Abstract
The lipid phosphorus of phosphatidylglycerol liposomes was found to diffuse extensively, after a lag time of 1 to 2 days, through a 0.1 μm pore size polycarbonate membrane in a two compartment system. Diffusion occurred when either multilamellar or large unilamellar vesicles were studied, even if they were sedimented to eliminate any smaller particles. The lipid of liposomes prepared under sterile conditions also diffused extensively. Diffusion appeared to be related to the age of the vesicles, and could be eliminated by incorporating antioxidants into the liposomes, or by using liposomes prepared from saturated phospholipids (C14 or larger). This indicated that diffusion was caused by phospholipid oxidation, which was confirmed by HPLC analysis. Phospholipid phosphorus that diffused through a membrane appeared more polar, as indicated by its capacity to distribute into the upper phase of a two phase extraction. Phospholipid phosphorus diffusion was preceded by the complete loss of liposomes contents, indicated by the complete diffusion of encapsulated carboxyfluorescein through the membrane. Oxidation of the lipid could be prevented by inclusion of either butylated hydroxytoluene or α-tocopherol in the membrane. The best retention of liposomal contents was achieved when both antioxidants and cholesterol were included in the liposome preparation. The antioxidant incorporated in the liposomes remained effective in protecting the phospholipids upon storage at 4°C for 2 months. The inclusion of EDTA in the suspension medium retarded the rapid oxidation, suggesting that the presence of trace amounts of heavy metal ions in the buffer catalyzed the oxidation. Phospholipid oxidation was most effectively inhibited by the presence of serum or chemically defined medium, suggesting that oxidation of liposomal lipids in a biological environment may be minimized if appropriate steps are taken.
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