Benjamin Gilbert

Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties.

Nanomaterials (NM) exhibit novel physicochemical properties that determine their interaction with biological substrates and processes. Three metal oxide nanoparticles that are currently being produced in high tonnage, TiO(2), ZnO, and CeO(2), were synthesized by flame spray pyrolysis process and compared in a mechanistic study to elucidate the physicochemical characteristics that determine cellular uptake, subcellular localization, and toxic effects based on a test paradigm that was originally developed for oxidative stress and cytotoxicity in RAW 264.7 and BEAS-2B cell lines. ZnO induced toxicity in both cells, leading to the generation of reactive oxygen species (ROS), oxidant injury, excitation of inflammation, and cell death. Using ICP-MS and fluorescent-labeled ZnO, it is found that ZnO dissolution could happen in culture medium and endosomes. Nondissolved ZnO nanoparticles enter caveolae in BEAS-2B but enter lysosomes in RAW 264.7 cells in which smaller particle remnants dissolve. In contrast, fluorescent-labeled CeO(2) nanoparticles were taken up intact into caveolin-1 and LAMP-1 positive endosomal compartments, respectively, in BEAS-2B and RAW 264.7 cells, without inflammation or cytotoxicity. Instead, CeO(2) suppressed ROS production and induced cellular resistance to an exogenous source of oxidative stress. Fluorescent-labeled TiO(2) was processed by the same uptake pathways as CeO(2) but did not elicit any adverse or protective effects. These results demonstrate that metal oxide nanoparticles induce a range of biological responses that vary from cytotoxic to cytoprotective and can only be properly understood by using a tiered test strategy such as we developed for oxidative stress and adapted to study other aspects of nanoparticle toxicity.

Use of a Rapid Cytotoxicity Screening Approach to Engineer a Safer Zinc Oxide Nanoparticle through Iron Doping

The establishment of verifiably safe nanotechnology requires the development of assessment tools to identify hazardous nanomaterial properties that could be modified to improve nanomaterial safety. While there is a lot of debate of what constitutes appropriate safety screening methods, one approach is to use the assessment of cellular injury pathways to collect knowledge about hazardous material properties that could lead to harm to humans and the environment. We demonstrate the use of a multiparameter cytotoxicity assay that evaluates toxic oxidative stress to compare the effects of titanium dioxide (TiO(2)), cerium oxide (CeO(2)), and zinc oxide (ZnO) nanoparticles in bronchial epithelial and macrophage cell lines. The nanoparticles were chosen on the basis of their volume of production and likelihood of spread to the environment. Among the materials, dissolution of ZnO nanoparticles and Zn(2+) release were capable of ROS generation and activation of an integrated cytotoxic pathway that includes intracellular calcium flux, mitochondrial depolarization, and plasma membrane leakage. These responses were chosen on the basis of the compatibility of the fluorescent dyes that contemporaneously assess their response characteristics by a semiautomated epifluorescence procedure. Purposeful reduction of ZnO cytotoxicity was achieved by iron doping, which changed the material matrix to slow Zn(2+) release. In summary, we demonstrate the utility of a rapid throughput, integrated biological oxidative stress response pathway to perform hazard ranking of a small batch of metal oxide nanoparticles, in addition to showing how this assay can be used to improve nanosafety by decreasing ZnO dissolution through Fe doping.

The effects of nanoparticle aggregation processes on aggregate structure and metal uptake.

Adsorption at the mineral-water interface is an important process governing metal ion concentration and mobility in aqueous systems. Ferric iron oxyhydroxide nanoparticles possess a large capacity for the adsorption of heavy metals but quantification of metal uptake and sequestration is challenging due to the tendency of natural nanoparticles to aggregate in natural waters. We studied the effects of aggregation via pH, ionic strength, drying, and freezing on the uptake and release of copper from ferrihydrite nanoparticles employing small-angle X-ray scattering (SAXS) studies of aggregate morphology, macroscopic Cu(II) sorption and desorption batch experiments, and extended X-ray absorption fine structure (EXAFS) spectroscopic studies of copper sorption geometries. Results show that the mechanism of aggregation has a large effect upon aggregate morphology and consequently on the net sorption/retention of ions from solution. While aggregation reduces the total amount of copper that can be adsorbed, it also may introduce physical constraints to desorption and/or increased proportions of higher strength binding sites that lead to greater retention, and hence more effective sequestration, of metal ion contaminants.

The fate of ZnO nanoparticles administered to human bronchial epithelial cells.

A particular challenge for nanotoxicology is the evaluation of the biological fate and toxicity of nanomaterials that dissolve in aqueous fluids. Zinc oxide nanomaterials are of particular concern because dissolution leads to release of the toxic divalent zinc ion. Although zinc ions have been implicated in ZnO cytotoxicity, direct identification of the chemical form of zinc taken up by cells exposed to ZnO nanoparticles, and its intracellular fate, has not yet been achieved. We combined high resolution X-ray spectromicroscopy and high elemental sensitivity X-ray microprobe analyses to determine the fate of ZnO and less soluble iron-doped ZnO nanoparticles following exposure to cultures of human bronchial epithelial cells, BEAS-2B. We complemented two-dimensional X-ray imaging methods with atomic force microscopy of cell surfaces to distinguish between nanoparticles that were transported inside the cells from those that adhered to the cell exterior. The data suggest cellular uptake of ZnO nanoparticles is a mechanism of zinc accumulation in cells. Following uptake, ZnO nanoparticles dissolved completely generating intracellular Zn(2+) complexed by molecular ligands. These results corroborate a model for ZnO nanoparticle toxicity that is based on nanoparticle uptake followed by intracellular dissolution.

Electron Small Polarons and Their Mobility in Iron (Oxyhydr)oxide Nanoparticles

Iron Hopping Iron oxide minerals shuttle electrons around in a wide range of biogeochemical processes. Katz et al. (p. 1200) used time-resolved x-ray absorption spectroscopy to take a closer look at how this happens. By using photoionized surface dyes to inject electrons into three different solid oxide phases, they found that electrons hop among iron centers at rates that depend more on structure in their immediate vicinity than on the extended ordering of the crystal lattice. These observations bolster the prevailing small polaron model in which charge carriers associate closely with individual metal sites. X-ray spectroscopy highlights the influence of local structure on electron transport in iron minerals. Electron mobility within iron (oxyhydr)oxides enables charge transfer between widely separated surface sites. There is increasing evidence that this internal conduction influences the rates of interfacial reactions and the outcomes of redox-driven phase transformations of environmental interest. To determine the links between crystal structure and charge-transport efficiency, we used pump-probe spectroscopy to study the dynamics of electrons introduced into iron(III) (oxyhydr)oxide nanoparticles via ultrafast interfacial electron transfer. Using time-resolved x-ray spectroscopy and ab initio calculations, we observed the formation of reduced and structurally distorted metal sites consistent with small polarons. Comparisons between different phases (hematite, maghemite, and ferrihydrite) revealed that short-range structural topology, not long-range order, dominates the electron-hopping rate.

Aggregation-induced growth and transformation of β-FeOOH nanorods to micron-sized α-Fe2O3 spindles

Intimate interconnection of crystal growth, (oriented) aggregation and phase transformation seem common in the formation of nano- and microcrystalline materials from solutions. Yet, the mechanistic linkages between the different processes have not been fully understood. In this work, we studied the hydrothermal growth of akaganeite (β-FeOOH) nanorods and their transformation to micron-sized hematite (α-Fe2O3) spindles using high-resolution cryogenic transmission electron microscopy (cryo-TEM). Only akaganeite particles and hematite spindles were detected in the samples. Further, cryo-electron 3D tomograms show that akaganeite nanorods were aggregated into loose three-dimensional networks with some embedded hematite spindles. Based on our cryo-TEM and additional X-ray diffraction, electron microscopy, and chemical data, we propose the following mechanism: first, formation of the early-stage hematite spindles is driven by phase stability change due to increase in size caused by oriented aggregation of akaganeite. Then, akaganeite particles continue to transform to hematite upon contact with and recrystallization onto hematite surfaces, making hematite grow with a constant aspect ratio and forming micron-sized nano-porous single-crystal spindles. Our growth model interprets experimental observations well and it resolves previous long-time debate over whether the hematite spindles are formed via classical Ostwald ripening or by oriented aggregation of hematite nanoparticles. Possibly, this aggregation-based concurrent growth and transformation model may also be applicable to crystal growth and phase transformation in other systems.

Kinetically controlled formation of a novel nanoparticulate ZnS with mixed cubic and hexagonal stacking

Nanoparticulate ZnS with mixed cubic and hexagonal close packed stacking was synthesized by reaction of zinc acetate with thioacetamide in weakly acidic solutions. The influences of temperature, reaction time, amounts of reagents and solution pH on the nanoparticle size and phase constitution were investigated. Experimental results suggest that the stacking in the nano-ZnS is controlled primarily by the precipitation kinetics. Factors that slow the precipitation rate favor the growth of nanoparticles with mixed stacking, probably because the probabilities of forming wurtzite-like layers and sphalerite-like layers under these conditions are approximately equal. Under conditions of rapid precipitation, the growth of sphalerite is favored, probably due to the aggregation of molecular clusters with sphalerite-like structure. UV–vis spectroscopy reveals that twins and stacking faults in nano-ZnS result in an electronic structure that differs from those of nano-scale sphalerite and wurtzite. New vibrational modes present in IR spectra of the nano-ZnS with mixed stacking indicate that the materials have novel optical properties. Control of defect microstructure may allow use of nano-ZnS in new technological applications.

Rate and mechanism of the photoreduction of birnessite (MnO2) nanosheets

Significance The photoreductive dissolution of Mn oxides governs the biogeochemical cycle of Mn and the fate of organic and inorganic species associated with Mn oxides in the euphotic zones of marine and freshwater systems. Mn oxide minerals also have garnered interest as water oxidation catalysts inspired by the Mn4CaO4 cluster of photosystem II. However, the mechanism of water oxidation by MnO2 and the rate limiting steps for this reaction are unknown. In this study, we couple flow-through experiments and ultrafast pump–probe optical and X-ray absorption spectroscopy to develop a photoreduction model that includes the mechanism and timescales for the initial electron transfer steps in the oxidation of water by MnO2. The photoreductive dissolution of Mn(IV) oxide minerals in sunlit aquatic environments couples the Mn cycle to the oxidation of organic matter and fate of trace elements associated with Mn oxides, but the intrinsic rate and mechanism of mineral dissolution in the absence of organic electron donors is unknown. We investigated the photoreduction of δ-MnO2 nanosheets at pH 6.5 with Na or Ca as the interlayer cation under 400-nm light irradiation and quantified the yield and timescales of Mn(III) production. Our study of transient intermediate states using time-resolved optical and X-ray absorption spectroscopy showed key roles for chemically distinct Mn(III) species. The reaction pathway involves (i) formation of Jahn–Teller distorted Mn(III) sites in the octahedral sheet within 0.6 ps of photoexcitation; (ii) Mn(III) migration into the interlayer within 600 ps; and (iii) increased nanosheet stacking. We propose that irreversible Mn reduction is coupled to hole-scavenging by surface water molecules or hydroxyl groups, with associated radical formation. This work demonstrates the importance of direct MnO2 photoreduction in environmental processes and provides a framework to test new hypotheses regarding the role of organic molecules and metal species in photochemical reactions with Mn oxide phases. The timescales for the production and evolution of Mn(III) species and a catalytic role for interlayer Ca2+ identified here from spectroscopic measurements can also guide the design of efficient Mn-based catalysts for water oxidation.

A disordered nanoparticle model for 6-line ferrihydrite

Abstract Much of the bioavailable and geochemically reactive iron in aerobic, circumneutral settings is frequently found in the form of nanoscale particles of a hydrated iron(III) oxyhydroxide phase known as ferrihydrite. Developing useful structural descriptions of defective nanophases such as ferrihydrite has long posed significant challenges. Recently, Michel et al. (2007, 2010) proposed a structural model for ferrihydrite in place of the long-accepted model of Drits et al. (1993). Both models reproduce to high accuracy certain forms of X‑ray scattering data from powdered ferrihydrite. However, discrepancies remain that we hypothesized are due to forms of structural disorder not easily represented by existing models. To test this hypothesis, we performed a novel structural analysis of total X‑ray scattering data acquired from 6-line ferrihydrite. We generated three candidate whole-nanoparticle models of ferrihydrite composed of a two-phase Drits model, the Michel model, and a hybrid phase based on a single-phase Drits model that incorporated tetrahedral Fe sites, creating a lattice in which the Michel model was one of many possible topologies. We implemented a reverse Monte Carlo (RMC) approach to explore alternative configurations of iron occupancies plus structural disorder, and to refine the nanoparticle structure using both the reciprocal and real-space forms of the X‑ray scattering data. We additionally used oxygen K-edge X‑ray absorption spectroscopy to semi-quantitatively assess the ratio of protonated:non-protonated oxygen sites in an iron(III) oxides. This analysis provides independent evidence for a significantly lower OH:O stoichiometric ratio for ferrihydrite than for goethite, further constraining the RMC models. The hybrid structure model gave better agreement to the experimental total scattering data than nanoparticles based upon either the Michel or Drits models. Models that incorporated tetrahedrally coordinated iron sites consistently achieved better matches to the data than models containing facesharing octahedra. Long-range vacancy disorder was essential for optimum fits to the scattering data, highlighting the utility of whole-nanoparticle models in place of unit-cell models with random distributions of iron vacancies. The RMC-derived structures do not satisfy all experimental constraints on composition and structure. Nevertheless this work illustrates that a suitably constrained RMC method applied to whole-nanoparticle models can be an effective approach for exploring disorder in nanocrystalline materials.

Belowground response to drought in a tropical forest soil. II. Change in microbial function impacts carbon composition

Climate model projections for tropical regions show clear perturbation of precipitation patterns leading to increased frequency and severity of drought in some regions. Previous work has shown declining soil moisture to be a strong driver of changes in microbial trait distribution, however, the feedback of any shift in functional potential on ecosystem properties related to carbon cycling are poorly understood. Here we show that drought-induced changes in microbial functional diversity and activity shape, and are in turn shaped by, the composition of dissolved and soil-associated carbon. We also demonstrate that a shift in microbial functional traits that favor the production of hygroscopic compounds alter the efflux of carbon dioxide following soil rewetting. Under drought the composition of the dissolved organic carbon pool changed in a manner consistent with a microbial metabolic response. We hypothesize that this microbial ecophysiological response to changing soil moisture elevates the intracellular carbon demand stimulating extracellular enzyme production, that prompts the observed decline in more complex carbon compounds (e.g., cellulose and lignin). Furthermore, a metabolic response to drought appeared to condition (biologically and physically) the soil, notably through the production of polysaccharides, particularly in experimental plots that had been pre-exposed to a short-term drought. This hysteretic response, in addition to an observed drought-related decline in phosphorus concentration, may have been responsible for a comparatively modest CO2 efflux following wet-up in drought plots relative to control plots.