Fluorescent protein chromophores modified with aromatic heterocycles for photodynamic therapy and two-photon fluorescence imaging

Abstract

In this paper, three fluorescent protein chromophore analogs PFPAr (PFPP, PFPC, and PFPT) were synthesized and proved to be useful for photodynamic therapy and two-photon fluorescence imaging. By adding five- or six-membered aromatic heterocycles to the photosensitizer PFP, we obtained three fluorescent protein photosensitizers PFPAr with better performances. As a demonstration, compared with the reported photosensitizer PFP, photosensitizer PFPP exhibits larger emission wavelengths (701 nm) and achieves a slight enhancement in the efficiency of singlet oxygen (Φ_Δ = 23%). Notably, PFPP can perform good two-photon fluorescence imaging with an 800 nm femtosecond laser in zebrafish. In in vitro cytotoxicity assays, PFPP shows good phototoxicity (IC₅₀ = 4.12 μM) and acceptable dark toxicity (cell viability assay >90%). The reactive oxygen imaging experiments and AO/EB double staining assay indicate that PFPP can generate singlet oxygen to eliminate A-549 tumor cells effectively with photoexcitation of 460 nm blue light (20 mW cm⁻²). Furthermore, PFPP can label the lysosomes of tumor cells with high specificity for lysosomes (Pearson's correlation coefficient of 0.91). Thus, our study demonstrated that the rational introduction of aromatic heterocycles into fluorescent protein photosensitizers can effectively enhance the key parameters of photosensitivity and pave the way for further two-photon photodynamic therapy.