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The objective of this study is to investigate the effect of lipopolysaccharide (LPS) addition on the gene transfection of human mesenchymal stem cells (hMSC). hMSC were treated with the LPS at different concentrations and the complex of spermine-introduced pullulan and luciferase plasmid DNA for 3 h. The maximum level of gene expression was observed for hMSC treated with a certain concentration range of LPS. In addition, the cytotoxicity, cellular internalization of complexes, and cell cycle after LPS treatment were investigated. The cytotoxicity increased with an increase in the LPS concentration treated. On the other hand, the cellular internalization of complexes increased with the increased LPS concentration, although the internalization was sharply reduced at the high concentration. The LPS treatment increased the actin polymerization of cells to allow to spread more. The enhanced cells spreading would enhance the cellular internalization of complexes. In addition, the LPS treatment increased the rate of cell cycle. It is possible that the balance of cytotoxicity, cellular internalization, and cell cycle caused by the LPS addition results in the enhanced gene transfection at a certain LPS concentration. It is concluded that LPS treatment positively modified the cellular internalization and the cell cycle, resulting in the enhanced gene transfection.

The objective of this study is to design biodegradable nanospheres of cationized gelatin as a carrier of cellular internalization of plasmid DNA. Ethylenediamine was chemically introduced into the carboxyl groups of gelatin to obtain cationized gelatin. The gelatin solution was filtered through a glass membrane under high pressure and dropped into 2-butanol, acetone or a mixture of the two to form nanospheres of cationized gelatin. The microspheres of cationized gelatin were prepared by the conventional water-in-oil emulsion method. The resulting nano- and microspheres of cationized gelatin were dehydrothermally treated at 160°C for different time periods to allow them to cross-link chemically. The size of nanospheres, prepared by the filtration method and changed by the type of solvents, was 1.86, 0.83 or 0.24 μm. The in vitro degradation of spheres became faster as the time period of dehydrothermal treatment was shorter. The degradation time of spheres in HCl solution linearly increased with an increase in the cross-linking time, irrespective of the sphere size. However, in the collagenase solution, when compared at the similar cross-linking density, the smaller spheres were degraded more slowly than the larger ones. The plasmid DNA incorporated in the nanospheres was released from the nanospheres with their degradation. The nanospheres incorporating plasmid DNA were internalized into cells, and intracellularly degraded with time to release plasmid DNA. The time period of plasmid DNA release was prolonged by increasing the nanosphere degradation time.

The objective of this study was to genetically engineer dendritic cells (DC) for biological activation and evaluate their anti-tumor activity in a tumor-bearing mouse model. Mouse DC were incubated on the surface of culture dishes which had been coated with the complexes of a cationized dextran and luciferase plasmid DNA complexes plus a cell adhesion protein, Pronectin, for gene transfection (reverse transfection). When compared with the conventional transfection where DC were transfected in the medium containing the complexes, the level of gene expression by the reverse method was significantly higher and the time period of gene expression was prolonged. Following the reverse transfection of DC by a plasmid DNA of mouse interleukin-12 (mIL-12) complexed with the cationized dextran, the mIL-12 protein was secreted at higher amounts for a longer time period. When injected intratumorally into mice carrying a mass of B16 tumor cells, the DC genetically activated showed significant anti-tumor activity.

The objective of this study is to prepare a non-viral carrier of gene expression from the polysaccharide dextran and evaluate the effect of amine compounds introduced to dextran on the level of gene expression. Dextran with a molecular weight of 74 x 10(3) was cationized by the chemical introduction of different amine compounds. The cationized dextran was complexed with a plasmid DNA and the vitro gene transfection was investigated for HeLa cells. The level of gene expression depended on the amine compound introduced to dextran. The highest level was observed for the complex of spermine-introduced dextran and plasmid DNA. The highest cellular internalization and the best buffering effect were observed among every cationized dextran. Every complex did not show any cytotoxicity. It is concluded that the superior properties of spermine-introduced dextran enabled the plasmid DNA to enhance the expression level to a great extent compared with other cationized dextrans. Cationized dextran is a promising non-viral carrier of plasmid DNA.

The objective of this study is to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Various amounts of spermine were chemically introduced into pullulan with molecular weights of 22,800, 47,300, and 112,000 to prepare cationized pullulan derivatives with different percentages of spermine introduced. Each cationized pullulan derivative was complexed with a plasmid DNA at various ratios and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the percent spermine introduced of cationized pullulan derivatives and the molecular weight of pullulan. However, when compared at the complexation molar ratio of pullulan derivative to the plasmid DNA, the expression level became maximum around the ratio of 10(2), irrespective of the pullulan molecular weight. Pre-treatment of cells with asialofetuin of asialoglycoprotein receptor ligand decreased the level of gene expression by the complexes. The cationized pullulan derivative with an appropriate physicochemical character is a promising non-viral carrier which promotes the receptor-mediated internalization of plasmid DNA and consequently enhances the expression level.

The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 x 10(3). Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion.

The objective of this study is to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver and evaluate the feasibility in gene transfection in the mice liver. Pullulan with a weight-average molecular weight of 22,800 was cationized by the chemical introduction of spermine (spermine-pullulan). The cationized pullulan derivative was complexed with a plasmid DNA and intravenously injected for in vivo gene transfection. The level of gene expression by the spermine-pullulan in the liver depended on the extent of spermine introduced and the highest level was observed for the spermine-pullulan with an introduction extent of 5.60. When a plasmid DNA coding NK4 of a hepatocyte growth factor (HGF)/scatter factor antagonist complexed with the spermine-pullulan was intravenously injected to mice 1 day before the inoculation of RLmale1 tumor cells, the tumor-bearing mice survived for a longer time period, while the GPT level and the number of tumor cells grown in the liver were low compared with those of free plasmid DNA injection. These findings indicate that the liver targeting of NK4 plasmid DNA by complexation with the spermine-pullulan specifically enhanced the liver expression level, resulting in augmented suppression effect on tumor growth therein.

The objective of this study is to design biodegradable nanospheres of cationized gelatin as a carrier of cellular internalization of plasmid DNA. Ethylenediamine was chemically introduced into the carboxyl groups of gelatin to obtain cationized gelatin. The gelatin solution was filtered through a glass membrane under high pressure and dropped into 2-butanol, acetone or a mixture of the two to form nanospheres of cationized gelatin. The microspheres of cationized gelatin were prepared by the conventional water-in-oil emulsion method. The resulting nano- and microspheres of cationized gelatin were dehydrothermally treated at 160°C for different time periods to allow them to cross-link chemically. The size of nanospheres, prepared by the filtration method and changed by the type of solvents, was 1.86, 0.83 or 0.24 μm. The in vitro degradation of spheres became faster as the time period of dehydrothermal treatment was shorter. The degradation time of spheres in HCl solution linearly increased with an increase in the cross-linking time, irrespective of the sphere size. However, in the collagenase solution, when compared at the similar cross-linking density, the smaller spheres were degraded more slowly than the larger ones. The plasmid DNA incorporated in the nanospheres was released from the nanospheres with their degradation. The nanospheres incorporating plasmid DNA were internalized into cells, and intracellularly degraded with time to release plasmid DNA. The time period of plasmid DNA release was prolonged by increasing the nanosphere degradation time. [ABSTRACT FROM AUTHOR]

The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 × 103. Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion. [ABSTRACT FROM AUTHOR]