Development of Piroxicam loaded SLN-based Hydrogel for Transdermal Delivery

Transcription

1 Verma: Development of Piroxicam loaded SLN-based Hydrogel for Transdermal Delivery 1 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 7 Issue 1 January March 2014 Research Paper IJPSN VERMA Development of Piroxicam loaded SLN-based Hydrogel for Transdermal Delivery Vinod Kumar Verma* and Ram Alpana SLT Institutes of Pharmaceutical Science, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chattishgarh, India. Received April 2, 2013; accepted August 5, 2013 ABSTRACT The aim of present study was to construct and investigate the efficacy of solid lipid nanoparticles (SLNs) based hydrogel for transdermal delivery of non-steroidal antiinflammatory drug (NSAID) piroxicam. Solid lipid nanoparticles (SLNs) of piroxicam were produced by solvent emulsification diffusion method in a solvent saturated system. The SLNs were composed of tripalmitin lipid, polyvinyl alcohol (PVA) as stabilizer, and solvent ethyl acetate. All the formulations were subjected to particle size analysis, zeta potential, drug entrapment efficiency, percent drug loading determination and in-vitro release studies. The SLNs formed were in nano-size range with maximum entrapment efficiency of 88.50±0.92. Further, Carbopol-934 was used as a gel matrix for preparation of hydrogel for improving the penetration rate across the skin and viscosity of piroxicam loaded SLNs for transdermal drug administration. The drug release behaviors from piroxicam loaded SLNs based hydrogel exhibited long duration and constant rate of drug release over 24 hr. Skin penetration of piroxicam loaded SLNs based hydrogel was assessed by confocal laser scanning microscopy (CLSM). KEYWORDS: Solid lipid nanoparticles (SLNs); Tripalmitin; Polyvinyl alcohol; Piroxicam, Drug entrapment efficiency; Hydrogel. Introduction Piroxicam is an anionic non-steroidal antiinflammatory drug (NSAID). It is a derivative of oxicam and widely used in the management and treatment of patients with rheumatoid arthritis, osteoarthritis disease. However, it has obvious adverse side effects upon oral administration. So, it is necessary to improve the skin uptake of piroxicam using a carrier with an ability of skin targeting. At the beginning of the 1990s, the advantages of solid particles, emulsions and liposomes were combined by the development of the solid lipid nanoparticles (SLNs). The SLNs were released by simply exchanging the liquid lipid (oil) of the emulsions by a solid lipid. There are two basic production methods for SLNs, the high pressure homogenization technique developed by Muller and Lucks (Muller et al., 1996) and the microemulsion technique invented by Gasco in Turin (Gasco 1993). The solid lipid nanoparticles (SLNs) are submicron colloidal carrier ( nm), which are composed of physiological lipid, dispersed in water or in an aqueous surfactant solution. SLNs as colloidal drug carrier combine the advantages of polymeric nanoparticles, fat emulsion and liposomes simultaneously and avoiding some of their disadvantages. In order to overcome the disadvantages associated with the liquid state of the oil droplets, the liquid lipid was replaced by a solid lipid, which eventually transformed into solid lipid Nanoparticles. Some of the advantages of SLNs as an alternative particulate carrier are cited in the literature. (Muller et al., 1995; ZurMuhlen et al., 1998). During the past several years, Solid lipid nanoparticles (SLNs) began to act as atypical carrier not only for pharmaceutical drugs but also for cosmetic active ingredients. (Muller et al., 2002). Compared with conventional carriers such as cream, tincture and emulsion, SLNs combine their advantages such as controlled release, good toleration in vivo and protection of active compounds from chemical degradation (Muller et al., 2000; Sylvia et al., 2003) especially, SLNs can favor drug penetration into the skin by producing occlusive properties which can increase the water content of the skin, maintain a sustained supply of the drug to the skin over a prolong period of time, and avoid systemic absorption, act as a UV sunscreen system and reduce irritation and improve the tolerance. (Jenning et al., 2000; Wissing et al., 2003; Zur Muhlen et al., 1998; Wissing et al., 2000; Maia et al., 2000; Sivaramakrishnan et al., 2004). A major problem in transdermal drug delivery is the low penetration rate of most substances through the barrier of the skin, the stratum corneum. One of the methods to increase the penetration rate across the skin is to encapsulate the drug in vesicular carriers, like liposomes. (Bouwstra et al., 1998). The transdermal route has been considered as a possible site for the systemic and localized delivery of drug. The possible benefits of topical drug delivery include that the drug can be delivered for a long duration 2338

2 Verma: Development of Piroxicam loaded SLN-based Hydrogel for Transdermal Delivery 2339 at a constant rate and drug can bypass hepatic first pass metabolism. The aqueous dispersion of solid lipid nanoparticles (SLNs) based hydrogel represents an alternative drug carrier system to emulsions and polymeric nanoparticles for topical application (Muller et al., 2000). The first report on this subject was by Mezei and Gulasekharam, in 1980, after which many other publications followed in this field (Schreier et al., 1994). The aim of the present study was to develop piroxicam loaded solid lipid nanoparticles based hydrogel for topical administration as single dose therapy, to determine the possibility of producing controlled drug release, drug targeting to the specific site, increase in the drug stability, high drug payload and penetration of the SLNs based hydrogel into the skin to investigate the thermodynamic state and flexibility of the SLNs. Materials and Methods Materials Piroxicam was provided as gift sample by m/s Ramdev Pharmaceutical Pvt. Ltd. Thane, Maharastra (INDIA). Tripalmitin lipid, Polyvinyl alcohol (PVA), Carbopol-934, Ethyl acetate and Rodamine B-6 was purchased from HiMedia Laboratory Pvt. Ltd. Mumbai Maharastra (INDIA). All other chemicals were of HPLC or analytical grade. Preparation of SLNs based hydrogel The piroxicam loaded SLN formulations were prepared using solvent emulsification diffusion method. A method adopted from Quintanar-Guerrero et al., 1996; 1998b; 1999a; 1999b). The method used with slightly modification and preparation of SLNs were described in previous work which were further used to form hydrogel was published. (Verma et al., 2010) Formulations were optimized on the basis of process variables viz. stirring rate, temperatue, concentration of lipid and concentration of stabilizer. The effect of these variables was observed on particle size, percent yield, drug entrapment efficiency and percent drug loading. The optimized formulations of piroxicam loaded SLNs based hydrogel were prepared using gel forming polymer according to a method. (Jenning and Gohla et. al., 2000) Ten percent glycerol (85%), 69.5% water and the gelling agent 0.5% (Carbopol-934) were weighed in a beaker and stirred at approximately 1000 rpm for 5 min. Finally, the SLNs dispersion (20%), respectively, was added under continuous stirring. The dispersion was neutralized with 1% w/v NaOH solution and the ph was adjusted to 6.0. The SLNs-loaded hydrogels were stored at room temperature for 1 month. Characterization of Piroxicam Loaded SLNs The SLNs characterization parameter like, particle size, shape, zeta potential, surface morphology drug entrapment efficiency (EE), percent drug loading (L), chemical and physical stabilities of selected piroxicam loaded SLNs and selected piroxicam loaded SLNs formulation in-vitro drug release studies were described in previously published article. (Verma et al., 2010) On continuation of study the piroxicam loaded SLNs used for the further study. Nature of drug in nanoparticles Differential Scanning Calorimetry (DSC) The in-vitro drug and lipid interaction was investigated by differential scanning calorimeter (DSC) (METTLER 305 Switzerland). The curve of piroxicam, piroxicam loaded tripalmitin SLNs and unloaded tripalmitin SLNs were recorded on a DSC equipped with a computerized data station. The instrument was calibrated with indium. All samples (about 5 mg) were heated in crimped aluminum pans (METTLER 305 Switzerland) at scanning rate of 10 ºC mn -1 using dry nitrogen flow (30 ml min 1 ). Characterization of Piroxicam Loaded SLNs based Hydrogel Rheological measurements The rheological measurements were performed on a rheometer Rheo Stress RS 100 (Haake Instruments, Karlsruhe, Germany) equipped with a cone-and-plate test geometry (plate diameter 20 mm, cone angle 48). All measurements were carried out at a temperature of 25 ± 0.18 ºC. The rheological properties of the developed piroxicam loaded SLN based hydrogels were studied by continuous shear investigations, which were performed in order to evaluate the shear rate [1/s] as a function of shear stress [Pa]. This study started applying 0 Pa up to a maximum shear stress of 40 Pa and the resulting shear rate was measured. In- vitro drug release from piroxicam loaded SLNs based hydrogel The in-vitro release studies were performed using modified Franz diffusion cell having a surface area of cm 2 and 75 ml capacity. Dialysis membrane (LA 401) having pore size 2.4 nm, molecular weight cutoff (HIMEDIA), was used. Membrane was soaked in distilled water for 12 hours before mounting in the cell. Selected Piroxicam loaded SLNs based Hydrogel formulations equivalent to 5 mg of drug was placed in the donor compartment and the recipient compartment was filled with dialysis medium (Phosphate buffer of ph 7.4, 75 ml). The content of the cell was stirred with the help of magnetic stirrer at 37 C. At fixed time interval of 0, 1, 2, 3, 4, 5, 6, 12, 18 and 24 hours 1 ml of sample was withdrawn from the receiver compartment through side tube. Fresh phosphate buffer solution (PBS) of ph 7.4 was placed to maintain constant volume. Samples were analyzed by UV-spectrophotometry at 326 nm. In vitro skin permeation studies The in- vitro skin permeation studies were carried out in excised Human Cadaver Skin (HCS). The skin collected was not older than 5 days after postmortem and stored at 4 ºC. The full-thickness abdomen skin from a single porcine was used for all the permeation

3 2340 Int J Pharm Sci Nanotech Vol 7; Issue 1 January March 2014 experiments. Hair was removed with a shaver, excised and examined for integrity and rinsed with physiological saline. The fat tissues below skin were carefully chopped. The abdomen skin from a single porcine was cut into many pieces because of a large area of abdomen skin. The thickness of each skin was similar. The skins pieces were clamped between the donor and the receptor chamber of vertical diffusion cell with an effective diffusion area of cm 2 and a cell volume of 100 ml. The receptor chambers were filled with freshly physiological saline. The diffusion cells were maintained at 37 ± 1 C using air-circulating water bath and the fluid in the receptor chambers was stirred continuously at 300 rpm. The piroxicam loaded SLNs based hydrogel formulations (1.0 g) were gently placed in the donor chambers. At 0, 1, 2, 3, 4, 5, 6, 12, 18 and 24 hr, 2 ml of the fluid in the receptor chambers were sampled for UV-spectrophotometric determination at 326 nm and replaced immediately with an equal volume of fresh physiological saline. Each preparation was studied three times. The result of each preparation is the average value of three experiments. The amount of piroxicam in the total receptor solution was determined from a calibration curve. The cumulative drug permeated (Qn) corresponding to the time of the n th sample was calculated from the following equation. n 1 Q =V C + V C n R n S i i=0 Where Cn and Ci are the drug concentrations of the receptor solution at the time of the n th sample and the i (the first) sample, respectively and VR and VS are the volumes of the receptor solution and the sample, respectively. The cumulative amounts of piroxicam from SLNs based hydrogel permeated through porcine skins were plotted as a function of time. The permeation rate of piroxicam from piroxicam loaded SLN based hydrogel at steady state flux (Jss, μg cm 2 h 1 ) was calculated from the slope of the linear portion of the cumulative amount permeated per unit surface area (μg/cm 2 ) versus time (h) plot. Permeability coefficient (Kp) was calculated using the relation derived from Fick s first law of diffusion, which described in the following equation: Jss = Kp/Co Where, Co is the initial drug concentration in the donor. (Williams et al., 2003). At the end of the experiments, the skins were removed and rinsed with distilled water. Then skins were respectively soaked in 5 ml of methanol for 24 hr and were subjected to five sonication cycles of 30 min each in an ultrasound bath, followed by centrifugal separation. The skin accumulative amount, namely the total amount of piroxicam extracted from skin at the end of the permeation study (at 24 hr) could be obtained from the concentration of piroxicam in supernatant methanol. (Baroli et al., 2000; Chen et al., 2006; Souto et al., 2004). The extracted piroxicam from skins was analyzed by UVspectrophotometer. It was observed that the sonication had no influence on the stability of piroxicam since no change in UV spectrometric peak signal of piroxicam in the residual extracted skins was observed. Skin sectioning and confocal laser scanning microscopy imaging studies Skin penetration of piroxicam loaded SLNs based hydrogel were assessed by confocal laser scanning microscopy (CLSM) after application of piroxicam loaded SLNs containing 0.05% Rodamine B6 homogeneously and non-occlusive containing probe. The experiments were run in Franz diffusion cells and the receiver contained PBS ph 5.5 solution. After 8 hr, the skin was removed and washed with PBS. A slicer (LEICA CM 1900) was used to prepare the cross-section of full skin perpendicular to the skin surface and horizontal section of skin parallel to the skin surface. The rinsed skin from the in vivo experiment were frozen at 20 C by liquid nitrogen and fixed by a viscous gel. (Baroli et al., 2000). This skin was fixed on the sample holder with the help of a skin frozen medium gel. (Gung LEICA, Instruments). Perpendicular sections of the skin (dermis to horny layer) 200 µm thickness were cut with the help of cryomicrotome (LEICA, Germany). The treated area was cutout and tested for probe penetration. The full skin thickness was optically scanned at µm increments through the Z-axis of a LEICA DMIRE2 confocal laser scanning microscope attached to a LEICA TCS SP2 fluorescence microscope used for visualizing the distribution of piroxicam loaded SLNs in skin. Optical excitation was carried out with a 488 nm argon laser-beam and Rodamine B-6 fluorescence emission was detected above 555 nm. No autofluoresence of skin was observed. Statistical Data Analysis All the Statistical analysis was performed with statistical (SPSS 13.0) software package and data were expressed as the mean value ± SD. The statistical significance were determined using the Student s t-test and analysis of variance (ANOVA) with p<0.05 as a minimal level of significance. Results and Discussion Differential Scanning Calorimetry (DSC) The in-vitro drug and lipid interaction was investigated by differential scanning calorimeter (DSC) shown in figure 1a & 1b, there was no interaction observe in the complex system of drug and polymer. Rheological measurements Rheological measurements are useful for the characterization of the viscoelastic properties of aqueous SLNs dispersions and of SLNs based hydrogels. Therefore continuous shear investigations were performed in the tested hydrogel formulations in order to evaluate the shear rate as a function of shear stress. (Souto et al., 2003) Conventional SLNs and NLC aqueous dispersions contain about 10 20% (w/w) of lipid matrix and 80 90% (w/w) of water. (Souto et al., 2003) As a

4 Verma: Development of Piroxicam loaded SLN-based Hydrogel for Transdermal Delivery 2341 result, solid lipid dispersions possess a low viscosity (approximately 100 mpa) and a yield value of practically zero. Therefore, the liquid solid lipid dispersions usually have to be incorporated inconvenient topical dosage forms like hydrogels or creams to obtain a topical application form having the desired semisolid consistency. (Lippacher et al., 2001; 2002) Incompatibilities with ingredients from the hydrogel or cream may occur due to interactions between the gel forming polymer, emulsifying agents, lipid and drug. These interactions can affect the semisolid consistency of the topical formulation and, therefore, its rheological status, which is a very important physical parameter in the development of a potential new drug delivery system for topical use. Shear rate was measured and low viscosities of selected piroxicam loaded SLNs based hydrogel were observed (Table 1). Fig. 1a. DSC curve of drug unloaded tripalmitin SLNs. Fig. 1b. The DSC curve of drug piroxicam loaded tripalmitin SLNs. TABLE 1 Rheological measurements and per cent drug loading of selected piroxicam loaded SLNs based hydrogel. S.No. Formulation Code of Optimized SLNs Hydrogel Viscosity of Hydrogel (Pa) Percent (%) Drug Loading 1 SLN-B5 H SLN-C5 H SLN-B8 H SLN-C8 H Values represent the mean ± S.D. (n = 3)

5 2342 Int J Pharm Sci Nanotech Vol 7; Issue 1 January March 2014 In vitro skin permeation studies In order to assess the skin uptake and penetration of piroxicam from SLNs, the in-vitro permeation ability through skin and into skin were performed using Franz diffusion cells. (Trotta et al., 2003). The permeation studies were performed for 24 hr according to the clinical application time shown in Table 2 and graphically represented in Figure 2. It indicated that piroxicam was not found in receptor chambers from piroxicam loaded SLNs and all the piroxicam-slns formulations could not penetrate through skin. But the amount of piroxicam in receptor chamber from the excised human cadaver skin (HCS) was increased with increase of time showing a steady permeation rate of ± 0.005, ± 0.008, ± 0.012, and ± μg/cm 2 /h of formulations SLN-B5 H, SLN-C5 H, SLN-B5 H and SLN-B8 H respectively (Table 3). The permeation followed zero order release kinetics. The high permeation rate of piroxicam from excised human cadaver skin (HCS) might be due to the significant permeation enhancement effect of gelling agent and ethanol. TABLE 2 Cumulative amount of drug release profiles of Piroxicam from SLNs based Hydrogel across Excised Human cadaver skin (HCS) S.No Time Duration (Hours) Cumulative amount of Drug Release (Per unit area) μg/cm 2 SLNs based Hydrogel Formulation Code SLN-B5 H SLN-C5 H SLN-B8 H SLN-C8 H ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.34 Values represent the mean ± S.D. (n=3) Values showed statically P < 0.05 significant ± ± ± ± ± ± ± ± ± Comparison of Cumulative amount of Drug Release ( μg/cm 2 ) SLN-B5 H SLN-C5 H SLN-B8 H SLN-C8 H Cumulative amount of Drug Release (μg/cm 2 ) Time (Hour) Fig. 2. Comparison of cumulative amount of drug release profiles of piroxicam loaded SLNs based hydrogel across excised human cadaver skin (HCS).

6 Verma: Development of Piroxicam loaded SLN-based Hydrogel for Transdermal Delivery 2343 TABLE 3 Steady state transdermal permeation flux of different SLNs based hydrogel formulation for the transport of piroxicam across excised human cadaver skin (HCS). SLNs based Hydrogel Formulation Code Steady State Transdermal Flux Jss (μg/cm 2 /h) Permeability Coefficient ( 10-5 cm 2 /h) SLN-B5 H ± ± 0.25 SLN-C5 H ± ± 0.82 SLN-B8 H ± ± 0.64 SLN-C8 H ± ± 0.67 Values represent the mean ± S.D. (n = 3), Values showed statically P < 0.05 significant. In addition, during the permeation studies, the loss of ethanol can enhance the concentration of piroxicam in excised human cadaver skin (HCS), also followed by the increase of the thermodynamic activity of piroxicam. (Wissing et al., 2001; 2002; 2003). These factors might contribute to the penetration through excised human cadaver skin (HCS). It is concluded that all the Piroxicam-SLNs formulations can avoid the systemic uptake of piroxicam and SLNs present a potential to avoid the systemic adverse side effect. The accumulative amounts of piroxicam in skins from the piroxicam loaded SLN based hydrogel formulations SLN-B5 H, SLN-C5 H, SLN-B8 H and SLN-C8 H were respectively ± 0.005, ± 0.008, ± 0.012, and ± μg/cm 2 /h. (Table 3) There is no significant difference of accumulative amount of piroxicam in skin between the above formulations and they still avoided systemic absorption of piroxicam and showed skin targeting. The piroxicam loaded SLNs based hydrogel SLN-B8 H significantly increased the accumulative uptake of piroxicam in skin, compared with the other formulations (P < 0.05). There are some similar results indicating that SLNs can increase the uptake of drug in skin. (Maia et al., 2000). The skin targeting effect was disclosed by fluorescence microscopy. (Baroli et al., 2000) SLN with small diameters are advantageous to improve the penetration of nanoparticles into skins and the controlled release of SLNs may induce the increase of drug accumulation. (Quintanar et al., 1998b). In the present work, the permeation studies also supported the previous research results. However, all the piroxicam-slns formulations had small diameters. The slight difference between the average particle sizes of piroxicam-slns formulations should not be the key factors influencing the uptake of drug in skin. The increase of solid lipid in formulations resulted in the decrease of the accumulative amount of piroxicam in skins. Additionally, the increase of PVPL in piroxicam loaded SLNs hydrogel formulations SLN-B5 H, SLN-C5 H, SLN-B8 H and SLN-C8 H led to the increase of the uptake of piroxicam in skin. It is concluded that the concentration of the ingredients of SLN influenced the uptake of drug in skin. In addition, Piroxicam-SLN- C8 had only EE of 88.0 ± 0.85, but SLN-C8 H showed a high skin targeting. EE might also act as an important factor for the uptake of Piroxicam in skin. Even though the occlusive effect succeeds to elucidate the penetration of drug into skin, the skin targeting mechanism is unclear and the relative mechanism need further investigation in future. (Williams et al., 2004; Cevc et al., 2004) Skin sectioning and confocal laser scanning microscopy imaging Skin penetration of piroxicam loaded SLNs based Rodamine B-6labeled hydrogel were assessed by confocal laser scanning microscopy (CLSM) as shown in Fig. 3. After the application period of 1 to 12 hr the piroxicam loaded SLNs based Rodamine B-6 labeled hydrogel could only be detected in the stratum corneum. However a small amount of the dyed hydrogel could be observed in the viable epidermis when applied on skin. After time duration 8-12 hr post application of piroxicam loaded SLNs based Rodamine B-6 labeled hydrogel did not penetrate further than the viable epidermis and the bright and weak fluorescence were observed in dermis after the administration of SLNs based labeled hydrogel of piroxicam. Thus the SLNs based Rodamine B-6 labeled hydrogel penetrate deeper and to a higher extent into human cadaver skin (HCS) this may be due to the similarity of hydrogel properties to the human skin. Fig. 3. Confocal laser scanning microscopy (CLSM) images of a cross section of human cadaver skin (HCS) treated with rodamine B-6 labeled piroxicam loaded SLNs based hydrogel. The fluorescence images of the skin sections at the different depths beneath the skin were represented around corneocytes. Stratum corneum (SC), viable epidermis (E) and dermis (D). Schatzlein and Cevc reported similar CLSM results, when they applied transferosomes TM and liposomes vesicles on skin. (Schatzlein et al., 1995; Cevc et al., 2004) However, they described an important discrepancy in penetration depth of the label, which was applied in transferosomes TM compared to application in conventional liposomes. The disagreements in the findings between the experiments carried out by Schatzlein and Cevc and the present study may be due to (i) different composition of the systems or (ii) differences between the murine skin and human cadaver skin (HCS). The fluorescence images of the skin sections at different depths beneath the skin were represented

7 2344 Int J Pharm Sci Nanotech Vol 7; Issue 1 January March 2014 around corneocytes. Stratum corneum (SC), viable epidermis (E) and dermis (D) after the application of hydrogel. The study revealed that when piroxicam loaded SLNs were incorporated in gelling agent (Carbopol-934) the piroxicam SLNs transport through the stratum corneum was increased as compared to normal piroxicam ointment. These studies suggested that the SLNs based hydrogel is advantageous for increasing the penetration depth of lipophilic gelling agent Carbopol-934. From the present study it can be concluded that the hydrogels are more favorable than a micellar solution or simple ointment. Furthermore also the thermodynamic state of the hydrogels plays an important role in transdermal penetration of lipophilic vesicles (SLNs loaded drugs). Conclusion Solid lipid nanoparticles represent a particular system, which can be produced with an established technique, solvent emulsification diffusion process for topical delivery of piroxicam with hydrogel system. It can be achieved after the selection of optimal formulation and process parameters. The size distribution of SLNs revealed a mono-dispersion profile in distilled water. In vitro release of piroxicam from SLNs in the phosphate buffer of ph 7.4, exhibited a biphasic pattern with an initial burst and prolonged release over 24 hr. The EE and the concentrations of the ingredients of formulations could influence the uptake of drug. The in vitro permeation studies showed all the formulations could avoid the systemic uptake of piroxicam. Piroxicam loaded SLNs based hydrogel formulation SLN-C8 H had high accumulative amount of piroxicam in skins and showed a significant skin targeting effect. The hydrogel plays an important role in transdermal penetration of lipophilic vesicles. The suitable mechanism for skin targeting still needs further studies. Acknowledgement The authors are thankful to the SLT Institutes of Pharmaceutical Science, Guru Ghasidas Vishwavidyalaya, Bilaspur, (C.G.) for providing facilities, encouragement and financial support throughout this work. References Baroli B, Lopez-Quintela MA, Delgado-Charro MB, Fadda AM and Blanco-Méndez J (2000). Microemulsions for topical delivery of 8-me thoxsalen. J. Control. Release 69: Bouwstra AJ, Mougin L, Junginger EH, Van Kuijk-Meuwissen and Marly EMJ (1998). Application of vesicles to rat skin in vivo: a confocal laser scanning microscopy study. J. Control. Release 56: Cevc G (2004). Lipid vesicles and other colloids as drug carriers on the skin. Adv. Drug Deliv. Rev. 56: Chen HB, Chang XL, Yang XL, Du DR, Liu W, Liu J, Weng T, Yang YJ and Xu HB (2006). Podophyllotoxin-loaded Solid lipid nanoparticles for epidermal targeting. J. Control. Release 110: Gasco MR (1993). Method for producing solid lipid microspheres having a narrow size distribution. US Patent 5: Jenning V, Gyslerb A, Schaefer-Kortingb M and Gohla SH (2000). Vitamin A loaded solid lipid nanoparticles for topical use: occlusive properties and drug targeting to the upper skin. Eur.J. Pharm. and Biopharm 49: Lippacher A, Muller RH and Maeder K (2001). Preparation of semisolid drug carriers for topical application based on solid lipid Nanoparticles. Int. J. Pharm. 214: Lippacher A, Muller RH and Maeder K (2002). Semisolid SLNs dispersion for topical application: influence of formulation and production parameters on viscoelastic properties. Eur. J. Pharm. Biopharm. 53: Maia CS, Mehnert W and Schafer-Korting M (2000). Solid lipid nanoparticles as drug carriers for topical glucocorticoids. Int. J. Pharm., 196: Mezei M and Gulasekharam V (1980). Liposome- A selective drug delivery system for the topical route of administration. I Lotion dosage form Life Sci. 26: Muller RH, Maeder K and Gohla S (2000). Solid lipid nanoparticles (SLN) for controlled drug delivery a review of the state of the art. Eur. J. Pharm. and Biopharm. 50: Muller RH, Radtke M and Wissing SA (2002). Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Adv. Drug Deliv. Rev. 54(1, Suppl): Muller RH and Lucks JS (1996). Medication Vehicles made of Solid Lipid Particles (Solid Lipid Nanospheres-SLN). Europ. Patent EP 0, B1. Muuller RH, Mehnert W, Lucks JS, Schwarz C, ZurMuhlen A, Weyhers H, Freitas C and Ruhl D (1995). Solid lipid nanoparticles (SLN)-an alternative colloidal carrier system for controlled drug delivery. Eur. J. Pharm. Biopharm. 41: Quintanar-Guerrero D, Allemann E, Fessi H and Doelker E (1999b). Pseudolatex preparation using a novel emulsion-diffudion process involving direct displacement of partially water-miscible solvents by distillation. Int. J. Pharm. 188: Quintanar-Guerrero D, Fessi H, Allemann E and Doelker E (1996). Influence of stabilizing agents and preparative variables on the formation of poly (d, l-lactic acid) nanoparticles by an emulsification-diffusion technique. Int. J. Pharm. 143: Quintanar-Guerrero D, Ganem-Quintanar A, Allemann E, Fessi H and Doelker E (1998b). Influence of the stabilizer coating layer on the purification and freeze-drying of poly (d, l-lactic acid) nanoparticles prepared by an emulsion-diffusion technique. J. Microencapsul. 15: Quintanar-Guerrero D, Gurny R, Allemann E, Fessi H and Doelker E (1999a). Method for producing aqueous colloidal dispersions of Nanoparticles. WO Patent 1(2): 87. Schatzlein A and Cevc G (1995). Proceeding of 6 th International Colloquium on Phospholipids; Phospholipids: Characterization, Metabolism and Noval Biological Application. Champaign, IIIinois USA: AOCS Schreier H and Bouwstra JA (1994). Liposomes and niosomes as topical drug carriers-dermal and transdermal drug delivery. J. Control. Release. 30: Sivaramakrishnan R, Nakamura C, Mehnert W, Korting HC, Kramer KD and Schafer-Korting M (2004). Glucocorticoid entrapment into lipid carriers -characterization by parelectic spectroscopy and influence on dermal uptake. J. Control. Release 97: Souto EB, Wissinga SA, Barbosab CM and Muller RH (2004). Evaluation of the physical stability of SLN and NLC before and after incorporation into hydrogel formulations. Eur. J. Pharm. Biopharm. 58: Souto EB, Wissing SA, Barbosa CM and Muller RH (2003). Preparation of Clotrimazole-loaded NLC and SLN. Int. Symp.Control. Release Bioact. Mater. 30: 321.

8 Verma: Development of Piroxicam loaded SLN-based Hydrogel for Transdermal Delivery 2345 Sylvia A and Muller RH (2003). The influence of solid lipid nanoparticles on skin hydration and viscoelasticity -in vivo study. Eur. J. Pharm. and Biopharm. 56: Trotta M, Ugazio E, Peira E and Pulitano C (2003). Influence of ion pairing on topical delivery of retinoic acid from microemulsion. J. Control. Release 86: Verma VK and Ram A (2010). Preparation Characterization and invitro release of Piroxicam loaded Solid Lipid Nanoparticles. Int. J. Pharm. Sci. Nanotech. 3(3): Williams A (2003). Transdermal and topical drug delivery from theory to clinical practice. Pharmaceutical Press, London. Williams AC and Barry BW (2004). Penetration enhancers. Adv. Drug. Deliv. Rev. 56: Wissing SA and Muller RH (2002). Solid lipid nanoparticles as carrier for sunscreens: in vitro release and in vivo skin penetration. J. Control. Release 81: Wissing SA and Muller RH (2003). Cosmetic applications for solid lipid nanoparticles (SLN). Int. J. Pharm. 254: Wissing SA, Lippacher A and Muller RH (2001). Investigations on the occlusive properties of solid lipid nanoparticles (SLN). J. Cosmet. Sci. 52: Wissing SA and Muller RH (2003). The influence of solid lipid nanoparticles on skin hydration and visco elasticity-in vivo study. Eur. J. Pharm. Biopharm. 56: Wissing SA, Mader K and Muller RH (2000). Solid lipid nanoparticles (SLN) as a novel carrier system prolonged release of the perfume. Int. Symp. Control. Release Bioact. Mater. 27: ZurMuhlen A and Mehnert W (1998). Drug release and release mechanism of prednisolone loaded solid lipid Nanoparticles. Pharmazie 53: ZurMuhlen A, Schwarz C and Mehnert W (1998). Solid lipid nanoparticles (SLN) for controlled drug delivery: drug release and release mechanism. Eur. J. Pharm. Biopharm. 45: Address correspondence to: Vinod Kumar Verma, SLT Institutes of Pharmaceutical Science, Guru Ghasidas Vishwavidyalaya Bilaspur, Chattishgarh, India. Phone: & ; vinodmod@yahoo.com