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Intracellular disposition and cytotoxicity of macromolecular conjugate of mitomycin C (MMC) with transferrin (TF) were examined in the human hepatoma cell line HepG2 cell and normal cultured rat hepatocyte. The conjugate (TF-MMC) was specifically bound to the HepG2 cell as well as TF. The number of the binding site and the association constant of TF-MMC in the HepG2 cell were 396000؎31000 molecules/cell and 3.24؋10 7 ؎0.58 ؋10 M ؊1, respectively. No difference in the binding parameters of TF-MMC and TF can be detected in the HepG2 cell. The association constant for the TF receptor was almost identical between HepG2 cell and hepatocyte, however, the numbers of the binding site of TF-MMC and TF in the HepG2 cell were from 40-times to 50-times greater than those in the hepatocyte. Furthermore, TF-MMC was internalized into the HepG2 cell and the hepatocyte as well as TF. The rates of internalization of TF-MMC and TF into the HepG2 cell were nearly identical to those into the hepatocyte. However, the levels of the internalization into the HepG2 cell were remarkably higher than those into the hepatocyte because the number of receptors in the HepG2 cell was larger than that in the hepatocyte, and the rate of release from the HepG2 cell was slower than that from the hepatocyte. TF-MMC inhibited the growth of the HepG2 cells. The 50% growth inhibition (GI 50 ) of TF-MMC against the HepG2 cell was 0.9 m mg MMC/ml, which was a little higher than that of MMC (GI 50 ‫5.0؍‬ m mg/ml). These results indicated that the TF-MMC might be useful for delivery of MMC to the HepG2 cell.

Polysaccharides are an attractive drug carrier directed to the liver because they are distributed in the liver after intravenous injection. Pullulan is a polysaccharide consisting of three a-1,4-linked glucose molecules, polymerized by a-1,6-linkages to the terminal glucose, and has been employed as a carrier of human interferon-b and plasmid DNA to the liver. 1,2) We previously investigated the biodisposition of pullulan in rats and demonstrated that the asialoglycoprotein receptor (ASGPR) contributes to hepatic distribution.3) Furthermore, intracellular disposition of polysaccharides has been examined in rat liver parenchymal and nonparenchymal cells and studies have shown that binding and internalization to parenchymal cells was inhibited by asialofetuin, indicating that ASGPR is involved in the intracellular disposition of pullulan. 4)In this study, we examined the uptake of pullulan, including the binding process followed by internalization in cultured rat liver parenchymal cells, and discuss the possibility of pullulan as a passive or active drug carrier via receptor mediated endocytosis (RME). MATERIALS AND METHODSMaterials Pullulan (MW 58200) was supplied by Kuraray (Japan). Arabinogalactan and asialofetuin were purchased from Sigma (St. Louis, MO. U.S.A.). Fluorescein isothiocyanate was obtained from Wako Pure Chemical (Osaka, Japan). All other reagents were the highest grade commercially available.Radio-Labeling of Pullulan The tyramine derivative of pullulan was prepared according to a previous report. 4)Briefly, pullulan was dissolved in 10% acetone in water and cooled in an ice bath. Then, 10% CNBr in acetone was added and the mixture stirred for 2 min. Tetraethylamine in acetone was added to the solution. After 2 min, tyramine in 0.1 M NaHCO 3 containing 0.5 M NaCl was added and the reaction mixture stirred overnight at 4°C. The mixture was dialyzed against water and the macromolecular fraction obtained as a white powder. Animals Wistar male rats (6 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The rats were maintained on a commercially balanced stock diet (Oriental Yeast Co. Ltd., Tokyo, Japan) with water ad libitum. Before experimentation, each rat was anesthetized with pentobarbital. All aspects of the study were performed according to the Fukuyama University guidelines for animal experiments.Isolation and Culture of Rat Liver Parenchymal Cells Hepatocytes were isolated from male Wistar rats (7 weeks old) by the collagenase perfusion method.6) The hepatocytes were centrifuged at 50ϫg and the resulting pellet washed with Hanks' medium containing 2.4 mM CaCl 2 and 10 mM HEPES (pH 7.4). The cells were fractionated on Percoll density gradients, which resulted in a greater than 98% viability, as verified by a trypan blue exclusion test. The cells were then resuspended in William's Medium E containing dexamethasone (1 µM), insulin (0.1 µM), and 10% FBS. The cells (1ϫ10 6 cells) were plated in a 35-mm collagen type I coated culture dish (Iwaki, Funabashi, Japan) and incubated in hu...

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