金纳米笼-量子点-Anti-AFP复合探针对肝癌细胞株的靶向光热治疗

Targeted light heat treatment of gold nano cage-quantum dots-Anti-AFP composite probe on HepG-2 hepatocellular carcinoma cells

目的热疗可靶向杀伤乳腺肿瘤细胞,但对肝肿瘤细胞的靶向热疗鲜见报道。本研究探讨金纳米笼-量子点-Anti-AFP复合探针对人肝癌细胞株HepG-2的靶向光热效应。方法用银立方纳米晶与次氯金酸通过置换反应法制备金纳米笼进行光热治疗,用水热合成法制备CnInS2-ZnS量子点进行细胞荧光显色,将金纳米笼、量子点和Anti-AFP三者耦合成金纳米笼-量子点-Anti-AFP复合探针。采用免疫组化的方法验证肿瘤细胞株是否表达AFP;用免疫荧光法验证复合探针与特定细胞株的靶向结合性;用功率密度为1.5W/cm2、波长为808nm激光照射进行光热治疗;用流式细胞术检测光热治疗后的细胞凋亡率;用荧光染色法检测光热治疗后的细胞死亡率。结果免疫组化实验发现,人肝癌细胞株HepG-2表达AFP,而乳腺癌细胞株Mcf-7不表达AFP。免疫荧光实验表明,复合探针中的Anti-AFP可与HepG-2结合并发出荧光,而不与Mcf-7细胞结合、细胞表面无荧光。光热实验表明,激光照射后,HepG-2细胞的升温明显高于Mcf-7,F=9.369,P<0.001;HepG-2的升温与探针浓度、激光照射时间成正相关、具有一定的时效量效关系。流式细胞术实验表明,光热治疗后,HepG-2的晚期凋亡率明显高于Mcf-7,t=12.551,P<0.001。荧光染色法显示,光热治疗后,95%的HepG-2细胞死亡,而Mcf-7细胞几乎均存活。结论金纳米笼-量子点-Anti-AFP复合探针可特异性与HepG-2细胞结合,并通过光热效应靶向杀伤HepG-2细胞。

OBJECTIVE Poor prognosis of liver tumor easy shift,heat treatment can be targeted to kill breast tumor cells,but the liver tumor cell targeting thermal therapy has not been reported.This study is to investigate the targeted photothermal effect of gold nano cage-quantum dots-Anti-AFP complex probe on hepatocellular carcinoma cell HepG-2and explore the killing effect of it on tumor cells.METHODS Silver cubic nanocrystalline and hypochlorous acid was used to prepare gold nanocages by replacement reaction method for photothermal therapy.Hydrothermal synthesis was performed to prepare CuInS2-ZnS quantum dots to conduct cell fluorescence staining.Coupling gold nanocages,quantum dot sum and anti-AFP into gold nanometer cage-quantum dot-Anti-AFP composite probe.The expression of AFP in tumor cell lines was examined by immunohistochemistry;The immunofluorescence method was used to validate the binding ability of the complex probe to specific cell lines.Photothermal treatment was performed with laser irradiation at apower density of 1.5 W/cm^2 and a wavelength of 808 nm.Flow cytometry was used to detect the apoptotic rate after photothermolysis.The cell death rate was measured by fluorescence staining.RESULTS The expression of AFP in human hepatocellular carcinoma cell line HepG-2was detected by immunohistochemistry,while that of breast cancer cell line Mcf-7could not be detected.Immunofluorescence assay showed that Anti-AFP could bind to HepG-2and fluoresce,but not to Mcf-7cells.The results of photothermal experiments showed that the temperature of HepG-2cells was significantly higher than that of Mcf-7cells（F=9.369,P〈0.001）,the temperature of HepG-2was positively correlated with the concentration of probe and laser irradiation time,and had a dose-effect relationship.Flow cytometry showed that the apoptotic rate of HepG-2was significantly higher than that of Mcf-7after photothermal therapy（t=12.551,P〈0.001）.Fluorescence staining showed that 95% of the HepG-2cells died after photothermal therapy,while the Mcf-7cells survived almost exclusively.CONCLUSION The gold nanocage-quantum dot-Anti-AFP composite probe can specifically bind to HepG-2cells and kill HepG-2cells through photothermal effect.