j. Soc. Cosmet. Chem., 44, 123-128 (March/April 1993) I. iposomes in cosmetics: Which kind of phospholipid? Which loading method? ADRIANA MEMOLI, LUISA G. PALERMITI, VALTER TRAVAGLI, and FRANCO ALHAIQUE, Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita di Roma (La Sapienza), Rome (A.M., L.G.P.), Dipartimento Farmaco Chimico Tecnologico, Universita di Siena, Siena (V. T. ), and Dipartimento Farmaco Chimico Tecnologico, Universita di Cagliari, Cagliari (F.A.), Italy. Received October 14, 1992. Synopsis Phospholipids of different origin (egg and soya) and purity were used to prepare liposomes by sonication. Loading of these vesicles was performed by means of two different techniques using a fluorescent lipophilic model molecule. The stability of the aggregated structures was checked by addition of increasing amounts of a surfactant to the liposome dispersion. No remarkable differences were observed in either the stability in regard to surfactant-induced breakage or the loading capacity of liposomes respectively prepared with 99% pure egg phosphatidylcholine or with the vegetable phospholipid, a commercial product that had a much lower purity. The comparison of the two loading methods indicated that incorporation of the model molecule within the vesicle structure was higher when the fluorescent marker was added before sonication. INTRODUCTION It is well known that double-chain amphiphiles, such as phospholipids, are capable of aggregating into bilayers that assume the form of liposomes: closed spheres of different structures and dimensions that can be loaded with active ingredients. Because of these properties, liposomes are present in several pharmaceutical preparations and are largely used in cosmetics. The origin, the extraction, and the purification method, and consequently the final composition and purity of the phospholipids used for the preparation of liposomes, can lead to dramatically different prices and at the same time to a great variety of loading capacity and stability structures (1). The aim of this work was to compare the behavior of a 99% pure egg phosphatidyl- choline (EPC) with that of a vegetable phospholipid that had a much lower purity and price (P90). In this sense it is also interesting to point out that, as far as the origin (egg or soya) is concerned, the vegetable phospholipid appears to be more appropriate for 123

124 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS topical applications in cosmetics and dermatology because of the high content of poly- unsaturated fatty acids (2), like linoleic acid, which are particularly valuable in cosmetic preparations (1). The effects of different methods of liposome loading were also consid- ered. Since the main expected differences must be related to the bilayer structure of the vesicles, 1,6-diphenyl-l,3,5-hexatriene (DPH) (3) was chosen for our studies. This fluorescent probe, whose interaction with phospholipid vesicles was recently reviewed (4), is localized within the lipid bilayers, and the phospholipid phase transitions induce only very small changes in DPH excited-state interconversion. Stability in regard to surfactant-induced breakage and loading capacity were respectively evaluated by means of turbidity measurements and fluorescence determinations of the probe incorporated or absorbed in the hydrophobic bilayer of the vesicles prepared by sonication. MATERIALS 99% pure L-ot-phosphatidylcholine from egg yolk (Sigma, type III-E, hexane solution, 100 mg/ml and type XI-E, chloroform solution, 100 mg/ml) and 90% pure enriched soya phosphatidylcholine (Phospholipon 90, Nattermann Phospholipids GmbH) were used for vesicle preparation. Crystalline DPH was purchased from Sigma. Solutions and dispersions of this marker were prepared just before use and handled as much as possible in the dark because of the photosensitivity of DPH (4). pH 7.5 HEPES solutions (10- 3 M), made with freshly distilled and aleaerated water, were used. Cholesterol, Triton X-100, and all other products used for the present investigation were of analytical grade. All solvents were tested for fluorescence at the wavelength of interest for our studies. Fluorescence measurements were carried out by means of a Perkin Elmer LS5 spectro- fluorometer using an excitation wavelength of 350 nm and an emission of 425 nm (slit 5/5 nm). Turbidity was evaluated with the same instrument, with excitation and emission wavelengths both set at 600 nm. Sonication was performed with a Soniprep 150 apparatus (MSE, Crowley) equipped with a 19-mm probe, operating at 23 KHz and with an amplitude of 6 •m. A phospholipids B test kit (Wako Chemicals GmbH) was used for quantitative deter- minations of these substances. METHODS Vesicles containing DPH were prepared according to two different techniques. METHOD A (MIXED FILM) The appropriate amount of phospholipid (80 mg O f P90 or 800 •1 of EPC solution), 5.6 mg of cholesterol, and 222 •1 of a 2 x 10 -4 M methanol solution of DPH were completely dissolved in 4-5 ml of methanol. The solvent was vacuum evaporated to