94 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Skin humidity is regulated to a large extent by lipids in the skin's horny layer. This complex lipid mixture is oriented, at least in part, as bimolecular leaflets (4). Liposomes have been employed in cosmetics and skin care products for several years with great success. However, few reports on the deposition of liposomal lipids into skin after topical application have been published (5-7). The objectives of the present study were to understand the nature of interaction of liposomal components with the skin by investigating the effect of liposome type and lipid concentration on deposition into the various strata of hairless mouse and pig skin using in vitro diffusion studies. MATERIALS AND METHODS Cholesterol (CH), cholesteryl sulfate (CS), and HEPES free acid were obtained from Sigma (St. Louis, MO). Egg lecithin (PC) was obtained from Avanti Polar Lipids (Birmingham, AL). ot-Tocopheral ((x-T) was obtained from Eastman Kodak (Rochester, NY). •4[C]-CS and 3[H]-CH were obtained from Amersham (UK). All other chemicals were of analytical grade. PREPARATION OF LIPOSOMES Multilamellar liposomes (MLV) containing PC:CH:CS at a molar ratio of 1:0.5:0.1 were prepared using the conventional film method (1). Briefly, the lipid mixtures-were dissolved in a 2:1 (v/v) mixture of chloroform and methanol. Trace amounts of 3[H]-CH and •4[C]-CS were incorporated in the phospholipid-based liposomes. The lipids and markers were deposited as a thin film in a round-bottomed flask by rotary evaporation under nitrogen. The flask containing the lipid film was stored in vacuum overnight to facilitate removal of residual solvents. The films were hydrated by the addition of an isotonic 0.05 M HEPES buffer, pH 7.4, with mild agitation at 45øC. The final concentrations of lipid were 50 mg/ml, 25 mg/ml, and 10 mg/ml. All of the liposomal preparations were examined with a Nikon Diaphot Light microscope to ensure liposomal quality and integrity. Large unilamellar vesicles were prepared by a modification of the reverse-phase evapo- ration method (REV) of Szoka and Papahadjopoulos (8). Appropriate amounts of the lipid mixtures, with trace amounts of radiolabeled CH and CS, were dissolved in 10 ml of a chloroform-methanol mixture (2:1 v/v). Five ml of 0.05 M HEPES buffer (pH 7.4) and enough additional methanol (up to 1.5 ml) were added to yield a clear solution after brief sonication. The organic solvents and a small amount of water were then removed under nitrogen at 45øC. Solvent removal was continued until all foaming ceased. The resulting liposomal suspension was stored at 4øC overnight before use in the diffusion experiments. Dehydration/rehydration liposomes (DRV) were prepared by a modification of the method reported by Kirby and Gregoriadis (9). Briefly, appropriate amounts of the various lipids, along with the radiolabeled lipid markers, were dissolved in chloroform/ methanol (2:1 v/v) in a round-bottomed flask. The solvents were removed using a rotoevaporator under vacuum, and the flask containing the film was dried overnight in a desiccator to remove residual solvent. An appropriate aliquot of 0.05 M HEPES buffer

LIPOSOMAL DEPOSITION 95 was then added, and the mixture was hydrated at 45øC. Intermittent vortexing was required for complete hydration. The resultant dispersion was then dehydrated at 50øC under vacuum using the rotoevaporator. When the liposomal suspension became very viscous, an amount of water, equivalent to that removed, was reintroduced into the viscous suspension. The rehydrated liposomes were allowed to equilibrate for about 45 min at 45øC, and the dispersion was stored at 4øC overnight before use in the diffusion experiments. DEPOSITION EXPERIMENTS Full-thickness hairless mouse skin was excised from fresh carcasses, and subcutaneous fat was carefully removed using a scalpel. Pig skin was obtained from a local abattoir and cleaned of any subcutaneous fat. The skin sections were mounted on Franz diffusion cells with a nominal surface area of 2 cm 2 and a receiver compartment with a 7-ml capacity (Crown Glass, Somerville, NJ). The epidermal side of the skin was exposed to ambient conditions while the dermal side was bathed by a 0.05 M isotonic HEPES buffer. The receiver solution was stirred continuously using a small Teflon-covered magnet. Care was exercised to remove any air bubbles between the underside of the skin and solution in the receiver compartment. The temperature of the receiver was maintained at 37øC. Following mounting of the section of skin, 200 txl of the test formulation were applied to the epidermal surface of the hairless mouse skin and 400 txl of the test formulation were applied to the pig skin. A smaller amount of formulation was found to be insuf- ficient to ensure uniform spreading across the entire exposed surface of the skin in the cell. A minimum of two cells was used for each formulation, using sections of skin from different skin specimens for each formulation. All experiments were carried out with non-occluded donor compartments. After 24 hr, the experiments were stopped and the diffusion set-up was dismantled for assay of radiolabeled lipids. ASSAY OF RADIOLABELED MARKERS Upon dismantling, the donor compartment of the cell was rinsed carefully five times with 0.5 ml buffer the skin was removed, and it too was rinsed twice with 3 ml of buffer. The washing procedure was found to be sufficient to remove more than 99 percent of the formulation when determined at time zero. All washings were collected and assayed for radiolabel. Following the rinsing procedure, the skin patch was mounted on a board, and a piece of adhesive tape (Scotch Magic Tape, 810, 3M Commercial Office Supply Division, St. Paul, MN), 1.9 cm wide and about 6 cm long, was used to strip the skin. The tape was of sufficient size to cover the full area of skin that was in contact with the formulation. Based on extensive investigations of the extent of strip- ping of the stratum corneum, as monitored using TEWL (transepidermal water loss) measurements (14), it was determined that nine strippings were required for complete removal of mouse stratum corneum. The skin after nine strippings appears glossy. A total of 18 strippings was needed for pig skin in order to remove the stratum corneum, as judged by the glossy appearance. Nine such strippings were carried out for mouse skin and 18 strippings for pig skin, and each strip was analyzed separately for radiolabeled lipid. The remaining skin, as well as the receiver compartment solution, was also assayed for lipid content. Assay of the donor, skin rinses, and receiver solutions were