靶向GPC3/TAMs载药纳米微泡肝癌诊疗一体化研究

Nanoscale contrast agent targeting GPC3 will be able to enhance the ultrasound diagnostic accuracy of hepatocellular carcinoma. Anti-tumor through deleting TAMs is a new approach. Bisphosphonates can delete macrophages and anti-tumor. However, bisphosphonates work on macrophages extensively and do not act specifically on tumor-associated macrophages(TAMs), therefore, it’s necessary to adopt a new preparation to encapsulate bisphosphonates delivering for anti-tumor. We hypothsized that the drug can be ferried to the tumor through targeted nanobubbles loading with drug, ultrasound can be used to break the drug loading nanobubbles, and the drug can be released locally after the nanobubbles cracked. Materials used for preparation of shells of nanobubbles and capsule of the anti-tumor medicine are similar or the same, and the materials which use for preparation of nanobubbles can be shared and integrated for preparation of nanobubbles and bisphosphonates. This program intended to formulate a small nanosized preparation targeting GPC3 by incorporating ligand L5 peptide onto shells of nanobubbles formulated using PLGA, perfluorocarbon, and encapsulating zoledronic acid in the shell. Its physical properties, stability, biocompatibility, and targeting ability in vitro and in vivo will be determined. The evaluation of its targeting enhanced sonography and effect on deleting TAMs will be accomplished in animals with tumor xenografts of hepatocellular carcinoma. Although the preparation is targeting the cells of the hepaticullar carcinoma, the zoledronic acid being contained in it can be carried into the tumor microenvironment where the TAMs inhabited, concentration of the zoledronic acid can be elevated in milieu of TAMs, and side effects on other macrophages can be reduced greatly. Therefore, the fabricating preparation will exibit dual functions that performing targeted enhanced ultrasound for the diagnosis and deleting TAMs aiming to anti-tumor. The potentials of this program are that to fabricate a practical nanoscale targeting contrast agent to improve ultrasound imaging diagnosis and to explore a therapeutic method for hepatocellular carcinoma by deleting TAMs through delivering drug with proper preparation.

靶向GPC3纳米微泡有望提高肝癌超声诊断准确性。削减TAMs是抗癌新途径。但能削减TAMs的双膦酸盐类药物无靶向性，靶向GPC3纳米微泡需减小粒径，故需开发新型制剂以适应需要。我们设想：用靶向纳米微泡运载抗癌药物到瘤体，用超声波击碎聚集到瘤体内的载药微泡使药物在瘤体定点释放；靶向GPC3微泡造影剂与抗TAMs药物制剂整合研究开发。本项目拟用L5为GPC3受体靶向配体，以聚乳酸-聚乙醇酸为微泡外壳材料，将唑来膦酸载于外壳中，用碳二亚胺法连接配体和微泡外壳制备以全氟化碳为核心的载药靶向声学微泡制剂，评估其物理特性、载药微泡制剂体外寻靶能力和抑制TAMs作用，通过动物移植瘤实验评估靶向成像效果和抗肝癌作用，研究其抗癌机制。靶向载药纳米微泡定点释放后对TAMs作用的药物浓度明显增高，可提高疗效和减少副作用，同时可根据需要造影诊断和观察治疗效果。该项目可能为开发诊疗一体化制剂提供一种方法和思路。

肝细胞癌的诊断和治疗水平有待进一步提高。开发新型制剂有望能够改进诊断和治疗水平。削减瘤体内肿瘤相关巨噬细胞（TAMs）具有抗癌潜能；靶向磷脂酰肌醇蛋白多糖-3（GPC3）纳米微泡有望提高肝细胞癌超声成像诊断的敏感性、特异性和准确性。本项目以含有羧基的脂质材料（DPPC、DPPG、DSPE-PEG2000）为基本材料，用薄膜水化法制备微泡外壳，将唑来膦酸（ZOL）包含于外壳材料中，用人工合成的肽[DHLASLWWGTEL]为GPC3受体靶向配体， 用碳二亚胺法连接配体和微泡外壳，以六氟化硫气体为微泡核心，制备载有药物唑来膦酸的靶向声学微泡制剂。主要研究内容：载唑来膦酸靶向声学微泡制备、物理特性评估、生物相容性评估、体外声学增强效应评估、体外寻靶能力和抑制TAMs作用评估、动物移植瘤靶向成像效果评估、削减瘤体TAMs抗癌作用评估。结果及关键数据：制备的微泡呈类圆体，微泡粒径平均值为380.9±176.5 nm；电位平均值为-51.4±10.4mV。制剂无细胞毒性，能够与细胞膜上含有GPC3亲和结合，能够抑制RAW264.7单核巨噬细胞增殖。体外声学增强效果与同浓度的商业用制剂注射用六氟化硫微泡（声诺维，SonoVue）类似，给予荷瘤动物静脉注射该制剂后，瘤体声学增强效果和模式与同浓度的声诺维类似，没有新的瘤体声学增强特征，无特别增强成像和延长增强成像。Cy5.5荧光染料标记的微泡体内实验显示瘤体无特别增强成像和延长增强成像效应。靶向载药纳米微泡有削减瘤体内巨噬细胞作用，但是对M1和M2型巨噬细胞无选择性。.科学意义：本项目研制的制剂在瘤体血管外不具备靶向成像能力。与核素成像等血管外靶向成像不同，超声靶向成像可能不能在肿瘤瘤体血管外实现。虽然这个结论是负面的，但是其提供的经验教训也可以为人们的科学探索借鉴。靶向载药纳米微泡中的唑来膦酸能否抗肝癌与瘤体间质内那一类细胞为主有关；如果瘤体内以促癌为主的M2巨噬细胞多时，则用药后对抗癌有积极作用；如果以抑癌为主的M1巨噬细胞多时，则用药不利于抗癌。