J. Cosmet. Sci., 61, 235–245 (May/June 2010) 235 Skin effi cacy of liposomes composed of internal wool lipids rich in ceramides R. RAMÍREZ, M. MARTÍ, C. BARBA, S. MÉNDEZ, J. L. PARRA, and L. CODERCH, Department of Chemical and Surfactant Technology, IQAC (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain. Accepted for publication November 16, 2009. Synopsis Ceramides from intercellular lipids of skin stratum corneum are known to play an essential role in maintain- ing and structuring the lipid barrier of the skin. Internal wool lipids (IWL), which are also rich in ceramides, have a composition similar to that of the stratum corneum lipids. IWL extracted with chloroform/methanol azeotrope at the laboratory scale have been shown to be capable of forming liposomes with a stable bilayer structure. Furthermore, topical application of these IWL liposomes on intact and compromised skin has been demonstrated to improve barrier skin properties. In this study we evaluated the effect on human skin repair of different IWL extract compositions obtained by two extraction methodologies. The formation and characteristics of the liposomes prepared were greatly infl uenced by the IWL composition, primarily the sterol sulfate content. The IWL liposomes improved skin barrier integrity and increased skin hydration when applied onto intact skin. These improvements were slightly enhanced in the case of IWL liposomes that were richer in polar lipids. INTRODUCTION Wool is a natural fi ber that is mainly comprised of protein. It contains external lipids (lanolin) and a small amount of internal lipids (1.5%). Internal wool lipids (IWL) arouse considerable interest given their high proportion of ceramides. IWL are rich in choles- terol, free fatty acids, cholesteryl sulfate, and ceramides and they resemble those from membranes of other keratin tissues such as human hair or stratum corneum (1–4). Intercellular lipids of stratum corneum, mainly ceramides, play an important role in the bar- rier function of the skin by preventing penetration of external agents and controlling transepi- dermal water loss to maintain the physiological skin water content (5). Recent studies have shown that formulations, especially ceramide supplementation, containing lipids that resem- ble the natural components of the skin can improve compromised skin conditions (6,7). IWL have been extracted from wool on account of their high proportion of ceramides. IWL extracted by Soxhlet extraction at the laboratory scale has been shown to be capable Address all correspondence to L. Coderch.

JOURNAL OF COSMETIC SCIENCE 236 of forming liposomes with a stable bilayer structure (8,9). This vesicular structure, which mimics the organized lipid structures of the stratum corneum, offers a suitable strategy for achieving an accurate vehiculization of a particular compound and for incorporating additional lipid content that may reinforce the barrier function of the skin (10–12). Fur- thermore, earlier studies (13,14) have demonstrated the ability of these IWL liposomes to improve skin barrier properties when applied onto intact and compromised skin. Slightly better results have been obtained with these liposomes, containing a mixture of natural ceramides, when compared with stratum corneum liposomes (modeling stratum corneum lipids using synthetic lipid mixtures), which had only one type of ceramide present in the formulation. Accordingly, IWL could be regarded as a new natural extract that is benefi - cial to topical application and suitable for incorporation into pharmaceutical or cosmetic formulations in the treatment and care of skin (15). Therefore, IWL have been extracted at the pilot plant scale both by organic solvent extrac- tion (OSE) using methanol or acetone (16) and by supercritical fl uid extraction (SFE) with CO2, using 10% methanol or ethanol as polarity modifi ers (17). In the present work, lipo- somes containing IWL extracts with different lipid compositions were formed. Vesicular diameter, polydispersity index, and stability were determined. The effi cacy of these IWL liposomes when applied topically onto intact skin on a long-term basis was studied. To this end, in vivo changes in transepidermal water loss (TEWL) were measured as an index of bar- rier repair, whereas the water-holding capacity was measured as changes in skin capacitance. Finally, the protection of intact skin against detergent action was evaluated after topical application of the IWL liposome samples by measuring the aforementioned parameters. MATERIALS AND METHODS EXTRACTION PROCEDURES Raw Spanish Merino wool samples supplied by SAIPEL (Terrassa, Spain) were used for lipid extraction. Prior to the extraction, raw wool was industrially cleaned following a procedure previously described (16). IWL were extracted at the pilot plant scale by OSE using metha- nol. The extraction procedure consisted of a pump-forced refl ow system. Five kilograms of wool were extracted for four hours at 56°C (16). IWL were also obtained at the pilot plant scale by SFE using a CSFF (Iberfl uid Instruments/ICP-CSIC, Spain). Fifty grams of wool were extracted with CO2 at 60°C and 160 atm using 10% methanol or ethanol as polarity modifi ers of CO2 (17). IWL extracts obtained at the pilot plant scale were concentrated and stored in chloroform/methanol (2:1, v/v) at −20°C until their analysis. LIPID ANALYSIS The quantitative analysis of the samples was performed by thin-layer chromatography coupled to an automated fl ame ionization detector (TLC-FID), an Iatroscan MK-5 ana- lyzer (Iatron, Tokyo, Japan). Samples were applied on silica gel S-III Chromarods using an SES (Nieder-Olm, Germany) 3202/15-01 sample spotter. The determination of the composition was made using an optimized TLC-FID protocol to analyze lipid content (16). The rods were developed initially to a distance of 10 cm with n-hexane/diethyl ether/formic acid (53:17:0.3, by vol) to separate apolar and polar lipids. After a partial