A Comparative Study on the Effect of Near-Infrared Fluorescent Dyes on the Pharmacokinetic Behavior of Nanobody-Based Tracers for In Vivo Imaging

Near-infrared fluorescent molecular imaging is increasingly being used as a tool to guide intraoperative decision-making. While research into novel fluorescent molecular tracers often prioritizes the targeting moiety, compelling evidence indicates that the choice of fluorophore can substantially influence tracer pharmacokinetics. In this study, tumor-specific Nanobodies were conjugated with four widely used dyes─IRDye800CW, ZW800-1, FNIR-Tag, and s775z-, and a side-by-side comparison of their in vivo biodistribution and tumor targeting was performed. Nanobodies labeled with FNIR-Tag or s775z showed markedly superior results compared to those labeled with IRDye800CW or ZW800-1, demonstrating strong tumor accumulation as early as 1 h postinjection and minimal background signal, particularly in the liver. The effect of increasing dye density on tracer pharmacokinetics was also assessed. Compared with Nbs labeled with a DOL of 1, those with an average of 2 dyes exhibited higher mean fluorescent tumor signals but no improvement in tumor-to-background ratios, owing to increased background signals.