Stability of poly(L-lysine)-complexed plasmid DNA during mechanical stress and DNase I treatment.
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 pin 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.
Capan
Y
Y
University of Kentucky, College of Pharmacy, Faculty of Pharmaceutical Sciences, Lexington 40536, USA.
Woo
B H
BH
Gebrekidan
S
S
Ahmed
S
S
DeLuca
P P
PP
D004247:DNA / Q000737:chemistry*; D003720:Densitometry; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004355:Drug Stability; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D010957:Plasmids / Q000737:chemistry*; D011107:Polylysine / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013314:Stress, Mechanical; D014465:Ultrasonics
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D012996:Solutions; D011107:Polylysine; D004247:DNA; D003850:Deoxyribonuclease I