【作者】 张岩；

【导师】 田文晶；

【作者基本信息】 吉林大学 ， 高分子化学与物理， 2015， 博士

【摘要】 生物成像技术是目前生物领域和生物医学领域中研究的热点之一，其中荧光生物成像技术是一种快速有效，灵敏度高，易于实现的生物成像技术。近年来，随着荧光成像技术的发展，这种技术已经成为生理过程观测和临床病理影像学的一个直接可视化的工具，对生命科学研究和疾病诊断具有非常重要的意义。荧光聚合物量子点作为一种新型的荧光成像材料，已引起了越来越多人的注意。然而由于传统荧光团的聚集淬灭现象，使得基于传统荧光团设计的荧光量子点的应用受到了极大的限制。近来，越来越多的具有聚集诱导发光（AIE）或聚集增强发射（AEE）性质的有机荧光分子被开发出来。基于AIE或AEE分子的聚合物纳米材料表现出优异的生物成像效果，并受到广泛的关注。为了提高成像的灵敏度，增强聚合物量子点的靶向成像能力，本论文利用AIE或AEE分子具有高固态荧光量子产率的性质，制备了不同的聚合物量子点，并探索了量子点的细胞成像的能力。1.基于DSA分子的聚合物量子点的制备及Hela细胞成像选取具有AEE性质的9,10-二苯乙烯基蒽分子（DSA）作为发光内核，利用兩亲性聚合物在水中能够自组装的性质，对DSA分子进行了包覆。通过使用两种不同的聚合物（PSMA和PIMA），获得了两种不同包覆基质的聚合物量子点，PSMA dots以及PIMA dots。这两种不同的聚合物量子点均具有均一的粒径分布、球型的外观以及很小的粒子尺寸（约23nm）。两种粒子在相同的条件下对比发现PSMA dots相较于PIMA dots具有更高的荧光量子产率，在PSMA与DSA的质量比为1:1时，具有最高的荧光量子产率（27.3%）。选择质量比为1:1的PSMA dots，在EDC/NHS的催化下，将叶酸修饰在量子点的表面，得到FA-dots。FA-dots具有优良的发光性能、稳定性以及生物相容性。由于叶酸基团的引入，使得FA-dots能通过与叶酸受体的相互作用特异性靶向叶酸受体过表达的肿瘤细胞。体外细胞实验证明量子点能够有效地被内吞到Hela细胞内，实现了靶细胞成像。2.基于DPPBPA分子的高效近红外聚合物量子点的制备及Hela细胞成像设计合成了一种新型的近红外发射的具有AEE性质的荧光小分子(2Z,2’Z)-3,3’-(2,5-di(piperidin-1-yl)-1,4-phenylene)bis(2-phenylacrylonitrile)(DPPBPA)。这种荧光小分子具有非常大的斯托克斯位移，并在近红外区有非常强的荧光发射，固态的荧光量子产率高达78%。利用具有马来酸酐的聚合物PSMA作为封包基质，DPPBPA作为高亮度的近红外发光核，通过自组装过程制备了高荧光量子产率的近红外聚合物量子点（DPPBPAdots）。这种DPPBPAdots具有球形的外观，粒径尺寸约为20nm。与DPPBPA分子类似，DPPBPAdots具有将近306nm的斯托克斯位移，并在近红外区具有很强的荧光发射，其水溶液的荧光产率高达20%。选择生物分子——链霉亲和素通过肽键连接到DPPBPA dots表面，得到SA-dots。通过链霉亲和素—生物素以及抗体—抗原之间的相互作用，实现了对MCF-7细胞的靶向标记。这种标记方法是非常有效的，并具有非常强的特异性。3. DSA分子共价结合的兩亲性嵌段共聚物的聚合物量子点的制备及细胞成像研究设计合成了一种新型的荧光兩亲性嵌段聚合物P4。P4包含AEE发光团DSA，з-己内酯（PCL）链段，聚乙二醇（PEG）链段以及叶酸基团。PCL是一种可降解且生物相容性非常好的材料，在这里将它作为聚合物的疏水链；生物相容性好且没有非特异性识别的PEG链段作为亲水端；具有癌细胞靶向性的叶酸基团作为靶向标记物。这种荧光兩亲性嵌段聚合物能够很容易的在水中自组装形成聚合物量子点，P4Pdots。P4Pdots具有非常好的胶体稳定性和化学稳定性，并具有均一的粒子尺寸，粒子的直径约为15nm。P4Pdots具有很亮的黄绿光发射，荧光量子效率高达29%。量子点具有很好的生物相容性，同时相比于3T3-L1细胞系，纳米粒子更容易被Hela细胞捕获，证明了该量子点具有良好的靶向作用。更多还原

【Abstract】 Bioimaging technology is one of the research hotspot of the areas in biotechnologyand biomedicine at present. Fluorescence imaging is a technique which can be easilyused for practical application with high effective and sensitivity. In recent years, withthe development of fluorescence imaging, it has become a crucial tool for directvisualization of physiological and pathological processes observation and clinicalimaging studies. To date, more and more fluorescent with aggregation-inducedemission (AIE) or aggregation enhanced emission (AEE) characteristics weresynthesized. Polymer nano materials, which based on AIE or AEE fluorescent exhibitexcellent result in biological imaging, have attracted great attention.Against this background, we prepared some novel polymer dots with highlyefficiency by using AIE or AEE molecules and the target cell imaging ability of thesepolymer dots were explored.1. Bright fluorescence polymer dots based on DSA for Hela cell imagingHere we prepared two fluorescence polymer dots by encapsulating AEEfluorescent9,10-divinyl anthracene (DSA) with amphiphilic polymer. By using twodifferent polymer, we obtained two polymer dots (PSMA dots and PIMA dots) withdifferent encapsulation matrix (PSMA and PIMA). These two kinds of polymer dotsboth have uniform particle size distribution, spherical appearance and small particlesize (about23nm). Comparison of these two kinds of dots, The PSMA dots havehigher fluorescence quantum yield. When the mass ratio of PSMA: DSA was1:1,PSMA dots reached the highest fluorescence quantum yield (about27.3%).Catalyzed by EDC/NHS, folic acid was conjugated to the surface of the PSMA dots(1:1) and FA-dots obtained. FA-dots possess good optical properties, stability andbiocompatibility. Due to the folate group from the surface of FA-dots, the FA-dotscould mark the cells where the folate receptor was over-expressing. Our in vitro cellexperiment, FA-dots were endocytosed into Hela cells and realized the cell imaging with specific.2. Highly efficient near-infrared polymer dots based on novel AEEfluorogen for specific cancer cell imagingWe report a new near-infrared emissive molecule (2Z,2’Z)-3,3’-(2,5-di(piperidin-1-yl)-1,4-phenylene)bis(2-phenylacrylonitrile)(DPPBPA) with very large stokesshift and high solid fluorescence quantum efficiency (Φ=78%). Near-infraredpolymer dots with a high fluorescence quantum efficiency were prepared by using anamphiphilic polymer poly(styrene-co-maleic anhydride)(PSMA) as theco-encapsulation matrix and AEE molecule DPPBPA as the high near-infraredemission core. DPPBPA dots are uniform spheres with small size of about20nm andshow large Stokes shift of306nm and strong emission in the near-infrared region.The fluorescence quantum efficiency (Φ) of DPPBPA dots dispersed in water canreach20%. Streptavidin was chosen as the biomolecule covalently linked into theDPPBPA dots to form streptavidin-dots (SA-dots) which can effectively label cellsby the biotin-streptavidin interaction. The SA-dots were applied to in vitro cellularimaging. The SA-dots can effectively and specifically label target cell without anynonspecific binding.3. Fluorescence amphiphilic polymer dots based on PCL-b-PEG fortargeted cell imagingHerein, new AIE Pdots for targeting HeLa cells were developed. Thesemonodispersed AIE Pdots can be easily prepared through a self-assembly process byusing AIE conjugated amphiphilic copolymers which contain an AIE fluorophore,9,10-bis(4-hydroxystyryl)anthracene (DSA), hydrophobic poly(з-caprolactone)segments, hydrophilic poly(ethylene glycol) segments and folate groups. Thepoly(з-caprolactone), a biodegradable material with good biocompatibility, was usedas the hydrophobic segment, and the biocompatible material poly(ethylene glycol)was used as the hydrophilic segment and folate groups acted as specifically targetedgroups. The AIE Pdots have good chemical stability and colloidal stability, whichindicates that no dye leakage phenomenon was found due to the stable chemical bondbetween DSA and PCL-b-PEG. These AIE Pdots possess strong emission and highsolid state fluorescence quantum efficiency. The biocompatibility and cell uptakebehavior of the folic acid modified AIE Pdots were further investigated using two cell lines (HeLa cells and3T3-L1cells), which indicated that the folic acid modifiedAIE Pdots pose little toxicity to living cells and can be applied for targeted HeLaintracellular imaging.更多还原