Molecular tuning of cyanine 5 dyes to improve the pharmacokinetic profile of Nanobody-based fluorescent tracers

Over the past two decades, it has become evident that fluorescence imaging holds substantial value in preclinical research and could also play a pivotal role in clinical applications such as intraoperative molecular imaging. The latter relies on applying targeted fluorescent agents designed to recognize specific biomarkers expressed in diseased tissues. Targeting moieties, such as camelid-derived nanobodies (Nbs), exhibit remarkable pharmacokinetics for molecular imaging owing to their robustness and compact size. However, the relatively small size of Nbs makes their pharmacokinetics sensitive to the chemical structure of attached fluorophores. In this study, we conducted a comparative analysis between Nbs labeled with three different sulfoCy5 derivatives (Cy5^2- (charge −2), Cy5^- (charge −1), and Cy5° (charge 0)). Nb-Cy5^2- and Nb-Cy5° allowed specific in vivo visualization of subcutaneous tumors in mice within 1 h with minimal background. Conversely, Nb-Cy5^- required at least 3 h to achieve sufficient contrast and exhibited nonspecific liver accumulation. Remarkably, Nb-Cy5^2- was able to overcome the renal retention typically observed for Nbs. Microscopy analyses of kidney sections revealed differential accumulation for Nb-Cy5^2- and Nb-Cy5° at the level of the proximal tubule cells, with only Nb-Cy5^2- showing internalization in lysosomes and endosomes and subsequent metabolization. In conclusion, this study underscores the significant influence of dye charges on the biodistribution profile and tumor-targeting capabilities of Nb tracers. Among the tested variants, the fluorescent dye Cy5^2- emerged as a promising choice to use in combination with Nbs for molecular imaging applications, in particular due to its low renal retention.