Uptake of Pullulan in Cultured Rat Liver Parenchymal Cells

Abstract

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...

Neuronal gene delivery by negatively charged pullulan–spermine/DNA anioplexes

“…3A, right panel), a carrier we previously synthesized for in vivo neuronal gene delivery [11], was not affected by asialofetuin pretreatment (p ¼ 0.10 and p ¼ 0.50, respectively; t-test). Asialofetuin is best known for competitively binding the asialoglycoprotein receptor (ASGPR), a C-type lectin, and it has indeed been shown that pullulan is internalized into liver parenchymal cells via the ASGPR [13]. In the present study, RT-PCR revealed that DRG sensory neurons do not express the ASGPR-1 and -2 isoforms (Fig.…”

“…Pitard et al (2004) showed that an amphiphilic poloxamine block copolymer could condense DNA into negatively charged nanospheres for efficient intramuscular gene delivery; however, their particles appeared to enter cells through a liposome-like membrane ''flip-flop'' mechanism that was likely derived from amphiphilicity rather than negative charge [22]. In our experiments, negatively charged but not positively charged pullulan-spermine/DNA appeared to enter cells via a receptormediated mechanism that could be competitively inhibited by asialofetuin, whose archetypal target is the ASGPR receptor [13].…”

“…Members of the ASGPR family were not expressed by the primary sensory neurons used in our studies; however we did detect expression of several galectins, which have also been shown to interact with asialofetuin [13]. We therefore consider that members of the galectin family including galectin-1, -3, or -5 may serve as cellular receptors or carriers for pullulan-spermine/DNA anioplexes.…”

“…These findings ran counter to expectations given the hypothesized cell entry mechanisms of positive charge-dependent nonspecific adsorptive endocytosis and receptor-mediated pullulan uptake [10,13]. We therefore subsequently further investigated the mechanisms of cell entry and intracellular trafficking for pullulanspermine/DNA anioplexes.…”

“…The infectious extracellular form of Chlamydia trachomatis is similar in size to our anioplexes (200-600 nm diameter [34] Mpiga and Ravaoarinoro. Microbiol Res 2006; 161: [9][10][11][12][13][14][15][16][17][18][19], and some serovars utilize caveolin-2-containing lipid rafts to gain entry to both phagocytic and nonphagocytic cells [32]. Notably, the cell entry of E. coli, P. aeruginosa, and C. trachomatis is blocked by treatment with methyl-beta-cyclodextrin [32], confirming the importance of cholesterol in the endocytosis of these large pathogens.…”