Nanoplastic characterization
Polystyrene-Europium (PS-Eu) Thermo Scientific™ Fluoro-Max™ Fluorescent Carboxylate-Modified Particles were purchased from Thermo Scientific. Subsequently they were characterized using different methods. This test material was used in collaborative work between different labs within the PlasticsFatE project (Grant Agreement n° 965367). Carboxylated PS-Eu nanoparticles were chosen as model particles in this study due to their ability to be synthesized as a highly monodisperse population, ensuring a uniform particle sample. The carboxylated surface enhances stability under culture conditions, minimizing aggregation and enabling researchers to investigate particle-induced effects without the confounding influence of aggregation-related artifacts. Furthermore, it has been demonstrated that during aging, PS particles can naturally form carboxyl groups on their surface [75].
Electron microscopy of particles via scanning electron microscopy (SEM)
5 µl of PS-Eu stock suspension were pipetted on a support and let dry overnight in a desiccator. The samples were coated with gold-palladium using the Gatan Precision Etching Coating System (PECS) 682 (USA), then they were imaged using a JSM-6500 F (JEOL) operated at 15 kV.
Size distribution Estimation via transmission electron microscopy (SEM)
To estimate the mean size distribution of PS-Eu particles, the diameter of 100 particles from different regions of the SEM grid were measured by ImageJ software.
Size distribution Estimation via nanoparticle tracking analysis (NTA)
Particle concentration and size distribution analysis in dispersion were performed by the ZetaView® PMX-120 (Particle Metrix GmbH, Germany) nanoparticle tracking analyser, equipped with a light source set to a wavelength of 488 nm. Before the measurements, the samples were diluted in water: stock PS-Eu nanoplastics (10 mg/ml) 50000 times, while PS-Eu exposed to complete culture medium (CCM) (final polymer concentration 100 µg/ml) and CCM alone were diluted 500 times. After optimization of the instrumental parameters, the sensitivity and the shutter, were set at 70 and 100, respectively, in 33 videos of 1 s recorded in 11 different positions, recording a total number of particles of around 2000 per single measurement, each measurement was performed in triplicate. The signal of CCM alone was used as blank and subtracted from the size distribution of PS-Eu exposed to CCM. For the dosimetry measurements PS-Eu NPs were incubated with CCM at 37 °C and kept steady. Sampling (1% of the volume) of the upper part of the dispersion was performed at 0 and after 18.5 h and 42.5 h. The sampled dispersions were diluted 500 times in water and measured by NTA.
Dynamic light scattering (DLS)
Size distribution of PS-Eu stock solution was measured by dynamic light scattering (DLS) at 37 °C using micro Vis cuvettes (Eppendorf, Hambourg, Germany) with a Zetasizer Nano-ZS (Malvern Instruments, Ltd, Worcestershire, UK). PS-Eu NPs were diluted to 0.1 g/L in 100% milliQ H2O. Measurements were based on the Smoluchowski model.
Electrophoretic light scattering (ELS) and Z-potential estimation
Z potentials on NPs were measured using a Zetasizer, Nano ZS electrophoretic light scattering analyser (Malvern Instruments, Malvern, UK) equipped with a light source wavelength of 632.8 nm and a fixed scattering angle of 173◦. The samples were measured in triplicate at 25 °C. PS-Eu particles incubated with CCM and M were purified by centrifugation and resuspended in water before been measured. All measurement were performed at pH 7.4 and with similar conductivity: 1.3 ± 0.2 mS/cm.
Dosimetry in culture medium using the ISDD model
The deposited dose in culture medium condition was calculated either theoretically or experimentally. Briefly, we first calculated the theoretical deposited mass using the in vitro sedimentation, diffusion and dosimetry model (ISDD) [32] assuming a concentration of 100 µg/ml, a diameter of 279 nm (values obtained with NTA, Fig. 1.C), and a density of 1.05 g/cm3, which is the density of polystyrene. Simulation time was set at 48 h (48 points); dish dept 0.00833 m; volume 0.25 ml; Temperature 310 K. The data obtained provided the theoretical deposited mass of PS-Eu particles in CCM without a protein corona and was equal to 2,93 µg over 48 h (blue line, Fig. 1.E). Then we modelled the deposited mass of PS-Eu particles assuming a core-shell structure of 316 nm composed by a polystyrene core of 279 nm, and a compact protein corona shell of 18.5 nm (values obtained with NTA, Fig. 1.C.) characterized by a density of 1.37 g/cm3 (the average density of proteins [76]). The final assumed density of the object was 1.15 g/cm3. The data obtained estimated a deposited mass in CCM of 6,26 µg over 48 h (red line, Fig. 1.E). However, often the corona is not compact, and consequently might have a density which is lower than 1.37 g/cm3. Thus, we measured experimentally with NTA the deposited number of PS-Eu particles at 0, 18 and 42,5 h – than converted to mass – and found a quite linear correlation between the time and deposited mass, with 4.5 µg deposited over 42,5 h equal to a deposited dose of ~ 5 µg at 48 h. Interestingly, the deposited mass measured experimentally lays perfectly between the deposited mass calculated theoretically with ISSD for PS-Eu Stock (2,93 µg) and PS-Eu CCM (6,26 µg), suggesting that a protein corona is formed, but that it is not fully compact as we assumed for PS-Eu CCM. In the experiments performed, cell treatment with particles is indicated in µg/ml, however in Table 1 we provide a dose table to allow an easy conversion of µg/ml in different formats.
TLR2 and TLR4 activation assay
This assay is based on human embryonic kidney (HEK293) reporter cells which upon exposure and activation of Toll-like receptor 2 (TLR2) and 4 (TLR4) by their respective microbial ligands (lipoteichoic acid, LTA and lipopolysaccharide, LPS), release the secreted embryonic alkaline phosphatase (SEAP) that produces a colorimetric reaction thanks to the presence of its substrate in the cell culture media. For the present study, exposure experiments with parental HEK293 cells, TLR2 and TLR4 reporter HEK293 cells (Invivogen, France) were conducted in a 96-well plate following the procedure previously reported by Afanou et al. 2023 [77]. Briefly, 50.400 cells/well resuspended in 180 µl enriched cell medium (DMEM + 10% fetal bovine serum + mandatory HEK Blue selection antibiotics) were exposed to 20 µl of the PS-Eu particles suspensions at doses ranging between 0 and 200 µg/ml (equal to 0 -1.33 × 109 particle/ml). The cells were then incubated at 37 °C, 5% CO2 and high relative humidity conditions for 24 h. Following the incubation, 20 µl of the cell supernatant were carefully transferred to new microplates and completed with 180 µl Quanti-Blue detection reagent (Invivogen, France). After 180 min incubation, the colour was spectrophotometrically measured at 649 nm using Synergy Neo 2 multimode microplate reader with Gen 5 data analysis software (BioTek Instruments GmbH, Bad Friedrichshall, Germany). Each sample was run in triplicate, and the whole experiment was repeated three times. The data are reported as mean fold change related to the parental cells (similar to the negative controls). Negative controls (Endotoxin free water) and positive controls for TLR2 (ultrapure LTA, lipoteichoic acid; InvivoGen, France) and TLR4 (ultrapure LPS, lipopolysaccharide; InvivoGen, France) activation were included in all experiments.
PS-Eu fluorescence emission and excitation spectra acquisition
100 µg/ml PS-Eu fluorescence emission and excitation spectra were collected using a Cytation 3 (Invivogen) after 48 h incubation in complete culture medium (CCM) at 5% CO2, 37 °C and humified conditions.
Transmission Electron microscopy Energy-Dispersive X-Ray spectroscopy (TEM-EDX)
For particle characterization, washed PS-Eu NPs were diluted in milliQ H2O, deposited on a Cu-C Formvar grid. 2% uranyl acetate was added, and NPs were air dried then observed with TEM EDX detector. PS-Eu NPs EDX spectra were acquired using an Xplore detector (Oxford Instruments).
Cell culture and exposure to particles
A549 cell culture and exposure to particles in 2D submerged conditions
100 µl of A549 cells in (ATCC) complete culture medium (DMEM, high glucose (Sigma), 4 mM L-glutamine (Sigma), 10% v/v FBS (Sigma)) were seeded at a concentration of 90,000 cell/ml in 96-well plates (TPP) and grown 24 h at 37 °C, and 5% CO2 in humified conditions. In parallel background controls wells containing exclusively complete medium were prepared. One day after seeding cells and medium wells were either left untreated or incubated with increasing concentrations of PS-Eu particles diluted in complete culture medium at final concentrations ranging from 3,125 to 100 µg/ml. An H2O2 positive control was also prepared by exposing cells to a final concentration of hydrogen peroxide of 0.05 mM. After 48 h Triton X-100 0,1% was added to half of the untreated wells and the plate was incubated for 30 min at 37 °C, and 5% CO2 in humified conditions. Cells response to particles was investigated by performing sequentially on the same plate: lactate dehydrogenase (LDH) assay, Resazurin assay, Neutral Red uptake (NRU) assay and Coomassie brilliant blue (CBB) assay. Alternatively, ROS production and particle uptake were estimated via flow cytometry after 30 min of treatment on the same type of cells (see Additional File 2).
A549 cell culture and exposure to particles in 24- and 12-well plate transwells
A549 cells in CCM were seeded at a concentration of 70,000 cell/ml in the top (apical) compartment of transwells (Sarstedt, PET, 3 μm pore size) and then grown for 10 days in submerged conditions in CCM (24-well plate transwells: top compartment: 250 µl, bottom (basolateral): 1000 µl. 12-well plate transwells: top 900 µl, bottom 1800 µl). The medium was replaced on day 3, 7 and 10 after seeding. On the same days, in 24-well plates grown cells, TEER, epithelial permeability, or tight junction expression were measured to monitor epithelial barrier formation over time for model characterization (see Additional File 2).
Starting from day 10 post seeding, samples were grown for an additional 7 days either in air-liquid interface (ALI) conditions (24 well-plate: top empty, bottom 1000 µl. 12-well plate: top empty, bottom:1800 µl) or submerged ones (24 well-plate: top 250 µl, bottom 1000 µl. 12-well plate: top 900 µl, bottom 1800 µl) and was changed every alternate day. In parallel to the stated samples, extra wells were prepared to serve as a control for future experiments: cell-free wells, and cell-containing wells.
On day 17, after measuring TEER and epithelial permeability, cells were exposed to plastic particles in duplicates. First, medium was changed in all bottom compartments. Then in the top compartment cells were either left untreated (medium change only) or incubated with particles at different concentrations (100 µg/ml, 50 µg/ml, 25 µg/ml) for 48 h in humified conditions at 37 °C, and 5% CO2. In submerged samples, particles were diluted in 250 µl (24-well-plate) or 900 µl (12-well plate) of CCM while in ALI-grown cells, cells were treated either with 20 µl (24-well plate) or 80 µl (12-well plate) of sterile-filtered milliQ water, the particle solvent (untreated control), or with water containing the same number of particles used for the submerged samples. This treatment is defined as quasi air-liquid interface (ALI). Medium was replaced also in control wells. After 48 h treatment, the response of cells to particles was investigated.
In 24-well plates first half of untreated control wells are incubated with Triton X-100 (0.1% v/v, Roche) for 30 min and then different assays were performed sequentially: LDH assay, lucifer yellow assay, TEER measurement and Resazurin assay. In 12-well plates instead cell ultrastructure (TEM), surface tension, particle translocation and surfactant protein production were investigated.
Preparation of PS-Eu for treatment of Calu-3
Initial PS-Eu NPs were washed twice by dilution at 1/5 in autoclaved miliQ water and centrifugation for 5 min at 15000 g at 4 °C to remove sodium azide from the dispersant (deionized water, 0.05% azide). The PS-Eu NPs pellet was then resuspended in autoclaved milliQ H2O. The final concentration of washed PS-Eu NPs was determined by DLS using the initial commercial suspension to establish a calibration curve after verification of similar HD.
Calu-3 2D and 3D cultures and exposure to particles
The Calu-3 human adenocarcinoma epithelial cell line (ATCC HTB-55) was used from passage 24 to 28 according to Sanchez-Guzman et al. 2021 [78]. Calu-3 cells were cultured in Eagle’s Minimum Essential Medium (MEM, Gibco, Thermo-Fisher, France) supplemented with 10% v/v foetal bovine serum (FBS) (F7524, Merck, France), 1% nn-essential amino acids (NEAA) 100×, 1% sdium pyruvate, 1% Gutamax, 1% pnicillin streptomycin 100 ×, and 1% HPES buffer 100 × (all from Gibco, Thermo-Fisher, France). Cells were cultured at a density of 40000 cells/cm² in 25–75 cm² culture flasks (Costar, Corning, France) at 37 °C in a humidified 5% CO2 atmosphere and passaged weekly before confluence. 2D cultures were done on round glass slides placed in a 24-wells plate (Costar) at a density of 200000 cells/cm² in 250 µl of MEM supplemented with 10% FBS. Two days later cells were exposed for 24 h to PS-Eu NPs diluted in MEM supplemented with 4% FBS (200 µl per well) and fixed for TEM. To reconstruct the epithelial barrier, Calu-3 cells were seeded on transwells polycarbonate inserts in 12-well plate with a 3 μm pores diameter (Costar) and a 1.12 cm² surface area. Before seeding, transwells inserts were hydrated with 1.5 ml of Hank’s balanced salt solution containing Ca and Mg (HBSS Ca2+/Mg2+, Gibco, Thermo-Fisher) in the lower compartment and 0.5 ml in the upper compartment for 15–30 min at room temperature. Cells are then seeded at a density of 50000 cells/transwell (apical: 0.5 ml of cell suspension, basolateral: 1.5 ml of MEM supplemented with 10% FBS), with medium change every 2–3 days. After 14 days, the apical medium was removed, and cells were cultured at the air-liquid interface for 1 week. The basolateral compartment was replaced by MEM supplemented with 4% FBS one day after creation of the air-liquid interface. At day 20, barrier permeability (lucifer yellow assay) and metabolic activity (AlamarBlue™ assay) were measured, and cells were exposed the next day. Exposures were done by adding droplets of PS-Eu suspensions diluted in HBSS Ca2+/Mg2+ at 70% in water on the apical compartment (18 µl/cm²) or by adding the positive controls staurosporine (STS, S4400 Merck, stock solution at 2mM in DMSO) at 0.75 µM or Triton X-100 (Tx, Euromedex, Souffelweyersheim France) at 0.01% t the culture media of the basal compartment. After 24–48 h, the apical secretome was collected by rinsing with 200 µl HBSS Ca2+/Mg2+, the basolateral media was collected, and both compartments were stored at -20 °C for further analysis. Subsequently, AlamarBlue and Ly assay were performed, and cells were then either lysed for mRNA quantification by RT-qPCR or lysed with milliQ H2O (100 µl) to quantify intracellular PS-Eu or fixed for TEM and Raman microscopy analysis. For more information see Additional File 4.
Assessment of cell response to particle treatment
Lactate dehydrogenase (LDH) assay on 2D-grown A549 cells
Cell necrosis was assessed via the quantification of lactate dehydrogenase (LDH) release using the Cytotoxicity Detection KitPLUS (Roche). Briefly, after 48 h of treatment, half of the untreated cells were lysed with 0.1% (v/v) Triton X-100. Afterward, 50 µl of supernatants from each well were transferred to a fresh plate and incubated with 50 µl of the reaction mix provided in the kit for 30 min, at RT, light protected. Then, 25 µl of stop solution were added to each well, and absorbance was measured at 490 nm (Tecan Infinite F Nano+). Triton-treated wells served as control for maximum LDH release while cell-free wells incubated with or without particles served as background control for the correspondent treatments.
Resazurin, neutral red uptake, and coomassie brilliant blue assays on 2D-grown A549 cells
After using part of the supernatant for the LDH assay, all wells were refilled 50 µl of fresh CCM and mitochondrial and acidic organelle activity was measured via Resazurin and Neutral Red uptake assays. Briefly, a mix of resazurin (0.15 µg/ml in sterile PBS, Sigma) and Neutral red (0.4 µg/ml in sterile PBS, filtered at 0.2 μm from Sigma) in a ratio 3:2 was prepared and 50 µl of it were added to each well. Samples were subsequently incubated under humidified conditions at 37 ºC and 5% CO2 for 4 h and then cell metabolic activity was measured at Ex: 560/ Em: 590 nm (Tecan Infinite F Nano+). The liquid present in each well was then aspirated and 60 µl of Neutral red (NR) solvent (50% EtOH, 49% dH20, and 1% acetic acid (Sigma-Aldrich)) were added to each well and samples incubated for 20 min at RT. Acidic organelle activity was then quantified using the same spectrophotometer set at Ex: 530/Em: 645 nm. Afterwards, protein content was estimated via Coomassie brilliant blue assay. Again, the supernatant was aspirated and 60 µl of Coomassie brilliant blue 6250 (CBB) staining (0.05% Coomassie blue 6250 (Merc) in 30% methanol (Honeywell), 10% acetic acid and 60% milliQ water, 0.2 μm filtered) was added to each well and samples were incubated for 30 min at RT. After eliminating the supernatant, 60 µl of CBB solvent (0.1 M NaOH (Signa-Aldrich) in dH20) were added to each well for 20 min. Then 60 µl of NR solvent were added on the top of all samples and absorbance was measured at 595 nm after carefully shaking the plate. In all assays Triton-treated wells served as control for minimum NRU, CBB or Resazurin signal while cell-free wells incubated with or without particles served as background control for the respective treatments.
ROS quantification and particle uptake estimation via flow cytometry on 2D-grown A549 cells
ROS production and particle uptake estimation were performed via flow cytometry using a BD FACS Melody. Briefly, A549 were first detached using Triple Select (Gibco) and then 32 × 104 cells were transferred in FACS tubes to be washed once with PBS (Sigma). Then samples were resuspended in a 20 mM solution of the general ROS indicator CM-H2DCFDA (Invitrogen Thermo Fisher), and incubated for 30 min at 37 °C, 5% CO2 in humified conditions. Once stained, samples were washed once more with PBS to be then either left untreated or incubated with increasing concentrations of PS-Eu particles (from 3,125 to 100 µg/ml) in CCM for additional 30 min at 37 °C, 5% CO2, humified conditions. 0.05 mM H2O2-treated cells served as control for ROS production. Finally, ROS production was quantified via flow cytometry after doublets exclusion and particle uptake was indirectly estimated by collecting side scatter values (SSC).
Preparation of transwells-grown A549 cells for confocal microscopy
The staining protocol used in this manuscript is a modification of the one published by Buckley et al. 2018 [79]. Briefly, sample fixation was performed for 10 min at − 20 °C directly on the 24-well plate plastic transwells using ice-cold 100% mthanol (Honeywell): acetone (Merk) 1:1, followed by 6 washing steps with PBS (Sigma) at RT (5 min each). To minimize non-specific binding of antibodies, a blocking solution containing 20% FBS (Sigma) and 1% BSA (Sigma) in PBS was added to cells for 30 min at RT. Samples were then incubated with anti-ZO-1 5 μg/ml Alexa Fluor 488 (Invitrogen, clone ZO1-1A12) and anti-Occludin 5 μg/ml Alexa Fluor 594 (Invitrogen, clone OC-3F10) in PBS for 1 h at RT, and washed with PBS for 50 min at RT. Again, during this period the buffer was replaced every 10 min. Once the washing step was completed, cell nuclei were stained with DAPI (1 µg/ml) in PBS for 10 min at RT. After discarding the liquid, membranes were cut from the transwells using a scalpel and mounted onto glass slides with natural mounting medium (Sigma). Confocal laser scanning microscopy (CLSM) was performed with an Axio Observer Z1 inverted microscope equipped with LSM 800 (ZEISS), using an immersion 100x magnification objective. The Alexa Fluor 488 signal was excited with a diode laser at a wavelength of 488 nm (green laser) with 3% intensity, Alexa Fluor 594 signal was excited with a diode laser at a wavelength of 561 nm (red laser) with 5% intensity, and the DAPI signal was excited with a diode laser at a wavelength of 405 nm (blue laser) with 3% intensity, and a pinhole aperture of 35 μm. At least three sets of images (512 × 512 pixels) were acquired per membrane. When over time confocal images were collected, cells were seeded at different time points and cultured for a given amount of days (e.g. 1, 3, 7, and 10) and then harvested, stained and imaged on the same day using this protocol.
Preparation of transwells-grown A549 cells for scanning electron microscopy (SEM)
The morphology of A549 cells grown for 17 days on 12-well plate transwells was investigated via SEM. Cells were immersed in Karnovski fixative (25% glutaraldehyde, 8% paraformaldehyde in Na-P buffer) and incubated overnight at 4 °C after the elimination of the cell medium. The day after, samples were washed 3 times with Na-P buffer (10 min each) before being incubated for 1 h with 1% OsO4 in dH2O. Osmium impregnation was then followed by 3 additional washing steps with dH2O before samples were dehydrated using ethanol series (10 min each, with 30%, 50% 70% and 90% EtOH). Finally, after two washes with absolute ethanol (10 min each) (Honeywell), transwells were incubated for 10 min with increasing concentrations of HDMS in ethanol (EtOH: HMDS ratios 7:3, 1:1 and pure HDMS). Lastly, pure HDMS was added to each well and let air dry overnight. Once ready, samples were detached from the transwells using a scalpel and mounted on supports. Samples were covered with a 5 nm gold/palladium layer using a Gatan PECS 682 operated at at 10 kV in argon atmosphere before they were imaged using a JSM-6500 F (JEOL) operated at 5 kV.
Lactate dehydrogenase (LDH) assay on transwells-grown A549 cells
Since LDH was measured in 24-well plate transwells, 250 µL of fresh medium were added to the apical compartment of quasi-ALI wells to equalize medium volumes with the submerged samples. Then, Triton X-100 (0.1% v/v, Roche) was added to half of untreated control cells. After 30 min of incubation at RT, supernatants from the apical and basolateral compartments were mixed and transferred into a fresh 96-well plate (TPP) (50 µL each, four replicates), where they were tested for LDH release as described in Sect. 5.3.1. This was done because LDH can be released, upon cell death, both in the apical and basolateral side through the membrane. Triton-treated samples served as control for maximum LDH release. LDH measurement was then followed in by lucifer yellow assay.
Lucifer yellow (Ly) assay performed on transwells-grown A549 and Calu-3 cells
In transwells-grown A549 cells, epithelial permeability was measured via lucifer yellow (Ly) assay either during cell culture (from day 1 to 17 after seeding) or upon 48 h treatment with particles, after performing LDH assay. (NOTE: if permeability was measured to monitor barrier formation over time during cell culture, the Triton X-100 incubation step mentioned in the previous section was not performed. This was done only after 48 h of incubation with the test sample). After apical and basolateral supernatants were mixed and used for LDH assay, transwells were washed twice with HBSSCa2+ Mg2+ (Gibco) and transferred to a fresh 24-well plate. Here 1000 µL of HBSS were added in the basolateral compartment while 200 µL of sterile-filtered Ly (0.1 mg/ml, Merk) in HBSS were added on the apical compartment. Samples were then incubated for 1 h at 37 °C, 5% CO2, in humified conditions. Subsequently, 150 µL of supernatant from the basolateral side were transferred in duplicates to a fresh 96-well plate. Fluorescence signal was acquired using a Tecan Infinite F Nano + plate reader (Ex: 485; Em: 538) with the gain set on optimal. Medium transwells served as controls for 100% permeability. After measurement cells were washed twice with HBSS and moved back to their original 24-well plate. Medium was refreshed and TEER measurement was performed.
In Calu-3 cells instead, which were grown on 12-well plates transwells, 500 µl of Ly at 0.1 mg/ml in HBSS Ca2+/Mg2+ were added to the apical compartment and 1 ml to the basolateral one. After 1 h incubation, 100 µl of the apical and basal solutions were transferred to a 96-well clear bottom microplate (µClear®) for fluorescence measurement on a FlexStation 3 multi-mode microplate reader (Ex: 485; Em: 538) and permeability was calculated according to (Sanchez-Guzman et al. 2021) [78].
Transepithelial electrical resistance (TEER) measurement on transwells-grown A549 cells
TEER was measured after performing Ly assay, using the Epithelial Volt/Ohm Meter 3 (EVOM3) (World Precision Instruments) following manufacturer’s instructions. EVOM3 was calibrated before each experiment, and cell-free transwells served as background control for TEER. Similarly, Triton X-100-treated samples served as a control for minimum electrical resistance. Untreated cells instead serve as control for maximum resistance. During data acquisition, the instrument was set on AUTO mode (resistance), and each well was measured twice. NOTE: prior measurement, when medium was changed in all samples after performing Ly assay, 250 µL of CCM were added in quasi-ALI cultures on the apical compartment to have a volume comparable to submerged samples. After measurement, when over time permeability/resistance measurements were performed (from day 0 to 17 after seeding), exceeding medium was discarded to restore quasi-ALI conditions, otherwise, after particle treatment, TEER measurement was followed by Resazurin assay.
Resazurin assay on transwells-grown A549 cells
Briefly, after measuring epithelial resistance (TEER), mitochondrial/metabolic activity was measured via Resazurin assay by adding 50 µl of resazurin 0.15 mg/ml (Roth) in PBS (Sigma) in the apical compartment of each well and incubating the cells for 2 h. Then 100 µl of supernatants from the transwells apical compartment were transferred in duplicates to a 96-well plate and fluorescence was recorded using a Tecan Infinite F Nano + microplate reader (Ex: 560; Em: 590) with the gain set on optimal. Triton X-100 treated samples (0.1% v/v, Roche) served as control for minimum viability and untreated cells as 100% viability.
Surface tension measurement on transwells-grown A549 cells
The surface tension in quasi-ALI and submerged samples was determined using the drop spreading method [80, 81], on cells grown on transwells in the 12-well plates format. Surface tension was measured both at 17 days after seeding and 48 h after treatment. Briefly, before performing the test, the supernatant of submerged cells was discarded, and cells were let air-exposed for 1 h at humified conditions. This equilibration period was important to exclude that medium leftover could affect drop spreading and consequently surface tension estimation. Then, small droplets of 5 µl composed by a mixture of dimethylphtalate (Sigma) and normal octanol (Honeywell) (4:1, v/v ratio, stained with 4 mg/ml of crystal violet (Sigma)) were dropped onto the epithelia surface. The surface tension of the hypophase was then calculated by the ratio of the diameter (d) of the deposited droplet (measured 30 s post deposition) and the diameter (d0) of the droplet prior deposition, while it was still hanging from the pipette: d/d0. The ST was quantified from a calibration curve obtained for thin liquid substrates, which gives the relationship between d/d0 and the ST of the film. The droplet diameter was calculated with the program ImageJ using photographs taken with a digital camera mounted on a Leica MZ FLIII stereomicroscope. Since deposited drops were sometimes not perfectly circular, the used diameters were calculated from the average of 4 different measurements in four different positions.
Particle translocation assessment on transwells-grown A549 and Calu-3 cells
Particle translocation potential was determined on A549 (submerged and quasi-ALI) and Calu-3 cells (quasi-ALI) grown on 12-well plates transwells using a fluorometric measurement. In A549, 150 µl of medium from the apical and basolateral compartments of treated and untreated A549 cells were transferred in duplicates to a 96-well plate. Then fluorescence emission of the Europium was quantified using a Tecan Infinite F Nano+ (Ex: 340; Em: 613) with gain set on optimal. Untreated and treated cell-free transwells served as background control and control from maximum translocation respectively. In quasi-ALI Calu-3 culture, basal media was collected and the apical compartment was collected by rinsing with 200 µL HBSSCa2+/Mg2+. A cell lysate was prepared by adding 100 µL of water for 15 min at -80 °C to the apical compartment. Lysates, medium from the apical and basolateral compartments of treated and untreated cells were stored at -20 °C before analysis. Then fluorescence was quantified in 96-well clear bottom microplate (µClear®, Greiner Bio-One, Dominique Dutscher, Bernolsheim France) using a FlexStation 3 multi-mode microplate reader (Ex: 264 nm; Em: 613 nm for the apical and basal media and Em: 614 nm for the cell lysate). A standard curve with PS-Eu was done in each corresponding media.
Surfactant protein (SP) quantification on transwells-grown A549 cells
Surfactant proteins quantification was performed both on the pellets and the supernatant of treated and untreated cells. Prior SP quantification via ELISA, proteins were isolated from the cell pellets. Briefly, treated and untreated A549 cells were washed once with PBS. The cells were then lysed with 300 µl Triton buffer containing 0.2% protease inhibitors and 0.2% phosphatase inhibitors and incubated on ice for 30 min. After centrifugation for 5 min at 13,000 rpm (4 °C), the supernatant was transferred to a new tube. Then protein concentration was determined using the Bradford assay. Then pellet samples, prior ELISA, were diluted to a comparable total protein concentration. A549 cell pellet and their supernatants were analysed using a quantitative sandwich ELISA. Assays were performed using SP-ELISA kits from MyBiosource (MBS4502605-96, SP-A; MBS2703500-96, SP-B; MBS4502613-96, SP-C; MBS4502615-96, SP-D; MBS1606900-96, SP-H; San Diego, CA, USA) and Cloud-Clone (SED755Hu, SP-G; Cloud-Clone Corp., Wuhan, China) according to manufacturer instructions. Absorbance was collected at 405 nm and 450 nm.
Preparation of transwells-grown A549 and Calu-3 cells for transmission electron microscopy (TEM)
After 48 h of treatment with PS-Eu particles, A549 cells grown on 12-well plate transwells were washed using PBS (Sigma) and fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 3 h at 4˚C. After an overnight rinsing in 0.33 M sucrose in 0.1 M cacodylate buffer at 4 ˚C, post-fixation was performed with 1% osmium tetroxide (OsO4) (Roth, Germany) and 0.8% potassium ferrocyanide (K4[Fe(CN)6] x 3 H2O) (Sigma-Aldrich, Germany) in 0.2 M cacodylate buffer for 30 min in the dark at room temperature. After post-fixation, cells were rinsed with 0.1 M cacodylate buffer (pH 7.3), dehydrated in an ethanol series (5 min in each ethanol concentration), first impregnated with a mixture (1:1) of ethanol and Epon resin (Serva Electrophoresis, Germany) and then with pure Epon resin. Polymerization was carried out for five days at temperatures ranging from 35 ˚C to 80 ˚C.
Semi-thin Sect. (1 μm thick) were stained with 1% toluidine blue and 2% borate in distilled water and viewed with a Nikon Eclipse TE bright-field microscope (Amsterdam, The Netherlands).
Ultrathin Sects. (50–60 nm thick) were collected on copper grids, contrasted with aqueous uranyl acetate for 20 min and lead citrate for 2 min, and examined at 80 kV with a Philips CM100 transmission electron microscope (Eindhoven, The Netherlands). An AMT CCD camera (USA) and AMT Digital Micrograph software were used to obtain digital images.
In Calu-3 instead, all steps were performed at room temperature, except when otherwise indicated. Cells were fixed sequentially in 0.1 M sodium cacodylate, pH 7.3 containing 1.5% paraformaldehyde and 2.5% glutaraldehyde for 1 h, then in the same buffer containing 1% OsO4 for 1 h (at 4 °C) and finally in 2% aqueous uranyl acetate for 1 h. Cells were dehydrated in an ascending series of ethanol (30%, 50%, 70%, 95%, and 3 × 100%, for 10 min each), then incubated sequentially in 1/3, 1/1 and 3/1 (v/v) ethanol/Epon mixtures (1 h each) before final embedding in pure Epon.
Ultrathin Sect. (80 nm) were imaged using a Jeol 1400 TEM (Jeol, Croissy-sur-Seine, France), operated at 120 keV and equipped with a RIO CMOS camera (Ametek SAS, Elancourt, France).
Alamar Blue™ assay on transwells-grown Calu-3 cells
AlamarBlue™ (AB, Thermo Fisher Scientific) was diluted (1:10 ratio) in HBSSCa2+/Mg2+ (Gibco, Thermo Fisher Scientific) and 1 ml was added to the basal compartment of control and treated cultures. After 2 h incubation, 100 µl of basal medium were transferred to a 96-well clear bottom microplate (µClear®). Fluorescence measurements were carried out on a FlexStation 3 multi-mode microplate reader (Molecular Devices) with Ex/Em wavelengths of 545 nm and 590 nm. To account for differences in cell number we compared the mitochondrial transformation of Alamar Blue of each transwell to the activity before treatment.
Enzyme-linked lectin assay (ELLA) on transwells-grown Calu-3 cells
Glycoproteins were quantified in the apical secretome by the ELLA according to Sanchez-Guzman and co-workers [78]. 96-well high binding plates were coated with lectin from Triticum vulgaris (Sigma-Aldrich) and washed with 0.5 M NaCl and 0.1% Teen 20 in PBS 1x. A standard curve was prepared with Porcine stomach mucin (Sigma-Aldrich) diluted in HBSSCa2+/Mg2+. Standards and samples were incubated for 1 h at 37 °C. Lectin detection was performed with Glycine max peroxidase conjugate (Sigma-Aldrich) by 1 h incubation at 37 °C. Tetramethylbenzidine (TMB) substrate reagent (R&D systems, Bio-Techne) was added for 1 h at room temperature and the enzymatic reaction was stopped with the 2 N Sulfuric Acid Stop Solution.
The absorbance was measured at 490 nm on a UV-vis plate reader (EL808 Biotek microplate reader).
Gene expression analysis – RT-qPCR on transwells-grown Calu-3 cells
Cell lysis and RNA extractions from control and treated cultures were done using the Nucleospin RNA/Protein Mini kit (Macherey-Nagel, Hoerdt, France) according to manufacturer’s instructions. Cells were lysed by adding 350 µl of the lysis buffer supplemented with 0.5 M dithiothreitol (Merck) and gently scraping with the pipette tip to detach and collect cells from the transwells membrane. Reverse transcription of the mRNAs was performed using multiscribe High-cDNA Capacity RT kits (Applied Biosystems, ThermoFisher Scientific) according to the manufacturer’s protocols. qPCR was done using LightCycler SYBR Green master mix (Roche Diagnostics, Meylan, France) with primers from Eurofins Genomics (Ebersberg, Germany). Reactions were carried out in sealed 384-well plates (Roche Diagnostics) in a LightCycler 480 thermocycler (Roche Diagnostics).
Gene expression was quantified using 2-∆∆Ct method to yield Log2 fold change of a gene relative to HPRT and UBC as housekeeping genes and normalized to the control at each timepoint. Table 2 shows the primers used.
Raman microscopy on transwells-grown Calu-3 cells
Calu-3 cells exposed at quasi-ALI to PS-Eu nanoplastics for 24 h at 45 µg/cm² were fixed with PFA 4% for 20 min at room temperature and rinsed with PBS before imaging. The transwell membrane was placed on fused silica substrate (ESCO Optics, UK) with cells facing the objective lens. Raman images were acquired on a WITec alpha300 RA inverted Raman microscope (Oxford Instruments) with a Zeiss 100x oil immersion objective (NA 1.3) using an excitation wavelength of 532 nm and a grating of 600 g/mm. Exposure time was set to 0.1 s and laser power to 10 mW. An area of 30 × 30 μm was analysed. 3D Raman images were taken using a depth step of 1 μm. Raman images were analysed using WITec Project 5 Plus software for cosmic ray removal, baseline subtraction, and spectral analysis.
Statistical analysis
Statistical tests were performed using the GraphPad Prism software version 10.0.2 (La Jolla, CA, USA). The normality of the data was tested with the Shapiro-Wilk test. For comparing different groups, one-way ANOVA was performed with Kruskal-Wallis test or Wilcoxon–Mann–Whitney test with Dunn’s multiple comparison for not normally distributed samples, or one-way ANOVA with Dunnett’s and Sidak tests for normally distributed samples. To compare different groups at different timepoints or different culture systems (quasi-ALI vs. submerged), two-way ANOVA was used with Dunnett’s multiple comparison test. Results were considered significant if p < 0.05 (*, #, §), p < 0.01(**, ##, §§), p < 0.001(***, ###, §§§), or p < 0.0001(****, ####, §§§§).