Construction of cathepsin B-responsive fluorescent probe and photosensitizer using a ferrocenyl boron dipyrromethene dark quencher.

A ferrocenyl boron dipyrromethene (BODIPY) has been developed and utilized as a dark quencher to construct a cathepsin B-responsive fluorescent probe and photosensitizer. The smart fluorescent probe and photosensitizer (Pc-FcQ) contains a zinc(II) phthalocyanine as the fluorescent and photosensitizing unit which is conjugated to the ferrocenyl BODIPY dark quencher via a cathepsin B-cleavable peptide substrate [Gly-Phe-Leu-Gly-Lys]. The photosensitizing properties of Pc-FcQ, including fluorescence and singlet oxygen generation, are significantly quenched through energy transfer to the BODIPY unit and subsequently by the photoinduced electron transfer from the nearby ferrocenyl moiety. Upon exposure of cathepsin B in human hepatocellular carcinoma HepG cells, the fluorescence emission of Pc-FcQ could be restored, indicating the cleavage of the peptide substrate and the separation of the phthalocyanine and ferrocenyl BODIPY unit. However, the intracellular fluorescence intensity of Pc-FcQ was largely diminished after the cells were pre-treated with cathepsin B inhibitor. Its intracellular fluorescence intensity was comparable to that of the control compound in which the peptide substrate was replaced by the non-cleavable one [Gly-Gly-Gly-Gly-Lys]. The singlet oxygen generation of Pc-FcQ was also examined in HepG2 cells as reflected by the cytotoxicity assay. The Pc-FcQ exhibited higher potency when compared with the non-cleavable analogue due to the cleavage of peptide substrate and the detachment of the BODIPY dark quencher from the phthalocyanine. The activation of the Pc-FcQ was also demonstrated in tumor-bearing nude mice. After intratumoral injection of Pc-FcQ, the fluorescence intensity at the tumor region increased gradually over 10 h as a result of the detachment of the dark quencher upon the action of cathepsin B. All the results suggest that this ferrocenyl BODIPY could serve as an efficient dark quencher and the resulting Pc-FcQ could act as the cathepsin B-responsive fluorescent probe and activatable photosensitizer.