NK4/甘草酸介导中药免疫脂质体肝肿瘤细胞靶向性及协同抗癌作用研究

Chinese herbs ingredients glycrrhizic acid (GA) can mediate drug targeting to the liver, but it fail to distinguish between normal and abnormal liver cells or tumor cells. c-Met is a high-affinity and specific binding receptor for hepatocyte growth factor (HGF), it is absent or expressed at low levels in normal tissues but is over-expressed in tumor cells, this molecule could be an important target for tumor cells recognition. On the basis of GA-mediated liposome liver targeted drug delivery system, this project intend to obtain c-Met specific ligand NK4 single chain antibody fragments from HGF cDNA by PCR, using NK4 as tumor cells targeted group, conjugation of NK4 and GA with the surface of liposomal nanoparticles by PEG-ylation and enzymatic catalysis reactions, construction of NK4/GA-mediated Chinese herbs immunoliposome drug delivery system, making the drug further targeted to tumor cells after accumulation in the liver tissue, so as to achieve the targeting of liver tumor cells. Meanwhile, using NK4 to block the activity of HGF/c-Met pathway, using GA to inhibit tumor cells growth and alleviate the toxicity of anti-cancer drug, drug carriers will have synergistic anti-cancer effect. NK4/GA’s dual effect of active targeting and synergistic anti-cancer will greatly improve the efficacy and reduce the toxicity of traditional Chinese medicine, it will provide us with a new technology platform for the treatment of malignant tumors.

中药成分甘草酸可介导药物靶向于肝脏，但不能区分正常细胞与异常或肿瘤细胞。c-Met是肝细胞生长因子(HGF)的特异性受体，在正常组织中c-Met 不存在或表达很低，而在肿瘤细胞则高度表达，是肿瘤细胞识别的一个重要靶点。本项目在甘草酸介导脂质体肝靶向的基础上，采用PCR技术从HGF cDNA中获取c-Met特异性配体NK4单链抗体片段，以NK4作为肿瘤细胞靶向基团，通过PEG偶联键合与酶促催化反应，使NK4与甘草酸连接于脂质纳米粒表面，构建NK4/甘草酸介导中药免疫脂质体给药系统，使药物聚集于肝组织之后进一步靶向于肿瘤细胞，从而达到对肝肿瘤细胞的靶向作用。同时，利用NK4阻断HGF/c-Met通路活性，甘草酸抑制癌细胞生长、缓和抗癌药毒性，药物载体将产生协同抗癌效果。NK4/甘草酸的主动靶向与协同抗癌双重效应将有力地改善中药药效不明显，毒副作用多等问题，为恶性肿瘤的治疗研究提供新的技术平台。

c-Met是肝细胞生长因子（HGF）的特异性受体，在正常组织中c-Met不存在或表达很低，而在肝癌细胞上高度表达，是肿瘤细胞识别的一个重要靶点。本项目以Anti-HGF antibody为原料，合成NK4基因（1341bp），并构建原核表达载体，经纯化后进行活性检测，NK4保留了其生物学活性。同时，基于肝细胞膜表面存在甘草次酸受体，中药甘草中的有效成分甘草酸、甘草次酸及其衍生物具有良好的趋肝性与肝细胞靶向性，因此项目分别以NK4和18-β-甘草次酸硬脂酸酯（甘草酸亭酸酯，GA）作为靶向配体，同时修饰于脂质体表面，构建NK4/GA介导脂质体给药系统（NK4-GA-LIP），使药物聚集于肝组织之后能进一步靶向于肿瘤细胞，从而达到对肝肿瘤细胞的靶向作用。研究结果表明：以羟基喜树碱为模型药物，制备的NK4-GA-LIP粒径均匀（100-200 nm），包封率可达80%以上，与羟基喜树碱注射剂相比，NK4-GA-LIP能显著提高药物的生物利用度，延长体内的滞留时间，具有明显的肝靶向性和抗肿瘤效果。NK4/甘草酸双重受体介导的脂质体主动靶向给药系统将为肝癌的治疗研究提供新的参考。