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ACS Nano. 2020 Mar 19. doi: 10.1021/acsnano.9b08818. [Epub ahead of print]

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

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Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense 5230, Denmark.
National Research Centre for the Working Environment, Copenhagen 2100, Denmark.
School of Geography Earth and Environmental Sciences, University of Birmingham, Edgbaston B15 2TT, United Kingdom.
Promethean Particles Ltd., Nottingham NG7 3EF, United Kingdom.
Department of Health Technology, Technical University of Denmark, Lyngby 2800, Denmark.


Despite broad application of magnetic nanoparticles in biomedicine and electronics, only a 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. Bronchoalveolar lavage fluid (BALF) samples were collected for proteome analyses by LC-MS/MS, testing Fe3O4 nanoparticles doped with increasing amounts of cobalt (Fe3O4, CoFe2O4 with an iron to cobalt ratio 5:1, 3:1, 1:3, Co3O4) at two doses (54 μg, 162 μg per animal) and two time points (day 1 and 3 days postinstillation). In discovery phase, in-depth proteome profiling of a 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. The highest expression increase could be found for a cluster of 39 proteins, which displayed strong dose-dependency to iron oxide and can be attributed to neutrophil extracellular trap (NET) formation. In-depth proteome analysis expanded the knowledge of in vivo NET formation. During screening, all BALF samples of the study (n = 166) were measured label-free as single-injections after a 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. Data are available via ProteomeXchange with identifier PXD016148.


LC−MS/MS; NETosis; bronchioalveolar lavage fluid; iron cobalt oxide nanoparticles; magnetic metal oxide nanoparticles; neutrophil extracellular trap formation; quantitative proteomics


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