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In recent years, there has been increased interest in the use of nanostructures in various industries, such as the food, textile, pharmaceutical, electronics, and chemical industries. Most of these applications require a proper preparation of specific nanomaterials. In this study, we characterized silver nanoparticles (AgNPs) stabilized with polyvinylpyrrolidone and prepared in aqueous suspensions using dynamic light scattering, atomic force microscopy, and transmission electron microscopy. We aimed to compare the influence of different AgNP preparation procedures, specifically autoclaving, sonication, and a combination of both, on the agglomeration of these nanoparticles. Additionally, the toxicity of the NPs after the selected sterilization methods toward the EA.hy926 endothelial cell line was determined using trypan blue labeling, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) tetrazolium salt reduction tests, and the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) tetrazolium bromide test. Based on the obtained results, we concluded that cells exposed to AgNPs after sonication had similar viability values above 80% across all cellular viability tests. Conversely, the cellular viability values of the EA.hy926 cell line exposed to the autoclaved AgNP solutions differed. From the XTT tests, we observed a falsely determined cellular viability value above 100% with a simultaneous increase in the XTT-measured absorbance for the cellular medium after autoclaving. However, the other viability tests showed a cellular viability value below 25%. The results prove the importance of selecting an appropriate method for measuring cell viability, especially for cells exposed to previously sterilized nanomaterials.
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