CONTRAST AGENTS FOR FAR RED AND NEAR INFARED IMAGING

需要有工具来监测免疫细胞相互作用及其在体外和体内的后果，以加速开发成功的针对癌症、艾滋病毒疾病和自身免疫性疾病的免疫疗法。细胞蛋白（CFSE）或膜（PKH染料）的稳定荧光标记就是这样的工具。然而，高水平的自身荧光和可见光区域的光谱重叠限制了它们在体外的使用，并且在完整动物体内的实时光学成像被高水平的组织吸收和散射所阻碍。我们假设，在远红色（FR）或近红外（NIR）发出荧光的稳定膜标记将：a)使用流式细胞术和共聚焦显微镜实现更敏感的细胞跟踪，b)为全动物免疫细胞运输的光学成像提供新的非放射性造影剂，c)有潜力成为未来的光诊断和/或光疗剂。在第一阶段，我们将合成两类探针，一类是FR （600-700 nm）激发/发射，另一类是MR （700-900 nm）激发/发射。每个探针的细胞毒性、膜保留、信号：噪声和与常用抗体标签的光谱重叠将使用流式细胞术和共聚焦显微镜进行表征。宏观成像的潜在效用将通过确定含有不同标记和未标记细胞混合物的组织“幻象”的图像质量与深度来评估。提出的商业应用：在远红或近红外发射的膜标记染料在三个市场具有显著的商业潜力：1)使用流式细胞仪服务于基础和临床研究人员的研究试剂市场；2)光学成像，特别是宏观或全身成像的类似但不断增长的市场；3)光诊断或光治疗。

Tools to monitor immune cells interactions and their consequences in vitro and in vivo are needed to accelerate development of successful immunotherapies for cancer, HIV disease, and autoimmune disorders. Stable fluorescent labels for cell proteins (CFSE) or membranes (PKH dyes) are such tools. However, high levels of autofluorescence and spectral overlap in the visible region limit their use in vitro, and real time optical imaging in intact animals is precluded by high levels of tissue absorption and scattering. We hypothesize that stable membrane labels that fluoresce in the far red (FR) or near infrared (NIR) will: a) enable more sensitive cell tracking using flow cytometry and confocal microscopy, b) provide novel non-radioactive contrast agents for optical imaging of immune cell trafficking in whole animals, and c) have potential as future photodiagnostic and/or phototherapeutic agents. In Phase I we will synthesize two classes of probes, one with FR (600-700 nm) excitation/emission and the other with MR (700-900 nm) excitation/emission. Each probe's cytotoxicity, membrane retention, signal:noise, and spectral overlap with commonly used antibody labels will be characterized using flow cytometry and confocal microscopy. Potential utility for macroscopic imaging will be assessed by determining image quality vs. depth in tissue "phantoms" containing varying admixtures of labeled and unlabeled cells. PROPOSED COMMERCIAL APPLICATIONS: Membrane labeling dyes emitting in the far red or near infrared have significant commercial potential in three markets: l) a research reagent market serving both basic and clinical investigators using flow cytometers; 2) a similar but growing market in optical imaging, especially macroscopic or whole body imaging; and 3) either photodiagnosis or phototherapy.