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Tissue specific and safety pinpoint targeting by bionano-capusele

通过生物纳米胶囊进行组织特异性和安全精确定位

基本信息

批准号：
15390383
负责人：
UEDA Masakazu
金额：
$ 9.41万
依托单位：
Keio 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 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390383/
关键词：
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

项目摘要

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 … More 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

简介：生物纳米胶囊(BNC)是我们通过生物技术程序设计和生产的人造空心纳米颗粒的概念。由于L蛋白氨基末端对人肝细胞的特异性亲和力，只有纳米尺寸的中空颗粒才显示出极强的选择性靶向性，作为一种新型的针对人肝脏的双链构象载体。由于其良好的潜力，我们提出了BNC的概念，作为一种高效的纳米机器来实现组织/细胞类型的基因、药物和蛋白质的特异性递送。结果：1.通过过度表达L蛋白，将其与分泌信号肽融合，有效地分泌了l个颗粒。在无血清、无抗生素选择压力的条件下，培养5d，L颗粒浓度可达1.7g/ml左右。L颗粒的分泌产物使其易于层析纯化。此外，它是d…进一步证明，纯化的L颗粒只能转染人肝细胞。因此，昆虫细胞表达系统是生产BNC的一种有吸引力的工具。2.用所得到的成纤维细胞生长因子和核糖核酸酶的原位融合蛋白(CL-RFN89)持续系统治疗可显著抑制体内人A431鳞癌的生长。与对照组相比，CL-RFN89治疗7天后，肿瘤生长抑制率为58.2%(p&lt；0.001)。此外，使用大鼠抗小鼠CD31抗体的免疫组织化学显示，CL-RFN89处理减少了肿瘤的血管形成。3.FITC标记的hRNase1-hEGF内化到EGFR高表达的A431细胞中。经人表皮生长因子和表皮生长因子受体缺陷的H69细胞处理的A431细胞未观察到内化。4.本研究设计了绿色荧光蛋白与S区域C-末端的融合基因。为了在Cos 7细胞中获得足够的表达水平，对C末端的截短进行了优化。通过在条件培养液和细胞提取液中的免疫反应性、荧光和密度来评价所产生的纳米颗粒。我们发现在体外展示EGFP的BNC中有两种不同的EGFP部分，而在BNC中只有一种取向占据一个颗粒，因此这两种类型的BNC-EGFP都可以通过抗体亲和柱有效地独立分离。结论：这种重组的BNC有望成为一种新型的选择性给药系统载体。较少

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Anti-angionenic effect of an insertional fusion protein of human basic fibroblast growth factor and ribonuclease-1.

人碱性成纤维细胞生长因子和核糖核酸酶 1 的插入融合蛋白的抗血管生成作用。

DOI：
发表时间：
2005
期刊：
Protein Engineering, Design & Selection 18(7)
影响因子：
0
作者：
Hayashida T.;Ueda M.;Aiura K.;Tada H.;Onizuka M.;Seno M.;Yamada H.;Kitajima M.
通讯作者：
Kitajima M.

Comparison of K-ras point mutation distributions in intraductal papillary-mucinous tumors and ductal adenocarcinoma of the pancreas