Preparation of novel double liposomes using the glass-filter method

The glass-filter method, a newly developed preparative method for liposomes, was applied for preparation of novel double liposomes. Double liposomes were prepared by filtering a suspension of liposomes prepared using a G4 filter (pore size: 10-16 microm) into a G3 filter (pore size: 40-100 microm) coated with a similar lipid layer. The morphological structure of the double liposomes was confirmed using scanning electron microscopy by the freeze-fracture method to be multivesicular vesicles consisting of small liposomes enveloped in larger liposomes. The diameter of liposomes prepared using the G4 filter was 0.8-2 microm and that of liposomes prepared using the G3 filter or double liposomes was 5-10 microm. These results suggested that the particle size of liposomes is dependent on the pore size of the glass-filter. The encapsulation efficiencies of double liposomes for brilliant blue FCF (BB) and erythrosine (ER) were higher than those of liposomes prepared by the standard Bangham method. Double liposomes showed suppressed release of BB or ER compared with normal liposomes. In particular, no release of BB was observed from the double liposomes prepared with stearylamine. These findings implied that the outer lipid layer protects the inner liposomes. The glass-filter method is the only method that we can get the double liposomes in a short period, and double liposomes prepared by this novel method had adequate size and good stability in vitro.