Leakage of a trapped fluorescent marker from liposomes: effects of eutectic crystallization of NaCl and internal freezing.

Leakage of trapped carboxyfluorescein from DL-alpha-dipalmitoylphosphatidylcholine multilamellar liposomes (diameter 1-2 microns) in NaCl solutions was measured after rapid freezing to temperatures between -15 and -55 degrees C. Leakage was low after freezing between -15 and -35 degrees C, but increased steeply between -35 and -45 degrees C. From DSC measurements it was found that the increase in leakage was associated with two crystallization processes: Eutectic crystallization of NaCl and freezing of undercooled solvent trapped in the interior of the liposomes ("internal freezing"). Damage caused by the former process could effectively be prevented by small amounts of trehalose (1% less than or equal to w less than or equal to 1.5%). Trehalose in these concentration also decreased damage due to internal freezing, but to a minor degree. In addition to these damaging transitions, a time-dependent process was found to cause leakage from the liposomes at -25 degrees C. The association between leakage and thermal activity suggests that DSC supplements cryomicroscopy and leakage measurements in the characterization of cryostability of liposomes.