The extensive use of silver nanoparticles (AgNPs) in over 30% of the currently registered nanomaterial-based products is associated with their antimicrobial properties. However, the numerous applications led to an increase in human exposure and environmental release of AgNPs along with the associated toxicities. Despite the reported alterations in key enzymatic and genetic moieties following sub-cellular localization of AgNPs, their toxicological profile (e.g., mechanism of toxicity, uptake, and stability in biological environment) is not clearly elucidated. According to a recent literature review by Tolaymat et al., Creighton AgNPs synthesized by the aqueous reduction of silver nitrate with sodium borohydride, is one of the most-heavily utilized bottom-up fabrication methods. The main goal of the current study is to quantify the concentration (0-300 µg mL-1) and time dependent (0-48 hr) uptake of negatively charged, Creighton AgNPs (average diameter of ~11 nm) in actively dividing African green monkey kidney (Vero 76) cells. For this, Creighton AgNPs were synthesized, characterized according to the U.S. Environmental Protection Agency (EPA) regulations, purified, size-selected and concentrated by tangential flow filtration (TFF). Vero 76 cells were chosen as an in vitro model because they are well established host carrier employed in several viral studies. After cellular exposure to AgNPs, the culture media, buffer washes (3x) and cells were harvested separately and analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES). Control groups included untreated cells and plain media. Results from three independent experiments showed that the concentrations of total Ag in the culture media were over an order of magnitude greater than the corresponding concentrations in cell fractions for all treatments and incubation times (e.g., Ag accumulation of 1.6 x 10-1 µg mL-1 in cells and 1.7 µg mL-1 in media for the lowest AgNP treatment of 3.0 µg mL-1 at 4 hr). The larger accumulation of Ag in cell culture media might be due to AgNP interactions with amino acid components of the culture media as demonstrated by UV-Vis absorption spectroscopy, Raman spectroscopy and TEM measurements. Overall, the Ag bioaccumulation was found to be concentration dependent. This study suggests that characterization of NPs in media needs to be practiced when reporting lethal concentrations of AgNPs in toxicological profiles.

Concentration and time dependent accumulation of Creighton silver nanoparticles in Vero 76 cells

249th National Meeting (2015)

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