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We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore-fluorophore or fluorophore-quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications.

pHLIP-FIRE, a Cell Insertion-Triggered Fluorescent Probe for Imaging Tumors Demonstrates Targeted Cargo Delivery In Vivo