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Assessment of in vitro particle dosimetry models at the single cell and particle level by scanning electron microscopy.

ABSTRACT: BACKGROUND:Particokinetic models are important to predict the effective cellular dose, which is key to understanding the interactions of particles with biological systems. For the reliable establishment of dose-response curves in, e.g., the field of pharmacology and toxicology, mostly the In vitro Sedimentation, Diffusion and Dosimetry (ISDD) and Distorted Grid (DG) models have been employed. Here, we used high resolution scanning electron microscopy to quantify deposited numbers of particles on cellular and intercellular surfaces and compare experimental findings with results predicted by the ISDD and DG models. RESULTS:Exposure of human lung epithelial A549 cells to various concentrations of differently sized silica particles (100, 200 and 500 nm) revealed a remarkably higher dose deposited on intercellular regions compared to cellular surfaces. The ISDD and DG models correctly predicted the areal densities of particles in the intercellular space when a high adsorption ("stickiness") to the surface was emulated. In contrast, the lower dose on cells was accurately inferred by the DG model in the case of "non-sticky" boundary conditions. Finally, the presence of cells seemed to enhance particle deposition, as aerial densities on cell-free substrates were clearly reduced. CONCLUSIONS:Our results further validate the use of particokinetic models but also demonstrate their limitations, specifically, with respect to the spatial distribution of particles on heterogeneous surfaces. Consideration of surface properties with respect to adhesion and desorption should advance modelling approaches to ultimately predict the cellular dose with higher precision.

SUBMITTER: Kowoll T

PROVIDER: S-EPMC6284276 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Project description:The protein is naturally dynamic and heterogeneous in solution. Protein dynamics involves both equilibrium fluctuations that regulate biological function and other non-equilibrium effects of biological motors, which convert chemical energy to mechanical energy. However, a single, unique structure of protein determined from X-ray crystal and conventional single-particle electron microscopy is insufficient to encompass the dynamic nature of proteins in solution. Structure determination of dynamic and heterogeneous protein is essentially required the determination of each individual particle of protein. Recently, Drs. Gang Ren and Lei Zhan published the first single molecule three-dimensional (3D) EM images of individual proteins ever obtained with enough clarity to determine their structure, an IgG antibody (14 Å resolution) and a 17nm HDL (36 Å resolution). These results depended upon four innovations: i) improved cryo-electron microscopy (cryoEM) sample preparation and Electron microscopy (EM) operation conditions resulted in the successful imaging of a 17 nm HDL particle (120-200kDa) by cryo-electron tomography (cryoET); ii) developed an optimized NS (OpNS) protocol that eliminates the rouleau artifact that has plagued EM research for three decades. This OpNS protocol provides high-contrast single lipoprotein images with similar size (<5%) and shape (<5%) to that seen by cryoEM; iii) developed a high-resolution and high contrast sample preparation protocol, cryo-positive-staining (cryoPS) that allows direct visualization of the secondary structure of a small protein, such as the β-strands in CETP and the helical double belt of apoA-I in spherical HDL; iv) developed a robust tomography reconstruction method, Individual Particle Electron Tomography (IPET) that is a high-resolution, high throughput reconstruction method that, to the best of our knowledge, is the only method for determining an individual protein structure. Remarkably, IPET went against the conventional wisdom that a single protein can NOT be reconstructed by EM and this opens a door for the study of protein dynamics via a particle-by-particle structural comparison.

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Assessment of in vitro particle dosimetry models at the single cell and particle level by scanning electron microscopy.

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