靶向性能量阻滞剂3-溴丙酮酸乙酯纳米粒介入治疗肝癌的实验研究

Transarterial chemoembolization therapy is an important method for interventional therapy of patients with unresectable liver tumor. Unfortunately, the defect of this therapy is that hypoxia of hepatocellular carcinoma (HCC) after embolization stimulates the remnants of HCC to growth and induces the overexpression hexokinaseⅡ(HK-Ⅱ) to improve the energy supply of the tumor cells, promote angiogenesis, and accelerated tumor invasion and metastasis. Literature and our experimental studies have shown that 3-bromopyruvate is a alkylating agent, which is a decomposition product of ethylbromopyruvate, 3-bromopyruvate can target HCC glycometabolism and energy metabolism by blocking HK-Ⅱpathway; at the same time, the inhibition of glyceraldehyde-3-phosphate dehydrogenase by 3-bromopyruvate enhance the sensitivity of tumor cells to chemotherapeutic agents owing to overcome tumor cells autophagy protection, eventually induce apoptosis. However, the current study also proved that 3-bromopyruvate is very unstable quality, insufficiently intake by tumor cells and inadequate targeting property in the vivo. On the basis of preliminary studies, we intend to create integrin ligands peptide-modified ethylbromopyruvate nanoparticles that maximize the efficacy of combined interventional treatment by improved the stability of the chemical structure and specially targeting in vivo. Molecular probe technology related to PET-CT, imageing , pathology and biochemistry methods are applied to assess the efficacy and explore its therapeutic mechanism of liver cancer in rat model. For the methodological innovation of the liver cancer treatment, this study may provide a new method for the combined interventional treatment.

介入栓塞术是治疗中、晚期肝癌的重要手段，但现有的研究证明：栓塞后可致肿瘤缺氧，诱导己糖激酶活化致线粒体供能，促新生血管形成等，短期内可致肿瘤复发。 文献和我们的实验研究表明：3-溴丙酮酸（3-溴丙酮酸乙酯的分解产物）是一种强效的能量阻滞剂，可选择性阻断己糖激酶Ⅱ途径，抑制肿瘤细胞ATP产生。同时，抑制甘油醛-3-磷酸脱氢酶，克服肿瘤细胞自噬保护，增强肿瘤细胞对化疗药物敏感性，最终诱导细胞凋亡。但3-溴丙酮酸在体内极不稳定，存在肿瘤细胞摄取不足、靶向性差等问题。 我们拟在前期研究的基础上，制备整合素配体偶联的3-溴丙酮酸乙酯纳米颗粒，使其在体内化学结构稳定，靶向性强，在与介入栓塞治疗结合后发挥最大疗效；并通过影像学及PET-CT分子探针技术、病理学和生物化学等手段评价其对大鼠肝癌的疗效，探索治疗机理。本研究为肝癌治疗方法学上的创新，有可能为肝癌综合性介入治疗提供一条新途径。

肝动脉栓塞是治疗中晚期肝癌的重要方法之一，目前临床试验表明：肿瘤往往栓塞不彻底致侧支血管建立、缺血缺氧促进血管内皮生长因子高度表达等，致肿瘤难以完全坏死。.能量阻滞剂3-溴丙酮酸是一种小分子烷化剂，丙酮酸盐类似物，通过抑制肿瘤细胞的糖酵解来抑制肿瘤细胞ATP产生，从而使肿瘤细胞凋亡。温敏纳米凝胶是一种新型液体栓塞材料，可作为药物的载体靶向输送药物并原位凝胶化缓释药物，提高局部药物浓度，减少毒副作用。基于以上理论，本实验第一步合成3-溴丙酮酸温敏纳米凝胶（3-BP-PIB）载药体系，检测不同浓度下载药体系物理表征。实验结果：当3-BP浓度为≤25mg/ml时，能在人体温附近（33-35℃）发生相转变，流动性佳，可作为肝动脉栓塞载体。第二步兔VX2肝癌动物实验，经肝动脉栓塞治疗兔VX2肝癌，14天后复查显示：3-BP-PIB栓塞效果（肿瘤体积、肿瘤生长率）与空白温敏纳米凝胶组（PIB）、3-溴丙酮酸组（3-BP）、生理盐水组（NS）有显著差异，与经典阿霉素碘油组（DOX-DY）无显著差异，但两者肝功能指标（ALT、AST）有显著差异。以上结果表明：3-BP-PIB经肝动脉栓塞化疗可有效抑制肿瘤的增长，较3-BP灌注化疗和空白PIB栓塞治疗肝癌效果佳；与经典DOX-DY相比，3-BP-PIB抑制肿瘤生长的效果与DOX-DY相当，但对肝脏毒性小。.CT灌注成像显示，肿瘤治疗前主要为肝动脉供血，栓塞治疗后肿瘤血液重新分配，栓塞术后3天，3-BP-PIB组、PIB组、DOX-DY组动脉血流（AF）值明显减小，术后7天及14天，3-BP-PIB组及DOX-DY组AF值继续下降，而PIB组AF值较术后3天有所回升。联合病理实验，肿块边缘高灌注区见存活的肿瘤细胞；低血流灌注部位病理示肿瘤细胞坏死。.以上实验表明，基于3-溴丙酮酸可以阻断肿瘤能量代谢的特性，结合可载药的新型栓塞材料，将两者有机的结合起来治疗肝癌，既可以起到靶向药物治疗的效果，使肿瘤部位持久高浓度药物释放，同时栓塞剂栓塞牢固，可以长久遏制肿瘤血管的再通。其为一种新的联合治疗方法，有望为肝癌综合治疗提供一条新的途径。