j. Soc. Cosmet. Chem., 43, 237-249 (September/October 1992) Development of a model of the lipid constituent phase of the stratum corneum: II. Preparation of artificial membranes from synthetic lipids and assessment of permeability properties using in vitro diffusion experiments D. KITTAYANOND, S. M. DOWTON, C. RAMACHANDRAN, G. L. FLYNN, and N. WEINER, College of Pharmacy, The University of Michigan, Ann Arbor, MI 48109-1065. Received April 28, 1992. Synopsis This paper describes the development of model membranes from liposomes using commercially available synthetic lipids. The composition of the lipid mixture used to prepare the liposomes was based on earlier studies to optimize the strength of interlipid interactions within the bilayers. Model membranes were prepared by extruding liposomes through a membrane filter below the phase transition temperature of the lipid mixture. The resulting membrane filter, with the deposited lipids, was then dried and treated with calcium chloride to allow transformation of the liposomal bilayers into extended sheet structures. The treated membranes were dried and mounted on diffusion cells. A variety of markers was then used to test the permeability characteristics of the model membrane. The results suggest that the membrane prepared using synthetic lipids is a suitable model for permeation characteristics of skin. INTRODUCTION In a previous paper (1), the optimization of the composition of liposomes prepared using commercially available semi-synthetic lipids suggested that the interlipid interactions of this mixture were similar to that obtained using lipids extracted from porcine stratum corneum. It was therefore of interest to determine if model membranes of the stratum corneum lipid compartment can be developed by the use of the optimized synthetic lipid mixture. The preparation of model membranes with synthetic or semi-synthetic lipids would also obviate excessive reliance on animal skin studies if they are shown to possess permeability characteristics similar to skin. The quest to circumvent problems associated with the use of human or animal skin to answer basic questions has prompted an extensive search for a reliable in vitro model. Attempts to adapt synthetic polymeric membranes such as cellulose acetate membranes 237

238 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (2,3) or dimethylpolysiloxane membranes (4-7) revealed that the nature of the materials comprising such membranes limits their use as a skin model system. A few studies described the formation of membranes by using the corneocytes and lipid extracts of porcine stratum corneum (8,9). Although reaggregation of stratum corneum compo- nents resulted in viable membranes, such an approach would not obviate the use of animal skin. Abraham and Downing (10) reported the in vitro formation of membranes by use of a mixture of epidermal ceramides, cholesterol, cholesterol sulfate, and fatty acids. Although the authors demonstrated that water transport across their model membrane was similar to that across the stratum corneum, the system suffers the constraint of requiring epidermal ceramides that involve difficult and time-consuming extraction and isolation procedures. Further, no permeation studies were reported with permeants other than water. Indeed, most of the studies on model membranes prepared from lipid mixtures have been solely tested by water transport characteristics (8-11). This study reports the preparation of model membranes from a mixture of synthetic lipids and investigates the permeability characteristics of a variety of markers with a range of physicochemical properties across this model membrane. The model mem- branes prepared possessed sufficient cohesive properties so as to be easily handled when employed in diffusion studies wherein the membrane was in intimate contact with solutions in the receiver compartment. MATERIALS AND METHODS MATERIALS Palmitic acid, stearic acid, ceramides type III from bovine brain, cholesterol, cholesterol sulfate, N-palrnitoyl-DL-dihydrogalactocerebroside, N-2-hydroxyethylpiperazine-N'- 2-ethanesulfonic acid (HEPES free acid), and bovine serum albumin (BSA) were pur- chased from Sigma Chemical Co. (St. Louis, MO). The lipids were of the highest purity available and were used as received. Carnauba fatty acid was a gift from Dr. D. T. Downing, Marshall Dermatology Research Laboratories, University of Iowa. The chain lengths of fatty acid components of this mixture have been reported to be in the range C24-C3o (12). The radioactive marker, [3H]-cortisol (specific activity 134 mCi/mg) was obtained ß . ß •4 .... from ICN Rad•ochermcals (Irwne, CA). [ C]-sucrose (specific acuwty 204 •Ci/rng), [•4C]-estradiol (specific activity 204 •Ci/mg), and [3H]-progesterone (spe- cific activity 151 mCi/mg) were purchased from Amersham (Arlington Heights, IL). All other chemicals were reagent grade, and all solvents used were of HPLC grade. Water was double-distilled and deionized using a Milli-Q ion-exchange system. METHODS Preparation of liposomes Liposomes were prepared by the reverse phase evaporation method by using a cera- mide:cholesterol:fatty acid:cholesterol sulfate mixture at a weight ratio of 37.7:28.3: 17.0:17.0. This ratio was found to exhibit the strongest interlipid interaction deter- mined by monolayer studies (1). Briefly, the lipid mixture contained in a one-liter