胞内还原环境响应型双靶点联合抗肿瘤治疗系统的构建及其抗肿瘤功效研究

The aim of the project is to build an anti-tumor combination therapy system based on dual targets for the suppression of proliferation and metastasis of malignant tumors. Hydrophobic anticancer drug paclitaxel (PTX) is conjugated to hyaluronic acid (HA) via bioreducible linkages (HA-ss-PTX) and PTX-loaded HA-ss-PTX micelles could be prepared by a simple amphiphilic self-assemble method. Since over expression of HA-binding receptors, such as CD44, has been found on the cell surface of several malignant tumors with high metastasis, receptor-mediated tumor targeting potential of HA-ss-PTX micelles could significantly enhance drug efficacy. Moreover, Esbp peptide (CDITWDQLWDLMK-CONH2) modified HA-ss-PTX conjugates (Esbp-HA-ss-PTX) are developed for active targeting tumor vascular endothelial cells via E-selectin and thus serve to induction of tumor vascular endothelial cell apoptosis, inhibit tumor angiogenesis and blocking tumor invasion and metastasis. The therapy system combined HA-ss-PTX and Esbp-HA-ss-PTX micelles has desirable characteristics, such as high safty, low toxicity, efficient drug-loading, targeted drug delivery and multiple mechanisms to inhibit tumor invasion and metastasis. The project focus on the design, redox-sensitivity evaluation, in vivo and in vitro anti-tumor effects and mechanism of inhibition of tumor invasion and metastasis.

本项目针对恶性肿瘤增殖及转移的关键问题，设计构建一种双靶点联合抗肿瘤治疗系统。通过含二硫键的连接臂将疏水性抗肿瘤药物紫杉醇（PTX）与靶向肿瘤细胞表面CD44受体的天然多糖透明质酸（HA）偶联，再物理负载PTX，构建主动靶向杀伤肿瘤细胞的胞内还原环境响应型纳米递释系统（HA-ss-PTX）。在此基础上，以Esbp肽（CDITWDQLWDLMK-CONH2）修饰HA-ss-PTX（Esbp-HA-ss-PTX），利用Esbp肽的导向功能在粘附分子E-选择素的介导下实现肿瘤血管内皮细胞靶向，从而诱导肿瘤血管内皮细胞凋亡、抑制肿瘤血管生成和阻断肿瘤转移。HA-ss-PTX 联合Esbp-HA-ss-PTX纳米胶束二元治疗系统具有安全低毒、高效运载、靶向释药及多机制抑制肿瘤侵袭转移等特点。项目将重点围绕该系统的构建、还原敏感性评价、体内外抗肿瘤作用、抑制肿瘤侵袭转移效果及机制展开研究。

本项目针对恶性肿瘤增殖及转移的关键问题，设计构建一种双靶点联合抗肿瘤治疗系统。通过含二硫键的连接臂将疏水性抗肿瘤药物紫杉醇（PTX）与靶向肿瘤细胞表面CD44受体的天然多糖透明质酸（HA）偶联，再物理负载PTX，构建主动靶向杀伤肿瘤细胞的胞内还原环境响应型纳米递释系统（HA-ss-PTX）。在此基础上，以Esbp肽（CDITWDQLWDLMK-CONH2）修饰HA-ss-PTX（Esbp-HA-ss-PTX），利用Esbp肽的导向功能在粘附分子E-选择素的介导下实现肿瘤血管内皮细胞靶向，从而诱导肿瘤血管内皮细胞凋亡、抑制肿瘤血管生成和阻断肿瘤转移。制备的HA-ss-PTX及Esbp-HA-ss-PTX胶束粒径在100nm左右，对模型药物紫杉醇（PTX）可实现高效负载。体外释放实验表明还原敏感型HA-ss-PTX及Esbp-HA-ss-PTX胶束具有明显的谷胱甘肽（GSH）触发式释药功能，在相对低的GSH缓冲液中（模拟血液）可以稳定存在，而在高浓度的GSH缓冲液中能够快速释放游离药物。细胞实验确证，HA-ss-PTX及Esbp-HA-ss-PTX胶束是通过HA受体介导的内吞进入肿瘤细胞，可增加对肿瘤细胞的特异性摄取，并增强体外抗肿瘤疗效；在模拟E-选择素表达的肿瘤血管内皮细胞（TNF-α诱导人脐静脉内皮细胞HUVECs）中，只有Esbp修饰后的胶束Esbp-HA-ss-PTX可被大量摄取，而HA-ss-PTX胶束摄取较少，表明Esbp与E-选择素间的相互作用为细胞摄取的主要机制。体外细胞实验同样表明，在细胞水平，还原敏感型胶束可将负载的荧光探针释放于细胞质，具有触发定位释放功能。活体成像实验证实胶束在荷瘤小鼠体内可明显蓄积于肿瘤部位，Esbp修饰后的胶束肿瘤靶向性强于未修饰胶束。药效学实验和肿瘤侵袭转移评价实验都表明，同时靶向肿瘤细胞和肿瘤血管内皮的细胞的HA-ss-PTX联合Esbp-HA-ss-PTX胶束二元治疗系统具有最强的抗肿瘤疗效和抑制肿瘤侵袭转移能力。实验结果表明，HA-ss-PTX联合Esbp-HA-ss-PTX胶束二元治疗系统是一种安全、高效的抗肿瘤侵袭转移药物传递系统。

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