NEAR INFRARED PH-SENSITIVE MOLECULAR PROBES FOR MICROSCOPY OF CELLS AND TISSUES

描述（由申请人提供）：许多生理过程在良好控制的pH范围内有效地发挥作用，并且与该范围的显著偏差通常指示病理状况。在细胞内，不同细胞器中pH梯度的维持允许差异化的 激活某些酶或产生蛋白质的活性构象以获得短暂或延长的活性。在全球范围内，细胞事件的总和可以反映组织和器官系统的pH。监测生物化学事件的pH的常见策略依赖于使用pH敏感的荧光分子探针来监测不同的生理和病理过程，包括与癌症、细胞增殖、内吞和其他生理过程相关的研究。目前，在分子细胞生物学中使用pH敏感性荧光染料与用于体内疾病成像之间存在重大脱节。这是因为这些染料中的大多数在可见波长内发荧光，其中天然荧光团和自发荧光对光的吸收限制了它们在组织和细胞成像中的使用。为了克服这些问题，我们建议开发一类新的pH敏感的近红外荧光分子探针，这是有用的成像分子过程的显微镜和全身动物成像的扩散光学断层扫描。这种方法的优点包括由于低自发荧光而具有高检测灵敏度，低光毒性， 这是因为使用了低能量辐射，并且光谱窗口扩展到900 nm，这允许使用400和900 nm波长之间的多个荧光团。更重要的是，从分子细胞生物学研究中收集的信息可以被翻译到小动物身上，并最终被翻译到人类身上，因为NIR光可以穿透到深层组织中。我们将通过以下方式实现这些目标：（1）设计和开发两类新的pH敏感性NIR染料;（2）证明它们对细胞中pH依赖性分子过程成像的实用性;（3）对体内实体瘤中酸中毒的发作进行成像。本项目产生的产品将分发给其他研究者用于研究目的。该项目的目标是（1）设计和开发两种新的pH敏感型NIR染料;（2）通过显微镜证明它们在细胞中pH依赖性分子过程成像中的实用性;（3）在体内实体瘤中成像酸中毒的发作。开发的技术平台将有助于各种基于pH的生物应用。

DESCRIPTION (provided by applicant): Many physiological processes function efficiently within a well-controlled pH range and significant deviations from this range typically are indicative of pathologic conditions. Within cells, maintenance of a pH gradient in different organelles allows for differential activation of certain enzymes or generation of active conformations of proteins for transient or prolonged activity. Globally, the sum of cellular events can reflect on the pH of tissues and organ systems. A common strategy to monitor the pH of biochemical events relies on the use of pH-sensitive fluorescent molecular probes to monitor diverse physiological and pathological processes, including studies related to cancer, cell proliferation, endocytotic and other physiological processes. Currently, there is a major disconnection between the use of pH-sensitive fluorescent dyes in molecular cell biology and for imaging diseases in vivo. This is because most of these dyes fluoresce in the visible wavelengths where absorption of light by native fluorophores and autofluorescence limits their use in both tissue and cell imaging. To overcome these problems, we propose to develop new classes of pH-sensitive near infrared fluorescent molecular probes that are useful for imaging molecular processes by microscopy and whole-body animal imaging by diffuse optical tomography. Advantages of this approach include high detection sensitivity because of low autofluorescence, low phototoxicity because of the use of low energy radiation, and the extension of the spectral window to the 900 nm which allows the use of multiple fluorophores between 400 and 900 nm wavelengths. More importantly, information gleaned from the molecular cell biology studies can be translated to small animals, and eventually to humans with the same pHsensitive NIR molecular probe because NIR light can penetrate into deep tissues. We will accomplish these goals by (1) designing and developing two new classes of pHsensitive NIR dyes; (2) demonstrating their utility to image pH-dependent molecular processes in cells; and (3) imaging the onset of acidosis in solid tumors in vivo. The products generated from this project will be distributed to other investigators for research purposes. Project Narrative The goals of this project are to (1) design and develop two new classes of pH-sensitive NIR dyes; (2) demonstrate their utility to image pH-dependent molecular processes in cells by microscopy; and (3) image the onset of acidosis in solid tumors in vivo. The technology platform developed will be useful for diverse pH-based biological applications.