193. Gene Transfection Using Inorganic Particle/PVA/DNA Complexes Prepared by Ultra High Pressure Technology
Authors: Tsuyoshi Kimura, Kwangoo Nam, Sihngo Mutsuo, Hidekazu Yoshizawa, Masahiro Okada, Tsutomu Furuzono, Toshiya Fujisato, Akio Kishida
Journal: Molecular Therapy (2006)

Various non-viral gene systems, such as naked DNA, lipoplexes, micelles and polyplexes, have been developed for effective and safe gene delivery into target cells. Although cationic compounds were employed as gene carriers due to the ability of complex formation with DNA electrostatically and effective gene transfer into cells, the intrinsic cytotoxicity of them is essential problem in non-viral gene delivery system. Therefore, we have tried the development of DNA complex with non-ionic, water soluble polymers via hydrogen bond using ultra high pressure (UHP) technology because the inter-, intra- molecular week hydrogen bonding interaction was empathized with high pressure process. Previously, polyvinyl alcohol (PVA) was utilized as the model hydrogen bonding polymers, and the PVA/ DNA complexes were obtained by UHP treatment. Although the PVA/DNA complexes were up-taken by cells, a little enhancement of gene expression was observed using them. In this study, we hypothesize that inorganic particles, such as calcium phosphate (CP), calcium carbonate and hydroxy apatite (HAp), promote the endosomal escape of transferred DNA because the inorganic particles are dissolved under low pH condition in endosome vesicles and then the rupture of endosome is induced by osmotic shock. We performed the development of inorganic particle/PVA/DNA complexes using UHP technology. Plasmid DNAs encoding enhanced green fluorescent protein (EGFP) gene or luciferase gene under CMV promoter were used. Nano-HAps having the average diameter of 50nm were synthesized by modified micro-emulsion method. Nano- HAp was dispersed ultrasonically in PVA solution and then mixed with DNA solution. CP/DNA complexes were prepared by general method and mixed with PVA solution. Their mixtures were treated under 10000 atmospheric pressures at 40 degree for 10min. By SEM observation, the irregular surface of inorganic particles/PVA/ DNA complexes was observed, indicating the encapsulation of inorganic particles in PVA/DNA particle. The nano-HAp/PVA/DNA complexes showed a higher transfection activity than DNA complexes with nano-HAp or PVA. With CP/PVA/DNA complexes, also, the transfection activity increased several fold than the PVA/ DNA complexes. These results indicate the utility of the inorganic particle/PVA/DNA complexes prepared by the UHP treatment for DNA delivery. Various non-viral gene systems, such as naked DNA, lipoplexes, micelles and polyplexes, have been developed for effective and safe gene delivery into target cells. Although cationic compounds were employed as gene carriers due to the ability of complex formation with DNA electrostatically and effective gene transfer into cells, the intrinsic cytotoxicity of them is essential problem in non-viral gene delivery system. Therefore, we have tried the development of DNA complex with non-ionic, water soluble polymers via hydrogen bond using ultra high pressure (UHP) technology because the inter-, intra- molecular week hydrogen bonding interaction was empathized with high pressure process. Previously, polyvinyl alcohol (PVA) was utilized as the model hydrogen bonding polymers, and the PVA/ DNA complexes were obtained by UHP treatment. Although the PVA/DNA complexes were up-taken by cells, a little enhancement of gene expression was observed using them. In this study, we hypothesize that inorganic particles, such as calcium phosphate (CP), calcium carbonate and hydroxy apatite (HAp), promote the endosomal escape of transferred DNA because the inorganic particles are dissolved under low pH condition in endosome vesicles and then the rupture of endosome is induced by osmotic shock. We performed the development of inorganic particle/PVA/DNA complexes using UHP technology. Plasmid DNAs encoding enhanced green fluorescent protein (EGFP) gene or luciferase gene under CMV promoter were used. Nano-HAps having the average diameter of 50nm were synthesized by modified micro-emulsion method. Nano- HAp was dispersed ultrasonically in PVA solution and then mixed with DNA solution. CP/DNA complexes were prepared by general method and mixed with PVA solution. Their mixtures were treated under 10000 atmospheric pressures at 40 degree for 10min. By SEM observation, the irregular surface of inorganic particles/PVA/ DNA complexes was observed, indicating the encapsulation of inorganic particles in PVA/DNA particle. The nano-HAp/PVA/DNA complexes showed a higher transfection activity than DNA complexes with nano-HAp or PVA. With CP/PVA/DNA complexes, also, the transfection activity increased several fold than the PVA/ DNA complexes. These results indicate the utility of the inorganic particle/PVA/DNA complexes prepared by the UHP treatment for DNA delivery.