Table 1.
Nanoparticles and concentrations used in vivo.
Fig 1.
Intravital lung imaging of silica nanoparticles in mice.
A-F: intravital microscopy of an alveolar region prior to instillation (A), 2 minutes after (C) and 1 h (E) after instilling Ø 50 nm red fluorescent amorphous silica particles, suspended in PFC (maximum intensity projection of a 60 μm-deep stack). The alveolar wall is auto-fluorescent in green. The particles are in agglomerates of variable size, similar to those observed by TEM (Fig 3). (B), (D), and (F) show the red channel only for better clarity. Agglomerates either have shrunk in size and intensity one hour after instillation, or have vanished entirely (red arrows). Note that the particles used do not photo bleach under the illumination chosen (S1H Fig). space bar: A-F = 20 μm shown in A.
Fig 2.
Quantification of in vivo silica nanoparticle clearance.
Plot (z, t) of particles numbers (A) and area covered by particles (B) within the z-stack of Fig 1, over time. The stack starts with the alveolar wall close to the pleura (0 μm) and progresses towards the alveolar ducts (60 μm). Most particles are found close to the pleura (within alveoli). Particle number and area decrease within the 60 minutes time frame. The data indicates no movement from the alveoli towards the alveolar ducts, indicating that the particles do not leave the area via the airways. C: Quantification of particle clearance (data are mean ± SD, n = 3), with the reduction in agglomerate numbers in red and area in maximum intensity projection in blue. About half of the particles are cleared from the field of view over the first 30 minutes after exposure. The remainder of particles persist over the observation time of one hour. Significant changes are determined by student's t-test with Bonferroni adjusted p-values corrected for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). D: Whole organ imaging. Overlay of a white light image of the organs and a heat map-display of the fluorescence in liver (top), spleen (middle), and kidney (bottom) 24 h after instillation of Ø 50 nm Cy-7-labelled silica particles into the lung (right). The control images (left column) shows a low level of false positive counts in the liver.
Fig 3.
Electron micrographs of lung sections after instillation of 50 nm-silica nanoparticles into the lungs of mice (3.8 μg/g body weight).
The micrographs show the progression of the nanoparticles from the alveolar lumen across the epithelium to the interstitial region or taken up by alveolar macrophages. Particles associated with- and inside epithelial cells (A-D) are in agglomerates, whereas particles that enter macrophages are entangled in tubular myelin (F, H). A-G: Particle movement over the first hour of exposure. A: Particle agglomerates adhere to type II cells. B: Particles have entered type II cells into large endosomes. C: The endosome containing the particles is surrounded by a membrane. For better visibility particles of 58 nm with silver core were used. D: Particles are found on both, the apical- and the basolateral side of the of type I epithelial cells. E: Particles have entered the interstitial region. F: Particle conglomerates are also taken up by alveolar macrophages. G: At the 1 h-timepoint, 25% of type II cells contained particles within regions of the lung that were exposed, whereas less than 10% of alveolar macrophages in the same region contained particles (data are mean ± SD, from three mice per condition and three sections per mouse. Significant changes are determined by student's t-test (**** p < 0.0001). H, I particles after 24 h: H: At this time point, we found no particles within alveolar epithelial cells. Particles were either in the alveolar lumen or I: inside alveolar macrophages. Particles inside macrophages and in the alveolar lumen were entangled in tubular myelin. Higher magnifications of uptake compartments with free agglomerates and with particles in a complex with tubular myelin are shown at high magnification in S4 Fig. space bars: A-B, F, H, I: 500 nm, C-E: 100 nm.
Fig 4.
Electron micrographs of low dose exposure (0.15 μg/g body weight) of silica nanoparticles in the lungs of mice.
Because particles were now individual rather that in agglomerates, we used ø 58 nm silica nanoparticle with 28 nm silver core for better visibility. After 1h, the lung was fixed and the left lower lobe sectioned for visualization. A: Particles between the epithelium and the endothelium, B,C: within the interstitium, D: inside a microvascular endothelial cell. E: Percentage of the alveolar epithelial cells (AECs) and of alveolar macrophages within TEM sections of mouse lungs that contained particles (0.15 μg/g body weight). Data are mean ± SD, from three mice per condition and three sections per mouse. Significant changes are determined by student's t-test (**** p < 0.0001). space bars: A-D = 100 nm.