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Interplay of phosphate and carbonate ions with flavin photosensitizers in photodynamic inactivation of bacteria.

ABSTRACT: Photodynamic inactivation (PDI) of pathogenic bacteria is a promising technology in different applications. Thereby, a photosensitizer (PS) absorbs visible light and transfers the energy to oxygen yielding reactive oxygen species (ROS). The produced ROS are then capable of killing microorganisms via oxidative damage of cellular constituents. Among other PS, some flavins are capable of producing ROS and cationic flavins are already successfully applied in PDI. When PDI is used for example on tap water, PS like flavins will encounter various ions and other small organic molecules which might hamper the efficacy of PDI. Thus, the impact of carbonate and phosphate ions on PDI using two different cationic flavins (FLASH-02a, FLASH-06a) was investigated using Staphylococcus aureus and Pseudomonas aeruginosa as model organisms. Both were inactivated in vitro at a low light exposure of 0.72 J cm-2. Upon irradiation, FLASH-02a reacts to single substances in the presence of carbonate or phosphate, whereas the photochemical reaction for FLASH-06a was more unspecific. DPBF-assays indicated that carbonate and phosphate ions decreased the generation of singlet oxygen of both flavins. Both microorganisms could be easily inactivated by at least one PS with up to 6 log10 steps of cell counts in low ion concentrations. Using the constant radiation exposure of 0.72 J cm-2, the inactivation efficacy decreased somewhat at medium ion concentrations but reached almost zero for high ion concentrations. Depending on the application of PDI, the presence of carbonate and phosphate ions is unavoidable. Only upon light irradiation such ions may attack the PS molecule and reduce the efficacy of PDI. Our results indicate concentrations for carbonate and phosphate, in which PDI can still lead to efficient reduction of bacterial cells when using flavin based PS.

SUBMITTER: Eckl DB

PROVIDER: S-EPMC8195418 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCandida albicans is the most prevalent fungal pathogen of the human microbiota, causing infections ranging from superficial infections of the skin to life-threatening systemic infections. Due to the increasing occurrence of antibiotic-resistant C. albicans strains, new approaches to control this pathogen are needed. Photodynamic inactivation is an emerging alternative to treat infections based on the interactions between visible light and photosensitisers, in which pheophorbide a (PPA) is a chlorophyll-based photosensitizer that could induce cell death after light irradiation. Due to PPA's phototoxicity and low efficiency, the main challenge is to implement photosensitizer cell targeting and attacking.MethodsIn this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway.ResultsA single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for C. albicans cells. Subsequently, PPa was integrated into JM phage to generate a combined nanoscale material, which was called PPA-JM-phage. After photodynamic inactivation, the growth of C. albicans was inhibited by PPA-JM-phage and apoptosis was observed. Scanning electron microscopy analysis revealed shrinking and rupturing of C. albicans. We also found that depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species levels were elevated significantly in C. albicans inhibited by PPA-JM-phage. Additionally, PPA-JM-phage also lead to S-phase arrest, and metacaspase activation resulting from mitochondrial dysfunction was also found to be involved in C. albicans apoptosis.ConclusionPPa-JM-phage may induce C. albicans apoptosis through a caspase-dependent pathway and the results herein shed light on the potential application of phtototherapeutic nanostructures in fungal inactivation.

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Interplay of phosphate and carbonate ions with flavin photosensitizers in photodynamic inactivation of bacteria.

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