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While it has been shown that high aspect ratio nanomaterials like carbon nanotubes and TiO(2) nanowires can induce toxicity by acting as fiber-like substances that damage the lysosome, it is not clear what the critical lengths and aspect ratios are that induce this type of toxicity. To answer this question, we synthesized a series of cerium oxide (CeO(2)) nanorods and nanowires with precisely controlled lengths and aspect ratios. Both phosphate and chloride ions were shown to play critical roles in obtaining these high aspect ratio nanostructures. High-resolution TEM analysis shows that single-crystalline CeO(2) nanorods/nanowires were formed along the [211] direction by an "oriented attachment" mechanism, followed by Ostwald ripening. The successful creation of a comprehensive CeO(2) nanorod/nanowire combinatorial library allows, for the first time, the systematic study of the effect of aspect ratio on lysosomal damage, cytotoxicity, and IL-1β production by the human myeloid cell line (THP-1). This in vitro toxicity study demonstrated that, at lengths ≥200 nm and aspect ratios ≥22, CeO(2) nanorods induced progressive pro-inflammatory effects and cytotoxicity. The relatively low "critical" length and aspect ratio were associated with small nanorod/nanowire diameters (6-10 nm), which facilitates the formation of stacking bundles due to strong van der Waals and dipole-dipole attractions. Our results suggest that both length and diameter components of aspect ratio should be considered when addressing the cytotoxic effects of high aspect ratio materials.

Most in vitro toxicity studies on engineered nanomaterials (ENMs) use transformed rather than primary cells for logistical reasons. However, primary cells may provide a more appropriate connection to in vivo toxicity because these cells maintain their phenotypic fidelity and are also capable of differentiating into lineages that may be differently affected by potentially hazardous ENM. Few studies to date have focused on the role of cellular differentiation in determining ENM toxicity. We compared the response of undifferentiated and differentiated primary human bronchial epithelial cells (NHBE) to cationic mesoporous silica nanoparticles (MSNP) that are coated with polyethyleneimine (PEI) since this polymer is known to exert differential cytotoxicity depending on its molecular weight and cationic density. The attachment of cationic PEI polymers to the MSNP surface was used to assess these materials' toxicological potential in undifferentiated and differentiated human bronchial epithelial cells (NHBE), using of a multi-parametric assay that screens for an integrated set of sub-lethal and lethal response outcomes. MSNP coated with high molecular weight (10 and 25 kD) polymers were more toxic in differentiated cells than particles coated with shorter length polymers. The increased susceptibility of the differentiated cells is in agreement with more abundant expression of a proteoglycan, syndecan-1, which contains copious heparin sulfate side chains. Pre-treatment with heparinase to remove the negatively charged sulfates decreased MSNP-PEI binding to the cell surface and lowered the cytotoxic potential of the cationic particles. These data demonstrate the importance of studying cellular differentiation as an important variable in the response of primary cells to toxic ENM properties.

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.

In vivo studies have demonstrated that the state of dispersion of carbon nanotubes (CNTs) plays an important role in generating adverse pulmonary effects. However, little has been done to develop reproducible and quantifiable dispersion techniques to conduct mechanistic studies in vitro. This study was to evaluate the dispersion of multiwalled carbon nanotubes (MWCNTs) in tissue culture media, with particular emphasis on understanding the forces that govern agglomeration and how to modify these forces. Quantitative techniques such as hydrophobicity index, suspension stability index, attachment efficiency, and dynamic light scattering were used to assess the effects of agglomeration and dispersion of as-prepared (AP), purified (PD), or carboxylated (COOH) MWCNTs on bronchial epithelial and fibroblast cell lines. We found that hydrophobicity is the major factor determining AP- and PD-MWCNT agglomeration in tissue culture media but that the ionic strength is the main factor determining COOH-MWCNT suspendability. Bovine serum albumin (BSA) was an effective dispersant for MWCNTs, providing steric and electrosteric hindrances that are capable of overcoming hydrophobic attachment and the electrostatic screening of double layer formation in ionic media. Thus, BSA was capable of stabilizing all tube versions. Dipalmitoylphosphatidylcholine (DPPC) provided additional stability for AP-MWCNTs in epithelial growth medium (BEGM). While the dispersion state did not affect cytotoxicity, improved dispersion of AP- and PD-MWCNTs increased TGF-β1 production in epithelial cells and fibroblast proliferation. In summary, we demonstrate how quantitative techniques can be used to assess the agglomeration state of MWCNTs when conducting mechanistic studies on the effects of dispersion on tissue culture cells.

The rate of expansion of nanomaterials calls for the consideration of appropriate toxicological paradigms in the safety assessment of nanomaterials. We advocate a predictive toxicological paradigm for the assessment of nanomaterial hazards. The predictive toxicological approach is defined as establishing and using mechanisms and pathways of injury at a cellular and molecular level to prioritize screening for adverse biological effects and health outcomes in vivo. Specifically as it relates to nanomaterials, a predictive approach has to consider the physicochemical properties of the material that leads to molecular or cellular injury and also has to be valid in terms of disease pathogenesis in whole organisms.

We developed a dispersal method for multi-walled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on pro-fibrogenic responses in tissue culture cells as well as in mouse lung. Here we demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells and alveolar macrophages (AM), and induced more prominent TGF-β1 and IL-1β production in vitro as well as TGF-β1, IL-1β and PDGF-AA production in vivo than non-dispersed tubes. Moreover, there was good agreement between the pro-fibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bio-processed by AM and induced less prominent collagen deposition at sites of non-granulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects pro-fibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

Zebrafish is an aquatic organism that can be used for high content safety screening of engineered nanomaterials (ENMs). We demonstrate, for the first time, the use of high content bright-field and fluorescence-based imaging to compare the toxicological effect of transition metal oxide (CuO, ZnO, NiO and Co3O4) nanoparticles in zebrafish embryos and larvae. High content bright-field imaging demonstrated potent and dose-dependant hatching interference in the embryos, with the exception of Co3O4 which was relatively inert. We propose that the hatching interference was due to the shedding of Cu and Ni ions, compromising the activity of the hatching enzyme, ZHE1, similar to what we previously proposed for Zn2+. This hypothesis is based on the presence of metal–sensitive histidines in the catalytic center of this enzyme. Co-introduction of a metal ion chelator, diethylene triamine pentaacetic acid (DTPA), reversed the hatching interference of Cu, Zn and Ni. While neither the embryos nor larvae demonstrated morphological abnormalities, high content fluorescence-based imaging demonstrated that CuO, ZnO and NiO could induce increased expression of the heat shock protein 70:enhanced green fluorescence protein (hsp70:eGFP) in transgenic zebrafish larvae. Induction of this response by CuO required a higher nanoparticle dose than the amount leading to hatching interference. This response was also DTPA sensitive. In conclusion, we demonstrate that high content imaging of embryo development, morphological abnormalities and HSP70 expression can be used for hazard ranking and determining the dose-response relationships leading to ENM effects on the development of the zebrafish embryo.

We have recently shown that the dissolution of ZnO nanoparticles and Zn(2+) shedding leads to a series of sublethal and lethal toxicological responses at the cellular level that can be alleviated by iron doping. Iron doping changes the particle matrix and slows the rate of particle dissolution. To determine whether iron doping of ZnO also leads to lesser toxic effects in vivo, toxicity studies were performed in rodent and zebrafish models. First, we synthesized a fresh batch of ZnO nanoparticles doped with 1-10 wt % of Fe. These particles were extensively characterized to confirm their doping status, reduced rate of dissolution in an exposure medium, and reduced toxicity in a cellular screen. Subsequent studies compared the effects of undoped to doped particles in the rat lung, mouse lung, and the zebrafish embryo. The zebrafish studies looked at embryo hatching and mortality rates as well as the generation of morphological defects, while the endpoints in the rodent lung included an assessment of inflammatory cell infiltrates, LDH release, and cytokine levels in the bronchoalveolar lavage fluid. Iron doping, similar to the effect of the metal chelator, DTPA, interfered in the inhibitory effects of Zn(2+) on zebrafish hatching. In the oropharyngeal aspiration model in the mouse, iron doping was associated with decreased polymorphonuclear cell counts and IL-6 mRNA production. Doped particles also elicited decreased heme oxygenase 1 expression in the murine lung. In the intratracheal instillation studies in the rat, Fe doping was associated with decreased polymorphonuclear cell counts, LDH, and albumin levels. All considered, the above data show that Fe doping is a possible safe design strategy for preventing ZnO toxicity in animals and the environment.

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.