Rationally Engineering a pH-Low Insertion Peptide Variant Probe for High-Sensitivity Imaging of Early Tumors

The detection of early stage tumors is essential in oncology but remains a formidable challenge. Here, we developed a fluorescent pH low insertion peptide (pHLIP) variant probe, 4 M pHLIP-Cy5.5, that can recognize the weak acidity (pH 6.8) of the early stage tumor microenvironment, enabling high-sensitivity imaging of small tumors in vivo. In vitro, 4 M pHLIP-Cy5.5 exhibited ultrasensitive pH-dependent conformational transitions and enhanced binding affinity to cancer cell membranes, achieving a 71-fold increase in the fluorescence intensity ratio (pH 6.8 to pH 7.4) over wild-type (WT) pHLIP-Cy5.5. In vivo, it detected tumors smaller than 20 mm³ in the BALB/c mouse model of breast cancer, showing a 7-fold higher signal than muscle tissue and a 2-fold improvement in the signal-to-background ratio over WT pHLIP-Cy5.5. Moreover, 4 M pHLIP-Cy5.5 exhibited prolonged tumor retention, with fluorescence in tumors remaining three times higher than in corresponding regions of healthy mice at 72 h postinjection. Owing to its superior pH sensitivity, rapid targeting capability, and excellent biosafety, 4 M pHLIP-Cy5.5 offers a promising strategy for early stage tumor imaging and cancer diagnosis.