摘要
目的合成并验证磁共振-荧光双模态对比剂Fe_3O_4-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Fe_3O_4-Cy5.5-NGR颗粒作为实验组对比剂,Fe_3O_4-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Fe_3O_4-Cy5.5-NGR和Fe_3O_4-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;Fe_3O_4-Cy5.5-NGR和Fe_3O_4-Cy5.5激发波为628.0 nm,发射波为675.2 nm;FITR中Fe_3O_4-Cy5.5-NGR和Fe_3O_4-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。Fe_3O_4-Cy5.5-NGR和Fe_3O_4-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Fe_3O_4-Cy5.5-NGR较Fe_3O_4-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 Fe_3O_4-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。
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. 0 TMR. 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. Thefluorescence 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.
出处
《首都医科大学学报》
CAS
北大核心
2018年第1期84-91,共8页
Journal of Capital Medical University
基金
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)~~
关键词
卵巢癌
荧光成像
体外靶向性
ovarian cancer
near infrared fluorescence
targeting ability
