Multiple regulatory agencies including the National Institute for Occupational Safety and Health (NIOSH), and the U.S. Environmental Protection Agency (EPA), have outlined the need to improve the risk assessment and control associated with the heavy use of nanomaterial-based consumer products. The Nanotechnology Product Database indicates that silver nanoparticles (AgNPs) are the main component of ~ 70% of the reported biomedical products. This work addresses this knowledge gap by combining CytoViva and Raman hyperspectral imaging to study the uptake, distribution, and toxicity of silver nanoparticles in human red blood cells. To achieve this, RBCs in 5% glucose solution were incubated at 37 °C, for 60 min, with 150 µg mL-1 of negatively charged, spherical AgNPs of an average diameter of ~ 20 nm, in the 10-35 nm size range. These citrate-capped AgNPs were selected as a nano-model due to their wide use, biocompatible capping agent, low cost, and simple fabrication. The physicochemical properties of AgNPs were characterized following the U.S. EPA recommendations after size-selecting, concentrating, and purifying them by tangential flow filtration (TFF) for improved homogeneity and reproducibility. CytoViva images showed that 48 ± 5 % of the total administered Ag was absorbed by cells and ~ 70% of the interacting AgNPs were present at the cell membrane. The deconvolution of the corresponding CytoViva hyperspectral data indicated changes in the content of RBC membrane spectrin (520 nm) and phospholipid (521 and 591 nm) components as a result of this interaction when compared to the negative controls. These toxicity effects of AgNPs were further confirmed through Raman hyperspectral imaging and a lipid peroxidation assay (two-fold increase in malondialdehyde). In addition, the spectral changes observed for the characteristic Raman vibrational modes of hemoglobin demonstrated the transformation of oxyhemoglobin (1226 cm-1) to deoxyhemoglobin (1214 cm-1) after AgNP exposure. Overall, the results of this study suggest that citrate-capped AgNPs may remain concentrated in the blood circulatory system despite their negative charge and biocompatible capping agent, and thereby raise major concerns with respect to the biomedical applications of AgNPs in humans.

Combining CytoViva and Raman hyperspectral imaging to study the uptake, distribution, and toxicity of silver nanoparticles in human red blood cells

ACS Fall 2021

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