Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: the particle isolation hypothesis

The instability of nonviral vectors in aqueous suspensions has stimulated a n interest in developing lyophilized formulations for use in gene therapy. previous work has demonstrated a strong correlation between the maintenance of particle size and retention of transfection rates. Our earlier work has shown that aggregation of nonviral vectors typically occurs during the fre ezing step of the lyophilization process, and that high concentrations of s ugars are capable of maintaining particle size. This study extends these ob servations, and demonstrates that glass formation is not the mechanism by w hich sugars protect lipid/DNA complexes during freezing. We also show that polymers (e.g,, hydroxyethyl starch) are not capable of preventing aggregat ion despite their ability to form glasses at relatively high subzero temper atures. Instead, our data suggest that it is the separation of individual p articles within the unfrozen fraction that prevents aggregation during free zing, i.e., the particle isolation hypothesis. Furthermore, we suggest that the relatively low surface tension of mono- and disaccharides, as compared to starch, allows phase-separated particles to remain dispersed within the unfrozen excipient solution, which preserves particle size and transfectio n rates during freezing. (C) 2000 Elsevier Science B.V. All rights reserved .