Abstract
Purpose Fluorescently labeled Nanobodies (Nbs) provide rapid, specific, and high-contrast molecular imaging capabilities, making them well suited for applications such as fluorescence-guided surgery. As dye properties can sbstantially influence tracer performance, this study evaluated three next-generation FNIR-Tag dyes, each conjugated to an anti-EGFR Nb. Procedures The anti-EGFR Nb 7D12 was conjugated to FNIR-Tag-1.0, FNIR-Tag-766 or FNIR-Tag-804, and characterized in vitro. In vivo imaging and biodistribution studies were performed to assess pharmacokinetics, tumor uptake, tumor-to-background ratios, contrast-to-noise ratios, and renal clearance. Results All three Nb-based tracers exhibited similar overall pharmacokinetic behavior, characterized by fast tumor accumulation, rapid clearance from blood and non-target tissues, and predominant renal elimination. 7D12-FNIR-Tag-1.0 demonstrated slightly higher tumor uptake, whereas 7D12-FNIR-Tag-766 produced marginally improved tumor-to-background and contrast-to-noise ratios. In contrast, 7D12-FNIR-Tag-804 yielded significantly lower tumor signal intensity, although its imaging contrast remained comparable due to proportionally reduced background fluorescence. Despite differences in dye net charge, no substantial variation in kidney retention was observed over 24 h. Microscopic analysis revealed distinct renal handling: 7D12-FNIR-Tag-766 showed partial endosomal internalization within proximal tubule cells, while 7D12-FNIR-Tag-1.0 remained primarily luminal. Conclusions FNIR-Tag-labeled Nbs enable effective tumor visualization as early as 1 h post-injection. Although overall pharmacokinetics were comparable across dyes, differences in tissue uptake, intrinsic brightness, and compatibility with imaging system optics can influence detection sensitivity and should inform dye selection for clinical imaging applications.