Metal Oxide Engineered Nanomaterials Research Articles

Metal Oxide Engineered Nanomaterials Modulate Rabbit Corneal Fibroblast to Myofibroblast Transformation.

PurposeCorneal keratocyte-fibroblast-myofibroblast (KFM) transformation plays a critical role in corneal stromal wound healing. However, the impact of engineered nanomaterials (ENMs), found in an increasing number of commercial products, on this process is poorly studied. This study investigates the effects of metal oxide ENMs on KFM transformation in vitro and in vivo.MethodsCell viability of rabbit corneal fibroblasts (RCFs) was tested following treatment with 11 metal oxide ENMs at concentrations of 0.5 to 250 µg/ml for 24 hours. Messenger RNA (mRNA) and protein expression of αSMA, a marker of myofibroblast transformation, were measured using RCFs after exposure to 11 metal oxide ENMs at a concentration that did not affect cell viability, in media containing either 0 or 10 ng/ml of TGF-β1. Additionally, the effect of topical Fe2O3 nanoparticles (NPs) (50 ng/ml) on corneal stromal wound healing following phototherapeutic keratectomy (PTK) was determined.ResultsV2O5, Fe2O3, CuO, and ZnO ENMs were found to significantly reduce cell viability as compared to vehicle control and the other seven metal oxide ENMs tested. V2O5 nanoflakes significantly reduced mRNA and protein αSMA concentrations in the presence of TGF-β1. Fe2O3 NPs significantly increased αSMA mRNA expression in the presence of TGF-β1 but did not alter αSMA protein expression. Topically applied Fe2O3 NPs in an in vivo rabbit corneal stromal wound healing model did not delay healing.ConclusionsFe2O3 NPs promote corneal myofibroblast induction in vitro but do not impair corneal stromal wound healing in vivo.Translational RelevanceThese experimental results can apply to human nanomedical research.

Toxicity assessment of metal oxide nanomaterials using in vitro screening and murine acute inhalation studies

Characterizations and in vitro toxicity screening were performed on metal oxide engineered nanomaterials (ENMs) independently comprising ZnO, CuO, CeO2, Fe2O3, WO3, V2O5, TiO2, Al2O3 and MgO. Nanomaterials that exhibited the highest toxicity responses in the in vitro screening assays (ZnO, CuO, and V2O5) and the lesser explored material WO3 were tested for acute pulmonary toxicity in vivo. Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide ENMs in a nose-only exposure system and toxicity outcomes (biomarkers of cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 h after the start of exposure were assessed. The studies were performed as part of the NIEHS Nanomaterials Health Implications Research consortium with the purpose of investigating the effects of ENMs on various biological systems. ENMs were supplied by the Engineered Nanomaterials Resource and Coordination Core. Among the ENMs studied, the highest toxicity was observed for CuO and ZnO NPs in both in vitro and in vivo acute models. Compared to sham-exposed controls, there was a significant increase in bronchoalveolar lavage neutrophils and proinflammatory cytokines and a loss of macrophage viability at both 4 h and 24 h for ZnO and CuO but not seen for V2O5 or WO3. These effects were observed in both female and male mice. The cell viability performed after in vitro exposure to ENMs and assessment of lung inflammation after acute inhalation exposure in vivo were shown to be sensitive endpoints to predict ENM acute toxicity.

Analysis of engineered nanomaterials (Ag, CeO2 and Fe2O3) in spiked surface waters at environmentally relevant particle concentrations.

Quantification of engineered nanomaterials (ENMs) concentrations in surface waters remains one of the key challenges in environmental nanoscience and nanotechnology. A promising approach to estimate metal and metal oxide ENM concentrations in complex environmental samples is based on the increase in the elemental ratios of ENM-contaminated samples relative to the corresponding natural background elemental ratios. This contribution evaluated the detection and quantification of Ag, CeO2, and Fe2O3 ENMs spiked in synthetic soft, or in natural river waters using the elemental ratio approach, and evaluated the effect of extractants including sodium hydroxide (NaOH), sodium oxalate (Na2C2O4) and sodium pyrophosphate (Na4P2O7) on the recovery of ENMs from the spiked waters. The extracted ENM concentrations were higher in Na4P2O7-extracted suspensions than in NaOH- and Na2C2O4-extracted suspensions due to the higher efficiency of Na4P2O7 to break up natural and engineered nanomaterial heteroaggregates. The size distributions of the extracted suspensions were determined by asymmetrical flow-field flow fractionation coupled to inductively coupled plasma-mass spectrometer (AF4-ICP-MS). These size distribution analysis demonstrated that Ag ENMs were extracted from the spiked river water as both primary particles and small (<100nm) aggregates, whereas CeO2 ENMs were extracted from the spiked river water as aggregates of particles in the size range 0-200nm. The number particle size distribution of the extracted suspensions confirmed that Ag ENMs were extracted as a mixture of primary and aggregated Ag ENMs. Small Ag ENMs (i.e. <20nm) were detected by AF4-ICP-MS, but these particles were not detected by single particle (sp)-ICP-MS due to high size detection limit of sp-ICP-MS. This study illustrates that the elemental ratio approach is a promising approach to detect and quantify ENMs in surface waters. This study also illustrates the need for a multi-method approach, including extraction, filtration, AF4-ICP-MS and sp-ICP-MS, to detect, quantify, and characterize ENMs in surface waters.

Mapping 2D- and 3D-distributions of metal/metal oxide nanoparticles within cleared human ex vivo skin tissues

Increasing numbers of commercial skincare products are being manufactured with engineered nanomaterials (ENMs), prompting a need to fully understand how ENMs interact with the dermal barrier as a major biodistribution entry route. Although animal studies show that certain nanomaterials can cross the skin barrier, physiological differences between human and animal skin, such as the lack of sweat glands, limit the translational validity of these results. Current optical microscopy methods have limited capabilities to visualize ENMs within human skin tissues due to the high amount of background light scattering caused by the dense, ubiquitous extracellular matrix (ECM) of the skin. Here, we hypothesized that organic solvent-based tissue clearing (“immunolabeling-enabled three-dimensional imaging of solvent-cleared organs”, or “iDISCO”) would reduce background light scattering from the extracellular matrix of the skin to sufficiently improve imaging contrast for both 2D mapping of unlabeled metal oxide ENMs and 3D mapping of fluorescent nanoparticles. We successfully mapped the 2D distribution of label-free TiO2 and ZnO nanoparticles in cleared skin sections using correlated signals from darkfield, brightfield, and confocal microscopy, as well as micro-spectroscopy. Specifically, hyperspectral microscopy and Raman spectroscopy confirmed the identity of label-free ENMs which we mapped within human skin sections. We also measured the 3D distribution of fluorescently labeled Ag nanoparticles in cleared skin biopsies with wounded epidermal layers using light sheet fluorescence microscopy. Overall, this study explores a novel strategy for quantitatively mapping ENM distributions in cleared ex vivo human skin tissue models using multiple imaging modalities. By improving the imaging contrast, we present label-free 2D ENM tracking and 3D ENM mapping as promising capabilities for nanotoxicology investigations.

Engineered metal oxide nanomaterials inhibit corneal epithelial wound healing in vitro and in vivo

Ocular exposure to metal oxide engineered nanomaterials (ENMs) is common as exemplified by zinc(II) oxide (ZnO), a major constituent of sunscreens and cosmetics. The ocular surface that includes the transparent cornea and its protective tear film are common sites of exposure for metal ENMs. Despite the frequency of exposure of the ocular surface, there is a knowledge gap regarding the effects of metal oxide ENMs on the cornea in health and disease. Therefore, we studied the effects of metal oxide ENMs on the cornea in the presence or absence of injury. Cell viability of immortalized human corneal epithelial (hTCEpi) cells was assessed following treatment with 11 metal oxide ENMs with a concentration ranging from 0.5 to 250 μg/ml for 24 h. An epithelial wound healing assay with a monolayer of hTCEpi cells was then performed using 11 metal oxide ENMs at select concentrations based on data from the viability assays. Subsequently, based on the in vitro results, in vivo testing of precorneal tear film (PTF) quantity and stability as well as a corneal epithelial wound healing were tested in the presence or absence ZnO or vanadium(V) oxide (V2O5) at a concentration of 50 μg/ml. We found that WO3, ZnO, V2O5 and CuO ENMs significantly reduced hTCEpi cell viability in comparison to vehicle control or the other metal oxide ENMs tested. Furthermore, ZnO and V2O5 ENMs also significantly decreased hTCEpi cell migration. Although ZnO and V2O5 did not alter PTF parameters of rabbits in vivo, corneal epithelial wound healing was significantly delayed by topical ZnO while V2O5 did not alter wound healing. Finally, hyperspectral images confirmed penetration of ZnO and V2O5 through all corneal layers and into the iris stroma. Considering the marked epithelial toxicity and corneal penetration of ZnO, further investigations on the impact of this ENM on the eye are warranted.

Solubility of metal oxide nanomaterials: cautionary notes on sample preparation

Eight metal oxides were obtained to investigate the dissolution behaviour of engineered nanomaterials (ENMs) dispersed in biologically relevant media. Identities of the metal oxide compounds, and their crystal form and size were checked using powder X-ray diffraction (XRD). Methods for sonication of metal oxide nanoparticles were optimized to achieve stable stock dispersions, and methods for separation of dissolved metal ions from dispersed nanoparticles were evaluated. The results of the optimization experiments showed that each metal oxide ENM required a different combination of sonication time and power (% amplitude). Optimized values for delivered sonication energy (J/mL) ranged from 24 for CuO to 833 for ɣ-Al2O3. Centrifugation at 20000G was found to be more effective and less prone to artefacts than using commercially available ultrafiltration devices for separation of dissolved metal fraction, under these experimental conditions. XRD results indicated that the composition of two metal oxide nanopowders (Mn2O3 and Fe2O3) did not meet the manufacturers’ claims, underscoring the importance of double-checking physical-chemical properties of commercial ENMs purchased for research.

Assessing the Risk of Engineered Nanomaterials in the Environment: Development and Application of the nanoFate Model.

We developed a dynamic multimedia fate and transport model (nanoFate) to predict the time-dependent accumulation of metallic engineered nanomaterials (ENMs) across environmental media. nanoFate considers a wider range of processes and environmental subcompartments than most previous models and considers ENM releases to compartments (e.g., urban, agriculture) in a manner that reflects their different patterns of use and disposal. As an example, we simulated ten years of release of nano CeO2, CuO, TiO2, and ZnO in the San Francisco Bay area. Results show that even soluble metal oxide ENMs may accumulate as nanoparticles in the environment in sufficient concentrations to exceed the minimum toxic threshold in freshwater and some soils, though this is more likely with high-production ENMs such as TiO2 and ZnO. Fluctuations in weather and release scenario may lead to circumstances where predicted ENM concentrations approach acute toxic concentrations. The fate of these ENMs is to mostly remain either aggregated or dissolved in agricultural lands receiving biosolids and in freshwater or marine sediments. Comparison to previous studies indicates the importance of some key model aspects including climatic and temporal variations, how ENMs may be released into the environment, and the effect of compartment composition on predicted concentrations.

Metal Nanomaterial Toxicity Variations Within the Vascular System.

Engineered nanomaterials (ENM) are anthropogenic materials with at least one dimension less than 100nm. Their ubiquitous employment in biomedical and industrial applications in the absence of full toxicological assessments raises significant concerns over their safety on human health. This is a significant concern, especially for metal and metal oxide ENM as they may possess the greatest potential to impair human health. A large body of literature has developed that reflects adverse systemic effects associated with exposure to these materials, but an integrated mechanistic framework for how ENM exposure influences morbidity remains elusive. This may be due in large part to the tremendous diversity of existing ENM and the rate at which novel ENM are produced. In this review, the influence of specific ENM physicochemical characteristics and hemodynamic factors on cardiovascular toxicity is discussed. Additionally, the toxicity of metallic and metal oxide ENM is presented in the context of the cardiovascular system and its discrete anatomical and functional components. Finally, future directions and understudied topics are presented. While it is clear that the nanotechnology boom has increased our interest in ENM toxicity, it is also evident that the field of cardiovascular nanotoxicology remains in its infancy and continued, expansive research is necessary in order to determine the mechanisms via which ENM exposure contributes to cardiovascular morbidity.

Germination and early plant development of ten plant species exposed to titanium dioxide and cerium oxide nanoparticles.

Ten agronomic plant species were exposed to different concentrations of nano-titanium dioxide (nTiO2 ) or nano-cerium oxide (nCeO2 ) (0 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL) to examine potential effects on germination and early seedling development. The authors modified a standard test protocol developed for soluble chemicals (OPPTS 850.4200) to determine if such an approach might be useful for screening engineered nanomaterials (ENMs) and whether there were differences in response across a range of commercially important plant species to 2 common metal oxide ENMs. Eight of 10 species responded to nTiO2 , and 5 species responded to nCeO2 . Overall, it appeared that early root growth may be a more sensitive indicator of potential effects from ENM exposure than germination. The observed effects did not always relate to the exposure concentration, indicating that mass-based concentration may not fully explain the developmental effects of these 2 ENMs. The results suggest that nTiO2 and nCeO2 have different effects on early plant growth of agronomic species, with unknown effects at later stages of the life cycle. In addition, standard germination tests, which are commonly used for toxicity screening of new materials, may not detect the subtle but potentially more important changes associated with early growth and development in terrestrial plants. Environ Toxicol Chem 2016;35:2223-2229. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.

Hyperspectral imaging of nanoparticles in biological samples: Simultaneous visualization and elemental identification.

While engineered nanomaterials (ENMs) are increasingly incorporated into industrial processes and consumer products, the potential biological effects and health outcomes of exposure remain unknown. Novel advanced direct visualization techniques that require less time, cost, and resource investment than electron microscopy (EM) are needed for identifying and locating ENMs in biological samples. Hyperspectral imaging (HSI) combines spectrophotometry and imaging, using advanced optics and algorithms to capture a spectrum from 400 to 1000 nm at each pixel in an enhanced dark-field microscopic (EDFM) image. HSI-EDFM can be used to confirm the identity of the materials of interest in a sample and generate an image "mapping" their presence and location in a sample. Hyperspectral mapping is particularly important for biological samples, where ENM morphology is visually indistinct from surrounding tissue structures. While use of HSI (without mapping) is increasing, no studies to date have compared results from hyperspectral mapping with conventional methods. Thus, the objective of this study was to utilize EDFM-HSI to locate, identify, and map metal oxide ENMs in ex vivo histological porcine skin tissues, a toxicological model of cutaneous exposure, and compare findings with those of Raman spectroscopy (RS), energy-dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM). Results demonstrate that EDFM-HSI mapping is capable of locating and identifying ENMs in tissue, as confirmed by conventional methods. This study serves as initial confirmation of EDFM-HSI mapping as a novel and higher throughput technique for ENM identification in biological samples, and serves as the basis for further protocol development utilizing EDFM-HSI for semiquantitation of ENMs.

Heteroaggregation of Cerium Oxide Nanoparticles and Nanoparticles of Pyrolyzed Biomass.

Heteroaggregation with indigenous particles is critical to the environmental mobility of engineered nanomaterials (ENM). We studied heteroaggregation of ceria nanoparticles (n-CeO2), as a model for metal oxide ENM, with nanoparticles of pyrogenic carbonaceous material (n-PCM) derived from pecan shell biochar, a model for natural chars and human-made chars used in soil remediation and agriculture. The TEM and STEM images of n-PCM identify both hard and soft particles, both C-rich and C,O,Ca-containing particles (with CaCO3 crystals), both amorphous and "onion-skin" C-rich particles, and traces of nanotubes. Heteroaggregation was evaluated at constant n-CeO2, variable n-PCM concentration by monitoring hydrodynamic diameter by dynamic light scattering and ζ-potential under conditions where n-PCM is "invisible". At pH 5.3, where n-CeO2 and n-PCM are positively and negatively charged, respectively, and each stable to homoaggregation, heteroaggregation is favorable and occurs by a charge neutralization-charge reversal mechanism (CNCR): in this mechanism, primary heteroaggregates that form in the initial stage are stable at low or high n-PCM concentration due to electrostatic repulsion, but unstable at intermediate n-PCM concentration, leading to secondary heteroaggregation. The greatest instability coincides with full charge neutralization. At pH 7.1, where n-CeO2 is neutral and unstable alone, and n-PCM is negative and stable alone, heteroaggregation occurs by a charge-accumulation, core-shell stabilization (CACS) mechanism: n-PCM binds to and forms a negatively charged shell on the neutral surface of the nascent n-CeO2 core, stabilizing the core-shell heteraggregate at a size that decreases with n-PCM concentration. The CNCR and CACS mechanisms give fundamental insight into heteroaggregation between oppositely charged, and between neutral and charged nanoparticles.

Advanced computational modeling for in vitro nanomaterial dosimetry.

BackgroundAccurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells “see,” during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition.MethodsHere we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, KD, and allows modeling of ENM dissolution over time.ResultsDose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for KD values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material.ConclusionsThe advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-015-0109-1) contains supplementary material, which is available to authorized users.

Tracking translocation of industrially relevant engineered nanomaterials (ENMs) across alveolar epithelial monolayers in vitro

Relatively little is known about the fate of industrially relevant engineered nanomaterials (ENMs) in the lungs that can be used to convert administered doses to delivered doses. Inhalation exposure and subsequent translocation of ENMs across the epithelial lining layer of the lung might contribute to clearance, toxic effects or both. To allow precise quantitation of translocation across lung epithelial cells, we developed a method for tracking industrially relevant metal oxide ENMs in vitro using neutron activation. The versatility and sensitivity of the proposed in vitro epithelial translocation (INVET) system was demonstrated using a variety of industry relevant ENMs including CeO2 of various primary particle diameter, ZnO, and SiO2-coated CeO2 and ZnO particles. ENMs were neutron activated, forming gamma emitting isotopes 141Ce and 65Zn, respectively. Calu-3 lung epithelial cells cultured to confluency on transwell inserts were exposed to neutron-activated ENM dispersions at sub-lethal doses to investigate the link between ENM properties and translocation potential. The effects of ENM exposure on monolayer integrity was monitored by various methods. ENM translocation across the cellular monolayer was assessed by gamma spectrometry following 2, 4 and 24 h of exposure. Our results demonstrate that ENMs translocated in small amounts (e.g. <0.01% of the delivered dose at 24 h), predominantly via transcellular pathways without compromising monolayer integrity or disrupting tight junctions. It was also demonstrated that the delivery of particles in suspension to cells in culture is proportional to translocation, emphasizing the importance of accurate dosimetry when comparing ENM–cellular interactions for large panels of materials. The reported INVET system for tracking industrially relevant ENMs while accounting for dosimetry can be a valuable tool for investigating nano–bio interactions in the future.

Nanomaterials in the Environment: From Materials to High-Throughput Screening to Organisms

One of the challenges in the field of nanotechnology is environmental health and safety (EHS), including consideration of the properties of engineered nanomaterials (ENMs) that could pose dangers to the environment. Progress in the field of nanomaterial development and nanotoxicology was presented at the International Conference on the Environmental Implications of Nanotechnology at the California NanoSystems Institute (CNSI) on the UCLA campus on May 11-14, 2010. This event was cohosted by the University of California Center for the Environmental Implications of Nanotechnology (UC CEIN) and the Center for the Environmental Implications of NanoTechnology (CEINT) based at Duke University. Participants included scientists and scholars from various backgrounds, including chemistry, biology, engineering, nanomaterial science, toxicology, ecology, mathematics, sociology, and policy makers. The topics of discussion included safety evaluation of ENMs from an environmental perspective, nanotoxicology, ecotoxicology, safe design of ENMs, environmental risk assessment, public perception of nanotechnology, application of ENMs in consumer products, and many more. The UC CEIN presented data on their predictive toxicological approach to the assessment of ENM libraries, which were designed and synthesized to develop an understanding of the material properties that could lead to hazard generation at the cellular and organismal levels in the environment. This article will focus on the first metal oxide ENM library that was introduced to harmonize research activities in the UC CEIN, with particular emphasis on the safety assessment of ZnO on cells and organisms. Methods of decreasing the observed toxic effects will also be discussed as an integral component of the UC CEIN's activity in developing safer nanomaterials to lessen their environmental impacts.