Activatable Near-Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha

Fibroblast activation protein-alpha (FAP alpha) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAP alpha has also been reported to promote. tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAP alpha inhibitors has led to the discovery of many FAP alpha-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAP alpha in vivo has largely lagged behind. Herein, we report an activatable near-infrared (NIR) fluorescent probe (ANP(FAP)) for in vivo optical imaging of FAPa. The ANP(FAP) consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAP alpha cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANP(FAP), high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAP alpha both in vitro and in vivo. In vitro assay on ANP(FAP) indicated the specificity of the probe to FAP alpha. The in vivo optical imaging using ANP(FAP) showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAP alpha expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAP alpha expression, demonstrating the in vivo targeting specificity of the ANP(FAP). Ex vivo imaging also demonstrated ANP(FAP) had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANP(FAP) could serve as a useful NIR optical probe for early detection of FAP alpha expressing tumors.