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Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties

Abstract

Cellular internalisation of industrial engineered nanoparticles is undesired and a reason for concern. Here we investigated and compared the ability of seven different mammalian cell cultures in vitro to incorporate six kinds of engineered nanoparticles, focussing on the role of cell type and particle properties in particle uptake. Uptake was examined using light and electron microscopy coupled with energy dispersive X-ray spectroscopy (EDX) for particle element identification. Flow cytometry was applied for semi-quantitative analyses of particle uptake and for exploring the influence on uptake by the phagocytosis inhibitor Cytochalasin D (CytoD). All particles studied were found to enter each kind of cultured cells. Yet, particles were never found within cell nuclei. The presence of the respective particles within the cells was confirmed by EDX. Live-cell imaging revealed the time-dependent process of internalisation of technical nanoparticles, which was exemplified by tungsten carbide particle uptake into the human skin cells, HaCaT. Particles were found to co-localise with lysosomal structures within the cells. The incorporated nanoparticles changed the cellular granularity, as measured by flow cytometry, already after 3 h of exposure in a particle specific manner. By correlating particle properties with flow cytometry data, only the primary particle size was found to be a weakly influential property for particle uptake. CytoD, an inhibitor of actin filaments and therewith of phagocytosis, significantly inhibited the internalisation of particle uptake in only two of the seven investigated cell cultures. Our study, therefore, supports the notion that nanoparticles can enter mammalian cells quickly and easily, irrespective of the phagocytic ability of the cells.

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Acknowledgements

This research was supported by the German Federal Ministry for Education and Research (BMBF) within the INOS project (Identification and Evaluation of Health and Environmental Effects of Technical Particles at the Nanoscale; Grant #03X0013C), WB was additionally supported by the Max Buchner Forschungsstiftung and by the Helmholtz Impulse and Networking Fund through Helmholtz Interdisciplinary Graduate School for Environmental Research (HIGRADE). Stefan Scholz (Department Bioanalytical Ecotoxicology, UFZ) is acknowledged for constructive discussions.

Author information

Correspondence to Wibke Busch.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Movie of HaCaT cells incorporating WCL particles. Photographs of HaCaT cells, exposed to 30 μg/mL WCL nanoparticles, were taken every 10 min during a time period of 2 days using an inverse microscope (Leica DMI 4000B, magnification 200×). The pictures are shown as a time flow movie in the Additional file 2. (MPEG 21380 kb)

Cell culture protocols. The cell lines and isolated cells used in the study and their standard treatments are explained in the Additional file 1. A table shows the numbers of seeded cells for the different experiments. (DOC 39 kb)

Movie of HaCaT cells incorporating WCL particles. Photographs of HaCaT cells, exposed to 30 μg/mL WCL nanoparticles, were taken every 10 min during a time period of 2 days using an inverse microscope (Leica DMI 4000B, magnification 200×). The pictures are shown as a time flow movie in the Additional file 2. (MPEG 21380 kb)

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Busch, W., Bastian, S., Trahorsch, U. et al. Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties. J Nanopart Res 13, 293–310 (2011). https://doi.org/10.1007/s11051-010-0030-3

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Keywords

  • Engineered nanoparticles
  • Uptake
  • Mammalian cells
  • Flow cytometry
  • Microscopy
  • Cytochalasin D
  • Health and safety