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Airborne microplastics (MPs) havebeen recently detected in human lung tissue, but their fate and impact on airways is stillunrevealed. Our study focuses on the impacts of polyethylene terephthalate(PET) or polystyrene (PS) alone or combined with BaP on an in vitro model of human bronchial epithelium that we recently developed to study short and long-term effects, on air-liquid interface (ALI) grown Calu-3 cell line (lung adenocarcinoma patient cells).

Additional file 4: Fig. S3. TiO2 and CB nanoparticles enhance Aβ40 production in 1C11 precursors and 1C115-HT neuronal cells. a ELISA-based quantification of Aβ40 peptides in 1C11 and 1C115-HT neuronal cells exposed to TiO2- or CB-NPs (1 µg cm−2) up to 24 h. b APP and BACE1 expression level as assessed by RT-qPCR and Western-blotting in 1C11 cells exposed to TiO2- or CB-NPs (1 µg cm−2) for 4 h. c ELISA-based quantification of Aβ40 peptides in 1C115-HT neuronal cells exposed to TiO2- or CB-NPs (1 µg cm−2) for 4 h in the presence or not of a siRNA toward PrPC (siPrP) or the PDK1 inhibitor, BX912 (1 µM). The experiments were performed three times in triplicates. Values are means ± SEM. * denotes p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus unexposed cells.

Additional file 2: Fig. S1. Hydrodynamic diameter (nm) of TiO2- and CB-NPs measured by DLS in DMEM/F12 at 37 °C. Diameter (nm) of aggregates of TiO2 and CB nanoparticles was measured by DLS following sonication of NPs, dilution in DMEM/F12, and centrifugation for 2 sec at 2000 g to remove large aggregates. At 10, 25 and 50 µg ml−1, TiO2-NPs displayed an average diameter of 130 nm (Polydispersity Index-PDI = 0.21), 206 nm (PDI = 0.53), and 375 nm (PDI = 0.22), respectively, and CB-NPs 171 nm (PDI = 0.13), 188 nm (PDI = 0.15), and 327 nm (PDI = 0.52), respectively. The experiments were performed in triplicates.

Additional file 3: Fig. S2. TiO2 and CB nanoparticles provoke TACE depletion and TNFR1 accumulation at the plasma membrane of serotonergic 1C115-HT neuronal cells in a PDK1-dependent manner. TNFR1 a and TACE b immunostaining at the cell surface of 1C115-HT neuronal cells exposed for 4 h to TiO2- or CB-NPs (1 µg cm−2) in the presence or not of the PDK1 inhibitor BX912 (1 µM) and related quantification histograms. Representative images of three experiments performed in triplicates are shown. Values are means ± SEM. * denotes p < 0.05 and **p < 0.01 versus unexposed cells.

Additional file 1: Table S1. Hydrodynamic diameter (nm) of TiO2- and CB-NPs in PBS at 22 °C measured by DLS. Diameter (nm) of aggregates of TiO2 and CB nanoparticles (5 up to 80 µg ml−1) measured by DLS after NP sonication, dilution in PBS, and centrifugation for 2 sec at 2000 g to remove large aggregates. The hydrodynamic diameter could not be measured for the 5 µg ml−1 NP concentration. The experiments were performed in triplicates.

Additional file 5: Fig. S4. Corruption of PrPC-coupled signaling pathways by TiO2- and CB-NPs in neuronal cells: toward a pro-Alzheimer effect of some TiO2 and CB nanoparticles. Cellular prion protein PrPC is a plasma membrane receptor recognized by TiO2- and CB-NPs in neuronal cells. The interaction between full-length PrPC and NPs mobilizes PrPC-coupled signaling pathways, leading to (i) the activation of NADPH oxidase and the production of ROS, and (ii) the activation of PDK1 that promotes the internalization of TACE α-secretase and thereby down-regulates TACE shedding activity at the root of plasma membrane TNFR1 accumulation and rise in Aβ40/42 production. Such NP interferences with the PrPC signaling network triggers molecular signs of Alzheimer’s disease: modification of cell redox equilibrium, neuronal priming to TNFα inflammatory stress, and accumulation of neurotoxic Aβ40/42 peptides (Image drawn using Servier medical art).

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Nanomaterials raise new questions in environmental and human toxicology and represent a novel interface with specific properties with the biological medium. Several unknown remain to explain all the mechanisms of toxicity, especially at the molecular lever. When they enter the biological medium, nanoparticles get covered by a protein corona. This corona yields to a new “biological identity” that controls the cellular response to nanoparticles and their fate in the organism. We studied the adsorption of model proteins on nanostructured silica. The first part is dedicated to the characterization of nanoporous silica and silica nanoparticles that we used. Then the adsorption of myoglobin, hemoglobin and protein mixture from yeast cells was studied to determine the thermodynamic and physical-chemical parameters of protein adsorption on silica. The enrichment of basic residues, gathered in charge clusters, favors the adsorption of proteins by the formation of electrostatic interactions with the charged surface of silica, independently of the global charge of the protein. On the contrary, the enrichment in aromatic residues is unfavorable to protein adsorption because they form π-π interactions that rigidify the protein structure. The identification of adsorbed and non-adsorbed proteins from a complex medium could also be used for cellular toxicity studies. From the study of the structure, the dynamics and the activity of myoglobin and hemoglobin adsorbed on silica nanoparticles, we tried to define the state of an adsorbed protein. The structural study, based on circular dichroism, fluorescence, infrared, X-ray and UV-visible spectroscopy and microcalorimetry, shows a substantial partial structure loss of adsorbed proteins together with a high conformational heterogeneity, without major modifications of the heme structure. Two potential interaction sites of myoglobin with silica nanoparticles have been identified by a footprinting technique. The study of adsorbed myoglobin dynamics by elastic and inelastic neutron scattering highlighted the important decrease of protein dynamics that occurs upon adsorption. However, despite the structure loss, adsorbed metmyoglobin retains almost all of its activity of ligand binding. Unexpectedly, adsorbed hemoglobin shows an increase of its oxygen affinity and a decrease of its cooperativity, without any dissociation of the tetramer. This effect can be reproduced on human hemoglobin, cross-linked DCL hemoglobin and variant S hemoglobin. Besides, two effectors allow modulating the affinity of adsorbed hemoglobin. Despite the extent of structural and activity changes, all these modifications are entirely reversible upon desorption in soft conditions. The adsorption of hemoproteins on silica nanoparticles depicts a new sort of stress with resilience for proteins in terms of structure, dynamics and activity relationship., Les nanomatériaux posent de nouvelles questions en termes de toxicologie humaine et environnementale et représentent une nouvelle interface avec le milieu biologique aux propriétés spécifiques. De nombreuses inconnues demeurent, en particulier à l’échelle moléculaire, pour permettre d’expliquer certains mécanismes de toxicité. Lorsqu’elles entrent en contact avec le milieu biologique, les nanoparticules se couvrent d’une couche de protéines adsorbées. Celle-ci leur confère une nouvelle « identité biologique » qui contrôle la réponse cellulaire et leur devenir au sein de l’organisme. Nous avons étudié l’adsorption de protéines modèles sur la silice nanostructurée. Après avoir caractérisé la silice nanoporeuse et les nanoparticules de silice utilisées, l’adsorption de la myoglobine, de l’hémoglobine et des protéines d’un extrait cellulaire de levure a été étudiée afin de déterminer les paramètres physico-chimiques et thermodynamiques de l’adsorption des protéines sur la silice. Un enrichissement en résidus basiques, regroupés en clusters de charge, favorise l’adsorption des protéines grâce à la formation d’interactions électrostatiques avec la surface chargée de la silice, indépendamment de la charge globale de la protéine. A l’inverse, un enrichissement en résidus aromatiques est défavorable à l’adsorption car ces résidus forment des interactions π-π qui rigidifient la structure de la protéine. L’identification des protéines adsorbées et non adsorbées à partir d’un milieu complexe pourrait également être utilisée pour les études de toxicité cellulaire. A partir de l’étude de la structure, de la dynamique et de l’activité de la myoglobine et de l’hémoglobine adsorbées sur les nanoparticules de silice, nous avons cherché à définir l’état d’une protéine adsorbée. L’étude de la structure, réalisée par dichroïsme circulaire, spectroscopie UV-visible, d’absorption X, infrarouge, fluorescence et microcalorimétrie, montre une perte partielle de structure importante des protéines adsorbées associée à une grande hétérogénéité de conformations, sans modification majeure de la structure de l’hème. Deux sites potentiels d’interaction entre myoglobine et nanoparticules de silice ont été identifiés à l’aide d’une technique de cartographie de surface par irradiation. L’étude de la dynamique de la myoglobine adsorbée par diffusion élastique et inélastique de neutrons a permis de montrer que l’adsorption s’accompagnait d’une diminution importante de la flexibilité de la protéine. Malgré la perte de structure, la metmyoglobine adsorbée conserve une activité de fixation de ligands très proche de celle de la protéine libre. L’hémoglobine adsorbée présente de façon inattendue une augmentation de son affinité pour l’oxygène et une diminution de sa coopérativité, sans dissociation du tétramère. Cet effet est reproductible lors de l’adsorption de l’hémoglobine humaine, de l’hémoglobine pontée DCL et de l’hémoglobine mutée S. Deux effecteurs permettent par ailleurs de moduler l’affinité de l’hémoglobine adsorbée. Aussi importantes soient-elles, les modifications de structure et d’activité observées sont entièrement réversibles après désorption dans des conditions douces. L’adsorption des hémoprotéines sur les nanoparticules de silice représente véritablement un nouveau type de stress avec résilience pour les protéines en termes de relations entre structure, dynamique et activité.