Prospects for the Use of Upconverting Nanoparticles as a Contrast Agent for Enumeration of Circulating Cells in vivo

Purpose: We recently developed a new fluorescence-based technique called "diffuse in vivo flow cytometry" (DiFC) for enumerating rare circulating tumor cells (CTCs) directly in the bloodstream. Non-specific tissue autofluorescence is a persistent problem, as it creates a background which may obscure signals from weakly-labeled CTCs. Here we investigated the use of upconverting nanoparticles (UCNPs) as a contrast agent for DiFC, which in principle could significantly reduce the autofluorescence background and allow more sensitive detection of rare CTCs. Methods: We built a new UCNP-compatible DiFC instrument (U-DiFC), which uses a 980 nm laser and detects upconverted luminescence in the 520, 545 and 660 nm emission bands. We used NaYF4:Yb,Er UCNPs and several covalent and non-covalent surface modification strategies to improve their biocompatibility and cell uptake. We tested U-DiFC with multiple myeloma (MM) and Lewis lung carcinoma (LLC) cells in tissue-mimicking optical flow phantoms and in nude mice. Results: U-DiFC significantly reduced the background autofluorescence signals and motion artifacts from breathing in mice. Upconverted luminescence from NaYF4:Yb,Er microparticles (U mu NP) and cells co-incubated with UCNPs were readily detectable with U-DiFC in phantoms, and from UCNPs in circulation in mice. However, we were unable to achieve reliable labeling of CTCs with UCNPs. Our data suggest that most (or all) of the measured U-DIFC signal in vitro and in vivo likely arose from unbound UCNPs or due to the uptake by non-CTC blood cells. Conclusion: UCNPs have a number of properties that make them attractive contrast agents for high-sensitivity detection of CTCs in the bloodstream with U-DiFC and other intravital imaging methods. More work is needed to achieve reliable and specific labeling of CTCs with UCNPs and verify long-term retention and viability of cells.