Red fluorescent protein dimer for singlet oxygen (1O2)/ superoxide radical (O2•−) simultaneously mediated photodynamic therapy and two-photon fluorescence imaging

Abstract

In this work, two lysosome-targeting red fluorescent protein analogues (APFP-lyso and DAPFP-lyso) were synthesized by phenolthiazine and anthracene anchoring strategies for singlet oxygen (1O2)/superoxide radical (O2•−) simultaneously mediated photodynamic therapy (PDT) in A-549 cells and two-photon fluorescence imaging in zebrafish. Compared with APFP-lyso of single-branch structure, fluorescent protein dimer DAPFP-lyso shows larger Stokes shift (Δλ = 5431 cm−1) with near-infrared emission at 664 nm and achieves 1.2- and 10.2-fold enhancement in 1O2 and O2•− production efficiency, respectively. In particular, effective two-photon absorption property of fluorescent protein dimer DAPFP-lyso (δ2PA = 81 GM) imparts the vivid two-photon fluorescence imaging in zebrafish under 800 nm excitation. Theoretical calculation indicated that fluorescent protein dimer DAPFP-lyso shows small singlet-triplet (ΔEST = 0.32 eV) to boost reactive oxygen species production efficiency. Intracellular PDT in A-549 cells, it is worth mentioning that DAPFP-lyso showed good biocompatibility, negligible cell dark toxicity (MTT assay >90%) and high phototoxicity (IC50 = 4.52 μM). The fluorescence imaging in A-549 cells and zebrafish manifests dimer DAPFP-lyso could generate 1O2 and O2•− under irradiation (460 nm, 23 mW cm−2). AO/EB dual staining assays and cell migration experiment in A-549 cells demonstrated that DAPFP-lyso could promote cancer cell apoptosis and migration under irradiation. The fluorescent protein dimer photosensitizer DAPFP-lyso had a precise lysosome-targeting ability (R = 0.95) could recognize lysosome specifically. These experimental results show that the fluorescent protein dimer design is of great significance for the development of new photosensitizers in the field of PDT.