磁共振-荧光双模态显像联合光热治疗在人表皮生长因子受体2阳性乳腺癌模型中的应用

Preclinical application of MR and fluorescent dual-modality imaging combined with photothermal therapy in HER-2 positive breast cancer

目的构建偶联有曲妥珠单抗和荧光染料吲哚菁绿（ICG）的超顺磁氧化铁纳米微粒。验证偶联后的纳米微粒（NPs）能否在体内外水平靶向乳腺癌细胞表面的人表皮生长因子受体2（HER-2）受体。方法构建纳米探针并检测其一系列表征。通过透射电子显微镜观察HER-2阳性乳腺癌细胞SK-BR-3对纳米探针的摄取能力。建立乳腺癌小鼠模型，经小鼠尾静脉注射纳米探针，通过磁共振成像（MRI）、荧光成像观察NPs能否聚集于肿瘤部位。待NPs聚集于肿瘤部位后，利用近红外热成像系统监测近红外光照射时肿瘤部位的升温情况。结果成功构建了Fe3O4-trastuzumab-ICG超顺磁氧化铁纳米微粒。NPs的粒径为（25.93±4.25）nm。偶联后的NPs在828 nm处出现明显的吸收峰，与ICG的荧光发射波长一致。NPs具有很高的弛豫率，为107.65 mM－1·s－1。近红外激光持续照射后，NPs溶液的最高温度可达到57.8℃。透射电子显微镜下可见，NPs可以靶向连接SK-BR-3细胞并进入内质网内。小鼠尾静脉注射NPs 24 h后，活体MRI肿瘤部位T2信号达到最低。空白对照组、Fe3O4-IgG-ICG组、曲妥珠单抗＋Fe3O4-trastuzumab-ICG组和Fe3O4-trastuzumab-ICG组小鼠肿瘤部位ΔT2值分别为（3.1±1.1）、（4.4±0.9）、（11.3±3.8）和（30.7±4.8）ms，Fe3O4-trastuzumab-ICG组高于其他组，差异均有统计学意义（均P〈0.05）。活体荧光成像显示，NPs在腹腔脏器及肿瘤组织一过性浓聚并经膀胱排泄，24 h后在肿瘤部位有明显聚集。近红外热成像实验显示，近红外光持续照射下，Fe3O4-trastuzumab-ICG组小鼠肿瘤部位的温度可以达到49.4℃。结论Fe3O4-trastuzumab-ICG纳米探针有潜力成为HER-2阳性乳腺癌多模态显像剂及光热治疗的工具。

Objective To construct superparamagnetic iron oxide nanoparticles （SPIONs） coated on trastuzumab and indocyanine green （ICG） and then investigate whether the coated nanoparticles （NPs） targeted to human epidermal growth factor receptor-2 （HER-2） receptors on breast cancer cells in vitro and in vivo.Methods The Fe3O4-trastuzumab-ICG NPs were constructed. And a series of characteristics of the NPs were evaluated. The uptake ability of SK-BR-3, a HER-2 positive breast cancer cell, was observed by transmission electron microscopy. Then the NPs were injected in the tail veins of SK-BR-3 xenograft tumor-bearing mice to observe the aggregation of NPs in the tumor sites by MRI and fluorescent imaging. Furthermore, when the NPs was gathered at the tumor sites, the near infrared thermal imaging system was used to monitor the tumor temperature after the near infrared radiation.Results The successfully constructed Fe3O4-trastuzumab-ICG NPs had the size of （25.93±4.25） nm. The absorption peak was 828 nm, which was as same as the emission wavelength of ICG. The NPs had a high relaxation rate of approximately 107.65 mM-1·s-1. The maximum temperature of NPs solution could reach to 57.8℃ after continuous near infrared laser irradiation. The transmission electron microscopy imaging revealed that the NPs could target and enter into the endoplasmic reticulum of SK-BR-3 cells. MRI analysis showed the lowest T2 relaxation time in the tumor sites 24 h after tail vein injection of the NPs. The △T2 of the tumor sites in the Fe3O4-trastuzumab-ICG group （30.7±4.8） ms was higher compared with that of control group （3.1±1.1） ms, Fe3O4-IgG-ICG group （4.4±0.9） ms and trastuzumab ＋ Fe3O4-trastuzumab-ICG group （11.3±3.8） ms., respectively, all showing statistically significant differences （P〈0.05）. The fluorescence imaging revealed that the NPs was concentrated transiently in the intraperitoneal organs and tumor sites, then excreted into the bladder. After 24 h, there was an obvious aggregation in the tumor sites. The near infrared thermal imaging experiments showed that the temperature of tumor sites in Fe3O4-trastuzumab-ICG group could go up to 49.4℃ after continuous near infrared light irradiation.Conclusion The newly constructed Fe3O4-trastuzumab-ICG NPs have the potential to act as a multifunctional imaging agent and a powerful tool for photothermal therapy for HER-2 positive breast cancer.