Size-Dependent Bioactivity of Silver Nanoparticles: Antibacterial Properties, Influence on Copper Status in Mice, and Whole-Body Turnover.

Purpose: The ability of silver nanoparticles (AgNPs) of different sizes to influence copper metabolism in mice is assessed.

Materials and methods: AgNPs with diameters of 10, 20, and 75 nm were fabricated through a chemical reduction of silver nitrate and characterized by UV/Vis spectrometry, transmission and scanning electronic microscopy, and laser diffractometry. To test their bioactivity, Escherichia coli cells, cultured A549 cells, and C57Bl/6 mice were used. The antibacterial activity of AgNPs was determined by inhibition of colony-forming ability, and cytotoxicity was tested using the MTT test (viability, %). Ceruloplasmin (Cp, the major mammalian extracellular copper-containing protein) concentration and enzymatic activity were measured using gel-assay analyses and WB, respectively. In vitro binding of AgNPs with serum proteins was monitored with UV/Vis spectroscopy. Metal concentrations were measured using atomic absorption spectrometry.

Results: The smallest AgNPs displayed the largest dose- and time-dependent antibacterial activity. All nanoparticles inhibited the metabolic activity of A549 cells in accordance with dose and time, but no correlation between cytotoxicity and nanoparticle size was found. Nanosilver was not uniformly distributed through the body of mice intraperitoneally treated with low AgNP concentrations. It was predominantly accumulated in liver. There, nanosilver was included in ceruloplasmin, and Ag-ceruloplasmin with low oxidase activity level was formed. Larger nanoparticles more effectively interfered with the copper metabolism of mice. Large AgNPs quickly induced a drop of blood serum oxidase activity to practically zero, but after cancellation of AgNP treatment, the activity was rapidly restored. A major fraction of the nanosilver was excreted in the bile with Cp. Nanosilver was bound by alpha-2-macroglobulin in vitro and in vivo, but silver did not substitute for the copper atoms of Cp in vitro.

Conclusion: The data showed that even at low concentrations, AgNPs influence murine copper metabolism in size-dependent manner. This property negatively correlated with the antibacterial activity of AgNPs.