After removal of nonaggregated lipids, the supernatants were lyophilized and solubilized in methanol in order to disrupt the nanostructure leading to the recovering of nonaggregated lipids which can be further analyzed by HPTLC as described in the experimental part. It is worth to note that no peak has been observed on the lane corresponding to the blank solution. Such result allowed us to conclude that peaks corresponding to the analyzed lipids (Egg-PC:

Rf = 0.04, PEG45-DSPE: Rf Inhibitors,research,lifescience,medical = 0.46 and PEG45-Tetraether: Rf = 0.79) were not overestimated because of the presence of other peaks having similar Rf values (Figure 3(a)). Calibration curves, based on either peak height or peak area, were plotted for each lipid (Figures 3(b) and 3(c)). From these calibration curves, amounts of lipids contained in each formulation studied were calculated (Table 2) and compared to initial amount of lipids used to prepare liposomes and archaeosomes (Table 2). Results given in Table 2 demonstrated that lipid composition of the prepared liposomes and Inhibitors,research,lifescience,medical Inhibitors,research,lifescience,medical archaeosomes are very similar to the initial lipid compositions: 88/12wt% for Egg-PC/PEG45-DSPE liposomes instead of an initial composition of 90/10wt% and 86/14wt% for Egg-PC/PEG45-Tetraether archaeosomes

instead of an initial composition of 90/10wt%. Figure 3 HPTLC measurements: (a) Scan of a plate at 366nm (fluorescence mode); (b and c) standard curves, based on peak height, for each lipid composing the prepared liposomes and archaeosomes. (AU = arbitrary unit). Table 2 Amounts of lipids contained in liposomes and archaeosomes

calculated from HPTLC data. Inhibitors,research,lifescience,medical The given values are an average Flavopiridol between peak height and peak area values. The values are reported to a volume of 1mL. 3.3. Carboxyfluorescein Encapsulation Inhibitors,research,lifescience,medical and Release Profile To assess vesicle stability, the kinetics of encapsulated CF release from PEG-bearing liposomes and archaeosomes was studied at 4°C (standard storage temperature of liposomal formulations) and 37°C (human physiological temperature). The percent release of CF was calculated from the formula described in the experimental part after evaluating the initial amount of encapsulated CF. Thus, a part of the sample containing the vesicle dispersion was treated with triton X-100 [36] for lipid membrane disruption. Then, the fluorescence analysis of the resulting sample allowed us to determine the almost CF concentration initially entrapped in the nanocarrier using a calibration curve beforehand established. The release profile of CF from vesicles at 4°C (Figure 4(a)) showed different rates of leakage between liposome and archaeosome formulations. Indeed, 45% CF release was found to be approximately 20h for the liposome sample and 100h for the archaeosome sample. This different behavior was dramatically increased when the formulations were studied at 37°C.