Stability of poly(L-lysine)-complexed plasmid DNA during mechanical stress and DNase I treatment


Capan Y. , Woo B., Gebrekidan S., Ahmed S., DeLuca P.

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY, vol.4, no.4, pp.491-498, 1999 (Journal Indexed in SCI) identifier identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 4 Issue: 4
  • Publication Date: 1999
  • Doi Number: 10.1081/pdt-100101386
  • Title of Journal : PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY
  • Page Numbers: pp.491-498
  • Keywords: complex, DNA, DNase, plasmid, poly(L-lysine), stability, DELIVERY, FORMULATIONS, QUANTITATION

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(R) nye and fluorescence densitometric assay methods.