Photoswitchable nanoprobes for in vivo flow cytometry

描述（由申请人提供）：前所未有的纳米技术进步有望彻底改变癌症诊断和治疗。然而，尽管为了解癌症生物学做出了大量努力，但导致高达90%的癌症死亡的转移仍然知之甚少。全面的研究已经证明了使用循环肿瘤细胞（CTC）的数量作为转移发展的标志物的巨大潜力。在不同的CTC测定中，体内光声（PA）流式细胞术（PAFC）展示了在自然生物环境中用功能化纳米颗粒（NP）标记的CTC的高通量、实时研究的独特能力。然而，尽管分子CTC靶向具有优势，但常规标记程序使得难以追踪单个CTC，这对于理解癌症转移的机制是重要的，包括鉴定CTC的起源（即，来自原发性肿瘤或来自转移）以及再接种或自接种过程的作用。该项目的目标是开发一个平台，用于工程化，表征和优化非荧光光谱可切换SNP，用作PA造影剂，可以在体内跟踪单个CTC。我们假设，可以通过使用特定波长的短激光脉冲直接在血流中进行NP的超快光谱切换来跟踪NP靶向的单个CTC，然后对含有切换的NP的CTC进行实时PA荧光监测。我们将通过以下具体目标测试该假设来完成我们的目标测试：（1）开发用于工程化和表征光谱可切换纳米颗粒作为光致光热和PA造影剂的平台;（2）测试优化的可切换纳米颗粒用于癌细胞的生物缀合和分子靶向的能力;以及（3）在体内研究可转换纳米颗粒的性质和通过超快光开关跟踪转移级联中的单个CTC。开发这项技术将允许对CTC进行体内研究，这将澄清早期转移性疾病的机制，并有助于开发先进的诊断技术和个性化治疗。跟踪单个标记的细胞可以提高我们对循环系统中细胞行为的理解，并提供跟踪任何循环细胞或细胞组的生命周期的独特方法。这种能力可以使研究界发现和评估与健康和疾病相关的生理和病理机制，包括在单细胞水平上研究免疫系统功能，菌血症，败血症和凝血。拟议的技术是用于临床前动物模型的先进研究工具，并有可能被批准用于人类，因为PA流式细胞术可安全用于人类，并且几种NP已被批准用于中试试验。

DESCRIPTION (provided by applicant): Unprecedented nanotechnological advances hold the promise to revolutionize cancer diagnostics and treatment. Nevertheless despite substantial efforts to understand cancer biology, metastases, which cause up to 90% of cancer deaths, are still poorly understood. Comprehensive studies have demonstrated the tremendous potential of using the number of circulating tumor cells (CTCs) as a marker of metastatic development. Among different CTC assays, in vivo photoacoustic (PA) flow cytometry (PAFC) demonstrates a unique capability for high-throughput, real-time study of CTCs labeled with functionalized nanoparticles (NPs) in the natural biological environment. However, despite the advantages of molecular CTC targeting, conventional labeling procedures make it difficult to track individual CTCs, which are important to understanding the mechanisms of cancer metastasis, including identification of the origin of CTCs (i.e., from primary tumor or from metastases) and the role of re-seeding or self-seeding processes. The goal of this project is to develop a platform for engineering, characterizing and optimizing nonfluorescent spectrally switchable SNPs to be used as PA contrast agents that can track individual CTCs in vivo. We hypothesize that individual CTCs targeted by NPs can be tracked through ultrafast spectral switching of NPs directly in the bloodstream using short laser pulses of specific wavelengths that are followed by real-time PA multicolor monitoring of CTCs containing the switched NPs. We will accomplish our goal test by testing this hypothesis through the following specific aims: (1) develop a platform fo engineering and characterizing spectrally switchable nanoparticles as multicolor photothermal and PA contrast agents; (2) test the capabilities of optimized switchable nanoparticles for bioconjugation and molecular targeting of cancer cells; and (3) study in vivo the properties of switchable nanoparticles and tracking of individual CTCs in the metastatic cascade via an ultrafast photoswitching. Developing this technology will allow in vivo study of CTCs that will clarify the poorly understood mechanisms of early metastatic disease and could help develop advanced diagnosis techniques and individualized therapies. Tracking individually labeled cells can improve our understanding of cell behavior in the circulatory system and provide a unique means of tracking the life cycle of any circulating cell or groups of cells. This ability can enabl the research community to discover and assess the physiological and pathological mechanisms related to health and diseases, including studies of immune system function, bacteremia, sepsis and clotting at the single cell level. The proposed technology is an advanced research tool for use in pre-clinical animal models and has the potential to be approved for human use because PA flow cytometry is safely used in humans and several NPs have been approved for pilot trials.