Engineering of an Enzyme-Activatable Ratiometric DNA Nanosensor for Spatially Resolved pH Imaging in Ischemic Stroke

Spatially resolved imaging of intracellular pH during ischemic stroke (IS) is critical for demarcating salvageable penumbral tissue and tracking lesion progression; however, engineering IS-specific pH sensors remains challenging. Herein, we engineered an apurinic/apyrimidinic endonuclease 1 (APE1)-activatable ratiometric DNA nanosensor (LPF) by encapsulating a dual-strand DNA probe (PF) within liposomes. The PF probe operates through a dual-strand molecular switch: the i-motif in the cytosine-rich F-strand undergoes pH-dependent folding, activating Cy3/Cy5 FRET for ratiometric pH sensing; simultaneously, the AP site on the P-strand blocks pH-sensing activity until cleaved by cytoplasmic APE1 upregulated in IS lesions, thereby enabling IS-specific activation. Liposomes enhance delivery to ischemic cells. In vitro studies identified 30-nt and 18-nt P-strand as optimal for protection and activation, respectively, with PF exhibiting high APE1 selectivity under acidic conditions. Confocal imaging demonstrated LPF's capability to specifically monitor pH changes in oxygen-glucose-deprived (OGD) cells, requiring cytoplasmic APE1 upregulation for activation. In MCAO mice, LPF achieved enzyme-mediated IS-specific activation and dynamically tracked pH gradients. This work establishes the first APE1-activated ratiometric nanosensor for spatially precise pH imaging in IS, providing a platform for stroke diagnosis and personalized intervention.