具靶向识别与肿瘤酸性微环境特异穿膜功能的新型药物-siRNA共传输体系的构建与评价

The overexpressed tumor cell surface GRP78 protein is one of the factors leading to cell migration, and the acidic microenvironment can further upregulate GRP78 expression. According to the above-mentioned characteristics of malignant tumors, a novel drug-siRNA co-delivery system was designed and fabricated in this project. It can selectively recognize GRP78 and has the ability of cell membrane-penetration in acidic tumor microenvironment which is mediated by pH low insertion peptide (pHLIP). First of all, the anticancer agent doxorubicin (DOX) and siRNA against pyruvate kinase M2 (PKM2) were simultaneously incorporated into nanocarriers self-assembled from pHLIP-PEI-PLA copolymer which is composed of pHLIP, branched polyethylenimine (PEI) and polylactide (PLA). Secondly, drug-siRNA-incorporated nanocarrier associated tightly with heterobifunctional polyethylene glycol (with heparin and GRP78 binding peptide conjugated at its two ends, Hep-PEG-GBP) through an electrostatic interaction between the cationic PEI and anionic heparin, obtaining the GBP-PEG-Hep/pHLIP-PEI-PLA@DOX+siRNA complex. This co-delivery system could accumulate in tumor tissue through enhanced permeability and retention (EPR) effect, specifically bind with tumor cell surface GRP78, enter cytoplasm via forming transmembrane α-helix in acidic microenvironment followed by releasing DOX and siRNA, and exert synergistic antitumor efficacy. Results of this project will provide a new strategy for effective and targeted therapy of malignant tumors, and lay the foundation for clinical application.

肿瘤细胞表面高表达的GRP78蛋白是造成细胞迁移的因素之一，而酸性微环境能增加GRP78表达。本项目依据恶性肿瘤上述特性设计并构建一种以GRP78为靶点、酸性微环境下可特异穿膜的低pH插入肽（pHLIP）介导的新型药物-siRNA共传输体系：首先以pHLIP与支化聚乙烯亚胺、聚乳酸的共聚物pHLIP-PEI-PLA为载体，同时负载抗癌药物阿霉素（DOX）与针对丙酮酸激酶PKM2的siRNA；其次通过静电相互作用与两端分别连接肝素和GRP78结合肽的PEG（Hep-PEG-GBP）相结合，构建复合物GBP-PEG-Hep/pHLIP-PEI-PLA@DOX+siRNA。该体系可通过EPR效应在肿瘤组织富集并特异结合肿瘤细胞表面GRP78，在酸性微环境下形成跨膜α螺旋直接进入细胞质，释放DOX和siRNA，发挥协同抗肿瘤功效。研究结果将为恶性肿瘤高效靶向治疗提供新策略，并为临床应用奠定基础。

传统化疗主要依赖小分子化合物，治疗效率低且伴有严重的毒副作用。新靶点的发现可为癌症治疗带来新希望，肿瘤细胞中高表达GRP78和PKM2，使得它们成为癌症治疗的新靶点。Warburg效应引起的肿瘤酸性微环境是恶性肿瘤的重要特征之一，已成为肿瘤诊断与治疗的一个新靶标。在酸性条件下，低pH 插入肽（pH low insertion peptide, pHLIP）可通过形成跨膜α螺旋将不能穿过细胞膜的物质递送至肿瘤细胞内。目前，本项目已取得的主要成果和关键数据包括以下四个方面。首先，我们以PEG为连接臂，在其两端分别连接抗肿瘤药物阿霉素和双功能多肽（由GRP78结合序列和核定位信号序列组成），合成了一种新型阿霉素原药，它可以通过识别肿瘤细胞表面过表达的GRP78受体内化进入肿瘤细胞，进而在核定位信号序列引导下进入细胞核发挥抗肿瘤活性。其次，我们首次设计并通过基因工程手段生物合成了一种Beclin 1功能蛋白Trx-pHLIP-Beclin 1（TpB），它由三部分组成：硫氧还蛋白标签（Trx）、低pH插入肽（pHLIP）和由20个氨基酸组成的Beclin 1保守序列。TpB在弱酸性环境下可通过形成跨膜α螺旋将其碳端的Beclin 1保守序列递送至肿瘤细胞内，启动自噬过程，诱发自噬性细胞死亡进而抑制肿瘤生长。再次，我们通过合成单宁酸（TA）功能化的Fe3O4纳米粒子，与细胞裂解液培养，发现PKM2是TA发挥作用的直接靶点。TA通过抑制PKM2的丙酮酸激酶活性而非其表达或蛋白激酶活性来抑制结肠癌细胞增殖。并且我们发现TA通过选择性结合至PKM2的赖氨酸433（K433）位点来诱导其四聚体解聚，进而抑制PKM2的代谢活性。最后，我们在大肠杆菌中高产率可溶表达了I型核糖体失活蛋白（RIPs）重组植物毒素Gelonin（rGel）。通过圆二色谱仪（CD）检测了rGel二级结构组成和热稳定性，发现其主要由β折叠和无规卷曲是其主要二级结构，它在60 °C以下具有很好的热稳定性。rGel可通过抑制细胞内蛋白质合成来发挥抗肿瘤活性。以上研究结果为以GRP78和PKM2为靶点以及基于肿瘤酸性微环境的新型药物递送系统的设计提供了新思路。