Development of an in vitro model of human bronchial epithelial barrier to study nanoparticle translocation

Toxicology in Vitro 29 (2015) 51–58

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Development of an in vitro model of human bronchial epithelial barrier to study nanoparticle translocation Isabelle George a,⇑,1, Sandra Vranic a,1, Sonja Boland a, Arnaud Courtois b, Armelle Baeza-Squiban a a b

Univ Paris Diderot, Sorbonne Paris Cité, Unit of Functional and Adaptive Biology (BFA) UMR 8251 CNRS, F-75205 Paris, France Centre de Recherche Cardio-Thoracique de Bordeaux, INSERM U1045 – Univ Bordeaux, 146, rue Léo Saignat, 33076 Bordeaux, France

a r t i c l e

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Article history: Received 8 November 2013 Accepted 10 August 2014 Available online 6 September 2014 Keywords: SiO2 nanoparticles Transwell filter TEER Calu-3 A549 NCI-H292

a b s t r a c t Inhalation is the most frequent route of unintentional exposure to nanoparticles (NPs). Our aim was to compare different in vitro models of human lung epithelial monolayers for their suitability to assess the translocation of 50 nm fluorescently labelled silica NPs (50 nm-SiO2-FITC-NPs). Human bronchial epithelial cell lines NCI-H292 and Calu-3 as well as human alveolar cell line A549 were seeded onto Transwell filters (TF) separating the well into an apical and a basal compartment. Measurements of the transepithelial electric resistance and monitoring the paracellular transport of a fluorescent marker (Lucifer Yellow) have shown that only Calu-3 cells formed a tight epithelium. In the absence of cells 4% of the initially applied NP concentration was found to cross the TF but the majority remained trapped inside the filter. After 24 h of treatment, 50 nm-SiO2-FITC-NPs were taken up by all cell types but their translocation was inversely correlated to the efficiency to prevent LY passage: translocation represented 3% of the initially apically applied NP concentration for Calu-3 cells, 9% for NCI-H292 cells and 35% for A549 cells. In conclusion, 50 nm-SiO2-FITC-NPs can cross different bronchial epithelial barriers, but the Calu-3 cell line appears to be the most relevant model for studying NP translocation. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Nanotechnologies are emerging technologies allowing the exploitation of unique, size-dependent properties of nanomaterials (NMs) compared to the corresponding bulk material. These new technologies are promising for future applications in different consumer products, medicine, electronics, etc. However, some important concerns are expressed regarding possible adverse effects on health and environment due to the increased use of NMs (Colvin, 2003; Hoet et al., 2004). It is thus of crucial importance to completely understand the interactions of NPs with living systems. Considering routes of NP entry in the organism, respiratory system is one of the principal routes due to the possible presence of NPs in the inhaled air (Hagens et al., 2007). For its protection, the Abbreviations: NPs, nanoparticles; FITC, fluorescein isothiocyanate; TF, Transwell filter; TEER, trans-epithelial electric resistance; LY, Lucifer Yellow. ⇑ Corresponding author. Address: Laboratoire des Réponses Moléculaires et Cellulaires aux Xénobiotiques, Unité Biologie Fonctionnelle et Adaptative, EAC CNRS 4413, Université Paris Diderot, Sorbonne Paris Cité, 5 rue Thomas Mann, 75205 Paris cedex 13, France. Tel.: +33 1 57 27 83 67. E-mail addresses: [email protected] (I. George), sandra.vranic@ univ-paris-diderot.fr (S. Vranic), [email protected] (S. Boland), arnaud. [email protected] (A. Courtois), [email protected] (A. Baeza-Squiban). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.tiv.2014.08.003 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.

respiratory tract is lined by an epithelium that acts as a physical barrier between the external environment and the closely regulated internal milieu. In order to keep the body free from NPs and other materials deposited after the inhalation, several protection mechanisms exist in the respiratory tract. Tight junctions formed between epithelial cells help to avoid the entrance of NPs by maintaining the integrity of the airway epithelial barrier and by preventing the passage of particles through the paracellular space. Conducting airways of the lungs are lined by a mucociliary epithelium that contributes to the clearance of particles from the airways through a ‘‘mucociliary escalator’’. This mechanism involves the secretion of mucus from the mucus producing cells that helps to trap the particles and microorganisms introduced with the inhaled air and move them towards the upper respiratory region where they are expelled from the trachea and swallowed (Geiser and Kreyling, 2010; Oberdörster et al., 2005). Finally, the most prevalent mechanism for solid particle clearance in the alveolar region is mediated by alveolar macrophages, through phagocytosis of deposited particles. However, the efficiency of described mechanisms is uncertain, as there is evidence about the NP retention in the lung and their potential translocation to the systemic circulation and secondary organs (Schleh et al., 2012; Semmler-Behnke et al., 2008, 2007).

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Due to a great diversity of manufactured NPs it would be very useful to develop a cellular model that faithfully represents human respiratory epithelium and that permits to perform a high throughput and low-cost evaluation of the potential translocation of NPs in vitro, thus replacing in vivo experimentation that is time and cost consuming and largely criticized because of ethical concerns. With this purpose, we firstly compared the potential of three human pulmonary cell lines to develop a tight epithelium in order to choose the best model. We used the human bronchial epithelial cell line NCI-H292 as a potential model to study the translocation of NPs in the bronchial region, the human alveolar epithelial cell line A549 in order to develop a model to study the translocation of NPs in the alveolar region and the Calu-3 cell line, a human bronchial epithelial cell line known for its ability to develop a tight epithelium and to produce mucus (Zhu et al., 2010). These cultures were established in two compartment chambers in order to treat cells at the luminal side and to recover the translocated NPs at the basolateral side. The tightness of the epithelial barriers was assessed by measuring two parameters: the increase of the transepithelial electric resistance (TEER) and the decrease of the passage of the fluorescent dye Lucifer Yellow (LY) through the epithelial barrier. Furthermore, we used 50 nm-SiO2-FITC-NPs as a model of NPs that were stably labelled with FITC to study their fate. Their internalization by the three cell lines was studied using confocal microscopy and their passage through the epithelium was quantified measuring the fluorescence intensity in the basal compartment.

2. Materials and methods 2.1. Nanoparticles Synthesis and characterization of fluorescein-doped 50 nmSiO2-FITC-NPs was already described by Vranic et al. (2013a). Briefly, fluorescein isothiocyanate (FITC) was covalently attached to (3-aminopropyl)-trimethoxysilane (APS) by reaction of the amino group with the isothiocyanate group. 5 mg of FITC was dissolved in 5 mL of 42.7 mM of APS in ethanol. The fluorescent silane was added to 250 mL ethanol, 5 mL TEOS (Tetraethyl orthosilicate), 7.6 mL ammonium hydroxide (28%) and 10.9 mL water after 12 h of stirring. The reaction was performed for 12 h at 50 °C in the dark under magnetic stirring. These particles have an average diameter of about 30 nm. Further silica growth was performed to obtain the expected diameter of 50 nm and to make the NP more spherical with a pure silica surface.

The human alveolar epithelial cell line (A549) was purchased from the ATCC and grown in DMEM/F12 culture medium with phenol red, containing 10% FCS, 1% penicillin–streptomycin (Life Technologies) and 1% glutaMAX. All experiments were performed with these cells from passages 85 to 88. All cell lines were grown in T75-flasks (Costar, Sigma–Aldrich) for cellular expansion and on 3 lm pore size polycarbonate Transwell filters (TF) in two compartment chambers (12 mm in diameter) for translocation experiments. Calu-3 cells were seeded at 500,000 cells/cm2 and NCI-H292 and A549 cells were seeded at 250,000 cells/cm2 on polycarbonate TF. TF with a porosity of 3 lm (Costar, Sigma–Aldrich) were used in order to promote NP translocation (George et al., 2013). 500 lL of complete culture medium were added in the apical chamber and 1500 lL in the basolateral chamber. Cell cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. 2.2.2. Assessment of cell confluence Cellular confluence was assessed by measurement of the transepithelial electric resistance (TEER) and by assessing the monolayer permeability to Lucifer Yellow (LY) (Sigma–Aldrich, Saint Quentin, France). These parameters were measured three times per week during 9–14 days. Complete cell culture medium was changed before each TEER measurements. After 1 h of incubation at 37 °C, TEER was determined using the STX2 electrode (World Precision Instruments, Hertfordshire, United Kingdom) and the electronic circuit of the Epithelial Voltohmmeter (EVOM). Mean TEER values of three wells were calculated. The mean resistance of a cell-free TF was subtracted from the resistance measured across each cellular monolayer to yield TEER value of the cell monolayer. To determine LY passage, cells were rinsed with HBSS (Sigma– Aldrich, supplemented with Ca2+ and Mg2+) and incubated for 1 h at 37 °C with 500 lL of LY (0.2 lg/mL final in HBSS). FluoStar Galaxy (BMG, Ortenberg, Germany) was used to determine fluorescence (485 nm/520 nm) of recovered apical and basolateral media. 2.2.3. Viability assay Calu-3 cells were seeded in 96-well plates at 50,000 cells/well. Cells were treated with 50 nm-SiO2-FITC-NPs in a concentration range from 0 to 50 lg/cm2 for 24 h. Metabolic activity was assessed using the WST-1 cell proliferation reagent (Roche, Meylan, France) according to the manufacturer’s recommendations. For this purpose, cells were rinsed after NP treatment and incubated for 2 h with WST-1 reagent. Using a microplate photometer Elx800 (Biotek, Colmar, France) we measured by spectrophotometry the absorbance of supernatants at 450 nm to avoid interference with internalized NPs.

2.2. Cell culture 2.3. Translocation studies 2.2.1. Human epithelial cell lines Human lung adenocarcinoma cells (Calu-3) were purchased from the American Type Culture Collection (ATCC, Sigma–Aldrich, Saint Quentin Fallavier, France) and grown in DMEM/F12 (Dulbecco’s Modified Eagle Medium) culture medium with phenol red (Life Technologies, Saint Aubin, France), containing 10% fetal calf serum (FCS, Life Technologies) and 1% glutaMAX (Life Technologies), subsequently referred to as complete cell culture medium. All experiments were performed with these cells from passages 21 to 33. The human bronchial epithelial cell line NCI-H292 from a pulmonary mucoepidermoid carcinoma (ATCC) was grown in RPMI (Roswell Park Memorial Institute medium) 1640 culture medium with phenol red supplemented with 1% glutaMAX and 10% FCS. All experiments were performed with these cells from passages 13 to 20.

9- or 14-days confluent cultures were rinsed one time with HBSS to eliminate trace amounts of FCS. Stock solutions of 50 nm-SiO2-FITC-NP (23.2 mg/mL in water) were shortly vortexed before making the final dilution at 5 lg/cm2 (corresponding to 10 lg/mL) in the cell culture medium without phenol red, glutaMAX and FCS. A serial dilution of 50 nm-SiO2-FITC-NPs (in a concentration range from 0 to 10 lg/cm2) as well as 100 lL of apical and basolateral media after 24 h of treatment were deposited inside a white 96-well plate with clear bottom (Greiner, Courtaboeuf, France). Fluorescence was quantified using Infinite 200 Pro (TECAN, Lyon, France). FITC coupled with NPs was excited at 488 nm and detected at 521 nm. Control fluorescence value (medium without NPs) was subtracted from obtained fluorescence values. Fluorescence values

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Fig. 1. Characterisation of 50 nm-SiO2-FITC-NPs. Observation by transmission electron microscopy (TEM) of 50 nm-SiO2-FITC-NP initial suspension (A) or diluted at 5 lg/cm2 in DMEM/F-12 medium (B), scale bar: 200 nm. Size distribution pattern by DLS measurements of 50 nm-SiO2-FITC-NPs immediately after dilution at 5 lg/cm2 (C) or 24 h later (D).

were converted into percentage using a standard curve. Values for the ‘‘filter’’ or ‘‘cells/filter’’ compartment were determined by subtraction of the apical and basal media from the initial NP concentration applied. Filters with/without cells were removed and used for confocal imaging experiments. The filter without cells was observed using a reflection mode.

alcohol mounting medium with DABCO (Sigma–Aldrich). Considering optical laws the theoretical resolution was calculated and instrument settings adapted to obtain the best possible resolution in our images. Image treatment was done with Image J software (Image J 1.42 NIH, USA). Z-stack merge and orthogonal views were obtained with the merge of respectively 5–10 z-stacks and 15–25 z-stacks with a stack-spread of 0.45 lm.

2.4. Immunofluorescence and confocal microscopy experiments Cells were fixed after 24 h of NP treatment using 4% paraformaldehyde in PBS (Santa-Cruz, Heidelberg, Germany) during 20 min at room temperature, then incubated with NH4Cl (50 mM, Sigma– Aldrich) for 10 min and permeabilized in 0.05% Tween 20 (Sigma–Aldrich) in PBS for 5 min. Fixed cells were incubated over night at 4 °C with human MUC5AC monoclonal primary antibodies (Life Technologies) diluted to 1:100 produce in mouse or with ZO-1 plolyclonal primary antibodies (Sigma Aldrich) diluted to 1:500 produce in rabbit. This incubation was followed by the incubation with secondary antibodies stained with Alexa 488 or 568 fluorochromes diluted to 1:500 (Life Technologies) for 2 h at room temperature. For actin filaments staining fixed cells were incubated for 40 min with rhodamine–phalloidin (final concentration at 0.9 nM in PBS, Life Technologies). Cell nuclei were stained for 2 min with DAPI (40 ,6-Diamidino-2-Phenylindole, Dihydrochloride, final concentration at 0.25 lg/mL in PBS, Life Technologies). Cells were examined under a Zeiss LSM710 confocal microscope using objectives 40 and 63 with a 1.5 zoom after mounting in Polyvinyl

2.5. Statistical analysis Every experiment was repeated at least twice with triplicates for each condition. Data are represented as means ± SD and were analyzed on commercially available software SigmaStat (Version 3.0, Systat software Inc., San Jose, California, USA) using analysis of variance (one-way ANOVA) followed by Dunnett’s test for multiple comparisons with p < 0.05 (two tailed) considered as significant. 3. Results 3.1. NP characterisation Transmission electron microscopy (TEM) observation for initial 50 nm-SiO2-FITC-NP suspension confirmed the expected diameter of 50 nm and the spherical shape of the silica (Fig. 1A). The same primary NP diameter of 50 nm was observed after dilution at

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H292 cell lines TEER values did not increase over time and were not significantly different from the first measurement performed at day 2. The maximal TEER value did not exceed 40 X/cm2. Lucifer Yellow passage slightly decreased at day 6 for NCI-H292 cells (Fig. 2B) and at day 8 for A549 cells (Fig. 2C) but the amount of fluorescence in the basal compartment remained too important to indicate the development of a tight epithelium. All cell cultures were stopped after 9 or 14 days to avoid the cellular decline. In addition, confocal microscopy experiments were performed to investigate the confluence, the presence of tight junctions, and the cellular integrity. As observed in Fig. 3, Calu-3 and NCI-H292 cells formed a confluent epithelium with an organized cortical actin filament network by contrast to A549 cells where the cortical actin filaments were not developed (Fig. 3A). Zonula-occludens 1 protein (ZO-1) associated to tight junctions was revealed by specific anti-ZO-1 antibody showing a difference of ZO-1 distribution between Calu-3 and NCI-H292 cells. The Calu-3 cell line expressed ZO-1 on the apical region clearly designing the cellular outlines whereas the NCI-H292 cell line expressed ZO-1 in a diffused-manner inside the cytoplasm (Fig. 3B). Calu-3 cells exhibited the capacity to produce mucus as revealed by the immuno-labelling of MUC5AC, a tracheo-bronchial mucin (Fig. 3C). 3.3. Behaviour of NPs inside cellular compartments

Fig. 2. Time course study of the transepithelial electric resistance and paracellular transport of Lucifer Yellow. Measurements of TEER and fluorescence of LY in the basal compartment were performed every 2–3 days during 9–14 days for (A) the Calu-3 cell line; (B) the A549 cell line; (C) the NCI-H292 cell line. *: Different from control (p < 0.05); n = 3.

5 lg/cm2 in DMEM/F-12 medium (Fig. 1B). Measurements of dynamic light scattering (DLS) and zeta potential (Pz) were performed by a Zetasizer (nano ZS, Malvern Instruments, USA) showing small agglomerates (242.7 nm/76.83 nm) and a negative surface charge (9.8 mV/15 mV) respectively in RPMI (Vranic et al., 2013a) and DMEM/F12 culture media (Fig. 1C). DLS measurements after 24 h of incubation in culture medium showed no modification of the state of NP agglomeration (Fig. 1D). No leakage of fluorescence was observed (data not shown).

A prerequisite to the study of NP translocation was to establish whether fluorescently labelled 50 nm-SiO2-FITC-NPs could be internalized by the three different cell lines. Cellular monolayers seeded onto 3 lm pore size polycarbonate TF were treated for 24 h with FITC-labelled 50 nm-SiO2-FITC-NPs at the concentration of 5 lg/cm2. WST-1 assay determined that this concentration was not cytotoxic for the studied cell lines (data not shown). After NP treatment cells were fixed and stained with phalloidin coupled to rhodamin to visualize actin filaments and with DAPI to reveal nuclei (Fig. 4). The actin filaments of Calu-3 cells were not affected by NP treatment (Fig. 4A) whereas in NCI-H292 and A549 cells, they seemed to be disorganized (Fig. 4B and C). Orthogonal views allowed appreciating NP localization inside the cells. 50 nm-SiO2FITC-NPs were located on the top and inside the cells mostly in the peri-nuclear region. For a better observation of NP distribution inside the monolayer only the green channel was presented on the second column of Fig. 4. We observed small NP aggregates distributed in a heterogeneous-manner inside the Calu-3 monolayer (Fig. 4A). The NP distribution inside NCI-H292 and A549 monolayers was more punctiform and less aggregated than the one observed in the Calu-3 epithelium (Fig. 4B and C). 3.4. NP translocation through Transwell filters in the absence of cells

3.2. Confluence and permeability of epithelial monolayers The confluence and the permeability of different epithelial monolayers grown in vitro on Transwell filters were assessed by two methods. The first one is the measurement of the TEER that increases with the development of tight junctions and with the absence of leakage of the monolayer via paracellular route. The second one is the evaluation of the fluorescence recovered from the basolateral medium when the fluorescent marker Lucifer Yellow was applied on the apical side. The passage of Lucifer Yellow was detected 1 h after application allowing the evaluation of the paracellular transport of the dye to the basolateral side. For the Calu-3 cell line, TEER values increased from 100 X/cm2 at day 4 to 1400 X/cm2 at day 14. Concurrently a decrease of the passage of Lucifer Yellow was observed over time, and at day 9 no Lucifer Yellow fluorescence was detectable in the basal compartment. The decrease of Lucifer Yellow passage was correlated with the increase of the TEER values over time (Fig. 2A). For the A549 and the NCI-

Before studying the 50 nm-SiO2-FITC-NP translocation across the cells, their ability to translocate through the 3 lm pore size polycarbonate TF was first characterized. For this purpose 3 lm pore size polycarbonate TF were exposed for 24 h to 50 nm-SiO2FITC-NPs dispersed in the culture medium at the concentration of 5 lg/cm2 in the apical chamber at 37 °C. The evaluation of 50 nm-SiO2-FITC-NP distribution allowed to determine that 4.1% of the NP concentration initially applied in the apical chamber was still present whereas 5.1% reached the basal chamber. A very important retention is observed inside the filter where 90.8% of the NP concentration initially applied remained trapped (Fig. 5A). These results were confirmed by confocal microscopy observations of the TF using a reflection mode where the z–y plane was revealed in red (Fig. 5B (ii)). 50 nm-SiO2-FITC-NPs were detected in green using fluorescence emission (Fig. 5B (i)). 50 nm-SiO2-FITC-NPs were clearly visualized on the top, inside and at the bottom of the filter (Fig. 5B (iii)).

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Fig. 3. Confocal microscopy observations of Calu-3, NCI-H292 and A549 monolayers seeded onto 3 lm pore size polycarbonate Transwell filters after 9–14 days of culture. For the three cell lines actin filaments were stained in red by phalloidin–rhodamin (A). ZO-1 was labelled in green by an anti-ZO-1 antibody except for the A549 cell line (B). The mucin MUC5AC was labelled in green using an anti-MUC5AC antibody only for the Calu-3 cell line (C). Nuclei were revealed in blue with DAPI for all images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. NP translocation through different cell lines NP translocation was evaluated in function of cell lines seeded onto 3 lm pore size polycarbonate TF. We have observed a translocation of 35%, 9% and 3% of the NPs initially applied to the apical chamber respectively for the A459, NCI-H292 and Calu-3 cell lines (Fig. 6A). We also noted a slight apical retention and an important trapping inside the cells and/or the filters. The human alveolar cell line allowed a very important NP translocation comparatively to human bronchial epithelial cell lines. The inability of A549 cells to form a confluent and tight epithelium can explain these results. Indeed, these important differences between cell lines are in line with the TEER values observed for different cultures as higher TEER led to a less significant NP translocation (Figs. 2 and 6A). These results were not due to a decrease of the TEER after NP treatment, as measurements of TEER before and after NP treatment revealed no significant modification whatever the cell line (Fig. 6B). The important NP translocation for the A549 cells was thus the consequence of a leaking epithelium. 4. Discussion Our aim was to develop the most suitable in vitro model of lung barrier in order to study NP translocation. Our specifications were

(i) to use human cells for a better representativeness, (ii) to use cell lines for their convenience, reproducibility and low cost and (iii) to determine cells able to form a tight epithelium when grown on a filter of large porosity that allows NP passage. In addition we aimed to develop a fast and easy method of quantification of NP translocation. For this purpose we chose to use SiO2-NPs stably labelled with FITC fluorochrome allowing not only their quantification by fluorescence measurement and their ability to pass through the epithelial barrier but also to investigate their interactions with cells by confocal microscopy. We used SiO2 as model NPs because of their occurrence not only in environmental and occupational exposure but also in nanomedicine as drug carriers. They have been thoroughly characterized in our previous studies (Vranic et al., 2013b). Different models of lung epithelial barriers have already been developed but were dedicated to assess the permeability of soluble drugs (Forbes and Ehrhardt, 2005). Calu-3 and A549 cell lines being among the most frequently used (Lanone et al., 2009; Madlova et al., 2009; Simon-Deckers et al., 2008), we investigated their behaviour towards NP translocation and used also the NCI-H292 lung carcinoma cell line (Heijink et al., 2010; van Schilfgaarde et al., 1995) that we previously used to fully characterize SiO2-NP uptake (George et al., 2013; Vranic et al., 2013a,b). It has been shown that the NCI-H292 cell line produces airway mucin and

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Fig. 4. Confocal microscopy observations of Calu-3 (A), NCI-H292 (B) and A549 (C) monolayers seeded onto 3 lm pore size polycarbonate Transwell filters treated with 50 nm-SiO2-FITC-NPs at 5 lg/cm2 for 24 h after 9 or 14 days of culture. Actin filaments were stained in red by phalloidin–rhodamin, nuclei were revealed in blue with DAPI and 50 nm-SiO2-FITC-NPs in green, and we focus on the 50 nm-SiO2-FITC-NP distribution inside the cellular monolayer by the observation of the green channel only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

responds in a similar manner to primary human lung epithelial cells to different inhaled agents (Newland and Richter, 2008). A thorough characterisation of the epithelial barrier formed with these three cell lines was necessary as the cultures were performed on filters of large porosity. Although we used 3 lm pore size polycarbonate Transwell filters in order to favour the passage of 50 nmSiO2-FITC-NPs, they were highly trapped inside the filter. This retention of 50 nm-SiO2-FITC-NPs is likely due to their specific characteristics as Geys et al. found higher passages with polystyrene NPs, especially for carboxyl-modified NPs (Geys et al., 2006). Epithelial cells form efficient monolayers by binding to each other through apically located adherens junctions and more basally located desmosomes. However their ability to restrict the movement of pathogens, large macromolecules or particles through the space between two cells is due to a complex molecular structure: the tight junctions. The formation of a tight epithelium was qualitatively characterized by an immunolabeling of the ZO-

1 protein that is associated to tight junctions, and a labelling of the cortical actin network that is physically and functionally connected to the tight junctions (Rodgers and Fanning, 2011). These observations already revealed a less organized cortical actin cytoskeleton in A549 cells and a more diffuse localization for the ZO1 protein in NCI-H292 cells. These morphological features were in accordance with results of the functional state of the tight junctions. A time course monitoring of both TEER to assess ion-selective permeability and of the flux of a labelled tracer molecule, Lucifer Yellow to assess size-selective molecule diffusion, revealed that only Calu-3 cells exhibit functional tight junctions. The inability of the A549 cell line to form an efficient barrier was not surprising, as it has been frequently reported in the literature (Forbes and Ehrhardt, 2005). However, no alternative human cell lines are currently commercially available to mimic the alveolar epithelium. With such a barrier model it is impossible to determine the respective contribution of the transcellular and the

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Fig. 5. 50 nm-SiO2-FITC-NP translocation across 3 lm pore size polycarbonate TF in absence of cells. NPs were applied at 5 lg/cm2 in DMEM/F12 at the apical compartment for 24 h. (A) Quantification of NP distribution in percentage inside the apical (in grey), basolateral (in black) and the filter (in white) compartments. (B) Confocal microscopy experiments showing (i) in green 50 nm-SiO2-FITC-NP distribution, (ii) the TF is detected in red, (iii) and the merge of the two channels. 50 nm-SiO2-FITC-NPs were detected on the top, inside and at the bottom of the TF. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. 50 nm-SiO2-NP translocation across 3 lm pore size polycarbonate TF in function of different cell lines. (A) Quantification of 50 nm-SiO2-FITC-NP distribution in percentage inside the apical (in grey), basolateral (in black) and the cells/filter (in white) compartments. Calu-3, NCI-H292 or A549 cells were cultured on TF for 9–14 days and treated with 50 nm-SiO2-FITC-NPs at 5 lg/cm2 for 24 h. (B) Modification of TEER measurements before 50 nm-SiO2-FITC-NP treatment (day 9 or 14, in grey) and after 50 nm-SiO2-FITC-NP treatment (day 10 or 15, in black) in function of the cell line.

paracellular passage due to the absence of efficient tight junctions. A549 epithelium sheet allowed an important NP passage towards the basal compartment as well as an apparently important uptake as large intracellular aggregates were observed. The A549 cell line is known to be able to synthesize lecithin and potentially to be in charge for pulmonary surfactant synthesis (Lieber et al., 1976). We can thus speculate that a corona has been formed around the NPs improving their cellular uptake and reducing their trapping inside the porous filter leading to an increased translocation. Furthermore we observed modulation of actin expression after 24 h of exposure. This could be related to the NP internalization by macropinocytosis, an actin dependent pathway (Nowak et al., 2014) or to the modulated expression of actin cytoskeleton genes observed in A549 cells treated with silicon-NPs (Okoturo-Evans et al., 2013; Tay et al., 2014). A change in actin expression could contribute to the increase of uptake and translocation. For NCI-H292 cells, morphological and functional studies of tight junctions lead to conclude on the lack of suitability of this cell

line to establish an efficient epithelial barrier. TEER was very low and similar to the one obtained with A549 cells suggesting a lack of ion-selective permeability. By contrast they better reduced the passage of Lucifer Yellow suggesting a control of the size-selective molecule diffusion. This was in accordance with the lower distribution of 50 nm-SiO2-FITC-NPs in the basolateral compartment compared to A549 cells. As previously described by previous studies NCI-H292 cells were known to exhibit low resistance (Heijink et al., 2010; van Schilfgaarde et al., 1995). After two weeks of culture, Calu-3 cells completely prevented the paracellular passage of the Lucifer Yellow and exhibited a TEER of 1400 X/cm2 that is in accordance with previous publications (Geys et al., 2007; Mura et al., 2011). While this cell line could be seeded onto 0.4 lm pore size polycarbonate TF (Florea et al., 2003) or cultivated at air–liquid interface (Ehrhardt et al., 2002; Foster et al., 2000; Grainger et al., 2006) our culture conditions have been revealed more relevant for our study as pores of 3 lm were more suitable to allow the crossing of NPs. Moreover, Calu-

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3 cells expressed MUC5AC mimicking the native phenotype in airways. They have previously been shown to express epithelial markers similar to primary cell lines making these cells an attractive alternative model (Stewart et al., 2012) to primary cultures. We provided evidence of a low translocation of 50 nm-SiO2-FITCNPs. The absence of cytotoxicity and no decrease of TEER after 24 h of treatment as well as the presence of internalized NPs without alteration of the actin cytoskeleton suggested that this translocation was not related to an alteration of the barrier integrity. Additional studies would be necessary to determine the mechanisms involved in the SiO2-NP transcytosis by elucidating their uptake in Calu-3 cells. Few studies were performed to determine the route of entry of NPs inside Calu-3 cells. A recent study provided evidence that Calu-3 cells could take up amphiphilic glycopolymer NPs of 300 nm through different mechanisms such as clathrin-mediated endocytosis or lipid raft/caveolae-mediated endocytosis but not by macropinocytosis (Zheng et al., 2013). To progress in this understanding as well as to further validate the model, we plan to use SiO2-FITC-NPs of different sizes and surface charges, and to develop a 3D-culture model in presence of macrophages and endothelial cells. To conclude, the comparison of different human pulmonary cell lines for their ability to form a tight epithelium when grown on filters with high pore size in order to develop a model to evaluate NP translocation revealed that the Calu-3 cell line is the most relevant model. These cells allowed reconstitution of an efficient barrier of differentiated cells where 50 nm-SiO2-FITC-NPs used as model NPs translocate to a low extent within 24 h. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

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