双模态对比剂Fe3O4-Cy5.5-NGR在卵巢癌的体外靶向效能研究

doi:10.3969/j.issn.1006-7795.2018.01.016

首都医科大学学报 ›› 2018, Vol. 39 ›› Issue (1): 84-91.doi: 10.3969/j.issn.1006-7795.2018.01.016

双模态对比剂Fe₃O₄-Cy5.5-NGR在卵巢癌的体外靶向效能研究

张紫欣, 孟颖, 梁宇霆

首都医科大学附属北京妇产医院放射科, 北京 100026

收稿日期:2016-11-14 出版日期:2018-01-21 发布日期:2018-01-27
通讯作者: 梁宇霆 E-mail:liangyuting688@sina.com
基金资助:
北京市自然科学基金面上项目（7162064），首都医科大学基础-临床合作基金重点研究项目（15JL07）。

MR-NIRF Fe₃O₄-Cy5.5-NGR bimodel contrast agents ovarian cancer cells

Zhang Zixin, Meng Ying, Liang Yuting

Department of Radiology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, China

Received:2016-11-14 Online:2018-01-21 Published:2018-01-27
Supported by:
This study was supported by Natural Science Foundation of Beijing(7162064), Basic-clinical Key Research Grant of Capital Medical University (15JL07).

摘要/Abstract

摘要： 目的合成并验证磁共振-荧光双模态对比剂Fe₃O₄-Cy5.5-NGR对卵巢癌ES-2细胞的体外靶向效能情况。方法化学方法合成Fe₃O₄-Cy5.5-NGR颗粒作为实验组对比剂，Fe₃O₄-Cy5.5颗粒作为对照组对比剂，在透射电镜（transmission electron microscopy，TEM）、红外光谱仪（far infrared spectrometer，FITR）和紫外分光光度仪（ultraviolet spectrophotometer-2600，UV-2600）等设备下检测对比剂的普通物理化学性质是否满足成像需要；选取ES-2人卵巢癌透明细胞株传代培养，细胞生长稳定后进行CD13免疫组织化学染色，观察细胞膜表面CD13表达量；两种对比剂与ES-2细胞共同孵育后检测粒子的细胞毒性，并用流式细胞仪验证细胞与粒子的体外特异性结合情况。在7.0TMR下验证磁性纳米颗粒Fe₃O₄-Cy5.5-NGR和Fe₃O₄-Cy5.5作为MR对比剂的磁敏感性。结果 TEM观察两种对比剂形态规则，水溶液中分散性良好，通过连接NGR短肽，实验组对比剂的平均直径达（8.930±0.773）nm，对照组对比剂直径约（7.480±0.695）nm；Fe₃O₄-Cy5.5-NGR和Fe₃O₄-Cy5.5激发波为628.0 nm，发射波为675.2 nm；FITR中Fe₃O₄-Cy5.5-NGR和Fe₃O₄-Cy5.5在2 882 cm^-1、1 632 cm^-1处出现特征性波峰，实验组对比剂在841 cm^-1处另外出现了波峰。细胞毒性实验（CCK-8）中对比剂与细胞共孵育后，细胞活性保持在90%左右。细胞免疫组织化学染色，荧光显微镜观察细胞呈侵袭性生长，细胞膜位置CD13高表达，细胞核及周围间质表达量很低。Fe₃O₄-Cy5.5-NGR和Fe₃O₄-Cy5.5对比剂r2值分别为155.49 mmol·L^-1·s^-1，180.74 mmol·L^-1·s^-1。流式细胞仪下观察到Fe₃O₄-Cy5.5-NGR较Fe₃O₄-Cy5.5对比剂的荧光强度高，在不同浓度（40、80和160μmol）分别为后者的3.1、1.65和1.26倍。结论 Fe₃O₄-Cy5.5-NGR作为磁共振-荧光双模态对比剂在ES-2细胞的体外靶向性能良好，可以满足体外成像需求，为进一步的裸鼠卵巢癌在体实验提供了实验基础。

关键词: 卵巢癌, 荧光成像, 体外靶向性

Abstract: Objective To synthesize magnetic resonance (MR) and near-infrared fluorescence (NIR) bimodel contrast agents Fe₃O₄-Cy5.5-NGR and verify the targeting ability in virto on ES-2 ovarian cancer cells.Methods Fe₃O₄-Cy5.5-NGR as experimental group and Fe₃O₄-Cy5.5 as control group, physical and chemical properties with transmission electron microscope (TEM), infrared spectroscopy (FITR) and ultraviolet spectrophotometer (UV-general physical and chemical properties 2600). ES-2 human ovarian cancer clear cell subculture lines for CD13 immunohistochemical staining and the expression of CD13 on cell surface. Co-incubate contrasting agents and ES-2 cells cytotoxicity and specific binding ability under flow cytometer. susceptibility of Fe₃O₄-Cy5.5-NGR and Fe₃O₄-Cy5.5 as magnetic nanoparticles on 7.0TMR.Results Fe₃O₄-Cy5.5-NGR is sharper than Fe₃O₄-Cy5.5, the mean diameter is (8.93 ±0.773)nm for NGR-connected agent and (7.48 ±0.695) nm for the other. Excitation wavelength for both agents is 628.0 nm and reflected wavelength is 675.2 nm; Fe₃O₄-Cy5.5-NGR and Fe₃O₄-Cy5.5 got specific peaks at 2 882 cm^-1 and 1 632 cm^-1; NGR-connected agents had another peaks at 841 cm^-1. No cytotoxicity displayed after co-incubate under CCK-8 text. High fluorescence on cell surface and slightly at the location of nucleus and fiber matrix with CD13 immunohistochemical staining. The values of r2 are 155.49 mmol·L^-1·s^-1, 180.74 mmol·L^-1·s^-1 for Fe₃O₄-Cy5.5-NGR Fe₃O₄-Cy5.5 seperatly. The fluorescence intensity of Fe₃O₄-Cy5.5-NGR is relatively high than Fe₃O₄-Cy5.5 in different concentrations(40, 80 and 160 μmol), the number were 3.1, 1.65 and 1.26 times, respectively.Conclusion Fe₃O₄-Cy5.5-NGR performed well on targeting ability as MRI-NIRF bimodal contrast agent in vitro on ES-2 cells, which may use in ovarian cancer xenografts on nude mice experiments.

Key words: ovarian cancer, near infrared fluorescence, targeting ability

中图分类号:

R737.31

张紫欣, 孟颖, 梁宇霆. 双模态对比剂Fe₃O₄-Cy5.5-NGR在卵巢癌的体外靶向效能研究[J]. 首都医科大学学报, 2018, 39(1): 84-91.

Zhang Zixin, Meng Ying, Liang Yuting. MR-NIRF Fe₃O₄-Cy5.5-NGR bimodel contrast agents ovarian cancer cells[J]. Journal of Capital Medical University, 2018, 39(1): 84-91.

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双模态对比剂Fe₃O₄-Cy5.5-NGR在卵巢癌的体外靶向效能研究

MR-NIRF Fe₃O₄-Cy5.5-NGR bimodel contrast agents ovarian cancer cells

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