| Project/Area Number |
15390383
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General surgery
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| Research Institution | Keio University |
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Principal Investigator |
UEDA Masakazu Keio University, School of Med., Associate Prof., 医学部, 助教授 (50142419)
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| Co-Investigator(Kenkyū-buntansha) |
JINNO Hiromjitsu Keio University, School of Med., Assistant Prof., 医学部, 講師 (20216261)
MIYATA Ryouhei Keio University, School of Med., Resident, 医学部, 助手 (20327571)
KIDO Hiroshi Keio University, School of Med., Resident, 医学部, 助手 (70327534)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2005: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2004: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2003: ¥6,900,000 (Direct Cost: ¥6,900,000)
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| Keywords | HBs antigen / L particle / DDS / cell-specificity / gene therapy / ミサイル療法 / ヒト肝細胞癌 / ヒト幹細胞癌 |
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| Research Abstract |
Introduction : The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. Due to the specific affinity to human hepatocytes localized in the amino-terminus of L-protein, only the nanometer size hollow particles, showed extremely selective targeting potential as a novel type of DDS vector to the directed to the human liver. Because of its favorable potential, we have proposed a BNC concept as an efficient nanomachine to achieve tissue-/cell-type specific delivery of genes, drugs and proteins.
Result : 1.L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 g/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was d
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emonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.
2.Continuous systemic treatment with resultant insetional-fusion protein of FGF and RNase (CL-RFN89) significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (p<0.001). Furthermore, immunohistochemistry using a rat anti-mouse CD31 antibody snowed that treatment with CL-RFN89 reduced tumor vascularization.
3.FITC-labelled hRNase 1-hEGF was internalized into EGFR-overexpressing A431 cells. The internalization was not observed In A431 cells pre-treated with hEGF and EGFR-deficient H69 cells.
4.The fusion of jellyfich green fluorescent protein (EGFP) to the C-terminus of the S region was designed in this study. Truncation of the C-terminus was optimized to obtain sufficient expression level in Cos 7 cells. The produced nanoparticles were evaluated by immunoreactivity, fluorescence, and density both in the conditioned media and in the cell extracts. We found two orientations of the EGFP moiety in BNCs that are displaying EGFP outside and are enclosing it inside, while only single orientation was found to occupy a particle, so that either type of BNC-EGFPs could be effectively and independently separated by antibody affinity column.
Conclusion : This recombinant BNC is now being developed as a novel platform of drug delivery system vector for selective delivery. Less
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