CD147ab介导联合PD-L1化疗/免疫协同ROS敏感型智能肿瘤靶向递送系统

It is well known that the concentration of the reactive oxygen species (ROS) in tumor tissues is much higher than that in normal tissues. It has been reported that boric acid ester compound could respond to the ROS concentration. Besides, it has been demonstrated that chemotherapeutic agent doxorubicin (DOX) could enhance the tumor intracellular ROS concentration level. In this work, a multi-functional targeted nano delivery system is developed for the co-delivery of functional siRNA and DOX, which is based on phenylboronic acid pinacol ester (BAPE) grafted chitosan wherein CD147ab is selected as target agent. The combination of DOX and ROS-responsive section can simultaneously enhance ROS signal and elicit ROS-triggered intracellular drug release. Functional PD-L1 siRNA is used to down-regulate the expression of PD-L1 in tumor cells to arouse and improve self-immune system, enhance the identification of tumors for further immunotherapy via the immune response of T cells against tumor. It is respected that the combination of chemotherapy and gene therapy can simultaneously inhibit the growth and metastasis of tumors. In this study, the tumor model in situ including the fluorescence self-luminous breast tumor model (Luc-4T1) and non-small cell lung tumor model (Luc-A549), and the in vitro 3D tumor spheroids model are developed to investigate the penetrating activity of the drug delivery system and evaluate the antitumor effect.

课题依据肿瘤细胞活性氧（reactive oxygen species， ROS）浓度高于正常组织，且硼酸酯类化合物对ROS浓度具有敏感性，联合化疗药物阿霉素（DOX）升高肿瘤细胞ROS的特性，以CD147ab为靶头，构建壳聚糖偶联苯硼酸酯（Benzeneboronic acid pinacol ester）实现ROS信号放大并触发释药的主动智能纳米靶向递药系统。同时，通过siRNA降低肿瘤细胞内的PD-L1表达水平，唤醒并提高机体自身的免疫对肿瘤的识别，维持T细胞抗肿瘤免疫应答发挥免疫治疗作用，实现化疗与基因联合抑制肿瘤增殖与转移。同时，采用荧光自发光乳腺癌细胞（Luc-4T1）和非小细胞肺癌（Luc-A549）等原位瘤模型，以及体外构建肿瘤3D模型评价纳米粒对肿瘤细胞的穿透机制及其药效学行为规律。

利用肿瘤组织所呈现的与正常组织活性氧浓度的差异，设计环境响应性纳米给药系统并对其进行靶向修饰，使其靶向递送至肿瘤并在肿瘤细胞内定点释放化疗药物与PD-1/PD-L1相关免疫基因，实现化疗的同时，改变肿瘤免疫特性，实现了化疗与基因协同抑制肿瘤增殖与转移的效果。本项目通过科学的分子设计，构建多种靶向智能纳米药物递送系统，主要包括：1）以羧甲基壳聚糖为骨架，修饰ROS敏感疏水性苯硼酸频哪醇酯构建两亲性聚合物羧甲基壳聚糖-苯硼酸频哪醇酯(CMCh-BAPE)聚合物，以阿霉素（Doxorubicin, Dox）为模型药物，并修饰CD147ab单克隆抗体 （CD147-CMCh-BAPE） 制备 ROS/pH 双重敏感型智能靶向型药物递送系统；体内外实验结果表明通过该药物递送系统的被动靶向、物理化学靶向和单抗介导的主动靶向作用实现了降低非靶器官毒性的同时增加了抗肿瘤疗效的效果。2）以ε-聚赖氨酸（ε-poly-L-lysine，PLL） 为骨架，连接活性氧ROS敏感基团缩硫酮（Thioketal, TK） 和顺乌头酸（Cis-aconitate, CA）自组装成CPT-NPs纳米粒，通过静电吸附作用实现对siPDL1 和siFGL1的同步负载。本课题构建的 ROS 敏感型纳米粒与肿瘤穿透肽 iRGD 联合应用后，能显著提高纳米粒递送效率，并且作为一种新型安全、高效的基因递送载体，能靶向肿瘤部位递送 siFGL1 和 siPD-L1，重新激活 T 细胞杀伤功能，为肿瘤免疫治疗提供一种新思路。.按照计划目标，我们完成了以上两个课题的载体构建，同时揭示了靶向纳米聚合物在分子、细胞、组织和整体动物水平上的摄取，转移和定位释放的机理及药效学评价。利用肿瘤微环境构建设计环境响应型纳米给药系统，并向肿瘤靶向递送化疗药物与免疫相关基因，实现了二者的协同抗肿瘤作用，为肿瘤的治疗提供了一种有前景的策略。