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

细胞内信号响应药物和基因传递系统的开发，开创DDS新概念

基本信息

批准号：
15300166
负责人：
KATAYAMA Yoshiki
金额：
$ 9.73万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15300166/
关键词：
gene therapy drug delivery system intracellular signal transduction gene delivery protein kinase apoptosis protease targeting

项目摘要

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

在这项研究中，我们研究了对细胞内蛋白质KINSE A（PKA）或caspase-3的反应的纳米药胶囊和基因输送系统的设计和表征。然后使用他们的结果，还开发了另一个对细胞内Rho激酶和HIV蛋白反应的基因载体。我们还开发了一种新的MRI对比剂，该药物有选择地识别血管杆。对于纳米毒胶囊的开发，我们基于PKA的底物肽序列合成了一个新的宏观经济器。使用这种单体甲基丙烯酰-PEG和N-异丙烯酰胺，用自由基聚合制备移植型共聚物。该材料形成具有聚异丙丙烯酰胺和亲水性PEG和底物肽的疏水核心的纳米颗粒。该粒子可以封装疏水性药物分子，并释放它们对PKA信号的反应。通过控制聚合物中的肽序列和含量，我们意识到了对PKA高责任的纳米颗粒……更多信号。在PKA反应性基因载体中，我们成功地稳定了DNA聚合物复合物并通过研究分子设计来控制复合物的大小。然后将复合物用HVJ的包膜封装到活细胞中。在该系统中，仅在细胞中激活转化的表达，其中细胞内PKA被连续激活。当用各种药物刺激细胞样品时，表达曲线完全与细胞内PKA活性的曲线完全重合，该活性通过CREB启动子测定法监测。这表明我们的系统实际上使用细胞内PKA控制转换表达。利用这个概念和结果，我们还成功地开发了对Rho激酶信号的反应，这是与血管疾病有关的另一种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，我们需要对靶血管疾病的监测程序。因此，我们使用有机染料Evans Blue选择性地监测了血管疾病的MRI对比剂。合成的MRI对比剂可以在大鼠中检测出因球囊损伤或动脉粥样硬化的血管病变。对于Caspase-3响应式载体的发展，我们设计和准备了各种肽 - 聚合物结合物，并将其对CASPASE-3的责任表征。结果，如果将TAT肽引入载体，获得的载体可以直接传递到活细胞。在这种情况下，转换的表达可以选择性地控制细胞内caspase-3信号。仅在用Staurosporin刺激细胞以激活凋亡信号级联反应的细胞后才能观察到该基因的表达。使用这些结果，我们还继承了另一个响应HIV蛋白酶的载体。载体将基因传递到T细胞样品中，但仅在HIV感染细胞中观察到基因激活。较少的