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Nanomaterials 2020, 10, 2255 7 of 11 reduction in ATP content compared to control. The x-axis represents the concentrations of silver nanoparticles. The different colors of the bars identify different silver nanoparticles (Citrate AgNPs, PEG AgNPs, PVP AgNPs, BPEI AgNPs, and Ag+). (C) EC50 of AgNPs to Hepa1c1c7 cells. The value represents the mean ± standard deviation of three replicates. The asterisks * p < 0.05, ** p < 0.001 comparing the untreated and treated (AgNPs, Ag+) cells. 4. Discussion 4.1. Media Impact on Citrate AgNPs’ Transformation Our results showed that the media impact on Citrate AgNPs is lowest in deionized water, highest in PBS and Serum, and α-MEM is in between. We found that Citrate AgNPs aggregated significantly in 10× PBS, where the color changed from yellow to gray (Figure 2d,D). The gray color may result from the formation of AgCl by the Cl− in PBS and dissolved Ag of Citrate AgNPs. Our results are consistent with others who conclude that silver ions are mainly precipitated as AgCl in the presence of chloride. Because phosphate is mostly protonated to hydrogen phosphate and dihydrogen phosphate in a moderate pH (around 7), silver phosphate was never detected [14]. We did not observe significant aggregation of Citrate AgNPs in saline (0.85%). The difference may be due to the lower concentration of Cl− (8 g/L) in saline than the Cl− (80g/L) in PBS. This result is consistent with the aggregation kinetics of the electrostatically stabilized AgNPs (e.g., citrate AgNPs). The ionic strength correlates with aggregation increases up to a certain ion concentration in the medium [15]. Nanomaterials can interact with proteins, peptides, and lipids (nanocorona) on their surface once the nanomaterials enter into biological systems [16,17]. The formation of a nanocorona depends on multiple characteristics of the biological media and the physicochemical properties of the nanomaterial [18–21]. For example, we found that the serum and α-MEM increased the hydrodynamic size of Citrate AgNPs and decreased the zeta potential of Citrate AgNPs, which increased the aggregation of Citrate AgNPs in media and may alter their bio-distribution, clearance, activity, and toxicity in organisms. By altering the zeta potential of Citrate AgNPs, the diversity of the proteins forming the protein corona may be affected, via changes in nonspecific electrostatic interactions with the proteins. Shannahan et al. (2013) found that that the number of amphiphilic proteins increases with the negativity of the AgNP’s zeta potential, indicating that electrostatic interactions play a major role in nanocorona formation [22]. Our results showed that citrate coating plays an important role in both AgNP stability and antibacterial activity. We found that the presence of salt (NaCl) increases the aggregation of Citrate AgNPs but the presence of serum decreases their aggregation in different media. Previous studies showed that the interaction of AgNPs with serum albumin had a significant effect on their antibacterial activity. Citrate AgNPs exhibited antibacterial properties due to minimized interactions with serum proteins [23]. However, uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins. 4.2. Impact of Aging on AgNPs’ Aggregation, Shape, Surface Charge, Size, and Dissolution Interaction of NPs with the environment significantly changes their physical and chemical properties because the high surface to volume ratio and reactivity of NPs makes them highly dynamic in environmental systems [3]. However, AgNPs’ aging may not only show changes in environmental media, but also during sample storage. 4.2.1. TEM Image Analysis for Aggregation and Shape of AgNPs The TEM images showed that Citrate AgNPs had substantial aggregation resulting in increased size, and change in shape from spherical particles to triangular or irregular shapes. Such shape transformations have been found in other studies [24–31]. Citrate AgNPs are stabilized by charge repulsion where the citrate is weakly bound to the core Ag. The aggregation of Citrate AgNPs afterPDF Image | Silver Nanoparticles Undergoing Long-Term Aging
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