A Tandem-Locked\nFluorescent Probe Activated by Hypoxia\nand a Viscous Environment for Precise Intraoperative Imaging of Tumor\nand Instant Assessment of Ferroptosis-Mediated Therapy

Noninvasive fluorescence detection of tumor-associated\nbiomarker\ndynamics provides immediate insights into tumor biology, which are\nessential for assessing the efficacy of therapeutic interventions,\nadapting treatment strategies, and achieving personalized diagnosis\nand therapy evaluation. However, due to the absence of a single biomarker\nthat effectively reflects tumor development and progression, the currently\navailable optical diagnostic agents that rely on “always-on”\nor single pathological activation frequently show nonspecific fluorescence\nresponses and limited tumor accumulation, which inevitably compromises\nthe accuracy and reliability of tumor imaging. Herein, based on intramolecular\ncharge transfer (ICT) and twisted intramolecular charge-transfer (TICT)\nhybrid mechanisms, we report a tandem-locked probe, <b>NTVI-Biotin</b>, for simultaneously specific imaging-guided tumor resection and\nferroptosis-mediated tumor ablation evaluation under the coactivation\nof nitro reductase (NTR)/viscosity. The dual-stimulus-responsive design\nstrategy ensures that <b>NTVI-Biotin</b> exclusively activates\nnear-infrared (NIR) fluorescence signals upon interaction with both\nNTR and elevated viscosity levels through triggering ICT on while\ninhibiting the TICT process. Meanwhile, functionalization with a tumor-targeting\nhydrophilic biotin-poly(ethylene glycol) moiety enhances tumor accumulation.\nThe probe’s dual-response and tumor-targeting design minimizes\nnonspecific tissue activation, allowing for precise tumor identification\nand lesion removal with a superior tumor-to-normal tissue (T/N >\n6)\nratio. More importantly, <b>NTVI-Biotin</b> was capable of evaluating\nferroptosis-mediated chemotherapeutics by real-time monitoring of\nthe alternations of NTR/viscosity levels. The results reveal that\nthe increased tumor signals of <b>NTVI-Biotin</b> following\nthe combination of ferroptosis and chemotherapy correlate well with\nthe tumor growth inhibition, demonstrating the potential of <b>NTVI-Biotin</b> to assess therapeutic efficacy.