THE PROTECTIVE EFFECT OF FREE AND MEMBRANE-BOUND CRYOPROTECTANTS DURING FREEZING AND FREEZE-DRYING OF LIPOSOMES

Liposomes were prepared from natural (EPC) and hydrogenated (HEPC) egg phosphatidylcholine, with and without cholesterol (CHOL), from sucrose fatty acid ester (SPS7; sucrose-palmitate/stearate) with CHOL and dicetylphosphate (DCP) or from EPC and HEPC with the mono-, di- and tri-ester of SPS7. The cryoprotective activity of sucrose or membrane-bound sucrose fatty esters was assessed. Vesicles were frozen and thawed, or freeze-dried and reconstituted, and retention of the encapsulated marker 5,6-carboxyfluorescein (CF) was monitored. CF retention decreased with decreasing freezing temperature, while increasing concentrations of sucrose provided increasing cryoprotection during freezing and thawing. SPS7 vesicles were fully protected by 0.6 M sucrose, whereas equimolar mixtures of EPC and HEPC with SPS7 required 1 M sucrose for complete protection. EPC/CHOL liposomes retained maximally 85% and HEPC/CHOL liposomes 95% marker at the highest sucrose concentration. Lyophilized liposomes without sucrose or in mixture with the SPS mono- or diester retained < 10% CF. Lyophilization of EPC and HEPC liposomes in the presence of 0.4 M sucrose resulted in 75% retention of originally encapsulated marker. Differential scanning calorimetry showed a significant reduction of the transition temperature (T(c)) of lyophilized HEPC liposomes in the presence of sucrose and the SPS monoester. Infrared spectroscopy indicated sucrose and the SPS monoester forming strong hydrogen bonds with phosphate head groups which supports the water replacement or 'pseudohydration' hypothesis.