Spatially Controlled Imaging of Proteases in Acidic Extracellular Milieu of Lesions Using DNA‐Peptide‐PNA Hybrid Probes

In situ detection of proteases has been extensively exploited for diverse biomedical applications; however, spatially controlled amplified imaging of extracellular proteases at diseased sites remains a significant challenge. Here, we report the design of DNA‐peptide‐PNA hybrid probes for sensitive imaging of protease activity within the acidic extracellular milieu of lesions, achieving enhanced spatial selectivity. In the strategy, both a pH‐sensitive peptide and a protease‐cleavable peptide substrate are rationally integrated into a DNA‐based signal amplification platform. Specifically, a pH (low) insertion peptide (pHLIP) enables controlled localization of a peptide‐substrate‐engineered DNA initiator on the cell membrane under acidic conditions. Subsequent protease‐mediated cleavage of the peptide substrate triggers signal amplification via initiator‐mediated hybridization chain reaction (HCR), resulting in markedly improved spatial controllability and detection sensitivity for extracellular protease profiling in diseased microenvironments. Furthermore, the molecular imaging capability of the DNA‐Peptide‐PNA hybrid system was validated for matrix metalloproteinase 2/9 (MMP2/9) in vivo using both 4T1 tumor and osteoarthritis models. These results highlight the potential of this strategy as a versatile platform for the specific imaging of diverse enzymes within extracellular environments characterized by pH dysregulation.