烯壳氮化铁纳米磁珠用于捕获肺癌循环肿瘤细胞的初步研究

doi:10.19586/j.2095-2341.2023.0037

生物技术进展 ›› 2023, Vol. 13 ›› Issue (4): 628-636.DOI: 10.19586/j.2095-2341.2023.0037

• 技术与方法 • 上一篇

烯壳氮化铁纳米磁珠用于捕获肺癌循环肿瘤细胞的初步研究

马文博¹(), 潘逸群¹, 王群², 马壮², 王明连¹(), 杨怡姝¹()

^1.北京工业大学环境与生命学部，北京 100124
^2.北京工业大学材料与制造学部，北京 100124

收稿日期:2023-03-24 接受日期:2023-04-28 出版日期:2023-07-25 发布日期:2023-08-03
通讯作者: 王明连,杨怡姝
作者简介:马文博 E-mail: mawenbo@emails.bjut.edu.cn；

Preliminary Study on the Application of Graphene-coated Iron Nitride Magnetic Beads to Capture Lung Cancer Circulating Tumor Cells

Wenbo MA¹(), Yiqun PAN¹, Qun WANG², Zhuang MA², Minglian WANG¹(), Yishu YANG¹()

^1.Faculty of Environmental and Life，Beijing University of Technology，Beijing 100124，China
^2.Faculty of Materials and Manufacturing，Beijing University of Technology，Beijing 100124，China

Received:2023-03-24 Accepted:2023-04-28 Online:2023-07-25 Published:2023-08-03
Contact: Minglian WANG,Yishu YANG

摘要/Abstract

摘要：

存在于血液中的循环肿瘤细胞（circulating tumor cell，CTC）在癌症的复发与转移中发挥重要作用，因此对循环肿瘤细胞的捕获已成为当前癌症研究的关键问题。抗体依赖性免疫磁珠捕获CTC的方法具有操作简便、快捷等特点，但存在样本量需求大和制备成本昂贵等问题。拟采用一种新型磁珠—等离子体修饰的烯壳氮化铁纳米磁珠（graphene-coated iron nitride magnetic beads, G@FeN-MB）开展捕获肺癌循环肿瘤细胞的初步研究。G@FeN-MB由多层石墨烯包覆，内核为氮化铁，与常用的氧化铁内核相比具有更强的磁响应性。石墨烯作为磁珠包覆壳层，相比市面上磁珠常用的SiO₂壳层具有更低的质量、更高的稳定性和更好的生物相容性。将氨基烯壳氮化铁磁珠与羧基烯壳氮化铁磁珠分别通过直接吸附法和碳二亚胺法制备2种链霉亲和素磁珠，并偶联CTC表面标志物上皮细胞黏附分子（epithelial cell adhesion molecule，EpCAM）的抗体制备氨基免疫磁珠（amino immuno magnetic bead, AIMB）与羧基免疫磁珠（carbaxylic immunomagnetic bead, CIMB）。结果显示，2种免疫磁珠均可捕获A549细胞，AIMB的捕获效率略高于CIMB；为了模拟CTC捕获环境，取健康人血与A549细胞混合制备不同浓度的细胞混液，并利用捕获效率更高的AIMB捕获人血A549细胞，计算被捕获的A549及捕获效率，结果显示，AIMB捕获效率从66.25%~81.50%不等。研究结果证明了基于G@FeN-MB制备免疫磁珠方法的可行性，得到的AIMB可以用于肺癌CTC分选，展现出其应用于临床研究的前景。

关键词: 循环肿瘤细胞, 烯壳氮化铁磁珠, 石墨烯, 细胞分选

Abstract:

Circulating tumor cells （CTCs） present in the blood play important roles in cancer recurrence and metastasis， so the capture of CTCs has become a key issue in current cancer research. The method of using antibody-dependent immunomagnetic beads to capture CTCs has the characteristics of simple operation and rapidity， but there are problems such as large sample size requirement and high preparation cost. This study intended to use a new type of magnetic bead-plasma-modified graphene-coated iron nitride magnetic beads （G@FeN-MB） to carry out a preliminary study on the capture of circulating tumor cells in lung cancer. G@FeN-MB is covered by multi-layer graphene， and the core is iron nitride， which has stronger magnetic response than the commonly used iron oxide core. Graphene used as a magnetic bead coating shell has lower mass， higher stability and better biocompatibility than the SiO₂ shell commonly used for magnetic beads on the market. In this study， two kinds of streptavidin magnetic beads were prepared by direct adsorption method and carbodiimide method respectively， and coupled with CTCs surface markers epithelial cell adhesion molecule （EpCAM） antibody preparation amino immunomagnetic beads （AIMB） and carboxylic immunomagnetic beads （CIMB）. The results showed that both kinds of immunomagnetic beads could capture A549 cells， and the capture efficiency of AIMB was slightly higher than that of CIMB； in order to simulate the CTCs capture environment， healthy human blood was mixed with A549 cells to prepare cell mixtures of different concentrations， and the capture efficiency was higher by using AIMB captures A549 in human blood， and calculated the captured A549 and capture efficiency. The results showed that the capture efficiency of AIMB ranged from 66.25% to 81.50%. The feasibility of the method of preparing immunomagnetic beads based on G@FeN-MB was proved， and the obtained AIMB can be used for the sorting of lung cancer CTCs， showing the prospect of its application in clinical research.

Key words: circulating tumor cells, graphene-coated iron nitride magnetic bead, graphene, cell sorting

中图分类号:

R734.2

马文博, 潘逸群, 王群, 马壮, 王明连, 杨怡姝. 烯壳氮化铁纳米磁珠用于捕获肺癌循环肿瘤细胞的初步研究[J]. 生物技术进展, 2023, 13(4): 628-636.

Wenbo MA, Yiqun PAN, Qun WANG, Zhuang MA, Minglian WANG, Yishu YANG. Preliminary Study on the Application of Graphene-coated Iron Nitride Magnetic Beads to Capture Lung Cancer Circulating Tumor Cells[J]. Current Biotechnology, 2023, 13(4): 628-636.

图/表 11

图1 GA@FeN-MB与GO@FeN-MB的透射电镜表征

Fig. 1 Transmission electron microscopy characterization of GA@FeN-MB and GO@FeN-MB

图2 烯壳氮化铁磁珠与其功能化后的SAMB的红外光谱分析A：GA@FeN-MB与GO@FeN-MB红外光谱分析对比；B：A-SAMB与GA@FeN-MB红外光谱分析对比；C：C-SAMB与GO@FeN-MB红外光谱分析对比

Fig. 2 FTIR of the magnetic beads and their functionalized SAMBs

图3 3种烯壳氮化铁磁珠吸附蛋白能力比较

Fig. 3 Comparison of the ability of three kinds of GO@FeN-MB to adsorb proteins

图4 2种磁珠与EpCAM抗体偶联的结果A:CIMB与抗体偶联结果；B：AIMB与抗体偶联结果；C：两种免疫磁珠荧光强度对比。ns表示在P>0.05水平上无统计学意义；****表示在P<0.0001水平上有统计学意义。

Fig. 4 Results of two magnetic beads coupled to EpCAM antibodies

图5 免疫磁珠捕获细胞后免疫荧光观察结果

Fig. 5 Immunofluorescence observations after cells were captured by IMB

图6 CCK-8法检测免疫磁珠捕获A549的能力注：***表示在P<0.001水平上有统计学意义；****表示在P<0.0001水平上有统计学意义。

Fig. 6 The CCK-8 method detected the ability of immunomagnetic beads to capture A549

图7 磁珠捕获细胞效率注：ns表示在P>0.05水平上无统计学意义；*表示在P<0.05水平上有统计学意义；**表示在P<0.01水平上有统计学意义。

Fig. 7 Efficiency of cells capture by magnetic beads

图8 免疫磁珠从小鼠细胞混液捕获A549后免疫荧光观察结果

Fig. 8 Immunofluorescence observation of A549 captured from mouse cells mixture by AIMBs

图9 磁珠从人血细胞混液捕获CTC后免疫荧光观察结果

Fig. 9 Immunofluorescence observation of CTCs captured from human blood cells mixture by AIMBs

表1 AIMBs从人血中捕获CTC数量及效率

Table 1 The quantity and efficiency of CTCs captured from human blood by AIMBs

CTC掺入数量 /（个·mL^-1）	捕获数量/个				捕获率/%		纯度/%
CTC掺入数量 /（个·mL^-1）	第1次	第2次	第3次	第4次	平均值	标准差	平均值	标准差
10	6	9	8	4	67.50	1.92	49.09	3.71
20	15	17	12	9	66.25	3.03	49.06	3.58
50	38	43	46	32	79.50	5.31	52.59	0.58
100	85	72	74	68	74.75	6.30	50.22	0.69
200	154	138	187	173	81.50	18.59	51.34	1.83

图10 AIMB捕获CTC数量线性关系

Fig. 10 Linear relationship of AIMBs captured CTCs number

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[1]	高先娟,汲霞,王清路,杜青青,王凤丹,王怀生. 羧基化石墨烯/半胱胺修饰金电极对多巴胺的电化学行为研究[J]. 生物技术进展, 2020, 10(3): 292-298.
[2]	王姣姣,耿圆圆,张家耀,姜伟. 肝细胞癌循环肿瘤细胞标志物及检测方法研究进展[J]. 生物技术进展, 2015, 5(2): 113-119.

烯壳氮化铁纳米磁珠用于捕获肺癌循环肿瘤细胞的初步研究

Preliminary Study on the Application of Graphene-coated Iron Nitride Magnetic Beads to Capture Lung Cancer Circulating Tumor Cells

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