Fragmentation into Small Vesicles of Dioleoylphosphatidylcholine Bilayers During Freezing and Thawing

Multilayered liposomes of some phosphatidylcholines progressively fragment into small vesicles when the electrolyte solution in which they are suspended is subjected to successive cycles of freezing and thawing. The fragmentation process, routinely monitored by absorbance measurements and verified by electron microscopy and dynamic light scattering, involves bilayer breakage and resealing. After 10 cycles of freezing and thawing in 0.1 M electrolyte solution, the result is a population of vesicles smaller than 200 nm diameter. Sucrose, a common cryoprotectant, completely inhibits fragmentation. Fragmentation is absolutely dependent upon the presence of an electrolyte. Those electrolytes most effective in promoting liposome fragmentation have large freezing point depressions and corresponding high solubilities at the freezing point. This, coupled with the observation that saturating concentrations of electrolyte are less effective than 0.1 M solutions indicates that an essential stage in the fragmentation process is osmotic extraction of water from the vesicles, i.e., ice formation in the external phase leads to a progressive increase in the electrolyte concentration of the residual external solution, which, in turn, dehydrates the vesicle. In addition, for maximal fragmentation, the minimum temperature must be at least as low as the solute eutectic temperature. Particular physical properties of the bilayer are also important, for dioleoyl and diphytanoyl derivatives are much more susceptible to fragmentation than are other phosphatidylcholines, and inclusion of 50 mol% cholesterol in dioleoylphosphatidylcholine completely inhibits membrane breakup. This system provides insight into mechanisms of freezing damage to membranes and may also offer a very simple and rapid assay for biological cryoprotectants.