Fumiyoshi Yamashita

Molecular weight-dependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver

To optimize a receptor-mediated and cell-selective gene transfer with polyethyleneimine (PEI)-based vector, we synthesized three galactosylated PEIs (Gal-PEI) with different molecular weights (PEI(1800), PEI(10,000), and PEI(70,000)) and investigated their potential as a targetable vector to asialoglycoprotein receptor-positive cells. All PEI derivatives formed complexes with plasmid DNA (pDNA), whereas the particle size of the complex became smaller on increasing the molecular weight of PEI. Transfection efficiency in HepG2 cells with PEI was highest with PEI(1800); efficiency was next highest with PEI(10,000), although the cellular association was similar. After galactosylation, Gal(19)-PEI(10,000)/pDNA and Gal(120)-PEI(70,000)/pDNA showed considerable agglutination with a galactose-recognizing lectin, but Gal(9)-PEI(1800) did not, suggesting that galactose units on the Gal(9)-PEI(1800)-pDNA complex are not sufficiently available for recognition. Gal(19)-PEI(10,000)-pDNA and Gal(120)-PEI(70,000)-pDNA complexes showed galactose-inhibitable transgene expression in HepG2 cells. Transfection efficiency was greatest with Gal(19)-PEI(10,000)/pDNA, a result that highlights the importance of obtaining a balance between the cytotoxicity and the transfection activity, both of which are found to be a function of the molecular weight of PEI. After intraportal injection, however, Gal(153)-PEI(70,000)/pDNA having a low N/P ratio was most effective, suggesting that additional variables, such as the size of the complex, are important for in vivo gene transfer to hepatocytes.

Inhibition of metastatic tumor growth in mouse lung by repeated administration of polyethylene glycol-conjugated catalase: quantitative analysis with firefly luciferase-expressing melanoma cells.

Purpose: To develop a novel and effective approach to inhibit tumor metastasis based on controlled delivery of catalase, we first evaluated the characteristics of the disposition and proliferation of tumor cells. Then, we examined the effects of polyethylene glycol-conjugated catalase (PEG-catalase) on tumor metastasis. On the basis of the results obtained, PEG-catalase was repetitively administered to completely suppress the growth of tumor cells. Experimental Design: Murine melanoma B16-BL6 cells were stably transfected with firefly luciferase gene to obtain B16-BL6/Luc cells. These cells were injected intravenously into syngeneic C57BL/6 mice. PEG-catalase was injected intravenously, and the effect was evaluated by measuring the luciferase activity as the indicator of the number of tumor cells. Results: At 1 hour after injection of B16-BL6/Luc cells, 60 to 90% of the injected cells were recovered in the lung. The numbers decreased to 2 to 4% at 24 hours, then increased. An injection of PEG-catalase just before inoculation significantly reduced the number of tumor cells at 24 hours. Injection of PEG-catalase at 1 or 3 days after inoculation was also effective in reducing the cell numbers. Daily dosing of PEG-catalase greatly inhibited the proliferation and the number assayed at 14 days after inoculation was not significantly different from the minimal number observed at 1 day, suggesting that the growth had been markedly suppressed by the treatment. Conclusions: These findings indicate that sustained catalase activity in the blood circulation can prevent the multiple processes of tumor metastasis in the lung, which could lead to a state of tumor dormancy.

Inhibition of experimental hepatic metastasis by targeted delivery of catalase in mice

AbstractBovine liver catalase derivatives possessing diverse tissue distribution properties were synthesized, and their effects on hepatic metastasis of colon carcinoma cells were examined in mice. An intraportal injection of 1 × 105 colon 26 cells resulted in the formation of more than 50 metastatic colonies on the surface of the liver at 14 days after injection. An intravenous injection of catalase (CAT; 35u2009000 units/kg of body weight) significantly (Pxa0< 0.001) reduced the number of the colonies in the liver. Galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT were also tested in the same system. Of these derivatives, Gal-CAT showed the greatest inhibitory effect on hepatic metastasis, and the number of colonies was significantly (Pxa0< 0.001) smaller than following treatment with catalase. High activities of matrix metalloproteinases (MMPs), especially MMP-9, were detected in the liver of mice bearing metastatic tumor tissues, which was significantly (Pxa0< 0.05) reduced by Gal-CAT. These results, combined with our previous finding that Gal-CAT can be efficiently delivered to hepatocytes, indicate that the targeted delivery of catalase to the liver by galactosylation is a promising approach to suppress hepatic metastasis. Decreased MMP activity by catalase delivery seems to be involved in its anti-metastatic effect.n Abbreviations: CAT – bovine liver catalase; ECM – extracellular matrix; Gal – galactosylated; HBSS – Hanks balanced salt solution; H2O2– hydrogen peroxide; Man – mannosylated; MMP – matrix metalloproteinase; ROS – reactive oxygen species; Suc – succinylated; SOD – superoxide dismutase

Targeted and sustained drug delivery using PEGylated galactosylated liposomes

To achieve a sustained and targeted delivery of liposomes to liver parenchymal cells (PC), we modified distearoyl-L-phosphatidylcholine (DSPC)/cholesterol (Chol) (60:40) (DSPC/Chol) liposomes with a galactosylated cholesterol derivative (Gal-C4-Chol), and polysorbate (Tween) 20 or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (PEG(x)-DSPE). After intravenous injection, DSPC/Chol/Gal-C4-Chol (60:35:5) (Gal) liposomes were rapidly eliminated from the blood circulation and mostly recovered in the liver. The blood elimination of DSPC/Chol/Gal-C4-Chol/Tween 20 (55:35:5:5) (Tween 20-Gal) liposomes was slightly reduced as compared to Gal-liposomes. In contrast, a significant reduction in the blood elimination was observed with DSPC/Chol/Gal-C4-Chol/PEG(2000)-DSPE (59:35:5:1) (PEG(2000)-Gal) liposomes. Hepatic uptake of DSPC/Chol/Gal-C4-Chol/PEG(350)-DSPE (59:35:5:1) (PEG(350)-Gal) liposomes was intermediate between PEG(2000)-Gal-liposomes and Tween 20-Gal-liposomes. The uptake of PEG(350)-Gal-liposomes by liver PC was 7.7-fold higher than that by non-parenchymal cells (NPC). These results suggest that PEG(350)-DSPE can control the delivery rate of Gal-liposomes to liver PC without losing its targeting capability.

Hepatocyte-targeted gene transfer by combination of vascularly delivered plasmid DNA and in vivo electroporation

To increase transgene expression in the liver, electric pulses were applied to the left lateral lobe after intravenous injection of naked plasmid DNA (pDNA) or pDNA/liver targeting vector complex prepared with galactosylated poly(L-lysine) or galactosylated polyethyleneimine. Electroporation (250u2009V/cm, 5u2009ms/pulse, 12 pulses, 4u2009Hz) after naked pDNA injection dramatically increased the expression up to 200u2009000-fold; the expression level obtained was significantly greater than that achieved by the combination of pDNA/vector complex and electroporation. We clearly demonstrated that the expression was dependent on the plasma concentration of pDNA at the time when the electric pulses were applied. Separation of liver cells revealed that the distribution of naked pDNA as well as transgene expression was largely selective to hepatocytes in the electroporated lobe. The number of cells expressing transgene product using vascularly administered naked pDNA followed by electroporation was significantly (P<0.01) greater and more widespread than that obtained by local injection of naked pDNA. These results indicate that the application of in vivo electroporation to vascularly administered naked pDNA is a useful gene transfer approach to a large number of hepatocytes.

Therapeutic effect of intravenous delivery of lipoplexes containing the interferon- β gene and poly I: poly C in a murine lung metastasis model

We have evaluated and compared the efficacy of systemic administration of lipoplex formulations containing plasmids encoding IFN-β or IFN-γ, and a synthetic double-strand RNA poly I:poly C (pI:pC), a type I IFN inducer, in a lung metastasis model in which colon carcinoma CT-26 cells were inoculated intravenously into immunocompatible mice. Injection of lipoplexes containing plasmid DNA, regardless of IFN gene insertion, stimulated a transient increase in the serum concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and IFN-γ, while injection of lipoplexes containing pI:pC led to a low level of TNF-α and undetectable IFN-γ production. Furthermore, injection of these lipoplexes containing plasmids resulted in the production of a mixture of type I and type II IFNs, partly derived from the inserted IFN genes, in lung tissue cultures. In tumor-prophylactic experiments, intravenous injection of lipoplexes containing plasmid, regardless of IFN gene insertion, showed a significant reduction in lung metastatic nodules probably due to proinflammatory cytokines such as TNF-α and IFN-γ nonspecifically induced by the CpG motifs in the plasmid and the type I IFNs produced. On the other hand, the antimetastatic effect of pI:pC-lipoplex seemed to be due mainly to IFN-β induced by pI:pC. In established lung metastasis experiments, a single intravenous administration of lipoplexes containing IFN-β gene or pI:pC, but not other lipoplexes, showed a significant therapeutic effect on the tumor metastasis: reduction in tumor nodules and prolongation of survival time of tumor-burden mice. The therapeutic effects were specifically impaired by anti-IFN-β antibody treatment, indicating that IFN-β produced by the lipoplexes played an important role in the suppression of established metastatic lung tumors. Thus, the local IFN-β in the lung delivered by intravenous administration of lipoplex containing IFN-β gene or pI:pC may be a convenient and useful method of inhibiting established metastatic lung tumors.

Analysis of Hepatic Disposition of Galactosylated Cationic Liposome/Plasmid DNA Complexes in Perfused Rat Liver

AbstractPurpose. To determine the intrahepatic disposition characteristics of galactosylated liposome/plasmid DNA (pDNA) complexes in perfused rat liver.nMethods. Galactosylated liposomes containing N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cholesterol (Chol), and cholesten-5-yloxy-N-{4-[(1-imino-2-D-thiogalactosylethyl)amino]butyl} formamide (Gal-C4-Chol) were prepared. The liposome/[32P]-labeled pDNA complexes were administered to perfused liver, and the venous outflow patterns were analyzed based on a two-compartment dispersion model.nResults. The single-pass hepatic extraction of pDNA complexed with DOTMA/Chol/Gal-C4-Chol liposomes was greater than that with control DOTMA/Chol liposomes. A two-compartment dispersion model revealed that both the tissue binding and cellular internalization rate were higher for the DOTMA/Chol/Gal-C4-Chol liposome complexes compared with the control liposome complexes. The tissue binding was significantly reduced by the presence of 20 mM galactose. When their cellular localization in the perfused liver at 30 min postinjection was investigated, it was found that the parenchymal uptake of the DOTMA/Chol/Gal-C4-Chol liposome complexes was greater than that of the control liposome complexes. The parenchymal cell/nonparenchymal cell uptake ratio was as high as unity.nConclusion. Galactosylation of the liposome/pDNA complexes increases the tissue binding and internalization rate via an asialoglycoprotein receptor-mediated process. Because of the large particle size of the complexes (∼150 nm), however, penetration across the fenestrated sinusoidal endothelium appears to be limited.

Liver targeting of catalase by cationization for prevention of acute liver failure in mice

To achieve hepatic delivery of CAT for the prevention of CCl4-induced acute liver failure in mice, two types of cationized CAT derivatives, HMD- and ED-conjugated CAT, were developed. Slight structural changes occurred during cationization and the number of increased free amino groups was 3.1 in HMD-CAT and 13.6 in ED-CAT. 111In-cationized CAT derivatives showed an increased binding to HepG2 cells, and were rapidly taken up by the liver. H2O2-induced cytotoxicity in HepG2 cells was significantly prevented by preincubation of the cells with cationized CAT derivatives. A bolus intravenous injection of the cationized CAT derivatives reduced the hepatotoxicity induced by CCl4 in mice. The ED-CAT, which showed more rapid and greater binding to the liver than the HMD-CAT, exhibited more beneficial effects as far as all the parameters examined (serum GOT, GPT, LDH and hepatic GSH) were concerned, suggesting that a high degree of cationization is effective in delivering CAT to the liver to prevent CCl4-induced hepatotoxicity. These results suggest that cationized CAT derivatives are effective in preventing acute liver failure, and ED-based cationization is a suitable method for developing liver-targetable cationized CAT derivatives, because it provides CAT with a high degree of cationization and a high remaining enzymatic activity.

Enhanced growth inhibition of metastatic lung tumors by intravenous injection of ATRA-cationic liposome/IL-12 pDNA complexes in mice.

Interleukin 12 (IL-12) is a proinflammatory cytokine with antitumor activity. All-trans–retinoic acid (ATRA) exerts antitumor effects by regulating a variety of gene expressions, including tumor necrosis factor receptor 1 (TNFR1), increases the number of TNFR1 and potentiates TNF-α-induced apoptosis in cancer cells. In this study, ATRA-incorporated cationic liposome (ATRA-cationic liposome)/IL-12 plasmid DNA (pDNA) complexes were prepared to improve therapeutic efficacy of cationic liposome/IL-12 pDNA complexes in a mouse model of metastatic lung tumor after intravenous injection. IL-12 production in lungs by ATRA-cationic liposome/IL-12 pDNA complexes was comparable with that by cationic liposome/IL-12 pDNA complexes. The number of metastatic tumor cells (colon26/Luc) was quantitatively evaluated by measuring luciferase activity. ATRA-cationic liposome/IL-12 pDNA complexes reduced the number of colon26/Luc cells and tumor nodules in lungs. ATRA-cationic liposome/IL-12 pDNA complexes significantly prolonged the survival time of mice, whereas cationic liposome/IL-12 pDNA only slightly prolonged it. ATRA-cationic liposome/IL-12 pDNA complexes increased the TNFR1 mRNA upregulation and the number of apoptotic cells in the lung. Moreover, reduced serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were observed in mice treated with ATRA-cationic liposome/IL-12 pDNA complexes. These results suggest that intravenous injection of ATRA-cationic liposome/IL-12 pDNA complexes is an effective method for the treatment of lung metastasis in mice.

Glycosylated cationic liposomes for carbohydrate receptor-mediated gene transfer.

Publisher Summary This chapter describes novel glycosylated cationic liposomes, consisting of a glycosylated cationic cholesterol derivative and several helper lipids, which increase the transfection efficiency to cells possessing the corresponding receptors, such as hepatocytes and macrophages. Glycosylated cationic liposomes are possible candidates for cell-specific nonviral vectors by means of a receptor-mediated mechanism, although their cell specificity and transfection efficiency need further improvement. The preparation of glycosylated liposomes is much easier than that of asialoglycoproteins as targeting ligands for liposomes may be less immunogenic. These glycosylated liposomes can be applied not only to gene delivery but also to targeted delivery of conventional drugs, such as prostaglandin E1 and probucol. Different criteria for the preparation of glycosylated liposomes are required to obtain efficient drug delivery.