NEW RESISTANT LIPOSOME COATED WITH POLYSACCHARIDE FILM FOR COSMETIC APPLICATION 229 concentrations from 1% to 10%. Regarding the salts resistance test, 70% of coated and noncoated liposome suspensions were mixed with 30% of various salt solutions. The fi nal concentrations of salts in liposome suspensions were set at 5%, 10%, 15%, 20%, 25%, and 30%. All fractions were expressed as w/w. During a period of 30 days, daily optical microscope observations were performed at a magnifi cation ×1000 at 25°C. We believe that the liposome suspension is unstable from the fi rst signs of destabilization, such as aggregation and destruction of the phospholipid bilayer. PERMEABILITY EVALUATION Magnesium chloride release from coated liposomes. Twelve percent of MgCl2 was entrapped in coated liposomes. The permeability of coated liposome membrane to MgCl2 was evalu- ated in acrylates/C10-30 alkyl acrylate cross-polymer hydrogels (0.8%, w/w) neutralized with sodium hydroxide. Being sensitive to electrolytes, the viscosity of hydrogels de- creases by increasing the concentration of MgCl2. The release of MgCl2 from coated lipo- somes was then evaluated by measuring the viscosity evolution of hydrogels containing 2% (w/w) of entrapped MgCl2 in coated liposomes (MCCL) and an equivalent amount of free MgCl2 (0.24% w/w). The viscosity measurement was performed using a rheometer apparatus (Rheomat RM 200, Lamy Rheology, Champagne au Mont d’Or, France). The effect of coated liposome dilution (1/2, 1/10) on the release of MgCl2 was carried out. We supposed that dilution of the external medium of coated liposomes (distilled water) could increase the diffusion of MgCl2 from their internal core. EVALUATION OF THE DELIVERY EFFICACY OF COATED LIPOSOMES In order to bring out the delivery effi cacy of coated liposomes, the latter were used to improve the detoxifi cation activity of MgCl2. Human skin explants were used to evaluate the effect of entrapped and free MgCl2 on P-gp transporter activity. Human normal skin explants were obtained from a piece of surgical resection of 37-year-old subjects. Hydro- gel composed of 1% (w/w) of acrylates/C10-30 alkyl acrylate cross-polymer neutralized with sodium hydroxide, 0.5% (w/w) of xanthan gum and preserved with 0.8% (w/w) of parabens and phenoxyethanol’s mixture was used. Deionized water was added to com- plete the formulation (100% w/w). Entrapped and free MgCl2 was incorporated into the hydrogel at 3% (i.e., 0.36% of MgCl2) and 0.36%, respectively. Hydrogel without coated liposomes and MgCl2 was used as placebo. A mixture of ascorbic acid (100 μg/ml) and glutathione (100 μg/ml) was tested as a positive control for the evaluation of P-gp activ- ity. Skin explants were incubated during 24 h at 37°C in contact with positive controls, entrapped MgCl2, nonentrapped MgCl2, and hydrogel as placebo. All formulations were applied to the skin explants’ surface except the reference product, which was directly diluted in the culture medium. At the end of the incubation period, skin explants were rinsed with a PBS and stored at -196°C. The assay was performed in triplicate. Proteins assay. At the end of the incubation period, proteins were quantifi ed in cell lysates by a spectrocolorimetric method using bicinchoninic acid assay (20).

JOURNAL OF COSMETIC SCIENCE 230 P-gp activity assay. At the end of the incubation period, P-gp activity was assessed in cell lysates using a sensitive and specifi c assay kit. For P-gp analysis, results are expressed as nmol of ATP consumed by P-gp per μg of proteins (mean ± S.D.). Statistical analysis. Levels of signifi cance were assessed using Student t-test (p ≤ 0.05). RESULTS AND DISCUSSION LIPOSOMES CHARACTERIZATION Structural characterization. Coated and noncoated liposomes used for the entrapment of MgCl2 (12%) were observed by optical microscope immediately after being prepared (Figure 1). Because of the high ionic strength of the medium, a decomposition and aggregation of liposome vesicles was observed attesting that noncoated liposomes are not resistant to 12% of MgCl2 (Figure 1A). These data are in agreement with those reported by Crom- melin (21), which supported that high ionic strength dispersions predicted an irrevers- ible aggregation of liposomes. However, we clearly observed in Figure 1B that coated liposomes are more resistant and can entrap 12% of MgCl2. Polysaccharide coating allowed the entrapment of high amount of electrolytes into liposomes represented by spherical vesicles with good membrane in- tegrity (Figure 1B). In Figure 2B, we confi rmed by freeze-fracture electron microscopy the spherical morphol- ogy of coated liposomes with entrapped MgCl2 (Figure 2B). These microscope images revealed that the coating procedure did not modify the vesicular structure and the mor- phology of liposomes (Figures 2B and C). Vesicle size measurement. The presence of polysaccharide coating was evaluated by vesicle size analysis of empty coated and noncoated liposomes. The mean vesicle size of non- coated liposomes was about 251.03 ± 15.56 nm. However, coated liposomes had a mean Figure 1. Optical microscope observation of liposomes (×1000): (A) Noncoated liposomes in the presence of 12% of magnesium chloride and (B) 12% of magnesium chloride entrapped in coated liposomes.