Legumain调控、分步释放药物载体逆转视网膜母细胞瘤多耐药性的研究

MRP1 (multidrug resistance-associated proteins 1) mediated drug resistance is a major impediment to chemotherapy of RB (retinoblastoma). As legumain was highly up-regulated in RB, we designed a series of legumain-responsive nano-carrier using hyaluronic acid, chitosan etc. as the skeleton structure. Doxorubicin was conjugated to the skeleton materials bridged with a legumain degradable peptide (PEP) and the resultant materials were further crosslinked to form nanoparticle. This nanoparticle was further subjected to load MRP1 inhibitor with physic force forming a step-released co-delivery system. The effect of PEP composition on the profile of legumain-mediated drug release was characterized. The in vitro and in vivo bio-effect of this co-delivery system was evaluated using drug resistance cancer cell line and nude mice cancer model. Furthermore, the mechanism of this co-delivery system reversed multidrug resistance and induced cell apoptosis will be studied. This work can be contributed to development a new strategy to RB treatment.

MRP1（耐药相关蛋白1）介导的RB（视网膜母细胞瘤）多耐药性是RB药物治疗失败的主要原因之一。本研究利用RB高表达Legumain的生物学特点，选用生物相容性好的大分子聚合物（透明质酸、壳聚糖等）作为基本骨架，合成Legumain响应释放的药物载体。用Legumain的底物肽段将阿霉素桥接（化学键合）至骨架结构上，并进一步将MRP1抑制剂通过物理作用包载，形成抑制剂与抗癌药物先后释放的共包载体系。研究底物肽段氨基酸组成对共载体系体外释药性能的影响，阐明载体中肽段的氨基酸组成与Legumain释药调控的关系。研究共包载体系抑制耐药型RB细胞的效果，及逆转RB细胞多耐药性的分子机制。构建RB的裸鼠模型，通过玻璃体注射给药，研究共包载体系逆转RB实体瘤多耐药性，诱导肿瘤凋亡的机制，为探索RB药物治疗新方法奠定基础。

耐药蛋白介导的肿瘤多耐药性和药物的对正常组织的毒副作用是肿瘤化学药物治疗失败的关键因素。由于姜黄素具有调节耐药蛋白表达和抑制耐药蛋白活性的双重功能，和肿瘤高表达Legumain的生物学特性，我选取生物相容性好壳聚糖和透明质酸为基本骨架；2）通过一段Legumain能够水解的肽段将抗癌药物（阿霉素，DOX）桥接（化学键合）至聚合物骨架上，交联形成纳米粒；3）将耐药蛋白抑制剂（姜黄素）包载通过疏水相互作用包载纳米粒子内核；用于耐药型肿瘤的靶向治疗。结果表明，这种共包载体系能够显著促进药物在耐药型细胞中的富集、增加耐药型肿瘤细胞对药物的敏感性，增强药物对肿瘤细胞的抑制效果。我们制备了基于裸鼠的耐药型肿瘤模型，并通过腹腔注射的给药方式研究体系对肿瘤细胞的靶向效果和对肿瘤生长的抑制效果。结果显示，载药体系能够显著增加药物的靶向性，延长药物系统循环时间、增强肿瘤的抑制效果。免疫组化分析表明，联合载药体系能够显著压制耐药相关蛋白的表达。药物的靶向运输是增加药物的治疗效果和减少药物的副作用的一项重要措施，本项目的实施对于探索恶性肿瘤药物治疗新方法具有重要的理论和实践意义。