H链铁蛋白介导的光磁双模式靶向探针的构建及肿瘤可视化成像研究

Aiming at the problems that multifunctional nanoparticles imaging system have the complicated preparation process and poor biocompatibility, with the characteristics that H chain ferritin nanocages can be used as a carrier and targeted molecular, a new dual mode imaging probe was developed. Using H chain ferritin nanocages as a carrier, loading magnetic ferroferric oxide nanoparticles internally, and coupling of carbon dots with near-infrared emission externally by covalence, not modifying targeting agents, realize the optical and magnetic resonance dual mode imaging. The carbon dots with near-infrared fluorescence properties were synthesized based on the chemical oxidation in a mixture of concentrated acids and surface modification. Using TEM instrument, particle size analyzer, nuclear magnetic resonance spectroscopy to characterize the structure and properties of the probe; Observing the uptake of the probe in the MCF-7 breast cancer cells by confocal laser scanning microscope; Detecting the toxicity of probe to MCF-7 cells with MTT colorimetric assay. The nude mice bearing breast cancer were selected as a model. After injection of imaging probes in the tail vein, we observe the tumor targeting imaging effect and distribution in mice through the vivo fluorescence imaging and magnetic resonance imaging at different time. The imaging probe have the simple preparation process and good biocompatibility, through H chain ferritin specific targeting to the tumor cells, realize the dual mode (near-infrared fluorescence/magnetic resonance) imaging and cell tracking, and provide new ideas and methods for the tumor imaging and diagnosis based on the ferritin nanocages.

针对目前多功能纳米粒成像系统存在制备过程复杂、生物相容性差的问题，利用H链铁蛋白纳米笼既可作载体，又具有靶向性的特点，构建一种新型双模式成像探针。以H链铁蛋白纳米笼为载体，内部装载磁性四氧化三铁纳米粒，外部通过共价键偶联近红外发光碳点，无需修饰靶向试剂，实现光磁双模式成像。以混酸氧化及表面修饰的方法合成近红外荧光碳点；采用透射电镜、粒度分析仪、核磁共振光谱等技术对探针结构及性能进行表征；通过激光共聚焦显微镜观察其在MCF-7乳腺癌细胞中的摄取情况；MTT比色法检测探针对MCF-7细胞的毒性。以荷瘤裸鼠为模型，尾静脉注射成像探针，在不同时间点通过活体荧光成像和磁共振成像观察其对肿瘤组织的靶向成像效果及在小鼠体内的分布。该探针制备过程简单，生物相容性好，在H链铁蛋白介导下特异性靶向肿瘤细胞，实现双模式（近红外荧光/磁共振）成像及细胞追踪，为基于铁蛋白纳米笼的肿瘤成像与诊断提供新的思路和方法。

双模态成像策略，即两种成像技术的结合与优势互补，可解决单一技术和成像模式的缺陷，为肿瘤诊断提供更准确和完整的信息。量子点、荧光染料分子和磁性纳米粒子等已经作为成像探针运用到了活体荧光成像和磁共振成像中。但是大多数量子点由有毒的重金属元素组成（如Cd等）；荧光染料容易聚集，其水溶液稳定性差，易产生光漂白现象；复杂的修饰往往导致功能化的纳米粒子高消耗、低纯度，生物相容性变差。基于此，本课题拟以H链铁蛋白纳米笼、碳点和磁性四氧化三铁为功能基元构建光/磁双模式靶向探针，以解决上述问题。首先设计合成近红外荧光碳点，其荧光强度高、光学稳定性和生物相容性好。以H链铁蛋白纳米笼为载体，内部合成磁性四氧化三铁纳米粒，外部通过共价键偶联近红外发光碳点，无需修饰靶向试剂，在H链铁蛋白介导下特异性靶向肿瘤细胞，实现体内和体外的双模式（近红外荧光/核磁共振）成像及细胞追踪，用于肿瘤的早期诊断。.采用一步水热合成法以柠檬酸为碳源，甲酰胺为氮源合成了氨基修饰的具有近红外发光特性的CDs。在绿光激发下发射明亮的红色荧光，生物相容性和稳定性良好。CDs的平均粒径为6 nm，且粒度大小分布均匀。实验发现在532 nm激光的照射下，CDs能够产生活性氧，通过对细胞核DNA的损伤抑制细胞的生长，发挥治疗作用。采用活性氧探针DPBF证明了CDs在532 nm激光照射下可以产生活性氧，而且在偶联至HFn之后并没有影响CDs活性氧的产生。通过分子生物学方法提取纯化得到HFn，HFn的粒径平均为12 nm，靶向性实验结果显示HFn具有良好的靶向性，M-HFn/CDs探针的体内成像实验结果显示纳米制剂主要在肿瘤部位聚集，再次证明了HFn的靶向能力。.本课题构建的M-HFn/CDs 光/磁双模式成像探针制备简单且可控性强。HFn纳米笼具有载体和肿瘤靶向的双重功能，减少了传统的表面靶向修饰，而且HFn纳米粒能够透过生物屏障（例如上皮、内皮以及血脑屏障）使探针到达靶向部位。该探针同时具有荧光成像和磁共振成像能力，用于肿瘤的早期诊断。