j. Soc. Cosmet. Chem., 45, 167-172 (May/June 1994) Liposomes in cosmetics. II. Entrapment of a hydrophilic probe 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) (A.M., L.G.P.), Dipartimento Farmaco Chimico Tecnologico, Universita di Siena (V. T. ), and Dipartimento Farmaco Chimico Tecnologico, Universita di Cagliari (F.A. ), Italy. Received January 6, 1994. Synopsis Phospholipids of different origin (egg and soya) and purity were used to prepare liposomes by sonication. Loading of these vesicles with a fluorescent hydrophilic model molecule (calcein) was carried out by means of two different methods. No appreciable differences were observed in the loading capacities of liposomes prepared with the two products. Obtained results indicated that the fraction of incorporated or absorbed calcein was always very small with respect to the total amount of this substance used for the preparation of the vesicles furthermore, the quantity of hydrophilic probe in the vesicle structure was remarkably affected by the loading method. The results of this investigation allowed us to consider the possible incorporation, in/on the vesicles, of additives that can be present in a formulation containing liposomes. INTRODUCTION It is well known that liposome entrapment capacity depends on several factors, such as the type of phospholipid, the physicochemical properties of the substance that must be loaded, and the technique used for the preparation of the vesicles. In a previous paper (1), in order to compare the behavior of a 99%-pure egg phosphati- dylcholine (EPC) with that of a mdch less expensive, but also less pure, vegetable phospholipid (P90), a lipophilic fluorescent probe (1,6-diphenyl-l,3,5-hexatriene DPH) (2), localized within the lipid bilayers, was chosen for our experiments. The results obtained (1) indicated that no remarkable differences were observed in either the stability in regard to surfactant-induced breakage or the loading capacity of liposomes respectively prepared with EPC or P90. It was also shown that the amount of lipophilic probe in the liposome structure was affected by the loading method. 167

168 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The aim of this work is to reproduce the same type of experiments with a hydrophilic fluorescent model molecule (calcein) and to compare the new results with those obtained with DPH. The choice of calcein is related to its self-quenching properties that make this substance a useful tool in liposome investigations (3). By means of this study we intend also to verify if empty liposomes, dispersed in a calcein solution, are capable of incorporating a certain amount of this substance. This last point acquires a fundamental importance if we consider that in the formulation of cosmetic preparations containing liposomes, hydrophilic substances can be present (e.g. preser- vatives, fragrances, coloring agents, etc.) and that their properties can be modified by incorporation in the vesicle structure furthermore, since liposomes have been claimed to enhance skin permeation, these substances could become, in the presence of lipo- somes, the cause of allergic or toxic effects. Studied liposomes were obtained by sonication, although other techniques (e.g. reverse- phase evaporation) may yield much higher trapping efficiency for hydrophilic substances (4). Sonication was chosen for this investigation because it is a procedure commonly used in cosmetics. MATERIALS 99%-pure L-ot-phosphatidylcholine from egg yolk (Sigma, type XI-E, chloroform so- lution, 100 mg/ml) and 90%-pure enriched soya phosphatidylcholine (Phospholipon 90, Nattermann Phospholipids GmbH) were used for vesicle preparation. Crystalline calcein was purchased from Sigma. HEPES pH 7.5 buffer solutions (10- 3 M), obtained 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. As in the previous work (1), fluorescence (Ex. 490 nm/Em. 520 nm) and turbidity (600/600 nm) measurements were carried out with a Perkin Elmer LS5 spectrofluorom- eter. Sonication was performed with a Soniprep 150 apparatus (MSE, Crowley). Lipo- some dimensions were evaluated with a Malvern Autosizer II. The Phospholipids B test kit (Wako Chemicals GmbH) was used for quantitative determinations of these sub- stances. Calcein solutions of two different concentrations (5 x 10-5 and 5 x 10 -2 M) were used for the present investigation these were obtained by dissolving an exactly weighed amount of the fluorescent probe in the minimum volume of a 1 M NaOH solution. The final calcein concentrations were then reached by addition of the HEPES solution (3). The pH (7.5) was checked each time by means of a potentiometer. The 5 x 10-5 M calcein solution was chosen because it had a concentration similar to that obtainable with DPH (1), while the more concentrated dye solution was used to constrain the hydro- philic probe within the liposomal structure. METHODS As in the previous study, vesicles containing the probe were prepared according to two different protocols.