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

Photodynamic therapy (PDT) utilizes photosensitizers (PSs) to generate reactive oxygen species (ROS) upon light irradiation, inducing cell death. Type II PDT, the dominant mechanism, relies on the generation of singlet oxygen (¹O2) through energy transfer from the excited PS to molecular oxygen (O2). However, the efficacy of Type II PDT is hampered by the hypoxic microenvironment frequently found in tumors and infected tissues, where oxygen is limited. Type I PDT, which involves electron transfer from the PS to a substrate, followed by electron transfer to oxygen to generate radical species like superoxide (O2−) and hydroxyl radicals (OH•), offers a potential solution to overcome this hypoxia limitation. Type I PDT is less oxygen-dependent and can even regenerate oxygen through disproportionation, Fenton, and Haber-Weiss reactions. While several strategies exist to design Type I PSs or enhance Type I ROS generation in Type II PSs, a universal molecular design principle remains elusive. This is because electron transfer is less efficient than energy transfer, and the two processes often compete. Efficient electron transfer requires close contact and appropriate redox potentials between the PS, substrate, and oxygen. This study proposes using appropriate substrates to facilitate this electron transfer process.

Current molecular design strategies for Type I PSs include cationization, heavy-atom regulation, and biotinylation of PSs. However, these lack universality. The challenge lies in the less efficient electron transfer compared to energy transfer. Previous work has shown that introducing substrates with suitable redox potentials can facilitate electron transfer and enhance Type I ROS generation. Studies have explored the use of various molecules and methods to overcome the limitations of oxygen-dependent PDT, including near-infrared light-initiated molecular superoxide radical generators, BODIPY-based photosensitizers generating superoxide radicals, and strategies to enhance singlet oxygen generation of nano-photosensitizers.

This study investigated the combination of carvacrol (CA), a natural substrate, with three classical Type II PSs (Chlorin e6 (PS1), Protoporphyrin IX (PS2), and Tetrakis(4-carboxyphenyl)porphyrin (TCPP, PS3)). The formation of CA/PS complexes was characterized using UV-Vis spectroscopy, scanning electron microscopy (SEM), and dynamic light scattering (DLS). ROS generation was assessed using various probes: 2,7-dichlorodihydrofluorescein (DCFH) for overall ROS, 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) for ¹O2, and dihydrorhodamine 123 (DHR 123) for O2−. The bactericidal activity of the CA/PS1 complex was evaluated against *Staphylococcus aureus* (*S. aureus*) under normoxic and hypoxic conditions using colony-forming unit (CFU) plate counting, live/dead staining, and SEM. Gas chromatography-mass spectrometry (GC-MS) was used to identify the oxidation by-products of CA. The role of thymoquinone (TQ), the identified oxidation product, was further investigated by characterizing TQ/PS1 complexes and assessing their ROS generation and bactericidal activity. Steady-state fluorescence quenching, fluorescence lifetime measurements, and photocurrent responses were used to study the electron transfer process between PSs and TQ. Density functional theory (DFT) calculations were performed to determine the HOMO and LUMO energy levels and simulate electrostatic potential maps. *In vivo* antibacterial activity was assessed using an MRSA infection mouse model. Biosafety and biocompatibility were evaluated *in vitro* and *in vivo*. Statistical analysis included one-way and two-way ANOVAs with Tukey's post hoc test.

The combination of CA and PS1 (Chlorin e6) resulted in a dramatic increase in overall ROS generation (433-fold compared to PS1 alone), mainly due to a significant increase in O2−. ¹O2 generation was actually reduced. GC-MS analysis revealed that CA was oxidized to TQ upon light irradiation. The TQ/PS1 complex showed enhanced O2− generation compared to PS1 alone. Fluorescence quenching experiments and fluorescence lifetime measurements confirmed electron transfer from PS1 to TQ. Photocurrent measurements showed enhanced charge transfer in the TQ/PS1 system. DFT calculations showed that the LUMO level of PS1 lies between those of TQ and CA, facilitating electron transfer from PS1 to TQ. The CA/PS1 and TQ/PS1 complexes showed significantly enhanced bactericidal activity against *S. aureus* both under normoxic and hypoxic conditions. *In vivo* experiments demonstrated that the TQ/PS1 complex exhibited a 99.6% bactericidal rate against MRSA in a mouse model, significantly higher than PS1 alone. *In vitro* and *in vivo* biosafety assessments demonstrated negligible toxicity.

This study demonstrates a simple, effective, and universal method to convert Type II PSs to Type I PSs by using TQ as an electron transfer mediator. The findings address the limitations of Type II PDT in hypoxic environments by leveraging the oxygen-independent nature of Type I PDT. The use of naturally occurring, biocompatible components like CA and TQ enhances the therapeutic potential and safety profile of this approach. The mechanism of action, involving the in situ generation of TQ and its role in facilitating electron transfer, is clearly elucidated. The success with multiple Type II PSs highlights the versatility of the strategy. The results open avenues for developing effective and hypoxia-tolerant PDT agents for various applications.

This research successfully demonstrated the conversion of Type II PSs to Type I PSs using thymoquinone (TQ) as an electron transfer mediator, significantly enhancing their efficacy in hypoxic conditions. The use of naturally occurring compounds like carvacrol and thymoquinone adds to the biocompatibility and potential for translation of this technology. Further research could focus on optimizing the formulation and exploring its application in diverse clinical settings.

While this study demonstrated excellent results, further investigation into long-term *in vivo* toxicity and the potential for off-target effects is warranted. The study focused on bacterial infections; the efficacy against other types of hypoxia-rich conditions may vary and needs further exploration. The precise concentration of TQ required for optimal efficacy might vary depending on the specific PS and the therapeutic context.