新型巨噬细胞膜包覆的载药纳米粒子在共振能量转移诱导下用于食管癌肺转移的靶向治疗

China is a region with high incidence of esophageal cancer. Esophageal cancer with lung metastases is usually found in advanced cancer patients and, it is difficult for the treatment of the lung metastases. Based on our previous work and international research frontier, in this project, we will conjugate the mutant of the bioluminescent protein, Renilla reniformis luciferase (Luc8) with hypocrellin A (HA), a photosensitizer, to develop a bioluminescence resonance energy transfer (BRET) system. The HA-Luc8 conjugates obtained catalyzed by coelenterazine (Coe) will be used for photodynamic therapy (PDT) of metastatic cancer based on the BRET. At the same time, cis-Dichlorodiamineplatinum(II) (DDP) will be conjugated with triphenylphosphonium (TPP). The TPP-DDP will target to mitochondria in cells, and the cell killing efficiency will be improved by the reactive oxygen species (ROS) generated by the HA during PDT. Furthermore, astragalus polysaccharides (APS), HA-Luc8, TPP-DDP and Coe will be encapsulated by a pH-sensitive liposome (Lip), and further coated with macrophage membrane (MM) to develop three novel drug loaded nanoparticles. These drug-loaded nanoparticles exhibit multifunctions: immunologic escape, targeting to metastatic foci, BRET-activated PDT, inducing M2 subtype to M1 macrophages, etc., which will significantly suppress the lung metastasis of esophageal cancer through novel targeted combination therapy: “immunotherapy + PDT + chemotherapy”. .The limitations of most common chemotherapy and PDT will be overcome by the methods developed by this project. The drug-loaded nanoparticles and therapy methods developed by this project are safe and highly efficient for cancer targeted therapy, which are very important for improving the therapy level of esophageal cancer with lung metastases.

我国是食管癌高发国家，晚期食管癌易发生肺转移且治疗困难。本项目拟在我们前期研究和国际前沿基础上，率先将高稳定性荧光素酶（Luc8）与竹红菌甲素（HA）连接，获得的HA-Luc8与腔肠素（Coe）可通过生物发光共振能量转移（BRET）激发HA对转移肿瘤进行光动力治疗（PDT）；同时，合成具有线粒体靶向和克服细胞耐药的顺铂前药TPP-DDP，PDT中的活性氧将协同TPP-DDP杀伤癌细胞。进而，以巨噬细胞膜包覆的pH敏感脂质体装载黄芪多糖及HA-Luc8、TPP-DDP和Coe，构建三种协同应用的新型纳米载药粒子，发挥巨噬细胞膜免疫逃逸和肿瘤靶向性、BRET诱导PDT、药物逆转巨噬细胞表型和靶向进攻线粒体等优越性，以全新的“免疫治疗+PDT+化疗”协同靶向摧毁食管癌肺转移灶。.本项目克服了常规化疗和PDT等存在的局限性，具有安全、靶向、高效等优势，对于推动食管癌肺转移靶向治疗水平发展意义重大。

食管癌靶向治疗是肿瘤治疗领域重要课题，为增强药物对肿瘤的靶向性以及治疗效果、克服光动力治疗因外源激发光照射深度不足的缺陷，本项目制备了荧光素酶Luc8，将竹红菌甲素（HA）与Luc8连接形成了HA-Luc8复合粒子，将顺铂（DDP）和三苯基膦（TPP）连接形成了DDP-TPP复合粒子，提取分离了巨噬细胞膜（MM），采用DOPE和DSPE两种磷脂制备了pH敏感脂质体（Lip），用该脂质体分别对HA-Luc8、DDP-TPP以及黄芪多糖（APS）进行了包覆，并进一步在脂质体上修饰了MM，形成了载药纳米粒子(HA-Luc8)/Lip/MM、(DDP-TPP)/Lip/MM、(DDP-TPP, HA-Luc8)/Lip/MM和APS/Lip/MM。在考察其性质的基础上，研究了这些载药纳米粒子在尾静脉注射后对食管癌小鼠肿瘤的生长抑制效果，并研究了其生物安全性。结果表明，所构建的HA-Luc8可通过与底物腔肠素反应产生的生物发光激发HA产生活性氧，可通过该共振能量转移杀伤食管癌细胞；DDP-TPP能进入细胞线粒体，较好地杀伤食管癌细胞；所构建的由Lip/MM包覆的载药纳米粒子，均可以靶向抑制食管癌肿瘤生长，抑制食管癌细胞在小鼠肺部成瘤，将两种或三种载药粒子同时注入荷瘤小鼠体内，能更好地抑制食管癌肿瘤生长；DDP-TPP经过Lip/MM包覆后，降低了DDP对小鼠的毒性，载体Lip/MM对小鼠未显示明显毒性。本项目制备的仿生纳米粒子及治疗方法，是一个平台技术，对于提高食管癌肿瘤靶向治疗效果有重要意义，也可为其它实体瘤的靶向治疗提供参考。