In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Macrophage Polarization Status Impacts Nanoceria Cellular Distribution but Not Its Biotransformation or Ferritin Effects.

The innate immune system is the first line of defense against external threats through the initiation and regulation of inflammation. Macrophage differentiation into functional phenotypes influences the fate of nanomaterials taken up by these immune cells. High-resolution electron microscopy was used to investigate the uptake, distribution, and biotransformation of nanoceria in human and murine M1 and M2 macrophages in unprecedented detail. We found that M1 and M2 macrophages internalize nanoceria differently. M1-type macrophages predominantly sequester nanoceria near the plasma membrane, whereas nanoceria are more uniformly distributed throughout M2 macrophage cytoplasm. In contrast, both macrophage phenotypes show identical nanoceria biotransformation to cerium phosphate nanoneedles and simultaneous nanoceria with ferritin co-precipitation within the cells. Ferritin biomineralization is a direct response to nanoparticle uptake inside both macrophage phenotypes. We also found that the same ferritin biomineralization mechanism occurs after the uptake of Ce-ions into polarized macrophages and into unpolarized human monocytes and murine RAW 264.7 cells. These findings emphasize the need for evaluating ferritin biomineralization in studies that involve the internalization of nano objects, ranging from particles to viruses to biomolecules, to gain greater mechanistic insights into the overall immune responses to nano objects.

Evaluation of enhanced darkfield microscopy and hyperspectral imaging for rapid screening of TiO2 and SiO2 nanoscale particles captured on filter media.

Here we report on initial efforts to evaluate enhanced darkfield microscopy (EDFM) and light scattering Vis-NIR hyperspectral imaging (HSI) as a rapid screening tool for the offline analysis of mixed cellulose ester (MCE) filter media used to collect airborne nanoparticulate from work environments. For this study, the materials of interest were nanoscale titanium dioxide (TiO2 ) and silicon dioxide (SiO2 ; silica), chosen for their frequent use in consumer products. TiO2 and SiO2 nanoscale particles (NPs) were collected on MCE filter media and were imaged and analyzed via EDFM-HSI. When visualized by EDFM, TiO2 and SiO2 NPs were readily apparent as bright spherical structures against a dark background. Moreover, TiO2 and SiO2 NPs were identified in hyperspectral images. EDFM-HSI images and data were compared to scanning transmission electron microscopy (STEM), a NIST-traceable technique for particle size analysis, and the current gold standard for offline analysis of filter media. As expected, STEM provided more accurate sizing and morphology data when compared to EDFM-HSI, but is not ideal for rapid screening of the presence of NPs of interest since it is a costly, low-throughput technique. In this study, we demonstrate the utility of EDFM-HSI in rapidly visualizing and identifying TiO2 and SiO2 NPs on MCE filters. This screening method may prove useful in expediting time-to-knowledge compared to electron microscopy. Future work will expand this evaluation to other industrially relevant NPs, other filter media types, and real-world filter samples from occupational exposure assessments.

Database of ab initio L-edge X-ray absorption near edge structure.

The L-edge X-ray Absorption Near Edge Structure (XANES) is widely used in the characterization of transition metal compounds. Here, we report the development of a database of computed L-edge XANES using the multiple scattering theory-based FEFF9 code. The initial release of the database contains more than 140,000 L-edge spectra for more than 22,000 structures generated using a high-throughput computational workflow. The data is disseminated through the Materials Project and addresses a critical need for L-edge XANES spectra among the research community.

Biological effects of inhaled hydraulic fracturing sand dust. II. Particle characterization and pulmonary effects 30 d following intratracheal instillation.

Hydraulic fracturing ("fracking") is used in unconventional gas drilling to allow for the free flow of natural gas from rock. Sand in fracking fluid is pumped into the well bore under high pressure to enter and stabilize fissures in the rock. In the process of manipulating the sand on site, respirable dust (fracking sand dust, FSD) is generated. Inhalation of FSD is a potential hazard to workers inasmuch as respirable crystalline silica causes silicosis, and levels of FSD at drilling work sites have exceeded occupational exposure limits set by OSHA. In the absence of any information about its potential toxicity, a comprehensive rat animal model was designed to investigate the bioactivities of several FSDs in comparison to MIN-U-SIL® 5, a respirable α-quartz reference dust used in previous animal models of silicosis, in several organ systems (Fedan, J.S., Toxicol Appl Pharmacol. 00, 000-000, 2020). The present report, part of the larger investigation, describes: 1) a comparison of the physico-chemical properties of nine FSDs, collected at drilling sites, and MIN-U-SIL® 5, a reference silica dust, and 2) a comparison of the pulmonary inflammatory responses to intratracheal instillation of the nine FSDs and MIN-U-SIL® 5. Our findings indicate that, in many respects, the physico-chemical characteristics, and the biological effects of the FSDs and MIN-U-SIL® 5 after intratracheal instillation, have distinct differences.

Nanoceria distribution and effects are mouse-strain dependent.

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. Objective: Assess nanoceria (cerium oxide, CeO2 nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. Methods: C57BL/6 and BALB/c female mice were i.p. injected with 10 mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1ß, and caspase-1 determination. Results: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. Conclusions: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences.

Tissue Specific Fate of Nanomaterials by Advanced Analytical Imaging Techniques - A Review.

A variety of imaging and analytical methods have been developed to study nanoparticles in cells. Each has its benefits, limitations, and varying degrees of expense and difficulties in implementation. High-resolution analytical scanning transmission electron microscopy (HRSTEM) has the unique ability to image local cellular environments adjacent to a nanoparticle at near atomic resolution and apply analytical tools to these environments such as energy dispersive spectroscopy and electron energy loss spectroscopy. These tools can be used to analyze particle location, translocation and potential reformation, ion dispersion, and in vivo synthesis of second-generation nanoparticles. Such analyses can provide in depth understanding of tissue-particle interactions and effects that are caused by the environmental "invader" nanoparticles. Analytical imaging can also distinguish phases that form due to the transformation of "invader" nanoparticles in contrast to those that are triggered by a response mechanism, including the commonly observed iron biomineralization in the form of ferritin nanoparticles. The analyses can distinguish ion species, crystal phases, and valence of parent nanoparticles and reformed or in vivo synthesized phases throughout the tissue. This article will briefly review the plethora of methods that have been developed over the last 20 years with an emphasis on the state-of-the-art techniques used to image and analyze nanoparticles in cells and highlight the sample preparation necessary for biological thin section observation in a HRSTEM. Specific applications that provide visual and chemical mapping of the local cellular environments surrounding parent nanoparticles and second-generation phases are demonstrated, which will help to identify novel nanoparticle-produced adverse effects and their associated mechanisms.

Preparation and characterization of multimodal hybrid organic and inorganic nanocrystals of camptothecin and gold.

We demonstrate a novel inorganic-organic crystalline nanoconstruct, where gold atoms were imbedded in the crystal lattices as defects of camptothecin nanocrystals, suggesting its potential use as simultaneous agents for cancer therapy and bioimaging. The incorporation of gold, a potential computed tomography (CT) contrast agent, in the nanocrystals of camptothecin was detected by transmission electron microscope (TEM) and further quantified by energy dispersive X-ray spectrometry (EDS) and inductively coupled plasma-optical emission spectrometers (ICP-OES). Due to gold's high attenuation coefficient, only a relatively small amount needs to be present in order to create a good noise-to-contrast ratio in CT imaging. The imbedded gold atoms and clusters are expected to share the same biological fate as the camptothecin nanocrystals, reaching and accumulating in tumor site due to the enhanced permeation and retention (EPR) effect.

Carboxylic acids accelerate acidic environment-mediated nanoceria dissolution.

Ligands that accelerate nanoceria dissolution may greatly affect its fate and effects. This project assessed the carboxylic acid contribution to nanoceria dissolution in aqueous, acidic environments. Nanoceria has commercial and potential therapeutic and energy storage applications. It biotransforms in vivo. Citric acid stabilizes nanoceria during synthesis and in aqueous dispersions. In this study, citrate-stabilized nanoceria dispersions (∼4 nm average primary particle size) were loaded into dialysis cassettes whose membranes passed cerium salts but not nanoceria particles. The cassettes were immersed in iso-osmotic baths containing carboxylic acids at pH 4.5 and 37 °C, or other select agents. Cerium atom material balances were conducted for the cassette and bath by sampling of each chamber and cerium quantitation by ICP-MS. Samples were collected from the cassette for high-resolution transmission electron microscopy observation of nanoceria size. In carboxylic acid solutions, nanoceria dissolution increased bath cerium concentration to >96% of the cerium introduced as nanoceria into the cassette and decreased nanoceria primary particle size in the cassette. In solutions of citric, malic, and lactic acids and the ammonium ion ∼15 nm, ceria agglomerates persisted. In solutions of other carboxylic acids, some select nanoceria agglomerates grew to ∼1 micron. In carboxylic acid solutions, dissolution half-lives were 800-4000 h; in water and horseradish peroxidase they were ≥55,000 h. Extending these findings to in vivo and environmental systems, one expects acidic environments containing carboxylic acids to degrade nanoceria by dissolution; two examples would be phagolysosomes and in the plant rhizosphere.

A case study of the translocation, bioprocessing and tissue interactions of EMP following inhalation exposure.

Human autopsied lung sections from a resident in the Quebec asbestos region were examined. The study utilized high resolution transmission electron microscopy, scanning transmission electron microscopy (HRTEM/STEM) with the analytical capabilities of electron energy loss spectroscopy (EELS) and energy dispersive spectroscopy (EDS) detectors. We report the first analytical ultrastructural characteristics of EMPs, detailing chemical concentration gradients inside the iron-protein coatings and lateral elemental gradients in the local tissue regions. It is shown that the EMPs are subjected to bioprocessing which involves physicochemical transformations and also an elemental transport mechanism that alters the inhaled EMP as well as the surrounding cellular matrix. At high resolution imaging the iron-rich coating around the EMP was observed to have a distinct channel-like nanostructure with some parallel aligned nanofibrils that are reminiscent of tooth enamel which consists of biomineralized nanocomposites with alternating organic/inorganic matrices.

High-throughput computational X-ray absorption spectroscopy.

X-ray absorption spectroscopy (XAS) is a widely-used materials characterization technique. In this work we present a database of computed XAS spectra, using the Green's formulation of the multiple scattering theory implemented in the FEFF code. With more than 500,000 K-edge X-ray absorption near edge (XANES) spectra for more than 40,000 unique materials, this database constitutes the largest existing collection of computed XAS spectra to date. The data is openly distributed via the Materials Project, enabling researchers across the world to access it for free and use it for comparisons with experiments and further analysis.

Assessing occupational erionite and respirable crystalline silica exposure among outdoor workers in Wyoming, South Dakota, and Montana.

Erionite is a naturally occurring fibrous mineral found in many parts of the world, including the western United States. Inhalational exposure to erionite fibers in some localities is associated with health effects similar to those caused by asbestos exposure, including malignant mesothelioma. Therefore, there is concern regarding occupational exposures in the western United States. Currently, there are no standard sampling and analytical methods for airborne erionite fibers, as well as no established occupational exposure limits. Due to the potential adverse health effects, characterizing and minimizing exposures is prudent. Crystalline silica also occurs naturally in areas where erionite is found, principally as the mineral quartz. Work activities involving rocks containing quartz and soils derived from those rocks can lead to exposure to respirable crystalline silica (RCS). The typically dry and dusty environment of the western United States can increase the likelihood of exposures to aerosolized rocks and soils, but inhalation exposure is also possible in more humid conditions. In this case study, we describe several outdoor occupational environments with potential exposures to erionite and RCS. We describe our method for evaluating those exposures and demonstrate: (1) the occurrence of occupational exposures to airborne erionite and RCS, (2) that the chemical make-up of the erionite mineral can be determined, and (3) that effective dust control practices are needed to reduce employee exposures to these minerals.

Analytical High-resolution Electron Microscopy Reveals Organ-specific Nanoceria Bioprocessing.

This is the first utilization of advanced analytical electron microscopy methods, including high-resolution transmission electron microscopy, high-angle annular dark field scanning transmission electron microscopy, electron energy loss spectroscopy, and energy-dispersive X-ray spectroscopy mapping to characterize the organ-specific bioprocessing of a relatively inert nanomaterial (nanoceria). Liver and spleen samples from rats given a single intravenous infusion of nanoceria were obtained after prolonged (90 days) in vivo exposure. These advanced analytical electron microscopy methods were applied to elucidate the organ-specific cellular and subcellular fate of nanoceria after its uptake. Nanoceria is bioprocessed differently in the spleen than in the liver.

From Dose to Response: In Vivo Nanoparticle Processing and Potential Toxicity.

Adverse human health impacts due to occupational and environmental exposures to manufactured nanoparticles are of concern and pose a potential threat to the continued industrial use and integration of nanomaterials into commercial products. This chapter addresses the inter-relationship between dose and response and will elucidate on how the dynamic chemical and physical transformation and breakdown of the nanoparticles at the cellular and subcellular levels can lead to the in vivo formation of new reaction products. The dose-response relationship is complicated by the continuous physicochemical transformations in the nanoparticles induced by the dynamics of the biological system, where dose, bio-processing, and response are related in a non-linear manner. Nanoscale alterations are monitored using high-resolution imaging combined with in situ elemental analysis and emphasis is placed on the importance of the precision of characterization. The result is an in-depth understanding of the starting particles, the particle transformation in a biological environment, and the physiological response.

In Vivo Processing of Ceria Nanoparticles inside Liver: Impact on Free-Radical Scavenging Activity and Oxidative Stress.

The cytotoxicity of ceria ultimately lies in its electronic structure, which is defined by the crystal structure, composition, and size. Despite previous studies focused on ceria uptake, distribution, biopersistance, and cellular effects, little is known about its chemical and structural stability and solubility once sequestered inside the liver. Mechanisms will be presented that elucidate the in vivo transformation in the liver. In vivo processed ceria reveals a particle-size effect towards the formation of ultrafines, which represent a second generation of ceria. A measurable change in the valence reduction of the second-generation ceria can be linked to an increased free-radical scavenging potential. The in vivo processing of the ceria nanoparticles in the liver occurs in temporal relation to the brain cellular and protein clearance responses that stem from the ceria uptake. This information is critical to establish a possible link between cellular processes and the observed in vivo transformation of ceria. The temporal linkage between the reversal of the pro-oxidant effect (brain) and ceria transformation (liver) suggests a cause-effect relationship.

Association of the sites of heavy metals with nanoscale carbon in a Kentucky electrostatic precipitator fly ash.

A combination of high-resolution transmission electron microscopy, scanning transmission electron microscopy, and electron energy-loss spectroscopy (HRTEM-STEM-EELS) was used to study fly ashes produced from the combustion of an eastern Kentucky coal at a southeastern-Kentucky wall-fired pulverized coal utility boiler. Fly ash was collected from individual hoppers in each row of the electrostatic precipitators (ESP) pollution-control system, with multiple hoppers sampled within each of the three rows. Temperatures within the ESP array range from about 200 degrees C at the entry to the first row to < 150 degrees C at the exit of the third row. HRTEM-STEM-EELS study demonstrated the presence of nanoscale (10 s nm) C agglomerates with typical soot-like appearance and others with graphitic fullerene-like nanocarbon structures. The minute carbon agglomerates are typically juxtaposed and intergrown with slightly larger aluminosilicate spheres and often form an ultrathin halo or deposit on the fly ash particles. The STEM-EELS analyses revealed that the nanocarbon agglomerates host even finer (< 3 nm) metal and metal oxide particles. Elemental analysis indicated an association of Hg with the nanocarbon. Arsenic, Se, Pb, Co, and traces of Ti and Ba are often associated with Fe-rich particles within the nanocarbon deposits.

Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery.

INTRODUCTION/OBJECTIVES: Adventitial application of FeCl(3) causes endothelial injury, platelet aggregation, and a rapid onset of thrombus formation. The transmigration pathway of the ferric ion has not been definitively identified. Using a combination of TEM and X-ray elemental analysis, this study aims to elucidate the endothelial pathway of ferric ion migration in carotid artery. METHODS AND RESULTS: Vascular injury was induced by placing a Whatman #1 filter paper strip saturated with 10% FeCl(3) over the common carotid artery in male C57BL/6 mice for 3 min. After rinsing in saline, the mice were terminated at 10 or 30 min. The FeCL(3) exposed segments of the common carotid artery were dissected, and processed for TEM. Thrombus formation was observed in all cases. Endothelial and smooth muscle injuries were observed in segments of the vessel in direct contact with the oxidant. The endothelial injury ranged from minimal damage to total denudation. The basal endothelial surface adjacent to the internal elastic lamina showed accumulation of electron opaque vesicles. The membrane enclosed particles transmigrated across the endothelium and exocytosed into the lumen. The nature of the particles shown by STEM/EDS was rich in ferric ion. Elemental analysis also showed that some ferric oxide aggregates formed near the developing thrombus in the vascular lumen. CONCLUSION: Our results showed the ferric ion permeated the endothelial basal lamina before entering the arterial lumen via endocytic-exocytic pathway. This study provides an ultrastructural framework for future analysis of the adluminal and luminal injuries in this model.

Elongated silica nanoparticles with a mesh phase mesopore structure by fluorosurfactant templating.

Mesoporous silica materials with pore structures such as 2D hexagonal close packed, bicontinuous cubic, lamellar, sponge, wormhole-like, and rectangular have been made by using surfactant templating sol-gel processes. However, there are still some "intermediate" phases, in particular mesh phases, that are formed by surfactants but which have not been made into analogous silica pore structures. Here, we describe the one-step synthesis of mesoporous silica with a mesh phase pore structure. The cationic fluorinated surfactant 1,1,2,2-tetrahydroperfluorodecylpyridinium chloride (HFDePC) is used as the template. Like many fluorinated surfactants, HFDePC forms intermediate phases in water (including a mesh phase) over a wider range of compositions than do hydrocarbon surfactants. The materials produced by this technique are novel elongated particles in which the layers of the mesh phase are oriented orthogonal to the main axis of the particles.

Kinetic precipitation of solution-phase polyoxomolybdate followed by transmission electron microscopy: a window to solution-phase nanostructure.

This study aimed to elucidate the structural nature of the polydisperse, nanoscopic components in the solution and the solid states of partially reduced polyoxomolybdate derived from the [Mo132] keplerate, [(Mo)Mo5]12-[Mo2 acetate]30. Designer tripodal hexamine-tris-crown ethers and nanoscopic molybdate coprecipitated from aqueous solution. These microcrystalline solids distributed particle radii between 2-30 nm as assayed by transmission electron microscopy (TEM). The solid materials and their particle size distributions were snap shots of the solution phase. The mother liquor of the preparation of the [Mo132] keplerate after three days revealed large species (r=20-30 nm) in the coprecipitate, whereas [Mo132] keplerate redissolved in water revealed small species (3-7 nm) in the coprecipitate. Nanoparticles of coprecipitate were more stable than solids derived solely from partially reduced molybdate. The TEM features of all material analyzed lacked facets on the nanometer length scale; however, the structures diffracted electrons and appeared to be defect-free as evidenced by Moiré patterns in the TEM images. Moiré patterns and size-invariant optical densities of the features in the micrographs suggested that the molybdate nanoparticles were vesicular.