High encapsulation efficiencies in sized liposomes produced by extrusion of dehydration-rehydration vesicles

In an attempt to produce sized liposomes with high encapsulation efficiency by a gentle procedure, we compared the encapsulation efficiency in liposomes of three different molecules: carboxyfluorescein (mol.wt 376 Da), protein A (mol. wt 40,000 Da), and a DNA-plasmid pN2 alpha 1AT (9.3 kb). Sized liposomes were obtained by extrusion of multilamellar vesicles through polycarbonate filters of defined pore size (0.2 micron). Multilamellar vesicles were formed by mechanical dispersion of dried egg phosphatidylcholine (PC) and cholesterol (CH) at molar ratio 1:1 in an aqueous buffer (MLVs) or by dehydration-rehydration procedure (DRVs) where small unilamellar vesicles (SUVs) obtained by probe sonication of PC:CH (1:1) in water were dried in the presence of the desired agent by lyophilization and then rehydrated in one tenth of the original volume. Our results show that, in all cases, the higher encapsulation efficiencies were obtained by dehydration-rehydration vesicles (DRVs) and that the ratio material/lipid in sized DRVs (S-DRVs) increased three- to four-fold with respect to sized liposomes (S-MLV) obtained by extrusion of MLVs. In addition, plasmid electrophoresis on agarose gels (0.8 per cent) showed that no detectable DNA fracture was produced in the encapsulation process. In summary, we showed that extrusion through polycarbonate filters of preformed DRVs is a gentle procedure which enhances the encapsulation efficiencies in sized liposomes of molecules with clearly different molecular weight and structure.