联合声动力、化疗与免疫疗法的多功能纳米治疗体系的构建及其靶向抗肝癌作用研究

In view that the molecular pathogenesis of hepatoma is very complex, the combination therapy of multiple methods is believed to be optimal in the clinical treatment of hepatoma. Sonodynamic therapy (SDT) can stimulate sonosensitizers to produce reactive oxygen species (ROS) to kill deep tumors by using the strong penetration of ultrasonic wave through biological tissues. The results of our preliminary experiments showed that the combination of SDT with chemotherapy could effectively inhibit the in vitro and in vivo growth of hepatoma cells and also activate the immune responses in tumor environment. Therefore, a multi-functional nano-therapeutic system is designed in this study to combine SDT, chemotherapy and immunotherapy, and exert their synergistic effects on hepatoma. Firstly, the nanocomplex, formed by π-π conjugation effect between porphyrin sonosensitizer and anthracycline agent, is encapsulated into the internal phase of cationic liposomes, and the agonist of toll-like receptor 7/8 (TLR 7/8) is loaded in the lipid bilayers of liposomes at the same time. Secondly, pH-responsive pullulan derivative is synthesized to be coated on the surfaces of above drug-loaded liposomes to enhance their in vivo security and stability, and to realize the hepatoma-targeted drug delivery. This novel nanocarrier system can produce rapid responses to the mildly acidic conditions of tumor microenvironment and endosomes, and the physical effects of focused ultrasound, thus cause the orderly releases of above three tumor therapeutic agents. ROS produced by sonosensitizer can activate the mitochondrial apoptosis pathway, anthracycline agent can inhibit the intracellular synthesis of DNA and RNA, and TLR 7/8 agonist can induce CD4+Th1 mediated immune responses in tumor microenvironment, thus is favor to exerting the optimal antitumor efficiency of combination therapy for hepatoma.

肝癌发病机制复杂，多种方法的联合是其治疗的最佳选择。声动力疗法（SDT）可利用超声波对生物组织的强穿透力，激活声敏剂产生活性氧，作用于深部肿瘤。前期研究发现SDT联合化疗能有效抑制肝癌生长，并激活宿主体内的免疫反应。为了增强这种抗肿瘤免疫能力，本课题拟构建一种多功能纳米体系，用于联合SDT、化疗与免疫疗法靶向治疗肝癌。卟啉声敏剂与蒽环化疗药物通过π-π共轭形成纳米复合物，而后包封于阳离子脂质体内水相，同时将Toll样受体7/8激动剂载于脂质双分子层，实现三种治疗剂的共载；合成pH-响应普鲁兰多糖，包裹脂质体以提高其体内安全性与稳定性，并实现靶向肝癌的药物递送。纳米体系能对肿瘤微环境与细胞内吞体的低pH条件以及超声的物理效应产生多重响应，实现三种治疗剂的有序释放，并主要通过激活肿瘤细胞线粒体凋亡通路、抑制DNA与RNA的合成、诱导肿瘤微环境CD4+Th1细胞免疫应答，实现对肝癌的联合治疗。

肝癌发病机制复杂，多种方法的联合是其治疗的最佳选择。声动力疗法（SDT）可利用超声波对生物组织的强穿透力，激活声敏剂产生活性氧，作用于深部肿瘤。我们的研究发现SDT联合化疗能有效抑制肝癌生长，并激活宿主体内的免疫反应。为了增强这种抗肿瘤免疫能力，本项目构建了能够高效联合SDT、化疗与/或免疫免疫疗法的多功能纳米体系，用于肝癌（和乳腺癌）的靶向治疗，取得的科研成果详述如下：一、以血卟啉作为声敏剂，阿霉素作为化疗药物，构建了能够高效联合SDT与化疗的纳米治疗体系，该体系结构简单，制备方法可重现，表现出显著的体内外SDT效应，能够高效联合SDT与化疗杀伤肿瘤，并间接地作用于肿瘤微环境（肿瘤血管和胶原沉积），发挥协同抗肝癌作用；二、在肝癌干细胞和乳腺癌耐药细胞中考察了该纳米治疗体系联合SDT与化疗对肿瘤多药耐药的逆转效应，结果显示纳米体系可通过高效介导声敏剂和化疗药物入胞，激活线粒体凋亡通路以及抑制P-gp活性来逆转耐药；三、探讨了基于普鲁兰多糖的免疫调节作用，发现普鲁兰多糖及其衍生物不仅具有显著的肝癌靶向性，还能够诱导人树突状CAL-1细胞的成熟与激活，其中氨基化普鲁兰多糖的激活效应最强，可以作为免疫佐剂用于抗肝癌免疫治疗；四、以普鲁兰多糖与Pluronic F68为材料开发了高度生物安全的纳米载体系统，用于靶向递送光敏剂IR780和化疗药物紫杉醇，并探讨了局部光热/光动力治疗和全身性系统化疗的序贯治疗方式对肝癌生长的抑制作用及其对肿瘤免疫耐受的逆转效应。综上可见，我们的研究为肝癌的精准与联合治疗提供了新方法与新策略。