LIPOSOMES IN COSMETICS 171 Table II Effect of the Loading Technique on the Ratio Between Probe and Phospholipid Concentrations Phospholipid Probe [Probe]/[Phospholipids] X 100 Method A Method B EPC DPH P90 DPH EPC Calcein 2 P90 Calcein 2 EPC Calcein 3 P90 Calcein 3 0.036 - 0.003 • 0.034 + 0.003 • 0.002 0.002 3.03 - 0.15 2.95 --- 0.15 0.018 + 0.003 • 0.016 -+ 0.0031 Undetectable Undetectable 0.78 -+ 0.05 0.80 --- 0.05 Calculated from the data reported in reference 1. Concentration of the loading solution: 5 X 10-5 M. Concentration of the loading solution: 5 X 10-2 M. The data reported in Table II clearly indicate that, within similar molarities of the probes in the loading solutions ([DPH] --• [calcein] = 5 X 10-5 M), the concentration of the hydrophilic dye in/on the liposomes, when detectable, was always much lower than that of the lipophilic DPH. Furthermore, when the 1000-times-more-concentrated calcein loading solution was used in order to "force" the capture of the probe by the vesicles, the corresponding increase of the molar ratio between calcein and phospholipids indicated that in all cases a very small amount of calcein is present in/on the vesicles. In this sense, it is interesting to point out that while in the case of DPH the percentage of marker in the liposomes was always very high with respect to the total amount present in the preparation [over 70% when the Method A was used and over 50% in the case of Method B (1)], when calcein was used, the fraction of this hydrophilic probe actually entrapped within the liposomal structure was always very small (ca. 2% and 0.1% for Methods A and B, respectively) and became almost negligible when empty liposomes were incubated in the calcein solution, even when the concentration of this fiuorophore was remarkably high (5 x 10-2 M). The comparison between a hydrophilic (calcein) and a lipophilic probe (DPH) indicates that, as expected, the fraction of substance entrapped or absorbed is much higher for DPH. Experimental results indicate also that the amount of probe found in the liposome structure, although affected by the loading method, is always very low with respect to the total amount present in the formulation and is not influenced by the type of phospholipid used in this study (99%-pure EPC or P90). As a consequence, the very slight reduction of"free" hydrophilic additive concentration, due to its incorporation in the vesicle structure, should not appreciably modify its properties in the preparation. REFERENCES (1) A. Memoli, L. G. Palermiti, V. Travagli, and F. Alhaique, Liposomes in cosmetics: Which kind of phospholipid? Which loading method?, J. Soc. Cosm. Chem., 44, 123-128 (1993). (2) B. J. Litman and Y. Barenholz, Fluorescent probe: Diphenyhexatriene, Methods Enzymol., 81, 678- 685 (1982). (3) T. M. Allen, in Liposome Technology, Vol. III, G. Gregoriadis, Ed. (CRC Press, Boca Raton Fl., 1984), pp. 177-182.

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