Gene delivery application of the polymer assembly interconnected by hydrogen bonding prepared by using ultra-high pressure

考察了利用超高压组装分子技术开发新型基因递送复合物的情况。本年度的课题是：1）用聚乙烯醇以外的聚合物组装DNA的研究，2）具有氢键的无机微粒的应用。通过使用多糖，包括葡聚糖，聚（丙烯酰胺），聚（丙烯酸），聚（乙二醇），和低分子量的聚（甲基丙烯酸羟甲酯）作为氢键聚合物与DNA的组装进行了检查。只有葡聚糖与聚乙二醇的组合使用超高压过程产生分子组装。对这两种聚合物与质粒DNA的组装进行了考察。澄清了组装体的形成受溶液状态的极大影响。空气中的二氧化碳一旦溶解于蒸馏水中，化合物就被阻碍了。在使用氢键比聚乙烯醇弱的聚合物的情况下，显然应该更严格地设定实验条件。研究了在无机微粒表面组装带有羟基的DNA。制备了在溶液中保持均匀分布、表面带有羟基的羟基磷灰石纳米晶，并考察了纳米晶与DNA的组装情况。DNA的化合物稳定下来，并且能够进行基因递送到细胞中的实验。质粒DNA的掺入量高，而编码蛋白的出现量低。这些结果表明，关于DNA在细胞内向细胞核的转运以及DNA与其他物质组装的条件的考察，必须考虑一种新的机制。作为候选材料，低分子量肽将被检查，因为肽的结构似乎不受超高压的影响。

The development of the complex for new gene delivery by the assembling molecule technology that utilizes the ultra high pressure was examined. This year, we examined following themes: 1) Examination of assembling DNA with polymers other than polyvinyl alcohol, 2) Application of inorganic micro particle that has hydrogen bond. The assembly with DNA was examined by using the polysaccharide including dextran, poly (acrylamide), poly (acrylic acid), poly (ethyleneglycol), and low molecular weight poly (hydroxymethylmethacrylate) as hydrogen bond polymers. Only the combination of dextran with poly (ethyleneglycol) gave molecular assembly using ultra-high pressure process. The assembly with plasmid DNA was examined about these two kinds of polymers. It was clarified that the formation of assembly was greatly influenced by the state of the solution. The compound was obstructed about the one that carbon dioxide in air dissolved to the distilled water once. In case of using the polymer to which the hydrogen bonds are weaker than poly (vinyl alcohol), it became clear that the experimental condition should be set more strictly. The assembling DNA with the hydroxyl group on the surface of the inorganic micro particle was examined. The hydroxyapatite nanocrystal that kept distributing in solution and had the hydroxyl groups on its surface was prepared, and the assembling with nanocrystals and DNA was examined. The compound of DNA stabilized, and was able to conduct the experiment on the gene delivery into the cell. The incorporation of plasmid DNA was high, however, the amount of appearance of the code protein was low. These results showed that a new mechanism had to be considered about the transportation to the nucleus in the cell and the examination of the condition of DNA assembling with other materials. As the candidate material, low molecular weight peptide will be examined because of that the structure of the peptide seemed not to be affected by the ultra-high pressure.