摘要
为了解决荧光成像中生物样品的自发荧光干扰和荧光探针的穿透深度不够这两个问题,本论文从上转换发光和长寿命发光两个角度出发,设计合成了一系列具有上转换发光特性的稀土纳米材料和具有微秒量级发光寿命的磷光铱(Ⅲ)配合物,并将这些发光探针用于细胞和小动物层次的靶向成像。主要研究内容包括以下两大部分。
Ⅰ.上转换发光活体成像系统的搭建以及稀土上转换发光纳米材料的肿瘤靶向活体成像和毒性研究
稀土上转换发光纳米材料(UCNPs)作为发光标记材料拥有极大优势如较高的光穿透深度以及无背景荧光干扰。但将UCNPs广泛用于生物成像仍然存在很大的障碍,主要的困难是目前商用的成像系统不适用于上转换发光成像。为此,我们搭建了第一台高对比度的稳态激光泵浦上转换发光小动物活体成像系统,并开展了上转换发光成像技术用于肿瘤靶向和活体成像的方法。结果表明上转换成像甚至在组织深度高达600μm仍没有自发荧光干扰。
更重要的是,我们还巧妙地运用了配体与受体之间的特异性作用,分别以整合素ανβ3受体、叶酸受体(FR)为靶点,以精氨酸-甘氨酸-天冬氨酸(RGD肽)、叶酸(FA)为识别位点,通过化学手段将RGD肽／FA与UCNPs结合起来,发展了一些具有靶向肿瘤功能的上转换发光探针,并首次将其成功地应用到小动物活体水平的肿瘤靶向成像上。结果表明RGD肽／FA标记的UCNPs具有很好的肿瘤靶向效果和高的信噪比(-24)。
迄今为止,只有少数研究涉及UCNPs的细胞毒性,关于UCNPs的长期活体毒性的研究尚未见文献报道。为此,我们设计合成了聚丙烯酸(PAA)包覆的NaYF4稀土上转换发光纳米材料(PAA-UCNPs),并将其用于长期的活体生物分布和毒性研究。结果表明PAA-UCNPs的摄取主要在肝脏和脾脏,且能以非常缓慢的方式通过肠道从小鼠体内排出体外。我们通过小鼠体重变化、组织学、血液学以及生化指标检测来考察PAA-UCNPs的长期活体毒性,结果表明15mg/kg剂量的PAA-UCNPs经尾静脉注射到小鼠体内长达115天,小鼠没有受到任何明显的毒性影响。
Ⅱ.磷光铱配合物用于肿瘤细胞的靶向成像以及活细胞内半胱氨酸、高半胱氨酸的比度成像
1)基于整合素ανβ3受体与RGD肽之间的特异性作用,我们设计合成了一个RGD肽偶联的、红光发射的磷光铱(Ⅲ)配合物,并将其应用于靶向成像整合素ανβ3表达的肿瘤细胞。结果表明该探针能快速(-15 min)、高灵敏度(2μM)、特异性地靶向成像整合素ανβ3过表达的U87MG细胞。此外,我们设计合成了一种RGD肽偶联的有机荧光探针用于靶向肿瘤细胞。光漂白实验显示该探针具有比有机染料吖啶橙更高的光稳定性。
2)半胱氨酸(Cys)和高半胱氨酸(Hcy)在生物系统中扮演许多重要的角色。我们合成了一个阳离子型的铱(Ⅲ)配合物,首次用于细胞内的Hcy和Cys的比度磷光成像。结果表明当探针孵育KB细胞30分钟后,发光强度比值(I586／I547)大于1；当用马来酰亚胺封闭了探针和Hcy/Cys的反应后,发光强度比值(I586／I547)小于l,表明该探针能对活细胞内Hcy和Cys的释放过程进行可视化观察。
In order to address two issues in fluorescence imaging:high autofluorescence background and limited light penetration. This thesis is focused on the upconversion luminescence and long lifetimes, synthesizing a series of rare-earth nanoparticles with upconversion luminescence and phosphorescent iridium complexes with-μs lifetimes for targeted imaging in vitro and in vivo.
I. Implementation of Upconversion Luminescence In Vivo Imaging System and Rare-Earth Upconversion Nanophosphors for Targeted Imaging of Tumors In Vivo and Toxicity Studies
Rare-earth upconversion nanophosphors (UCNPs) exhibit unique luminescence properties such as remarkable light penetration depth and no background fluorescence. Up until now, the broad application of UCNPs in bioimaging suffers a hindrance resulting from the absence of a commercially available system for upconcersion luminescence (UCL) imaging. Therefore, we set up the first high contrast upconversion luminescence in vivo imaging system, and developed the methods of targeted imaging of tumors in vivo using upconversion luminescence imaging protocol. Results indicated that UCL possess unique features in vivo imaging:the remarkable light penetration depth (-600μm) and no background fluorescence.
More importantly, based on the high specificity between the ligand and receptor: folic acid (FA) and folic acid receptor (FR), arginine-glycine-aspartic peptide (RGD) peptide andαvβ3 integrin receptor, we synthesized the RGD/FA-labeled UCNPs through the chemical methods, and for the first time developed UCL probes for targeted imaging of tumor in vivo. Results showed that RGD/FA-labeled UCNPs can successfully target imaging of tumors in vivo with a high signal-to-noise ratio (-24).
To date, there are only few studies concerning the cytotoxicity of UCNPs. No reports on long-term toxicological studies of UCNPs using animal models. Therefore, we demonstrated the long-term in vivo biodistribution and toxicity studies using polyacrylic acid-coated NaYF4 upconversion nanophosphors (PAA-UCNPs) as near infrared (NIR)-to-near infrared (NIR) luminescence probes. In vivo biodistribution imaging studies indicated that PAA-UCNPs originated predominantly from the liver and spleen and can be excreted from the body of the mice through the intestinal tract in a very slow manner. In vivo toxicity studies results indicated that mice intravenously injected with 15mg/kg of PAA-UCNPs survived for 115 days without any evident (body weight of the mice, histological, hematological and biochemical) toxic effects.
Ⅱ. Phosphorescent Iridium(Ⅲ) Complexes for Targeted Imaging of Tumor Cells and Ratiometric Imaging of Intracellular Homocysteine and Cysteine
1) Based on the high affinity between the integrinαvβ3 and the amino acid sequence Arg-Gly-Asp (RGD), we synthesized RGD-conjugated phosphorescent iridium complex with red emissions for targeted imaging of integrinαvβ3 expressions. Results showed that this complex can specifically target imaging integrinαvβ3 over expressioned U87MG cells with high sensitivity (2μM) and a rapid response time (-15 min). Furthermore, we designed an organic fluorescence probe for targeted imaging of integrinαvβ3 expressions. Photobleaching experiment established that this probe has higher photostability than acridine orange.
2) Thiol-containing amino acids, homocysteine (Hcy) and cysteine (Cys) play many crucial roles in biological systems. Herein, we demonstrated ratiometric phosphorescence imaging of intracellular Hcy and Cys using a cationic iridium(III) complex as a luminescent probe. Importantly, cell imaging experiments demonstrated that the probe can monitor the changes of Hcy/Cys within living cells in a ratiometric mode. When KB cells incubated with iridium complex for 30 min at 37℃showed an emission ratio (I586/I547) of> 1, indicating the reaction of iridium complex with Hcy/Cys. In a competition assay, KB cells were pre-incubated with N-ethylmaleimide (as a thio-reactive compound) at 37℃for 1 h and then incubated with iridium complex at 37℃for 30 min. As a result, the phosphorescence intensity of the green channel increased and that of the red channel decreased, so the ratio (I586/I547) was reduced to< 1.
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