A GSH and β-Galactosidase Sequential-Activated Dual-Locked Fluorescent Nanoprobe for Precise Liver Cancer Diagnosis and Intraoperative Navigation

Abstract Accurate intraoperative navigation for solid tumors is often hindered by the nonspecific activation of imaging probes in non-target tissues. In this work, a near-infrared dual-locked fluorescent nanoprobe HCy-M@MSN@PEG was developed that can be sequentially activated by glutathione (GSH) and β-galactosidase (β-Gal), enabling highly specific imaging of liver tumors. This nanoprobe is constructed by encapsulating a β-Gal-responsive small-molecule probe (HCy-M) within PEGylated mesoporous silica nanoparticles (MSN@PEG). Upon exposure to the first biomarker GSH, the nanoprobe undergoes rapid disassembly, releasing the encapsulated HCy-M, which then responds to the second biomarker β-Gal, producing a fluorescence turn-on signal that enables selective imaging of liver tumors. Through meticulous material optimization, including the incorporation of disulfide bonds into both the MSNs framework and surface PEG chains to accelerate GSH responsiveness, as well as the modification of the structure of small-molecule probe to balance loading efficiency and release rate, the nanoprobe achieved a release efficiency of 42.9% within 3 h under GSH stimulation, and the released HCy-M responded to β-Gal within 20 min with a 10.25-fold fluorescence enhancement. The probe efficiently discriminated between normal, senescent, and liver cancer cells. In vivo, it exhibited excellent tumor targeting with a signal-to-background ratio of 4.47 and successfully guided precise resection of liver tumors via fluorescence imaging in mouse models. This work provides a promising tool for precise liver cancer diagnosis and intraoperative navigation, with potential for advancing fluorescence-based theranostics.