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The freeze-induced fusion and leakage of small unilamellar vesicles (SUV) of natural and synthetic phosphatidylcholines and the suppression of these processes by sucrose was studied by electron microscopy, by high-resolution NMR, and by ESR techniques. During slow freezing of SUV suspensions in water, the lipid was compressed into a small interstitial volume and transformed into a multilamellar aggregate without vesicular structure. When frozen in sucrose solution, the lipid also was compressed between the ice crystals but remained in the form of vesicles. The fractional amount of lipid remaining as SUV after freezing was found to increase significantly only at sucrose/lipid molar ratios above 0.4. Eu3+ displaced sucrose from the lipid by competitive binding. During freezing in the absence of sucrose, the vesicles became transiently permeable to ions. ESR studies showed that fusion of vesicles in the absence of sucrose is far more extensive when they are frozen while above their phase-transition temperature (tQ) than when frozen while below their tc. It is concluded that the extent of membrane disruption depends on the membrane mobility at the moment of freezing and that sucrose exerts its protective effect by binding to the membrane interface and/or by affecting the water structure.Zwitterionic phospholipids, such as phosphatidylcholine (PtdCho), which exhibit only limited swelling in excess water (1), form large (>100 nm) multilamellar vesicles (MLV) as the thermodynamically most-stable form at room temperature. These can be transformed into small unilamellar vesicles (SUV) (<80 nm) by sonication or other dispersion methods. Small vesicles are an ideal test system for the study of membrane stability because they undergo fusion and revert to MLV when subjected to certain processes, such as passage through the gel-to-liquid-crystal phase-transition temperature (tc), dehydration, freezing/thawing, freeze-drying/ rehydration, or, in the case of negatively charged vesicles, addition of divalent cations. The factor common to these diverse processes is the formation of two lipid phases, the liquid-crystalline and the gel state, whose coexistence leads to defects in molecular packing (2,3). Membrane stability is also dependent on the degree of hydration in the sense that hydration can prevent interbilayer contact (4-6). By measuring the resistance of SUV to effects of phase transition or dehydration, the stabilizing or destabilizing effect of various additives can be tested. Known stabilizers include mono-and disaccharides (7) and certain ions such as tetraalkylammonium and perchlorate (8), whereas membranes become destabilized on addition of univalent metal ions above 0.5 M (9) or multivalent ones at 1-10 mM (10).The stabilizing effect of sucrose during freezing of liposomes had been noted (11); systematic investigations of the stabilizing effect of various sugars on the lipid bilayers of cellular components were prompted by the observation that certain organisms that can survive dehydration are rich in treha...

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