Chitosan nanoparticle enhances the efficiency of antimicrobial Radachlorin-mediated photodynamic therapy against Staphylococcus aureus

BACKGROUND AND OBJECTIVESStaphylococcus sp. displays an important group of Gram-positive bacteria among which asignificant resistance against antibiotics is quickly spreading. Antimicrobial photodynamictherapy (aPDT) is a novel strategy to eliminate bacterial infections. In this method by usingof a photosensitizer, molecular oxygen, and light of a specific wavelength to excite thephotosensitizer, reactive oxygen species (ROS) are produced which lead to microbial celldeath. Radachlorin is a natural photosensitizer which is a mixture of sodium salts of chlorine۶, chlorin p۶ and purpurin ۵ with a high quantum yield of the singlet oxygen productionupon red light excitation. The incorporation of Radachlorin into polymeric nanoparticles isconsidered as a promising strategy for enhancing its aPDT potency. Therefore, the objectiveof this work was to preparation of biocompatible chitosan nanoparticles with the inclusion ofRadachlorin and an evaluation of the possibility of their use for aPDT against Staphylococcusaureus ATCC۶۵۳۸ as a model of Gram-positive bacteria.MATERIALS AND METHODSRadachlorin-containing chitosan nanoparticles (Ra-Ch-NPs) were prepared using ionotropicgelation method and was then characterized using Fourier Transform Infrared spectroscopy,UV–vis spectroscopy, scanning electron microscopy, and dynamic light scattering. For PDT,bacterial suspensions of Staphylococcus aureus were exposed to either free Radachlorin orRa-Ch-NPs at different concentrations for ۱ h and then both two groups were irritated withred light (۶۴۰ nm and ۲۰ J/cm۲). The control groups were exposed to red light alone,Radachlorin alone, and or neither Radachlorin nor light irradiation. Then well diffusionmethod and counting of colony forming units (CFU) were used to determine the minimuminhibitory concentrations (MIC) of the samples and antibacterial activities in each subgroup.DPBF assay was performed to determine the generation of singlet oxygen after lightirradiation. The cytotoxicity of compounts was assessed on normal fibroblast cells usingMTT assay.as well as using red light irradiation alone did not have a significant antibacterial effect, whileirradiation of Staphylococcus aureus cells cultured with Ra-Ch-NPs and Radachlorin,significantly induced cell death and colony growth inhibition with inhibition zones ۲۳ ± ۱,and ۱۶ ± ۲ mm, respectively. MIC values for Radachlorin and Ra-Ch-NPs after illuminationwere ۶۰۰ μg / mL and ۲۰۰ μg / mL, respectively.Comparatively, Ra-Ch-NPs indicated higher photodynamic inactivation than free Radachlorinwith ۴.۷ and ۳.۲ log۱۰ (CFU/mL) reductions in Staphylococcus aureus, respectively. TheRadachlorin (۴۶.۰۶ ± ۲.۲۳%) and Ra-Ch-NPs (۵۱.۱۴ ± ۱.۴۴%) displayed remarkable singletoxygen production which was comparable to methylene blue (۵۵.۳۶ ± ۰.۲۷%). None of thetested compounds either in the absence of light or after light irradiation showed toxicity onnormal fibroblast cells.CONCLUSIONConsidering the results of this work, aPDT with Ra-Ch-NPs and Radachlorin is effective inkilling Staphylococcus aureus, although Ra-Ch-NPs are more efficient than free Radachlorin.It will be intriguing to affirm these data by further in vivo researches