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Nanomaterials 2020, 10, 2255 3 of 11 2.2. Media Impact on Physicochemical Properties of AgNPs To identify media impact on AgNPs’ physicochemical properties, Citrate AgNPs (1.14 mg/mL) were added to 1 mL of the different media, respectively. The media were deionized water, saline (0.85%, NaCl: 8.5 g/L), PBS (10× phosphate-buffered saline, NaCl: 80 g/L, KCl: 2 g/L, Na2HPO4: 14.4 g/L, and KH2PO4: 2.4 g/L, pH 7.4), serum (Fetal Bovine Serum), and α-MEM (Minimum Essential Media α with 10% FBS and 1% antibiotics). After shaking by hand for about half a minute, the samples were analyzed by TEM and dynamic light scattering (DLS). 2.3. Aging of AgNPs in 4 ◦C Storage To identify the aging of AgNPs during storage, Citrate AgNPs, PEG AgNPs, PVP AgNPs, and BPEI AgNPs were kept for 1 year in a fridge at 4 ◦C. The aggregation, size, and shape were analyzed by TEM and DLS as described above. The dissolution of AgNPs was analyzed by ultracentrifugation (rpm using a Beckman 45Ti rotor, 30 min, Beckman Coulter Life Sciences, Brea, CA, USA) and ICP-MS. The toxicity of AgNPs to Hepa1c1c7 cell lines was analyzed before and after storage in the fridge. 2.4. Cell Lines and Cell Culture The Department of Environmental Toxicology, University of California, Davis provided Hepa1c1c7 cells. They were maintained at 37 ◦ C with a humidified atmosphere of 5% CO2 in an incubator. Cells were cultured in α-MEM (Gibco® Minimum Essential Media α) with 10% FBS (Gibco® Fetal Bovine Serum) and antibiotics. 2.5. Cell Viability CellTiter-Glo® luminescent assay (Promega, Madison, WS, USA) was used to measure cell viability. Detergents were added to the reagent to break cell membranes. This resulted in the release of ATPase inhibitors to media and their stabilization. ATPases are a group of enzymes that catalyze the hydrolysis of a phosphate bond in adenosine triphosphate (ATP) to form adenosine diphosphate (ADP). The assay is based on the conversion of beetle luciferin to oxyluciferin by a recombinant luciferase in the presence of ATP. The quantity of ATP in cells is proportional to the observed luminescence. The observed luminescence was measured by a microplate reader machine (Spark® multimode microplate reader, Tecan Group Ltd. Männedorf, Switzerland). White, opaque, walled, 96-well plates (Nunc, Myriad industries, San Diego, CA, USA) were used for experiments. The ATP assay was conducted by platin, 5 × 103 cells per well. Different concentrations (0, 0.1, 1, 5, 10, 25, and 50 μg/mL) of Citrate AgNPs, PVP AgNPs, PEG AgNPs, BPEI AgNPs, and AgNO3, for 24 h, were used for treatment. 2.6. Statistical Analysis Statistical analyses for the cell viability were done using analysis of variance (ANOVA) and Tukey’s test with significance level at p < 0.05. Levene’s test was used to check homogeneity of variances. SPSS version 16 was used for the analyses. 3. Results 3.1. Characterization of AgNPs All studied AgNPs were monodispersed with similar primary size (around 30 nm), as shown by TEM images (Figure 1A–D). ζ potential varied from −22.9 mV to +46.5 mV in the four types of AgNPs.PDF Image | Silver Nanoparticles Undergoing Long-Term Aging
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