuvant InducedArthritis Rat

Effect on Joint DiameterThree days after intra-articular administration of m-BSA antigen, the first sign of inflammation in theform of joint swelling became apparent. Anothersignificant feature was reduced mobility in rightlimb due to the onset of arthritis. Seven days laterafter antigen induction, the disease reachedmaximum severity (assumed to be 100%). Thetreatment was started on eighth day (wellestablishes) in all the groups. One group was treatedwith plain capsaicin, another with SLN-5 and lastwith gel containing no capsaicin. The jointdiameters were measured using digital micrometer.A significant reduction in joint inflammation in ratstreated with SLN-5 as compared to treated withplain gel and gel without drug was observed. Thepercentage inflammation rate in all the groups fromday 7 (time of m-BSA challenge) to day 28 is shownin (Figure 8). The treatment with plain capsaicingel did not show much reduction in jointinflammation within the study period of sevendays, whereas the treatment with drug loaded SLNsshowed 25% remission in inflammation rate afterthe 15 days and 51% at the end of study.

Histopathology ResultsHistological view of saggital sections of the kneejoint samples taken from normal rats, rats treatedwith SLN-5 gel and rats treated with plain capsaicingel for both the limbs shown in (Figure 9). The kneejoint section of normal rats (without induction ofFigure 7: (a) The Amount of Capsaicin in Epidermis after 2, 6,

and 12 hr Respectively

Figure 7: (b) The Amount of Capsaicin in Dermis after 2, 6,and 12 hr, Respectively

Figure 8: Comparison of Inflammation Behavior betweenThree Groups of Antigen-Adjuvant Induced Arthritisafter Treatment



arthritis) shows normal surface of joint cartilage andadequate joint space, whereas in case of rats withinduced arthritis treated with plain SLNs gelwithout capsaicin, erosion of cartilage, papillaryinfoldings of synovial membrane and irregularouter margin of cortex of the bone and alsoinflammatory infiltrate was seen with proliferationof fibroblast around the cortex of the bone. The kneejoint sections of rats treated with drug loaded SLNsgel showed minimal papillary foldings, adequatejoint space and shows possible recovery of jointcartilage as the surface of bone from both the sides.However the results obtained from the studiesshows that certain damage could not be rectified asthe treatment was started at the stage of wellestablished arthritis. The results are in consonantlywith early findings [49].

Anti-inflammatory ActivityIt has been reported that carrageenan-inducededema can be divided into 2 phases. The first phaseoccurs throughout the first hour after carrageenaninjection. It derives from the release of cytoplasmicenzymes and serotonin from mast cells and theincrease of prostaglandin in the inflammatory area.The second phase occurs 3 to 5 hours aftercarrageenan injection. In this phase, themacrophages in carrageen and insulted dermaltissue release interleukin-1 to induce accumulationof polymorphic nuclear cells into the inflammatoryarea. This then releases the lysosomal enzymes andactive oxygen to destroy connective tissues andinduce paw swelling. In this study, the progress of

the paw edema test was compatible with that foundin the literature. Induction of acute inflammationin control rats resulted in a prominent increase inpaw thickness throughout the first hour afterintraplantar injection of carrageenan and reacheda peak of inflammation after 4 hours (Figure). Theresults of the paw edema test were evaluated usingrepeated-measures ANOVA, and the interactionwas found to be significant between Factor 1 andFactor 2. This means that the paw edema differencesamong formulations for each hour were not similar.The difference in the increase of paw thicknessbetween hours was significant. Because ofinteraction between factor 1 and factor 2,formulations were compared for each hour using1-way ANOVA. Homogeneity of variance,analyzed using the Levene test, was observed at allhours. The Duncan test, used as post hoc analysis,found that when all formulations were comparedwith the control, a significant difference was found.The differences in paw thickness increase amongformulations against time are shown in (Figure 9).In vitro and in vivo studies were compared.According to in vitro studies, SLN-5 hadbetter penetration than NIO-2 gel, cream (Zostrix®),and plain capsaicin gel. The result of betterpermeation of SLN-5 was also supported by in vivostudies.

Skin Irritation Evaluation by PathologicBiopsyTo evaluate the influence of ingredients on the skinirritation, the skin was pathologically investigatedafter application of the capsaicin gels. Figure 10represents microscopic photographs of rat skin at10 h after with or without treatment with SLN-5gel and the commercially available product,respectively. After 10 hr of formulationapplication, the treatment side was observed andskin scores awarded by visual observation. Thetotal irritation scores (TSI) of skin are listed inTable 2. The results revealed that the damagedegree of skin (TSI) tended to increase slightlywith the increases in penetration rate (PR). Therewas no significant difference (P±0.05) in TISbetween the SLN-5, the penetration of whichwas highest, and the commercial product,indicating that the damage degree of SLN-5 wasacceptable.

Figure 9: Percentage Increase of Paw Thickness after SubPlantar Injection ofcarrageenan. Values are Meansof 3 Determinations ± SD.



Figure 10: Microscopic Photos of Rat Skin at 10 h after with andwithout Treatment with SLN-5 gel and theCommercial Available Product. A: without treatment;B: treated with SLN-5 gel; C: treated with theCommercial Available Product

Table 2Skin Irritation Scores Following Application of Different Formulations

Rabbit No. Plain drug solution SLN-5(Gel) Cream(Zostrix®) NIO-2(Gel)Erythema Edema Erythema Edema Erythema Edema Erythema Edema

1 0 0 0 0 0 0 0 02 0 0 1 0 0 0 1 03 0 0 1 0 1 0 1 14 0 0 0 0 1 0 1 05 1 0 1 0 0 0 1 06 1 0 0 0 1 0 1 1Ave. 0.83 0.33 0.50 0 0.5 0 0.66 0.16Scores are as defined 0 – no erythema, 1 – very slight erythema, 2 – well defined erythema, 3 – moderate to severeerythema. Similarly defined as edema

CONCLUSIONCapsaicin loaded SLNs can be preparedsuccessfully by using modified high shearhomogenization and ultrasound techniques asproved in previous studies. The surface structureof the formulation was spherical and rough. Theencapsulation efficiencies were over 60% and themean size was in the range of 100-150nm. Therelease rate of SLN-5 was much slower than thatthan those of NIO-2, cream (Zostrix®). The releasepattern of the formulation was found to be of thenon- fickian. Skin irritation studies indicatedinsignificant damage by the prepared formulationas compared to commercial formulation. Accordingto both in vitro and in vivo studies, SLN-5 showedthe best permeation and effectiveness. Our studiesprovided evidence that SLNs developed werevaluable as topical delivery carrier to enhance thepenetration of lipophillic drug capsaicin.

ACKNOWLEDGEMENTSThe authors are grateful to the Department ofpharmaceutical sciences and drug research Punjabiuniversity Patiala for carrying out research work. We arethankful SAIF Punjab University Chandigarh and NationalInstitute of Pharmaceutical Sciences and research (NIPER)INDIA for carrying out SEM, TEM and DSC studies.

REFERENCES[1] Fang J. Y., Fang C. L., Liu C. H., Su Y. H. Lipid

Nanoparticles as Vehicles for Topicalpsoralen Delivery:Solid Lipid Nanoparticles (SLN) versus Nanostructuredlipidcarriers (NLC). Eur. J. Pharm Biopharm 2008; 70:633–640.

[2] Mei Z., Chen H., Wang T., Yang Y., Yang X. SolidLipid Nanoparticle and Microemulsion for TopicalDelivery of Triptolide. Eur. J. Pharm Biopharm 2003; 56:189–196.



[3] Jenning V., Gysler A., Schäfer-Korting M., Gohla S.Vitamin A-loaded Solid lipid Nanoparticles for TopicalUse: Occlusive Properties and Drug Targeting to theUpper Skin. Eur J Pharm Biopharm 2000a; 49: 211–218.

[4] Wissing S. A., Müller R. H. The Influence of Solid LipidNanoparticles on Skin Hydration and Viscoelasticity-in Vivo Study. Eur. J. Pharm Biopharm 2003; 56: 67–72.

[5] Cevc G. Lipid Vesicles and other Colloids as DrugCarriers on the Skin. Adv Drug Deliv. Rev. 2004; 56: 675–711.

[6] Schäfer-Korting M., Mehnert W., Korting H. LipidNanoparticles for Improved Topical Application ofDrugs for Skin Diseases. Adv Drug Deliv Rev. 2007; 59:427–443.

[7] Chen H., Chang X., Du D., Liu W., Liu J., Weng T., YangY., Xu H., Yang X. Podophyllotoxin-loaded Solid LipidNanoparticles for Epidermal Targeting. J. ControlRelease 2006; 110: 296–306.

[8] Maia C. S., Mehnert W., Schäfer-Korting M. Solid LipidNanoparticles as Drug Carriers for TopicalGlucocorticoids. Int. J. Pharm. 2000; 196: 165–167.

[9] Maia C. S., Mehnert W., Schaller M., Korting H. C.,Gysler A., Haberland A., Schäfer-Korting M. DrugTargeting by Solid Lipid Nanoparticles for Dermal Use.J. Drug Target 2002; 10: 489–495.

[10] Liu J., Gong T., Wang C., Zhong Z., Zhang Z. Solid LipidNanoparticles Loadedwith Insulin by Sodium Cholate-phosphatidylcholine-based mixed Micelles:Vpreparation and Characterization. Int. J. Pharm. 2007a;340: 153–162.

[11] Mei Z., Chen H., Wang T., Yang Y., Yang X. Solid LipidNanoparticle and Microemulsion for Topical Deliveryof Triptolide. Eur. J. Pharm Biopharm 2003; 56: 189–196.

[12] Sivaramakrishnan R., Nakamura C., Mehnert W.,Korting H. C., Kramer K. D., Schäfer-Korting M.Glucocorticoid Entrapmentinto Lipid Carriers-characterization by Parelectric Spectroscopy andInfluence on Dermal Uptake. J. Control Release 2004; 97:493–502.

[13] Tominack R. L., Spyker D. A. Capsaicin and Capsaicin.A Review: Case Report of the use of Hot Preppers inChild Abuse. ClinToxicol 1987; 25: 591–601.

[14] Hayes A. G., Skingler M., Tyers M. B. Effects of SingleDose of Capsaicin on Nociceptive Thresholds in theRodents. Neuropharmacology 1981; 20: 505–511.

[15] Wang J. P., Hsu M. F., Teng C. M. Antiplatelet Effect ofCapsaicin. Thromb Res. 1984; 36: 497–507.

[16] Clozel J. P., Roberts A. M., Hoffman JI. E, Coleridge H.M., Coleridge JCG. Vagal Chemoreflex CoronaryVasodilation Evoked by Stimulating Pulmonary C-Fibers Indogs. Circ Res. 1985; 57: 450–460.

[17] Fusco M. M., Giacovazzo M. Peppers and Pain: ThePromise of Capsaicin. Drugs 1997; 53: 909–914.

[18] Pershing L. K., Reilly C. A., Corlett J. L., Crouch D. J.Effects of Vehicle on the Uptake and EliminationKinetics of Capsaicinoids in Human Skin in Vivo. ToxicolAppl. Pharmacol. 2004; 200(1): 73–81.

[19] Rains C., Bryson H. M. Topical Capsaicin. A Review ofits Pharmacological Propertiesband TherapeuticPotential in Post-herpetic Neuralgia, DiabeticNeuropathy and Osteoarthritis. Drugs Aging 1995; 7(4):317–328.

[20] Kasting G. B. Kinetics of Finite Dose Absorptionthrough skin 1. Vanillylnonanamide. J. Pharm Sci. 2001;90(2): 202–212.

[21] Fang J. Y., Lin H. H., Hus L. R., Tsai Y. H.Characterization and Stability of Various Liposome-encapsulated Enoxacin Formulation. Chem Pharm. Bull.1997; 45: 1504-1509.

[22] Hou D. Z., Xie C. S., Huang K. J., Zhu C. H. TheProduction and Characteristicsof Solid LipidNanoparticles (SLNs). Biomaterials 2003; 24: 1781–1785.

[23] Sun W., Zhang N., Li A., Zou W., XuW. Preparationand Evaluation of N3-Otoluyl- fluorouracil-loadedliposomes. Int. J. Pharm. 2008; 353: 243–250.

[24] Fang J. Y., Wu P. C., Huang Y. B. Tsai Y. H. In VitroPermeation Study of Capsaicin and its SyntheticDerivatives from Ointment bases using Various SkinTypes. Int. J. Pharm. 1995; 126: 119–128.

[25] Hassonville S. H., Chiap P., Liégeois J., Evrard B.,Delattre L., Crommen J., Piel G., Hubert P.Development and Validation of a High-performanceLiquid Chromatographicmethod for the Determinationof Cyproterone Acetate in Human Skin. J. Pharm BiomedAnal. 2004; 36: 133–143.

[26] Christophers E., Kligman A. M. Preparation of IsolatedSheets of Human Stratumcorneum. Arch Dermatol 1963;88: 702–705.

[27] Puglia C., Bonina F., Trapani G., Franco M., Ricci M.Evaluation of in Vitro Percutaneous Absorption ofLorazepam and Clonazepam from Hydro-alcoholic gelformulations. Int. J. Pharm. 2001; 228; 79–87.

[28] Puglia C., Blasi P., Rizza L., Schoubben A., Bonina F.,Rossi C., Ricci M. Lipid Nanoparticles for ProlongedTopical Delivery: an in vitro and in vivo investigation.Int. J. Pharm. 2008; 357: 295–304.

[29] Bianca B., Quintela MAL, Charro MAD, Fadda M. B.,Fadda A. M., Mendez J. B. Microemulsions for TopicalDelivery of 8-methoxsalen. J. Control Release 2000; 69:209–218.

[30] Matsumo II, Yudoh K, Monta I. Apoptosis is a NovelTherapeutic Strategy for RA: Investigations using anExperimental Arthritis Animal Model. J. Rheumatol1999; 34: 215-226.

[31] Sjostrom B., Bergenstahl B.. Preparation of SubmicronDrug Particles in lecithin-stabilized o/w emulsions. I.



Model Studies of the Precipitation of CholesterylAcetate. Int. J. Pharm. 1992; 88: 53–62.

[32] Winter C. A., Risley E. A., Nuss G. V. Carrageenan-induced Edema in Hind Paw of the Rat as an Assay forAnti-inflammatory Drugs. Proc. Soc. Exp. Biol. Med.1962; 111: 544-547.

[33] Escribano E., Calpena A. C., Queralt J., Obach R.,Doménech J. Assessment of Diclofenac Permeation withDifferent Formulations: Anti-inflammatory Study of aSelected Formula. Eur. J. Pharm Sci. 2003; 19: 203-210.

[34] Muller R. H., Schwarz C., Mehnert W., Lucks J. S.Production of Solid Lipid Nanoparticles for ControlledDrug Delivery. Proc Int Symp Control Rel Bioact Mater1993; 20: 480–487.

[35] Liedtke S., Wissing S., Muller R. H., Mader K. Influenceof High Pressure Homogenization Equipment onNanodispersions Characteristics. Int. J. Pharm 2000; 196:183–185.

[36] Jenning V., Gysler A., Korting M. S., Gohla S. VitaminA Loaded Solid Lipid Nanoparticles for Topical Use:Occlusive Properties and Drug Targeting to the UpperSkin. Eur. J. Pharm. Biopharm. 2000; 50: 189–97.

[37] Muller R. H., Mader K., Gohla S. Solid LipidNanoparticles (SLN) for Controlled Drug Delivery —A Review of the State of the Art. Eur J. Pharm Biopharm2000; 50: 161–177.

[38] Trotta M., Debernardi F., Caputo O. Preparation ofSolid Lipid Nanoparticles by a Solvent Emulsification–diffusion Technique. Int. J. Pharm 2003; 257: 153–160.

[39] Muller R. H., Jacobs C., Kayser O. Nanosuspensions asParticulate Drug Formulations in Therapy Rationale forDevelopment and what we can Expect for the Future.Adv Drug Deliv Rev. 2001; 47: 3–19.

[40] Muller R. H., Bohm BHL. In: Muller R. H., Benita S.,Bohm B., editors. Emulsions and Nanosuspensions for

the Formulation of Poorly Soluble Drugs. Stuttgart:Medpharm Scientific, 1998. 149–173.

[41] Liversidge G. C., Cundy K. C. Particle Size Reductionfor Improvement of Oval Bioavailability ofHydrophobic Drugs.1. Absolute Oral Bioavailability ofNanocrystalline Danazole in Beagle Dogs. Int. J. Pharm1995; 127: 91–97.

[42] Liversidge G. C. Drug Nanocrystals for Improved DrugDelivery. Int Symp Control Rel Bioact Mater 1996; 23: 74–81.

[43] Levy M. Y., Schutze W., Fuhrer C., Benita S.Characterization of Diazepam Submicron EmulsionInterface: Role of Oleic Acid. J. Microencapsul 1994; 11:79-92.

[44] Yang S. C., Zhu J. B. Preparation and Characterizationof Solid Lipid Nanoparticles. Drug Dev. Ind. Pharm.2002; 28: 265–274.

[45] Venkateswarlu V., Manjunath K. Preparation,Characterization and in Vitro Release Kinetics ofClozapine Solid Lipid Nanoparticles. J. Control Release2004; 95: 627–638.

[46] Bhadra D., Bhadra S., Jain S., Jain N. K. A PEGylatedDendritic Nanoparticulate Carrier of Fluorouracil. Int.J. Pharm 2003; 257: 111–124.

[47] Schäfer-Korting M., Mehnert W., Korting H. LipidNanoparticles for Improved Topical Application ofDrugs for Skin Diseases. Adv Drug Deliv. Rev. 2007; 59:427–443.

[48] Cevc G. Lipid Vesicles and other Colloids as DrugCarriers on the Skin. Adv Drug Deliv Rev. 2004; 56: 675–711.

[49] William A. S., Camilleri J. P., Goodfellow R. M.,Williams B. D. A Single Intra-articular Injection ofLiposomally Conjugated Methotrexate Suppresses JointInflammation in Rat Antigen Induced Arthritis. Br. J.Pharmacol 1996; 35: 719-724.

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