title: Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry.
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.
  
  
  CONCLUSIONS
  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.
authors: 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. sandra.vranic@univ-paris-diderot.fr
authors: Sandra
authors: S
authors: Vranic
authors: Nicole
authors: N
authors: Boggetto
authors: Vincent
authors: V
authors: Contremoulins
authors: Stéphane
authors: S
authors: Mornet
authors: Nora
authors: N
authors: Reinhardt
authors: Francelyne
authors: F
authors: Marano
authors: Armelle
authors: A
authors: Baeza-Squiban
authors: Sonja
authors: S
authors: Boland
mesh_terms: D000327:Adsorption; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry / Q000633:toxicity; D010316:Particle Size; D012822:Silicon Dioxide / Q000737:chemistry / Q000633:toxicity; D013499:Surface Properties; D014343:Trypan Blue / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D012822:Silicon Dioxide; D014343:Trypan Blue
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