Determination of protection from serum nuclease activity by DNA-polyelectrolyte complexes using an electrophoretic method.
Polyelectrolyte complexes between cationic polymers and DNA have emerged as potential nonviral vectors for DNA delivery. For successful in vivo delivery, methods for analyzing their ability to prevent digestion of the DNA payload by serum nucleases are essential. We report here a simple assay to determine degradation of DNA in these complexes using standard electrophoretic techniques. The assay is based on a high pH buffer which can dissociate the complexes under standard electrophoretic conditions. This assay can be used qualitatively to determine the time taken for degradation to occur. Alternatively, with a standard gel analysis program it can be used quantitatively to investigate rates of DNA degradation from complexes in the presence of serum nucleases. We have shown that it can distinguish between different formulations with the same polymer, and also to distinguish between the time taken to degradation and the rates of degradation of DNA in complexes formed with two structurally related, linear polyamidoamine polymers. The assay could also distinguish between the time to degradation using poly-l-lysine complexes, although these were less well dissociated by the electrophoresis buffer, and could not be analyzed quantitatively. This assay will be of value in investigating and developing polyelectrolyte formulations for parenteral administration.
Hill
I R
IR
School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom
Garnett
M C
MC
Bignotti
F
F
Davis
S S
SS
D002021:Buffers; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000097:blood*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000097:blood / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000097:blood / Q000235:genetics*; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D015203:Reproducibility of Results
D002021:Buffers; D004247:DNA; D003851:Deoxyribonucleases; D011073:Polyamines; D000071228:Polyelectrolytes