Factors affecting the size distribution of liposomes produced by freeze–thaw extrusion

This paper describes the development of a protocol for the production of liposomes using a freeze–thaw extrusion methodology. Laser diffraction particle size analysis showed that the median diameter of freeze–thawed egg phosphatidylcholine multilamellar vesicles (eggPC MLVs) was increased when cholesterol was included in the bilayers. Using a freeze–thaw cycle of 3 min freezing in liquid nitrogen at −196°C followed by 3 min thawing at 50°C resulted in an anomalously large particle size for eggPC/cholesterol formulations. When liposomes were repeatedly freeze–thawed a maximum size was achieved after five freeze–thaw cycles. Dispersion of liposomes in sodium chloride solutions promoted size increases following freeze–thawing, suggesting that vesicles had aggregated or fused. Poloxamers P338 and P407 inhibited the size increases observed during freeze–thawing for eggPC MLVs dispersed in 1.0 M NaCl, probably through steric prevention of aggregation and fusion.