Molecular Probes for Biomembrane Recognition

项目总结/摘要 目的是生产新型近红外荧光多价分子探针， 膜受体聚集的高级细胞显微镜研究以及范式转换应用 在荧光引导手术中。有证据表明携带多个受体拷贝的多价探针 拮抗剂可以触发探针的受体聚集和细胞内吞，这是许多生物学研究所高度期望的。 成像应用。但目前不可能通过合理的设计来生产有效的多价 分子探针用于可靠的体内靶向。第一个具体目标将利用一个新的可编程预- 快速产生本征明亮、光稳定、近红外荧光库的组装方法 具有系统性改变的多价结合性质的分子探针， 距离和PEG化程度来控制生物分布。将构建35个成员的多价探针文库。 用携带不同拷贝数的环状五肽cRGDfK的每个成员制备， 整合素受体的配体。荧光探针将针对广泛的癌症，但这项提议将 卵巢癌是卵巢癌的一种常见类型。探针库将 用卵巢癌细胞筛选促进广泛整合素介导的内吞作用的能力。细胞 显微镜实验将使用来自文库的最佳探针来可视化和量化多价聚类 整联蛋白受体，一个重要但定义不清的细胞生物学过程，如信号传导， 增殖、迁移和癌症转移。第二个特定的目标将产生两个不同的近红外 用于荧光引导手术的荧光多价探针。一个子目标将产生一个体内 荧光探针，可以成像上皮卵巢癌在临床相关的小鼠模型。一种新型 将开发成像方案以确定观察到的探针信号是否来源于肿瘤结节， 表面或深埋在组织中。荧光探针将使外科医生能够识别肿瘤结节 小于1毫米，比目前在最佳治疗期间切除的结节尺寸小十倍以上。 细胞减灭术将切除的卵巢癌结节的大小降低一个数量级， 预计会增加患者术后的存活率。第二个子目标将产生高性能， 荧光多价肽探针，可以帮助外科医生可视化薄和埋藏的神经， 伤害他们。该提案的高整体影响力来自于多功能预处理器的创新， 一种快速制备近红外荧光多价分子库的组装合成方法 几乎任何细胞表面生物标志物的探针，以及两个提出的荧光分子的意义， 用于荧光引导手术中的单独和临床重要应用的探针。

Project Summary/Abstract The objective is to produce new classes of near-infrared fluorescent, multivalent molecular probes for advanced cell microscopy studies of membrane receptor clustering and also for paradigm shifting applications in fluorescence guided surgery. There is evidence that multivalent probes bearing multiple copies of a receptor antagonist can trigger receptor clustering and cell endocytosis of the probe, which is highly desired for many imaging applications. But presently it is not possible to produce, by rational design, an effective multivalent molecular probe for reliable in vivo targeting. The first specific aim will utilize a new programmable pre- assembly method to rapidly produce libraries of intrinsically bright, photostable, near-infrared fluorescent molecular probes with systematically altered multivalent binding properties such as ligand loading, linker distance, and degree of PEGylation to control biodistribution. A 35-member library of multivalent probes will be prepared with each member bearing a different copy number of the cyclic pentapeptide, cRGDfK, a targeting ligand for integrin receptors. The fluorescent probes will target a broad range of cancers, but this proposal will focus on epithelial ovarian carcinoma, the most common clinical form of ovarian cancer. The probe library will be screened with ovarian cancer cells for ability to promote extensive integrin-mediated endocytosis. Cell microscopy experiments will use the best probes from the library to visualize and quantify multivalent clustering of integrin receptors, a centrally important but poorly defined process in cell biology events such as signaling, proliferation, migration, and cancer metastasis. The second specific aim will produce two different near-infrared fluorescent multivalent probes for use in fluorescence guided surgery. One sub-aim will produce an in vivo fluorescent probe that can image epithelial ovarian cancer in a clinically relevant mouse model. A novel imaging protocol will be developed to determine if an observed probe signal originates from a tumor nodule on the surface or buried deep in the tissue. The fluorescent probe will enable surgeons to identify tumor nodules that are <1 mm, which is more than ten times smaller than the nodule size currently removed during optimal cytoreductive surgery. Lowering the size of resected ovarian cancer nodules by an order of magnitude is predicted to increase patient survivorship after surgery. A second sub-aim will produce a high performance, fluorescent multivalent peptide probe that can help a surgeon to visualize thin and buried nerves and avoid damaging them. The high overall impact of the proposal derives from the innovation of the versatile pre- assembly synthesis method to rapidly prepare libraries of near-infrared fluorescent multivalent molecular probes for virtually any cell surface biomarker, and the significance of the two proposed fluorescent molecular probes for separate and clinically important applications in fluorescence guided surgery.