Highly efficient singlet oxygen generation, two-photon photodynamic therapy and melanoma ablation by rationally designed mitochondria-specific near-infrared AIEgens

Abstract

Photosensitizers (PSs) with multiple characteristics, including efficient singlet oxygen (¹O₂) generation, cancer cell-selective accumulation and subsequent mitochondrial localization as well as near-infrared (NIR) excitation and bright NIR emission, are promising candidates for imaging-guided photodynamic therapy (PDT) but rarely concerned. Herein, a simple rational strategy, namely modulation of donor-acceptor (D-A) strength, for molecular engineering of mitochondria-targeting aggregation-induced emission (AIE) PSs with desirable characteristics including highly improved ¹O₂ generation efficiency, NIR emission (736 nm), high specificity to mitochondria, good biocompatibility, high brightness and superior photostability is demonstrated. Impressively, upon light irradiation, the optimal NIR AIE PS (DCQu) can generate ¹O₂ with efficiency much higher than those of commercially available PSs. The excellent two-photon absorption properties of DCQu allow two-photon fluorescence imaging of mitochondria and subsequent two-photon excited PDT. DCQu can selectively differentiate cancer cells from normal cells without the aid of extra targeting ligands. Upon ultralow-power light irradiation at 4.2 mW cm^-2, in situ mitochondrial photodynamic activation to specifically damage cancer cells and efficient in vivo melanoma ablation are demonstrated, suggesting superior potency of the AIE PS in imaging-guided PDT with minimal side effects, which is promising for future precision medicine.

Fig. 1. (A) Molecular structures of AIEgens (CPy, CQu, DCPy and DCQu) and their molecular orbital amplitude plots of HOMOs and LUMOs calculated by TD-DFT at the B3LYP/6-31G (d,p) basis set. (B) Absorption spectra of CPy, CQu, DCPy and DCQu in DMSO. (C) PL spectra of DCQu in DMSO/toluene mixtures with different toluene fractions. [DCQu] = 15 μM, λ_ex = 500 nm. (D) Plot of relative PL intensity (I/I₀) vs. the composition of the DMSO/toluene mixtures of CPy, CQu, DCPy and DCQu, where I₀ = PL intensity in pure DMSO solution. Inset: fluorescent photographs of DCQu in the DMSO solution and in a DMSO/toluene mixture with 99% toluene fraction taken under 365 nm UV irradiation. (E) Normalized PL spectra of the fluorogens in the solid state. Inset: fluorescence photographs of (i) CPy, (ii) CQu and (iii) DCPy taken under 365 nm UV irradiation and (iv) DCQu taken using a CRI in vivo imaging system. (F) (i) Single-crystal structure of DCQu. (ii) Molecular stacking structure along the long molecular axis and (iii) the short molecular axis. The hydrogen atoms in (ii) and (iii) are omitted for clarity.

Fig. 2. (A) Co-localization images of HeLa cells stained with CPy, CQu, DCPy and DCQu and MitoTracker Green, and Merged images. The displayed Pearson's correlation coefficient (R) denotes the goodness of colocalization. [AIEgens] = 1 μM, [MitoTracker Green] = 0.5 μM, and λ_ex = 488 nm. Scale bar = 20 μm. (B) Fluorescence images of different normal cells (HLF and LX2) and cancer cells (HepG2, B16, A549 and HeLa) stained with DCQu for 30 min. [DCQu] = 1 μM. (C) Relative fluorescence intensity of different cells incubated with DCQu for 30 min. The intensity data were measured by image J. [DCQu] = 1 μM.

Fig. 3. (A) Absorption spectra of ABDA in the presence of DCQu under white light (4.2 mW cm⁻²) irradiation. [DCQu] = 5 μM, [ABDA] = 50 μM, and the time interval for UV measurement = 20 s. (B) Decomposition of ABDA in the presence of different PSs under light irradiation, where A₀ and A are the absorbance of ABDA at 378 nm before and after irradiation. [PSs] = 5 μM, [ABDA] = 50 μM, and the time interval for UV measurement = 20 s. (C) Detection of intracellular ROS generation using H2DCF-DA in HeLa cells incubated with DCQu followed by irradiation with white light irradiation for different times. (D) Cell viability of HeLa cancer cells and HLF normal cells stained with different concentrations of DCQu in the absence or presence of white light irradiation.

Fig. 4. (A) The upconversion PL spectra of crystalline powder of DCQu at different input powers at 900 nm. Inset: Fluorescent photograph of DCQu crystals taken under a fluorescence microscope. (B) The corresponding linear relationship between the output fluorescence intensity and the square of input laser power (W²). (C) The two-photon excitation window of crystalline powder of DCQu.

Fig. 5. Two-photon excited fluorescence (top row) and bright-field (bottom row) images of HeLa cells stained with DCQu (5 μM) followed by different two-photon (900 nm, fs Ti:sapphire laser) scans.

Fig. 6. (A) Cell viability of melanoma cancer B16 stained with different concentrations of DCQu or Ce6 in the absence or presence of white light irradiation. (B) Tumor growth curves of B16 melanoma-bearing mice after different treatments. (C) Calculated tumor inhibition ratios of each treatment group. (D) Survival rate of mice after different treatments. (E) Histological sections of tumor tissues stained with hematoxylin and eosin. Scale bar = 100 μm.