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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

在本研究中，我们研究了响应于细胞内蛋白激酶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的MRI造影剂，可以选择性识别血管群。为了开发纳米药物胶囊，我们合成了一种基于PKA底物肽序列的大分子单体。以该单体甲基丙烯酰-PEG和N-异丙基丙烯酰胺为单体，通过自由基聚合制备了接枝共聚物。该材料形成具有聚异丙基丙烯酰胺和亲水性PEG的疏水核和底物肽的纳米颗粒。这种微球可以包裹疏水性药物分子，并响应PKA信号释放药物分子。通过控制聚合物中肽段的序列和含量，实现了对PKA高度响应的纳米粒子 ...更多信息在PKA应答基因载体中，我们成功地稳定了DNA-聚合物复合物，并通过分子设计的研究控制了复合物的大小。然后将复合物通过HVJ微囊导入活细胞。在该系统中，转基因的表达仅在细胞中被激活，其中细胞内PKA被持续激活。当细胞样本用各种药物刺激时，表达谱与细胞内PKA活性谱完全一致，并用CREB启动子测定来监测。这表明我们的系统实际上是通过细胞内PKA来控制转基因表达的。利用这一概念和结果，我们还成功地开发了响应Rho激酶信号的gone控制系统，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，我们需要对靶血管疾病进行监测。因此，我们开发了一种选择性使用有机染料伊文思蓝监测血管疾病的MRI造影剂。合成的MRI造影剂可用于检测球囊损伤或动脉粥样硬化大鼠血管病变。为了开发caspase-3响应性载体，我们设计并制备了多种肽-聚合物偶联物，并表征了它们对caspase-3的响应性。结果表明，如果将达特肽引入载体，所获得的载体可以将转基因直接递送至活细胞。在这种情况下，转基因的表达可以通过细胞内caspase-3信号选择性地控制。该基因的表达仅在用激活凋亡信号级联的星形孢菌素刺激细胞后观察到。利用这些结果，我们还成功地另一个载体响应HIV蛋白酶。载体将基因导入T细胞样品，但仅在HIV感染细胞中观察到基因激活。少