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Impact of serum as a dispersion agent for in vitro and in vivo toxicological assessments of TiO2 nanoparticles

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Abstract

Nanoparticles (NP) have a tendency to agglomerate after dispersion in physiological media, which can be prevented by the addition of serum. This may however result in modification of the toxic potential of particles due to the formation of protein corona. Our study aimed to analyze the role of serum that is added to improve the dispersion of 10 nm TiO2 NPs on in vitro and in vivo effects following the exposure via the respiratory route. We characterized NP size, surface charge, sedimentation rate, the presence of protein corona and the oxidant-generating capacity after NP dispersion in the presence/absence of serum. The effect of serum on NP internalization, cytotoxicity and pro-inflammatory responses was assessed in a human pulmonary cell line, NCI-H292. Serum in the dispersion medium led to a slower sedimentation, but an enhanced cellular uptake of TiO2 NPs. Despite this greater uptake, the pro-inflammatory response in NCI-H292 cells was lower after serum supplementation (used either as a dispersant or as a cell culture additive), which may be due to a reduced intrinsic oxidative potential of TiO2 NPs. Interestingly, serum could be added 2 h after the NP treatment without affecting the pro-inflammatory response. We also determined the acute pulmonary and hepatic toxicity in vivo 24 h after intratracheal instillation of TiO2 NPs in C57BL/6N mice. The use of serum resulted in an underestimation of the local acute inflammatory response in the lung, while a systemic response on glutathione reduction remained unaffected. In conclusion, serum as a dispersion agent for TiO2 NPs can lead to an underestimation of the acute pro-inflammatory response in vitro and in vivo. To avoid potential unwanted effects of dispersants and medium components, we recommend that the protocol of NM preparation should be thoroughly tested, and reflect as close as possible realistic exposure conditions.

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Acknowledgments

This study was funded by the E.C. FP7 ENPRA (No. NMP4-SL-2009-228789) grant and the Danish Centre for Nanosafety, Grant No. 20110092173 from the Danish Working Environment Research Foundation. The authors would like to acknowledge the ImagoSeine facility, member of the France BioImaging infrastructure, supported by the French National Research Agency (ANR-10-INSB-04 “Investments for the future”). We are very grateful to K. Moreau and B. Crobeddu for important technical assistance. We gratefully acknowledge the skilled assistance by Y. Kembouche and E. Christiansen in development of the generic batch dispersion protocol as well as DLS and viscosity measurements at NRCWE. We would like to thank Dr. E. H. J. M. Jansen, D. L. A. C. Leseman, P. K. Beekhof, L. J. J. de la Fonteyne and A. J. F Boere for their valuable technical assistance.

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    Authors and Affiliations

    1. Univ Paris Diderot (Sorbonne Paris Cité), UMR 8251 CNRS, Unit of Functional and Adaptive Biology (BFA), Laboratory of Molecular and Cellular Responses to Xenobiotics, Univ Paris Diderot, 5 rue Thomas Mann, 75205, Paris cedex 13, France

      Sandra Vranic, Armelle Baeza-Squiban & Sonja Boland

    2. Nanomedicine Lab, Faculty of Medical and Human Sciences, University of Manchester, AV Hill Building, Upper Brook Street, Manchester, M13 9PT, UK

      Sandra Vranic

    3. Centre for Sustainability, Environment and Health, National Institute for Public Health and the Environment, Bilthoven, The Netherlands

      Ilse Gosens & Flemming R. Cassee

    4. Danish Centre for Nanosafety, National Research Centre for the Working Environment, Copenhagen, Denmark

      Nicklas Raun Jacobsen & Keld A. Jensen

    5. Centre for Safety of Substances and Products, National Institute for Public Health and the Environment, Bilthoven, The Netherlands

      Bas Bokkers

    6. School of Life Sciences, Heriot-Watt University, John Muir building, Edinburgh, UK

      Ali Kermanizadeh & Vicki Stone

    7. Department of Public Health, Section of Environmental Health, University of Copenhagen, Copenhagen, Denmark

      Ali Kermanizadeh

    8. Institute of Occupational Medicine, Edinburgh, UK

      Lang Tran

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    1. Sandra Vranic
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    Correspondence to Sonja Boland.

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    Sandra Vranic and Ilse Gosens have equally contributed to this work.

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    Vranic, S., Gosens, I., Jacobsen, N.R. et al. Impact of serum as a dispersion agent for in vitro and in vivo toxicological assessments of TiO2 nanoparticles. Arch Toxicol 91, 353–363 (2017). https://doi.org/10.1007/s00204-016-1673-3

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