Impact of agglomeration state of nano- and submicron sized gold particles on pulmonary inflammation

Background: Nanoparticle (NP) toxicity testing comes with many challenges. Characterization of the test substance is of crucial importance and in the case of NPs, agglomeration/aggregation state in physiological media needs to be considered. In this study, we have addressed the effect of agglomerated versus single particle suspensions of nano-and submicron sized gold on the inflammatory response in the lung. Rats were exposed to a single dose of 1.6 mg/kg body weight (bw) of spherical gold particles with geometric diameters of 50 nm or 250 nm diluted either by ultrapure water or by adding phosphate buffered saline (PBS). A single dose of 1.6 mg/kg bw DQ12 quartz was used as a positive control for pulmonary inflammation. Extensive characterization of the particle suspensions has been performed by determining the zetapotential, pH, gold concentration and particle size distribution. Primary particle size and particle purity has been verified using transmission electron microscopy (TEM) techniques. Pulmonary inflammation (total cell number, differential cell count and pro-inflammatory cytokines), cell damage (total protein and albumin) and cytotoxicity (alkaline phosphatase and lactate dehydrogenase) were determined in bronchoalveolar lavage fluid (BALF) and acute systemic effects in blood (total cell number, differential cell counts, fibrinogen and C-reactive protein) 3 and 24 hours post exposure. Uptake of gold particles in alveolar macrophages has been determined by TEM. Results: Particles diluted in ultrapure water are well dispersed, while agglomerates are formed when diluting in PBS. The particle size of the 50 nm particles was confirmed, while the 250 nm particles appear to be 200 nm using tracking analysis and 210 nm using TEM. No major differences in pulmonary and systemic toxicity markers were observed after instillation of agglomerated versus single gold particles of different sizes. Both agglomerated as well as single nanoparticles were taken up by macrophages. Conclusion: Primary particle size, gold concentration and particle purity are important features to check, since these characteristics may deviate from the manufacturer's description. Suspensions of well dispersed 50 nm and 250 nm particles as well as their agglomerates produced very mild pulmonary inflammation at the same mass based dose. We conclude that single 50 nm gold particles do not pose a greater acute hazard than their agglomerates or slightly larger gold particles when using pulmonary inflammation as a marker for toxicity.