DISPOSITION CHARACTERISTICS OF PLASMID DNA IN THE SINGLE-PASS RAT-LIVER PERFUSION SYSTEM

Purpose. To define the hepatic uptake mechanism of a plasmid DNA, we q uantitated the uptake of pCAT (plasmid DNA encoding chloramphenicol ac etyltransferase reporter gene fused to simian virus 40 promoter), a mo del plasmid, after a single pass through the perfused rat liver using albumin- and erythrocyte-free Krebs-Ringer bicarbonate buffer (pH 7.4) . Methods. [P-32]pCAT was introduced momentarily into this system from the portal vein as a bolus input or constant infusion mode, and the o utflow patterns and hepatic uptake were evaluated using statistical mo ment analysis. Results. The venous outflow samples had electrophoretic bands similar to that of the standard pCAT, suggesting that the plasm id is fairly stable in the perfusate during liver perfusion. In bolus experiments, pCAT was largely taken up by the liver and the uptake was decreased with increase in injected dose. Statistical moment analysis against outflow patterns demonstrated that the apparent volume of dis tribution of pCAT was greater than that of human serum albumin, indica ting a significant reversible interaction with the tissues. The result s of collagenase perfusion experiments suggest that the hepatic accumu lation of pCAT occurred preferentially in the nonparenchymal cells (NP C). The amount of total recovery in the liver decreased substantially by preceding administration of polyinosinic acid, dextran sulfate, suc cinylated bovine serum albumin, but not by polycytidylic acid. This su ggests that pCAT is taken up by the liver via scavenger receptors for polyanions on the NPC. In constant infusion experiments, the presence of 2,4-dinitrophenol and NH4Cl caused a significant increase in the ou tflow concentration of [P-32]pCAT and decrease by half in the total he patic recovery than that of plasmid DNA administered alone, suggesting that plasmid DNA may undergo internalization by the NPC. Conclusions. The liver plays an important role in the elimination of plasmid DNA a nd a successful delivery system will be required to avoid its recognit ion by the scavenger receptors on the liver NPC.