一种光/酶双重敏感的“特洛伊木马”式肿瘤靶向递药系统的构建与评价

To solve the poor effect of tumor-targeted drug delivery system (DDS) on tumors lacking EPR effect and tumorous deep regions, a trojan horse-like DDS was designed. Mononuclear cells with cRGD peptides on the cell membrane surface (MCRGD) were utilized as the “trojan horse” and gold nanorods (GNRs) and DOX prodrugs were loaded into them. This DDS could escape the monitoring of MPS and be targeted to the tumor microvascular wall. MCRGD crossed the endothelial cell wall and migrated to the necrotic center by a chemo-attractive gradient. When drug loaded MCRGD penetrated into tumor tissues, the intracellular GNRs generated ROS. Lysosome membranes and MC membranes were damaged by ROS, hence GNRs and prodrugs were released from the leaking MC cells. The active DOX was co-actived by 2 kinds of enzymes which were specifically expressed in tumor microenvironment. The photo/enzyme dual-sensitive targeting strategy can significantly improve the tumor targeting and the safety to normal tissues of drugs. Then, the combination of the chemotherapy of DOX with the photodynamic therapy of GNRs offered synergistic effects against the tumor cells. The method of preparation and purification, physicochemical property, drug release behavior, and safety of this GNRs/HAD@MCRGD DDS will be studied. Specific emphasis is placed on exploring the chemotactic responsiveness, the light responsiveness, the enzyme responsiveness, the tumor-targeting in vitro and in vivo, and the antitumor efficiency of it. The implementation of this project will provide a valuable exploration to improving the level of tumor targeting therapy.

为解决靶向递药载体对EPR效应缺失肿瘤与肿瘤深层病灶疗效差的难题，设计了一种“特洛伊木马”式靶向DDS，以表面修饰RGD环肽的单核细胞（MCRGD）为“木马”，内含金纳米棒（GNRs）与阿霉素（DOX）前药。该DDS可逃避MPS吞噬并靶向至肿瘤血管壁；MCRGD受肿瘤分泌的趋化因子作用穿透血管壁并向肿瘤病灶中心迁移；当MCRGD深入肿瘤后，内部的GNRs在激光照射下产生活性氧（ROS），ROS使MC细胞膜泄漏，释放出抗肿瘤前药与GNRs；前药在2种肿瘤特异性酶的共同作用下激活为原药。这种光/酶双重敏感靶向策略可显著提高药物对肿瘤的靶向性和对正常组织的安全性；利用药物的细胞毒性与GNRs的PDT作用深层次联合治疗肿瘤。拟对该靶向DDS的制备纯化、理化性质、释药行为、安全性等进行研究，重点探索其趋化作用、光、酶响应性，体内外靶向性及抗肿瘤功效。本课题实施将为提高肿瘤靶向治疗水平提供有益探索。

利用AAN肽将DOX与HA共价结合，制备前药HA-AAN-DOX（HAD）；制备吸收峰800 nm左右的纳米金棒（GNRs）及其PEG修饰产物（PEG-GNRs）。制备膜融合脂质体cRGD-MFL，与小鼠单核细胞共孵育得到表面修饰cRGD的单核细胞MCRGD。再将PEG-GNRS，HAD与MCRGD共孵育得同时包载GNRs与HAD的GNRs/HAD@MCRGD。稳定性实验表明GNRs/HAD@MCRGD在12h内不会发生药物泄露。GNRs/HAD@MCRGD在无红外激光照射下与RAW巨噬细胞共培养，不会对RAW产生毒性也不受RAW吞噬。GNRs/HAD@MCRGD在1200mw/cm2的红外激光（808nm）照射3min后，可产生明显的ROS。倒置荧光显微镜显示红外激光照射不仅使HAD从MCRGD细胞中释放，且释放的HAD可再次被肿瘤细胞摄取。酶响应释放试验表明在pH7.4的PBS中DOX几乎不能从HAD中释放；而在同时含有HA酶与Legumain酶的pH6.5的PBS中，DOX快速释放，24h累积释放78%。肿瘤细胞杀伤性实验结果表明，HAD@MCRGD、GNRs@MCRGD、GNRs/HAD@MCRGD在无红外激光照射时，对细胞均无明显杀伤作用；GNRs@MCRGD在辅以红外激光照射时，具有一定的肿瘤细胞杀伤力；GNRs/HAD@MCRGD在辅以红外激光照射时，杀伤性显著好于DOX阳性对照组与GNRs@MCRGD+红外激光照射组。共聚焦显微镜断层扫描肿瘤细胞球显示GNRs/HAD@MCRGD有较强的肿瘤内部渗透能力。HeLa荷瘤裸鼠的肿瘤生长曲线和抑瘤率结果显示，GNRs/HAD@MCRGD+照射组抑瘤率显著高于DOX对照组。安全性试验证明GNRs/HAD@MCRGD对正常细胞与巨噬细胞没有毒性。本课题成功构建了一种以表面人工修饰cRGD肽的单核细胞为载体，同时包载GNRs与阿霉素前药HAD的新型递药系统GNRs/HAD@MCRGD，在体内外表现出良好的肿瘤靶向性与抑瘤功效，毒副作用相比阿霉素盐酸盐有显著降低，是一种前景广阔的抗肿瘤药物递药载体。