Cyanine-Based Near Infrared-II Aggregation-Caused-Quenching Probes for Particokinetic Analysis

Aggregation-caused quenching (ACQ) probes provide an integrity-reporting strategy for tracking nanocarriers by emitting strong fluorescence when molecularly dispersed within the hydrophobic core of intact carriers and becoming quenched after release into aqueous biological environments. However, previously reported near-infrared-II (NIR-II) ACQ probes suffer from limited effective emission in the NIR-II window and potential fluorescence reillumination after probe release, which may compromise nanoparticle quantification and thereby particokinetic analysis. Here, we developed a series of Flav7-derived cyanine ACQ probes with strong absorption and bright emission in the NIR-II region. By modifying terminal substituents, introducing methoxy groups to modulate intermolecular π-π stacking, and screening counterions with different sizes and hydrophobicity, ACQ-F4 was identified as the optimized probe. ACQ-F4 achieved a favorable balance between efficient nanocarrier labeling, water-triggered quenching, and suppressed reillumination in biological environments. Using ACQ-F4 as the probe, the particokinetics and biodistribution of polymeric nanoparticles (PNs) were investigated after intravenous or intraperitoneal administration. Multi-compartment integration of blood, liver, and spleen yielded approximately 90.2% systemic particle exposure after intraperitoneal administration compared with the intravenous reference. In conclusion, this study presents rationally engineered NIR-II ACQ probes for reliable bioimaging and quantification of PNs in vivo, enabling more comprehensive particokinetic assessment beyond blood kinetics alone.