SOCIETY OF CRITICAL CARE MEDICINE 32ND CRITICAL CARE CONGRESS SAN ANTONIO, TEXAS, USA JANUARY 28-FEBRUARY 2, 2003: ORAL/SANDWICH PRESENTATIONS: Poster Presentation: Clinical Science: Sepsis Infection: PDF Only

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.

Polycations have been used for gene delivery in vitro quite successfully, however, in vivo applications suffered from serum effects that lower the overall gene drug efficiency. PEG polymers have been used extensively to minimize serum effects and create "stealth liposomes", biocompatible materials, and proteins with extended circulation. Here, we report our efforts towards creating "stealth polyplexes". A comb-type polycation, poly-L-lysine-graft-PEG copolymers were successfully prepared by ring opening of PEG-epoxide with E-amino lysine groups of linear poly-L-lysine. The ratios of PEG-epoxide to poly-L-lysine, PEG-epoxide size (2K, 3K, and 5B), and poly-L-lysine size (10K, 26K, and 38K) were varied. Copolymers with as little as 2% grafted PEG chains sterically stabilized DNA/copolymer complexes (polyplexes) even at charge neutrality. These polyplexes, formed with copolymers with various size of PEG chains grafted on various lenghths of poly-L-lysine backbone, remained relatively small, approximately 100 nm in saline With higher degree of grafting, the binding was significantly diminished. In addition, the morphology of polyplexes changed from thoroidal to more elongated, worm-like forms. Some globular structures were detected in cases of a lower degree of grafting. Finally, DNA release form polyplexes when exposed to negatively charged macromolecules, poly-L-aspartic acid sodium salt, is very structure dependant. Enhanced levels of luciferase expression observed with PLL-PEC polyplexes versus either free DNA or PLL polyplexes are encouraging and warrant further optimization of the polymeric gene delivery system.