Grantee Research Project Results

In the past year, the potential effects of manufactured nanoparticles on human health have been investigated. In particular, a series of acute and sub-chronic exposures of titanium dioxide (TiO₂) nanoparticles with a primary particle size of 5 nm have been completed. Although there have been earlier inhalation studies on TiO₂ ultrafine particles, these have been done on particles with a particle size of 20 nm and above, there is some evidence that particles with a primary particle size less than 10 nm may behave differently than particles with a primary particle size greater than 10 nm. TiO₂ nanoparticles with a primary size of 2-5 nm have not previously been studied in inhalation exposure models and represent some of the smallest manufactured nanoparticles. The properties of TiO₂ nanoparticles as well as the characteristics of aerosols of these particles were evaluated. The purpose of this study was to assess the toxicity of these nanoparticles using a murine model of lung inflammation and injury. Mice were exposed to TiO₂ nanoparticles in a whole-body exposure chamber acutely (4 hr) or sub-acutely (4 hr/day for 10 days). (See Figure 1 showing the schematic of the exposure chamber.) Toxicity in exposed mice was assessed by enumeration of total and differential cells, determination of total protein, LDH activity and inflammatory cytokines in bronchoalveolar fluid. Lungs also were evaluated for histopathological changes. Mice exposed acutely to 0.77 or 7.22 mg m^-3 of nanoparticles demonstrated minimal lung toxicity or inflammation. Mice exposed sub-acutely (8.88 mg m^-3) and necropsied immediately, 1 or 2 weeks post exposure had higher counts of total cells and macrophages in the broncheoalveolar lavage fluid compared to sentinels. However, mice recovered by third week post exposure. Other indicators were negative. From this study, we concluded that mice sub-acutely exposed to 2-5 nm TiO₂ nanoparticles showed a significant but moderate inflammatory response among animals 0, 1 or 2 weeks after exposure that resolved 3 weeks post exposure.

 

In another study, instillation and acute inhalation exposures of different size TiO₂ particles to mice are run to examine how nanoparticle size and exposure method affect inflammatory response. Through the use of a whole body exposure chamber and nasal instillation technique along with bulk and surface characterization techniques, it was found that the smaller 5 nm particles did not create a larger inflammatory response than 20 nm particles. The aggregation states of the particles were shown using TEM to behave very different in the instillation solution compared to the powder delivery in the acute inhalation exposure. One conclusion from this study is that the aggregation state of the particles, along with other surface and physical characteristics, is an important factor in the toxicity of nanoparticles.

 

Figure 1. The powder XRD pattern of iron nanoparticles is shown along with reference spectra of Fe metal, Fe₃O₄ and g-Fe₂O₃. (B) The powder XRD pattern of copper nanoparticles is shown along with reference spectra of Cu metal, Cu₂O and CuO. Based on the XTD data as well as TEM measurements and surface analysis using X-ray photoelectron spectroscopy, pictorial representations of the iron and copper nanoparticles with these different phases are shown.

 

Finally, we have completed a study of acute and sub-acute exposures of 25 nm iron and copper nanoparticles. Based on the physicochemical characterization data, which include powder X-ray diffraction, TEM (from whole body chamber, vide infra) and X-ray photoelectron spectroscopy for these commercial materials, these nanoparticles consist of a metallic core and an oxide coating that contains two phases with the most oxide phase being on the surface of the nanoparticles (see Figure 1 which shows XRD data). The toxicity data shown below in Figure 2 give a comparison of the total number of cells and differential cell counts in the bronchoalveolar lavage fluid for control mice and for mice sub-acutely exposed to Fe and Cu nanoparticles. It can be seen from these data that Cu nanoparticles produce the greatest response and give rise to the highest cell count and the greatest percentage of neutrophils. Copper nanoparticle exposed mice show significantly larger response as well as signs of perivasculitis and aveolitis in lungs. After 3 weeks post-exposure, all inflammatory markers decreased and lungs were evaluated as normal for both Fe and Cu nanoparticle-exposed mice. In vitro dissolution studies showed that Cu nanoparticles displayed enhanced propensity for dissolution compared to Fe nanoparticles at biologically relevant pHs. Nanoparticle dissolution results in both dissolved ions and nanoparticles of decreased size. As such, it is surmised that both the formation of dissolved ions and the concomitant production of smaller nanoparticles below 10 nm that form during the dissolution process following inhalation of Cu nanoparticles, may be the cause of the increased inflammatory response in exposed mice.

 

Figure 2. Number of cells in BAL fluid from animals acutely sub-acutely exposed to Fe and Cu nanoparticles

 

We also began studies on Ag nanoparticle inhalation toxicity under this EPA grant that is being continued in other studies that now currently are funded by NIOSH.