Development of intracellular signal-responsive drug and gene delivery system, and creation of new concept on DDS

在这项研究中，我们研究了一种响应细胞内蛋白激酶A(PKA)或caspase-3的纳米药物胶囊和基因递送系统的设计和表征。然后利用他们的结果，还开发了另一种响应细胞内Rho激酶和HIV蛋白酶的基因载体。我们还开发了一种新型的选择性识别血管军团的MRI造影剂。为了开发纳米药物胶囊，我们合成了一种新的基于底物肽序列的PKA大分子单体。该单体与N-异丙基丙烯酰胺通过自由基聚合法合成了接枝型共聚物。这种材料形成了以聚异丙基丙烯酰胺和亲水性聚乙二醇酯为疏水核的纳米粒子和底物多肽。这种颗粒可以包裹疏水药物分子，并响应PKA信号将其释放。通过控制聚合物中的多肽序列和含量，我们实现了对pka…具有高响应性的纳米粒子在PKA响应型基因载体中，我们通过分子设计的研究，成功地稳定了DNA-聚合物复合体，并控制了复合体的大小。然后用HVJ的包膜-胶囊将该复合体送入活细胞。在这个系统中，转基因的表达只在细胞中被激活，其中细胞内的PKA被持续激活。当细胞样本被不同的药物刺激时，表达谱与细胞内PKA活性的谱完全一致，这是通过CREB启动子实验来监测的。这表明我们的系统实际上是通过细胞内的PKA来控制转基因的表达。利用这个概念和结果，我们还成功地开发了响应Rho激酶信号的Gone调控系统，这是另一种与血管疾病相关的非常重要的蛋白激酶。为了将这种Rho激酶反应系统应用于动物，我们需要对目标血管疾病进行监测。因此，我们开发了一种使用有机染料伊文斯蓝选择性监测血管疾病的磁共振造影剂。为了开发caspase-3反应载体，我们设计并制备了不同的多肽-聚合物结合物，并表征了它们对caspase-3的响应性。结果表明，将TAT多肽引入载体，可将转基因基因直接输送到活细胞中。在这种情况下，转基因的表达可以被细胞内的caspase-3信号选择性地调控。只有在细胞受到星形孢菌素刺激后，才能观察到该基因的表达，星孢菌素激活了凋亡信号级联反应。利用这些结果，我们还成功地获得了另一种对HIV蛋白酶有反应的载体。载体将基因导入T细胞样本，但仅在HIV感染细胞中观察到基因激活。较少

In this research, we investigated a design and characterization of nano-drug capsule and gene delivery system that respond to intracellular protein kinse A (PKA) or caspase-3. Then using their results, another gene carrier responding to intracellular Rho kinase and HIV protease were also developed. We also developed a new class of MRI contrast agent that recognizes vascular legion selectively.For the development of nano-drug capsule, we synthesized a new macro-monomer based on substrate peptide sequence for PKA. Using this monomer methacryloyl-PEG and N-isopropylacrylamide, graft-type copolymer was prepared with radical polymerization. This material formed nano-particle having hydrophobic core of poly isopropylacrylamide and hydrophilic PEG and the substrate peptide. This particle can encapsulate hydrophobic drug molecules, and release them responding to PKA signal. By controlling the peptide sequence and content in the polymer, we realized nano-particle with high responsibility to PKA … More signal.In the PKA-responsive gene carrier, we succeeded to stabilize the DNA-polymer complex and to control the size of the complex through the investigation of molecular design. Then the complex was delivered into living cell with envelop-capsule of HVJ. In this system, expression of the transgene was activated only in the cell, in which intracellular PKA was continuously activated. When the cell sample was stimulated with various drugs, the expression profile completely coincided to the profile of intracellular PKA activity, which was monitored with CREB promoter assay. This indicated that our system actually control the transgene expression using intracellular PKA. Using this concept and results, we also succeeded the development of gone control system responding to Rho kinase signal, that is another very important protein kinase related to vascular disease. To apply this Rho kinase-responsive system to animal, we needed the monitoring procedure of the target vascular disease. Thus, we developed a MRI contrast agent monitoring vascular disease selectively using organic dye, Evans blue. Synthesized MRI contrast agent could detect vascular lesion with balloon injury or atheroscurelosis in Rat.For the development of caspase-3 responsive carrier, we designed and prepared various peptide-polymer conjugates, and characterized their responsibility to caspase-3. As the result, obtained carrier could deliver the transgene directly to living cell, if Tat peptide was introduced to the carrier. In this case, expression of the transgene could be controlled by intracellular caspase-3 signal selectively. The expression of the gene was observed only after the cell was stimulated with staurosporin that activates the apoptotic signal cascade. Using these results, we also succeeded another carrier responding to HIV protease. The carrier delivered to gene into T-cell sample, but the gene activation was observed only in HIV-infectious cell. Less