Stabilization of lipid bilayer vesicles by sucrose during freezing

Abstract

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. Eu(3+) 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 (t(c)) than when frozen while below their t(c). 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.