Functionalized Near-Infrared Cyanine Dyes for Fluorescence Imaging and Diagnostics

The utilization of fluorescent cyanine dyes that emit within the near-infrared window significantly enhances biological imaging due to enhanced light penetration and minimal background fluorescence. This thesis describes efforts to develop new classes of functionalized cyanine dyes for imaging and diagnostics. Chapter 1 provides an overview of cyanine dye structure and the major concepts underlying the rational design of bioresponsive fluorescent cyanine probes. The development of heptamethine cyanine probes for imaging hypoxic cancer cells is described in Chapters 2 and 3. A first generation probe (Chapter 2) included a reactive p-nitrobenzyl group that produced "turn on" fluorescence when it entered hypoxic cells. A second-generation version (Chapter 3) has additional appendages that optimize the probe photophysics for eventual operation in living subjects. The probe design in Chapter 4 is a functionalized cyanine dye with targeting ligands that enhance affinity for receptors on the surface of cancer cells in culture and within tumor spheroids. A comparison of mono- versus tri-valent targeting systems found the tri-valent version to exhibit greater cell imaging contrast. In Chapter 5, esterase-responsive near-infrared probes are harnessed for photodynamic therapy of cancer cells. One molecular platform was a heptamethine cyanine probe that showed dual activation of fluorescence and singlet oxygen production. A second esterase-responsive near-infrared probe was a derivative of 5-aminoevulinic acid and found to produce high amounts of protoporphyrin IX in cancer cells for more effective photodynamic therapy. Chapter 6 turns to the topic of fluorescence sensing and describes a fluorescent ratiometric supramolecular tandem assay for detecting phosphatase and phytase enzymes. The study capitalizes on the selective binding of a calixpyridiniumn quencher molecule to a polyanionic pyrene fluorophore with no binding to a cationic near-infrared pentamethine cyanine. The three-component system enabled the creation of ratiometric fluorescence enzyme assays that detected the presence of alkaline phosphatase and phytase.

发射于近红外窗口（near-infrared window）的荧光菁染料（fluorescent cyanine dyes），可凭借更强的光线穿透性与极低的背景荧光，大幅提升生物成像性能。本论文围绕开发用于成像与诊断的新型功能化菁染料（functionalized cyanine dyes）展开研究工作。第1章概述了菁染料的结构，以及合理设计生物响应型荧光菁探针（bioresponsive fluorescent cyanine probes）的核心原理。第2章与第3章阐述了用于成像缺氧癌细胞（hypoxic cancer cells）的七甲川菁染料（heptamethine cyanine）探针的开发过程：第2章介绍的第一代探针引入了反应性对硝基苄基（p-nitrobenzyl）基团，该基团在进入缺氧癌细胞后可触发荧光"点亮"（turn on）效应；第3章的第二代探针则新增了修饰性侧链，用于优化探针的光物理性能，以适配最终的活体成像应用。第4章的探针设计采用了带有靶向配体（targeting ligands）的功能化菁染料，该配体可提升探针与培养体系内及肿瘤球（tumor spheroids）表面癌细胞受体的结合亲和力；通过对比单价与三价靶向体系，研究发现三价靶向体系可实现更高的细胞成像对比度。第5章聚焦酯酶响应型（esterase-responsive）近红外探针在癌细胞光动力疗法（photodynamic therapy）中的应用：其中一款分子平台为七甲川菁染料探针，可同时激活荧光发射与单线态氧（singlet oxygen）生成；第二款酯酶响应型近红外探针为5-氨基乙酰丙酸（5-aminoevulinic acid）的衍生物，研究发现其可在癌细胞中高效生成原卟啉IX（protoporphyrin IX），从而实现更高效的光动力疗法。第6章转向荧光传感领域，介绍了一种用于检测磷酸酶（phosphatase）与植酸酶（phytase）的荧光比率型超分子串联检测法（supramolecular tandem assay）：该研究利用杯吡啶鎓（calixpyridinium）猝灭剂分子对聚阴离子芘荧光团（polyanionic pyrene fluorophore）的选择性结合特性，且该猝灭剂不会与阳离子型近红外五甲川菁染料（pentamethine cyanine）结合；这套三组分体系可构建出比率型荧光酶检测法，用于检测碱性磷酸酶（alkaline phosphatase）与植酸酶的存在。