NEW RESISTANT LIPOSOME COATED WITH POLYSACCHARIDE FILM FOR COSMETIC APPLICATION 227 1,3-propanediol was purchased from DuPont Tate & Lyle (Loudon, TN). Stearoyl Inulin was obtained from Miyoshi (Saitama, Japan), and α-tocopherol from VitaeCaps (Talavera de la Reina, Spain). Dermosoft® 1388 and polyglyceryl-10-laurate were supplied by Dr. Straetmans (Hamburg, Germany). Natural MgCl2 was purchased from Celnat (Saint-Germain Laprade, France) and acry- lates/C10-30 alkyl acrylate cross-polymer from Lubrizol (Wickliffe, OH). Sodium lauryl ether sulfate (SLES) was purchased from Cognis (Monheim, Germany) and Polysorbate 20 was obtained from Kolb (Hedingen, Switzerland). Behenylalcoholethoxyl- ate, Triton X-100, and sodium chloride (99%) were provided by Prolabo (Darmstadt, Germany). Phosphate-buffered solution (PBS), ascorbic acid, bicinchoninic acid, glutathi- one, and sodium dodecyl sulfate (SDS) (≥99%) were purchased from Sigma (Saint-Quentin Fallavier, France). Pgp-Glo™ Assay Systems were purchased from Promega (Charbon- nieres, France). Parabens and phenoxyethanol were provided from Jan Dekker (Langenfeld, Germany). Xanthan gum and sodium hydroxide were purchased from Cargill (Hamburg, Germany) and Le Comptoir Français Interchimie (Compans, France), respectively. LIPOSOME PREPARATION Natural polysaccharides are chemically hydrophobized by grafting stearic acid chains via an ester bond. Coated liposomes were prepared by dissolving phospholipids in 1,3- propanediol (7:20 w/w) at 70°C. Then, polysaccharide-fatty acid complex (Stearoyl Inulin) was incorporated at 75°–80°C to the homogenous mixture. After adding 0.1% of α-tocopherol to the previous mixture, the aqueous phase was vigorously homogenized with lipid phase using a rotor stator for 20 min at 1500 rpm. Finally, the coated liposome suspension was homogenized using Turrax for 5 min at 3000 rpm to reduce the polydispersity of vesicles. At the end of the process, 0.5% of Dermosoft® 1388 was added as antimicrobial system. Noncoated or classical liposomes were prepared under the same conditions (process and composition) without adding polysaccharide-fatty acid complex. MAGNESIUM CHLORIDE ENTRAPMENT Coated and noncoated liposomes entrapped MgCl2 were prepared by adding 12% (w/w) of MgCl2 into the aqueous phases before their homogenization with the lipid phases. LIPOSOME CHARACTERIZATION Structural characterization. Optical microscope and freeze-fracture electron microscopy were used to determine the morphology of liposomes. Vesicles were observed under an optical microscope (Nikon Eclipse 50i from Nikon, Kanagawa, Japan) immediately after their preparation. Coated and noncoated lipo- somes were deposited between two glass lamellas and observed using a phase contrast

JOURNAL OF COSMETIC SCIENCE 228 mode at a magnifi cation ×1000. Immersion oil was deposited between the objective lens and the upper glass lamella. Coated and noncoated liposomes were observed in parallel using freeze-fracture electron microscopy. About 1 μl of liposome suspension was placed on a copper base. The sample was rapidly frozen with a cryoprotectant (glycerol) in liquid nitrogen (-196°C) cooled propane. After storage in liquid nitrogen, samples were placed on the support immersed in liquid nitrogen which was inserted into the cryoscouring device (BAL-TEC BAF 060 (Nanterre, France)) and then cooled to -150°C. When a vacuum of 10-7 torr was reached, samples were fractured using a scalpel blade cooled to -150°C. The fracture surface was shadowed by a spray of platinum, and a layer of 4 nm was fi lled in one direction at an angle of 45°C to reveal the relief and surface structures. The thin layer was consolidated with vertically sprayed carbon, which is transparent to electrons. A quartz oscillating measuring device measured the fi nal thickness it was about 30 nm. The replicas were cleaned with a mix- ture of solvents and/or sulfochromic acid, rinsed with distilled water, and examined under a transmission electron microscope (FEI CM120, Philips, Cambridge, UK). Vesicle size measurement. The empty noncoated liposomes size was measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instruments, Worces- ter, UK). The samples were diluted (1:400) with ultrafi ltrated distilled water. Droplets sizes were obtained from the correlation function calculated by the dispersion technology software using various algorithms. The apparatus is equipped with a 4 mW He/Ne laser, emitting 633 nm, measurement cell, photomultiplier, and correlator. The refractive in- dex and absorbance were set at 1.47 and 0.01 respectively at 25°C. The measurements were performed in fi ve replicates (19). DLS is not adapted to polydisperse suspensions and important size vesicles. Therefore coated liposomes sizes were determined using static light scattering. The size distribution of empty coated liposomes and MCCL was determined using static light scattering (Beckman Coulter LS230 instrument, Brea, CA) according to Fraunhofer diffraction theory. By applying the polarization intensity differential scattering, this ap- paratus can detect particles from 0.04 to 2000 μm using up to 116 size classes and a he- lium neon laser with a wavelength of 750 nm. Empty coated liposomes and MCCL suspensions were dispersed in distilled water under agitation into the measuring cell to be in the optimal obscuration range (8%–20%). Par- ticle size distribution was expressed in volume units. STABILITY EVALUATION Resistance to surfactants and electrolytes. Since most cosmetic formulations are emulsions, the improvement of liposome stability against surfactants is a very important aspect for their use in such cosmetic formulations. Empty coated and noncoated liposomes were incubated for 30 days at 25°C with different percentages of ionic surfactants (SDS, SLES), nonionic surfactants (Triton X-100, Poly- sorbate 20, polyglyceryl-10-laurate, Behenyl alcohol ethoxylate), and salts (NaCl, MgCl2). As high as 90% of liposome suspensions were mixed with 10% of surfactant solutions at different concentrations. Surfactant solutions were prepared in distilled water at different