Phase behavior of freeze-dried phospholipid-cholesterol mixtures stabilized with trehalose

SATOSHI OHTAKE; CAROLINA SCHEBOR; JUAN J. DE PABLO

Puerto Vallarta, México

Congreso; V CONGRESO IBEROAMERICANO DE INGENIERÍA DE ALIMENTOS (CIBIA V).; 2005

Introduction. Achieving long-term stability in biological systems has been a long-standing goal of the food, pharmaceutical, and biomedical industries. Avoiding the need for refrigeration would reduce production and storage costs drastically. The desiccation of phospholipidic vesicles has been studied in efforts to understand biological membranes under low-water content conditions. Trehalose is effective in protecting biological membranes upon freeze-drying, and has been widely used to preserve the integrity of phospholipid liposomes. The effects of trehalose on dehydrated cholesterol-containing liposomes, however, have not been examined in detail. The aim of this work is to understand how cholesterol-containing liposomes behave upon lyophilization.  Methodology. Liposomes were obtained by extrusion of mixtures consisting of 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE) and cholesterol (Ch). Samples were freeze-dried, and the phase transitions were studied by DSC.  Results and discussion. The phase transition temperature (Tm) for dehydrated DPPC is 105°C, which is higher than that observed in the fully hydrated state (42°C). Upon the addition of Ch, the peak for the transition observed at 105°C decreases both in magnitude and in temperature. A second, broad endothermic transition at 70°C becomes observable at 23 mol% Ch and its peak intensity increases as the cholesterol proportion is increased. The intercalation of cholesterol between the lipids destabilizes the phospholipid packing in the gel phase, leading to a depression in the Tm of the dehydrated samples. The presence of the two endothermic peaks can be explained by the existence of Ch-rich and Ch-poor domains, as described for these same systems in the fully hydrated state (1). Two transitions at 52° and 94°C are observed for dehydrated DPPE. All of the DPPE-Ch mixtures exhibit multiple transitions. A freeze-dried DPPE-DPPC mixture (1:1 molar ratio) exhibits a single transition at 83°C which suggests that the two phospholipids are miscible.  As the Ch proportion is increased in the DPPE-DPPC-Ch mixtures, the Tm at 83°C decreases both in temperature and in magnitude.  A new transition at 60°C appears above 9 mol% Ch and grows in magnitude with increasing cholesterol content. The two peaks can also be ascribed to Ch-rich and Ch-poor regions. The Tm of freeze-dried pure DPPC decreases from 105°C to 25°C upon the addition of trehalose. Trehalose also reduces the Tm of pure DPPE from 52°C to 39°C, and that of the DPPE-DPPC (1:1 molar ratio) mixture from 83°C to 21°C.  The Tm depression is explained by the ability of trehalose to restrict the contraction of the lipids that would otherwise occur upon dehydration (2).  The addition of cholesterol influences the Tm depending on the phospholipids present in each mixture.  Cholesterol does not affect significantly the Tm of DPPC-Ch mixtures.  For DPPE-Ch mixtures, however, the Tm decreases from 39°C (pure DPPE) to 21°C as increasing amounts of cholesterol are incorporated into the liposomes.   Conclusion. In this work, we have analyzed the phase behavior of various freeze-dried mixtures of DPPE, DPPC, and cholesterol and have examined the effects of trehalose addition to these liposomes. The main focus of the use of trehalose has been to reduce the Tm of phospholipids upon dehydration. This is an important aspect since liposomes undergoing phase transition during rehydration can lead to leakage of encapsulated components. In this study we show that for dehydrated systems containing cholesterol, however, trehalose is also necessary to limit phase separation. Despite the abundance of reports examining the Ch-containing liposomes in the fully hydrated state, there is a shortage of studies on these systems in the dried state. This work provides strong evidence for the effectiveness of trehalose in stabilizing cholesterol-containing membranes upon lyophilization and could be useful in the design of liposomes for entrapment and delivery of pharmaceuticals.  Acknowledgment. The authors are thankful for financial support from the National Science Foundation and the Defense Advanced Research Projects Agency.  Carolina Schebor thanks CONICET (PIP2734).  References. 1. McMullen, T. Lewis, R., and McElhaney, R. (1993). Differential scanning calorimetric study of the effect of cholesterol on the thermotropic phase behavior of a homologous series of linear saturated phosphatidylcholines. Biochemistry 32:516-522. 2. Crowe, L., and Crowe, J. (1988). Trehalose and dry dipalmitoylphosphatidylcholine revisited, Biochim. Biophys. Acta 946:193-201.