Biodegradable nanoparticles composed of dendrigraft poly-L-lysine for gene delivery.

We developed novel gene vectors composed of dendrigraft poly-L-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-L-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, γ-polyglutamic acid (γ-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/γ-PGA complexes) were shown to be stable nanoparticles, and those with γ-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/γ-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/γ-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/γ-PGA complexes for clinical gene therapy.

Effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to liver surface in rats.

Objectives The aim was to study the effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to the liver surface in rats. Methods 5-FU solution with or without viscous additives was applied to the rat liver surface with a cylindrical diffusion cell. Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites and assayed for 5-FU content. Key findings The absorption rate of 5-FU from the liver surface was decreased in the presence of carboxymethylcellulose sodium (CMC-Na) and polyvinyl alcohol (PVA) as compared with the control. The k(a) values of PVA 15% and CMC-Na 1% were reduced to about 80 and 67% of the control. The maximum plasma concentration of 5-FU was decreased by incorporation of viscous additives. The 5-FU concentration at the diffusion cell attachment site of the liver (site 1) plateaued at 180 min in the absence of viscous additives. On the other hand, the concentration of 5-FU at site 1 increased in a time-dependent manner until 360 min in the presence of viscous additives. Conclusion Viscous additives might be useful for retaining drugs at their application site and controlling the rate of absorption from the liver surface.

Efficient in vivo gene transfer by intraperitoneal injection of plasmid DNA and calcium carbonate microflowers in mice.

Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for in vivo experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers.

Pretreatment with epidermal growth factor enhances naked plasmid DNA transfer onto gastric serosal surface in mice.

We have developed a simple administration method, which is gastric serosal surface instillation of naked plasmid DNA (pDNA) in experimental animals. The purpose of this study was to improve gastric gene transfer efficiency by pre-treatment with a macropinocytosis enhancer, such as fetuin or epidermal growth factor (EGF), in mice. A series of concentrations of fetuin were instilled onto gastric serosal surface prior to instillation of naked pDNA in mice; however, fetuin did not improve transgene expression in the stomach 6 h after administration of pDNA. EGF also did not affect transgene expression in the stomach when pDNA was instilled immediately after EGF instillation. On the other hand, when pDNA was instilled onto gastric serosal surface 24 h after EGF treatment, transgene expression in the stomach was significantly improved by 2.6-fold. In addition, transgene-positive cells were increased 5.3-fold by EGF pre-treatment. High transgene expression in the stomach lasted for 48 h in the EGF pre-treatment group in comparison with that in the no pre-treatment group. These findings are valuable to develop an effective method of in vivo gene transfer to the stomach.

Multiple components in serum contribute to hepatic transgene expression by lipoplex in mice.

BACKGROUND: Interaction of cationic liposome/plasmid DNA complex (lipoplex) with serum was not a limiting factor for in vivo transfection. After intraportal injection of lipoplex, hepatic transgene expression was enhanced by interaction with serum in mice. In the present study, we analyzed the mechanism of enhanced hepatic transgene expression of lipoplex by interaction with serum components. METHODS: Lipoplexes were incubated with several serum components for 5 min at 37 ° C before administration. Transfection efficiency of lipoplexes was measured 6 h after intraportal injection of lipoplex in mice. RESULTS: Depletion of divalent cation from serum decreased hepatic transgene expression. The addition of calcium ion to divalent cation-depleted serum restored transgene expression. Heat-inactivated serum and bovine serum albumin diminished the enhancing effect of serum on hepatic transgene expression. On the other hand, removal of anionic proteins from serum using an anion-exchanging column was critical for the enhancing effect of serum on transgene expression. Among the serum components tested, fibronectin and complement component C3 enhanced hepatic transgene expression. CONCLUSIONS: Hepatic transgene expression by lipoplex was enhanced by interaction with multiple components in serum. Interaction of lipoplex with serum could be an important factor for successful in vivo gene transfer. Hence, the information obtained in the present study is valuable for the future development of effective gene carriers.

Rubbing gastric serosal surface enhances naked plasmid DNA transfer in rats and mice.

We have developed in vivo gene transfer to mesothelial cells on the peritoneal organs, including the stomach. Simple instillation of naked plasmid DNA onto the gastric serosal surface in mice resulted in effective but transient transgene expression. Here, we developed a simple method to improve not only the transfection efficiency but also the duration of transgene expression. Rubbing the gastric serosal surface using a medical spoon immediately after instillation of naked plasmid DNA onto the gastric serosal surface resulted in 59-fold higher transgene expression 24 h after administration in rats. Without rubbing, transgene expression decreased under the detection limit 7 d after administration. On the other hand, rubbing the gastric serosal surface with a medical spoon after instillation of plasmid DNA prolonged transgene expression for one month. Mechanistic study in mice revealed that improved transfection should not be due to stimulation of cell function such as macropinocytosis by rubbing because rubbing before instillation of plasmid DNA did not improve transfection. Plasmid DNA should enter effectively into cells during rubbing. These findings are valuable to develop an effective method of in vivo gene transfer into peritoneal organs.