复合机制介导自组装共输送药物传递载体的构建及其机制研究

Based on the important question of designing active target drug delivery carrier arised from low response of tumor microenvironment and low selectivity of organize tageting, the aim of this project is to construct a new type of multi-mechanism mediated and tumor microenvironment response and self-assemble co-delivery drugs carrier, in which gene drug with inversion muti-drug resistance(MDR) of tumor and chemotherapeutic drug are carried. A new type of anti-EphA10 monoclone antibody with target recognition specificity and proliferation inhibition is used to modify the carrier. Under the condition of increasing co-delivery efficency of drugs in carrier, the in vivo-process of the carrier with target recognition specificity, singnal transduction interruption, pH-trigger PEG broken, electric charge reversed and inversion of MDR are realised and the effectiveness of targeted therapy is improved. The project is also to research and investigate the machanism of carrier transport, cells targeting, endocell matabolism and tumor inhibition. With applying the methods of pharmaceutics, cytobiology, molecular biology and oncology, the relationship among co-delivery carrier of structure, properties and function will be investigated. Besides, the mechanism of inversing MDR of tumor will also be investigated. We hope we can reach the aim of sciencific evaluating, exploring regularity, revealing entitative of the co-delivery carrier in a strong sicentific and highly neoteric style.

基于目前肿瘤主动靶向药物传递载体设计中面临的由于载体在肿瘤微环境响应能力不强和组织选择性不理想而导致的药物递送效率不高的重要问题，本课题旨在构建一种新型复合机制介导自组装共输送药物传递载体，同时运载具有逆转肿瘤多药耐药(MDR)的基因药物和化疗药物。采用新型只针对肿瘤细胞兼具特异识别和增殖抑制作用的抗EphA10单克隆抗体（Ep10mAb）修饰载体，在提高载体对共输送药物传递效率的同时，实现该载体在体内特异性靶向识别、信号传导阻断、pH触发PEG链断裂致电荷反转、逆转肿瘤多药耐药从而提高肿瘤靶向治疗效果。研究和探讨载体转运、细胞靶向、胞内代谢及肿瘤抑制的机制。采用药剂学、细胞生物学、分子生物学、肿瘤学等手段，开展共输送药物传递载体结构、性质和功能三者的关系及逆转肿瘤MDR机制的研究，以期达到科学评价、探索规律、揭示本质的目的，具有很强的科学性和新颖性。

基于目前肿瘤主动靶向药物传递载体设计中面临的由于载体在肿瘤微环境响应能力不强和组织选择性不理想而导致的药物递送效率不高的重要问题，本课题构建了一种新型复合机制介导自组装共输送药物传递载体，用于逆转肿瘤多药耐药的治疗。合成一种新型具有肿瘤微环境响应性的pH敏感胆固醇衍生物PEG-BIS-Chol，通过1H NMR对化合物结构进行表征，证明了目标的产物的合成；成功构建新型EphA10抗体修饰的pH敏感基因传递载体EphA10-PEG2000-BIS-Chol/Liposomes/MDR1-siRNA(EPSLR），结果表明载体的粒径与Zeta电位随着质量比（DOTAP/siRNA, w/w）改变而变化，当质量比为15时载体的平均粒径为180±15nm，Zeta电位为接近零。siRNA可以与载体紧密结合，并能明显提高siRNA在血清中的稳定性。包封率考察表明siRNA包封率较高，实验组均在80%以上。细胞毒实验结果表明所制备的载体与阿霉素共同作用后可使阿霉素对耐药细胞的细胞毒增强; 采用流式细胞术和激光共聚焦（CLSM）进行细胞摄取实验测定，结果表明经EphA10修饰后的载体靶向性明显增强并且具有溶酶体逃逸功能。Western blot实验考察EPSLR入胞后基因沉默效率，结果表明EPSLR具有显著的MDR1基因沉默效果；以DIR染料作为荧光探针采用荷瘤小鼠体内活体成像技术测定脂质体在体内分布结果表明，EPSLR具有显著的肿瘤靶向效果，且在心脏中几乎无荧光。建立了H22荷瘤小鼠模型，并以此模型研究了各组制剂的抑瘤效果。体内药效学实验结果表明， EPSLR联合阿霉素的体内抑瘤效果明显强于阿霉素溶液以及其他制剂组。