Stability of liposomes on storage: freeze dried, frozen or as an aqueous dispersion

For various types of liposomes carboxyfluorescein (CF) latency and physical stability on storage were investigated. Three regimens were compared: storage at 4-6°C in an iso-osmotic aqueous buffer, freezing of the dispersions at -5 or -30°C, or freeze drying of the dispersions. Reverse phase evaporation vesicles (REV) were used with mean diameters between 0.2 and 0.3 µm. Liposomes consisted of egg phosphatidylcholine (PC) and phophatidylserine (PS) without or with cholesterol (chol) (9/1 and 10/1/4, respectively) or of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylglycerol (DPPG) without or with chol (10/l/[5]). PC/PS liposome dispersions lost 25% of the entrapped CF within 10 days. The leakage rate decreased with the inclusion of cholesterol in the bilayer. Both after a freezing/thawing cycle and after freeze-drying no acceptable CF latency could be obtained. The cryoprotectants that were tested failed to raise CF latency significantly. However, the physical integrity of the liposomes could be maintained by proper choice of the cryoprotectant. When stored at 4-6°C, DSPC/DPPG/(chol) dispersions were stable for at least 6 months. Upon freezing/thawing less than 10 % CF was lost. Freeze drying without cryoprotectants reduced CF latency dramatically on rehydration. The physical structure was maintained and maximum latencies of 70 % could be obtained with the use of lactose as a cryoprotectant.