Pro-Inflammatory versus Immunomodulatory Effects of Silver Nanoparticles in the Lung: The Critical Role of Dose, Size and Surface Modification

Abstract

The growing use of silver nanoparticles (Ag-NPs) in consumer products raises concerns about their toxicological potential. The purpose of the study was to investigate the size- and coating-dependent pulmonary toxicity of Ag-NPs in vitro and in vivo, using an ovalbumin (OVA)-mouse allergy model. Supernatants from (5.6-45 µg/mL) Ag50-PVP, Ag200-PVP or Ag50-citrate-treated NR8383 alveolar macrophages were tested for lactate dehydrogenase and glucuronidase activity, tumor necrosis factor (TNF)-α release and reactive oxygen species (ROS) production. For the in vivo study, NPs were intratracheally instilled in non-sensitized (NS) and OVA-sensitized (S) mice (1-50 µg/mouse) prior to OVA-challenge and bronchoalveolar lavage fluid (BALF) inflammatory infiltrate was evaluated five days after challenge. In vitro results showed a dose-dependent cytotoxicity of Ag-NPs, which was highest for Ag50-polyvinilpyrrolidone (PVP), followed by Ag50-citrate, and lowest for Ag200-PVP. In vivo 10-50 µg Ag50-PVP triggered a dose-dependent pulmonary inflammatory milieu in NS and S mice, which was significantly higher in S mice and was dampened upon instillation of Ag200-PVP. Surprisingly, instillation of 1 µg Ag50-PVP significantly reduced OVA-induced inflammatory infiltrate in S mice and had no adverse effect in NS mice. Ag50-citrate showed similar beneficial effects at low concentrations and attenuated pro-inflammatory effects at high concentrations. The lung microbiome was altered by NPs instillation dependent on coating and/or mouse batch, showing the most pronounced effects upon instillation of 50 µg Ag50-citrate, which caused an increased abundance of operational taxonomic units assigned to Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. However, no correlation with the biphasic effect of low and high Ag-NPs dose was found. Altogether, both in vitro and in vivo data on the pulmonary effects of Ag-NPs suggest the critical role of the size, dose and surface functionalization of Ag-NPs, especially in susceptible allergic individuals. From the perspective of occupational health, care should be taken by the production of Ag-NPs-containing consumer products.

Keywords: adjuvant; allergic inflammation; silver nanoparticles; surface modification.

Figure 1

In vitro effects of Ag50-polyvinilpyrrolidone (PVP), Ag200-PVP and Ag50-citrate nanoparticles (NPs) on alveolar macrophages (NR8383). Four concentrations (5.6, 11.25, 22.5 and 45 µg·mL⁻¹) were applied for 16 h (a,b,d) or 90 min (c). Supernatants were tested for activity of lactate dehydrogenase (LDH) (a) and glucuronidase (GLU) (b) relative to the positive control (Triton X-100). Release of H₂O₂ (c) is expressed relative to the positive control (zymosan). Bioactive tumor necrosis factor (TNF)-α was measured as percent lysis of L-929 reporter cells (d). Data are means±standard deviation (SD) from three independent experiments; dashed lines indicate mean values of untreated control cells; * p < 0.05, ** p < 0.01, *** p < 0.001. Uptake of silver NP (45 µg/mL, 16 h) is shown in (e). Note that while the bottom of the culture dish is clear, Ag-NPs appear as black grains localized intracellularly; bars: 20 µm.

Figure 2

Ag50-PVP NPs exert dose-dependent effects in vivo. Bronchoalveolar lavage fluid (BALF) cell analysis. In each graph, on the left are non-sensitized mice (NS) and on the right sensitized (S) mice. White plots represent particle-free supernatant (SUP) controls and filled plots depict NPs-treated lungs. The dose is given above. AMs, alveolar macrophages; Neu, neutrophils; Ly, lymphocytes and Eos, eosinophils. (n = 5–8/group) * p < 0.05, ** p < 0.01; ns, not significant.

Figure 3

Ag50-PVP NPs exert dose-dependent effects in vivo. Histological analysis of representative lung sections of S mice 5 days after SUP (a), 50 μg Ag50-PVP (b) or 1 μg Ag50-PVP (c) and ovalbumin (OVA) challenge. Periodic acid Schiff (PAS) staining: inflammatory infiltrate (arrows) and mucus hypersecretion (arrowheads). Scale bar: 100 μm.

Figure 4

Ag-NPs size and surface modification significantly affects their in vivo response. BALF cell analysis following intratracheal (i.t.) instillation of (a) Ag200-PVP and (b) Ag50-citrate prior to OVA challenge. In each graph, on the left are non-sensitized mice (NS) and on the right sensitized (S) mice. White plots represent particle-free supernatant (SUP) controls and filled/colored plots depict NP-treated lungs. The dose is given above. AMs, alveolar macrophages; Neu, neutrophils; Ly, lymphocytes and Eos, eosinophils. (n = 5–10/group) * p < 0.05, ** p < 0.01; ns, not significant; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. respective groups treated with Ag50-PVP.

Figure 5

Ag-NPs size and surface modification significantly affects BALF total protein and cytokines and the expression of inflammatory genes. Total protein (A) and IL-5 (B) were analyzed in BALF (n = 5–6/group) and the expression of Arg1 (C) and Muc5ac (D) was analyzed in lung homogenates (n = 3–4/group); lungs were retrieved on day 53, 24 h after NPs or particle-free supernatant (SUP) instillation and OVA challenge. The groups are depicted underneath. White plots represent SUP controls and filled/colored plots represent NP-treated groups. * p < 0.05, *** p < 0.001 vs. NS SUP; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. 50 μg Ag50-PVP.

Figure 6

Lung microbiome beta: diversity analysis. Principal Component Analysis (PCA) plot generated from partial 16S ribosomal ribonucleic acid (rRNA) gene sequences clustered in operational taxonomic units (OTUs) with a 97% similarity level obtained from lung samples of two mice batches treated with either Ag50-PVP (batch 1) or Ag50-citrate (batch 2) NPs in different doses (1 µg, 50 µg) and the respective controls (SUP). The 10 most abundant OTUs are shown. Significant differences between the factors: coating/batch (Ag50-PVP/batch-1 vs. Ag50-citrate/batch-2) and dose (SUP, 1 µg, 50 µg), are indicated by p values < 0.05 (bold letters).

Figure 7

Experimental protocol. Sensitized (S) mice were intraperitoneally injected with 1 µg OVA in phosphate-buffered saline (PBS)/alum (black arrows). Non-sensitized (NS) mice were injected with PBS/alum. On day 52, mice were intratracheally instilled with Ag-NPs or with SUP (white arrow) and subsequently challenged with OVA aerosol (arrowhead). Mice were sacrificed on day 53 or on day 57 (stars).