INTRODUCTION:

Solid-lipid nanoparticles (SLNs) have recently gained significant attention as potential alternate colloidal drug delivery system over conventional dosage form.

Isotretinoin, a derivative of retinoic acid (13-cis-retinoic acid), has been commonly used for the treatment of severe acne and other dermatological diseases (1,2). However, it has obvious adverse side-effects by oral administration.

The launched topical preparations such as cream also show systemic absorption and significant skin irritation (3). So, it is necessary to improve the skin uptake and reduce systemic absorption of isotretinoin using a carrier with an ability of skin targeting. Compared with conventional carriers such as cream, tincture and emulsion, SLN combine their advantages such as controlled release, in vivo good toleration and protection of active compounds(4,5). Especially, SLN can favor drug penetration into the skins(6,7), maintain a sustained release to avoid systemic absorption(8), act as a UV sunscreen system(9), reduce irritation(10,11). Moreover, SLNs are composed of physiological and biocompatible lipids and are free from the risk of acute or chronic toxicity. Various research teams(12-17) have studied the effect of lipid type on the final particle size of the SLN dispersions formed. Particle size is an important factor for topical route. SLN was found to have a skin targeting. The topical route has various advantages over other pathways, including avoiding hepatic first pass effect, delivering drugs or phytoconstituents continuously, fewer side-effects, and improving patient compliance(18). However, stratum corneum is the main barrier of the skin; it not only prevents dehydration but also hinders the penetration of various drugs. The intercellular lipids of stratum corneum play a key role in establishing the permeability barrier of the skin(19).

The aim focuses on the formulation of topical preparations for the treatment of severe acne that could be localized in the stratum corneum, viable epidermis, and appendages but have reduced systemic absorption. It has been reported that topical isotretinoin reduce acne, fine wrinkling, hyperpigmentation, roughness, and sallowness associated with photoaging(20).

In this study four chemically different lipids were selected: Glyceryl monostearate (GMS), Precirol ATO5 (PRE 5), Glyceryl tristearate (GTS), and Dimethyl phosphor ethanol amine (DMPE). The rationale was to select a mono glyceride (GMS), a diglyceride (PRE 5), a triglyceride (GTS) that are found in mammals and a bacterial lipid DMPE and study the effect of lipid matrix type on the final SLN particle size, entrapment efficiency, in vitro release pattern and physical stability on short-term storage.

The present study focused on the preparation, characterization, skin targeting evaluation and in-vitro release pattern of isotreinoin-loaded SLN (IT-SLN).

MATERIALS:

Isotretinoin was a gift from Ipca Laboratories Ltd. (Ratlam, M.P.), GMS (monoester of 16-carbon fatty acid [stearic acid] and glycerol) and GTS (triglyceride derivative of 16-carbon fatty acid [stearic acid] and glycerol) were purchased from Sigma chemicals. Precirol ATO5 (mono-, di- and triglycerides of palmitostearic acid with the diester fraction predominating was purchased from Colorcon India Ltd. (Mumbai, India), Dimyristoyl phosphoethanolamine and soybean lecithin were purchased from Sigma chemicals. Tween 80 was purchased from Loba Chemie, Mumbai. All other chemicals used were of analytical grade. Water was distilled and filtered before use through a 0.22µm nylon filter. Female mice (20±2g) were obtained from animal house of B.N. College of Pharmacy. All the experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) B.N. College of Pharmacy, Udaipur, Rajasthan and conducted according to the Indian National Science Academy Guidelines for the use and care of experimental animals.

METHODS:

Partitioning nature of isotretinoin between the lipids and pH 7.4 Phosphate Buffer:

Ten milligrams of isotretinoin was dispersed in a mixture of melted lipid (1g) and 1ml of hot pH 7.4 phosphate buffer saline (PBS) and shaken for 30 minutes, incorporating a mechanical shaker using a hot water bath maintained at 10⁰C above the melting point of the lipid under investigation (21). The aqueous phase of the above mixture was separated from the lipid by centrifugation at 25000 rpm for 20 minutes in a high-speed centrifuge machine (Remi, India). The clear supernatant obtained after centrifugation was suitably diluted (to a concentration of 5µg/ml of isotretinoin) with 0.1N HCl, and the isotretinoin content was determined in a UV-visible spectrophotometer (UV Thermospectronic) at 353nm against a solvent blank. The partition coefficient of isotretinoin in lipid/pH 7.4 phosphate buffer saline was calculated using Equation I.

PC = (C iso- C isa) / C isa……………... (I)

Where, C iso = the initial amount of isotretinoin added (10mg), and C isa = the concentration of isotretinoin in pH 7.4 Phosphate Buffer Saline.

Preparation and optimization of SLNs:

The isotretinoin-loaded solid-lipid nanoparticles (IST-SLN) formulations of different lipids were prepared using Hot Homogenization method (22). Briefly, the lipid, soybean lecithin and isotretinoin were dissolved in ethanol. Then, ethanol as the organic solvent was completely removed using rotary evaporator (ESRA, India) at 80⁰C. The melt residue was added to the hot water containing Tween 80. Then, the preemulsion was prepared using a high speed stirrer for 20 minutes. The pre-emulsion subsequently homogenized in a high-pressure homogenizer at 800 bar in a water bath maintained at 10⁰C, above the melting point of the lipid. After the homogenization, the dispersion was cooled at 5-8⁰C, the SLN dispersion stabilized by Tween 80 and soybean lecithin was obtained.

Freeze-Drying/Lyophilization of SLNs

The prepared SLN dispersions were diluted with cryoprotectant solution and then rapidly frozen at temperatures ranging from -40⁰C to -60⁰C in liquid nitrogen in a freeze dryer (Micro Modulo Edwards) for 24hrs.

Characterization of SLNs Particle Size, Its Distribution and Zeta Potential:

The average particle size was measured by Malvern Instruments Corp, U.K. at 25⁰C. For measurement of particle size, the samples were diluted to diminish droplet interactions. All samples were diluted 25 times with distilled water and filtered through 0.22µm Millipore filters. The effect on size of particles was checked after first and second weeks to see the changes. The initial particle size and zeta potential of the isotretinoin-loaded SLN dispersions were measured immediately after high-pressure homogenization using the Malvern zetasizer. This batch was divided into 2 sample sets, one stored at 4⁰C (in a refrigerator) and the other stored at 40⁰C (in a temperature regulated oven). All samples were stored in plain glass vials (USP type I) that were sealed and wrapped with aluminium foil. Samples were withdrawn after 15, 30, 60, 90 and 120 days and subjected to particle size and zeta potential measurements.

Surface Morphology by Transmission Electron Microscopy:

TEM was used to characterize the microstructure of SLN. The SLN was placed on a carbon coated copper grid and then a drop of 1% phosphotungustic acid covered on SLN. The superfluous phosphotungustic acid on SLN was wiped-off by filter paper. The TEM images were obtained using Philips CM 200 (U.K.).

Drug Entrapment Efficiency Determination:

Entrapment efficiency is the percentage of the initial drug or active constituent incorporated into particulate systems. Entrapment efficiency was estimated by centrifugation method (23).The prepared formulations were placed in centrifugation tube and centrifuged at 10,000 rpm for 30minutes. The supernatant (1ml) was withdrawn and diluted with phosphate buffer solution (PBS) pH 7.4 or distilled water. The entrapped drug was determined by UV Spectrophotometer at 353nm. The samples from the supernatant were diluted 100 times before taking absorbance measurement (24). The free drug in the supernatant gives us the total amount of unentrapped drug by Equation II

Total drug- Diffused drug x 100

Percent entrapment =----------------------------------…. ..(II)

Total drug

In-Vitro Skin Permeation Studies:

The modified Franz Diffusion Cell was fabricated from borosilicate glass and consisted of two compartments i.e. receptor and donor. The cell has an effective receptor volume of 30ml and skin surface area of 2cm². The two halves of the cell were secured in place with the help of strong metallic clips. The receptor solution was stirred by a Teflon bead of 12mm length on a magnetic stirrer. The temperature of cell contents were maintained at 37±1⁰C, with the help of thermostat attached to the magnetic stirrer assembly. For in-vitro evaluation of isotretinoin-loaded-SLNs (IST-SLNs) and isotretinoin-loaded-SLNs dispersed in cream base (IST-SLNs-Cream), rat and goat were selected. The Full-thickness abdomen skin from rat and goat was used for all the permeation experiments. After hair was removed with a shaver, excised and examined for integrity using a microscope (20X, magnification power). The skin was rinsed with physiological saline. The fat tissues below skin were carefully removed; washed with distilled water and then equilibrated for an hour in pH 7.4 Phosphate Buffer Saline, before running the experiment. The skin was trimmed into a circular section of about 2cm diameter. The skin of both the rat and goat, one at a time were clamped between the donor and the receptor chamber of vertical diffusion cell with an effective diffusion area of 2cm² and cell volume of 30ml. The receptor chambers were filled with freshly mixture of Phosphate Buffer Saline pH 7.4 and 95% ethanol (7:3v/v). Ethanol was used to solubilize isotretinoin. The diffusion cells were maintained at 37⁰C using a re-circulating water bath and the fluid in the receptor chamber was stirred continuously at 300rpm. Both the formulations, one at a time (1.0gm) were gently placed in the donor chamber. At 1-8 hours, 1.0ml of the fluid in the receptor chambers were sampled for U.V.-Spectrophotometer at 353nm and replaced immediately with an equal volume of freshly mixture of PBS (pH 7.4) and 95% ethanol (7:3v/v).The cumulative amounts of isotretinoin permeated through rat and goat skins were plotted as a function of time. The permeation rate of isotretinoin at steady state (J, µg/cm²/h) through skins was calculated from the slope of the linear portion of the cumulative amount permeated per unit area versus time plot. The in-vitro permeation data were subjected to zero-order kinetics.

Skin Accumulative Amount:

Drug remain on skin was found out by washing skin after release study with diffusion medium and filtered this solution. These samples were analysed by diluting it with methanol and measured absorbance at 353nm. The % drug present into skin was also found out after washing skin with PBS. Skin was cut into small pieces. All pieces were collected with solution and homogenized into tissue homogenizer (Remi, India).The sample was centrifuged at 10,000rpm for 20minutes in centrifuge machine (Remi, India). Supernatant was taken and filtered. The filtrate was collected and analysed by diluting it with methanol. Absorbance was taken for these samples at 353nm using methanol as blank.

Stability of the Isotretinoin-Loaded-SLN:

The chemical and physical stabilities of IST-SLN were evaluated at 2-8⁰C and room temperature for 120days via clarity, particle size, zeta potential and HPLC analysis of isotretinoin.

Skin Irritation Study:

All the experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) B.N. College of Pharmacy, Udaipur, Rajasthan and conducted according to the Indian National Science Academy Guidelines for the use and care of experimental animals. The mice (20±2g) were divided into 4 groups (n=3). On the previous day of the experiment, the hairs on the backside area of mice were removed. The animals of group I was served as normal, without any treatment. One group of animals (group II, control) were applied with SLN dispersion without drug while group III, group of animals were applied with drug containing SLN. A 0.8% v/v aqueous solution of formalin was applied as a standard irritant (group IV). The animals were applied with cream each day upto 7 days and finally the application sites were graded according to a visual scoring scale, always by the same investigator. The erythema scale was as follows: 0, none; 1, slight; 2, well-defined; 3, moderate; and 4, scar formation.

In-Vitro Occlusion Studies:

The occlusivity of formulations is determined by occlusion factor. First investigations were performed by de vringer (25). The in-vitro model consists of a beaker (capacity 25ml) of water covered by a filter paper, 200mg of the formulation is spread on a filter surface of 18.8cm²; a reference control is maintained, which is a beaker having filter paper but no formulation. An occlusion factor is calculated by the formula:

F = 100[(A-B)/A]……... (III)

Where, A = Water loss without sample (reference). B = Water loss with sample.

RESULTS:

In order to disperse isotretinoin homogenously in the melted lipid, ethanol was used as solvent. A high speed stirring was employed using over-head stirrer to obtain a pre-emulsion before homogenization. A hot water bath was used for maintaining the pre-emulsion above the melting point. In order to select a suitable lipid for isotretinoin-loaded SLN, Glyceryl monostearate (GMS), Glyceryl tristearate (GTS), Precirol ATO 5/ Glyceryl palmitostearate (PRE) and 1,2-Dimyristoyl-sn-glycero-3-phospho ethanol amine (DMPE) were respectively used as the lipids of SLN. It was found that all the lipids could result in translucent dispersion. Glyceryl tristearate based SLN had a large particle size and the Glyceryl monostearate based SLN had a wide size distribution. The Glyceryl palmitostearate and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine based SLN showed a suitable size, size distribution, zeta potential and physical stability.

Then, PRE and DMPE were selected as the optimum lipid for isotretinoin-loaded SLN. The particle size of different SLN formulations prepared with all the four lipids were respectively measured by Malvern Particle size Analyzer. The particle size was shown in (Table II). The average size of all the formulations is located between 21nm to 500nm. SLNs prepared with PRE had the most narrow size distribution and those prepared with DMPE had the small particle size, as compared with other formulation. The particle size was decreased with the increase of the concentration of Tween 80. But soybean lecithin showed an optimum concentration when the concentration of soybean lecithin in SLN was increased. On the basis of particle size, particle morphology, zeta-potential and physical stability (Table II), it was found that GMS-based SLN had a wide size distribution ranges from (100-500nm), not having spherical shape with high particle charge (50-55mv) and form gel during 1 week study. The GTS-based SLNs had a large particle size (greater than 500nm), having irregular shape, high particle charge (70-75mv) and very low physical stability, as the particle aggregation occurs due to large size and very high particle charge. Therefore, both the SLNs prepared from GTS and GMS showed poor stability due to large particle size and wide size distribution. So, they were discarded and further characterization were carried out only on PRE and DMPE based SLNs as they possess acceptable size for penetration into the skin, low charge, spherical shape and very good stability. It was found that GTS based SLNs are not spherical in shape in case of both drug-loaded (200nm and 500nm) and unloaded SLNs (200nm and 500nm).So, it was concluded that GTS was not found to be a suitable lipid for IT-SLN, also that drug loading did not result difference in particle size. The TEM imaging of Precirol ATO5 showed spherical shape and had a narrow size distribution (Figure 1) in case of both drug loaded and unloaded SLNs. The imaging showed that PRE-4-IT SLN exhibited a spherical shape and particle size of 21nm, PRE-3-IT SLN exhibited a spherical shape and particle size of 50nm, and found to be the best lipid for SLN preparation (Figure 2and3).

Table I: The Composition of Drug-Loaded Solid-Lipid Nanoparticles

Formulation code	Isotretinoin (%)	Lipid (wt %)	Soybean lecithin (wt %)	Tween 80 (wt %)	Water (wt %)
GTS-F1	0.05	3.00	6.00	3.00	87.95
GTS-F2	0.05	3.00	6.00	4.50	86.45
GTS-F3	0.05	3.00	6.00	6.00	84.95
GTS-F4	0.05	3.00	4.00	4.50	88.45
GTS-F5	0.05	3.00	8.00	4.50	84.45
GMS-F1	0.05	3.00	6.00	3.00	87.95
GMS-F2	0.05	3.00	6.00	4.50	86.45
GMS-F3	0.05	3.00	6.00	6.00	84.95
GMS-F4	0.05	3.00	4.00	4.50	88.45
GMS-F5	0.05	3.00	8.00	4.50	84.45
PRE-F1	0.05	3.00	6.00	3.00	87.95
PRE-F2	0.05	3.00	6.00	4.50	86.45
PRE-F3	0.05	3.00	6.00	6.00	84.95
PRE-F4	0.05	3.00	4.00	4.50	88.45
PRE-F5	0.05	3.00	8.00	4.50	84.45
DMPE-F1	0.05	3.00	6.00	3.00	87.95
DMPE-F2	0.05	3.00	6.00	4.50	86.45
DMPE-F3	0.05	3.00	6.00	6.00	84.95
DMPE-F4	0.05	3.00	4.00	4.50	88.45
DMPE-F5	0.05	3.00	8.00	4.50	84.45

Also, it was found that Myristyl phosphoethanolamine (MP-2-IT) showed comparable results same as Precirol ATO 5 as shown in (Figure 4). The GMS based SLNs (Figure 5) were found to be better than GTS based SLNs (Figure 6) regarding morphology and size. They were found to be slightly spherical. According to morphology Precirol ATO5 based and Myristly phophoethanolamine based SLNs were found to be the best.

Characterization of Isotretinoin-Loaded SLNs:

The isotretinoin-loaded solid-lipid nanoparticles were characterized for Morphology by TEM, Particle size, Zeta potential, Entrapment efficiency (Table II) and In-vitro release profiling.The particle size of selected isotretinoin-loaded SLN was measured at 4⁰C and 40⁰C by particle size analyzer (Malvern Instruments, UK). A significant increase in particle size and zeta potential was observed for GTS based and GMS based nanoparticles, when stored at 40⁰C. The particle size of GTS-3-IT at 40⁰C increased from 198nm to 908nm in 120 days and at 4⁰C increased from 198nm to 728nm. Another observation was formation of a rigid gel in the stability samples of isotretinoin-loaded GMS-3-IT and GMS-5-IT stored at 4⁰C after 30 days.

Samples kept at 40⁰C did not gel. There was an increased in particle size of GMS-3-IT and GMS-5-IT formulations at 40⁰C as shown in (Figure 7) after 60 days. Furthermore, a clear drug phase separation was observed in GTS-3-IT SLN

formulations. A thin layer of solid was found to sediment at the base of the glass vials in case of PRE-1-IT and PRE-2-IT formulations in 120 days at 4⁰C (Figure 7). Also, DMPE-1-IT and DMPE-4-IT were found to be unstable as the drug expulsion from the lipid matrix occured. Inspite of it, there was a very little increase in the size of the SLNs prepared from Precirol ATO5 and Myristoyl phosphoethanolamine. Similar, gelling and drug phase separation was also observed by Westesen et al during their study of the stability of phospholipid, Tyloxapol trimyristin, Witepsol H 42, and Witepsol H 35 nanoparticles.It was observed that the temperature of gel formation depends mainly on the lipid matrix of the colloidal lipid emulsions, could be prevented by using a co-surfactant. The zeta potential was found to increase with longer duration of storage and higher temperature in case of GTS-3-IT. The changes in the zeta potential were more prominent for all the isotretinoin-loaded SLN samples except PRE-4-IT, PRE-3-IT and DMPE-2-IT formulations stored at 40⁰C when compared with those stored at 4⁰C. The Entrapment efficiencies were found to be in the order of (Table II):

PRE (97.70%) > DMPE (95.50%) > GMS (80.02)> GTS (75.56%)

The Precirol ATO based SLNs exhibited the highest entrapment of isotretinoin (97.70%), while the Glyceryl tristearate based SLNs showed the poor entrapment efficiency (75.56%). Myristoyl ethanolamine based SLNs exhibited the entrapment of isotretinoin (95.50%). Glyceryl monostearate based SLNs showed the entrapment efficiency of (80.02%), which is also good as compared to GTS based SLNs.It was found that entrapment efficiency depends upon the partition coefficient. The lipid with highest partition coefficient for drug was found to have highest entrapment efficiency. The partition coefficient followed the order:

PRE (2.24) > DMPE (2.19) > GMS (2.12) > GTS (1.71)

The partition coefficient values obtained for the different lipids correlated with the entrapment efficiency of SLNs. Hence, an initial study of the partitioning nature of the drug between melted lipid and aqueous media can provide some clues about the entrapment in the SLN formulation.

The in-vitro occlusion studies and skin irritability studies were carried out; occlusion factor was found to be greatest for IST-SLN cream i.e 50. The skin irritability tests results showed score 0 for all the formulations, means no erythma. It was found that isotretinoin-loaded SLN dispersed in cream base increased the skin hydration.

Particle size distribution and zeta potential graphs were represented in (Figures 8 and 9) for PRE-4-IT SLNs.

Table II: Characterization of Drug-Loaded Solid-Lipid Nanoparticles (n=3)(mean±S.D.)

Formulation code	Particle size (nm)	Zetapotential (mV)	Entrapment Efficiency (%)
GMS-1	500±3.71	-60.73±4.09	75.49±0.46
GMS-2	524±2.57	-65.97±4.13	78.59±0.39
GMS-3	200±2.37	-64.73±4.32	79.03±0.39
GMS-4	200±2.53	-63.13±4.76	70.62±0.40
GMS-5	198±2.82	-68.93±5.32	80.02±0.41
GTS-1	520±5.32	-68.96±5.19	70.03±0.46
GTS-2	500±4.92	-70.17±5.31	73.13±0.42
GTS-3	500±5.01	-69.70±5.21	75.56±0.41
GTS-4	200±5.21	-71.23±5.33	66.69±0.48
GTS-5	200±4.82	-75.43±5.45	74.83±0.43
PRE-1	42.7±1.10	-10.94±1.51	91.89±0.36
PRE-2	26.33±1.0	-17.13±1.21	92.88±0.32
PRE-3	32.4±1.2	-16.12±2.23	95.05±0.29
PRE-4	21±0.99	-15.97±1.51	90.92±0.39
PRE-5	50.0±1.32	-20.97±2.63	97.70±0.24
DMPE-1	60±1.2	-19.45±2.45	87.02±0.44
DMPE-2	50±1.3	-21.03±2.61	90.01±0.33
DMPE-3	52.3±1.1	-20.83±2.43	93.31±0.31
DMPE-4	42.7±1.0	-20.81±2.42	80.01±0.42
DMPE-5	60.83±1.33	-22.03±2.58	95.50±0.28

*GMS indicates glyceryl monostearate; GTS, glyceryl tristearate; PRE, Precirol ATO5; DMPE, 1,2-dimyristoyl-sn-glycero-3 phosphoethanolamine; S.D., standard deviation; n, number of observations.

Studies of In Vitro Permeation:

In order to assess the skin uptake and penetration of isotretinoin from SLN, the in vitro permeation ability through skins and into skins were performed using Franz diffusion cells (26). The in vitro permeation studies were performed using rat and goat skins in order to draw comparison on the basis of different species. The permeation studies were performed for 8h according to the clinical application time. It indicated that isotretinoin was not found in receptor chambers from IT-SLN formulations and all the IT-SLN formulations could not penetrate through skin. The diffusion data on rat and goat skins were subjected to zero-order, I-order, higuchi and korsemeyer data curves. The permeation followed zero order release kinetics. The % drug present in different compartments after 8h in case of goat and rat skins were tabulated in (Table IIIandIV) respectively. From these observations it is concluded that all the IT-SLN formulations can avoid the systemic uptake of isotretinoin in comparison with the isotretinoin cream and SLN present a potential to avoid the systemic adverse side-effect. The % drug into skins from PRE-1-IT, PRE-2-IT, PRE-3-IT, PRE-4-IT and DMPE-2-IT were respectively 13.03±0.982, 16.42±0.992, 20.01±1.003, 22.09±1.010 and 18.01±1.006 in case of rat skin and 17.03±0.972, 20.02±1.003, 24.33±1.011, 33.02±1.032 and 22.03±1.010 in case of goat skin (Table III). Even though IT-SLN PRE-1, 2, 3 and DMPE-2 failed to increase the uptake of drug in rat skins significantly when compared with goat skins due to greater thickness, they still avoided systemic absorption of isotretinoin and showed skin targeting. The IT-SLN PRE-4 significantly increased the accumulative uptake of isotretinoin in both the skin, compared with the other formulations. PRE-4-IT showed 22.09±1.010% (rat skin) and 33.02±1.032% (goat skin) penetration of drug in skin over other formulations, and had an enhanced skin targeting effect. However, all the IT-SLN of Precirol ATO5 and Myristyl phosphoethanolamine formulations had small diameters. The slight difference between the average particle size of IT-SLN formulations should not be the key factors influencing the uptake of drug in skins. According to (Table I), PRE-4-IT SLNs had the lowest concentration of soybean lecithin, but had the highest uptake of isotretinoin in skins when compared with PRE-2 and PRE-5. The increase of soybean lecithin in formulations resulted in the decrease of the accumulative amount of isotretinoin in skins (Figure 10). Additionally, the increase of Tween 80 in PRE-1,2 and 3 led to the increase of the uptake of isotretinoin in skins.The accelerated studies were carried out for selected DMPE-2-IT, PRE-4-IT and PRE-3-IT for a period of 4 weeks. The degradation is subjected to zero-order and I-order.The degradation followed zero-order kinetic data.The decomposition of isotretinoin from solid-lipid nanoparticles dispersed in cream base was shown by Arrhenius plot.The shelf-life of 72.8 days was found for PRE-3 and PRE-4 formulations respectively.

DISCUSSION:

Acne vulgaris is a chronic inflammatory disease of the pilosebaceous units. The etiologic factors include increased sebum production, ductal hyperkeratosis, abnormality of the microbial flora within the pilosebaceous unit, and chemomediators of inflammation. The dermal inflammation is not due to presence of bacteria, but from biologically active chemomediators produced by P. acnes and the microenvironment within the pilosebaceous unit, is probably more important than the absolute number of P. acnes organisms. Indeed, the major role of the sebaceous gland appears to be supplying nutrients needed by P. acnes and moreover, the microbiologic principle of biofilms appear to be applicable to P. acnes in acne (27). Isotretinoin, a derivative of retinoic acid (13-cis-retinoic acid), has been commonly used for the treatment of severe acne and other dermatological diseases. However, it has obvious adverse side-effects by oral administration. The launched topical preparations such as cream also show systemic absorption and significant irritation. so, it is necessary to improve the skin uptake and reduce systemic absorption of isotretinoin using a carrier with an ability of skin targeting.

Acne is mainly a disease of pilosebaceous unit, isotretinoin acts by decreasing the sebum, which in turn inhibits the growth of Propionibacterium acnes. But in dermatologic treatment, improving the efficacy demands high drug levels in the skin. In an experiment with nanoparticle dispersion, it was found that a greater quantity of drug remained localized in the skin, with lesser amounts penetrating into the receptor compartment as compared with conventional creams. Thus, drug localizing effect in the skin seems possible with novel colloidal particulate drug carriers, such as solid-lipid nanoparticles. The colloidal carrier, being submicron in size, enhances the drug penetration into the skin, and because of its lipoidal nature, the penetrated drug concentrates in the skin and remain localized for a longer period of time, thus enabling drug targeting to the skin. Compared with conventional carriers such as cream, tincture and emulsion, SLN combine advantages such as, controlled release, in vivo good toleration and protection of active compounds. In this work, SLN was used for topical delivery of isotretinoin and the long-term is to explore a novel formulation with skin targeting effect for the treatment of severe acne.

The purpose of the study was to construct isotretinoin-loaded SLN (IT-SLN) with skin targeting for topical delivery of isotretinoin and to avoid the systemic uptake of isotretinoin in skins. In order to avoid the local dryness caused by drug, the IT-SLNs were dispersed in suitable herbal cream base containing vitamin E, aloe-vera gel and oils. In this work, SLNs were prepared with four different lipids viz Glyceryl monostearate, Glyceryl tristearate, Glyceryl palmitostearate and Myristoyl phosphoethanolamine by hot homogenization technique for topical delivery of isotretinoin. The first three lipids, GMS, GTS and PA5 are the mammalian lipids, while DMPE is found in the bacteria. Such type of selection was made to assess the effect of selected lipids. Out of four, PA5 and DMPE were selected as the suitable lipid for SLNs depending upon the particle size, zeta-potential, surface morphology, skin accumulative amounts and drug content. Tween 80 and soybean lecithin were used as surfactants (in five different concentrations) to stabilize SLN.

Table III: % Drug present in different compartments after 8 hours (Goat skin) (mean±S.D., where n=3)

Formulation code	% drug release after 8 hours	% drug above skin after 8 hours	% drug into skin after 8 hours	Mass balance
PRE-1-IT	0.443±0.005	74.41±3.320	17.03±0.972	91.89±4.307
PRE-2-IT	0.440±0.003	72.42±3.290	20.02±1.003	92.88±4.306
PRE-3-IT	0.441±0.004	70.27±3.201	24.33±1.011	95.05±4.220
PRE-4-IT	0.386±0.001	57.71±2.960	33.02±1.032	90.92±4.0
DMPE-2-IT	0.447±0.006	67.55±3.020	22.03±1.010	90.01±2.004

* PRE indicates Precirol ATO5; IT, isotretinoin; DMPE, 1,2-dimyristoyl-sn-glycero-3 phosphoethanolamine; S.D., standard deviation; n, number of observations.

*Table IV: % Drug present in different compartments after 8 hours (Rat skin) (mean±S.D., where n=3)

Formulation code	% drug release after 8 hours	% drug above skin after 8 hours	% drug into skin after 8 hours	Mass balance
PRE-1-IT	0.889±0.009	77.97±3.301	13.03±0.982	91.89±4.3
PRE-2-IT	0.881±0.008	75.57±3.301	16.42±0.992	92.88±4.3
PRE-3-IT	0.883±0.009	74.1±3.208	20.01±1.003	95.05±4.22
PRE-4-IT	0.773±0.007	68.05±3.083	22.09±1.010	90.92±4.1
DMPE-2-IT	0.894±0.010	71.10±5.129	18.01±1.006	90.01±4.113

* PRE indicates Precirol ATO5; IT, isotretinoin; DMPE, 1,2-dimyristoyl-sn-glycero-3 phosphoethanolamine; S.D., standard deviation; n, number of observations^.

CONCLUSION:

The various IT-SLN formulations were prepared by hot homogenization method for topical delivery of isotretinoin. The in-vitro permeation data showed that all the IT-SLN formulations can avoid the systemic uptake of isotretinoin in skins. The IT-SLN consisting of 3.0% PRE 5, 4.0% soybean lecithin, 4.5% Tween 80 and IT-SLN consisting of 3.0% DMPE, 6.0% soybean lecithin and 4.5% Tween 80 could significantly increased the accumulative uptake of isotretinoin in skin and showed a significantly enhanced skin targeting effect. It is concluded that the concentration of the ingredients of SLN had no influence on the uptake of drug in skins. In addition PRE-4-IT SLNs had only entrapment efficiency of 90.92±0.39%, but showed a high skin targeting. The entrapment efficiency is correlated with the partition coefficient of drug in lipid. It was found that the drug with high partition coefficient showed high entrapment efficiency. Entrapment efficiency might also act as an important factor for the uptake of isotretinoin in skins. Even though the occlusive effect succeeds to elucidate the penetration of drug from into skins. The permeation of drug followed zero-order kinetics and mechanism of drug release was found to be diffusion controlled. The future perspectives open a wide area for SLN as potential new adjuvant for vaccines, for parenteral and pulmonary administration.

ACHNOWLEDGEMENT:

Authors would like to thanks SAIF, IIT Mumbai for their help in conducting the Transmission Electron Microscopy for SLN formulation. They also thank Ipca Laboratories Ltd. Ratlam (India) for providing Isotretinoin as gift sample for this work. Humble thanks goes to Dr. O.P. Mahatma, Principal, B.N. Girls College of Pharmacy, Udaipur (India) for providing required facilities for carrying out this research work.

REFERENCES:

1. Katsambas A, Papakonstantinou A. Acne: systemic treatment. Clin Dermatol. 2004;22:412-418.

2. Klasco RK. Editor. Drugdex System, vol. 128, Green Wood Village (CO): Thomson Micromedex, 2006.

3. Queille-Roussel C, Poncet M, Mesaros S, Clucas A, Baker M, Soloff A. Comparison of the cumulative irritation potential of Adapalene gel and cream with that of Erythromycin/Tretinoin solution and gel and Erythromycin/Isotretinoin gel. Clin Therap. 2001;23:205-212.

4. Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery- review of the state of the art. Eur J Pharm Biopharm. 2000;50:161-177.

5. Sylvia A, Muller RH, Wissing SA. Cosmetic applications for solid-lipid nanoparticles (SLN). Int J Pharm. 2003;254:65-68.

6. Jenning V, Gysler A, Schafer-Korting M, Gohla SH. Vitamin A loaded solid lipid nanoparticles for topical use: occlusive properties and drug targeting to the upper skin. Eur J Pharm Biopharm. 2000;49:211-218.

7. Wissing SA, Muller RH. The influence of solid lipid nanoparticles on skin hydration and viscoelasticity- in vivo study. Eur J Pharm Biopharm. 2003;56:67-72.

8. Zur Muhlen A, Schwarz C, Mehnert W. Solid lipid nanoparticles (SLN) for controlled drug delivery- drug release and release mechanism. Eur J Pharm Biopharm. 1998;45:149-155.

9. Wissing SA, Muller RH. Solid lipid nanoparticles as carriers for sunscreens: in vitro release and in vivo skin penetration. J Control Release. 2002;81:225-233.

10. Maia CS, Mehnert W, Schafer-Korting M. Solid lipid nanoparticles as drug carriers for topical glucocorticoids. Int J Pharm. 2000;196:165-167.

11. Sivaramakrishnan R, Nakamura C, Mehnert W, Korting, HC, Kramer KD, Scafer-Korting M. Glucocorticoid entrapment into lipid carriers- characterization by parelectric spectroscopy and influence on dermal uptake. J Control Release. 2004;97:493-502.

12. Heiati H, Tawashi R, Philips NC. Drug retention and stability of solid lipid nanoparticles containing azidothymidine palmitate after autoclaving, storage and lyophilization. J Microencapsul. 1998;15:173-184.

13. Bunjes H, Westesen K, Koch MHJ. Crystallization tendency and polymorphic transitions in triglyceride nanoparticles. Int J Pharm. 1996;129:159-173.

14. Westesen K, Bunjes H, Koch MHJ. Physicochemical characterization of lipid nanoparticles and evaluation of their drug loading and sustained release potential. J Control Release. 1997;48:223-236.

15. Cavalli R, Gasco MR, Morel S. Behaviour of timolol incorporated in lipospheres in the presence of a series of phosphate esters. STP. Pharma Sci. 1992;2:514-518.

16. Mehnert W, Mader K. Solid lipid nanoparticles production, characterization and application. Adv Drug Deliv Rev 2001;47:165-196.

17. Siekmann B, Westesen K. Submicron-sized parenteral carrier systems based on solid lipids. Pharm Pharmacol Lett. 1992;1:123-126.

18. El Maghraly GM, Williams AC, Barry BW. Skin delivery of oestradiol from lipid vesicles: importance of liposome structure. Int J Pharm. 2000;204:159-169.

19. Elias PM. Lipids and the epidermal permeability barrier. Arch Dermatol Res. 1981;270:95-117.

20. Griffiths CE, Maddin S, Weidow O, Marks R, Donald AE, Kahlon G. Treatment of photoaged skin with a cream containing 0.05% isotretinoin and sunscreens. J Dermatolog Treat. 2005;16:79-86.

21. Venkateshwarlu V, Manjunath K. Preparation, characterization and in vitro release kinetics of clozapin solid lipid nanoparticles. J Control Release. 2004;95:627-638.

22. Reddy LH, Vivek K, Bakshi N. Tamoxifen citrate loaded solid lipid nanoparticles (SLN™): preparation, characterization, in vitro drug release and pharmacokinetic evaluation. Pharm Dev Technol. 2006;11:167-177.

23. Fry DW, White JC, Goldman, ID. Rapid separation of low molecular weight solutes from liposomes without dilution. Anal Biochem. 1978;90(2):809-815.

24. Dodov MG, Simonoska M, Goracinovo K. Formulation and characterization of topical liposome bearing lidocaine hydrochloride. Bull Chem Technol Medona. 2005;24:59-65.

25. Vringer T. Topical preparation containing a suspension of solid lipid nanoparticles. Eur Pat Appl 1992; EP 0506, 197 Al. 1992 In European Patent No. 91200664.

26. Trotta M, Ugazio E, Peira E, Pulitano C. Influence of ion pairing on topical delivery of retinoic acid from microemulsion. J Control Release. 2003;86:315-321.

27. Burkhart CN, Gottwald L. Assessment of etiologic agents in acne pathogenesis. Skinmed. 2003;2(4):222-228.

Received on 18.09.2012 Modified on 26.09.2012

Research J. Pharm. and Tech. 5(11):November, 2012; Page 1375-1384