2002 ANNUAL SCIENTIFIC SEMINAR 297 NOVEL METHOD OF FORMULATING SKIN CARE PRODUCTS WITH LIPOSOMES Vitthal S. Kulkarni, Ph.D., Michael Ross, Barbara Brockway, Ph.D., James Wilmott and James Hayward, Ph.D. The Collaborative Group, Ltd., 3 Technology Drive, East $etauket, NY 11733 vitthal. kulkarni @ collabo. com INTRODUCTION: Liposomes are composed of !ipids that are self-assembled into a bilayer membrane forming closed ball like microstructures. Liposomes are kno•vn to enhance skin penetration and also are reported to deliver the actives to hair follicles. In the presence ofsurfactants, ho•vever, the liposomes loose their bilayer integrity making the encapsulated active to leak out or under excessive presence ofsurfactants, the bilayer structure may completely disintegrate to form micelies. Thus, stabilizing liposomes in a finished skin care product is challenging because the surfactants that are commonly used in producing a cream or lotion destabilize the liposomes. Therefore, to take advantages of !iposomes and make them •vork in real skin care products it is necessary to have a carrier system that is compatible •vith the liposomes. Lecithin has long been used as a dispersing agent for pigments, particularly in liquid makeup, however, the phospholipid content of commercial lecithin varies •videly and it also contains free-fatty acids, oils, glycolipids, and sugars. Moreover, the commercial varieties of lecithin are often fortified •vith surfactants including Polysorbate 2 I, PEG-6 Laurate and Sulfated Caster Oil to render lecithin water soluble. As mentioned above, the presence of surfactants is harmful to the stability of liposomes therefore •ve developed novel systems consisting of oil-in-•vater nano-dispersions using pure phospholipids and tested its compatibility with liposomes. MATERIALS AND METHODS: By applying very high energy, through high-pressure high-shear (HPHS) processing, to the mixture of oil, water and pure phospholipids we have produced stable oil-in- water nano-dispersions (particle size typically 500nm) of a variety of silicone or natural oils. The hydrophobic tails of phospholipids implant themselves into the oil droplet where as the hydrophilic headgroups remain at the oil/water interface. We studied the integrity of !iposomes in the presence of nano-dispersion and in cosmetic gels by 3Sp NMR (Varian UNITY 300 300 MHz) and fluorescence spectroscopy (Hitachi, F-2000). RESULTS AND DISCUSSION: The surface tension (measured using a Langmuir Film Balance, Kibron, Inc.) of water was almost unchanged (reduced by about 2 nuN/m) when a typical nano-dispersion (AM 100), produced as mentioned above, svas mixed with svater (at I%) suggesting that the nano-dispersions are practically "surfactant-free" (see Fig. I). Figure 1: Comparison of surface tension values of 1% AM I00 and 1% dispersion of a cream prepared using conventional emulsifiers ("conventional cream"). The data indicates that the surface tension of 1% AM i00 (70 mN/m) is almost same as that of pure water (72 raN/m) where as that of 1% conventional cream is significantly lower (28 I% AM 100 I%Co•vcmional disp•nion Crtam (Bl•k) A typical surfactant-free nano-dispersion (AM 400) was fully dispersed in •vater then mixed at 1:1 ratio with phospholipid liposomes (10mM !ipid) and the integrity of the !iposomes was monitored by 3•p NMR spectroscopy. The NMR data indicated that 100% of the lipid remained in the bilayer phase (see Fig. 2A) suggesting that the nano-dispersion did not &stabilize the liposomes. We also followed the stability of phospholipid liposomes in two types of gels (25% liposomes in gel) by 3•P NMR. In the gel containing carbomer and long alkyl chain acrylate crosspolymer (pemulen TR 2) the NMR data indicated that 100% of the !ipid remained in the bilayer phase after 8 months of storage at room temperature. Although there was increase in the lyso-lipid content over time, the NMR showed that all of the !ipid molecules remained in the bilayer phase suggesting that the !iposomes remained intact after 8 months of storage at room temperature. However, in a gel without carbomer or pemulen, the !iposomes degraded in 1 week at room temperature. Only 62.7% of the !ipid remained in the bilayer phase while remaining formed miceliar phase consisting of lyso-lipid, which is suspected to be formed as a result of !ipid hydrolysis (see Fig. 2B). Our data suggest that a gel that forms a good matrix structure is essential for long term stability

298 JOURNAL OF COSMETIC SCIENCE of liposomes. Williams' et al. have indicated 2 year stability of pro-liposomes (a concentrated lipid/glycol mix that forms multi-lamellar liposomes upon hydration) in 2% carbomer gel. Using a fluorescence spectrophotometer we studied the intensity of the emission peak ()•Ex=465, 3.Em=512 rim) of calcein (Sigma.) encapsulated in liposomes. A dilute solution of COC12 (10rnM) was added (50pl) to the sample (2ml) that quenched the fluorescence arising from the non-encapsulated dye. The integrity of liposomes was tested by addition of a dilute solution of conventional cream made by using non-ionic emulsifiers. Soon after the addition of conventional cream (a non-ionic emulsion), the emission peak height decreased considerably, where as under the similar conditions, upon addition of a dilute nano- dispersion, the emission peak remained almost unchanged (see Fig. 2C). This experiment suggests that the surfactants in conventional cream disturbed the integrity of liposomes, forcing the dye to leak out of liposomes which was quenched by COC12 resulting in decreased intensity of the emission peak. Formulating finished skin care products by combining actives with various surfactant-free nano-dispersions and gels is a novel approach one of the many advantages of this new approach over the traditional skin care formulation method includes its cofi•patibility with liposomes. St•blll• of Llpo•,m•t in C_dls i week s mooms [mc, c[ w/CarbemtaP•u• mc, c[ w/o CarbemcaP•u• ] Stability of Liposomes i •1 I:1%AMI00 j •o 2: nv tionlcream o $ to 15 •o 25 Figure 2A: 3,p NMR spectra: Top: Liposomes+AM 400 (1:1), Middle: Liposomes alone Bottom: nao- dispersion, AM 400 alone 2B: Integrity of liposomes as tested by 3'P NMR in two different gels 2C: Stability of liposomes in the presence of nano-dispersion or conventional cream (an emulsion made using non-ionic surfactants in convention manner) as studied by using a fluorescence spectrophotometer CONCLUSIONS: The NMR data suggested that the liposomes remained intact in the presence of AM 400, a surfactant-free nano-dispersion. The NMR data also revealed that all of the lipid remained in bilayer phase (liposomes) when mixed with gel containing carbomer/pemulen and other polymers and stored at room temperature for 8 months. Fluorescence spectroscopy studies indicated that liposomes were vulnerable to the surfactants in the emulsion where as the liposomes did not get perturbed in the presence of surfactant-free nano-dispersion. Our data suggests that a combination of surfactant-free nano-dispersions and an appropriate gel will provide a matrix system that is compatible with liposomes and that it may offer long term stability to the liposomes. REFERENCES: 1. Williams, W. P., Perrett, S., Golding, M., Amaud, J-P., and Michez, M. "The pro-liposome method: A practical approach to the problem of the preparation and utilization of liposomes suitable for topical applications" published in "Phospholipids: Characterization, Metabolism, and Novel Biological Applications", Eds. Cevc, G., and Paltauf, F., AOCS Press, Champaign, IL, 1995, pp.181-188