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传递系统的设计和表征。然后利用他们的结果，另一种对细胞内Rho激酶和HIV蛋白酶有反应的基因载体也被开发出来。我们还开发了一种新的MRI造影剂，可以选择性地识别血管军团。为了开发纳米药物胶囊，我们合成了一种新的基于PKA底物肽序列的大分子单体。以该单体甲基丙烯酰- peg和n -异丙基丙烯酰胺为原料，采用自由基聚合法制备接枝型共聚物。该材料形成了具有疏水核心的聚异丙基丙烯酰胺和亲水性PEG及底物肽的纳米颗粒。该颗粒可以包封疏水药物分子，并根据PKA信号释放药物分子。通过控制聚合物中多肽的序列和含量，我们实现了对PKA具有高责任的纳米颗粒。在pka反应性基因载体中，我们通过分子设计研究成功地稳定了dna -聚合物复合物，并控制了复合物的大小。然后用HVJ包膜胶囊将复合物送入活细胞。在该系统中，转基因仅在细胞内被激活表达，其中细胞内PKA持续被激活。当细胞样品受到各种药物刺激时，表达谱与细胞内PKA活性谱完全一致，用CREB启动子试验监测PKA活性谱。这表明我们的系统实际上是通过细胞内PKA来控制转基因的表达。利用这一概念和结果，我们还成功开发了响应Rho激酶信号的gone - control系统，Rho激酶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，我们需要目标血管疾病的监测程序。因此，我们开发了一种MRI造影剂，选择性地使用有机染料埃文斯蓝监测血管疾病。合成MRI造影剂可检测大鼠血管损伤及动脉粥样硬化。为了开发caspase-3响应载体，我们设计并制备了多种多肽-聚合物偶联物，并表征了它们对caspase-3的作用。结果表明，所获得的载体只要在载体上引入Tat肽，就能将转基因直接传递到活细胞中。在这种情况下，转基因的表达可以由细胞内caspase-3信号选择性地控制。只有在staurosporin刺激细胞激活凋亡信号级联后才能观察到该基因的表达。利用这些结果，我们还成功地获得了另一个对HIV蛋白酶有反应的携带者。载体将该基因送入t细胞样本，但仅在hiv感染细胞中观察到该基因的激活。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