共载基因和药物的纳米胶束逆转乳腺癌耐药治疗机理

The synchronous inhibition for pump resistance and nonpump resistance path of breast cancer cells is key to reverse effectively breast cancer multidrug resistance, this aim can be expected to achieve by using delivery carrier to synchronously deliver two pathway inhibitors and chemotherapy drug to breast cancer multidrug resistance cells. For this, on the basis of initial research, we design and synthesize HA/PLL-PBA delivery carrier, and construct HA/Bcl-2 siRNA/PLL-PBA-p-gp siRNA-DOX micelle with these properties of targeting MCF-7/DOX breast cancer multidrug resistant cells, endosome escape, gene and drug release triggered by ATP responsively, using p-gp siRNA, Bcl-2 siRNA and DOX as therapeutical gene and drug. Afterwards, we research these properties and relative mechanisms of HA/Bcl-2 siRNA/PLL-PBA-p-gp siRNA-DOX micelle loading and releasing gene and drug, cytotoxicity, cell cloning, cell migration and invasion, endosome escape, gene and drug release triggered by ATP in vitro. Meanwhile, targeting distribution property，therapeutical efficiency and mechanism of reversing breast cancer multidrug resistant, as well as toxicity of HA/Bcl-2 siRNA/PLL-PBA-p-gp siRNA-DOX micelle in MCF-7/DOX breast cancer bearing-mice are also further investigated. This study contributes to lay a foundation for solving the bottleneck problem of ineffective treatment for breast cancer multidrug resistant.

同步抑制泵耐药和不依赖泵耐药通路是有效逆转乳腺癌耐药治疗关键，但这需借助载体将两个通路抑制剂和化疗药同步递送到耐药细胞才有望实现。为此本项目在前期研究基础上，合成透明质酸修饰的聚赖氨酸-苯基硼酸（HA/PLL-PBA）递送载体，以能有效抑制泵耐药和不依赖泵耐药的p-gp siRNA和Bcl-2 siRNA及阿霉素（DOX）为治疗基因和药物，构建具有主动靶向MCF-7/DOX乳腺癌耐药细胞、内涵体逃逸和ATP响应性触发释放基因和药物特性的HA/Bcl-2 siRNA-PLL-PBA-p-gp siRNA-DOX纳米胶束；研究纳米胶束荷载、释放基因和药物特性及机理；研究纳米胶束细胞毒性、克隆形成、迁移和侵袭、内涵体逃逸、ATP响应性释放等特性及机理；研究纳米胶束在荷MCF-7/DOX乳腺癌裸鼠体内靶向分布、逆转耐药治疗效率和机理及纳米胶束的毒性；从而为解决乳腺癌耐药治疗无效的瓶颈问题奠定基础。

乳腺癌治疗耐药性的产生是影响其疗效的重要因素，而实现同步抑制泵耐药和不依赖泵耐药是逆转乳腺癌耐药治疗的有效方法。因此本项目中合成透明质酸修饰的聚赖氨酸-苯基硼酸（HA/PLL-PBA）递送载体，以能有效抑制泵耐药和不依赖泵耐药的p-gp siRNA和Bcl-2 siRNA及阿霉素（DOX）为治疗的基因和药物，构建具有特异性靶向MCF-7/DOX乳腺癌耐药细胞、内涵体逃逸和ATP响应性触发释放基因和药物特性的HA/Bcl-2 siRNA-PLL-PBA-p-gp siRNA-DOX纳米胶束，该纳米胶束可将双基因有效递送至乳腺癌细胞，并发挥效应逆转其耐药，从而最大效率发挥纳米胶束中DOX对肿瘤的杀伤效率。细胞水平实验证明，与对照组相比，HA/Bcl-2 siRNA-PLL-PBA-p-gp siRNA-DOX纳米胶束可以有效抑制耐药性乳腺癌细胞中Bcl-2及p-gp基因表达，显著降低耐药性乳腺癌细胞的增值、侵袭、迁移及克隆形成，高效杀伤耐药性乳腺癌细胞。动物水平实验证明，与对照组相比，HA/Bcl-2 siRNA-PLL-PBA-p-gp siRNA-DOX纳米胶束对荷瘤裸鼠乳腺癌的增长显示出最高效的抑制效率，随后的免疫组化实验结果进一步证实该纳米胶束并没有对实验动物的心肝脾肺肾等组织器官造成损伤，展现了良好的生物相容性。本项目构建的载体有效实现了同步抑制耐药和不依赖泵耐药的通路，显著逆转了DOX对乳腺癌耐药治疗的效果，为乳腺癌的耐药治疗提供了新的技术方法。