A Comparative Study on the Effect of Near-Infrared Fluorescent Dyes on the Pharmacokinetic Behavior of Nanobody-Based Tracers for In Vivo Imaging
Noemi B. Declerck, Sofie Declercq, Pieterjan Debie, Julie Du Ville, Łukasz Mateusiak, Jelena Saliën, Marcus C. M. Stroet, Michael Luciano, Dong‐Hao Li, Martin J. Schnermann, Bradley D. Smith, Sophie Hernot
Abstract
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.