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 intracellul ar degradation of plasmid DNA, When a non-viral vector is used, it is not k nown to what extent the plasmid DNA catabolism is subordinated to the catab olism of the vector. In the work reported here, the problem was approached by following the intracellular fate in rat liver, of plasmid [S-35]DNA comp lexed with a cationic peptide poly-L-lysine that can be hydrolyzed by cellu lar peptidases or with its stereoisomer, poly-D-lysine, that cannot be spli t by these enzymes. Complexes of DNA with poly-L-lysine and poly-D-lysine a re taken up to the same extent by the liver, mainly by Kupffer cells, but t he intracellular degradation of nucleic acid molecules is markedly quicker when poly-L-lysine is injected. The association of DNA, with the polycation s inhibits DNA hydrolysis in vitro by purified lysosomes but similarly for poly-L-lysine and poly-D-lysine, The intracellular journey followed by [S-3 5]DNA complexed with poly-L- or poly-D-lysine was investigated using differ ential and isopycnic centrifugation, Results indicate that [S-35]DNA is tra nsferred more slowly to lysosomes, the main site of intracellular degradati on 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 vec tor in a prelysosomal compartment is required to allow endocytosed plasmid DNA to rapidly reach lysosomes, Such a phenomenon could explain why injecte d plasmid DNA is more stable in vivo when it is associated with poly-D-lysi ne, (C) 1999 Federation of European Biochemical Societies.