Hybrid hydrogel-nanocarrier antimicrobial photodynamic therapy with Allura red against clostridium perfringens under oxygen-limited (hypoxic) conditions: An in vitro proof-of-concept.

BACKGROUND: Photodynamic therapy (PDT) has emerged as a promising approach against bacterial biofilms. However, its efficiency is usually limited under oxygen-deficient conditions such as in Clostridium perfringens infections. OBJECTIVE: To design and evaluate an in vitro hybrid hydrogel-nanocarrier antimicrobial photodynamic platform and to determine whether measurable antibacterial, antibiofilm, and anti-α-toxin effects can be maintained against Clostridium perfringens under verified oxygen-limited conditions. METHODS: The hybrid formulation was prepared by combining a sodium alginate-alum hydrogel with Allura Red AC-chitosan nanocarriers. C. perfringens biofilms were treated with a 630 nm diode laser at fluences of 5, 10, 15, and 20 J/cm² under strictly oxygen-limited (hypoxic) conditions verified using a resazurin indicator. Reactive oxygen species (ROS), including singlet oxygen (¹O₂), were quantified using SOSG and HPF probes, while biofilm biomass and viability were assessed via crystal violet, Congo red, and CFU assays. Photoluminescence quantum yield (the ratio of emitted to absorbed photons) was determined using standard spectrofluorometric procedures. RESULTS: The hybrid system produced measurable ROS even in low-oxygen environments, indicating a shift toward Type I photochemical pathways. Significant biofilm disruption and bacterial inactivation (p < 0.05) were achieved, particularly at 15 J/cm². The hydrogel provided prolonged retention and more uniform illumination, whereas chitosan nanocarriers enhanced dye-bacteria contact and localized ROS delivery. Allura Red served only as a model chromophore to test platform performance under stringent oxygen-limited conditions and is not intended for clinical antimicrobial PDT. CONCLUSION: This study demonstrates an in vitro, platform-level proof-of-concept for an oxygen-tolerant antimicrobial photodynamic therapy system under oxygen-limited conditions. Allura Red was used exclusively as a conservative stress-test chromophore and is not proposed as a clinical photosensitizer. The findings indicate that platform design-hydrogel retention and nanocarrier-mediated bacterial contact/targeting-rather than chromophore efficiency alone, plays a central role in sustaining measurable photodynamic activity under oxygen-limited conditions.