基于肿瘤免疫-基因联合治疗策略构建的靶向仿生纳米体系抗转移性黑素瘤研究

Malignant melanoma (MM), which is not sensitive to radiotherapy or chemotherapy, is highly invasive and easily metastatic with poor prognosis. Immunotherapy and gene therapy are expected to be effective treatment methods for metastatic MM. In this study, we aim to construct a targeted co-delivery biomimetic nano system siRNA-Dexo-(PD-L1 mAb) for immuno-gene therapy according to the immunotolerance and metastasis mechanism of MM, based on biologically exosomes derived from dendritic cell (Dexo) with unique immunity and drug loading functions. Without the introduction of other exogenous vectors and immunological adjuvants, we integrate three key strategies including “effective activation and enhance of immune effects of T-cell with Dexo, relieve the immune inhibitory state of tumors by blocking PD-1/PD-L1 pathway through PD-L1 mAb as well as suppress MM metastasis from the source by gene silencing (BRAF siRNA)” simply and skillfully, to achieve “multiple breakthroughs” and procedural boosting of the body's ability to kill and identify MM Cells, which is expected to effectively suppressing invasion and transfer of MM. Meanwhile, we will study the properties of targeted co-delivery biomimetic nano system, such as targeted drug delivery, immunomodulation and inhibition of invasion and metastasis as well as synergistic mechanisms of restoring and activating body’s anti-tumor immunity and regulating invasion and metastasis of MM from multiple levels. This study can provide new strategies and ideas for the biological comprehensive therapy of MM and other tumors.

恶性黑素瘤(MM)对放化疗均不敏感，极易侵袭转移，预后很差。免疫治疗和基因治疗可望成为转移性MM的有效治疗方法。本课题拟针对MM的免疫耐受和转移机制，基于生物来源的具独特免疫和载药功能的树突状细胞外泌体(Dexo)，构建免疫-基因治疗联用的靶向共递送仿生纳米体系siRNA-Dexo-(PD-L1 mAb)。在不引入其它外源性载体和免疫佐剂前提下，简单巧妙地融入“用Dexo有效激活和增强T细胞免疫效应、通过PD-L1 mAb阻断PD-1/PD-L1途径解除肿瘤免疫抑制状态、用基因沉默方式(BRAF siRNA)从根源上抑制MM转移”等三个关键策略，实现“多点突破”，程序性放大机体对MM细胞的杀伤和识别能力，有效抑制MM侵袭转移。并从靶向给药、免疫调节、抑制侵袭转移等多层级研究其恢复和激活自身抗肿瘤免疫及调控MM侵袭转移的协同作用机制，为MM及其他肿瘤的生物综合治疗提供新策略和思路。

本课题首先在诱导DC细胞成熟的基础上，提取了生物来源的具有独特免疫功能和载药功能的成熟树突状细胞外泌体（mDexo），并以兼具有靶向作用和免疫治疗效果的免疫调节剂PD-L1单克隆抗体（PD-L1）对其进行特异性修饰，得到一种兼具靶向性和免疫功能的仿生递送载体PD-L1-mDexo。然后通过电穿孔技术将基因药物siRNABRAF导入进PD-L1-mDexo内腔里，构建出靶向共递送免疫-基因治疗药物仿生纳米体系PD-L1-mDexo-siRNABRAF。采用该最佳制备工艺制得的PD-L1-mDexo-siRNABRAF外观圆整，平均粒径为117.39 ±15.6 nm，Zeta电位为-6.9 ±1.4 mV。体外实验证明其对siRNA的保护作用较强，注射入体内后siRNABRAF不易被血液中的RNA酶降解，且溶血率低于5%，生物相容性良好。PD-L1-mDexo-siRNABRAF组的细胞存活率显著低于游离药物组（PD-L1和siRNABRAF）和单载制剂组（PD-L1-mDexo和mDexo-siRNABRAF），另外，siRNABRAF、mDexo-siRNABRAF和PD-L1-mDexo-siRNABRAF制剂的IC50分别为771.9、529.5和173.5 nM，PD-L1-mDexo-siRNABRAF组的IC50值显著低于其他两组，以上结果均说明我们制得的PD-L1-mDexo-siRNABRAF具有最强的黑色素瘤细胞杀伤作用，达到了预期目标。体内实验结果证明PD-L1-mDexo-siRNABRAF组的肿瘤体积显著低于生理盐水组、游离药物组和单载药组的肿瘤体积（P<0.05）；其相对肿瘤增殖率也最低，且显著低于其他各组（P<0.05），由此说明，PD-L1-mDexo-siRNABRAF的体内抗黑色素瘤效果最佳，可以显著的降低黑色素瘤的增殖。体内安全性结果显示，所有制剂均对小鼠血常规各指标无影响，除PD-L1组具有明显的肝脏和肾脏毒性外，其他各组均对小鼠肝脏和肾脏功能没有影响，且我们制得的PD-L1-mDexo-siRNABRAF的各肝脏指标最低，最接近未荷瘤小鼠组，由此说明其体内安全性较高。因此，我们构建的靶向共递送免疫-基因治疗药物仿生纳米体系PD-L1-mDexo-siRNABRAF对黑色素瘤具有良好的治疗效果，且安全性高。