Bright and Stable Near-IR dyes for Bioimaging

描述（由申请人提供）：光学成像是一种先进的技术，用于成像生物技术产品，如凝胶，传感器和微阵列。它也越来越多地用于监测动物中的特定分子途径。后一种技术使用吸收和发射近红外（NIR）辐射（700-900 nm）的荧光染料，这是血红蛋白和水吸收最小的光谱窗口，因此允许光子穿透组织几厘米。基于花菁的近红外染料（Cy 7）是在极少数可商购的显示近红外物理特征的荧光染料中流行的那些。不幸的是，它们在生物条件下非常容易降解，并且还已知会发生光化学异构化，导致非荧光产物。这个建议建立在我们最近发现的方酸轮烷（SR）是世界上最明亮和最稳定的荧光染料之一。目前这一代的SR染料显示出接近Cy 5染料的吸收和发射曲线。在此，我们提出了新一代NIR SR染料的合成，其可以是Cy 7染料的上级替代品。我们的计划是开发近红外吸收明亮和稳定的方酸轮烷染料，可以很容易地进行共轭与各种生物分子。它们很可能成为流行的花青（Cy 7）染料的上级替代品，并成为体外和体内成像应用中非常有用的探针。第一阶段的总体工作计划是制备新的明亮和稳定的NIR-SR染料，并比较用NIR-SR或Cy 7标记的各种生物缀合物的性质。本提案的两个具体目标是：1）将在MTTI进行的SA 1-合成新型明亮且稳定的NIR SR NHS酯并与3种不同类型的生物分子、锌配位的吡啶二甲基胺配体（Zn-DPA）、抗体和蛋白质缀合，以与Cy 7类似物进行比较。第一个目标是生产明亮和稳定的近红外SR染料，并根据化学稳定性和亮度确定最佳的两种。随后将方酸轮烷探针以及作为对照探针的Cy 7与Zn-DPA、IgG蛋白和链霉亲和素缀合。2)将在UND进行的SA 2-生物缀合物的体外评价。将使用标准凝胶电泳和链霉亲和素/生物素方案表征MTTI开发的NIR-SR和Cy 7荧光IgG和链霉亲和素生物缀合物的体外分子识别性能。将使用荧光显微镜研究NIR-SR-和Cy 7-Zn-DPA探针与金黄色葡萄球菌和大肠杆菌细菌膜的相互作用及其光稳定性。目标是证明与Cy 7染料相比，在3种不同系统中使用NIR-SR染料标记的分子探针的优势。公共卫生相关性：本研究开发的荧光探针将在生物技术中用作体外诊断设备的染色剂，并在医学科学中用作活体探针，以成像活体动物的疾病。这种光学成像技术将帮助研究人员发现新的治疗方法，并最终帮助医生为特定患者选择最合适的治疗方法。后一种应用是光学成像如何有助于个性化医疗概念发展的一个例子。

DESCRIPTION (provided by applicant): Optical imaging is an advanced technique to image biotechnology products like gels, sensors, and micro arrays. It is also used increasingly to monitor specific molecular pathways in animals. This latter technique uses fluorescent dyes that absorb and emit near-infrared (NIR) radiation (700-900 nm) a spectral window where hemoglobin and water absorb minimally and therefore allow photons to penetrate several centimeters through tissue. Cyanine based NIR dyes (Cy7) are the popular ones among the very few commercially available fluorescent dyes showing NIR photophysical features. Unfortunately they are highly susceptible to degradation under biological conditions, and also known to undergo photochemical isomerization leading to nonfluorescent products. This proposal builds on our recent discovery that squaraine rotaxanes (SRs) are among the world's brightest and most stable fluorescent dyes. Current generation of SR dyes show absorption and emission profiles close to the Cy5 dyes. Herein we are proposing the synthesis of new generation NIR SR dyes which can be a superior substitute for Cy7 dyes. Our plan is to develop NIR absorbing bright and stable squaraine rotaxane dyes which can readily undergo conjugation with various biomolecules. They are likely to become superior replacements for the popular cyanine (Cy7) dyes and become extremely useful probes for both in vitro and in vivo imaging applications. The overall workplan for Phase I is to prepare novel bright and stable NIR-SR dyes and compare the properties of various bioconjugates that have been labeled with NIR-SR or Cy7. The two Specific Aims of the present proposal are: 1) SA1 to be performed at MTTI - Synthesize novel bright and stable NIR SR NHS esters and conjugate to 3 different types of biomolecules, a zinc coordinated dipicolylamine ligand (Zn-DPA), an antibody and a protein for comparison with Cy7 analogues. The first goal is to produce bright and stable NIR SR dyes and identifying the best two based on the chemical stability and brightness. This will be followed by conjugation of the squaraine rotaxane probes as well as Cy7 as a control probe to Zn-DPA, IgG protein and streptavidin . 2) SA2 to be perfomed at UND - In vitro evaluation of bioconjugates. In vitro molecular recognition performance of the NIR-SR and Cy7 fluorescent IgG and streptavidin bioconjugates developed at MTTI will be characterized using standard gel electrophoresis and streptavidin/biotin protocols. NIR-SR- and Cy7-Zn-DPA probes will be investigated for their interaction with S aureus and E coli bacteria membranes and their photostability using fluorescence microscopy. The goal is to demonstrate the advantage of using molecular probes labeled with an NIR-SR dye in 3 different systems compared with Cy7 dye. PUBLIC HEALTH RELEVANCE: The fluorescent probe developed by this research will be used in biotechnology as a stain for in vitro diagnostic devices, and in medical science as an in vivo probe to image disease in living animals. This optical imaging technology will help researchers discover new therapies and eventually it will help physicians choose the most appropriate therapy for a specific patient. This latter application is an example of how optical imaging will contribute to the evolving concept of personalized medicine.