Deposition and biokinetics of inhaled nanoparticles

Abstract

Particle biokinetics is important in hazard identification and characterization of inhaled particles. Such studies intend to convert external to internal exposure or biologically effective dose, and may help to set limits in that way. Here we focus on the biokinetics of inhaled nanometer sized particles in comparison to micrometer sized ones.The presented approach ranges from inhaled particle deposition probability and retention in the respiratory tract to biokinetics and clearance of particles out of the respiratory tract. Particle transport into the blood circulation (translocation), towards secondary target organs and tissues (accumulation), and out of the body (clearance) is considered. The macroscopically assessed amount of particles in the respiratory tract and secondary target organs provides dose estimates for toxicological studies on the level of the whole organism. Complementary, microscopic analyses at the individual particle level provide detailed information about which cells and subcellular components are the target of inhaled particles. These studies contribute to shed light on mechanisms and modes of action eventually leading to adverse health effects by inhaled nanoparticles.We review current methods for macroscopic and microscopic analyses of particle deposition, retention and clearance. Existing macroscopic knowledge on particle biokinetics and microscopic views on particle organ interactions are discussed comparing nanometer and micrometer sized particles. We emphasize the importance for quantitative analyses and the use of particle doses derived from real world exposures.

Figure 1

Concept of quantitative nanoparticle biokinetics. Nanoparticles (NP) are administered at time t = 0. From this time point on, the entire urinary and fecal excretions are collected separately. Animals are euthanized at times t₁, t₂, t₃, etc., and organs and tissues of interest as well as the entire remaining carcass are sampled (100% balanced sampling). Samples require special preparation before chemical analysis, e.g. by inductive-coupled-plasma mass spectroscopy (ICP-MS). When nanoparticles are radio-labeled, samples will be analyzed directly, without any further preparation. Reprinted with permission from [21].

Figure 2

Micrographs of horse trachea fixed by immersion in non-polar fixative (A, B). Note that the lung lining layer (aqueous phase [asterix] and surfactant film at the air liquid interface [arrow]), has been preserved with this fixation technique. EP = epithelium, CI = cilia. Bars: (A) = 2 μm, (B) = 0.2 μm. Reprinted with permission from [21].

Figure 3

Nanoparticle morphology. Section profiles (transects) of TiO₂ nanoparticles collected from the aerosol on filters, embedded in Epon and cut perpendicularly to the filter. Note that the as 20 nm measured TiO₂ aerosol particles are already agglomerates of smaller primary particle structures of 3 - 5 nm formed immediately after spark ignition and condensation. Bar: 200 nm.

Figure 4

Elemental microanalysis of a particle in lung tissue by electron spectroscopic imaging (ESI, three window method), demonstrating that the nanoparticles (arrows) consists of titanium, adapted from [21]. The first image (A), taken at 0 eV, shows the structural details; it is slightly shifted as compared to the images used for elemental microanalyses (B-D). For elemental microanalysis, three images are taken: two below the element-specific edge in order to extrapolate a background image, at ΔE = 390eV (not shown) and ΔE = 440eV (B), respectively, and one image within the maximum of the element specific signal at ΔE = 464eV (L_2,3 edge of titanium) (C). The net titanium signal (D) is calculated by subtraction of the extrapolated background image from the titanium specific signal. ESI images have reversed contrast as solely inelastically scattered electrons are used with an energy loss producing a dark field image. Hence, the obtained image reflects the titanium distribution in white pixels. Bar: 500 nm.

Figure 5

Multistage tissue sampling design for EFTEM analysis of nanoparticles, adapted from [21]. Stage 1 - Lung slices: exhaustive cutting of agar embedded lung lobes (with random start) into equally thick slices [38], followed by systematic sampling of slices, e.g. every second (with random start) and Epon embedding. Stage 2 - Tissue blocks and ultrathin sections: systematic sampling (with random start) of tissue blocks from lung slices using a point counting test system and cutting of ultrathin (≤ 50 nm) sections, which are placed on 600-mesh hexagonal copper grids and stained with lead citrate and uranyl acetate. Stage 3 - Quadrats on ultrathin sections: generation of a virtual field, completely contained within the ultrathin section, at 80× magnification. Field subdivision into a predetermined number of uniform quadrats and systematic subsampling of quadrats thereof (marked in grey). Stage 4 - Fields for nanoparticle analysis: Subsampling of a group of seven adjacent fields, delimited by the hexagonal TEM grid bars, within each quadrat at 6300× magnification, using a point counting test system. Tissue analysis within these hexagonal fields for (i) the presence of particles with matching nanoparticle characteristics and (ii) particle localization within the compartments of interest. Stages 3 and 4 can equally be applied on ultrathin sections of cell pellets. Alternative to stages 3 and 4 - Fields for nanoparticle analysis are sampled by picking a random hexagonal field on the ultrathin section as starting point at 80× magnification. From there on systematic tissue analysis in horizontal and vertical direction, using the automated goniometer of the microscope.

Figure 6

The respiratory tract (A) and particle deposition in a normal adult mouth breathing male human subject at rest, as a function of particle size (B). Data of bronchi are the sum of the deposition in bronchi and bronchioles. Adapted from [105] and [39] and reprinted with permission from [21].

Figure 7

Components of the inner surface of the lungs (A); particle deposition and immediate wetting (B) and complete displacement (C) of the deposited particle (white sphere) into the lung lining layer by surfactant. Reprinted with permission from [21].

Figure 8

TEM micrographs (A) and (B) of inhaled TiO₂ nanoparticles (arrows) located in large phagolysosomes, which contain other phagocytosed material, in mouse surface macrophages. Bars: 200 nm.