Adapalene loaded solid lipid nanoparticles gel: An effective approach for acne treatment

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Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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dapalene loaded solid lipid nanoparticles gel: An effective approachor acne treatment

mit K. Jaina,d, Ashay Jaina,b, Neeraj K. Garga,b, Abhinav Agarwala, Atul Jaina,b,om Akshay Jaina,d, Rajeev K. Tyagic, Rakesh K. Jaina,d, Himanshu Agrawale,ovind P. Agrawala,∗

Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar, MP 470003, IndiaDrug Delivery Research Group, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh 160014,

ndiaDepartment of Periodontics, College of Dental Medicine Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USABhagyoday tirth Pharmacy College, Khurai Road, Sagar, MP 470001, IndiaPharmaceutics Research Laboratory, M. S. University of Baroda, Vadodara, India

r t i c l e i n f o

rticle history:eceived 12 January 2014eceived in revised form 27 May 2014ccepted 29 May 2014vailable online 6 June 2014

eywords:dapaleneolid lipid nanoparticles (SLNs)cne

a b s t r a c t

Salient features such as controlled release, target ability, potential of penetration, improved physicalstability, low cost compared to phospholipids, and ease of scaling-up makes solid lipid nanoparticles(SLNs) a viable alternative to liposomes for effective drug delivery. Adapalene (ADA) is a second generationretinoid effective in treating various dermatologic disorders such as Acne vulgaris with a few noticeabledose-mediated side effects. The present study was aimed at developing and characterizing ADA loadedSLNs for effective topical delivery. The formulated SLN system was characterized for particle size, polydispersity index, entrapment efficiency and drug release properties. The resultant formulation (ADAloaded SLNs incorporated into carbopol hydrogel) was evaluated for in vitro drug release, skin permeationand bio-distribution, rheological behaviour, and texture profile analysis. The SLNs based ADA gel has

pidermal targetingheologyopical delivery

shown its potential in targeting skin epidermal layer, and reducing systemic penetration. The developedsystem can avoid systemic uptake of ADA in skin layers, and can localize drug in skin epidermis asconfirmed by rat skin model. Our results advocate potential of SLNs as a novel carrier for topical deliveryof ADA in topical therapeutic approaches. This study open new avenues for drug delivery which bettermeets the need of anti-acne research.

. Introduction

Acne vulgaris (AV) is the most common dermatological disorderarely posing a serious threat, but affecting overall performanceillions of individuals [1]. AV is usually associated with inflamma-

ion of pilosebaceous units caused by the gram-positive organism,ropionibacterium acnes on mainly face skin, neck, chest and upperack [2,3]. The microenvironment of sebaceous follicles undergoeselective changes that leads plugging of pilosebaceous follicles andevelopment of micro-comedo resulting in to acne lesions, includ-

ng non-inflammatory as well as inflammatory nodules [4]. There

re effective treatments available such as topical and oral antibi-tics, topical and oral retinoids. The retinitis is one of the regularlyrescribed classes of medicine. The topical treatment is the first

∗ Corresponding author. Tel.: +91 9981338997.E-mail address: [email protected] (G.P. Agrawal).

ttp://dx.doi.org/10.1016/j.colsurfb.2014.05.041927-7765/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

choice in mild and moderate acne, whereas systemic therapy isapplied to treat severe and moderate cases [5]. The topical treat-ment of mild to moderate acne with all trans retinoic acid (RA) hasbeen effective in acne therapeutic [6].

Retinoids, natural or synthetic derivatives of vitamin A, due totheir ability to modify abnormal follicular keratinization are highlyeffective in Acne vulgaris therapeutics [4]. The topical application ofRA follows high incidences of skin irritation, photosensitivity, andlow patient compliance. The systemic therapy with antibiotics hasits own disadvantages such as nausea, vomiting, and contraceptivefailure in pregnant women [7]. The administration of a drug viatopical route is a better option than systemic route using noveldrug delivery systems, and present potential to reduce side effectswithout having an effect on drug efficacy [8].

Solid lipid nanoparticles (SLNs) as novel nano-particulate car-rier systems have drawn considerable attention due to improveddelivery and stability of drugs. SLNs consist of biocompatible lipidcore and an amphiphilic surfactant at the outer shell [9]. They have

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hown advantages over fat emulsions, polymeric nanoparticles andiposomes. They circumvent limitation observed with other car-iers, and safeguarding drug to a greater extent against chemicalegradation compared to that seen with liposomes [10–12]. More-ver, these systems may be industrialized because of their virtuef no or minimal requirement of organic solvents [13].

Adapalene (ADA), 6-[3-(1-adamantyl)-4-methoxy-phenyl]aphthalene-2-carboxylic acid, is a topical anti-acne agent with

few clinical effects similar to tretinoin as well as iso-tretinoin.owever, ADA have shown better acceptability than retinoids

14], and thus considered an appropriate first-line therapy for allases of acne with a few exceptions [15]. The role of ADA playedn reducing sebum production by sebaceous glands remains to beemonstrated [16]. One day treatment therapy by ADA reversesncharacteristic follicular conditions caused by the comedoneormation and cutaneous inflammatory reactions involved inathogenesis of acne [14,17]. It has been previously reportedhat ADA selectively binds to RAR subtypes b and g [17] andorms ADA-RAR complex. The retinoids perform their biologicalunctions by an interacting with specific nuclear retinoic acidRAR) and retinoid X (RXR) receptors [18]. Eventually, ADA-RARomplex binds to RXR, and ADA/RAR/RXR mediating regulation ofranscription [17].

The current study was designed to develop and explore deliv-ry potential of SLNs based hydrogel for targeted and sustained

DA delivery to affected sites. The SLN loaded ADA system was for-ulated and characterized for their size, entrapment efficiency and

urface charge distribution. The characterized SLNs were incorpo-ated into 1% Carbopol® 934 gel and formulations were investigatedor in vitro drug release, stability study, and in vitro permeationnd biodistribution into different layers of skin. In brief, our resultsalidate the suitability of the delivery vehicle and set platform tostablishing an effective treatment for acne.

. Materials and methods

.1. Materials

The ADA was a generous gift from Glenmark pharmaceut-cals Ltd. (Nasik, India). Hydrogenated soya phosphatidylcholineHSPC) was a kind gift from Lipoid, Ludwigshafen, Germany. Tris-earin, Triton X-100 was procured from Sigma Aldrich (Germany).ellulose dialysis bag (MWCO 10 kDa) and G-50 Sephadex wereurchased from Himedia (Mumbai, India). Nylon membrane filter0.22 and 0.45 �m) was acquired from Pall Gelman Sciences (USA).he deionised and filtered water was used all over the study.

.2. Fabrication of ADA loaded solid lipid nanoparticles (SLNs-A)

The SLN was prepared by the solvent injection method aseported by elsewhere [19] with slight modifications. In brief, theristearin (1%, w/v), soya lecithin (PC; 0.3%, w/v) and ADA (0.1%,/v) were dissolved in 10 ml acetone and ethanol mixture (1:1,

/v), while temperature was maintained 70 ◦C on a water bathith continuous stirring. The heated lipid phase was added into

queous phase (with 0.2% (w/v) Tween 80) drop by drop using

Entrapment Efficiency (%) = Total a

syringe at a constant flow rate of 5 ml/min at said tempera-ure with stirring. The dispersion was stirred by mechanical stirrerRemi Instrument, Mumbai, India) at 4000 rpm for 1 h followedy sonication for 1 min by using probe sonicator (Lark innovative

iointerfaces 121 (2014) 222–229 223

technology, Chennai) to generate nano-size suspension. The unen-trapped or free drug was removed by cellulose dialysis bag (MWCO10 kDa) and resulting dispersion was filtered through membranefilter (0.45 �m) to remove excess lipid. The suspension was sub-jected to FTIR spectroscopic studies by KBr pellet method afteradsorption of small amount of suspension on KBr pellet using an IRspectroscope (Perkin-Elmer, USA). The separated SLNs-A suspen-sion was lyophilized (VirTis AdVantage) and stored.

2.3. Drug content determination

The SLNs-dispersion was filled into the cellulose dialysis bag(MWCO 10 kDa), and was extensively dialyzed with magnetic stir-ring (50 rpm) against double distilled water (DDW) under sinkconditions for 10 min to remove un-entrapped drug from formula-tion. The samples were collected in HPLC vials and diluted withthe solvent (methanol and dimethyl formamide). The ADA wasestimated by HPLC method as reported earlier [20] with minormodifications. Briefly, HPLC analysis was isocratically performedusing Merck RP-8 column (250 mm × 4.6 mm i.d., particle size5 �m) and acetonitrile–water (65:35, v/v; the pH was adjusted to2.5 with ortho-phosphoric acid) as the mobile phase (flow rate,1.3 ml/min) and previously degassed by bath sonicator for 15 min.The injectable volume was 20 �L for all solutions, and detectionwavelength was set at 321 nm [20]. The entrapment efficiency (EE)was calculated according to the following equation:

nt of drug added − Amount of drug in collected sampleTotal amount of drug added

× 100

2.4. Fabrication of SLNs-A gel

SLNs-A dispersion was incorporated into concentratedCarbopol® 934 gel base so that the final concentration of Carbopol®

934 remained 1% (w/v) and gel was assented to hydrate for 24 h. Theresulting mixture was stirred for 3–5 h at room temperature withmagnetic stirrer followed by neutralization with Tri-ethanolamineto obtain an adequate semisolid carbopol gel matrix at pH 6.0. Thecarbopol gel was appropriately viscous when neutralized to adjustpH 6.0 [21].

2.5. Characterization of SLNs-A and SLNs-A gel

2.5.1. Particle sizeThe average particle size and polydispersity index of SLNs-A

were determined by photon correlation spectroscopy using zeta-sizer (PCS, Nano ZS90 zetasizer, Malvern Instruments Corp, UK). Thesample was diluted with filtered deionized water in polystyrenecuvettes and was observed at a fixed angle of 90◦ at 25 ± 0.1 ◦C.

2.5.2. Zeta potentialThe zeta potential of the SLNs-A was determined in folded cap-

illary cells by laser Doppler anemometry using Malvern zetasizerwhich is also called as Doppler-Electrophoretic Light Scatter Ana-lyzer. The zeta potential was measured on samples well-dispersedin deionised water at temperature, 25 ± 0.1 ◦C and electric field,15.24 V/cm.

2.5.3. Transmission electron microscopy (TEM)The SLNs-A was characterized for size and morphology by TEM

using a Philips CM 10 electron microscope with an acceleratingvoltage of 3 kV (Morgani, 268D; Holland). A drop of sample wasplaced on a carbon coated copper grid to leave a thin film on thegrid. Sample was negatively stained with 1% phosphotungustic acid

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efore the film got dried, and photomicrographs were taken at5,000× magnification.

.5.4. Rheological behaviour and SLN-A gel texture studyThe rheological behaviour of SLNs-A gel was determined using

dynamic rheometer (Anton Paar, Germany) equipped with a conend plate test geometry (cone diameter, 75 mm, cone angle 0.999◦)t 25 ◦C. Samples were put on to the plate and parameter wasdjusted according to equipment recommendations. The apparentiscosity of the samples was recorded using instrument softwareheoplus, and flow behaviour of sample was determined by flowurve test. Further, test was performed by measuring the viscos-ty (Pa s) and shear stress (�) as a function of shear rate (� in s−1,ange from 0.1 to 100 s−1). The viscoelastic behaviour of samplesas recorded using a cone and plate geometry. The linear viscoelas-

ic region (LVR) was determined through amplitude sweep test byeasuring G′ (storage modulus), G′′ (loss modulus) and shear stress

�). The dynamic moduli (G′ and G′′ in Pa) and shear stress (Pa)ere determined as function of shear stress ranging from 0.05 to

00 Pa and at a constant frequency of 1 Hz [22]. The LVR providesnformation about the minimum strain required in the oscillationrequency sweep test. The oscillation frequency sweep test wasarried-out by measuring G′ (storage modulus) and G′′ (loss mod-lus), and complex viscosity (�*) as a function of frequency (Hz)anging 100–0.01 Hz at a constant strain amplitude of 1% in lin-ar viscoelastic region. This test was carried out to monitor sampleehaviour at constant strain and on changing frequency.

Prepared SLNs-A gel was investigated for textural profile anal-sis (TPA) characteristic using TA-XTPlus texture analyzer (Stableicrosystems, UK). Briefly, equipment was equilibrated and main-

ained at 32 ◦C, and recommended volume of SLNs-A gel was putver lower stage of equipment to note down readings on samples.

.6. In vitro drug release study

In vitro drug release of entrapped drug from SLNs-A and SLNs-el formulations was studied by employing dialysis tube diffusionechniques. 5 ml of SLNs-A dispersion free from unentrapped drugnd weighed amount of SLNs-A gel with an equivalent amountf ADA was individually kept in a dialysis membrane (MWCO0–12 kDa Himedia, India) which was tied at both ends and placed

n separate beaker containing 20 ml solvent mixture of 80% (v/v)ethanol and DMF (50:1) in PBS (pH 5.6). The beakers were assem-

led above a magnetic stirrer in order to have continuous stirring at00 rpm and maintained constant temperature, 32 ± 1 ◦C. One mlf sample was withdrawn intermittently (0, 5, 10, 15, 30, 45 min, 1,, 4, 6, 8, 10, 12, 16, 24, 36 and 48 h) and was replaced with sameolume of solvent mixture in receptor compartment [13]. Samplesere analyzed to quantify the ADA by HPLC method as described

n Section 2.3.

.7. Skin permeation study: in vitro

Permeation study was carried out using shaved skin of Wistarats procured from CDRI, Lucknow, India. All experimental proto-ols were approved by the Institutional Animals Ethical Committeef Dr. Hari Singh Gour University, Sagar. All animal experimentsere carried out in accordance with guidelines of Council for the

urpose of Control and Supervision of Experiments on AnimalsCPCSEA), ministry of social justice and empowerment, Govern-

ent of India.The fully thick skin from abdominal region of shaved rat

as excised and examined for integrity using a lamp inspectionethod. It was properly rinsed with saline followed by chopping

f all fat tissues below the skin [23]. Skin was clamped on a verticalranz diffusion cell in such a manner so that stratum corneum side

iointerfaces 121 (2014) 222–229

facing upwards into the donor compartment, and dermal side wasfacing downwards into the receptor compartment. The receivercompartment was filled with 30 ml of PBS (pH 5.6): methanol(with DMF) 1:4 stirred continuously at 300 rpm and cells weremaintained at 32 ± 0.5 ◦C using a recirculating water bath, allowingskin samples to equilibrate for 30 min before closing doors [24].Plain drug solution, SLNs-A and SLNs-A gel (with an equivalentADA to SLNs-A) were applied gently in the donor compartment.0.5 ml sample from receiver compartment was collected peri-odically (0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h) and same volume ofPBS: methanol solution was added in receptor compartment touphold a constant volume throughout the study [25]. The completeexperiment was carried out for 24 h. All samples were filteredthrough an aqueous 0.22 �m pore size cellulose membrane filterand cumulative volumes of ADA permeated through rat skins wereanalyzed.

2.8. Skin distribution study: in vitro

Following in vitro permeation study, skin was removed andmounted carefully on diffusion cell. The formulation was scrappedto retrieve most of adhered cells with the help of a scraper. The cleanskin tissue was washed three times with deionised water and letit dried. Further, epidermal and dermal layers were manually sep-arated using tweezers, and these skin layers were chopped intopieces and macerated in 5 ml methanol: DMF (50:1) to extract ADA[26]. Solutions were filtered through a membrane (0.45 �m) andfiltrate was analyzed for ADA concentration as discussed earlier inSection 2.6.

2.9. Histopathology study

The fluorescein isothiocyanate (FITC) marker was encapsulatedinto optimized formulations of SLNs and incorporated into carbopolgel to investigate deposition pattern of SLNs. FITC loaded SLNs andSLNs-gel was applied uniformly on shaved skin of wistar rats. Ani-mals were euthanized, skin specimens were excised, and sampleswere frozen at -20 ◦C. The sections of 5 �m diameter were preparedby cryostat from every specimen and fixed in 10% formalde-hyde solution for at least 72 h. All slices were then dehydratedusing ethanol followed by paraffin embedding. The sections wereviewed under fluorescence microscope and microphotographswere taken from different areas (TE2000-U, Nikon, Melville,NY, USA).

2.10. Stability study of SLNs-A and SLNs-A gel

The physical and chemical stabilities of SLNs-A and SLNs-A gelwere involved in short term observations of different attributes,viz., possible changes in physical appearance like de-colouration,gel consistency, change in odours and appearance of drug crystalsor precipitates. The formulations were evaluated at three differ-ent storage conditions, i.e. in refrigerated condition (RF; 5 ± 3 ◦C),room temperature (RT; 25 ± 2 ◦C/60 ± 5% RH) and elevated tem-perature (HT; 40 ± 2 ◦C/75 ± 5% RH) over a period of 3 monthsin order to see clarity of formulations, particle size and zetapotential.

2.11. Data analysis

Statistical analysis of in vitro data and all the skin permeationexperiments of each preparation were performed three times, and

data were expressed mean value S.D. The statistical data were ana-lyzed using non-parametric tests with a Wilcoxon test. Statisticaldifferences are denoted as p ≤ 0.05 (NS = not significant), p ≤ 0.01(significant) and p ≤ 0.001 (highly significant).

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ication

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that has most narrow size distribution of 0.169. This formulationwas considered optimized formulation with average particle size(148.3 ± 2.5 nm) (Table 1) and highest EE (89.90 ± 1.2) as com-pared to that seen with other formulations. The zeta potential is an

Table 1Comparison of % drug entrapped, particle size and polydispersity index (PDI) (n = 6).

SLNs-A formulationcode

Surfactantconc. (%)

Particle size PDI EE

Fig. 1. Fabr

. Result and discussion

.1. Formulation of ADA loaded solid lipid nanoparticles (SLNs-A)nd SLNs-A gel

The SLNs were fabricated by hot homogenization techniquey employing solvent injection method which involves intenseiffusion of solvent across solvent–lipid phase in aqueous phase fol-

owed by evaporation of solvent and resulting in enhanced rigidityf lipid nanoparticles. High speed stirring was deployed to obtain

pre-emulsion phase prior to homogenization, ADA was dispersedomogeneously in the molten lipid, and acetone and ethanol sol-ent was used. A hot water bath was used to maintain pre-emulsionn or above the melting temperature (Fig. 1). Furthermore, prepa-ation of ADA loaded SLNs was characterized by FTIR spectroscopydata not shown). The prepared SLNs were incorporated into 1%arbopol® 934 gel. The gel neutralized with triethanolamine and arizzled, homomorphic viscous SLNs-A gel was produced.

.2. Estimation of drug entrapment efficiency

The greater entrapment efficiency (89.90 ± 1.9%) of SLNs-A wasound at 1% surfactant concentration (Table 1). However, furtherncrease (greater than 1%) in surfactant concentration has shown a

oss of entrapment efficiency and increased Poly Dispersity indexPDI). We believe loss in entrapment efficiency is probably due tourface leaching of the entrapped drug. The high lipophilicity andetter compatibility between drug and lipid may result in high EE

of SLNs-A.

of SLNs formulations, and might prove beneficial in reducing skinirritation due to minimal or no contact of drug to skin surface.

3.3. Particle size, zeta potential and morphology

The average particle size and poly dispersity index of differ-ent SLNs-A formulations were measured by photon correlationspectroscopy using zeta-sizer (ZS 90, Malvern Instruments, UK)(Table 1). The increased surfactant concentration resulted in adecrease in particle size at a certain threshold. The average sizeof all formulations falls between 140 and 220 nm range. The sur-factant concentration dependent change (decrease in particle size)also showed an impact on polydispersity index and entrapmentefficiency of optimized formulation. The chylomicron mimickingbehaviour of SLNs might achieve size nearly equivalent to SLNs-A

A 0.5 219.1 ± 3.2 0.417 74.90 ± 1.2B 1.0 148.3 ± 2.5 0.169 89.90 ± 1.2C 1.5 141.5 ± 6.5 0.218 80.90 ± 1.2D 2.0 197.5 ± 5.2 0.283 69.90 ± 1.2

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Fig. 2. TEM photomicrographs of SLNs-A.

ipp(rtp

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observed in linear mode. This pattern delineates higher efficiency ofsample (increase in modulus) with a decrease in measurement time

Fpt

mportant criterion for examining the storage stability of lipidarticles and their cellular demeanour in drug release. The zetaotential of optimized formulations of SLNs-A was −12.0 mVTable 1). The higher negative value of zeta potential providesepulsive interaction between SLNs, and thus prevents aggrega-ion of nanoparticles. In addition, tween 80 used in this study alsorovided stearic stability to achieve stable formulation.

TEM results confirmed spherical shape of prepared SLNs-A, and

hotomicrographs (Fig. 2) of prepared formulation are suggestivef their nanometric size range and narrow size distributions.

ig. 3. (a) Flow curve graph shows that if increase in shear rate, viscosity of gel initially inlot between G′ , G′′ at Y1, shear stress at Y2 and linear strain at X1. (c) Frequency dependhe storage modulus and loss modulus increases whereas complex viscosity decreases. (d

iointerfaces 121 (2014) 222–229

3.4. Analysis of rheological and textural profile of SLNs-A gel

A rheological study demonstrates flow behaviour of SLNs-Agel. The value of flow behaviour index obtained for SLNs-A gelwas found less than unity (n = 0.89613), which indicates shear-thinning behaviour of SLNs-A. Herschel–Bulky model was preferredfor SLNs-A gel due to its suitability for current study [27]. Therheograms represent values of shear stress and viscosity obtainedat varying shear rates, these have been delineated in Fig. 3a–c. Thepresent results show an initial increase in viscosity are directly cor-related to increased shear rate, and started revealing decrease insecond phase. The graph was plotted on log scale between viscosityat Y1, shear stress at Y2 and shear rate at X1 (Fig. 3a). The consis-tency value was found 1.146 Pa.s, and high yield stress value wascalculated as high as 23.473 Pa. The result obtained from ampli-tude LVR test revealed that storage modulus (G′) is quite high ascompared to loss modulus (G′′) which further assures high elas-ticity of gel that retains virtue of less dissipation of energy, andloss in modulus will be larger only when sample is predominantlyviscous [28]. The graph plotted between G′, G′′ at Y1, shear stressat Y2 and strain at X1 has been found linear (Fig. 3b). The loss intangent is the measure of the energy, lost to stored energy, in thecyclic deformation (tan ı = G′′/G′). A value of tan ı < 1 does mean aprevalent elastic demeanour [29]. The outcome of frequency sweepstudy made possible to determine the internal alteration in thestructure of the gel. The test was conducted at 1% strain underchanging frequency sweep i.e. 0.1 to 100 Hz at room temperature(25 ± 0.5 ◦C). The values for the storage modulus (G′), loss modulus(G′′) and complex viscosity (�*) were laid down across frequencyrange. As is shown in Fig. 3c logarithm graph, no cross-over wasseen at ambient temperature. Furthermore, storage and loss mod-ulus saw an increase where decrease in complex viscosity was

(frequency = 1/time). In brief, if gel formulation has attain a partic-ular phase and viscosity, it can be easily transported and stored

creases and at the second point it begin to decrease. (b) Amplitude LVE test: linearency test at 1% strain: no crossover was found at ambient room temperature, also) Textural behaviour of SLNs-A gel.

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Fig. 5. Skin distribution of ADA delivered via SLNs-A and SLNs-A gel into differentskin strata and receptor fluids. On the Y-axis, the units of ADA permeation (#) are:(�g/cm2) of tissue surface area for ADA in the epidermis and dermis, and (�g/ml)

ig. 4. In vitro drug release study in 80% (v/v) methanol:DMF (50:1) in PBS (pH 5.6)n = 6; p ≤ 0.05*). *Significant difference between SLNs-A and SLNs-A gel.

t ambient temperature provided it is not subjected to any shearhanges which might alter their viscosity and in turn stability andtructure.

Other rheological characteristics, i.e. firmness, stickiness andork of adhesion were further characterized by textural profile

nalysis (TPA). Texture curve (Fig. 3d) shows uniformity, smooth-ess and absence of any grittiness or lumps in the SLNs-A gel. TheLNs-A gel confirmed fairly good gel strength, diffusion of sam-le at ease and extrusion from the tube. The gel was discoveredo have shown adequate cohesiveness which is pre-requisite toustain formulation adhering to the site of application. In con-lusion, developed SLNs-A gel was met with pre-requisite criteriaiz., cohesiveness, spread-ability, ease of extrusion, absence ofrittiness and particulate matter as required for any topical prepa-ation.

.5. In vitro drug release study

The drug release was studied intermittently starting from 0 h to8 h. The initial burst release of 25.57 ± 0.4% and 16.82 ± 0.04% fromLNs-A and SLNs-A gel was an interesting observation respectivelynd followed slower and sustained release (Fig. 4). The initial bursts probably due to rapid release of drug adsorbed on the surfacer presence of drug just underneath the stratum of the SLNs. Thelower and sustained release, however, attributed to the diffusionf drug molecules through lipid matrix of SLNs. Furthermore, anbundant reduction in cumulative release of ADA from SLNs for-ulation observed is the virtue of existence of lipid matrix hinders

rug to release. However, a substantial decrease in the cumulativeelease of ADA from SLN-A gel was observed in comparison to SLN-

formulation. Consistent to similar study conducted by Jain et al.11], our results describe the presence of gel matrix surroundingLN-A, and impede the drug release.

.6. Ex vivo skin permeation studies

Franz diffusion cells were used to evaluate the skin uptake andkin targeting potential of SLNs. The permeation ability of ADA fromLNs formulation into rat skin was determined by sampling ratrom 0 to 8 h (clinical application time) [30]. 0.15% ADA tincturen Methanol: DMF was used as a reference to evaluate skin tar-eting ability of SLNs-A and SLNs-A gel. The insignificant amountf ADA in the receptor chamber for SLNs-A and SLNs-A gel wasbserved even after 8 h which indicated the inability of ADA fromLNs-A and SLNs-A gel in penetrating skin. The amount of ADAn the receptor chamber from reference tincture showed a steady

ncrease with an increase in time (Fig. 5), and a permeation rate wasalculated, 76 ± 0.3 �g cm−2 h−1 (permeation followed zero orderelease kinetics). The low inadequate concentration of ADA in epi-ermis of samples treated with tincture is due to rapid diffusion

for ADA measured in the receptor compartment. The statistical data is expressed asmean ± SE (n = 6).

(because of penetration-enhancement properties of methanol) ofADA in the acceptor medium. Moreover, rapid permeation and lossof methanol due to evaporation might increase concentration ofADA in tincture which in turn increases thermodynamic activityof ADA [31]. Aforementioned are a few important factors whichmight contribute to the permeation of ADA through skin from alco-holic solution. It is therefore concluded that SLNs-A and SLNs-A gelminimizes or avoids systemic uptake of ADA when compared totincture, and presents robust system(s) to avoid or minimize sys-temic adverse side effect. This may be due to reduced or no skindehydration by SLNs-A and SLNs-A gel, and as a consequence ofwhich hydrated stratum corneum facilitates drug penetration intodeeper strata of skin [32]. In brief, the components of SLNs, mayfuse in skin and mix with lipids to loosen up their structure whichhindered lamellar arrangement of lipids with increased thicknessof the stratum corneum [33,34].

3.7. Skin distribution study

These results are suggestive of the efficiency of SLNs-A andSLNs-A gel to allow ADA permeation into skin, and quantificationof ADA content in epidermis, dermis, and receptor fluids (Fig. 5).SLNs formulations deliver quantifiable amounts of ADA into epi-dermal layer of skin, but minimal ADA quantity was observed inthe dermis. Results also obviated no effect of extended viscosity ofSLNs-A gel on the deposition of ADA into skin at each investigatedskin layer. However, it showed significant increase in the retentiontime of formulation at the site of application. The current resultsalso revealed that ADA comprehensively remains in the epidermallayer because higher hydrophilicity of dermal layer circumscribevaluable partitioning of hydrophobic drug [26]. The outcome fromskin distribution study showed advancement of SLNs based ADAformulations, i.e. an efficient topical drug delivery system with leasttransdermal localization and avoids systemic absorption. Consis-tent to what has been reported earlier [35,36], small sized SLNsshowed their merit in improving penetration ability of nanoparti-cles into the skin, and higher drug accumulation due to sustainedrelease from SLNs. The interaction of SLNs lipids and surfactantswith skin lipids is presumably an important factor leading to greaterpenetration [37]. Moreover, occlusive effect is also an important

factor affecting penetration of drug into skins (due to small particlesize of carrier), which might have more effect controlling penetra-tion of nano-actives into the skin.

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ig. 6. Histopathology study shown qualitative uptake and the distribution of the fl

.8. Histopathology study

We decided to perform fluorescence microscopy study to con-rm and to determine the qualitative uptake and distributionattern of lipid nano-carriers into the skin. FITC loaded SLN-Ael and SLNs with no gel loaded was used in order to determinextent of penetration of SLNs into the skin. The photomicrographseflect green fluorescence intensity in the different layers of skin.he fluorescence was observed throughout the skin (Fig. 6a and) suggesting lipophilic nature of SLNs improved in vivo transder-al delivery, and also allowing stockpiling and transportation of

ioactive through the vasculature in dermis and epidermis.

.9. Stability study

SLNs-A and SLNs-A gel has shown the stability of formulationver the period of three months. No considerable variations inlarity, and phase separation were observed, demonstrating goodhysical stability of SLNs. Moreover, SLNs-A was found stable inentrifuge test, and the stability might inherit from the lethargicransition of dispersed lipid from metastable forms to the stableorm in SLNs, low particles size, and steric effect of Tween 80 [38].he minimal or non-significant degradation of ADA in SLNs andLNs gel was seen with stable transparency for over 3 months.

. Conclusions and future perspectives

Very recently several studies have been reported for the effec-ive topical delivery of different kinds of anti-acne molecules. The

arious colloidal carriers like liposomes and mixed vesicles [39],ipid-nanoparticles [40] and microemulsion [41] were employedo either improve the physicochemical properties of drugs oro improve their pre-clinical efficacy. However, there are no

cent marker loaded nanocarriers throughout skin section. (A) SLN-A; (B) SLN-A gel.

studies have been reported on ADA loaded SLNs. SLNs of ADAwere prepared by hot homogenization method followed by theincorporation into carbopol gel for topical delivery. The currentstudy indicates targeting prospective, spatial delivery and elevatedretention potential of the formulation in epidermal tissues. Thein vitro permeation study illustrates a low or no systemic uptakeof ADA when compared with reference. The small diameter ofSLNs and increased permeation effect of soya lecithin on stratumcorneum may contribute to the penetration of SLNs-A into thestratum corneum, and shows a better skin targeting effect. Thus,the small diameter of SLNs, and soya lecithin might open a wayto facilitate targeted delivery of ADA into the dermis layer. TheseNPs accumulated particularly in the epidermis, and maintained inthe skin layer at significant limits. In conclusion, the present studyopens new vista for SLNs as efficient vectors to carry large and sys-temic doses of ADA as evidenced from in vitro studies, showinga sustained release nature of formulation. The developed SLNs-A nanoparticulate system demonstrated the optimal therapeuticresponse, improved therapeutic efficacy and minimal penetrationacross epidermis with an interception of minimal side effects. Thecurrent study is an important step towards skin acne and drugdelivery, and might be a step forward in unravelling skin targetingmechanism and help addressing acne problems.

Acknowledgements

The authors are grateful for the fellowship and grant provided

EMR-I), New Delhi, India. The authors also thank Glenmarkpharmaceuticals Ltd. (Nasik, India) for providing ADA as gift sample,Lipoid, Ludwigshafen, Germany for providing Hydrogenated soyaphosphatidylcholine (HSPC).

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