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 an 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 freezing step of the lyophilization process, and that high concentrations of sugars are capable of maintaining particle size. This study extends these observations, and demonstrates that glass formation is not the mechanism by which sugars protect lipid/DNA complexes during freezing. We also show that polymers (e.g., hydroxyethyl starch) are not capable of preventing aggregation despite their ability to form glasses at relatively high subzero temperatures. Instead, our data suggest that it is the separation of individual particles within the unfrozen fraction that prevents aggregation during freezing, 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 transfection rates during freezing.