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Open Access Research

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

Sandra Vranic1*, Nicole Boggetto2, Vincent Contremoulins2, Stéphane Mornet3, Nora Reinhardt3, Francelyne Marano1, Armelle Baeza-Squiban1 and Sonja Boland1

Author Affiliations

1 Univ Paris Diderot, Sorbonne Paris Cité, Laboratory of Molecular and Cellular Responses to Xenobiotics, Unit of Functional and Adaptive Biology (BFA) EAC CNRS 4413, 5 rue Thomas Mann, Paris 75 013, France

2 Institut Jacques Monod, Sorbonne Paris Cité, ImagoSeine BioImaging Core Facility, CNRS, UMR 7592, Univ Paris Diderot, Paris, France

3 Institut de Chimie de la Matière Condensée de Bordeaux, UPR CNRS 9048, Université Bordeaux 1, 87 Avenue du Docteur A. Schweitzer, Pessac cedex F-33608, France

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Particle and Fibre Toxicology 2013, 10:2  doi:10.1186/1743-8977-10-2

Published: 6 February 2013

Abstract

Background

The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface.

Results

Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells.

Conclusion

The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.

Keywords:
Lung epithelial cells; ImageStreamX; Endocytosis; Clathrin; Macropinocytosis; TiO2; SiO2