Hirotaka Miyamoto

Rac-mediated macropinocytosis is a critical route for naked plasmid DNA transfer in mice.

We have recently discovered the potential for in vivo naked plasmid DNA (pDNA) transfer into gastric serosal surface cells in mice. As pDNA are huge molecules, the mechanism of gene transfer without carriers and physical forces is of great biological interest. The endocytic route for naked pDNA transfer into gastric mesothelial cells was not clathrin- or caveolae-mediated endocytosis, but macropinocytosis. Naked pDNA transfer required both actin polymerization and myosin-based contraction. Upstream kinases of Rho family GTPases, Syk, Src family kinases and PI-3K were involved in naked pDNA transfer. Furthermore, the intracellular signaling pathway was not mediated via the Rho pathway, but by the Rac pathway. Downstream molecules of Rac, PAK and WAVE2 co-operated with naked pDNA transfer. Overall, it was demonstrated that the Rac signaling pathway regulated the macropinocytosis of naked pDNA. The information in this study would be helpful to clarify in vivo cell functions and to improve in vivo transfection efficiency.

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.

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

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.

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

Objectivesu2002 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.

Evaluation of changes in hepatic disposition of phenolsulfonphthalein, indocyanine green and fluorescein isothiocyanate-dextran at low temperatures using a rat liver perfusion system

Objectivesu2002 The aim of this study was to determine the factor changing the hepatic disposition of a drug during hypothermia using a rat liver perfusion system.

One-step formation of lipid-polyacrylic acid-calcium carbonate nanoparticles for co-delivery of doxorubicin and curcumin

Abstract A doxorubicin (Dox) and curcumin (Cur) combination treatment regimen has been widely studied in pre-clinical research. However, the nanoparticles developed for this combination therapy require a consecutive drug loading process because of the different water-solubility of these drugs. This study provides a strategy for the “one-step” formation of nanoparticles encapsulating both Dox and Cur. We took advantage of polyacrylic acid (PAA) and calcium carbonate (CaCO3) to realise a high drug entrapment efficiency (EE) and pH-sensitive drug release using a simplified preparation method. Optimisation of lipid ratios and concentrations of CaCO3 was conducted. Under optimal conditions, the mean diameter of PEGylated lipid/PAA/CaCO3 nanoparticles with encapsulated Cur and Dox (LPCCD) was less than 100u2009nm. An obvious pH-sensitive release of both drugs was observed, with different Dox and Cur release rates. Successful co-delivery of Cur and Dox was achieved via LPCCD on HepG2 cells. LPCCD altered the bio-distribution of Dox and Cur in vivo and decreased Dox-induced cardiotoxicity. The current investigation has developed an efficient ternary system for co-delivery of Dox and Cur to tumours, using a “one-step” formation resulting in nanoparticles possessing remarkable pH-sensitive drug release behaviour, which may be valuable for further clinical studies and eventual clinical application.

Influence of vasomodulators and tumor transplantation on the disposition of 5-fluorouracil after application to the liver surface in rats

This study investigated the effect of epinephrine (a vasoconstrictor) and hydralazine (a vasodilator) on the hepatic disposition of 5‐fluorouracil (5‐FU) after application to the surface of the liver in rats. Normal livers were compared with a Walker 256 carcinoma cell tumor model. A cylindrical diffusion cell was attached to the liver surface. 5‐Fluorouracil was added into the diffusion cell in combination with vasomodulators or after pretreatment with epinephrine. After selected treatment times, the 5‐FU concentrations were assayed at three sites in the excised livers. The 5‐FU concentration in the region under the cell attachment site (site 1) was significantly higher after concomitant application of 5‐FU and epinephrine, compared with 5‐FU alone, and increased in an epinephrine dose‐dependent manner. On the other hand, preferential distribution of 5‐FU at site 1 was not seen when applied in combination with hydralazine. After 10 min of epinephrine pretreatment, the concentration of 5‐FU at site 1 was approximately two times higher than that for the control. Furthermore, the 5‐FU concentration at site 1 of the tumor model was greatly increased compared with the normal liver. These results suggest that application of epinephrine to the liver surface might enhance the accumulation of 5‐FU at the desired target site.

Evaluation for effect of hypothermia on the disposition of 4-nitrophenol in rats by in-vitro metabolism study and rat liver perfusion system.

The aim of this study was to evaluate the effect of hypothermia on the in‐vivo pharmacokinetics of 4‐nitrophenol (4NP) using rat liver homogenate and rat liver perfusion system.

Edaravone, a cytoprotective drug, enhances transgene expression mediated by lipoplexes in HepG2 cells and mice

ABSTRACT A requirement of gene therapy is efficient nucleic acid delivery. However, the application of cationic liposomes to gene therapy is restricted by their inefficient transfection capacity, which may be caused by cytotoxicity. This cytotoxicity is highly dependent on cationic lipid‐induced reactive oxygen species (ROS). Here, to provide cellular protection, we used edaravone, an efficacious anti‐oxidative drug, to scavenge ROS during transfection using cationic liposome/plasmid DNA complexes (lipoplexes). Both free edaravone and edaravone‐loaded liposomes (EDLPs) enhanced transgene expression in the human hepatoma cell line, HepG2, while EDLPs decreased the effective dose of edaravone. The cellular protective effect of edaravone was found to decrease the cytotoxicity of cationic liposomes. Edaravone was also effective in the commercial product, Lipofectamine® 3000, which may expand the application of edaravone to promote transfection efficiency. Compared with free edaravone, EDLPs also showed superior transgene expression in mice. Our findings will promote the development of efficient and safe gene therapy.

Effect of Metabolic Inhibitors on the Hepatic Disposition of 5-Fluorouracil after Application to the Rat Liver Surface.

We evaluated the effects of 5-fluorouracil (5-FU) metabolic inhibitors, gimeracil or uridine, on the hepatic disposition of 5-FU after application to the liver surface in rats, aiming to enhance the availability of 5-FU in the liver. 5-FU solution with or without metabolic inhibitors was applied to the rat liver surface using a cylindrical diffusion cell. The liver, blood and the remaining solution in the diffusion cell were collected at specified times, and assayed for 5-FU content. 5-FU absorption properties were not altered by addition of gimeracil and uridine. The 5-FU concentration in the diffusion cell attachment site of the rat liver (site 1) at 0.1-0.4u2009M ratios of gimeracil to 5-FU was significantly higher than that of the control. On the contrary, the addition of uridine did not increase the 5-FU concentration at site 1. At a 0.1u2009M ratio of gimeracil to 5-FU, the maximum 5-FU plasma concentration was the lowest, and the area under the 5-FU concentration-time curve at site 1 was 3.4 times greater than that of the control. We demonstrated that applying 5-FU with gimeracil to the rat liver surface could increase the availability of 5-FU in the liver.