A GSH-responsive NIR-BODIPY fluorophore with large Stokes-shift for tumor specific fluorescence imaging and surgical guidance

The tumor microenvironment (TME)-activatable probes have proven effective in enhancing the signal-to-background ratio (SBR) for precise fluorescence imaging in tumor diagnosis. However, many fluorophores have suboptimal emission spectra and a short Stokes shift, which may lead to overlap with bio-autofluorescence, excitation, and emission spectra, limiting their use in intraoperative guidance. Herein, a γ-glutathione (GSH) responsive near-infrared (NIR) BODIPY probe, named “Pro-Dye” was synthesized with a large Stokes shift of 91 nm. The Pro-Dye can be rapidly and specifically activated by high concentrations of GSH both in solution and inside cancer cells, while remaining inactive in normal cells (Human umbilical vein endothelial cells, HUVECs). The Pro-Dye was further encapsulated by 1,2-distearoyl-sn‑glycero-3-phosphoethanolamine-N-(polyethylene glycol)-5000 (DSPE-PEG₅₀₀₀) to form Pro-Dye nanoparticles (NPs), making it water-dispersible for in vivo application. in vivo fluorescence imaging demonstrated that Pro-Dye NPs can accumulate at the tumor and exhibit an improved SBR compared to the “always-on” probe (Dye NPs). Moreover, the tumor can be precisely resected under the real-time guidance of fluorescence imaging of Pro-Dye NPs, showing a well-defined tumor margin.