Multicolor cyanine dendritic dots for real-time, high-resolution tracking of nanoparticle biodistribution

Fluorescence imaging has become a powerful tool for visualizing nanoparticles in drug delivery systems, and multicolor fluorescence imaging offers an intuitive approach to exploring complex biological systems. Here, we present multicolor cyanine-based polylysine (PLL) dendritic dots (Cy3/Cy5/Cy7-PLL; up to eight generations) with a well-defined single-molecule structure and bright, photostable fluorescence. Using these probes, we achieved low-crosstalk three-channel tracking in live mice, enabling high-resolution, real-time visualization of nanoparticle biodistribution and within-subject comparisons that reduce inter-animal variability. By systematically modulating size and surface chemistry, we investigated how these parameters govern biodistribution, encompassing their efficacy in <i>in vitro</i> and <i>in vivo</i> tumor imaging, as well as nanoparticle tracking at both cellular and organ levels. We demonstrate that these multicolor dendritic dots serve as an advanced method for simultaneous, real-time tracking of nanoparticles with different traits, offering significant potential for future applications in various biomedical fields.