共载siRNA/PTX 序贯释放“双管齐下”抗前列腺癌耐药研究

Castration-resistant, chemotherapy resistance and metastasis are the key issues during the clinical treatment of prostate cancer. A sequential release PTX and Pooled siRNA delivery system was designed and attempted to resolve these issues. This delivery system was composed with a core and shell structure, modified with targeting-PSMA aptamer. PTX and PE-PEG micelles was assembled in the core of the delivery system, pooled siRNA which interfesthe expression of AR、β-Tubulin、CXCR4 genes was constituted with CaP in the shell. The core-shell structure of this delivery system facilitates the sequential release of pooled siRNA and PTX, further sensibilizationof PTX chemotherapy. The active targeting group, an aptamer targeting-PSMA which overexpressed both in primary prostate cancer and metastases could improve the tumor targeting delivery efficiency. PTX resistant LNCaP cell line was used in this study. The stability and biocompatibility of the delivery system were evaluated both in vitro and in vivo. The targeting delivery efficiency, binding and cellular uptake mechanism caused by the active targeting-PSMA apatmer was assessed. We also elaborated the molecular mechanisms of the inhibited thecastration-resistant, reduced PTX resistance and metastasis of the delivery system. The tumor-target delivery efficiency and synergistic treatment effects were confirmed both in the primary prostate cancer and metastasis animal models. We look forward to provide certain theoretical foundation for selectingand designing novel drug delivery system, and expect to provide a new therapy for solving these problems during the clinical chemotherapy of prostate cancer.

本项目拟构建一种双靶向壳核组装序贯释药的递送系统，尝试解决前列腺癌临床治疗遇到的激素耐受、化疗耐药和易转移的关键问题。构建以PE-PEG为骨架，核内包裹紫杉醇（PTX），壳层组装能分别靶向沉默AR、β-Tubulin、CXCR4三种基因的siRNA，靶向PSMA适配子修饰的递送系统（siRNA/PTX@NPs-Apt）。通过siRNA的沉默抵抗前列腺癌的激素耐受和转移；序贯释放siRNA/PTX，增敏化疗耐药；PSMA适配子靶向表达于前列腺癌原位瘤和转移瘤实现“双重靶向”。本项目以PTX耐药的LNCaP细胞系（LNCaP/PTX）为研究模型，明确构建的递送系统抵抗前列腺癌激素耐受、化疗耐药和转移的分子机制；构建移植瘤和转移瘤动物模型，从动物水平证实递送系统对前列腺原发瘤和转移瘤的靶向递送和协同效果；从分子水平阐明其抗肿瘤作用机理。期待为解决前列腺癌临床化疗中存在的相关问题提供新的治疗模式。

紫杉醇 (PTX) 是治疗前列腺癌（PCa）的一线化疗药物癌症，但大量患者在短期治疗后获得耐药性。为了开发联合疗法来克服前列腺癌的 PTX 耐药性，我们建立耐 PTX的 LNCaP细胞 (LNCaP/PTX)，并发现 LNCaP/PTX细胞表现出上皮间质转化 (EMT) 和增强转移的特征。我们揭示了β-tubulin III、AR和CXCR4 在 LNCaP/PTX 细胞中表达显着增加并直接有助于耐药性增强和 EMT特征。因此，我们开发了前列腺癌特异性膜抗原适配体 (Apt) 功能化的壳核纳米粒(PTX/siRNAs NPs-Apt)。疏水性 DSPE 形成了致密的内核包载 PTX，亲水性 Apt-PEG2K和磷酸钙 (CaP) 静电吸附 siRNA形成外壳，依次释放 siRNA 和 PTX，其中 CaP 可以触发溶酶体逃逸以确保多种 siRNAs 有效释放到细胞质中以逆转EMT并使耐药细胞重新对PTX敏感，而位于核心的PTX随后释放发挥化疗杀伤作用，以达到最佳的协同作用。 PTX/siRNAs NPs-Apt 表现出增强的肿瘤靶向能力，并在皮下和原位 前列腺癌模型中取得了卓越的疗效，且副作用最小。