A Morphologic Study on the Fate of Ultrafine Silver Particles: Distribution Pattern of Phagocytized Metallic Silver in Vitro and in Vivo

Abstract

The distribution pattern of inhaled particles is an important factor for the evaluation of health effects. In this study, we morphologically investigated the fate of agglomerated ultrafine particles in macrophages in vitro and in vivo. Metallic silver (Ag) was chosen as a test particle, since it can be easily produced and detected by elemental and morphologic analyses. Ultrafine Ag particles generated by an electric spark generator in an argon atmosphere were collected on PTFE filters. The particles were suspended in distilled water and adjusted to different concentrations (10 μg/ml to 1 mg/ml) with phosphate-buffered saline (PBS). For the in vitro study, 1774 macrophage cell suspensions (200,000 cells in 400 μl medium) were plated in small chambers. Six hours later, 100 μl of the silver-PBS suspension was added to each chamber. For the next 9 days, the chamber slides were examined daily with an inverted microscope in order to detect agglomerated particles in the cell. The medium was changed every day, and Ag in the medium was checked by inductively coupled plasma mass spectrometry (ICP-MS). On days 1, 3, 5, 7, and 9, cells in the chambers were fixed with 2.5% buffered glutaraldehyde and examined ultrastructurally. For the in vivo study using F344 rats, 50 μg Ag particles were instilled intratracheally. On days 1, 4, and 7 following instillation, rats were sacrificed and the lungs were examined morphologically. The Ag content in the lung, liver, and lung-associated lymph nodes was analysed by ICP-MS. In the in vitro study, the dose-dependent presence of agglomerated particles was observed in 1774 cells. The size and form of particles remained unchanged throughout the observation period. Electron microscopy with x-ray microanalysis showed that both single and agglomerated Ag particles were observed in the dilated phagolysosome of 1774 cells. In the in vivo study, focal accumulation of Ag-particle-laden alveolar macrophages was found. Ag particles were also observed in the alveolar wall. Ag content in the lung was constant between day 1 and day 7, indicating that no rapid particle translocation from the lung to other organs had taken place in this time period. In vitro and in vivo studies suggested that agglomerated Ag particles remained in targets for a given period of time—at least up to 7 days.