CytoCy5S™, a compound of many structures. in vitro and in vivo evaluation of four near-infrared fluorescent substrates of nitroreductase (NTR)

Abstract CytoCy5S™, a quenched, red-shifted fluorescent probe, has been used to exploit the imaging potential of the nitroreductase (NTR) reporter gene platform. Its use has been reported in a number of publications, however there are discrepancies in both the reported structure and its physicochemical properties. Herein, we aim to highlight these discrepancies and to define the best candidate of the four substrates under study for preclinical work in NTR reporting by optical applications. We report the synthesis, purification and characterisation of four NTR substrates, including alternately described structures currently referred by the name CytoCy5S. A comparative NTR enzymatic assay was performed to assess the spectroscopic characteristics of the different reductively activated probes. The NTR expressing triple-negative breast carcinoma cell line, MDA-MB-231 NTR+, was employed to compare, both in vitro and in vivo, the suitability of these fluorescent probes as reporters of NTR activity. Comparison of the reporting properties was achieved by flow cytometry, fluorescence microscopy and optical imaging, both in vivo and ex vivo. This study evaluated the different spectroscopic and biological characteristics of the four substrates and concluded that substrate 1 presents the best features for oncological in vivo preclinical optical imaging.