Preservation of dry liposomes does not require retention of residual water.

Abstract

Certain sugars, particularly trehalose, dramatically alter physical properties of dry phospholipids in ways that mimic the presence of water. As a result, these sugars are capable of preserving the integrity of dry liposomes and membranes. Since these effects could conceivably be due to the presence of small amounts of water in the dry preparations of sugar and lipid, we have done careful measurements of the residual water contents in the dry samples and report the results here. Lyophilized liposomes composed of palmitoyloleoylphosphatidylcholine and phosphatidylserine (9:1) contain at most 0.2 mol of H20 per mol of lipid. When the trehalose concentration in the dry mixtures is increased, there is no increase in the apparent water content of the samples over a wide range of sugar concentrations. Over the same range of trehalose contents the maximal effect of trehalose on physical properties of the lipids and on stabilization of liposomes is achieved. We conclude that the stabilization does not require retention of residual amounts of water in the dry trehalosephospholipid preparations. Similar studies with other sugars show a relationship between the amount of sugar interacting with the lipid and the ability of the same sugar to stabilize dry liposomes.In view of their practical potential as vehicles for delivery of water-soluble drugs to cells in whole organisms (e.g., ref. 1), stabilization of liposomes for long-term storage is an important, practical problem. We (2, 3) and others (4-6) have addressed this problem and have found that liposomes can be preserved quite effectively by freezing (5-8) or freeze-drying (2-5) in the presence of certain sugars. For example, when liposomes were freeze-dried in the presence of adequate amounts of trehalose, they were found upon rehydration to have retained as much as 100% of their original contents (2-4). By contrast, when they were freeze-dried without sugar, upon rehydration they leaked essentially all of their contents. Some of the same sugars that are effective at preserving dry liposomes are also remarkably effective at stabilizing structure and function in dry biological membranes (9).In an effort toward understanding the mechanism by which sugars preserve dry liposomes and membranes, we have studied over the past few years the effects of sugars on physical properties of phospholipids. Those studies have shown that the sugars that are the best preservation agents also most strongly inhibit fusion during freezing (5,6,8,10) or freeze-drying (3). However, the amount of sugar required to inhibit fusion is much less than that required to prevent leakage, so inhibition of fusion alone is clearly not the sole mechanism of preservation (3). The same sugars are very effective at reducing the transition temperature (Tm) for the dry lipids, thus potentially maintaining them in a fluid phase in the absence of water (3,6,11). We previously suggested that the dry lipids were in liquid crystalline phase above Tm, but more recent evidence (12) suggests that it is a fluid ...

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