Maintenance of nonviral vector particle size during the freezing step of the lyophilization process is insufficient for preservation of activity: insight from other structural indicators.

The instability of nonviral vectors as liquid formulations has stimulated considerable interest in developing dehydrated formulations that would be resistant to shipping stresses and could be stored at room temperature. Recently, we reported that high sucrose/DNA ratios are capable of maintaining particle size during the freezing step of the lyophilization process and we suggested that the separation of individual particles within sugar matrices is responsible for the reported protection of nonviral vectors during the freezing step of a typical lyophilization protocol. The purpose of this study was to extend these observations to other nonviral vectors that incorporate different cationic components. Cationic lipid-based complexes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), with helper lipid cholesterol (Chol) or dioleoylphosphatidyl-ethanolamine (DOPE), showed similar protection by sucrose. Formulations of a polyethylenimine (PEI)-based vector required much higher excipient/DNA ratios for size protection compared with protamine- and lipid-based vectors. At low sucrose/DNA ratios, zeta potentials for all complexes were significantly lowered during freezing. Similar results were obtained at high sucrose/DNA ratios, except for DOTAP-DOPE-containing vectors which maintained zeta potential values comparable to unfrozen controls. The changes in zeta potential values indicate that complexes are altered during freezing despite the maintenance of particle size as determined by light scattering. Furthermore, these changes might explain the observed reduction in transfection activity and provide new information about the effects of physicochemical changes of nonviral vectors during the freezing step of lyophilization. Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1445-1455, 2001