Disposition characteristics of plasmid DNA in the single-pass rat liver perfusion system.
OBJECTIVE
To define the hepatic uptake mechanism of a plasmid DNA, we quantitated the uptake of pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene fused to simian virus 40 promoter), a model plasmid, after a single pass through the perfused rat liver using albumin- and erythrocyte-free Krebs-Ringer bicarbonate buffer (pH 7.4).


METHODS
[32P]pCAT was introduced momentarily into this system from the portal vein as a bolus input or constant infusion mode, and the outflow patterns and hepatic uptake were evaluated using statistical moment analysis.


RESULTS
The venous outflow samples had electrophoretic bands similar to that of the standard pCAT, suggesting that the plasmid 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 distribution of pCAT was greater than that of human serum albumin, indicating a significant reversible interaction with the tissues. The results of collagenase perfusion experiments suggest that the hepatic accumulation of pCAT occurred preferentially in the nonparenchymal cells (NPC). The amount of total recovery in the liver decreased substantially by preceding administration of polyinosinic acid, dextran sulfate, succinylated bovine serum albumin, but not by polycytidylic acid. This suggests 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 outflow concentration of [32P]pCAT and decrease by half in the total hepatic 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 and a successful delivery system will be required to avoid its recognition by the scavenger receptors on the liver NPC.
Yoshida
M
M
Department of Drug Delivery Research, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Mahato
R I
RI
Kawabata
K
K
Takakura
Y
Y
Hashida
M
M
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000008:administration & dosage / Q000493:pharmacokinetics*; D016264:Dextran Sulfate / Q000494:pharmacology; D016503:Drug Delivery Systems; D004587:Electrophoresis, Agar Gel; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy; D006801:Humans; D007275:Injections, Intravenous; D007291:Inosine Monophosphate / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010477:Perfusion; D010957:Plasmids / Q000235:genetics*; D011108:Polymers; D011169:Portal Vein / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000737:chemistry
D002021:Buffers; D011108:Polymers; D012709:Serum Albumin; D007291:Inosine Monophosphate; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D004247:DNA; D016264:Dextran Sulfate; D015500:Chloramphenicol O-Acetyltransferase