Influence of particle size on persistence and clearance of aerosolized silver nanoparticles in the rat lung

Abstract

The growing use of silver nanoparticles (AgNPs) in consumer products raises concerns about potential health effects. This study investigated the persistence and clearance of 2 different size AgNPs (20 and 110 nm) delivered to rats by single nose-only aerosol exposures (6 h) of 7.2 and 5.4 mg/m(3), respectively. Rat lung tissue was assessed for silver accumulations using inductively-coupled plasma mass spectrometry (ICP-MS), autometallography, and enhanced dark field microscopy. Involvement of tissue macrophages was assessed by scoring of silver staining in bronchoalveolar lavage fluid (BALF). Silver was abundant in most macrophages at 1 day post-exposure. The group exposed to 20 nm AgNP had the greatest number of silver positive BALF macrophages at 56 days post-exposure. While there was a significant decrease in the amount of silver in lung tissue at 56 days post-exposure compared with 1 day following exposure, at least 33% of the initial delivered dose was still present for both AgNPs. Regardless of particle size, silver was predominantly localized within the terminal bronchial/alveolar duct junction region of the lung associated with extracellular matrix and within epithelial cells. Inhalation of both 20 and 110 nm AgNPs resulted in a persistence of silver in the lung at 56 days post-exposure and local deposition as well as accumulation of silver at the terminal bronchiole alveolar duct junction. Further the smaller particles, 20 nm AgNP, produced a greater silver burden in BALF macrophages as well as greater persistence of silver positive macrophages at later timepoints (21 and 56 days).

Keywords: engineered nanomaterials; inhalation; inhaled; lung; macrophage.

FIG. 1.

Silver nanoparticle aerosol generation and exposure system. Particles in suspension are aerosolized into water droplets using a 6-jet collison nebulizer. Water is removed using diffusion dryers and particle charge is neutralized using a krypton-85 beta emission source. Dry particles are directed to a nose-only exposure chamber.

FIG. 2.

Aerosol AgNPs concentration and morphology of aerosolized particles. Mass concentration comparison of exposure to citrate buffer, 20 or 110 nm AgNP (A) determined by gravimetric filter (gray bars) and XRF filter analysis (black bars). Transmission electron micrographs (TEM) of 20 (B) and 110 nm AgNP (C). Bar = 100 nm.

FIG. 3.

Aerosol AgNPs characterization. Characterization of AgNPs aerosol was performed using a scanning mobility particle sizer (A, D), cascade impactor (B, E), and dynamic light scattering (DLS) (C, F). SMPS peak particle number at 77.4 nm for 20 nm AgNP and (E) 78.2 nm for 110 nm AgNP. Note the shoulder in the 110 nm AgNP trace at 120 nm (D). Mass concentration of size fractions was determined using a Merced type cascade impactor type (B, E). This indicates that there were some agglomerates of particles in the sample. Particle size in suspension was determined by DLS (C, F). Samples from manufacturer sealed containers (circles) and particles recovered from nebulizer at the end of exposure (diamonds) are very similar indicating lack of degradation. SMPS data are geometric mean ± σ_g (geometric standard deviation), cascade impactor data are mean ± SD, and DLS is Z-average diameter.

FIG. 4.

Total and silver positive macrophages recovered from bronchoalveolar lung lavage at 1, 7, 21, and 56 days following exposure. Number of macrophages recovered from BALF (A). Percent of silver positive macrophages that are positive for silver staining based on autometallography and light microscopy at × 40 magnification (B). Macrophages were scored depending on the intensity of silver staining as light, moderate, and heavy as shown in Supplementary Figure 1 (C). Compared with the 20 nm 1 day score, the 20 nm 7 day had a P = 0.096 and the 20 nm 21 day was P = 0.068. Asterisk (*), significantly different than citrate buffer at same timepoint; dagger (†), significantly different than 1 day timepoint for same particle type; double dagger (‡), significantly different than 20 nm AgNP at same timepoint; and delta (§), significantly different than 7 day timepoint for same particle type (P < 0.05) (n = 6).

FIG. 5.

Silver quantification using ICP-MS in the lung tissue following exposure. The amount of silver retained in the right middle lobe at 1, 7, 21, and 56 days post-exposure (C). Asterisk (*), significantly less than T₀ time-point for same particle type (P < 0.05); dagger (†), significantly less than T₀ and T₁ time-point for same particle type (P < 0.05) (n = 6).

FIG. 6.

Autometallography of silver localization from 20 nm AgNP at the alveolar duct junction. Lung tissue stained for silver at 1 day (A, B), 7 days (C, D), 21 days (E, F), and 56 days (G, H) post-exposure to aerosolized 20 nm AgNP. Silver positive macrophages (arrows) were detected at 1, 7, and 21 days. Deposition to the epithelium (open arrows) at 1 day. Staining of the subepithelial basement membrane zone is observed at 7 and 21 days, and to a lesser extent at 56 days (arrowheads). Bars = 50 µm.

FIG. 7.

Autometallography of silver localization from 110 nm AgNP at the alveolar duct junction. Lung tissue stained for silver at 1 day (A, B), 7 days (C, D), 21 days (E, F), and 56 days (G, H) post-exposure to aerosolized 110 nm AgNP. Silver positive macrophages (arrows) are detected at 1, 7, and 21 day timepoints. Silver localized to the epithelium (open arrows) at 1 day. Staining of the subepithelial basement membrane zone is observed at 7 and 21 days, and to a lesser level at 56 days (arrowheads). Bars = 50 µm.

FIG. 8.

Cytoviva-enhanced dark field images of AgNPs in unstained paraffin embedded lung sections. Cellular AgNPs uptake within the lung was qualitatively evaluated by the assessment of lung sections following exposure to 20 nm AgNP at (A) 1 day, (B) 7 days, and (C) 21 days or 110 nm AgNP at (D) 1 day, (E) 7 days, and (F) 21 days by Cytoviva-enhanced dark field microscopy. (G) AgNPs within lung epithelial cells at 21 days following exposure to 20 nm AgNP. Alterations in mean spectral profiles for (H) 20 nm AgNP and (I) 110 nm AgNP. Mean spectral profiles of AgNPs (black), AgNPs within alveolar macrophages at 24 h (red), and following 24 h incubation with artificial lysosomal fluid (ALF) (blue). Bar = 10 µm.

FIG. 9.

TEM images of AgNPs in BALF macrophages. Small silver particles have clumped together to form a larger spherical particle from the original 20 nm AgNP (A, B). Lesser number of the smaller particles from the original 20 nm AgNP are less organized (C, D). Small particles from the original 110 nm AgNP are along the membrane of a vesicle (E).