近红外聚合物量子点的设计制备及活体荧光成像研究

发布时间：2018-05-03 11:09

本文选题：近红外聚合物量子点 + 细胞毒性　；参考：《吉林大学》2017年博士论文

【摘要】：活体荧光成像技术是生命科学研究和医学基础研究的重要手段。活体荧光成像很大程度上依赖于显像剂的光学特性。以高亮度的近红外荧光材料作为显像剂的活体荧光成像具有深的组织穿透性、高的成像信噪比及灵敏度。因此,发展性能优异的近红外荧光材料对于推动活体荧光成像的发展具有重要意义。半导体聚合物量子点(Semiconducting polymer dots,Pdot)作为一种新型的有机荧光纳米材料,具有光学吸收截面大、荧光亮度高、斯托克斯位移大、稳定性好、生物相容性好等优良特性,已在生物传感、药物输送及肿瘤治疗等领域获得广泛研究。然而,目前在近红外区域发光的Pdot非常少,并且普遍发光较弱,限制了其在活体成像中的应用。因此,本研究致力于设计制备具有优良光学性能的近红外Pdot,并通过对Pdot的纯化来提高其生物相容性。通过在特异性细胞标记、干细胞示踪及对小鼠肿瘤和淋巴结成像方面的研究探索,证明近红外Pdot是一种理想的荧光成像试剂,在生物医学领域具有巨大应用潜力。本文的研究结果如下:1、我们将近红外染料掺杂于Pdot,实现了Pdot在近红外区域的高亮度、窄带荧光发射。由于共轭聚合物与近红外染料间高效的F?rster共振能量转移,使得Pdot的量子效率可高达22%,发射峰的半峰全宽仅为20 nm。同时该Pdot具有高的单粒子荧光亮度及优异的稳定性。2、我们发现利用凝胶过滤层析法对Pdot纯化,可降低Pdot对间充质干细胞(Mesenchymal stem cells,MSCs)的毒性。纯化的Pdot对MSCs增殖无影响,并且在长期储存后对MSCs无毒性增强趋势。以高浓度纯化的Pdot孵育MSCs,MSCs的多能性标志物表达不受影响。3、我们通过在Pdot表面修饰细胞穿膜肽——八聚精氨酸(Octa-arginine,R8),促进了MSCs对Pdot的摄取,低浓度Pdot在较短的孵育时间内即可高亮度标记干细胞,体外可长期示踪到MSCs的荧光信号。另外,多项分析结果表明摄取大量Pdot的MSCs,其生物学功能不受影响。4、我们首次将近红外Pdot标记的MSCs移植到肝切除模型鼠体内,实现了Pdot对MSCs的体内示踪。研究表明MSCs可促进肝脏再生,并且可降低肝脏的炎性反应。因此,近红外Pdot可作为一种非常理想的荧光纳米材料,用于基于干细胞的再生医学研究。5、我们利用Suzuki偶联法将近红外卟啉单元连接到共轭聚合物骨架上,首次合成在~800 nm处具有窄带发光的共轭聚合物。以该共轭聚合物制备得到小尺寸、高亮度的Pdot(NIR800 Pdot),在特异性细胞标记应用上,与商品化的近红外探针PE-Cy7相比,展现出更好的标记效果。6、我们制备了混杂Pdot,与NIR800 Pdot相比,其吸收光谱发生红移,同时荧光亮度得到显著提高;将聚乙二醇(PEG)基团修饰到混杂Pdot表面,有效降低网状内皮系统对Pdot的摄取,显著延长其体内停留时间;以PEG-Pdot作为荧光造影剂对淋巴结成像,与传统的蓝染法比较,前者展现了更高的成像信噪比及更长的示踪窗口时间;PEG-Pdot在荷瘤小鼠肿瘤及淋巴结部位大量蓄积,以Pdot作为造影剂的荧光成像,可指导临床精准的外科切除。
[Abstract]:Living fluorescent imaging is an important means of life science and basic medical research. Living fluorescent imaging relies heavily on the optical properties of imaging agents. The living fluorescence imaging with high luminance near infrared fluorescence as imaging agent has deep tissue penetration, high imaging signal to noise ratio and sensitivity. Excellent near infrared fluorescence materials are of great significance for promoting the development of living fluorescent imaging. Semiconducting polymer dots (Pdot), as a new kind of organic fluorescent nanomaterials, has high optical absorption cross section, high luminance, large Stokes displacement, good stability and good biocompatibility. The excellent properties have been widely studied in the fields of biosensing, drug delivery and cancer treatment. However, there are few Pdot luminescence in the near infrared region, and the widespread luminescence is weak, which restricts its application in vivo imaging. Therefore, this study is devoted to the design and preparation of near infrared Pdot with excellent optical properties and through to Pdo T is purified to improve its biocompatibility. Through the study of specific cell markers, stem cell tracers and imaging of tumor and lymph nodes in mice, it is proved that near infrared Pdot is an ideal fluorescent imaging reagent and has great potential for application in the field of biomedicine. The results of this study are as follows: 1, we are nearly infrared dyes. Doped with Pdot, the high luminance and narrow band fluorescence emission of Pdot in the near infrared region are achieved. The quantum efficiency of Pdot can be as high as 22% because of the efficient F? Rster resonance energy transfer between the conjugated polymer and the near infrared dye. The half peak width of the emission peak is only 20 nm. and the Pdot has high single particle luminance and excellent stability.2, and the Pdot has high luminance and excellent stability. We found that the purification of Pdot by gel filtration chromatography can reduce the toxicity of Pdot to Mesenchymal stem cells (MSCs). The purified Pdot has no effect on the proliferation of MSCs, and increases the nontoxic tendency to MSCs after long-term storage. The MSCs of Pdot is incubated with high concentration of Pdot, and the expression of the pluripotent marker of MSCs is not affected. We improve the uptake of Pdot by modifying the membrane peptide - eight polyarginine (Octa-arginine, R8) on the surface of the Pdot. The low concentration Pdot can mark the stem cells with high brightness in the short incubation time. In vitro, the MSCs can be traced to the fluorescent signal of MSCs in vitro. In addition, the multiple analysis results show that a large amount of Pdot is absorbed in a large amount of Pdot. The function is not affected by.4, and our first near infrared Pdot labeled MSCs has been transplanted into the hepatectomy model rat and the Pdot has been traced to MSCs in vivo. The study shows that MSCs can promote liver regeneration and reduce the inflammatory response of the liver. Therefore, near infrared Pdot can be used as an unusually ideal fluorescent nano material based on stem cells. In the regenerative medicine study.5, we use the Suzuki coupling method of near infrared porphyrin unit to connect to the conjugated polymer skeleton and synthesize the conjugated polymer with narrow band luminescence at ~800 nm for the first time. The conjugated polymer is prepared to obtain small size, high brightness Pdot (NIR800 Pdot), and in the application of specific cell marking, and commercialized near red. Compared with the external probe PE-Cy7, we showed a better labeling effect of.6. We prepared a hybrid Pdot. Compared with NIR800 Pdot, the absorption spectrum was red shift and the luminance was significantly improved. The modification of the polyethylene glycol (PEG) group to the mixed Pdot surface could effectively reduce the uptake of Pdot in the network endothelial system and prolong the retention time in the body significantly. PEG-Pdot was used as a fluorograph for lymph node imaging, compared with the traditional blue staining method, the former showed a higher imaging signal to noise ratio and longer tracer window time; PEG-Pdot was accumulated in tumor and lymph nodes of tumor bearing mice, and Pdot was used as a fluorograph of contrast agent, which could guide clinical surgical excision.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3

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