Atg4B-Activated Near-Infrared Nanoprobe for Precise Imaging of Autophagy <i>In Vivo</i>

Autophagy plays a crucial role in maintaining cellular homeostasis, and its dysregulation is implicated in various diseases. High-precision imaging of autophagy activity in deep tissues is vital for elucidating the mechanisms of autophagy-related disorders, yet existing near-infrared (NIR) fluorescent probes often lack sufficient accuracy. To overcome this limitation, we developed an Atg4B-activated NIR nanoprobe for highly selective detection of autophagy. The "dual quenched" fluorescent probe, <b>IR780-CBT NP</b>, was synthesized <i>via</i> a CBT-Cys click condensation reaction using the fluorescent precursor Cys(StBu)-Thr-Phe-Gly-Lys(IR780)-CBT (<b>IR780-CBT</b>) under reducing conditions. Upon cellular uptake by autophagy-active cells, Atg4B-specific hydrolysis induces disassembly of <b>IR780-CBT NPs</b>, restoring NIR fluorescence. In autophagy-activated MDA-MB-231 cells and tumor-bearing mice, <b>IR780-CBT NPs</b> exhibited 4.5-fold and 3.1-fold enhanced fluorescence signals, respectively, compared to the "RAPA + Inh."-treated controls. These findings highlight the potential of <b>IR780-CBT NPs</b> for precise autophagy imaging <i>in vivo</i>, offering a promising tool for early diagnosis and mechanistic studies of autophagy-related diseases.