新型靶向肿瘤细胞的药物运输穿膜肽设计与跨膜运输机理分析

The study of cell-penetrating peptides (CPPs) has become one of the hot topics in the field of intracellular delivery of pharmaceutical molecules. Our research has previously characterized a new CPP, HBD, which was derived from human C-terminal heparin binding domain of EC-SOD. Based on the overexpression of the EGF receptors on tumor cell surface as well as the structure characteristics of HB-EGF and its high expression level in many tumor cell lines, we constructed a novel tumor-homing CPP (S3-HBD) by fusing HBD domain with an ErbB-binding domain (S3) derived from vaccinia virus growth factor for the intracellular drug delivery. Our previous work showed that S3 domain intensified the internalization efficiency of HBD and the targeting ability for tumor cells. This phenomenon is probably related with the synergetic effects of the cooperative adsorption access of the two functional domains with their targeting molecules, cell-surface receptors and heparin polysaccharide. In this research project, we will analyze the interaction mechanism of S3-HBD with its targeting molecules on cell surface, clarify the molecular basis of the synergetic effects, evaluate the priority differences of S3-HBD between normal cells and cancer cells, reveal the intracellular distribution and final destination of the transported drug and other properties of S3-HBD according to translocation mechanism analysis, which would provide a solid scientific support for developing S3-HBD as an efficient in vivo drug targeting delivery tool with independent intellectual property rights.

肿瘤靶向细胞穿膜肽研究是药物分子胞内运输的热点。HBD是我组发现的一种源于EC-SOD肝素结合域的人源性穿膜肽。本研究针对肿瘤细胞普遍存在表皮生长因子受体（ErbB）高表达的现象，以在多种肿瘤细胞中高表达的HB-EGF结构特征为启示，将HBD与牛痘病毒生长因子ErbB受体结合区域（简称S3）融合，构建一种新型肿瘤细胞靶向性药物运输载体（S3-HBD）。前期工作结果显示嵌合S3的HBD对肿瘤细胞的穿膜效率和靶向运输能力显著提高，推测与S3-HBD的两种结构域同细胞表面受体和肝素多糖间的相互协同吸附作用有关。本项目将通过分析S3-HBD与膜上靶位点间相互识别吸附的机制，阐明协同作用的分子基础，诠释其对正常细胞和不同肿瘤细胞间选择的差异性，通过穿膜运输机理分析揭示这种新型靶向穿膜肽转运药物分子进入胞内的定位和去向等特性，为其作为一种拥有自主知识产权的高效体内药物靶向运输工具的应用提供科学依据。

本研究针对肿瘤细胞普遍存在表皮生长因子受体（ErbB）高表达的现象，以在多种肿瘤细胞中高表达的HB-EGF结构特征为启示，将我们前期研究得到的人源性穿膜肽HBD与牛痘病毒生长因子ErbB受体结合区域（简称S3）融合，构建一种新型肿瘤细胞靶向性药物运输载体（S3-HBD）。抗体掩蔽实验和配体竞争性抑制实验研究结果证实了S3和HBD与肿瘤细胞表面的ErbB和肝素样组分专一性靶向结合的协同效应赋予了S3-HBD对肿瘤细胞的专一性穿膜活性。通过对S3的结构模拟分析和突变改造获得与受体更高结合活性的突变体ELBD，其与ErbB结合的效率比S3提高了3倍；通过对不同人源性肝素结构域的筛选，得到与ELBD有更好协同结合能力的EHB，其对HeLa细胞穿膜活性提高了约15倍。EHB与肿瘤细胞的结合和药物靶向递送能力与肿瘤细胞表面的ErbB数量呈正相关。内吞抑制剂分析揭示EHB介导的内吞主要是依赖于网格蛋白，其以内吞体进入细胞，然后释放进入细胞质或转化由内吞体转化成溶酶体被降解。EHB可大大提高MAP30的抗肿瘤活性，与单独的MAP30相比可提高20倍，但对正常细胞的毒性仍保持不变，具有很好的靶向抗肿瘤能力。荷瘤小鼠的体内实验表明，融合靶向肽S3的Apoptin-HBD抑瘤率达70%，而没有融合S3的Apoptin-HBD抑瘤率为40%，表明引入S3的药物蛋白在体内能靶向抑制ErbB过表达的肿瘤细胞生长，发挥治疗作用。本研究有望促进以ErbB为特异性靶标的肿瘤靶向治疗。

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