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Photoswitchable nanoprobes for in vivo flow cytometry

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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.

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