A Cascade Recognition of Activatable Probe for Fluorescence Navigation Glioblastoma Surgery: Overcoming Blood‐Brain Barrier and Off‐Target Limitations

Precise imaging and complete resection of glioblastoma (GBM) remain critically challenging due to two major obstacles: the inefficient delivery of probes across the blood-brain barrier (BBB) and the lack of tumor-specific activation, leading to poor contrast and inadequate tumor margin delineation. To address these challenges, we developed ANG-hCy-MC, a cascade recognition of activatable probe designed for high-fidelity visualization of GBM. This probe leverages Angiopep-2-mediated targeting of LRP1 receptors to facilitate efficient BBB crossing, followed by a cascaded activation process triggered specifically by the tumor-associated enzymes cathepsin B (Cat B) and monoamine oxidase (MAO). This dual-enzyme cascade mechanism ensures ultra-selective fluorescence turn-on exclusively within tumor cells, thereby eliminating off-target signals and providing exceptional tumor-to-normal tissue contrast, enabling the precise navigation and resection of orthotopic glioblastoma in live mice. Notably, in ex vivo human GBM specimens, ANG-hCy-MC achieved a remarkable tumor-to-normal fluorescence ratio of 7.83 at the invasive edge, enabling clear identification of even single infiltrating tumor cells. This probe allows real-time, high-contrast intraoperative guidance with unprecedented resolution, offering a powerful and clinically translatable strategy for achieving complete tumor resection and improved patient outcomes in GBM surgery.