Tissue specific and safety pinpoint targeting by bionano-capusele

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中空纳米粒子的概念。由于对人肝细胞的特异性亲和力定位在l蛋白的氨基端，因此只有纳米尺寸的中空颗粒，才显示出极具选择性的靶向潜力，作为一种新型的针对人肝脏的DDS载体。由于其良好的潜力，我们提出了BNC概念作为一种高效的纳米机器来实现基因，药物和蛋白质的组织/细胞类型特异性递送。结果：1；L蛋白通过过表达与分泌信号肽融合而有效分泌L颗粒。在无血清培养基中无抗生素选择压力培养5天后，L颗粒浓度达到约1.7 g/ml。L粒子的分泌产生使其易于用色谱法纯化。进一步证实纯化后的L颗粒仅能转染人肝细胞。因此，昆虫细胞表达系统是一种有吸引力的生产BNC.2的工具。用FGF - RNase融合蛋白（CL-RFN89）持续全身治疗可显著抑制体内人A431鳞状细胞癌的生长。用CL-RFN89治疗7天后，与对照组小鼠相比，肿瘤生长抑制率为58.2% （p<0.001）。此外，使用大鼠抗小鼠CD31抗体的免疫组织化学发现，CL-RFN89治疗可减少肿瘤血管化。fitc标记的hRNase 1-hEGF内化到egfr过表达的A431细胞中。在hEGF和缺乏egfr的H69细胞预处理的A431细胞中未观察到内化。本研究设计了水母绿色荧光蛋白（EGFP）在S区c端的融合。优化c端截断，使其在Cos - 7细胞中获得足够的表达水平。制备的纳米颗粒在条件培养基和细胞提取物中通过免疫反应性、荧光和密度进行评估。我们发现在bnc中EGFP片段有两个取向，在外面显示EGFP，在里面封闭EGFP，而只有一个取向占据一个粒子，因此两种类型的bnc -EGFP都可以通过抗体亲和柱有效独立地分离。结论：该重组BNC可作为一种新型的靶向给药载体。少

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