Evaluating pulmonary toxicity of engineered metal-based nanoparticles using in vivo and in vitro models

The overall goals of this doctoral dissertation were to 1) assess effects of nanoparticle (NP) exposure on host defense in a murine pulmonary infection model, 2) evaluate an integrated dynamic in vitro exposure system (DIVES) that overcomes limitations of submerged exposure systems for NP toxicity testing and 3) provide information on the rank of NP toxicity and assess the potential of the DIVES as a screening tool for NP toxicity. To achieve the first goal, we used Klebsiella pneumoniae (K.p.) in a murine lung infection model to determine if pulmonary bacterial clearance is enhanced or impaired by copper (Cu) NP exposure. Cu NP exposure induced strong inflammatory responses and an impairment in host defense against bacterial lung infections in both inhalation and instillation exposure studies even though there was an upregulation of pro-inflammatory cytokines and recruitment of neutrophils to the lungs. Thus, Cu NP exposure may lead to increased risk of pulmonary infection by impairing host defense against bacteria. In the second study, we integrated the DIVES capable of generating NP aerosols and depositing NPs directly onto cells grown at the air-liquid interface (ALI) to mimic a more realistic in vivo pulmonary exposure to inhaled NPs. Furthermore, we characterized the efficiency of NP delivery, the distribution of particle deposition and the effects of exposure conditions in the DIVES on the viability of A549 cells (human alveolar type-II-like cancer cells) as a precursor to studies of NP toxicity. The DIVES was shown to provide efficient, uniform and controlled dosing of particles to epithelial cells grown at the ALI. In addition, this exposure system delivered a continuous airborne-exposure of NPs to lung cells without loss of cellular viability. Lastly, to assess the DIVES as a means to rank NP toxicity and prioritize NPs for in vivo testing, we compared in vitro measurements obtained using the DIVES and the submerged exposure system to in vivo results obtained using a murine model of lung inflammation. Exposure to Cu NPs induced a significant increase in cytotoxicity and inflammatory responses compared to Fe NPs at the ALI in the DIVES. The results of this comparison suggest that air-delivery of NPs to lung cells using the DIVES can provide evidence of toxicity at a lower concentration of NPs compared to responses in the submerged condition. More importantly, our in vitro results presented in this dissertation are in agreement with our in vivo findings showing that Cu NPs have a higher propensity for NP dissolution and this may contribute to the greater toxicity of Cu NPs than Fe NPs. Thus, the results of these comparisons suggest that the DIVES has a significant potential for screening NP toxicity and allows for a higher throughput than in vivo studies. Overall, we found that exposure of lung cells at the ALI using the DIVES is preferable to submerged exposure for in vitro NP toxicity testing and provides useful information on the rank of NP toxicity and prioritization of NPs for in vivo testing.