基于还原氧化石墨烯的新型超声—荧光双模态靶向纳米微泡的制备及其肝癌可视化光热治疗研究

Thermal ablation such as radiofrequency ablation and microwave ablation is an important option for the treatment of HCC, and our clinical findings showed that improving local ablation therapeutic range and precision became difficult and hot in HCC treatment. Photothermal therapy (PTT) as a non-invasive thermal ablation technique, which can realize the selective killing of cancer cells, can reduce the normal cell and tissue injury. Reduced graphene oxide photothermal therapy was identified to be effective in the tumor therapy, but RGO cannot be an independent imaging and no ability of active targeting. Our previous study suggested that targeted nanoscale microbubbles were ideal ultrasound molecular probes and visible targeted therapy carriers due to their characteristics of ultrasound imaging, gene carriers and ultrasound-activated gene release. This study intends to regard skilled in preparation of nanoscale microbubbles and reduced graphene oxide as the premise, over-expressed GPC3 receptors of hepatomacell membranes as the target, PLGA as the shell material and Cy7 as the luminescent material, and synthesize the complex of hepatoma glypican 3 antibody targeted PLGA- Cy7 nanoscale microbubbles with reduced graphene oxide. The nanoscale microbubbles are both photothermal therapy agents and contrast agents with ultrasound - fluorescence bi-modal imaging, combined with ultrasound-targeted microbubble destruction(UTMD) to realize targeted RGO delivery , which can monitor real-time photothermal therapy and realize the visible photothermal therapy. It is expected that novel ultrasound-fluorescence bi-modal targeted nanoscale microbubbles with reduced graphene oxide are prepared successfully, evaluated qualitatively and quantitatively the effect of targeted therapy and ultrasound molecular imaging in vivo and in vitro experiments, which provides a new method for visible photothermal therapy in hepatocellular carcinoma.

热消融是治疗肝癌的一种重要手段，我们临床研究发现如何提高其疗效和精准度是工作的难点和热点。光热治疗（PTT）作为一种非侵入性热消融技术，可实现癌细胞的选择性杀伤，降低对正常细胞和组织的损伤。还原氧化石墨烯（RGO）的光热治疗疗效肯定，但RGO无法独立显像且无主动靶向能力。本课题组前期研究表明，靶向纳米微泡具有成像、载基因及超声释放基因等特性，可实现超声监控下的靶向治疗。课题组设想以熟练掌握的纳米微泡及还原氧化石墨烯制备方法为前提，以肝癌细胞膜高表达的GPC3受体为靶点，以PLGA为成膜材料，Cy7为荧光染料，创新性提出制备携载RGO的抗GPC3靶向PLGA-Cy7纳米微泡复合体，具有光热治疗剂和超声—荧光双模态成像双重功能。通过超声靶向微泡破坏技术（UTMD）介导RGO靶向投递，实时监控PTT进程，实现肝癌的靶向可视化光热治疗，通过体内外实验评价其诊疗效果，为肝癌热消融治疗提供一种新手段。

还原氧化石墨烯（RGO）是一种新型的二维碳纳米材料，有近红外区（NIR）的光吸收特性，广泛应用于光热治疗。本项目在前期工作基础上，应用超声联合靶向载RGO纳米微泡治疗肝癌，将肝癌细胞膜高表达的磷脂酰肌醇蛋白聚糖3（GPC3）作为配体制备靶向性纳米微泡，并将还原氧化石墨烯包裹入其中构成仅有纳米级大小的载光热材料复合物。该复合物在超声的辐照下，经过通透性增大的细胞膜靶向递送到肿瘤细胞内，有效地解决光热材料没有主动靶向能力这一难题。首先以磷脂复合物作为原材料、以GPC3作为靶向配体，利用薄膜水化法和机械振荡法制备纳米微泡，再借助生物素-亲和素系统，将GPC3和纳米微泡偶联，合成载RGO的GPC3靶向纳米微泡。体外检测超声辐照下纳米微泡破坏后释放RGO的能力，结果显示实验组明显高于对照组。之后进行细胞实验，采用人源肝癌细胞系HepG2，扫描电子显微镜下证实载RGO的GPC3靶向纳米微泡成功连接HepG2细胞。同时我们也发现合成的纳米微泡在808nm激光辐照下会产生活性氧（ROS），ROS是光动力治疗的核心，这也有助于提高光热治疗的效果。最终我们用CCK-8试剂盒来验证超声联合载RGO的GPC3靶向纳米微泡治疗肝癌的效果，结果表明相比于其他对照组，实验组细胞活力明显降低。基于上述研究结果课题组得出结论，载RGO的GPC3靶向纳米微泡联合超声可以高效地递送光热材料进入肿瘤位点。课题组成员还尝试将光热材料替换成药物，制备具有肝癌靶向性的载阿帕替尼纳米微泡，提高药物的递送效率从而增强化疗效果并减少全身毒副作用。项目资助发表SCI收录论文6篇。资助发表国家核心期刊论文3篇，授权国家发明专利1项，获得黑龙江省科技进步二等奖1项，培养硕士研究生2名，均已顺利获得硕士学位。项目经费各项支出基本与预算相符合，剩余经费用于后续文章发表。