Engineering of a GSH activatable photosensitizer for enhanced photodynamic therapy through disrupting redox homeostasis

Abstract

Although disrupted redox homeostasis has emerged as a promising approach for tumor therapy, most existing photosensitizers are not able to simultaneously improve the reactive oxygen species level and reduce the glutathione (GSH) level. Therefore, designing photosensitizers that can achieve these two aspects of this goal is still urgent and challenging. In this work, an organic activatable near-infrared (NIR) photosensitizer, CyI-S-diCF₃, is developed for GSH depletion-assisted enhanced photodynamic therapy. CyI-S-diCF₃, composed of an iodinated heptamethine cyanine skeleton linked with a recognition unit of 3,5-bis(trifluoromethyl)benzenethiol, can specifically react with GSH by nucleophilic substitution, resulting in intracellular GSH depletion and redox imbalance. Moreover, the activated photosensitizer can produce abundant singlet oxygen (¹O₂) under NIR light irradiation, further heightening the cellular oxidative stress. By this unique nature, CyI-S-diCF₃ exhibits excellent toxicity to cancer cells, followed by inducing earlier apoptosis. Thus, our study may propose a new strategy to design an activatable photosensitizer for breaking the redox homeostasis in tumor cells.

Scheme 1. (A) Chemical structure and proposed activation mechanism of CyI-S-diCF₃ mediated by GSH. (B) Illustrations of CyI-S-diCF₃ for GSH depletion-assisted enhanced photodynamic therapy through breaking the redox homeostasis of tumor cells.

Fig. 1. (A) The UV-vis absorption spectra and (B) fluorescence spectra of CyI-S-diCF₃ (10 μM) after incubation with different concentrations of GSH (0–100 μM) in PBS (10 mM, pH 7.4, 30% DMSO). (C) Fluorescence responses of CyI-S-diCF₃ (10 μM) with the addition of various amino acids (100 μM) in PBS (10 mM, pH 7.4, 30% DMSO) at 37 °C for 5 min (blank, GSH, Cys, Hcy). (D) The NIR-I fluorescence spectra of CyI-S-diCF₃ (10 μM) in the presence of GSH (100 μM) with or without addition of NMM (100 μM) in PBS (10 mM, pH 7.4, 30% DMSO) at 37 °C for 5 min.

Fig. 2. (A) ROS generation from CyI-S-diCF₃ (10 μM) with or without GSH (100 μM) using DPBF as a probe under 808 nm irradiation (0.33 W cm⁻², 3 min). (B) Compare the attenuation of the maximum absorbance of DPBF at 60 s of irradiation with 808 nm laser through various power density. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (C) GSH-depletion abilities in the absence and presence of CyI-S-diCF₃ (4.0 μM) with indicator DTNB, respectively. (D) Schematic illustration of oxidative stress strengthened by GSH depletion and enhanced singlet oxygen production efficiency.

Fig. 3. In vitro antitumor effects of CyI-S-diCF₃. (A) Cell viabilities of 4T1 cells treated with CyI-S-diCF₃ at various concentrations in the dark or under 808 nm laser irradiation (1.0 W cm², 5 min). Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (B) Calcein AM (green) and propidium iodide (red) co-staining fluorescence imaging of 4T1 cells after different treatments. 808 nm light irradiation (1.0 W cm², 5 min) was conducted after cells were incubated with CyI-S-diCF₃ (4.0 μM) for 4 h. Scale bar: 100 μm.

Fig. 4. (A) Schematic illustration of cell apoptosis induced by GSH-depleted photosensitizers and light-triggered singlet oxygen production to amplify the oxidative stress. (B) Cellular GSH levels of 4T1 cells after treatment of CyI-S-diCF₃ for 4 h. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (C) Detection of intracellular ROS generation by DCFH-DA in 4T1 cells after different treatments: (1) PBS only, (2) PBS with 808 nm laser, (3) CyI-S-diCF₃ only, (4) CyI-S-diCF₃ with 808 nm laser. (D) Mitochondrial membrane potential monitored by using JC-1 dye after incubation without and with CyI-S-diCF₃ (4.0 μM) for 4 h, followed by 808 nm laser irradiation (1.0 W cm², 5 min). Scale bar: 50 μm.