Disposition Characteristics of Plasmid DNA in the Single-pass Rat Liver Perfusion System
- 53 Downloads
Purpose. 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 pC AT 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 NH4C1 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.
Unable to display preview. Download preview PDF.
- 1.E. Wagner, M. Cotten, R. Foisner, and M. L. Birnstiel. Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. USA. 88:4255–4259 (1991).Google Scholar
- 2.R. I. Mahato, K. Kawabata, Y. Takakura, and M. Hashida. In vivo disposition characteristics of plasmid DNA complexed with cationic liposomes. J. Drug Target. 3:149–157 (1995).Google Scholar
- 3.Y. Takakura, T. Fujita, H. Furitsu, M. Nishikawa, and M. Hashida. Pharmacokinetics of succinylated proteins and dextran sulfate in mice: Implications for hepatic targeting of protein drugs by direct succinylation via scavenger receptors. Int. J. Pharm. 105:19–29 (1994).Google Scholar
- 4.K. Kawabata, Y. Takakura, and M. Hashida. The fate of plasmid DNA after intravenous injection in mice: Involvement of scavenger receptors in its hepatic uptake. Pharm. Res. 12:825–830 (1995).Google Scholar
- 5.K. Nishida, C. Tonegawa, S. Nakane, T. Kakutani, M. Hashida, and H. Sezaki. Statistical moment analysis of hepatobiliary transport of phenol red in the perfused rat liver. Pharm. Res. 6:140–146 (1989).Google Scholar
- 6.T. Kakutani, K. Yamaoka, M. Hashida, and H. Sezaki. A new method for assessment of drug disposition in muscle: Application of statistical moment theory to local perfusion systems. J. Pharmacokinet. Biopharm. 13:609–631 (1985).Google Scholar
- 7.K. Nishida, C. Tonegawa, S. Nakane, Y. Takakura, M. Hashida, and Sezaki H. Effect of electric charge on the hepatic uptake of macromolecules in the rat liver. Int. J. Pharm. 65:7–17 (1990).Google Scholar
- 8.K. Yamaoka, T. Nakagawa, and T. Uno. Statistical moments in pharmacokinetics. J. Pharmacokinet. Biopharm. 6:547–558 (1978).Google Scholar
- 9.R. Blomhoff, H. K. Blomhoff, H. Tolleshaug, T. B. Christensen, and T. Berg. Uptake and degradation of bovine testes beta-galactosidase by parenchymal and nonparenchymal rat liver cells. Int. J. Biochem. 17:1321–1328 (1985).Google Scholar
- 10.W. Emlen and M. Mannik. Effect of DNA size and strandedness on the in vivo clearance and organ localization of DNA. Clin. Exp. Immunol. 56:185–192 (1984).Google Scholar
- 11.M. S. Brown, S. K. Basu, J. R. Falck, Y. K. Ho, and J. L. Goldstein. The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages. J. Supramol. Struct. 13:67–81 (1980).Google Scholar
- 12.S. Acton, D. Resnick, M. Freeman, Y. Ekkel, J. Ashkenas, and M. Krieger. The collagenous domains of macrophage scavenger receptors and complement component C1q mediate their similar, but not identical, binding specificities for polyanionic ligands. J. Biol. Chem. 268:3530–3537 (1993).Google Scholar
- 13.K. Nishida, K. Mihara, T. Takino, S. Nakane, Y. Takakura, M. Hashida, and H. Sezaki. Hepatic disposition characteristics of electrically charged macromolecules in rat in vivo and in the perfused liver. Pharm. Res. 8:437–443 (1991).Google Scholar
- 14.M. Naito, T. Kodama, A. Matsumoto, T. Doi, and K. Takahashi. Tissue distribution, intracellular localization, and in vitro expression of bovine macrophage scavenger receptors. Am. J. Pathol. 139:1411–1423 (1991).Google Scholar
- 15.J. M. Backer, C. R. Kahn, and M. F. White. Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: Studies in 2,4-dinitrophenol-treated cells. Proc. Natl. Acad. Sci. USA. 86:3209–3213 (1989).Google Scholar