Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo

In vitro assays have demonstrated the capability of poly-L-lysine to protec t plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysin-DNA polyplexes after in travenous injection into mice. Polyplexes consisted of P-32-radiolabeled pl asmid DNA complexed with poly-L-lysine at specified charge ratios. Variatio ns in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro s tudies, the systemically administered polyplexes exhibited marked DNA degra dation in the vascular compartment within 5 min. Substitution of poly-L-lys ine E-amino sites with polyethylene glycol or hydrocarbon chains resulted i n faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradati on rates only slightly. An analysis was made of the strength of the poly-L- lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes w ith the strongest binding between conjugate and DNA in the competition assa y were also the most stable in blood. However, tighter binding was not enou gh to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate wit h improved in vivo transfection efficiency. (C) 2000 Elsevier Science B.V. All rights reserved.