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Non-viral Liver-targeted Gene Delivery Using Chitosan-DNA PEI-DNA Nanoparticles In Vitro And In Vivo

Posted on:2005-11-01Degree:DoctorType:Dissertation
Country:ChinaCandidate:H DaiFull Text:PDF
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Background/Aim: INTRODUCTION:Liver is one of the most important targets for gene medicine applications; because it is the site of many genetic metabolic disorders and the transgene expressed in the liver can easily access systemic circulation. The anatomy of the liver at the microscopic level has an important characteristic that favors gene delivery. The hepatic endothelium possesses fenestrae of a couple hundred nanometers in diameter which occupy about 6 to 8% of the sinusoidal surface area. These sinusoidal and bile canaliculus fenestrae permit easy transport of macromolecules and nanoparticles to hepatocyte directly.Intravenous injection of naked plasmid DNA shows no gene expression even in the liver where the highest uptake is observed. High gene expression can be achieved only by a hydrodynamic-based procedure or mechanical massage method through a rapid injection of naked DNA in large volume (-8% body weight). The concerns are that such a procedure may alter the physiological conditions of the liver, and it is not feasible in clinical settings. Reducing the injection rate and volume of the naked DNA solution to physiologically acceptable range will abolish the gene expression. A vector or carrier that efficiently protects DNA from degradation in serum, as well as facilitates its transport to the liver and the binding and uptake by parenchymal cells is clearly required for meaningful transgene expression in the liver,but gene expression is low and transient. A number of gene carriers/vectors have been studied for gene delivery to the liver, including viral and non-viral gene carriers. Viral vectors, particularly adenovirus and lentivirus, have demonstrated high level of transgeneexpression. Viral vectors have the ability to infect a wide variety of cell types in vivo, therefore, liver-targeted gene delivery can only be achieved through localized injection through the hepatic artery or portal vein,or by conjugating a polymer with hepatocyte-specific ligand. However, viral vectors are associated with severe problems such as host immune response against the vectors leading to the rapid rejection of transduced cells, intrinsic toxicity of the viral proteins, and limited packaging size. Non-viral gene carriers have been increasingly proposed as safer alternatives, Nanosphere encapsulating DNA gene delivery methods was established by prof Leong in 1998,because of ease of synthesis, flexibility in the size of the transgene to be delivered, and most importantly, the low toxicity and minimum host immune response. In addition, they can better address the pharmaceutical issues such as storage stability and scale-up synthesis. With a more localized gene delivery route, gene carriers without specific ligand have shown to mediate high level of transgene expression. Otsuka et al prepared multilamellar liposomes encapsulating DNA to deliver DNA to rat and pig livers. When delivered through portal vein injection, only very low transient gene expression was observed even when a high DNA dose (10 mg/kg) was used, and gene transfection was mainly found in Kupffer cells.Expression in lung and spleen was also observed. In contrast, intrabiliary injection of the complexes containing low dose DNA produced higher transgene expression and lasted for at least 6 days, and gene expression was restricted to the liver hepatocytes.In this study, we examine the feasibility of achieving rat hepatocyte transfection in vitro and in vivo,and liver-targeted gene delivery by chitosan-DNA nanoparticles through bile duct and portal vein infusions. The time-course of transgene expression in rat liver following administration of chitosan-DNA nanoparticles was compared with that of PEI-DNA nanoparticles or naked DNA.Aim:To investigate the feasibility, safety, transfection efficiency, and delivery method of a novel Nanosphere complexes-Based gene therapy, compared with plasmid DNA. Methods: After Reconstructing plasmid luciferase-VR1255, EGFP,Non-virual carriers was used to transfect theluciferase-VR1255,EGFP gene into the HepG2, Hep3B, Hu...
Keywords/Search Tags:Nanoparticles, gene therapy, Liver-targeted
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