Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Adapalene loaded solid lipid nanoparticles gel: An effective approach for acne treatment Amit K. Jain a,d , Ashay Jain a,b , Neeraj K. Garg a,b , Abhinav Agarwal a , Atul Jain a,b , Som Akshay Jain a,d , Rajeev K. Tyagi c , Rakesh K. Jain a,d , Himanshu Agrawal e , Govind P. Agrawal a,∗ a Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar, MP 470003, India Drug Delivery Research Group, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh 160014, India c Department of Periodontics, College of Dental Medicine Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA d Bhagyoday tirth Pharmacy College, Khurai Road, Sagar, MP 470001, India e Pharmaceutics Research Laboratory, M. S. University of Baroda, Vadodara, India b a r t i c l e i n f o Article history: Received 12 January 2014 Received in revised form 27 May 2014 Accepted 29 May 2014 Available online 6 June 2014 Keywords: Adapalene Solid lipid nanoparticles (SLNs) Acne Epidermal targeting Rheology Topical delivery a b s t r a c t Salient features such as controlled release, target ability, potential of penetration, improved physical stability, 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 generation retinoid effective in treating various dermatologic disorders such as Acne vulgaris with a few noticeable dose-mediated side effects. The present study was aimed at developing and characterizing ADA loaded SLNs for effective topical delivery. The formulated SLN system was characterized for particle size, poly dispersity index, entrapment efficiency and drug release properties. The resultant formulation (ADA loaded SLNs incorporated into carbopol hydrogel) was evaluated for in vitro drug release, skin permeation and bio-distribution, rheological behaviour, and texture profile analysis. The SLNs based ADA gel has shown its potential in targeting skin epidermal layer, and reducing systemic penetration. The developed system can avoid systemic uptake of ADA in skin layers, and can localize drug in skin epidermis as confirmed by rat skin model. Our results advocate potential of SLNs as a novel carrier for topical delivery of ADA in topical therapeutic approaches. This study open new avenues for drug delivery which better meets the need of anti-acne research. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Acne vulgaris (AV) is the most common dermatological disorder rarely posing a serious threat, but affecting overall performance millions of individuals [1]. AV is usually associated with inflammation of pilosebaceous units caused by the gram-positive organism, Propionibacterium acnes on mainly face skin, neck, chest and upper back [2,3]. The microenvironment of sebaceous follicles undergoes selective changes that leads plugging of pilosebaceous follicles and development of micro-comedo resulting in to acne lesions, including non-inflammatory as well as inflammatory nodules [4]. There are effective treatments available such as topical and oral antibiotics, topical and oral retinoids. The retinitis is one of the regularly prescribed classes of medicine. The topical treatment is the first ∗ Corresponding author. Tel.: +91 9981338997. E-mail address: agrawal.gp.dops@gmail.com (G.P. Agrawal). http://dx.doi.org/10.1016/j.colsurfb.2014.05.041 0927-7765/© 2014 Elsevier B.V. All rights reserved. choice in mild and moderate acne, whereas systemic therapy is applied to treat severe and moderate cases [5]. The topical treatment of mild to moderate acne with all trans retinoic acid (RA) has been effective in acne therapeutic [6]. Retinoids, natural or synthetic derivatives of vitamin A, due to their ability to modify abnormal follicular keratinization are highly effective in Acne vulgaris therapeutics [4]. The topical application of RA follows high incidences of skin irritation, photosensitivity, and low patient compliance. The systemic therapy with antibiotics has its own disadvantages such as nausea, vomiting, and contraceptive failure in pregnant women [7]. The administration of a drug via topical route is a better option than systemic route using novel drug delivery systems, and present potential to reduce side effects without having an effect on drug efficacy [8]. Solid lipid nanoparticles (SLNs) as novel nano-particulate carrier systems have drawn considerable attention due to improved delivery and stability of drugs. SLNs consist of biocompatible lipid core and an amphiphilic surfactant at the outer shell [9]. They have A.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 shown advantages over fat emulsions, polymeric nanoparticles and liposomes. They circumvent limitation observed with other carriers, and safeguarding drug to a greater extent against chemical degradation compared to that seen with liposomes [10–12]. Moreover, these systems may be industrialized because of their virtue of no or minimal requirement of organic solvents [13]. Adapalene (ADA), 6-[3-(1-adamantyl)-4-methoxy-phenyl] naphthalene-2-carboxylic acid, is a topical anti-acne agent with a few clinical effects similar to tretinoin as well as iso-tretinoin. However, ADA have shown better acceptability than retinoids [14], and thus considered an appropriate first-line therapy for all cases of acne with a few exceptions [15]. The role of ADA played in reducing sebum production by sebaceous glands remains to be demonstrated [16]. One day treatment therapy by ADA reverses uncharacteristic follicular conditions caused by the comedone formation and cutaneous inflammatory reactions involved in pathogenesis of acne [14,17]. It has been previously reported that ADA selectively binds to RAR subtypes b and g [17] and forms ADA-RAR complex. The retinoids perform their biological functions by an interacting with specific nuclear retinoic acid (RAR) and retinoid X (RXR) receptors [18]. Eventually, ADA-RAR complex binds to RXR, and ADA/RAR/RXR mediating regulation of transcription [17]. The current study was designed to develop and explore delivery potential of SLNs based hydrogel for targeted and sustained Entrapment Efficiency (%) = 223 technology, Chennai) to generate nano-size suspension. The unentrapped or free drug was removed by cellulose dialysis bag (MWCO 10 kDa) and resulting dispersion was filtered through membrane filter (0.45 ␮m) to remove excess lipid. The suspension was subjected to FTIR spectroscopic studies by KBr pellet method after adsorption of small amount of suspension on KBr pellet using an IR spectroscope (Perkin-Elmer, USA). The separated SLNs-A suspension 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 stirring (50 rpm) against double distilled water (DDW) under sink conditions for 10 min to remove un-entrapped drug from formulation. The samples were collected in HPLC vials and diluted with the solvent (methanol and dimethyl formamide). The ADA was estimated by HPLC method as reported earlier [20] with minor modifications. Briefly, HPLC analysis was isocratically performed using Merck RP-8 column (250 mm × 4.6 mm i.d., particle size 5 ␮m) and acetonitrile–water (65:35, v/v; the pH was adjusted to 2.5 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 detection wavelength was set at 321 nm [20]. The entrapment efficiency (EE) was calculated according to the following equation: Total amount of drug added − Amount of drug in collected sample × 100 Total amount of drug added ADA delivery to affected sites. The SLN loaded ADA system was formulated and characterized for their size, entrapment efficiency and surface charge distribution. The characterized SLNs were incorporated into 1% Carbopol® 934 gel and formulations were investigated for in vitro drug release, stability study, and in vitro permeation and biodistribution into different layers of skin. In brief, our results validate the suitability of the delivery vehicle and set platform to establishing an effective treatment for acne. 2. Materials and methods 2.1. Materials The ADA was a generous gift from Glenmark pharmaceuticals Ltd. (Nasik, India). Hydrogenated soya phosphatidylcholine (HSPC) was a kind gift from Lipoid, Ludwigshafen, Germany. Tristearin, Triton X-100 was procured from Sigma Aldrich (Germany). Cellulose dialysis bag (MWCO 10 kDa) and G-50 Sephadex were purchased from Himedia (Mumbai, India). Nylon membrane filter (0.22 and 0.45 ␮m) was acquired from Pall Gelman Sciences (USA). The deionised and filtered water was used all over the study. 2.2. Fabrication of ADA loaded solid lipid nanoparticles (SLNs-A) The SLN was prepared by the solvent injection method as reported by elsewhere [19] with slight modifications. In brief, the tristearin (1%, w/v), soya lecithin (PC; 0.3%, w/v) and ADA (0.1%, w/v) were dissolved in 10 ml acetone and ethanol mixture (1:1, v/v), while temperature was maintained 70 ◦ C on a water bath with continuous stirring. The heated lipid phase was added into aqueous phase (with 0.2% (w/v) Tween 80) drop by drop using a syringe at a constant flow rate of 5 ml/min at said temperature with stirring. The dispersion was stirred by mechanical stirrer (Remi Instrument, Mumbai, India) at 4000 rpm for 1 h followed by sonication for 1 min by using probe sonicator (Lark innovative 2.4. Fabrication of SLNs-A gel SLNs-A dispersion was incorporated into concentrated Carbopol® 934 gel base so that the final concentration of Carbopol® 934 remained 1% (w/v) and gel was assented to hydrate for 24 h. The resulting mixture was stirred for 3–5 h at room temperature with magnetic stirrer followed by neutralization with Tri-ethanolamine to obtain an adequate semisolid carbopol gel matrix at pH 6.0. The carbopol gel was appropriately viscous when neutralized to adjust pH 6.0 [21]. 2.5. Characterization of SLNs-A and SLNs-A gel 2.5.1. Particle size The average particle size and polydispersity index of SLNs-A were determined by photon correlation spectroscopy using zetasizer (PCS, Nano ZS90 zetasizer, Malvern Instruments Corp, UK). The sample was diluted with filtered deionized water in polystyrene cuvettes and was observed at a fixed angle of 90◦ at 25 ± 0.1 ◦ C. 2.5.2. Zeta potential The zeta potential of the SLNs-A was determined in folded capillary cells by laser Doppler anemometry using Malvern zetasizer which is also called as Doppler-Electrophoretic Light Scatter Analyzer. The zeta potential was measured on samples well-dispersed in 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 accelerating voltage of 3 kV (Morgani, 268D; Holland). A drop of sample was placed on a carbon coated copper grid to leave a thin film on the grid. Sample was negatively stained with 1% phosphotungustic acid 224 A.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 before the film got dried, and photomicrographs were taken at 15,000× magnification. 2.5.4. Rheological behaviour and SLN-A gel texture study The rheological behaviour of SLNs-A gel was determined using a dynamic rheometer (Anton Paar, Germany) equipped with a cone and plate test geometry (cone diameter, 75 mm, cone angle 0.999◦ ) at 25 ◦ C. Samples were put on to the plate and parameter was adjusted according to equipment recommendations. The apparent viscosity of the samples was recorded using instrument software Rheoplus, and flow behaviour of sample was determined by flow curve test. Further, test was performed by measuring the viscosity (Pa s) and shear stress () as a function of shear rate ( in s−1 , range from 0.1 to 100 s−1 ). The viscoelastic behaviour of samples was recorded using a cone and plate geometry. The linear viscoelastic region (LVR) was determined through amplitude sweep test by measuring G′ (storage modulus), G′′ (loss modulus) and shear stress (). The dynamic moduli (G′ and G′′ in Pa) and shear stress (Pa) were determined as function of shear stress ranging from 0.05 to 100 Pa and at a constant frequency of 1 Hz [22]. The LVR provides information about the minimum strain required in the oscillation frequency sweep test. The oscillation frequency sweep test was carried-out by measuring G′ (storage modulus) and G′′ (loss modulus), and complex viscosity (*) as a function of frequency (Hz) ranging 100–0.01 Hz at a constant strain amplitude of 1% in linear viscoelastic region. This test was carried out to monitor sample behaviour at constant strain and on changing frequency. Prepared SLNs-A gel was investigated for textural profile analysis (TPA) characteristic using TA-XTPlus texture analyzer (Stable Microsystems, UK). Briefly, equipment was equilibrated and maintained at 32 ◦ C, and recommended volume of SLNs-A gel was put over lower stage of equipment to note down readings on samples. 2.6. In vitro drug release study In vitro drug release of entrapped drug from SLNs-A and SLNsgel formulations was studied by employing dialysis tube diffusion techniques. 5 ml of SLNs-A dispersion free from unentrapped drug and weighed amount of SLNs-A gel with an equivalent amount of ADA was individually kept in a dialysis membrane (MWCO 10–12 kDa Himedia, India) which was tied at both ends and placed in separate beaker containing 20 ml solvent mixture of 80% (v/v) methanol and DMF (50:1) in PBS (pH 5.6). The beakers were assembled above a magnetic stirrer in order to have continuous stirring at 100 rpm and maintained constant temperature, 32 ± 1 ◦ C. One ml of sample was withdrawn intermittently (0, 5, 10, 15, 30, 45 min, 1, 2, 4, 6, 8, 10, 12, 16, 24, 36 and 48 h) and was replaced with same volume of solvent mixture in receptor compartment [13]. Samples were analyzed to quantify the ADA by HPLC method as described in Section 2.3. 2.7. Skin permeation study: in vitro Permeation study was carried out using shaved skin of Wistar rats procured from CDRI, Lucknow, India. All experimental protocols were approved by the Institutional Animals Ethical Committee of Dr. Hari Singh Gour University, Sagar. All animal experiments were carried out in accordance with guidelines of Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), ministry of social justice and empowerment, Government of India. The fully thick skin from abdominal region of shaved rat was excised and examined for integrity using a lamp inspection method. It was properly rinsed with saline followed by chopping of all fat tissues below the skin [23]. Skin was clamped on a vertical Franz diffusion cell in such a manner so that stratum corneum side facing upwards into the donor compartment, and dermal side was facing downwards into the receptor compartment. The receiver compartment was filled with 30 ml of PBS (pH 5.6): methanol (with DMF) 1:4 stirred continuously at 300 rpm and cells were maintained at 32 ± 0.5 ◦ C using a recirculating water bath, allowing skin samples to equilibrate for 30 min before closing doors [24]. Plain drug solution, SLNs-A and SLNs-A gel (with an equivalent ADA to SLNs-A) were applied gently in the donor compartment. 0.5 ml sample from receiver compartment was collected periodically (0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h) and same volume of PBS: methanol solution was added in receptor compartment to uphold a constant volume throughout the study [25]. The complete experiment was carried out for 24 h. All samples were filtered through an aqueous 0.22 ␮m pore size cellulose membrane filter and cumulative volumes of ADA permeated through rat skins were analyzed. 2.8. Skin distribution study: in vitro Following in vitro permeation study, skin was removed and mounted carefully on diffusion cell. The formulation was scrapped to retrieve most of adhered cells with the help of a scraper. The clean skin tissue was washed three times with deionised water and let it dried. Further, epidermal and dermal layers were manually separated using tweezers, and these skin layers were chopped into pieces and macerated in 5 ml methanol: DMF (50:1) to extract ADA [26]. Solutions were filtered through a membrane (0.45 ␮m) and filtrate was analyzed for ADA concentration as discussed earlier in Section 2.6. 2.9. Histopathology study The fluorescein isothiocyanate (FITC) marker was encapsulated into optimized formulations of SLNs and incorporated into carbopol gel to investigate deposition pattern of SLNs. FITC loaded SLNs and SLNs-gel was applied uniformly on shaved skin of wistar rats. Animals were euthanized, skin specimens were excised, and samples were frozen at -20 ◦ C. The sections of 5 ␮m diameter were prepared by cryostat from every specimen and fixed in 10% formaldehyde solution for at least 72 h. All slices were then dehydrated using ethanol followed by paraffin embedding. The sections were viewed under fluorescence microscope and microphotographs were 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 gel were 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 crystals or precipitates. The formulations were evaluated at three different storage conditions, i.e. in refrigerated condition (RF; 5 ± 3 ◦ C), room temperature (RT; 25 ± 2 ◦ C/60 ± 5% RH) and elevated temperature (HT; 40 ± 2 ◦ C/75 ± 5% RH) over a period of 3 months in order to see clarity of formulations, particle size and zeta potential. 2.11. Data analysis Statistical analysis of in vitro data and all the skin permeation experiments of each preparation were performed three times, and data were expressed mean value S.D. The statistical data were analyzed using non-parametric tests with a Wilcoxon test. Statistical differences are denoted as p ≤ 0.05 (NS = not significant), p ≤ 0.01 (significant) and p ≤ 0.001 (highly significant). 225 A.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 Fig. 1. Fabrication of SLNs-A. 3. Result and discussion 3.1. Formulation of ADA loaded solid lipid nanoparticles (SLNs-A) and SLNs-A gel The SLNs were fabricated by hot homogenization technique by employing solvent injection method which involves intense diffusion of solvent across solvent–lipid phase in aqueous phase followed by evaporation of solvent and resulting in enhanced rigidity of lipid nanoparticles. High speed stirring was deployed to obtain a pre-emulsion phase prior to homogenization, ADA was dispersed homogeneously in the molten lipid, and acetone and ethanol solvent was used. A hot water bath was used to maintain pre-emulsion on or above the melting temperature (Fig. 1). Furthermore, preparation of ADA loaded SLNs was characterized by FTIR spectroscopy (data not shown). The prepared SLNs were incorporated into 1% Carbopol® 934 gel. The gel neutralized with triethanolamine and a grizzled, homomorphic viscous SLNs-A gel was produced. of SLNs formulations, and might prove beneficial in reducing skin irritation 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 different SLNs-A formulations were measured by photon correlation spectroscopy using zeta-sizer (ZS 90, Malvern Instruments, UK) (Table 1). The increased surfactant concentration resulted in a decrease in particle size at a certain threshold. The average size of all formulations falls between 140 and 220 nm range. The surfactant concentration dependent change (decrease in particle size) also showed an impact on polydispersity index and entrapment efficiency of optimized formulation. The chylomicron mimicking behaviour of SLNs might achieve size nearly equivalent to SLNs-A that has most narrow size distribution of 0.169. This formulation was considered optimized formulation with average particle size (148.3 ± 2.5 nm) (Table 1) and highest EE (89.90 ± 1.2) as compared to that seen with other formulations. The zeta potential is an 3.2. Estimation of drug entrapment efficiency The greater entrapment efficiency (89.90 ± 1.9%) of SLNs-A was found at 1% surfactant concentration (Table 1). However, further increase (greater than 1%) in surfactant concentration has shown a loss of entrapment efficiency and increased Poly Dispersity index (PDI). We believe loss in entrapment efficiency is probably due to surface leaching of the entrapped drug. The high lipophilicity and better compatibility between drug and lipid may result in high EE Table 1 Comparison of % drug entrapped, particle size and polydispersity index (PDI) (n = 6). SLNs-A formulation code Surfactant conc. (%) Particle size PDI EE A B C D 0.5 1.0 1.5 2.0 219.1 148.3 141.5 197.5 0.417 0.169 0.218 0.283 74.90 89.90 80.90 69.90 ± ± ± ± 3.2 2.5 6.5 5.2 ± ± ± ± 1.2 1.2 1.2 1.2 226 A.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 3.4. Analysis of rheological and textural profile of SLNs-A gel Fig. 2. TEM photomicrographs of SLNs-A. important criterion for examining the storage stability of lipid particles and their cellular demeanour in drug release. The zeta potential of optimized formulations of SLNs-A was −12.0 mV (Table 1). The higher negative value of zeta potential provides repulsive interaction between SLNs, and thus prevents aggregation of nanoparticles. In addition, tween 80 used in this study also provided stearic stability to achieve stable formulation. TEM results confirmed spherical shape of prepared SLNs-A, and photomicrographs (Fig. 2) of prepared formulation are suggestive of their nanometric size range and narrow size distributions. A rheological study demonstrates flow behaviour of SLNs-A gel. The value of flow behaviour index obtained for SLNs-A gel was found less than unity (n = 0.89613), which indicates shearthinning behaviour of SLNs-A. Herschel–Bulky model was preferred for SLNs-A gel due to its suitability for current study [27]. The rheograms represent values of shear stress and viscosity obtained at varying shear rates, these have been delineated in Fig. 3a–c. The present results show an initial increase in viscosity are directly correlated to increased shear rate, and started revealing decrease in second phase. The graph was plotted on log scale between viscosity at Y1 , shear stress at Y2 and shear rate at X1 (Fig. 3a). The consistency value was found 1.146 Pa.s, and high yield stress value was calculated as high as 23.473 Pa. The result obtained from amplitude LVR test revealed that storage modulus (G′ ) is quite high as compared to loss modulus (G′′ ) which further assures high elasticity of gel that retains virtue of less dissipation of energy, and loss in modulus will be larger only when sample is predominantly viscous [28]. The graph plotted between G′ , G′′ at Y1 , shear stress at Y2 and strain at X1 has been found linear (Fig. 3b). The loss in tangent is the measure of the energy, lost to stored energy, in the cyclic deformation (tan ı = G′′ /G′ ). A value of tan ı < 1 does mean a prevalent elastic demeanour [29]. The outcome of frequency sweep study made possible to determine the internal alteration in the structure of the gel. The test was conducted at 1% strain under changing 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 frequency range. As is shown in Fig. 3c logarithm graph, no cross-over was seen at ambient temperature. Furthermore, storage and loss modulus saw an increase where decrease in complex viscosity was observed in linear mode. This pattern delineates higher efficiency of sample (increase in modulus) with a decrease in measurement time (frequency = 1/time). In brief, if gel formulation has attain a particular phase and viscosity, it can be easily transported and stored Fig. 3. (a) Flow curve graph shows that if increase in shear rate, viscosity of gel initially increases and at the second point it begin to decrease. (b) Amplitude LVE test: linear plot between G′ , G′′ at Y1 , shear stress at Y2 and linear strain at X1 . (c) Frequency dependency test at 1% strain: no crossover was found at ambient room temperature, also the storage modulus and loss modulus increases whereas complex viscosity decreases. (d) Textural behaviour of SLNs-A gel. A.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 227 Fig. 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. at ambient temperature provided it is not subjected to any shear changes which might alter their viscosity and in turn stability and structure. Other rheological characteristics, i.e. firmness, stickiness and work of adhesion were further characterized by textural profile analysis (TPA). Texture curve (Fig. 3d) shows uniformity, smoothness and absence of any grittiness or lumps in the SLNs-A gel. The SLNs-A gel confirmed fairly good gel strength, diffusion of sample at ease and extrusion from the tube. The gel was discovered to have shown adequate cohesiveness which is pre-requisite to sustain formulation adhering to the site of application. In conclusion, developed SLNs-A gel was met with pre-requisite criteria viz., cohesiveness, spread-ability, ease of extrusion, absence of grittiness and particulate matter as required for any topical preparation. 3.5. In vitro drug release study The drug release was studied intermittently starting from 0 h to 48 h. The initial burst release of 25.57 ± 0.4% and 16.82 ± 0.04% from SLNs-A and SLNs-A gel was an interesting observation respectively and followed slower and sustained release (Fig. 4). The initial burst is probably due to rapid release of drug adsorbed on the surface or presence of drug just underneath the stratum of the SLNs. The slower and sustained release, however, attributed to the diffusion of drug molecules through lipid matrix of SLNs. Furthermore, an abundant reduction in cumulative release of ADA from SLNs formulation observed is the virtue of existence of lipid matrix hinders drug to release. However, a substantial decrease in the cumulative release of ADA from SLN-A gel was observed in comparison to SLNA formulation. Consistent to similar study conducted by Jain et al. [11], our results describe the presence of gel matrix surrounding SLN-A, and impede the drug release. 3.6. Ex vivo skin permeation studies Franz diffusion cells were used to evaluate the skin uptake and skin targeting potential of SLNs. The permeation ability of ADA from SLNs formulation into rat skin was determined by sampling rat from 0 to 8 h (clinical application time) [30]. 0.15% ADA tincture in Methanol: DMF was used as a reference to evaluate skin targeting ability of SLNs-A and SLNs-A gel. The insignificant amount of ADA in the receptor chamber for SLNs-A and SLNs-A gel was observed even after 8 h which indicated the inability of ADA from SLNs-A and SLNs-A gel in penetrating skin. The amount of ADA in the receptor chamber from reference tincture showed a steady increase with an increase in time (Fig. 5), and a permeation rate was calculated, 76 ± 0.3 ␮g cm−2 h−1 (permeation followed zero order release kinetics). The low inadequate concentration of ADA in epidermis of samples treated with tincture is due to rapid diffusion Fig. 5. Skin distribution of ADA delivered via SLNs-A and SLNs-A gel into different skin 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) for ADA measured in the receptor compartment. The statistical data is expressed as mean ± SE (n = 6). (because of penetration-enhancement properties of methanol) of ADA in the acceptor medium. Moreover, rapid permeation and loss of methanol due to evaporation might increase concentration of ADA in tincture which in turn increases thermodynamic activity of ADA [31]. Aforementioned are a few important factors which might contribute to the permeation of ADA through skin from alcoholic solution. It is therefore concluded that SLNs-A and SLNs-A gel minimizes or avoids systemic uptake of ADA when compared to tincture, and presents robust system(s) to avoid or minimize systemic adverse side effect. This may be due to reduced or no skin dehydration by SLNs-A and SLNs-A gel, and as a consequence of which hydrated stratum corneum facilitates drug penetration into deeper strata of skin [32]. In brief, the components of SLNs, may fuse in skin and mix with lipids to loosen up their structure which hindered lamellar arrangement of lipids with increased thickness of the stratum corneum [33,34]. 3.7. Skin distribution study These results are suggestive of the efficiency of SLNs-A and SLNs-A gel to allow ADA permeation into skin, and quantification of ADA content in epidermis, dermis, and receptor fluids (Fig. 5). SLNs formulations deliver quantifiable amounts of ADA into epidermal layer of skin, but minimal ADA quantity was observed in the dermis. Results also obviated no effect of extended viscosity of SLNs-A gel on the deposition of ADA into skin at each investigated skin layer. However, it showed significant increase in the retention time of formulation at the site of application. The current results also revealed that ADA comprehensively remains in the epidermal layer because higher hydrophilicity of dermal layer circumscribe valuable partitioning of hydrophobic drug [26]. The outcome from skin distribution study showed advancement of SLNs based ADA formulations, i.e. an efficient topical drug delivery system with least transdermal localization and avoids systemic absorption. Consistent to what has been reported earlier [35,36], small sized SLNs showed their merit in improving penetration ability of nanoparticles into the skin, and higher drug accumulation due to sustained release from SLNs. The interaction of SLNs lipids and surfactants with skin lipids is presumably an important factor leading to greater penetration [37]. Moreover, occlusive effect is also an important factor affecting penetration of drug into skins (due to small particle size of carrier), which might have more effect controlling penetration of nano-actives into the skin. 228 A.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 Fig. 6. Histopathology study shown qualitative uptake and the distribution of the fluorescent marker loaded nanocarriers throughout skin section. (A) SLN-A; (B) SLN-A gel. 3.8. Histopathology study We decided to perform fluorescence microscopy study to confirm and to determine the qualitative uptake and distribution pattern of lipid nano-carriers into the skin. FITC loaded SLN-A gel and SLNs with no gel loaded was used in order to determine extent of penetration of SLNs into the skin. The photomicrographs reflect green fluorescence intensity in the different layers of skin. The fluorescence was observed throughout the skin (Fig. 6a and b) suggesting lipophilic nature of SLNs improved in vivo transdermal delivery, and also allowing stockpiling and transportation of bioactive through the vasculature in dermis and epidermis. 3.9. Stability study SLNs-A and SLNs-A gel has shown the stability of formulation over the period of three months. No considerable variations in clarity, and phase separation were observed, demonstrating good physical stability of SLNs. Moreover, SLNs-A was found stable in centrifuge test, and the stability might inherit from the lethargic transition of dispersed lipid from metastable forms to the stable form in SLNs, low particles size, and steric effect of Tween 80 [38]. The minimal or non-significant degradation of ADA in SLNs and SLNs gel was seen with stable transparency for over 3 months. studies have been reported on ADA loaded SLNs. SLNs of ADA were prepared by hot homogenization method followed by the incorporation into carbopol gel for topical delivery. The current study indicates targeting prospective, spatial delivery and elevated retention potential of the formulation in epidermal tissues. The in vitro permeation study illustrates a low or no systemic uptake of ADA when compared with reference. The small diameter of SLNs and increased permeation effect of soya lecithin on stratum corneum may contribute to the penetration of SLNs-A into the stratum corneum, and shows a better skin targeting effect. Thus, the small diameter of SLNs, and soya lecithin might open a way to facilitate targeted delivery of ADA into the dermis layer. These NPs accumulated particularly in the epidermis, and maintained in the skin layer at significant limits. In conclusion, the present study opens new vista for SLNs as efficient vectors to carry large and systemic doses of ADA as evidenced from in vitro studies, showing a sustained release nature of formulation. The developed SLNsA nanoparticulate system demonstrated the optimal therapeutic response, improved therapeutic efficacy and minimal penetration across epidermis with an interception of minimal side effects. The current study is an important step towards skin acne and drug delivery, and might be a step forward in unravelling skin targeting mechanism and help addressing acne problems. 4. Conclusions and future perspectives Acknowledgements Very recently several studies have been reported for the effective topical delivery of different kinds of anti-acne molecules. The various colloidal carriers like liposomes and mixed vesicles [39], lipid-nanoparticles [40] and microemulsion [41] were employed to either improve the physicochemical properties of drugs or to improve their pre-clinical efficacy. However, there are no The authors are grateful for the fellowship and grant provided by the UGC, AICTE, and CSIR HRDG(Award # 09/135(0667)/2012EMR-I), New Delhi, India. The authors also thank Glenmark pharmaceuticals Ltd. (Nasik, India) for providing ADA as gift sample, Lipoid, Ludwigshafen, Germany for providing Hydrogenated soya phosphatidylcholine (HSPC). A.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 121 (2014) 222–229 References [1] Z.D. Draelos, E. Carter, J.M. Maloney, B. Elewski, Y. Poulin, C. Lynde, et al., Two randomized studies demonstrate the efficacy and safety of dapsone gel, 5% for the treatment of Acne vulgaris, J. Am. Acad. Dermatol. 56 (2007), http://dx.doi.org/10.1016/j.jaad.2006.10.005, 439.e1–439.e10. [2] K. Raza, B. Singh, P. Singal, S. Wadhwa, O.P. Katare, Systematically optimized biocompatible isotretinoin-loaded solid lipid nanoparticles (SLNs) for topical treatment of acne, Colloids Surf. B: Biointerfaces 105 (2013) 67–74, http://dx.doi.org/10.1016/j.colsurfb.2012.12.043. [3] M. Wainwright, H. Smalley, C. Flint, The use of photosensitisers in acne treatment, J. Photochem. Photobiol. B 105 (2011) 1–5, http://dx.doi.org/ 10.1016/j.jphotobiol.2011.06.002. [4] R. Berger, R. Rizer, A. Barba, D. Wilson, D. Stewart, R. Grossman, et al., Tretinoin gel microspheres 0.04% versus 0.1% in adolescents and adults with mild to moderate Acne vulgaris: a 12-week, multicenter, randomized, double-blind, parallel-group, phase IV trial, Clin. Ther. 29 (2007) 1086–1097, http://dx.doi.org/10.1016/j.clinthera.2007.06.021. [5] D. Thiboutot, H. Gollnick, V. Bettoli, B. Dréno, S. Kang, J.J. Leyden, et al., New insights into the management of acne: an update from the Global Alliance to Improve Outcomes in Acne group, J. Am. Acad. Dermatol. 60 (2009) S1–S50, http://dx.doi.org/10.1016/j.jaad.2009.01.019. [6] O.H. Mills, R.S. Berger, Irritation potential of a new topical tretinoin formulation and a commercially-available tretinoin formulation as measured by patch testing in human subjects, J. Am. Acad. Dermatol. 38 (4) (1998) S11–S16. [7] J.J. Leyden, A. Shalita, D. Thiboutot, K. Washenik, G. Webster, Topical retinoids in inflammatory acne: a retrospective, investigator-blinded, vehicle-controlled, photographic assessment, Clin. Ther. 27 (2005) 216–224. [8] A. Kovacevic, S. Savic, G. Vuleta, R.H. Müller, C.M. Keck, Polyhydroxy surfactants for the formulation of lipid nanoparticles (SLN and NLC): effects on size, physical stability and particle matrix structure, Int. J. Pharm. 406 (2011) 163–172, http://dx.doi.org/10.1016/j.ijpharm.2010.12.036. [9] R.H. Müller, R.D. Petersen, A. Hommoss, J. Pardeike, Nanostructured lipid carriers (NLC) in cosmetic dermal products, Adv. Drug Deliv. Rev. 59 (2007) 522–530, http://dx.doi.org/10.1016/j.addr.2007.04.012. [10] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release 70 (2001) 1–20. [11] A. Jain, A. Agarwal, S. Majumder, N. Lariya, A. Khaya, H. Agrawal, et al., Mannosylated solid lipid nanoparticles as vectors for site-specific delivery of an anti-cancer drug, J. Control. Release 148 (2010) 359–367, http://dx.doi.org/ 10.1016/j.jconrel.2010.09.003. [12] N.K. Garg, P. Dwivedi, C. Campbell, R.K. Tyagi, Site specific/targeted delivery of gemcitabine through anisamide anchored chitosan/poly ethylene glycol nanoparticles: an improved understanding of lung cancer therapeutic intervention, Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 47 (2012) 1006–1014, http://dx.doi.org/10.1016/j.ejps.2012.09.012. [13] A. Zur Mühlen, C. Schwarz, W. Mehnert, Solid lipid nanoparticles (SLN) for controlled drug delivery – drug release and release mechanism, Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Fur Pharm. Verfahrenstechnik eV 45 (1998) 149–155. [14] C.E. Irby, B.A. Yentzer, S.R. Feldman, A review of adapalene in the treatment of Acne vulgaris, Drugs 43 (2004) 421–424. [15] M. Kawashima, S. Harada, C. Loesche, Y. Miyachi, Adapalene gel 0.1% is effective and safe for Japanese patients with Acne vulgaris: a randomized, multicenter, investigator-blinded, controlled study, J. Dermatol. Sci. 49 (2008) 241–248, http://dx.doi.org/10.1016/j.jdermsci.2007.09.012. [16] T. Sato, N. Akimoto, K. Kitamura, H. Kurihara, N. Hayashi, A. Ito, Adapalene suppresses sebum accumulation via the inhibition of triacylglycerol biosynthesis and perilipin expression in differentiated hamster sebocytes in vitro, J. Dermatol. Sci. 70 (2013) 204–210, http://dx.doi.org/10.1016/j.jdermsci.2013.02.003. [17] B. Shroot, S. Michel, Pharmacology and chemistry of adapalene, J. Am. Acad. Dermatol. 36 (1997) S96–S103. [18] J. Bastien, C. Rochette-Egly, Nuclear retinoid receptors and the transcription of retinoid-target genes, Gene 328 (2004) 1–16, http://dx.doi.org/10.1016/ j.gene.2003.12.005. [19] M.A. Schubert, C.C. Müller-Goymann, Solvent injection as a new approach for manufacturing lipid nanoparticles – evaluation of the method and process [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] 229 parameters, Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Fur Pharm. Verfahrenstechnik eV 55 (2003) 125–131. L.A. Martins, L.Z. Meneghini, C.A. Junqueira, D.C. Ceni, A.M. Bergold, A simple HPLC-DAD method for determination of adapalene in topical gel formulation, J. Chromatogr. Sci. 49 (2011) 796–800. S.C. Shin, J.Y. Kim, I.J. Oh, Mucoadhesive and physicochemical characterization of Carbopol-Poloxamer gels containing triamcinolone acetonide, Drug Dev. Ind. Pharm. 26 (2000) 307–312. B. Chen, H. Li, Y. Ding, H. Suo, Formation and microstructural characterization of whey protein isolate/beet pectin coacervations by laccase catalyzed crosslinking, LWT – Food Sci. Technol. 47 (2012) 31–38, http://dx.doi.org/10.1016/ j.lwt.2012.01.006. J. Liu, W. Hu, H. Chen, Q. Ni, H. Xu, X. Yang, Isotretinoin-loaded solid lipid nanoparticles with skin targeting for topical delivery, Int. J. Pharm. 328 (2007) 191–195, http://dx.doi.org/10.1016/j.ijpharm.2006.08.007. W. Tiyaboonchai, W. Tungpradit, P. Plianbangchang, Formulation and characterization of curcuminoids loaded solid lipid nanoparticles, Int. J. Pharm. 337 (2007) 299–306. Z. Mei, H. Chen, T. Weng, Y. Yang, X. Yang, Solid lipid nanoparticle and microemulsion for topical delivery of triptolide, Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Fur Pharm. Verfahrenstechnik eV 56 (2003) 189–196. B.E. Kilfoyle, L. Sheihet, Z. Zhang, M. Laohoo, J. Kohn, B.B. Michniak-Kohn, Development of paclitaxel-TyroSpheres for topical skin treatment, J. Control. Release 163 (2012) 18–24, http://dx.doi.org/10.1016/j.jconrel.2012.06.021. R. Basu, U.S. Shivhare, G.S.V. Raghavan, Time dependent rheological characteristics of pineapple jam, Int. J. Food Eng. 3 (2007). S. Tamburic, D. Craig, An investigation into the rheological, dielectric and mucoadhesive properties of poly(acrylic acid) gel systems, J. Control. Release 37 (1995) 59–68, http://dx.doi.org/10.1016/0168-3659(95)00064-F. F. Madsen, K. Eberth, J.D. Smart, A rheological examination of the mucoadhesive/mucus interaction: the effect of mucoadhesive type and concentration, J. Control. Release 50 (1998) 167–178. M. Trotta, E. Ugazio, E. Peira, C. Pulitano, Influence of ion pairing on topical delivery of retinoic acid from microemulsions, J. Control. Release 86 (2003) 315–321. S. Wissing, A. Lippacher, R. Müller, Investigations on the occlusive properties of solid lipid nanoparticles (SLN), J. Cosmet. Sci. 52 (2001) 313–324. G. Cevc, Lipid vesicles and other colloids as drug carriers on the skin, Adv. Drug Deliv. Rev. 56 (2004) 675–711. Q. Lv, A. Yu, Y. Xi, H. Li, Z. Song, J. Cui, et al., Development and evaluation of penciclovir-loaded solid lipid nanoparticles for topical delivery, Int. J. Pharm. 372 (2009) 191–198, http://dx.doi.org/10.1016/j.ijpharm.2009.01.014. Y. Yokomizo, H. Sagitani, Effects of phospholipids on the percutaneous penetration of indomethacin through the dorsal skin of guinea pigs in vitro, J. Control. Release 38 (1996) 267–274. E.B. Souto, S.A. Wissing, C.M. Barbosa, R.H. Müller, Development of a controlled release formulation based on SLN and NLC for topical clotrimazole delivery, Int. J. Pharm. 278 (2004) 71–77. A.C. Williams, B.W. Barry, Penetration enhancers, Adv. Drug Deliv. Rev. 56 (2004) 603–618. R.H. Müller, M. Radtke, S.A. Wissing, Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations, Adv. Drug Deliv. Rev. 54 (Suppl. 1) (2002) S131–S155, http://dx.doi.org/ 10.1016/S0169-409X(02)00118-7. V. Venkateswarlu, K. Manjunath, Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles, J. Control. Release 95 (2004) 627–638. A. Bhatia, B. Singh, K. Raza, S. Wadhwa, O. Prakash, Tamoxifen-loaded lecithin organogel (LO) for topical application: development, optimization and characterization, Int. J. Pharm. 444 (2013) 47–59, http://dx.doi.org/ 10.1016/j.ijpharm.2013.01.029. V. Chawla, S.A. Saraf, Rheological studies on solid lipid nanoparticle based carbopol gels of aceclofenac, Colloids Surf. B: Biointerfaces 92 (2012) 293–298, http://dx.doi.org/10.1016/j.colsurfb.2011.12.006. G. Bhatia, Y. Zhou, A.K. Banga, Adapalene microemulsion for transfollicular drug delivery, J. Pharm. Sci. 102 (2013) 2622–2631, http://dx.doi.org/10.1002/ jps.23627.