Persistence of silver nanoparticles in the rat lung: Influence of dose, size, and chemical composition

Abstract

Increasing silver nanoparticle (AgNP) use in sprays, consumer products, and medical devices has raised concerns about potential health effects. While previous studies have investigated AgNPs, most were limited to a single particle size or surface coating. In this study, we investigated the effect of size, surface coating, and dose on the persistence of silver in the lung following exposure to AgNP. Adult male rats were intratracheally instilled with four different AgNPs: 20 or 110 nm in size and coated with either citrate or polyvinylpyrrolidone (PVP) at 0.5 or 1.0 mg/kg doses. Silver retention was assessed in the lung at 1, 7, and 21 d post exposure. ICP-MS quantification demonstrated that citrate-coated AgNPs persisted in the lung to 21 d with retention greater than 90%, while PVP-coated AgNP had less than 30% retention. Localization of silver in lung tissue at 1 d post exposure demonstrated decreased silver in proximal airways exposed to 110 nm particles compared with 20 nm AgNPs. In terminal bronchioles 1 d post exposure, silver was localized to surface epithelium but was more prominent in the basement membrane at 7 d. Silver positive macrophages in bronchoalveolar lavage fluid decreased more quickly after exposure to particles coated with PVP. We conclude that PVP-coated AgNPs had less retention in the lung tissue over time and larger particles were more rapidly cleared from large airways than smaller particles. The 20 nm citrate particles showed the greatest effect, increasing lung macrophages even 21 d after exposure, and resulted in the greatest silver retention in lung tissue.

Keywords: Clearance; intratracheal instillation; macrophage; respiratory.

Figure 1

Total macrophages (A, C, E and G) and percentage of silver positive macrophages (B, D, F and H). in BALF 1, 7 or 21 days after i.t. instillation of 4 types of silver nanoparticles of two doses (0.5 and 1.0 mg/kg). (A, B) 20nm citrate, (C, D) 110nm citrate, (E, F) 20nm PVP and (G, H) 110nm PVP. * significantly greater than control at same time-point (p < 0.05). † significantly different from same dose at 1 day time point (p < 0.05) (n = 6) NT = not tested.

Figure 2

BALF Macrophage silver uptake scores. Macrophages were scored based on intensity of silver staining (light, moderate or heavy) as shown in Figure S1 and adjusted for the total number of silver positive macrophages that were recovered from BALF to generate a macrophage silver uptake score for each treatment group and timepoint. * significantly different than 1 d time-point for same AgNP size, coating and dose, † significantly difference between citrate and PVP coated AgNP at same timepoint, AgNP size and dose, ‡ significantly difference between 20nm and 110nm AgNP at same timepoint, AgNP coating and dose (p < 0.05) (n = 5-6).

Figure 3

TEM image of macrophages containing silver particles. An (A) overview and (B) detail of a AgNP (open arrow) in a vacuole of a macrophage recovered from BALF. (C) An example of a AgNP (open arrow) in the lysosome of a BALF recovered macrophage.

Figure 4

Silver quantification in the right middle lung lobe by ICP-MS following 1.0 mg/kg dose of AgNPs. (A) is 20nm AgNPs treated animals and (B) is 110nm treated animals. Black bars are PVP coated AgNPs and grey bars are citrate coated AgNPs. * significantly different than 1 d time-point for same AgNP size and coating (p <0.05) (n = 6).

Figure 5

Autometallography (dark staining) of silver localization on and within the epithelium of the proximal airways (intrapulmonary lobar bronchus) 1 day post exposure to 1.0 mg/kg AgNPs. (A) 20nm citrate, (B) 110nm citrate, (C) 20nm PVP and (D) 110nm PVP. Note the staining on the ciliated cell (arrows) as well as within Clara cells (arrowheads) Bars = 50μm.

Figure 6

Autometallography (dark staining) of silver localization in the terminal bronchiole alveolar duct junction at 1 days post exposure to 1.0 mg/kg AgNPs. (A, B) 20nm citrate, (C, D) 110nm citrate, (E, F) 20nm PVP and (G, H) 110nm PVP. Clara cells (arrowheads) and macrophages (arrows) as well as the subepithelial basement membrane zone (open arrows) contained silver. Bars = 50μm.

Figure 7

Autometallography (dark staining) of silver localization to the terminal bronchiole alveolar duct junction at 7 day post exposure to 1.0 mg/kg AgNPs. (A, B) 20nm citrate, (C, D) 110nm citrate, (E, F) 20nm PVP and (G, H) 110nm PVP. Macrophages (arrows), basement membrane zone (open arrows), alveolar walls (arrowheads) and vessels (V) contained heavy localization of silver. Clara cells were not stained. Bars = 50μm.

Figure 8

Semi-quantitative scoring of silver distribution in the terminal bronchiole region. (A) 20 nm citrate, (B) 110 nm citrate, (C) 20 nm PVP and (D) 11 0nm PVP. * significantly less than 1 Day post treatment (p < 0.05), † significantly less than 7 days post treatment (p < 0.05) (n = 6).