John M. Pettibone

Inhalation Exposure Study of Titanium Dioxide Nanoparticles with a Primary Particle Size of 2 to 5 NM

Background Nanotechnology offers great promise in many industrial applications. However, little is known about the health effects of manufactured nanoparticles, the building blocks of nanomaterials. Objectives Titanium dioxide (TiO2) nanoparticles with a primary size of 2–5 nm have not been studied previously in inhalation exposure models and represent some of the smallest manufactured nanoparticles. The purpose of this study was to assess the toxicity of these nanoparticles using a murine model of lung inflammation and injury. Materials and Methods The properties of TiO2 nanoparticles as well as the characteristics of aerosols of these particles were evaluated. Mice were exposed to TiO2 nanoparticles in a whole-body exposure chamber acutely (4 hr) or subacutely (4 hr/day for 10 days). Toxicity in exposed mice was assessed by enumeration of total and differential cells, determination of total protein, lactate dehydrogenase (LDH) activity and inflammatory cytokines in bronchoalveolar lavage (BAL) fluid. Lungs were also evaluated for histopathologic changes Results Mice exposed acutely to 0.77 or 7.22 mg/m3 nanoparticles demonstrated minimal lung toxicity or inflammation. Mice exposed subacutely (8.88 mg/m3) and necropsied immediately and at week 1 or 2 postexposure had higher counts of total cells and alveolar macrophages in the BAL fluid compared with sentinels. However, mice recovered by week 3 postexposure. Other indicators were negative. Conclusions Mice subacutely exposed to 2–5 nm TiO2 nanoparticles showed a significant but moderate inflammatory response among animals at week 0, 1, or 2 after exposure that resolved by week 3 postexposure.

Adsorption of Organic Acids on TiO2 Nanoparticles: Effects of pH, Nanoparticle Size, and Nanoparticle Aggregation

In this study, the adsorption of two organic acids, oxalic acid and adipic acid, on TiO2 nanoparticles was investigated at room temperature, 298 K. Solution-phase measurements were used to quantify the extent and reversibility of oxalic acid and adipic acid adsorption on anatase nanoparticles with primary particle sizes of 5 and 32 nm. At all pH values considered, there were minimal differences in measured Langmuir adsorption constants, K ads, or surface-area-normalized maximum adsorbate-surface coverages, Gamma max, between 5 and 32 nm particles. Although macroscopic differences in the reactivity of these organic acids as a function of nanoparticle size were not observed, ATR-FTIR spectroscopy showed some distinct differences in the absorption bands present for oxalic acid adsorbed on 5 nm particles compared to 32 nm particles, suggesting different adsorption sites or a different distribution of adsorption sites for oxalic acid on the 5 nm particles. These results illustrate that molecular-level differences in nanoparticle reactivity can still exist even when macroscopic differences are not observed from solution phase measurements. Our results also allowed the impact of nanoparticle aggregation on acid uptake to be assessed. It is clear that particle aggregation occurs at all pH values and that organic acids can destabilize nanoparticle suspensions. Furthermore, 5 nm particles can form larger aggregates compared to 32 nm particles under the same conditions of pH and solid concentrations. The relative reactivity of 5 and 32 nm particles as determined from Langmuir adsorption parameters did not appear to vary greatly despite differences that occur in nanoparticle aggregation for these two different size nanoparticles. Although this potentially suggests that aggregation does not impact organic acid uptake on anatase particles, these data clearly show that challenges remain in assessing the available surface area for adsorption in nanoparticle aqueous suspensions because of aggregation.

Environmental implications of nanoparticle aging in the processing and fate of copper-based nanomaterials.

Copper nanomaterials are being used in a large number of commercial products because these materials exhibit unique optical, magnetic, and electronic properties. Metallic copper nanoparticles, which often have a thin surface oxide layer, can age in the ambient environment and become even more oxidized over time. These aged nanoparticles will then have different properties compared to the original nanoparticles. In this study, we have characterized three different types of copper-based nanoparticle (NP) samples designated as Cu(new) NPs, Cu(aged) NPs, and CuO NPs that differ in the level of oxidation. The solution phase behavior of these three copper-based nanoparticle samples is investigated as a function of pH and in the presence and absence of two common, complexing organic acids, citric and oxalic acid. The behavior of these three copper-based NP types shows interesting differences. In particular, Cu(aged) NPs exhibit unique chemistry including oxide phases that form and surface adsorption properties. Overall, the current study provides some insights into the impacts of nanoparticle aging and how the physicochemical characteristics and reactivity of nanomaterials can change upon aging.

Inflammatory response of mice to manufactured titanium dioxide nanoparticles: Comparison of size effects through different exposure routes

TiO2 is a widely used manufactured nanomaterial and the opportunity for human exposure makes it necessary to study its health implications. Using murine models for inflammation, size effects of inflammatory response in instillation and acute inhalation exposures of TiO2 nanoparticles with manufacturers’ average particles sizes of 5 and 21 nm were investigated. The properties of the primary nanoparticles, nanoparticle agglomerates aerosol and instillation solution for both sized nanoparticles were evaluated. Mice were acutely exposed in a whole-body exposure chamber or through nasal instillation and toxicity was assessed by enumeration of total and differential cells, determination of total protein, LDH activity and inflammatory cytokines in BAL fluid. Lungs were also evaluated for histopathological changes. Results show the larger TiO2 nanoparticles were found to be moderately, but significantly, more toxic. The nanoparticles had different agglomeration states which may be a factor as important as the surface and physical characteristics of the primary nanoparticles in determining toxicity.

Rational strategy for characterization of nanoscale particles by asymmetric-flow field flow fractionation: A tutorial

This tutorial proposes a comprehensive and rational measurement strategy that provides specific guidance for the application of asymmetric-flow field flow fractionation (A4F) to the size-dependent separation and characterization of nanoscale particles (NPs) dispersed in aqueous media. A range of fractionation conditions are considered, and challenging applications, including industrially relevant materials (e.g., metal NPs, asymmetric NPs), are utilized in order to validate and illustrate this approach. We demonstrate that optimization is material dependent and that polystyrene NPs, widely used as a reference standard for retention calibration in A4F, in fact represent a class of materials with unique selectivity, recovery and optimal conditions for fractionation; thus use of these standards to calibrate retention for other materials must be validated a posteriori. We discuss the use and relevance of different detection modalities that can potentially yield multi-dimensional and complementary information on NP systems. We illustrate the fractionation of atomically precise nanoclusters, which are the lower limit of the nanoscale regime. Conversely, we address the upper size limit for normal mode elution in A4F. The protocol for A4F fractionation, including the methods described in the present work is proposed as a standardized strategy to realize interlaboratory comparability and to facilitate the selection and validation of material-specific measurement parameters and conditions. It is intended for both novice and advanced users of this measurement technology.

Inflammatory response of mice following inhalation exposure to iron and copper nanoparticles

We examined pulmonary inflammatory responses of mice following whole-body inhalation exposure to copper and iron nanoparticles in acute and sub-acute studies. Concentrations for sub-acute copper and iron exposures were 3.6 mg m−3. No significant pathology was found following acute exposure. Immediately following sub-acute exposure, both iron- and copper-exposed mice showed increased inflammation compared to sentinels. Copper nanoparticle-exposed mice had significantly higher lavage cytokines as well as perivasculitis and alveolitis. Three weeks post-exposure, all inflammatory markers decreased for iron nanoparticle-exposed mice, however, some remained elevated for copper-exposed mice. At biologically relevant pHs, in vitro studies showed that copper nanoparticles displayed a greater propensity for dissolution compared to iron. We conclude that the presence of dissolved ions, the concomitant formation of smaller nanoparticles and the absence of particles in stained lung sections immediately postexposure (inferring either translocation or more dispersed aerosol distribution) contributed to the increased inflammation observed in copper nanoparticle-exposed mice.

Discriminating the states of matter in metallic nanoparticle transformations: what are we missing?

A limiting factor in assessing the risk of current and emerging nanomaterials in biological and environmental systems is the ability to accurately detect and characterize their size, shape, and composition in broad product distributions and complex media. Asymmetric flow field-flow fractionation (A4F) is capable of separation without stationary phase interactions or large applied forces. Here, we demonstrate unprecedented A4F fractionation of metallic nanoclusters with core diameters near 1 nm and with high resolution. The isolated nanocluster populations were characterized online with UV-vis absorption and inductively coupled plasma mass spectrometry (ICP-MS). We apply our methodology to a model system, poly(N-vinyl-2-pyrrolidone)-protected silver nanoparticles with an excess of tripeptide-glutathione (GSH). The temporal evolution of the initial silver nanoparticle distribution in the presence of excess GSH results in the appearance and persistence of a continuum of matter states (e.g., Ag(+) nanoclusters and nanoparticles) that could be fractionated with A4F, characterized by their optical signatures and diffusion coefficients, and quantified with ICP-MS. The results suggest that our methodology is generally applicable to metallic systems when appropriate online detection is coupled to the A4F. Because we extend the capability of the coupled A4F system to reliably detect, characterize, and quantify metallic populations in the sub-5 nm regime, the opportunity exists to survey the formation and transformation products of nanomaterials in more relevant biological and environmental systems. Thus, individually assessing the risks associated with specific ion, nanocluster, and nanoparticle populations is achievable, where such populations may have previously been misrepresented.

Predictive gold nanocluster formation controlled by metal-ligand complexes.

The formation of ligand-protected gold nanoclusters during size-selective syntheses is seemingly driven by the inherent properties of the protecting ligands, but a general description of the product formation has not been presented. This study uses diphosphine-protected Au clusters as a model system to examine i) control of metal-ligand complex distributions in methanol-chloroform solutions, ii) role of solution perturbations, e.g., oxidation, and iii) nanocluster formation through reduction of characterized complex distributions. By selectively reducing complexes and monitoring cluster formation with electrospray ionization mass spectrometry and UV-vis, data show the distribution of complexes can be controlled through ligand exchange, and the reduction of specific complexes produce characteristic ligated gold clusters based on ligand class. Specifically, 1,n-bis(diphenylphosphino)n-alkane ligands, L(n), where n = 1 through 6, are classified into two distinct sets. The classes represent ligands that either form mainly [AuL(n)(2)](+) (Class I, n = 1-3) or bridged [Au(2)L(n)(2)](2+) (Class II, n = 4-6) complexes after complete ligand exchange with AuClPPh(3). Selectively reducing gold-phosphine ligand complexes allows mapping of product formation, resulting collectively in a predictive tool for ligated gold cluster production by simply monitoring the initial complex distribution prior to reduction.

NanoEHS – defining fundamental science needs: no easy feat when the simple itself is complex

Nanotechnology is no longer in its infancy and has made significant advances since the implementation of the National Nanotechnology Initiative (NNI) in 2000. Incorporation of nanotechnology in many fields including information technology, medicine, materials, energy, catalysis and cosmetics has led to an increase in engineered nanomaterial (ENM) production, and consequently, increased nanomaterial use. In comparison, the generation of concrete and consistent evidence related to the environmental health and safety of nanomaterials (NanoEHS) is lacking. The main factors contributing to the slower progress in NanoEHS versus conventional EHS are related to the complexity, property transformations, life cycles and behavior of nanomaterials even in carefully controlled environments. Therefore, new systematic, integrated research approaches in NanoEHS are needed for overcoming this complexity and bridging current knowledge gaps. A workshop on “NanoEHS: Fundamental Science Needs” brought together scientists and engineers to identify current fundamental science challenges and opportunities within NanoEHS. Detailed discussions were conducted on identifying the fundamental properties that are critical in NanoEHS, differentiating between conventional and NanoEHS studies as well as understanding, the effect of dynamic transformations on nanometrology, role of dosimetry and mechanistic data gaps in nanotoxicology. An important realization that even simple nanoscale materials can be complex when considering NanoEHS implications was noted several times during the workshop. Despite this fact, a number of fundamental research areas to further the scientific foundation to address NanoEHS needs are suggested.

Reaction network governing diphosphine-protected gold nanocluster formation from nascent cationic platforms

We identify the reaction network governing gold monolayer protected cluster (MPC) formation during the reduction of Au(PPh(3))Cl and L(5) (L(5) = 1,5-bis(diphenylphosphino)pentane) in solutions. UV-vis spectroscopy and electrospray ionization mass spectrometry (ESI-MS) monitored the formation of ligated Au(x): 6 ≤ x ≤ 12 clusters, which comprise the reaction intermediates and final products. Initially, predominantly [Au(2)L(5)(2)](2+) complexes form through dissolution of Au(PPh(3))Cl. These complexes control the reduction and nucleation reactions that form nascent phosphine-ligated Au(8) and Au(10) ionic clusters. [Au(10)L(5)(4)](2+) is an observed growth platform for ligated Au(11) and Au(12) clusters. The data for syntheses of Au : L(5) systems evidence that the nascent reaction products (t < 3 days) are less dependent on the chosen reducing agent (borane tert-butylamine complex or NaBH(4)); instead, after reduction ceases, subsequent solution phase processing provides greater control for tuning cluster nuclearity.