Understanding silver nanoparticle effects on hepatocytes using 3D electron microscopy imaging combined with molecular and cellular approaches.
Laboratory: Laboratoire de Chimie et Biologie des Métaux (UMR 5249)
Team: Biology of Metals (BioMet)
Name and status of scientist in charge of the project: Deniaud Aurélien (MCF)
Adress: LCBM UMR 5249 CEA-CNRS-UGA BIG/ CEA/Grenoble, Bât K 17 rue des Martyrs 38054 Grenoble Cedex 09
Contact: aurelien.deniaud@cea.fr
Keywords: Electron Microscopy, Silver Nanoparticles, Hepatocytes
Candidate profile: We are looking for a highly motivated master’s student interested by a project at the interface between biology and physics.
Possibility of PhD: Yes, this is a new project recently funded by the CEA.
Context of the project :
AgNPs are widely used in consumer products for their biocidal properties but their impact on humans are poorly understood. In a recent study, we analyzed in detail AgNP fate in hepatocytes (liver cell line) using various approaches including electron microscopy and synchrotron- based cellular imaging (Veronesi*, Deniaud* et al., accepted by Nanoscale). In order to go further, we will now use a cutting-edge imaging technique: the Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM). This approach is quite new in biology and allows the 3D reconstruction of a biological sample at a resolution of a few nanometers in all 3 space dimensions. The preliminary acquisitions that we have performed are promising and we will pursue this work in the context of a project funded by the CEA.
This project is in partnership between three teams at the CEA-Grenoble that possess complementary skills. The BioMet team at the Laboratory of Chemistry and Biology of Metals in the BIG institute is expert in metal homeostasis and nanoparticle toxicity studies. This team studies metallic nanoparticle fate and impact in hepatocytes (using cell culture models) with a specific focus on metal homeostasis. To address these questions, we perform intracellular metal quantifications, qPCR and biochemical experiments. The INAC-MEM team is part of the “Institut Nanosciences et Cryogénie (INAC)”, and is located on the nanocharacterization platform (PFNC) at Minatec. They are experts in FIB-SEM imaging with a pioneer role in France for its development for biological sample imaging. They recently published a detailed analysis of organelle organization in a diatom cell (Flori et al, Protist, 2016). At the “Institut de Biologie Structurale (IBS)”, the IBS-MEM team masters electron microscopy and 3D reconstructions of biological samples: proteins as well as cells. The complementarity of these teams should allow us to break new ground in understanding AgNP fate inside hepatocytes.
Student’s project:
The objective of this project is to obtain the first 3D reconstructions of hepatocytes exposed to silver nanoparticles (AgNP) using FIB-SEM (@PFNC-Minatec). This will allow us to properly analyze AgNP fate within exposed cells and cell ultrastructure alterations. During this project, we will first optimize FIB-SEM acquisition conditions in order to be able to visualize AgNP entry processes, NP distribution inside the cell and their intracellular transformation. This method may also allow us to visualize putative excretion of AgNPs into bile canaliculi and cell ultrastructure modifications (impact on the mitochondrial network for example). We will combine these analyses with synchrotron-based imaging as well as cellular and molecular experiments in order to get an integrated view of AgNP fate inside hepatocytes as well as their impact on hepatocyte physiology.
Methods:
Cell culture, Metal quantification by ICP-AES, Sample preparation for electron microscopy (@ IBS), FIB-SEM as well as TEM and SEM coupled with Energy Dispersive X-ray spectroscopy (@ Minatec), Volume reconstruction and segmentation.
Relevant publications of the team:
- Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions. M. Cuillel, M. Chevallet, P. Charbonnier, C. Fauquant, I. Pignot-Paintrand, J. Arnaud, D. Cassio, I. Michaud-Soret* and E. Mintz*, Nanoscale 2014, 6 (3), 1707-15.
- Visualization, quantification and coordination of Ag+ ions released from silver nanoparticles in hepatocytes. G. Veronesi*, A. Deniaud*, T. Gallon, PH. Jouneau, J. Villanova, P. Delangle, M. Carrière, I. Kieffer, P. Charbonnier, E. Mintz, I. Michaud-Soret*, Nanoscale, accepted.
Documents needed to apply: – Cover letter – Resume
