Assessment of Silver-Nanoparticles-Induced Erythrocyte Cytotoxicity through Ion Transport Studies

Silver nanoparticles (AgNPs) are the most frequently used nanomaterials in industrial and biomedical applications. Their functionalization significantly impacts their properties and potential applications. Despite the need to produce, investigate and apply them, not much is known about the toxicity of silver nanoparticles to and their interaction with blood components, such as erythrocytes. Here, we report on the effect of two negatively charged AgNPs (Creighton, and Lee-Meisel) on ion transport in human red blood cells (HRBCs).

HRBCs were obtained from blood of adult donors, which was either expired, fresh or refrigerated for variable lengths of time, and from fresh or refrigerated cord blood. Rb⁺ and K⁺ ions were measured by atomic emission and absorption spectrophotometry, respectively. Erythrocyte hemoglobin optical density (Hb_c OD), was determined at 527 nm to estimate RBC volume in the same tubes in which Rb⁺ and K⁺ were measured. Cellular Rb⁺ uptake and intracellular K⁺ concentrations, [K]_i, were calculated in mmol/L of original cells (LOC) per time. Rubidium, a potassium ion (K⁺) congener used to measure K⁺ influx, [K]_i, and Hb_c ODs were determined in the presence and absence of several concentrations (0-150 µg mL^-1) of spherical AgNPs of an average diameter of 10 nm, at different time points (0-60 min).

Creighton AgNPs inhibited Rb⁺ influx and depleted the cells of K⁺ independently of the source and in a time and dose-dependent manner. In contrast, Lee-Meisel AgNPs caused ~ 50 % Rb⁺ influx inhibition and ~ 15 % K⁺ loss with larger interindividual variability than Creighton AgNPs. The loss of K⁺ from HRBCs was entirely accounted for by an increase in extracellular K⁺ concentration, [K]_o. Enhanced dark field optical microscopy in conjunction with CytoViva® hyperspectral imaging helped visualize AgNPs internalized by HRBCs, thus pointing to a potential cause for their cytotoxic effects.

These findings indicate that HRBC K⁺ homeostasis is an early and sensitive biomarker for AgNPs toxicity and is a function of their surface functionalization.