COSMETIC LIPOSOMES 125 form a thin film of lipids and additives inside the vessel. 2.5 ml of HEPES buffer were added the mixture was kept in the dark for 2 h, then gently shaken for I h and sonicated, under a nitrogen stream, for 40 min (8 times for 5 min). The temperature was maintained at 15-20øC by means of a water bath. The liposome dispersion was finally diluted 1:1 with HEPES. METHOD B (ABSORBED FLUOROPHORE) SUV were prepared according to the same procedure described above, but no marker was added until the final dilution. This 1:1 dilution at the end of the vesicle preparation was performed with a DPH dispersion prepared as follows: 4-5 ml of methanol were added --4 to 222 Ixl of the 2 X 10 M methanol soluuon of DPH, the solvent was vacuum evaporated, 2.5 ml of HEPES were added to the residue, and the mixture was then vortexed and sonicated to obtain a homogeneous dispersion of DPH. Unmarked lipo- somes were kept in the dark overnight with the fluorophore dispersion. Longer times did not significantly increase the amount of absorbed DPH. It has been pointed out (5) that sonication of phospholipid dispersions leads mainly to small unilamellar vesicles (SUV, 10-100 nm), but according to the aim of this study, actual liposome sizes were not determined. Liposome separation from the "free" phospholipids and non-incorporated DPH was performed on 1-ml samples with Sephadex G200. Columns were eluted with HEPES and all the vesicles were collected (liposomes were eluted with the void volume and their presence was checked by means of a turbidity test) to reach a final volume of 5 mi. The phospholipids B test was performed before and after the passage through the columns in order to verify the percentage of aggregated form with respect to the total amount used. All final preparations containing the vesicles were tested for turbidity. The reproduci- bility of these last measurements, performed on the different preparations, indicated that the average dimensions and concentration of liposomes were to be considered as constant (e.g., for all liposomal dispersions corresponding to a phospholipid concentra- tion of 0.3 mg/ml, turbidity = 71.8 + 2.1). DPH fluorescence was initially determined on intact purified liposomes in order to verify once more the reproducibility among the various preparations of the same kind. The vesicle structure was then broken by dilution (1:9) with methanol for the deter- mination of the total amount of DPH present in the vesicles. Quantitative DPH determinations were obtained from appropriate calibration curves of the marker in methanol. In order to study and compare the resistance of liposomes, the change in turbidity by progressive addition of a surfactant (Triton X-100) was measured (6,7). RESULTS AND DISCUSSION In Table I the fluorescences of DPH in the vesicle dispersion are reported for the different types of phospholipids and for the two methods of vesicle loading. In the same table the fluorescence measured when vesicles were broken with methanol is also given.

126 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table I Fluorescence Values of DPH-Loaded Liposomes Prepared With Phospholipids of Different Type and Fluorescence Determined on Vesicles Broken With Methanol Phospholipid Method A Method B EPC 170.4 + 8.5 148.5 + 8.0 EPC (broken SUV) 47.9 + 2.5 34.7 + 2.0 P90 160.0 -+ 8.2 144.3 + 8.0 P90 (broken SUV) 45.5 + 2.5 33.7 + 2.0 Reported results represent the mean values obtained from five separate experiments. Reproducibility can be evaluated by the low range of fluorescence fluctuations in the different preparations ("•5%). As it is possible to observe, only a small difference in DPH fluorescence between EPC and P90 liposomes has been detected. When compared with intact vesicles, broken liposomes in methanol always gave a much lower fluores- cence because of the presence of the organic solvent (4). From the fluorescence values reported in this table, it is also possible to observe that less DPH was present in the "Method B" formulations because of the smaller amount of the probe that can be liposomally incorporated by means of this technique. The marker actually present in the vesicles was calculated from the values determined in methanol (i. e., after the breakage of the aggregated structure), where fluorescence is linearly dependent on DPH concen- tration. In this sense, it must also be pointed out that surfactants are often used to disaggregate liposome structures, but their presence leads to higher fluorescence values in water dispersions, gives non-linear calibration curves, and can induce fluorescence changes in the fluorophore (8-10) that can yield uncorrect or misleading results. The phospholipid test indicated that over 95% of the initial amount of phospholipids was recovered as SUV after the passage through Sephadex nevertheless, for a correct comparison among the different preparations, these minor variations have been consid- ered and the percentage of entrapped or absorbed DPH was calculated according to the following expression: [DPH]a % DPH - x K x 100 [DPH]b where the subscripts a and b indicate the DPH concentrations (mmoles x ml-•) after and before purification, respectively, and K is the ratio between phospholipid concen- trations (mg/ml) before and after the passage through Sephadex. The coefficient K allows comparison of the different preparations by considering the small loss of phos- pholipid during purification and by correcting at the same time the dilution factor. In Table II the percentage of directly entrapped (Method A) or absorbed DPH on empty vesicles (Method B) is given for both EPC and P90. No variations between type III-E and XI-E EPC were detected. Reported results are the average values obtained from five separate experiments. As it can be observed from obtained results, the difference in loading capacity between EPC and P90 vesicles, although detectable, is always below 4%. In order to compare the stability of EPC and P90 liposomes, the changes in turbidity of the vesicle dispersion by addition of increasing amounts of Triton X-100 were