Abstract
Differential scanning calorimetry (DSC) was used to determine the amount of water that freezes in an aqueous suspension of multilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes. The studies were performed with dehydrated suspensions (12-20 wt% water) and suspensions containing an excess of water (30-70 wt% water). For suspensions that contained > or = 18 wt% water, two ice-formation events were observed during cooling. The first was attributed to heterogeneous nucleation of extraliposomal ice; the second was attributed to homogeneous nucleation of ice within the liposomes. In suspensions with an initial water concentration between 13 and 16 wt%, ice formation occurred only after homogeneous nucleation at temperatures below -40 degrees C. In suspensions containing < 13 wt% water, ice formation during cooling was undetectable by DSC, however, an endotherm resulting from ice melting during warming was observed in suspensions containing > or = 12 wt% water. In suspensions containing < 12 wt% water, an endotherm corresponding to the melting of ice was not observed during warming. The amount of ice that formed in the suspensions was determined by using an improved procedure to calculate the partial area of the endotherm resulting from the melting of ice during warming. The results show that a substantial proportion of water associated with the polar headgroup of phosphatidylcholine can be removed by freeze-induced dehydration, but the amount of ice depends on the thermal history of the samples. For example, after cooling to -100 degrees C at rates > or = 10 degrees C/min, a portion of water in the suspension remains supercooled because of a decrease in the diffusion rate of water with decreasing temperature. A portion of this supercooled water can be frozen during subsequent freeze-induced dehydration of the liposomes under isothermal conditions at subfreezing storage temperature Ts. During isothermal storage at Ts > or = -40 degrees C, the amount of unfrozen water decreased with decreasing Ts and increasing time of storage. After 30 min of storage at Ts = -40 degrees C and subsequent cooling to -100 degrees C, the amount of water associated with the polar headgroups was < 0.1 g/g of DPPC. At temperatures > -50 degrees C, the amount of unfrozen water associated with the polar headgroups of DPPC decreased with decreasing temperature in a manner predicted from the desorption isotherm of DPPC. However, at lower temperatures, the amount of unfrozen water remained constant, in large part, because the unfrozen water underwent a liquid-to-glass transformation at a temperature between -50 degrees and -140 degrees C.
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