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

Photodynamic therapy (PDT) relies on photosensitizers (PSs) to produce reactive oxygen species upon light irradiation. The dominant Type II pathway transfers energy from excited PSs to O2 to produce singlet oxygen (1O2), but efficacy is compromised in hypoxic microenvironments common in solid tumors and infected tissues. Type I PDT, which proceeds via electron transfer to generate radical species such as superoxide (O2−) and hydroxyl radicals, offers improved performance under hypoxia by regenerating oxygen through disproportionation and related reactions. However, a universal molecular design principle for efficient Type I PSs is lacking because electron transfer is generally less efficient and competes with energy transfer. The study asks whether introducing an appropriate substrate with suitable redox potential can mediate electron transfer from classical Type II PSs to oxygen, converting them into effective Type I generators under hypoxia for antibacterial therapy.

The authors note mature guidance for designing Type II PSs, while strategies to achieve Type I activity include cationization, heavy-atom regulation, and biotinylation. These approaches can endow PSs with some Type I ROS generation but do not constitute a universal principle. The challenge arises from the inherently less efficient, competitive electron transfer process that requires close contact and matched redox potentials among PS, substrate, and oxygen. Prior work has explored supramolecular systems, TADF-based "electron pumps," and specialized molecular designs for superoxide generation, yet a broadly applicable, simple method for Type I conversion remains elusive.

• Formation of carrier-free self-assembled complexes by mixing chlorin e6 (PS1) with carvacrol (CA) to form CA/PS1, and PS1 with thymoquinone (TQ) to form TQ/PS1; characterization by UV-Vis absorption, SEM, DLS; colloidal stability in PBS assessed over 7 days at 4 °C.
• ROS measurements under controlled light irradiation: total ROS with DCFH; singlet oxygen with ABDA; superoxide with DHR 123. Vitamin C (VC) used as a radical scavenger to validate O2− involvement.
• Mechanistic probing: GC-MS analysis of photoinduced oxidation products of CA in the presence of PS1 to identify TQ; steady-state fluorescence quenching of PS1 by TQ and Stern–Volmer analysis; time-resolved fluorescence lifetimes; photocurrent measurements in a three-electrode setup to evaluate charge separation; DFT calculations of HOMO/LUMO levels, electrostatic potential maps, and adsorption energies for PS1/CA and PS1/TQ complexes.
• In vitro antibacterial assays: CFU plate counting against Staphylococcus aureus and MRSA under normoxic and hypoxic conditions (hypoxia via AnaeroPack), with and without light (60 mW/cm², 10 min); VC quenching at bacterial level; live/dead staining (SYTO-9/PI); SEM imaging of bacterial morphology.
• Universality tests with other PSs: protoporphyrin IX (PS2) and TCPP (PS3) combined with TQ to assess O2− generation, fluorescence quenching, lifetimes, and energy level alignment.
• In vivo antibacterial assessment: subcutaneous MRSA infection in BALB/c mice; hypoxia verification by Hypoxyprobe; subcutaneous administration of TQ/PS1, PS1, TQ, or PBS; light irradiation (60 mW/cm², 10 min); CFU counting from infected tissue; histology (H&E, Masson trichrome).
• Biosafety: NIH 3T3 cell viability (MTT) under dark/light; in vivo blood biochemistry and hematology at days 1 and 7; major organ histology; cytokine levels (IL-6, TNF-α) in an open-wound model.

• CA/PS1 self-assembles into ~111.7 ± 21.2 nm spherical nanoparticles with good colloidal stability.
• Under light, CA/PS1 yields a ~433-fold increase in DCFH fluorescence (overall ROS), about 5.5-fold higher than PS1 alone. Singlet oxygen generation (ABDA) is reduced versus PS1 alone, consistent with CA quenching 1O2, whereas superoxide generation (DHR 123) is strongly enhanced and quenched by vitamin C, confirming O2− as the dominant increased ROS.
• GC-MS reveals photoinduced oxidation of CA to TQ in the presence of PS1 and light; TQ appears at 2.28 min retention time matching the TQ standard.
• TQ/PS1 complexes markedly enhance O2− generation; TQ alone shows negligible O2−. An optimal TQ concentration of ~20 μg/mL maximizes O2− generation in the PS1 system; VC quenches the signal.
• Antibacterial activity in vitro: Under normoxia, CA/PS1 reduces S. aureus CFU by ~5000-fold compared to PS1 alone. Under hypoxia, CA/PS1 achieves 99.9998% killing, with a ~67,000-fold CFU difference versus PS1. TQ/PS1 similarly achieves near-complete bacterial elimination; TQ alone has negligible activity. VC suppresses antibacterial effects in a dose-dependent manner, indicating Type I PDT-driven killing.
• Photophysical mechanism: TQ efficiently quenches PS1 fluorescence with Stern–Volmer constant reported as 1.92 × 10^10 M^−1 s^−1; PS1 fluorescence lifetime shortens from 5.36 ns to 3.60 ns in the presence of TQ; photocurrent is highest for TQ/PS1, indicating enhanced charge separation.
• DFT energy levels: LUMO (eV): TQ −3.31, PS1 −2.61, PS3 −2.60, PS2 −2.13, CA −0.26, supporting electron transfer from excited PSs to electron-deficient TQ; adsorption energies suggest spontaneous association (TQ/PS1 −2526.45 kJ/mol; CA/PS1 −2452.46 kJ/mol). Electrostatic potential maps indicate favorable charge transfer pathways.
• Generality: TQ enhances O2− generation with PS2 and PS3; fluorescence quenching shows linear Stern–Volmer behavior with Ksv values of 7.93 × 10^6 and 8.05 × 10^6 M^−1 s^−1 for PS2 and PS3, respectively; lifetimes shorten in the presence of TQ.
• In vivo MRSA model: Hypoxia confirmed in infected tissue by Hypoxyprobe. TQ/PS1 plus light achieves ~99.6% antibacterial rate versus ~48.8% for PS1 alone. Histology shows reduced inflammation and improved tissue structure after TQ/PS1 treatment.
• Biosafety: 3T3 viability remains >80% in dark; light induces cytotoxicity, suggesting need for selectivity strategies. In vivo, no significant changes in blood parameters or organ histology; no elevated IL-6 or TNF-α at 24 h post-treatment, indicating negligible acute toxicity.

The study demonstrates that a simple co-assembly of a classical Type II PS with a natural small molecule (CA) can yield a Type I-capable system under light by in situ conversion of CA to TQ, which functions as an electron transfer mediator. This mediator bridges electron transfer from the excited PS to oxygen, boosting superoxide generation and enabling efficient PDT under hypoxic conditions. The reduced dependence on molecular oxygen, via superoxide disproportionation and related reactions, mitigates hypoxia-associated limitations. Mechanistic data (fluorescence quenching/lifetimes, photocurrent, DFT energy alignment and electrostatics) support the electron transfer pathway facilitated by TQ. The approach generalizes across multiple Type II PSs (PS1–PS3), and translates to strong antibacterial efficacy in vitro (including hypoxia) and in vivo against MRSA, with supportive biosafety data. These findings address the lack of a universal design strategy for Type I PSs by introducing a broadly applicable, mediator-enabled electron transfer strategy that converts Type II PSs into effective Type I generators.

The work introduces a simple, effective, and broadly applicable method to convert Type II photosensitizers into Type I by leveraging thymoquinone as an electron transfer mediator formed in situ from carvacrol via 1O2 oxidation. This strategy enhances electron transfer from PSs to oxygen to generate O2−, alleviates hypoxia limitations, and provides potent antibacterial effects against S. aureus and MRSA in vitro and in vivo. Mechanistic experiments and computations corroborate the electron transfer pathway and generality across different PS scaffolds. The use of readily available phytochemicals and clinically relevant porphyrin PSs underscores translational potential. Future work could explore broader pathogen models, optimization of delivery and selectivity in complex tissues, and applications beyond antibacterial therapy.

The paper does not explicitly enumerate limitations. Potential considerations include: the scope is demonstrated with three porphyrinic Type II PSs and bacterial infection models (S. aureus/MRSA), so generalizability to other PS classes and pathogens remains to be validated; dependence on light delivery may constrain treatable sites; although hypoxia tolerance is improved, initial 1O2 generation is needed to convert CA to TQ; observed light-induced cytotoxicity toward mammalian cells suggests the need for targeting/selectivity strategies to minimize off-target effects in clinical contexts.