Fast and Robust Proteome Screening Platform Identifies Neutrophil Extracellular Trap Formation in the Lung in Response to Cobalt Ferrite Nanoparticles

Despite broad application of magnetic nanoparticles in biomedicine and electronics only few in vivo studies on biocompatibility are available. In this study, toxicity of magnetic metal oxide nanoparticles on the respiratory system was examined in vivo by single intratracheal instillation in mice. BALF samples were collected for proteome analyses by LC-MS/MS, testing Fe3O4 nanoparticles doped with increasing amount of cobalt (Fe3O4, CoFe2O4 with iron to cobalt ratio 1:3, 1:1, 3:1, Co3O4) at two doses (54µg, 162µg per animal) and two time points (day 1 and 3 post instillation). In a discovery phase, in-depth proteome profiling of few representative samples allowed for comprehensive pathway analyses. Clustering of the 681 differentially expressed proteins (FDR < 0.05) revealed general as well as metal oxide specific responses with an overall strong induction of innate immunity and activation of the complement system. Highest expression increase could be found for a cluster of 38 proteins, which displayed strong dose-dependency to iron oxide and can be attributed to neutrophil extracellular trap (NET) formation. During screening, all BALF samples of the study (n=166) were measured label-free as single-injections after short gradient (21 min) LC separation using the Evosep One system, validating the findings from the discovery and defining protein signatures which enable discrimination of lung inflammation. We demonstrate a proteomics-based toxicity screening with high sample throughput easily transferrable to other nanoparticle types.