Long term stability of poly((2-dimethylamino)ethyl methacrylate)-based gene delivery systems

Purpose. To study the stability of polymer-plasmid complexes (polyplexes) b oth as an aqueous dispersion and in their lyophilized form. Methods. The characteristics of the polyplexes (size, charge and transfecti on potential) were monitored at different temperatures. Moreover, we studie d possible changes in the secondary and tertiary structure of the plasmid b y agarose gel electrophoresis and by CD spectroscopy to gain insight into t he mechanism of polyplex degradation. Results. The polyplexes preserved almost their full transfection potential after aging in an aqueous solution of 20 mM Hepes (pH 7.4) containing 10% s ucrose at 4 and 20 degrees C for 10 months. On the other hand, the polyplex es aged at 40 degrees C were rather unstable and lost their transfection ca pability with a half-life of around 2 months. During storage, conformationa l changes in the secondary and tertiary structure of DNA were observed. Whe n naked plasmid DNA was aged at 40 degrees C as an aqueous solution and com plexed with polymer just before the transfection experiment, a slower drop in its transfection capability was observed. The freeze-dried polyplexes us ing sucrose as lyoprotectant almost fully retained their transfection effic iency, even when aged at 40 degrees C for 10 months. Conclusions. This study provides information about polyplex stability in aq ueous dispersions on storage and demonstrates that freeze-drying is an exce llent method to ensure long term stability.