| Co-Investigator(Kenkyū-buntansha) |
NIIDOME Takuro Kyushu University, Department of Applied Chemistry, Faculty of Engineering, Associate Professor, 大学院・工学研究院, 助教授 (20264210)
MURATA Masaharu Kyushu University, Department of Applied Chemistry, Faculty of Engineering, Assistant Professor, 大学院・工学研究院, 助手 (30304744)
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| Budget Amount *help |
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2004: ¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 2003: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Research Abstract |
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
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