Thymoquinone as an electron transfer mediator to convert Type II photosensitizers to Type I photosensitizers

Abstract

The development of Type I photosensitizers (PSs) is of great importance due to the inherent hypoxic intolerance of photodynamic therapy (PDT) in the hypoxic microenvironment. Compared to Type II PSs, Type I PSs are less reported due to the absence of a general molecular design strategy. Herein, we report that the combination of typical Type II PS and natural substrate carvacrol (CA) can significantly facilitate the Type I pathway to efficiently generate superoxide radical (O₂^-•). Detailed mechanism study suggests that CA is activated into thymoquinone (TQ) by local singlet oxygen generated from the PS upon light irradiation. With TQ as an efficient electron transfer mediator, it promotes the conversion of O₂ to O₂^-• by PS via electron transfer-based Type I pathway. Notably, three classical Type II PSs are employed to demonstrate the universality of the proposed approach. The Type I PDT against S. aureus has been demonstrated under hypoxic conditions in vitro. Furthermore, this coupled photodynamic agent exhibits significant bactericidal activity with an antibacterial rate of 99.6% for the bacterial-infection female mice in the in vivo experiments. Here, we show a simple, effective, and universal method to endow traditional Type II PSs with hypoxic tolerance.

Fig. 1. Schematic illustration of the facilitated generation of O₂^–• through electron transfer strategy.

a Photoinduced conversion from CA to electron transfer mediator TQ and the subsequent electron transfer process for boosting the Type I pathway. b The PDT pathway in the absence or presence of electron transfer mediator TQ. c Chemical structures of Type II PSs and TQ.

Fig. 2. ROS generation properties of CA/PS1 complex.

a Comparison of the PL intensity of DCFH alone and DCFH with CA/PS1 (PS1 4 µg/mL, CA 150 µg/mL), PS1 (4 µg/mL), and CA (150 µg/mL) under light irradiation for overall ROS detection. b Comparison of the decomposition of ABDA alone and ABDA with CA/PS1 (PS1 4 µg/mL, CA 1 mg/mL), PS1 (4 µg/mL), and CA (1 mg/mL) under light irradiation for ¹O₂ detection. c Comparison of the PL intensity of DHR 123 alone and DHR 123 with CA/PS1 (PS1 4 µg/mL, CA 150 µg/mL), PS1 (4 µg/mL), and CA (150 µg/mL) under light irradiation for O₂^−• detection. d Comparison of the PL intensity of DHR 123 with or without radical scavenger under light irradiation. e Summary of different types of ROS generation in the presence of ROS detection probes alone and probes with different treatments. Data in (a–e) are presented as mean ± SD derived from n = 3 independent samples. Statistical significance was analyzed via one-way ANOVA test with a Tukey post hoc test.

Fig. 3. Bactericidal activities of CA/PS1 complex.

Photographs of the LB agar plates after the inoculation and overnight incubation of S. aureus cultures when treated under normoxic (a) or hypoxic (b) conditions with or without light irradiation (60 mW/cm², 10 min) in different groups. c, d CFU counts from the antibacterial assay as shown in (a, b), respectively. Data in (c, d) are presented as mean ± SD derived from n = 3 independent biological samples. Statistical significance was analyzed via two-way ANOVA test with a Tukey post hoc test. e Representative live/dead bacterial staining images of S. aureus treated with or without light irradiation in different groups. Green fluorescence: live bacteria, red fluorescence: dead bacteria. Scale bar = 10 µm. f Representative SEM images of S. aureus treated with or without light irradiation in different groups. Scale bar = 500 nm. Different groups include CA/PS1 (PS1 4 µg/mL, CA 150 µg/mL), PS1 (4 µg/mL), and CA (150 µg/mL).

Fig. 4. ROS generation properties and bactericidal activities of TQ/PS1 complex.

a GC-MS analysis of photoinduced oxidization of CA in the presence of PS1 with or without light irradiation. b Comparison of the PL intensity of DHR 123 with the addition of different concentrations of TQ (0-100 µg/mL) in the presence of PS1 (4 µg/mL) under light irradiation. c Comparison of the PL intensity of DHR 123 with or without radical scavenger under light irradiation. CFU counts of S. aureus treated with TQ/PS1 (PS1 4 µg/mL, TQ 8 µg/mL), PS1 (4 µg/mL), or TQ (8 µg/mL) under normoxic (d) or hypoxic (e) conditions in the presence or absence of light irradiation (60 mW/cm², 10 min). f Abolishment of the antibacterial activities by the addition of O₂^−• scavenger VC in the presence of TQ/PS1 complex (PS1 4 µg/mL, TQ 8 µg/mL). Data in (b–f) are presented as mean ± SD derived from n = 3 independent samples. Statistical significance was analyzed via one-way (b, c, f) or two-way (d, e) ANOVA test with a Tukey post hoc test. ns no significance.

Fig. 5. The study of electron transfer between PS and TQ.

a The emission spectra change of PS1 (10 μM) with increasing concentrations of TQ (0–30 mM) under excitation at 405 nm in DMF. b Stern–Volmer plot of fluorescence intensity change of PS1 against TQ in DMF. c Fluorescence decay curves of PS1 and TQ/PS1 at a peak of 670 nm. d Photocurrent responses of PS1, TQ, and TQ/PS1 systems. e HOMO and LUMO energy levels of CA, PS1, and TQ. f Simulated electrostatic potential map of TQ/PS1.

Fig. 6. In vivo bactericidal activities of TQ/PS1 complex.

a Detection of hypoxic conditions in both normal and infected tissues using the Hypoxyprobe Plus kit. Green fluorescence represents hypoxyprobe, blue fluorescence corresponds to DAPI. Scale bar = 100 μm. Data repeated independently (n = 3) with similar results. b Photographs of MRSA colonies in infected tissues treated with different groups upon light irradiation (60 mW/cm², 10 min) displayed on agar plates. c CFU counts of MRSA colonies from the antibacterial assay. Data presented as mean ± SD derived from n = 5 independent biological samples. Statistical significance was analyzed via one-way ANOVA test with a Tukey post hoc test. d H&E and e Masson-trichrome staining images of infected tissue slices at 7 d post-treatment in different groups. Scale bar = 200 μm. Data presented in (d, e) repeated independently (n = 5) with similar results. Different groups include TQ/PS1 (PS1 0.03 mg/kg, TQ 0.06 mg/kg), PS1 (0.03 mg/kg), TQ (0.06 mg/kg), and PBS.