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

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.

Fig 1. Transmission electron microscopy images (TEM) and size distributions of PS nano-beads.

NPs were suspended in RPMI 1640 cell culture media supplemented with 5% (v/v) FBS and 1% (v/v) penicillin/streptomycin. TEM images (column 1) show non-functionalized (A and D), carboxylated (B and E) and aminated (C and F) polystyrene nanobeads (scale bars = 100 nm). Size distributions (column 2) of each nanobeads were determined by dynamic light scattering at t₀ at room temperature (blue curves) and after 24h in an incubator at 37°C (green curves).

Fig 2. Cell index real-time monitoring of Calu-3 epithelial cells and THP-1 macrophages exposed to PS nanobeads.

Cell index real-time monitoring of Calu-3 epithelial cells and THP-1 macrophages are reported on column 1 and 2 respectively. One representative experiment among three independents experiments were carried out for 48h and cell indexes were normalized at time 0 to ensure non inter-wells variability prior to the addition of nanoparticles. Calu-3 epithelial cells and THP-1 differentiated macrophages were exposed 48 h to the three kinds of PS nanobeads, PS-NF (A and D), PS-COOH (B and E) and PS-NH₂ (C and F).

Fig 3. Fluorescence follow-up of Calu-3 cells and THP-1 macrophages exposed to PS nanobeads.

Total sum intensity of fluorescence performed by 24 h real-time video-microscopy follow-up on Calu-3 cells (A) and THP-1 macrophages (B) exposed to PS-NF (blue curve), PS-COOH (red curve) and PS-NH₂ (green curve) nanobeads.

Fig 4. Confocal microscopy images of Calu-3 cells and THP-1 macrophages exposed to PS-NH₂ nanobeads.

Confocal microscopy images of Calu-3 cells are reported on (A) and in (B) for THP-1 macrophages. For both cell types, times of exposure are as follows: 1 (line 1), 2 (line 2), 4 (line 3) and 24 h (line 4). Cytoplasm staining (actin) is represented on column 1, nanobeads on column 2, DNA staining on column 3 and column 4 represents overlay of the three stainings. White arrows indicate nanobeads location on overlay images (column 4). White bars correspond to 10 μm.