Phlip-Fire: A High-Contrast, Insertion-Triggered Fluorescent Probe for Targeting Tumors In Vivo

pHLIP-FIRE (Fluorescence Insertion REporter) is a novel diagnostic tool based on pHLIP (pH Low Insertion Peptide), a water soluble membrane peptide that interacts with membranes by forming a transmembrane helix at low pH. The fluorescence of pHLIP-FIRE is activated by separation of mutually quenching or donor-quencher fluorescent groups via the cleavage of a disulfide-linker when the C-terminus moves into the highly reducing environment inside the cell. Targeting studies in mice bearing breast tumors showed strong targeting of pHLIP-FIRE in vivo, giving a contrast index of 20 with respect to background. This contrast is a significant enhancement in comparison with traditional fluorescent pHLIP constructs. Experiments in vitro and in cultured cells show that the time-course of dequenching in pHLIP-FIRE is pH-dependent and quite slow ( ∼ 1 - 2 days). Direct imaging with pHLIP-FIRE gives the first demonstration of pHLIP insertion and cargo translocation in vivo. Our technology shows promise as a tool for cancer diagnosis or image-guided surgery, and the demonstration of cargo delivery into tumors may lead to a new generation of targeted chemotherapeutic delivery.