Development and characterization of a Versatile Engineered Nanomaterial Generation System (VENGES) suitable for toxicological studies

Abstract

A novel system for generation of engineered nanomaterials (ENMs) suitable for in situ toxicological characterization within biological matrices was developed. This Versatile Engineered Nanomaterial Generation System (VENGES) is based on industry-relevant, flame spray pyrolysis aerosol reactors that can scaleably produce ENMs with controlled primary and aggregate particle size, crystallinity, and morphology. ENMs are produced continuously in the gas phase, allowing their continuous transfer to inhalation chambers, without altering their state of agglomeration. Freshly generated ENMs are also collected on Teflon filters for subsequent physicochemical and morphological characterization and for in vitro toxicological studies. The ability of the VENGES system to generate families of ENMs of pure and selected mixtures of iron oxide, silica, and nanosilver with controlled physicochemical properties was demonstrated using a range of state-of-the-art-techniques. Specific surface area was measured by nitrogen adsorption using the Brunauer-Emmett-Teller method, and crystallinity was characterized by X-ray diffraction. Particle morphology and size were evaluated by scanning and transmission electron microscopy. The suitability of the VENGES system for toxicological studies was also shown in both in vivo and in vitro studies involving Sprague-Dawley rats and human alveolar-like monocyte derived macrophages, respectively. We demonstrated linkage between physicochemical ENM properties and potential toxicity.

Figure 1

Schematic diagram of the VENGES system.

Figure 2

TEM images of Fe₂O₃ nanoparticles generated at different liquid precursor to dispersing gas ratios: a) 4/6 ratio, b) 7/3 ratio.

Figure 3

XRD spectra for (γ-Fe₂O₃) nanoparticles for different liquid precursor to dispersing gas ratios (Miller indices, hkl, according to the ICSD 8721 are indicated in parentheses)

Figure 4

Fe₂O₃ specific surface area (SSA) (triangles), corresponding particle diameter (d_BET, circles), and crystal size (d_XRD, diamonds) as a function of FSP liquid precursor to dispersion O₂ flow ratio. Increasing ratios increase particle concentration and high temperature particle residence time resulting in large primary particles and crystals.

Figure 5

SiO₂ TEM images at different liquid precursor to dispersing O₂ flow ratios: a) 3/7, b) 5/5, c) 6/4, and d) 7/3.

Figure 6

SiO₂ specific surface area (SSA, triangles) and the corresponding particle diameter (d_BET, squares) as a function of FSP liquid precursor to dispersion O₂ flow ratio.

Figure 7

STEM image of 10% Ag on SiO₂ and the corresponding Ag particle size distribution.

Figure 8

XRD of Ag deposited on the surface of SiO₂ nanoparticles for different % wt of Ag. The d_XRD size of silver is indicated that comes mostly from the large mode of bimodal Ag size distribution arising from FSP of Ag nitrate solutions (Heine and Pratsinis, 2007). In parenthesis the Miller indices (hkl) of the crystal planes according to the ICSD 64997 is indicated.

Figure 9

Cellular and biochemical parameters of lung injury and inflammation. A. Differential cell counts in BAL at 24 hours post-exposure. There were significant increases in lavaged neutrophils when rats were instilled with 1 mg/kg iron oxide or 10% wt Ag on SiO₂. B. Biochemical parameters of injury/inflammation. Instillation of suspension of iron oxide (D_DLS 1. 76 μm) or 10% wt Ag on SiO₂ (D_DLS 0. 19 μm) significantly increased myeloperoxidase (MPO), lactate dehydrogenase (LDH) and albumin compared with control. No significant change was observed in BAL hemoglobin levels. In addition, 10% wt Ag on SiO₂ induced higher albumin levels compared with iron oxide. (* P < 0.05, vs. control, * P < 0.05, ^# iron oxide vs. 10% wt Ag on SiO₂).

Figure 10

Membrane integrity cytotoxicity assay for 0.05 or 0.5 mg/ml solutions of sonicated ENMs. The images show particle and concentration dependent toxicity as green nuclear fluorescence; the mean % positive cells is indicated within each image.