Stability of Poly(l-Lysine)-Complexed Plasmid DNA During Mechanical Stress and DNase I Treatment

Abstract

The aim of this study was to investigate the formation and stability of complexes between plasmid DNA (pDNA) and poly(l-lysine) (PLL). Formation of pDNA/PLL complexes with various ratios was determined by a fluorescence spectrophotometric method using fluorescamine. The effects of sonication, vortexing, and exposure to DNase I on the stability of free pDNA and pDNA/PLL complexes are discussed. A linear correlation between PLL added and PLL bound was obtained with overall reaction efficiency of 84.2–92.6%. Sonication degraded both free and PLL-complexed pDNA within 15 sec of vortexing. However, vortexing did not alter the stability of free and complexed pDNA. Dramatic increase in the protection of pDNA in pDNA/PLL complexes was observed in the DNase I digestion experiment; 68.1–89.0% of total pDNA in the pDNA/PLL complexes was protected from DNase I digestion compared to only 19.2% of total pDNA that remained undegraded after DNase I treatment of free pDNA. An increase in the PLL/pDNA ratio led to an increase in the protection of supercoiled pDNA; 15.5–38.2% of supercoiled pDNA in PLL/pDNA complexes was protected after DNase I treatment. The results show that complexation of pDNA with PLL can stabilize the supercoiled structure of pDNA for the development of biodegradable microspheres as a delivery system for pDNA. Stability of pDNA/PLL complex can be monitored by PicoGreen® dye and fluorescence densitometric assay methods.