Stabilization of Liposomes in Frozen Solutions Through Control of Osmotic Flow and Internal Solution Freezing by Trehalose

The purpose of this study was to elucidate the effect of trehalose distribution across the membrane on the freeze-related physical changes of liposome suspensions and their functional stability upon freeze-thawing. Cooling thermal analysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposome suspensions showed exotherm peaks of bulk (−15°C to −25°C) and intraliposomal (approx. −45°C) solution freezing initiated by heterogeneous and homogeneous ice nucleation, respectively. The extent of the intraliposomal solution freezing exotherm depended on liposome size, lipid composition, cosolutes, and thermal history, suggesting that osmotic dehydration occurred due to the increasing difference in solute concentrations across the membrane. A freeze-thawing study of carboxyfluorescein-encapsulated liposomes suggested that controlling the osmotic properties to avoid the freeze-induced intraliposomal solution loss either by rapid cooling of suspensions containing trehalose in both sides of the membrane (retention of the intraliposomal supercooled solution) or by cooling of suspensions containing trehalose in the extraliposomal media prior to freezing (e.g., osmotic shrinkage) led to higher retention of the water-soluble marker. Evaluation and control of the osmotically mediated freezing behavior by optimizing the formulation and process factors should be relevant to the cryopreservation and freeze-drying of liposomes.