多级响应性双硒交联纳米材料共载双靶点siRNA治疗三阴性乳腺癌的研究

Triple-negative breast cancer (TNBC) has the poorest prognosis, and is badly in need of therapeutic regimen. In our previous studies, the epidermal growth factor receptor (EGFR) inhibitor was specifically delivered to tumor and exhibited significant superiority on anti-tumor effects, indicating that small inference RNA (siRNA) targeting to the EGFR has shown great potential for the treatment of TNBC. However, aiming at one target with limited effects and low efficiency of intracelluar release both hinder the application of siRNA. To overcome these obstacles, “combination therapy” and “sequential response” are applied together in this project to construct a novel gene carrier, which could be triggered by “dual pH/Redox”. Diselenium(Se-Se)-containing and PEG-modified poly (2-(N,N-dimethylamino) ethyl methacrylate) (PEG- hydrazone-PDMAEMA), was chosen to be the cationic polymer in this study. Then for combination therapy, siRNA drugs targeting EGFR and BRD4, are encapsulated into nanoparticles modified with CREKA and pH-sensitive GALA. CREKA peptide helps nanoparticles for tumor targeting. Then cell-penetrating peptide GALA, which was originally protected by PEG, are exposed after the hydrazone broken in acidic environment around tumors to enhance the lysosomal escape of nanoparticles. Finally, the fast release of siRNA is mediated by the diselenium broken, which was induced by the high intracellular concentration of reduced glutathione (GSH). This project would be not only a beneficial exploration in the combination therapy of siRNA for TNBC, but also an new idea for delivering gene drugs in vivo.

三阴性乳腺癌（TNBC）预后差，临床急需治疗方案。申请者前期研究发现将EGFR抑制剂靶向递送后抑瘤效果显著，可见靶向EGFR的小干扰RNA（siRNA）对TNBC治疗具有潜力，但仍需克服单靶疗效有限、载体封装后胞内释放效率低等缺陷。本项目将“联合给药”和“多级响应”相结合，构建一种“双pH/氧化还原”级联触发的共载基因药物载体。首次将双硒键与DMAEMA单体交联制备新型材料，并以EGFR和BRD4为靶点，将两种siRNA包载于修饰有“程序式脱落”的 CREKA和GALA肽的纳米粒中。CREKA肽实现肿瘤靶向，微酸环境裂解腙键促使PEG层脱落以便入胞，并暴露出掩盖的GALA肽；入胞后溶酶体中GALA肽恢复α-螺旋，其膜致孔效应加速纳米粒逃逸；最后胞质中高浓度的谷胱甘肽诱导载体材料中双硒键断裂，实现药物快速释放。本项目既是双靶点联合用药治疗TNBC的有益探索，又为体内递送基因药物提供了新思路。

三阴性乳腺癌约占所有乳腺癌的15%，由于ER、PR、HER2受体均为阴性表达而缺乏有效的治疗药物，已经成为死亡率最高的一个亚型。本项目构建了一种新型的基于肿瘤微环境多级响应性的纳米递释系统，有望提高对三阴性乳腺癌的治疗效果。本项目通过CREKA级联GALA功能基修饰纳米材料，包载针对EGFR和BRD4蛋白两个关键治疗靶点的siRNA药物形成核-壳结构的siRNA复合物（GC-NP），平均粒径为79.45±3.77 nm，包封率为91.64±3.72%，具有良好的体外稳定性（保护siRNA不被酶解）和氧化还原敏感（适合在肿瘤微环境触发释药）的特性。载药系统可显著促进三阴性乳腺癌MDA-MB-231细胞对siRNA的摄取和体内肿瘤靶向递送、GALA肽加速纳米粒的溶酶体逃逸，有利于靶向基因的沉默。体外细胞实验显示GC-NP显著提高了转染效率，具有较强的抑制细胞增殖、横向迁移和侵袭能力；联合给药不仅沉默EGFR和BRD4两个关键蛋白，还协同抑制了PI3K、p-PI3K、Akt、p-Akt、BRD4和c-Myc等相关蛋白，有利于发挥最佳的协同增效作用。荷瘤小鼠的体内药效学评价也证实了GC-NP表现出明显的肿瘤靶向性，具有最强的抑瘤效果和较高的安全性，同时实现“联合给药”、“靶向递送”和“触发释药”，提高了对三阴性乳腺癌TNBC的治疗效果，今后还有望用于其他基因类药物的体靶向递送。