Chem. Pharm. Bull. 54(2) 242—244 (2006)

been shown to form bilayer vesicles (cationic liposomes) and have been applied to gene delivery. Cationic vesicles designed for that purpose are generally intrinsically unstable. Although application of cationic liposomes has also been anticipated for the delivery of drugs, peptides, vitamins and lipids and other biologically active compounds to biological cells and tissues, liposomes used for that purpose need to be intrinsically stable in order to retain their contents. In a previous study we revealed that phosphatidylcholines enriched with unsaturated acyl chains stabilized the liposomes of dimethyldialkylammoniums such as dimethyldipalmitylammonium at physiological temperature. In contrast, 1,2-dioleoyl3-trimethylammonium propane (DOTAP) formed stable vesicles at physiological temperature with phosphatidylcholines containing either saturated acyl chains such as dimyristoylphosphatidylcholine (DMPC) or unsaturated acyl chains such as dilinoleoylphosphatidylcholine (DLPC). We also revealed that these cationic liposomes interacted with erythrocytes as cationic vesicles and induced the formation of tightly aggregated structures of several erythrocytes which might also induce fusion of the erythrocytes. The lipid lamella of the stratum corneum of the skin contains a high ratio of negatively charged lipids, which are expected to interact with cationic liposomes. Transfer of some of the bilayer components of the liposomes to the skin is then possibly induced. Therefore, in this work we prepared cationic liposomes containing retinoic acid (all-trans-retinoic acid) as a bilayer component, and examined the incorporation of retinoic acid into skin by the cationic liposomes. Although topical retinoic acid has been clinically used with success for the treatment of several cutaneous diseases such as psoriasis or ichthyosis and mild acne, its efficient delivery into skin has not been established yet. We compared the incorporation efficiency by cationic liposomes with those by electrically neutral liposomes and elastic liposomes as well as that by an organic solvent solution. Experimental Materials Chloride salt of DOTAP was purchased from Avanti Polar Lipids (Alabaster, AL, U.S.A.). Bromide salt of dimethyldipalmitylammonium was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dimyristoryphosphatidylcholine (DMPC, L-a-phosphatidylcholine dimyristoyl) and Dipalmitoylphosphatidylcholine (DPPC, L-a-phosphatidylcholine dipalmitoyl) were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All-trans-retinoic acid, egg yolk phosphatidylcholine (egg yolk PC), heptaethylene glycol mono-n-dodecyl ether and all other reagents were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Preparation of Sonicated Liposomes Consisting of Phosphatidylcholines, Double-Chained Cationic Surfactants and Retinoic Acid Liposomes consisting of phosphatidylcholines, DOTAP (or dimethyldipalmitylammonium) and retinoic acid were prepared as described previously. The liposome constituents were dissolved in chloroform, and the solvent was evaporated under a nitrogen stream. The dried films were prepared by removing the solvent under vacuum evaporation, then hydrated and suspended by vortex mixing in phosphate-buffered saline (PBS) (150 mM NaCl, 10 mM phosphate buffer, pH 7.4) containing 10 mM sodium ascorbate. The final concentration of retinoic acid was 2.5 mM, and the total concentrations of double-chained cationic surfactants and phosphatidylcholines were 10 mM. The suspension was then sonicated with a probe-type sonicator for 10 min at an output power of 80 W at 30 °C under a stream of nitrogen. The vesicular sizes were determined with quasielastic light scattering using an ELS-800 laser particle analyzer (Otsuka electronics, Japan) at a scattering angle of 90°. Measurement of the Intradermal Concentration of Retinoic Acid Full thickness dorsal skin was excised from male guinea pigs and subcutaneous fat and other extraneous tissues were trimmed. In vitro study on the skin incorporation of retinoic acid was examined as described previously. The skin was mounted in a Franz cell with water jackets (37 °C). The available diffusion area was about 0.64 cm, and the lower cell volume was about 4.5 ml. The upper cells were filled with 1 ml PBS and the receiver cells were also filled with PBS. The lower cells were stirred at 450 rpm by a magnetic stirrer during 12 h pretreatment of the skin. After washing both cells, 1 ml liposome suspension containing 2.5 mM retinoic acid was added to the upper compartments, and the incubation experiment was started. A control experiment was also carried out by the addition of 2.5 mM retinoic acid dissolved in isopropyl myristate. After 18 h treatment, the skins were removed from the cells and washed three times with ice-cold methanol. Following room temperature drying, each skin was weighed, minced and placed in 10 ml of methanol, then homogenized using a tissue homogenizer Polytoron (Kinematica AG, Switzerland). The samples were then centrifuged and the supernatant layer was used to determine the concentration of retinoic acid by HPLC. The concentration of retinoic acid was determined by HPLC (L-6000; Hitachi, Tokyo, Japan) with an L-4000 UV detector (Hitachi) at 356 nm. Separation was achieved on a reversed242 Notes Chem. Pharm. Bull. 54(2) 242—244 (2006) Vol. 54, No. 2