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

ABSTRACT 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.