ASGPR介导的pH敏感型肝癌靶向载药磁性脂质纳米粒的构建及其对肝癌MRI可视化靶向治疗的研究

Targeting therapeutics and real-time diagnosis is crucial for hepatocellular carcinoma (HCC) therapy. A novel targeting drug delivery system for hepatocellular carcinoma – ASGPR mediated pH-sensitive magnetic lipid nanoparticles loading doxorubicin will be constructed and its potential for MRI visualization combined with targeted therapy on hepatocellular carcinoma will be explored. In this study, doxorubicin (DOX), used as a model drug, is conjugated to the molecular imaging probes superparamagnetic iron oxide nanoparticles (SPIO) by acid sensitive hydrazone linkage via glutamic acid (Glu) to construct the pH sensitve drug loaded magnetic nanocarriers (DOX-Glu-SPIO). And then, DOX-Glu-SPIO is coated with lipid to improve their in vivo stability. Finally, asialoglycoprotein receptor (ASGPR) tageting ligand DSPE-PEG2000-Lf (Lf：Lactoferrin) is incorporated onto the surface of the magnetic drug loaded lipid nanoparticles. So that the targeted drug loaded magnetic lipid nanoparticles (DOX-Glu-SPIO@Lipid-Lf) could deliver both DOX and SPIO into the hepatic tumor sites with ASGPR mediated endocytosis. And DOX and SPIO might also be released from the nanocarriers via pH-triggered bonds hydrazone in the acidic environment of the hepatic tumor cells after accumulation. The novle drug dilevery system is expected to achieve anticancer targeting therapy combined with real-time monitor under MRI visualization on hepatocellular carcinoma. Then, self-assembly properties and characteristics of the DOX-Glu-SPIO@Lipid-Lf are studied. And the in vitro and in vivo targeting efficiencies and the antitumor effect for liver cancer therapy are also evaluated. Furthermore, the ability of MRI visualization combined with targeting therapy and its function mechanism on HCC will be investigated. Therefore, the research design and implementation might provide a brand new method for the development of targeting drug delivery system for MRI visualization combined with targeted therapy on hepatic cancer.

肝癌靶向治疗及实时疗效监测是肝癌治疗的关键。本项目拟利用谷氨酸（Glu）为桥梁，将化疗药物阿霉素（DOX）通过pH敏感的腙键与分子影像探针超顺磁性四氧化三铁纳米粒（SPIO）共价结合，形成载药磁性纳米粒。纳米粒外层被脂膜包被，可进一步提高纳米载体稳定性，并利用膜表面插入的无唾液酸糖蛋白受体（ASGPR）靶向分子DSPE-PEG2000-Lf（Lf：Lactoferrin）在ASGPR受体介导下，实现纳米粒（DOX-Glu-SPIO@Lipid-Lf）对化疗药物与分子探针的靶向联合递送。纳米粒的腙键在肿瘤酸性环境下断裂，释放DOX与SPIO，以期同步实现肝癌化学药物靶向治疗与实时MRI成像监测。并表征纳米粒的理化性质，评价其体内外肿瘤靶向性和抑瘤效应，探讨纳米粒在肝癌MRI可视化靶向治疗中的应用价值及其靶向诊断治疗一体化的作用机制。本项目的研究成果将为肝癌靶向诊断治疗一体化研究提供新的思路。

肝癌靶向治疗及实时疗效监测是肝癌治疗的关键。本项目成功构建了pH敏感的载药磁性纳米粒（DOX-CA-SPIO）和肝癌靶向长循环脂质体（Lf-PLS）传递系统。并将传递系统Lf-PLS装载化疗药物DOX与MR对比剂SPIONs，探索了其在肝癌靶向诊疗应用的可行性。装载DOX的Lf-PLS，在透射电镜下观察为圆形或类圆形囊泡，粒径约100 nm，Zeta电位约-10 mV，包封率 > 90%。装载SPIONs的Lf-PLS，为圆形或类圆形粒子，粒径为（138 ± 2）nm，PDI为0.131 ± 0.019，Zeta电位为 （-23.8 ± 0.3）mV，包封率为（93.88 ± 3.21）%。磁学性能评价结果表明， Lf-PLS具有较好的顺磁性，其磁饱和强度为92.04 emu/g Fe。体外MRI成像结果表明，Lf-PS的弛豫率为611.3 mM-1s-1。激光共聚焦显微镜与流式细胞术结果表明，ASGPR阳性细胞HepG2对Lf-PLS的摄取显著性高于PLS和BSA-PLS；ASGPR阴性细胞NIH 3T3对三种载体的摄取无显著性差异。载体的靶向机制可能在于，Lf与HepG2细胞膜表面ASGPR进行特异性识别，Lf-PLS通过ASGPR受体介导的内吞作用主动转运药物进入细胞。MTT结果显示，载DOX的Lf-PLS对HepG2、BEL7402、SMMC7721等ASGPR阳性表达肝癌细胞的抑制作用显著性优于未被修饰的PLS。小动物活体成像结果发现，与PLS相比，Lf-PLS在肿瘤部位的蓄积显著性高于PLS。药效学实验结果表明，Lf-PLS的肿瘤抑制效果显著性高于PLS组和游离药物DOX组（P<0.05）。本项目实施期间，培养本科生5名，发表高质量学术论文4篇，其中SCI论文1篇（IF：4.383，他引次数：11次），中文核心期刊论文3篇。指导药学专业本科生获得校级科研立项3项。本项目的研究成果，为肝癌主动靶向给药系统与靶向分子影像探针的研发提供新的设计思路，为探索MRI可视化靶向治疗的研究提供了理论指导，对肝癌靶向诊断治疗一体化的研究与发展具有潜在的应用价值。