Synthesis, toxicity evaluation and determination of possible mechanisms of antimicrobial effect of arabinogalactane-capped selenium nanoparticles.

Background: The elemental selenium nanoparticles (SeNPs) find application in biology and medicine due to wide spectrum of their biological activity combined with low toxicity. For instance, SeNPs are promising antimicrobial agents for plant treatment against the bacterial phytopathogen Clavibacter michiganensis sepedonicus (Cms). Careful characterization of possible mechanisms of antimicrobial action of SeNPs as well as the assessment of their biosafety for plant and animal organisms represents urgent challenge.

Methods: AG-stabilized SeNPs (AG/SeNPs) were synthesized by oxidation of selenide-anions by molecular oxygen dissolved in the reaction medium in the presence of AG macromolecules. The antimicrobial activity of AG/SeNPs against Cms was investigated both by observing the change in optical density of bacterial suspension and directly evaluating the cell death using fluorescent microscopy with propidium iodide staining. The effect of AG/SeNPs on the dehydrogenase activity was studied by determination of Cms enzymes ability to reduce colorless TTC to formazan. The effect of AG/SeNPs nanocomposite on the respiration rate of Cms cells was examined by polarographic method. For qualitative visualization of the potential on the inner membrane of Cms mesosomes, the potential-dependent TMRM dye and fluorescence microscopy were used. The toxicity of the AG/SeNPs was investigated on white mice by the Litchfield-Wilcoxon method. The effect of AG/SeNPs on plant organisms (potato plants) was studied on healthy and Cms-infected plants by determining the level of chlorophyll and lipid peroxidation products (LPO) in their leaves when treated with nanoparticles.

Results: Spherical Se° nanoparticles with an average size of 94 nm were obtained using the stabilizing potential of AG. It was found that these nanoparticles exhibited the pronounced (up to 60 %) bacteriostatic action (in 6.25 μg/mL concentration) against the bacterial phytopathogen Cms. It was shown and experimentally confirmed for the first time that the probable causes of the bacteriostatic action of AG/Se°NPs against Cms are non-reversible inhibition of Cms cell respiration, a decrease of the transmembrane potential with a change in the cell wall permeability for H protons and a decrease in their dehydrogenase activity. It was revealed that the treatment of healthy and Cms-infected potato plants with an aqueous solution of AG/Se°NPs involved no significant changes in the content of LPO and negative effect on the chlorophyll content, thus contributing to the saving of these values at the level of control intact plants.

Conclusion: Using a complex of complementary methods, we have found that antimicrobial activity of AG/SeNPs is apparently due to their ability to inhibit the dehydrogenase activity of Cms cells, as well as to disrupt the integrity of the cell membrane, resulting in a decrease of transmembrane potential and reduction of cellular respiration. The antimicrobial and antibiofilm activity of AG/SeNPs, together with their nontoxicity and safety for plant and animal organisms, determine the prospects for design of AG/SeNPs-based drugs for the rehabilitation of plants from the Cms.