Yoshida M, Mahato R I, Kawabata K, Takakura Y, Hashida M
Department of Drug Delivery Research, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Pharm Res. 1996 Apr;13(4):599-603. doi: 10.1023/a:1016058407671.
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).
[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.
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.
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.
为了确定质粒DNA的肝脏摄取机制，我们使用无白蛋白和红细胞的Krebs-Ringer碳酸氢盐缓冲液（pH 7.4），在灌注大鼠肝脏单次通过后，对模型质粒pCAT（编码与猿猴病毒40启动子融合的氯霉素乙酰转移酶报告基因的质粒DNA）的摄取进行了定量。
[32P]pCAT以推注输入或持续输注模式从门静脉瞬间引入该系统，并使用统计矩分析评估流出模式和肝脏摄取。
静脉流出样本的电泳带与标准pCAT相似，表明质粒在肝脏灌注期间在灌注液中相当稳定。在推注实验中，pCAT大部分被肝脏摄取，并且摄取量随着注射剂量的增加而减少。对流出模式的统计矩分析表明，pCAT的表观分布容积大于人血清白蛋白，表明与组织有显著的可逆相互作用。胶原酶灌注实验结果表明，pCAT在肝脏中的积累优先发生在非实质细胞（NPC）中。预先给予聚肌苷酸、硫酸葡聚糖、琥珀酰化牛血清白蛋白后，肝脏中的总回收率显著降低，但聚胞苷酸则没有。这表明pCAT通过NPC上的多阴离子清道夫受体被肝脏摄取。在持续输注实验中，2,4-二硝基苯酚和NH4Cl的存在导致[32P]pCAT的流出浓度显著增加，并且总肝脏回收率比单独给予质粒DNA时降低一半，表明质粒DNA可能被NPC内化。
肝脏在质粒DNA的消除中起重要作用，并且需要一个成功的递送系统来避免其被肝脏NPC上的清道夫受体识别。
