Fluorescent protein chromophore-based photosensitizer modified with anthracenyl for lysosome-targeted photodynamic therapy and two-photon fluorescence imaging

Abstract

In this work, two cis-anthracenyl fluorescent protein chromophore analogs, AnFP-1 and AnFP-2 were synthesized by [2 + 3] cycloaddition for photodynamic therapy and two-photon fluorescence imaging. We combine the anthracenyl group and fluorescent core imidazolinone to form the dye AnFP-1, and then the morpholine indole structure was introduced via Knoevenagel reaction to obtain the dye AnFP-2. The heavy-atom-free photosensitizing dyes AnFP-1 and AnFP-2 have satisfied yields of singlet oxygen, measured to be 30% and 21% in methanol solution, respectively. The tetrazolium blue (MTT) experiment showed that the survival rate of A549 cells was over 92% without light exposure and under 460 nm light exposure for 10 min, the half-inhibitory concentrations (IC50) of the photosensitizing dyes AnFP-1 and AnFP-2 were 3.7 μmol•L−1 and 4.1 μmol•L−1, respectively. Acridine orange (AO)/Ethidium bromide (EB) double staining experiments showed that the two photosensitizing dyes could generate singlet oxygen and induce apoptosis in cancerous cells under irradiation. In addition, the photosensitizing dye AnFP-2 has high specificity to lysosomes which improve the photodynamic effect. Its Pearson co-localization correlation coefficient is 93%. In biological imaging, AnFP-1 and AnFP-2 showed satisfactory two-photon fluorescence imaging in zebrafish with 800 nm femtosecond laser excitation. According to these results, AnFP-1 and AnFP-2 can be used in photodynamic therapy and two-photon fluorescence imaging, which lays the foundation for developing new photosensitizing dyes.