Specific uptake and genotoxicity induced by polystyrene nanobeads with distinct surface chemistry on human lung epithelial cells and macrophages.

Vincent Paget,Sylvie Chevillard,Patrice Rat,Ghislaine Lacroix,Samir Dekali,Kelly Blazy,Romain Grall,Anne Braun,Thierry Kortulewski,Olivier Aguerre-Chariol,Christelle Gamez

doi:10.1371/journal.pone.0123297

Abstract

Nanoparticle surface chemistry is known to play a crucial role in interactions with cells and their related cytotoxic effects. As inhalation is a major route of exposure to nanoparticles, we studied specific uptake and damages of well-characterized fluorescent 50 nm polystyrene (PS) nanobeads harboring different functionalized surfaces (non-functionalized, carboxylated and aminated) on pulmonary epithelial cells and macrophages (Calu-3 and THP-1 cell lines respectively). Cytotoxicity of in mass dye-labeled functionalized PS nanobeads was assessed by xCELLigence system and alamarBlue viability assay. Nanobeads-cells interactions were studied by video-microscopy, flow cytometry and also confocal microscopy. Finally ROS generation was assessed by glutathione depletion dosages and genotoxicity was assessed by γ-H2Ax foci detection, which is considered as the most sensitive technique for studying DNA double strand breaks. The uptake kinetic was different for each cell line. All nanobeads were partly adsorbed and internalized, then released by Calu-3 cells, while THP-1 macrophages quickly incorporated all nanobeads which were located in the cytoplasm rather than in the nuclei. In parallel, the genotoxicity study reported that only aminated nanobeads significantly increased DNA damages in association with a strong depletion of reduced glutathione in both cell lines. We showed that for similar nanoparticle concentrations and sizes, aminated polystyrene nanobeads were more cytotoxic and genotoxic than unmodified and carboxylated ones on both cell lines. Interestingly, aminated polystyrene nanobeads induced similar cytotoxic and genotoxic effects on Calu-3 epithelial cells and THP-1 macrophages, for all levels of intracellular nanoparticles tested. Our results strongly support the primordial role of nanoparticles surface chemistry on cellular uptake and related biological effects. Moreover our data clearly show that nanoparticle internalization and observed adverse effects are not necessarily associated.

Highlights

The increasing production of engineered nanoparticles (NPs) for applications in a wide range of industrial processes and consumer products raise the problem of their effects on human health [1]
Inhalation is a major route for NPs exposure and, in contrast to large particles, NPs can be deposited by diffusion mechanisms in all structures along the respiratory tract, from the head airways to the alveoli, entering into cells and possibly inducing cytotoxic effects [3,4,5,6,7]
PS-NF and PS-COOH nanobeads were indirectly sonicated with a cup-horn at room temperature, while PS-NH2 nanobeads were just vortexed before use

Summary

Introduction

The increasing production of engineered nanoparticles (NPs) for applications in a wide range of industrial processes and consumer products (such as drugs, food, cosmetics, surface coating, etc.) raise the problem of their effects on human health [1]. Inhalation is a major route for NPs exposure and, in contrast to large particles (normally cleared by the upper airways), NPs can be deposited by diffusion mechanisms in all structures along the respiratory tract, from the head airways to the alveoli, entering into cells and possibly inducing cytotoxic effects [3,4,5,6,7]. Cells were grown for at least 24 h for THP-1 and 48 h for Calu-3, with impedance measured every 5 min during 6 h, every 15 min until addition of nanobeads. Normalized cell index was calculated by the software at the selected normalization time point, which was chosen as time just before the addition of nanoparticles in order to minimize inter-wells variability before the beginning of exposure

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