Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification.

R. P. Goodrich and co-workers (1989, U.S. Patent 4,874,690; 1992, Proc. Natl. Acad. Sci. USA 89,967-971) have reported that red blood cells can be preserved in the dry state by addition of mixtures of hydroxyethyl starch (HES) and glucose. More recently, Spieles and co-workers (1996, Cryo-Lett. 17, 43-52) found that HES alone is insufficient to preserve the dry cells and concluded on this basis that the studies of Goodrich et al. were incorrect. In the present paper we revisit that suggestion, using liposomes as a model to study effects of HES and glucose on membrane stability. In previous studies we and others have established that liposomes can be stabilized in the dry state if they are dried in the presence of disaccharides. Monosaccharides have not been effective. Measurements of effects of glucose on phase transitions in the dry lipids and vibrational frequency of the phosphate headgroup suggest that glucose shows an interaction with dry egg phosphatidylcholine similar to that seen with disaccharides. Nevertheless, glucose does not inhibit fusion in liposomes during drying, and it does not prevent leakage. Hydroxyethyl starch, which has a very high glass transition (Tg), inhibits fusion in the dry liposomes, but it does not depress the liquid crystalline to gel phase transition temperature (Tm) in the dry phospholipids, does not cause a shift in the phosphate vibration indicative of hydrogen bonding of the sugar to the phosphate, and does not stop leakage of trapped carboxyfluorescein. However, if glucose is added to the HES-containing samples, the liposomes are stabilized, so long as the samples are maintained below the Tg of the mixture. If they are heated above that Tg they fuse and leak their contents. We conclude that both glass formation and depression of Tm in the dry lipids are required. The role of glass formation in stabilization during drying of liposomes appears to be inhibition of fusion.