Fullerene C60-chitosan conjugate applied in the photoinactivation of Staphylococcus aureus

Abstract

A novel fullerene/chitosan (C60-CS) conjugate was synthesized. The addition reaction of the amine groups of chitosan (CS) to fullerene (C60) was carried out in N,N-dimethylformamide (DMF), and triethylamine at room temperature. The conjugate C60-CS was purified by dialysis in water. C60-CS showed an absorption spectrum characteristic of modified fullerenes. The ability to produce O2(1Δg) was determined by the decomposition of 9,10-dimethylanthracene (DMA) in DMF, finding a quantum yield value of 0.07. Besides, to obtain information about the contributions of the photoreaction mechanisms, the L-tryptophan photodecomposition was studied in the presence of reactive oxygen species (ROS) scavengers, such as D-mannitol and sodium azide. The results indicated a significant contribution of the type II mechanism to the photooxidation of the amino acid. This effect was confirmed by studies of the kinetics of nitrotetrazolium blue photoreduction in the presence of nicotinamide adenine dinucleotide. This conjugate´s photodynamic inactivation (PDI) capacity was investigated in Staphylococcus aureus. The photodynamic effect induced a 4 log decrease in cell viability when the cells were treated with 10 μM C60-CS and 30 min of irradiation with white light. The photoreaction mechanisms involved in the photosensitized inactivation of S. aureus by C60-CS were studied with the addition of azide ions, deuterated saline water, D-mannitol, and potassium iodide. It was confirmed that the photodynamic action of C60-CS was mainly mediated by type II photoprocess. Photooxidation of DMA induced by C60-CS in S. aureus cells indicated the production of O2(1Δg) in this biological medium. Finally, PDI studies were performed on agar surfaces, treating the cells with C60-CS before deposition on the surface. The subsequent inactivation of S. aureus sensitized by C60-CS after 30 min of irradiation with white light produced a marked elimination of bacteria as a function of increasing concentration. Therefore, this conjugate is a promissory photosensitizing agent to inactivate S. aureus.