文献：High-Specific Isolation and Instant Observation of Circulating Tumour Cell from HCC Patients via Glypican-3 Immunomagnetic Fluorescent Nanodevice

文献链接：

作者：Qihui Chu,Weiwei Mu ,Chuanjin Lan Yang Liu，Tong Gao，Li Guan，Yuxiao Fang，Zipeng Zhang， Yingchao Liu， Yongjun Liu， Na Zhang

相关产品：Fe3O4 四氧化三铁

原文摘要：

Purpose: Specific targeting receptors for efficiently capturing and applicable nanodevice for separating and instant observing of circulating tumour cells (CTC) are critical for early diagnosis of cancer. However, the existing CTC detection system based on epithelial cell adhesion molecule (EpCAM) was seriously limited by low expression and poor specificity of targeting receptors, and not instant observation in clinical application.Methods: Herein, an alternative glypican-3 (GPC3)-based immunomagnetic fluorescent system (C6/MMSN-GPC3) for high-specific isolation and instant observation of CTC from hepatocellular carcinoma (HCC) patients’ peripheral blood was developed. The high-specific HCC targeting receptor, GPC3, was employed for improving the sensitivity and accuracy in CTC detection. GPC3 monoclonal antibody (mAb) was linked to immunomagnetic mesoporous silica for specific targeting capture and separate CTC, and fluorescent molecule coumarin-6 (C6) was loaded for instant detection of CTC.Results: The cell recovery (%) of C6/MMSN-GPC3 increased in 106 HL-60 cells (from 49.7% to 83.0%) and in whole blood (from 42% to 80.3%) compared with MACS® Beads. In clinical samples, the C6/MMSN-GPC3 could capture more CTC in the 13 cases of HCC patients and the capture efficiency was improved by 83.3%–350%. Meanwhile, the capture process of C6/MMSN-GPC3 was harmless, facilitating for the subsequent culture. Significantly, the C6/MMSN-GPC3 achieved the high-specific isolation and instant observation of CTC from HCC patients’ blood samples, and successfully separated CTC from one patient with early stage of HCC (Stage I) and one post-surgery patient, further indicating the potential ability of C6/MMSN-GPC3 for HCC early diagnosis and prognosis evaluation.Conclusion: Our study provides a feasible glypican-3 (GPC3)-based immunomagnetic fluorescent system (C6/MMSN-GPC3) for high-specific isolation and instant observation of HCC CTC.

Fe3O4：一种铁的氧化物，也称为四氧化三铁。从化学结构上看，它可以看作是由FeO和Fe2O3组成的复合物，其化学式可以写成。铁元素在其中呈现出两种不同的价态，其中三分之一的铁为 +2 价，三分之二的铁为 +3 价。这种特殊的结构赋予了Fe3O4一些物理和化学性质。它具有反尖晶石结构。在这种结构中，氧离子（O2-）形成立方最密堆积，而铁离子（Fe2+和Fe3+）则填充在氧离子形成的四面体和八面体空隙中。这种晶体结构使得Fe3O4具有较高的稳定性。基于Fe3O4的性质，该文献合成路线如下：

图：合成线路示意

MMSN的合成：

在去离子水中加入CTAB和三甲胺。然后，在高温的条件下剧烈搅拌。Fe3O4纳米颗粒溶液用超声处理，滴加入三颈烧瓶中。最后，加入TEOS，在高温下再反应，完成溶胶-凝胶反应。沉淀物由外部磁场分离，用去离子水和乙醇洗涤几次，并在室温下真空下干燥过夜。产物在含硝酸铵的乙醇溶液中回流，以去除CTAB标品。

C6/MMSN-GPC3的制备：

GPC3抗体与Traut试剂在PBS缓冲液（含 EDTA）下进行硫代化。硫化GPC3抗体的纯化采用Zeba Desalt旋转柱。然后，将 C6/MMSN-PEG2000-Mal超声分散于PBS缓冲液（含 EDTA）中，加入上述溶液中。这个反应在一夜之间进行了。离心去除游离硫化GPC3抗体。最终得到的沉淀量为C6/MMSN-GPC3。

图：电镜图像

结论：

该文献成功制备了基于Fe3O4合成的C6/MMSN-GPC3，这是一种基于甘聚糖-3（GPC3）的免疫磁荧光系统。结果显示，C6/MMSN-GPC3的细胞回收率增加，还能捕获更多的CTC并且捕获效率提高，同时，C6/MMSN-GPC3的捕获过程是无害的，便于后续培养。综上，该文献提供了一种可行的基于甘聚糖-3（GPC3）的免疫磁性荧光系统（C6/MMSN-GPC3），可用于HCC CTC的高特异性分离和即时观察。