Uptake by rat liver and intracellular fate of plasmid DNA complexed with poly-L-lysine or poly-D-lysine.

Efficiency of transfection is probably dependent on the rate of intracellular degradation of plasmid DNA. When a non-viral vector is used, it is not known to what extent the plasmid DNA catabolism is subordinated to the catabolism of the vector. In the work reported here, the problem was approached by following the intracellular fate in rat liver, of plasmid [35S]DNA complexed with a cationic peptide poly-L-lysine that can be hydrolyzed by cellular peptidases or with its stereoisomer, poly-D-lysine, that cannot be split by these enzymes. Complexes of DNA with poly-L-lysine and poly-D-lysine are taken up to the same extent by the liver, mainly by Kupffer cells, but the intracellular degradation of nucleic acid molecules is markedly quicker when poly-L-lysine is injected. The association of DNA with the polycations inhibits DNA hydrolysis in vitro by purified lysosomes but similarly for poly-L-lysine and poly-D-lysine. The intracellular journey followed by [35S]DNA complexed with poly-L- or poly-D-lysine was investigated using differential and isopycnic centrifugation. Results indicate that [35S]DNA is transferred more slowly to lysosomes, the main site of intracellular degradation of endocytosed macromolecules, when it is given as a complex with poly-D-lysine than with poly-L-lysine. They suggest that the digestion of the vector in a prelysosomal compartment is required to allow endocytosed plasmid DNA to rapidly reach lysosomes. Such a phenomenon could explain why injected plasmid DNA is more stable in vivo when it is associated with poly-D-lysine.

Cationic lipids destabilize lysosomal membrane in vitro.

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N-(1-(2,3-dioleoxy)propyl)-N,N,N,-trimethylammonium methyl-sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of beta-galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.