Conditionally Activated ROS Generation by MMP2/9‐Specific C ₆₀ ‐Based Fluorescence Probes

Abstract A C ₆₀ ‐coumarin conjugate, covalently connected via a matrix metalloproteinase (MMP2/9)‐cleavable peptide linker (Pro‐Leu‐Gly‐Val‐Arg‐Gly), is developed as a probe for both imaging and photodynamic treatment of MMP2/9‐expressing malignant cells. In the synthesized probe, the coumarin fluorescence is completely suppressed intramolecularly by the C ₆₀ moiety, while an intensive fluorescence increase is observed in the presence of MMP2/9 dependent on cleavage of the peptide linker. The specificity of the probe to detect MMP2 is confirmed by control experiments resulting in no emission 1) with a control probe bearing a shuffled peptide (Val‐Arg‐Leu‐Gly‐Pro‐Gly) or 2) in the presence of an MMP2 inhibitor (1,10‐phenantheoline). The probe is added to three types of tumor cell lines with different MMP2/9‐expression levels to perform in vitro cellular imaging. MMP2/9 is successfully detected by the probe in DAOY cells with the highest expression level of MMP2/9. Upon cleavage of the probe by MMP2/9, photoinduced ¹ O ₂ generation is much enhanced, resulting in the MMP‐dependent induction of higher photocytotoxicity of the probe in DAOY compared to HCT116 with much lower expression. The results suggest that the probe can serve as (I) a fluorescent reporter for MMP2/9 and (II) a photosensitizer for selective photodynamic treatment of malignant cells with MMP2/9 overexpression.