叶酸受体靶向的近红外荧光共轭聚合物纳米材料的构建及应用

Fluorescent conjugated polymers (FCP) have emerged as a novel class of promising fluorescent probes for bioimaging with high fluorescence, good photostability and low cytotoxicity. However, as most of the reported FCP nanoparticles have low emission intensity in the near-infrared (NIR) region, it greatly restricted their application in in vivo imaging. Exploration of FCP nanoparticles containing energy deficient units with NIR emission signature in the polymer backbones can be a promising strategy to fabricate FCP nanoparticles with bright NIR emission. Based on this, we have developed NIRFCP nanoparticles with emission at 778 nm and the quantum yield of the 778nm emission in water was around 0.09. And this nanoparticle can be further used for targeted imaging integrin αvβ3 receptor high-expressed tumors in vivo after conjugation with RGD peptide. As a target agent, the non-specific uptake in the liver, spleen and skin greatly restricted its application in targeted imaging smaller size tumors. Therefore, how to reduce the non-specific uptake of probes is an urgent problem needsed to be solved. In this project, based on our previous study, we purpose to improve the tumor uptake of folate conjugated NIRFCP nanoparticles by investigating the effects of particle size, surface charge, and PEG linkage length on the in vivo non-specific uptake of NIRFCP nanoparticles. The result of this study will expand the application of NIRFCP nanoparticles in the tumor molecular imaging and provide a unique approach to construct novel tumor-targeted fluorescent nanoparticles.

荧光共轭聚合物（FCP）光稳定性好、毒性小，是新一代荧光纳米探针的发展方向。但其发射波长在可见光范围内，限制了其在活体成像中的应用。发展近红外光（NIR）发射的FCP纳米探针是一个关键难点。针对于此，申请人前期研究设计合成了发射波长在778nm、NIR量子产率为0.09的NIRFCP纳米探针，耦联RGD肽后，可靶向成像高表达整合素αvβ3受体的肿瘤。但作为靶向试剂，该探针在肝脾皮肤的非特异摄取阻碍了其对较小肿瘤的靶向成像。如何减少探针的体内非特异性摄取是一个亟待解决的关键问题。本项目拟在已有工作基础上，构建叶酸受体靶向的NIRFCP纳米探针，并探讨粒径大小、表面电荷、以及PEG链长度对NIRFCP纳米探针的体内非特异性摄取的影响,旨在提高NIRFCP纳米探针在肿瘤部位的摄取量，拓展NIRFCP纳米探针在肿瘤分子影像领域中的应用。本项目的结果将为构建更为有效的肿瘤靶向荧光纳米探针提供新思路。

荧光共轭聚合物光稳定好，毒性小，是新一代荧光纳米探针的发展方向，在分子成像领域的应用也引起了广泛关注。但是已报道的荧光共轭聚合物纳米材料中，由于其发射光的波长主要在可见光的范围内，为此这类材料用于小动物活体成像的例子还非常有限。针对于此，申请人前期设计合成了粒径大小约30nm的近红外荧光共轭聚合物纳米材料用于小鼠淋巴结成像及脑胶质瘤U87MG靶向成像。.本项目在已有工作基础上，深入研究了不同粒径大小、NIR775染料参杂的、表面PEG-COOH修饰的MEH-PPV近红外荧光共轭聚合物纳米材料的制备及体内成像应用。结果显示这类材料在778nm近红外发射峰的半峰宽为20nm，Stokes位移大于300nm，在4度保存具有长时间（大于9个月）的胶体稳定性和光稳定性，经尾静脉注射到U87MG肿瘤裸鼠体内可用于肿瘤血管显像（Bioconjug Chem. 2015;26(5):817-21.）。而且，该类材料在HeLa细胞活体示踪中也显示出其独有的特点：低毒性、持续的光亮度和光稳定性（大于20天）、以及高灵敏度（5 ug）（J. Mater. Chem. B, 2016;4:202-6）。.此外，采用共沉淀方法分别合成了PEG-COOH和PEG-NH2修饰的C7-叶酸功能化的PFBT纳米材料，粒径大小分别为50-60nm和90-105nm。细胞成像及流式细胞仪数据显示PEG-COOH修饰的叶酸功能化的材料（FA-PFBT-COOH）的荧光强度及细胞标记率均高于PEG-NH2修饰的材料（FA-PFBT-NH2）。且与没有叶酸功能化的材料对比，叶酸功能化能增强材料对U87MG, H1299及SKOV3细胞的摄取。活体成像实验中，材料FA-PFBT-COOH经尾静脉注射H1299肿瘤裸鼠体内，在肿瘤部位能检测到较强的近红外荧光信号。结果表明FA-PFBT-COOH这类纳米材料可用于肿瘤细胞的体内外成像（Chinese Journal of Chemistry, 2016，DOI: 10.1002/cjoc.201500780）。