RESUMEN
Development of technology to deliver foreign gene(s) to a specific organ/tissue is one of the major challenges in gene therapy. Here, we show liver- and lobe-specific gene transfer following the continuous microinstillation of plasmid DNA (pDNA) onto the liver surface in mice. Naked pDNA was continuously instilled onto the right medial liver lobe using syringe pump in male ddY mice. Our previous studies showed liver- and lobe-selective gene expression after instillation of 30 mul of pDNA solution onto the liver surface, but gene expression was also found in the other liver lobe, kidney and spleen. To improve target site selectivity of gene expression, the instillation volume was decreased; however, non-specific gene expression in the other liver lobe and diaphragm was still detected. To prevent immediate diffusion of the pDNA solution, we performed continuous microinstillation of pDNA using a syringe pump; as a result, target site selectivity was greatly improved. As for instillation speed, 5 min infusion was enough to prevent diffusion of pDNA solution. Furthermore, transfection efficiency in the target site was maintained when instillation speed was slowed. Wiping off residual pDNA solution from the applied liver lobe resulted in a further improvement in selectivity, suggesting not only immediate diffusion, but also gradual diffusion, are important factors for successful target site-specific gene transfer. Information in this study will be useful for further development of an effective gene delivery system targeted to a specific organ/tissue by use of other non-viral or viral vectors.
Asunto(s)
ADN/administración & dosificación , Expresión Génica , Técnicas de Transferencia de Gen , Hígado/metabolismo , Plásmidos/administración & dosificación , Animales , ADN/genética , Vectores Genéticos , Instilación de Medicamentos , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Endogámicos , Especificidad de Órganos , Plásmidos/genéticaRESUMEN
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.
Asunto(s)
ADN/administración & dosificación , Mucosa Gástrica/metabolismo , Técnicas de Transferencia de Gen , Pinocitosis/fisiología , Plásmidos/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Membrana Serosa/metabolismo , Actinas/metabolismo , Animales , Caveolas/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Dosificación de Gen , Técnicas para Inmunoenzimas , Masculino , Ratones , Contracción Muscular , Miosinas/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND: The purpose of this study was to achieve stomach-selective gene transfer in rats by our simple and novel administration method, which is gastric serosal surface instillation of naked plasmid DNA (pDNA). METHODS: Naked pDNA encoding firefly luciferase as a reporter gene was instilled onto the gastric serosal surface in male Wistar rats. As controls, we performed intraperitoneal, intragastric and intravenous administration of naked pDNA. At appropriate time intervals, we measured luciferase activities in the stomach and other tissues. RESULTS: Gene expression in the stomach 6 h after gastric serosal surface instillation of naked pDNA (5 microg) was significantly higher than that after using other administration methods. The present study is the first report on stomach-selective gene transfer following instillation of naked pDNA onto the gastric serosal surface in rats. Also, the gene expression level in the stomach 6 h after gastric serosal surface instillation of naked pDNA was markedly higher than that in other tissues. In a dose-dependent study, the gene expression level was saturated over 5 microg. Gene expression in the stomach was detected 3 h after gastric serosal surface instillation of naked pDNA. The gene expression level peaked 12-24 h after instillation of naked pDNA, then decreased to a level similar to 3 h at 48 h. CONCLUSIONS: Gastric serosal surface in stillation of naked pDNA can be a highly stomach-selective gene transfer method in rats.
Asunto(s)
ADN/administración & dosificación , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Plásmidos/administración & dosificación , Animales , Mucosa Gástrica/metabolismo , Dosificación de Gen , Regulación de la Expresión Génica , Genes Reporteros , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Masculino , Ratas , Ratas Wistar , Membrana Serosa/metabolismo , Factores de TiempoRESUMEN
Gene therapy for gastric cancer and gastric ulcer is a rationalized strategy since various genes correlate with these diseases. Since gene expressions in non-target tissues/cells cause side effects, a selective gene delivery system targeted to the stomach and/or cancer must be developed. The route of vector transfer (direct injection, systemic, intraperitoneal, gastric serosal surface and oral administration) is an important issue which can determine efficacy and safety. Strategies for cancer gene therapy can be categorized as suicide gene therapy, growth inhibition and apoptosis induction, immunotherapy, anti-angiogenesis, and others. Combination of the target gene with other genes and/or strategies such as chemotherapy and virotherapy is promising. Candidates for treatment of gastric ulcer are vascular endothelial growth factor, angiopoietin-1, serum response factor, and cationic host defense peptide cathelicidin. In this review, we discuss stomach- and cancer-targeted gene transfer methods and summarize gene therapy trials for gastric cancer and gastric ulcer.
Asunto(s)
Terapia Genética/métodos , Gastropatías/terapia , Animales , Apoptosis , Vectores Genéticos , Humanos , Inmunoterapia/métodos , Ratones , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapia , Úlcera Gástrica/terapiaRESUMEN
The present study was undertaken to elucidate the absorption and distribution characteristics of 5-fluorouracil (5-FU) after its application to the liver surface in rats to examine the possibility of reducing the systemic side effects of this agent. 5-FU was applied to the surface of the liver by employing a cylindrical diffusion cell. Approximately 69% of the dose was absorbed in 360 min. The time course of the change in the amount of 5-FU remaining in the diffusion cell obeyed first-order kinetics. Also, a linear relationship was observed between the apparent permeability coefficient, P app, and the reciprocal of the square root of the molecular weight of several compounds including 5-FU. The estimated P app value of 5-FU was in good agreement with the experimental value. The plasma concentration of 5-FU was low (<1.2 microg/ml) until 360 min after the application. Following i.v. administration, 5-FU was rapidly eliminated from the plasma and could not be detected at 120 min. In the analysis of tissue distribution, the liver was divided into three sites; the region under the diffusion cell attachment site (site 1), the treated lobe excluding site 1 (site 2), and untreated lobes (site 3). After being administered i.v., 5-FU mainly distributed in the kidney, and the concentration in the liver was significantly lower than that in kidney, spleen, or heart. After its application to the liver surface, however, 5-FU preferentially distributed at site 1, and was not detected at the other sites or in other tissues. Thus, these results suggested the possibility of a reduction in the systemic side effect of 5-FU on its application to the liver surface.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Fluorouracilo/farmacocinética , Hígado/metabolismo , Absorción , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/efectos adversos , Cámaras de Difusión de Cultivos , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Inyecciones Intravenosas , Riñón/metabolismo , Masculino , Ratas , Ratas Wistar , Distribución TisularRESUMEN
Stomach-selective gene transfer is a promising approach as a therapeutic strategy for refractory gastric diseases. In this study, we improved the stomach selectivity of gene expression following microinstillation of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. pDNA encoding firefly luciferase was used as a reporter gene. It was confirmed that the gene expression level in the stomach 6h after gastric serosal surface microinstillation of pDNA was significantly higher than after intragastric, intraperitoneal and intravenous administration. Regarding selectivity of gene expression, the gene expression level in the stomach after gastric serosal surface microinstillation of 1 microg/1 microL (dose/volume) pDNA was 5.7 times higher than that in the spleen. In our previous study (30 microg/30 microL), the expression level in the stomach was 2.7 times higher than that in the spleen; therefore, the selectivity was 2.1 times higher in this study. When we investigated gene expression at various pDNA solution concentrations, the ratio of the gene expression level in the stomach to that in the spleen was the highest as 1 microg/1 microL of pDNA, which was considered the optimal concentration. Information in this study is useful for further development of target organ-selective gene delivery systems.
Asunto(s)
ADN/administración & dosificación , Mucosa Gástrica/metabolismo , Técnicas de Transferencia de Gen , Plásmidos/administración & dosificación , Animales , ADN/química , Expresión Génica , Genes Reporteros/genética , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Intubación Gastrointestinal , Luciferasas/genética , Masculino , Ratones , Plásmidos/química , Plásmidos/genética , Distribución TisularRESUMEN
PURPOSE: As peritoneal damage in long-term peritoneal dialysis therapy is a major problem correlated to patient prognosis, diagnosis of peritoneal damage is important. To develop a diagnostic method for peritoneal damage, we focused on hyperpermeability across the peritoneum in a pathogenic peritoneal damage condition. In this study, disposition characteristics of an intraperitoneally injected marker substance in peritoneal damaged rats were analyzed. MATERIALS AND METHODS: Peritoneal damaged rats were prepared by intraperitoneal injection of a glucose degradation product, methylglyoxal (MGO), for five or ten consecutive days. Phenolsulfonphthalein (PSP), as a marker substance, was intraperitoneally or intravenously injected into MGO-treated rats. Subsequently, the PSP disposition characteristics were pharmacokinetically analyzed. RESULTS: In both cases of 5 and 10 days treatment of MGO, absorption of PSP after intraperitoneal injection was significantly enhanced. Plasma concentration and urinary excretion of PSP in MGO-treated rats were also higher than those in saline-treated rats in the early phase. On the contrary, there was no significant difference in terms of the pharmacokinetic parameters of intravenously injected PSP in saline- or MGO-treated rats. These results indicated that intraperitoneally injected MGO primarily acts on the peritoneal membrane; therefore, the peritoneal permeability of the marker substance was enhanced. CONCLUSION: We demonstrated that pharmacokinetic analysis of peritoneum permeability might be a potent diagnostic method for peritoneal damage in experimental animals and patients receiving peritoneal dialysis.
Asunto(s)
Indicadores y Reactivos/farmacocinética , Enfermedades Peritoneales/diagnóstico , Enfermedades Peritoneales/metabolismo , Fenolsulfonftaleína/farmacocinética , Animales , Indicadores y Reactivos/administración & dosificación , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Masculino , Enfermedades Peritoneales/inducido químicamente , Permeabilidad , Fenolsulfonftaleína/administración & dosificación , Piruvaldehído , Ratas , Ratas Wistar , Distribución TisularRESUMEN
The purpose of present study was to examine spleen-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the spleen surface in mice. Gene expression in the spleen and other tissues was evaluated based on firefly luciferase activity. Six hours after spleen surface instillation of naked pDNA, high gene expression in the spleen was observed. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the spleen surface, gene expression in the spleen was significantly higher than those in other tissues. Six hours after instillation of naked pDNA onto the spleen surface, gene expression in the spleen reached the peak value, and thereafter decreased gradually. By utilizing a glass-made diffusion cell that is able to limit the contact dimension between the spleen surface and naked pDNA solution administered, site-specific gene expression in the spleen was found. This novel gene transfer method is expected to be a safe and effective strategy for DNA vaccine against serious infectious diseases and cancers.
Asunto(s)
ADN/administración & dosificación , Expresión Génica , Técnicas de Transferencia de Gen , Plásmidos , Bazo/metabolismo , Animales , ADN/genética , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Endogámicos , Especificidad de ÓrganosRESUMEN
The purpose of the present study was to examine unilateral lung-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the pulmonary pleural surface in mice. Naked pDNA was administered intravenously, intraperitoneally, and instilled onto the right pulmonary pleural surface. Four hours later, right pulmonary pleural surface instillation of naked pDNA resulted in high gene expression in the right lung. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the right or left pulmonary pleural surface, gene expressions in the applied lung were significantly higher than those in the other lung and tissues. In addition, gene expressions were detected only in the intrathoracic tissues, not in the intraperitoneal tissues. Four hours after instillation of naked pDNA onto the right pulmonary pleural surface, gene expression in the right lung was the highest, and thereafter gene expression in the right lung decreased gradually. This novel gene transfer method is expected to be a safe and effective treatment against serious lung diseases.
Asunto(s)
ADN/administración & dosificación , Técnicas de Transferencia de Gen , Pulmón/metabolismo , Plásmidos/metabolismo , Pleura/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos , Plásmidos/administración & dosificación , Factores de TiempoRESUMEN
The purpose of the present study was to achieve a stomach-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. Gene expression in the stomach and other tissues was evaluated by firefly luciferase activity. Six hours after gastric serosal surface instillation of naked pDNA, high gene expression in the stomach was observed. On the contrary, intravenous and intraperitoneal injection of naked pDNA exhibited no detectable gene expression. Following instillation of naked pDNA onto the gastric serosal surface, gene expression in the stomach was significantly higher than in other tissues. Gene expression in the stomach was highest 12 h after the instillation and thereafter decreased gradually. Utilizing a glass-made diffusion cell that is able to limit the contact dimension between the gastric serosal surface and the naked pDNA solution administered, site-specific gene expression in the stomach was achieved. This novel gene transfer method is expected to be a safe and effective treatment against serious stomach diseases.
Asunto(s)
ADN/administración & dosificación , Mucosa Gástrica/metabolismo , Terapia Genética/métodos , Plásmidos , Gastropatías/terapia , Animales , Masculino , RatonesRESUMEN
The present study was undertaken to elucidate the stomach- and site-selective delivery of 5-fluorouracil (5-FU) following its application on the gastric serosal surface in rats. An experimental system utilizing a cylindrical diffusion cell attached to the gastric serosal surface was established. To evaluate the gastric distribution of 5-FU, the stomach was separated into the site under the diffusion cell (site 1) and the site not under the diffusion cell (site 2). Furthermore, the mucosal side at site 1 was separated from the serosal side. After intravenous and oral administration of 5-FU, the 5-FU concentrations at sites 1 and 2 until 240 min were similar. After gastric serosal surface application of 5-FU, however, the concentration of 5-FU at site 1 until 240 min was approximately 10-fold higher than that at site 2, and was sustained. Furthermore, the 5-FU concentration on the mucosal side at site 1 and the serosal side at site 1 were comparable after gastric serosal surface application. The blood concentration of 5-FU was low (<4.4 microg/ml) until 240 min after gastric serosal surface application. The maximum blood concentration of 5-FU after gastric serosal surface application was significantly lower than after intravenous administration. Thus, the stomach- and site-selective delivery system following application on the gastric serosal surface could be applied with anticancer drugs for the treatment of gastric cancer.