Preparation of ENMs. Neutral-, amine-, and carboxyl-FLG were manufactured via dielectric barrier discharge of mined graphite by Perpetuus Carbon Technologies (PCT, UK). CB particles were sourced from (FLAMMRUSS 101, Lamp Black #8235102) Evonik Degussa Inorganic Materials, Frankfurt. All ENMs were supplied as powder and were suspended at a stock concentration of 10 mg/ml in double distilled water, which were then titrated to final concentrations with cell culture medium. Prior to exposures ENMs were sonicated in a 90W Ultrasonic Bath (Fisher Scientific #FB15046) for 20 minutes at 37 °C to encourage destabilisation of agglomerate material.
ENM characterisation. Each test ENM had previously been characterised by Burgum et al (Burgum et al., 2020). Briefly, neutral-FLG, amine-FLG, carboxyl-FLG (as well as, to a lesser extent, CB particles) particle and agglomerate sizes were investigated with several techniques including; plunge-freeze scanning electron microscopy (SEM), dynamic light scattering (DLS), atomic force microscopy (AFM) and Raman spectroscopy. The agglomerate analysis suggested a difference in size between the particle types when suspended in cell culture media with the additional surface groups of amine-FLG and carboxyl-FLG increasing the average diameter by ~ 300 and ~ 400 nm respectively. The addition of surface groups reduced the average thickness of FLG layer number when measured by AFM, the greatest effect produced by carboxyl groups which lowered the calculated layer number from 50 to 4 atomic layers of graphene.
Cell culture. The TT1 cell line was kindly donated by Professor Terry Tetley of Imperial College London, UK. This alveolar cell line had been previously transformed, immortalised and characterised by Kemp and colleagues as a model for ATI cells (Kemp et al., 2008). TT1 cells were thawed from liquid nitrogen and transferred to T25 flasks to initiate proliferation for 48 hours or until reaching 70–80% confluency before sub-culturing into a T75. TT1 cells were cultured with DCCM-1 Media (Geneflow Ltd, UK) supplemented with 1% Penicillin/Streptomycin/L-Glutamine (Sigma, UK) and 10% New-born Calf Serum (NCS) (Sigma, UK). Sub-culturing of TT1 cells began with removing existing media and discarding, washing the adherent cells twice with pre-warmed PBS before adding 4 ml of thawed Trypsin-EDTA and incubated flat at 37 °C for 5 minutes to disassociate. Once cells detached, complete media was added (6 ml) to de-activate the Trypsin-EDTA solution and the cells transferred to 15 ml Falcon tubes to be centrifuged at 340 g for 10 minutes. The resulting supernatant was discarded, cells re-suspended and seeded at 1 × 105 and returned to the incubator at 37 °C. When seeding TT1 cells for toxicological assays, cells were counted by re-suspending and taking 10 µl and adding to 90 µl of fresh media then pipetting 10 µl into a Neubauer chamber. Cells in the four corners of the chamber were counted, averaged and multiplied by 104 for the number of cells per ml. TT1 cells were sub-cultured every 3 days, reaching 90–100% confluency in that time. TT1 cells were not used in toxicological assays once exceeding 20 passages. THP-1 cells and differentiation were sourced and cultured as described by Evans and colleagues (Evans et al., 2019). Briefly, THP-1 monocytes were differentiated with 50 nM of PMA, incubated for 24 hours before a complete media change was performed allowing the now differentiated THP-1 macrophages to recover. On the day of seeding co-cultures, d.THP-1 macrophages were resuspended at 1 × 105 cells/ml in DCCM-1 media.
THP-1 differentiation. To differentiate, THP-1 cells were centrifuged at 130 g for 5 minutes at room temperature, following this the cells were resuspended in a 15 ml Falcon tube at a concentration of 5 × 105 cells/ml with 50 nM of phorbol-12-myristate-13-acetate (PMA). This suspension was transferred to a T75 flask and incubated at 37 °C for 24 hours. Cells were checked for morphology via light microscopy; differentiation was evident when cells did not detach from flask over gentle agitation.
Construction of co-culture model. The alveolar lung epithelial co-culture model was comprised of TT1 alveolar cells (seeded at 1 × 106 cells/ml) and d.THP-1 macrophages (1 × 105 cells/ml per 6-well insert). These co-cultures were constructed upon the apical side of PET track-etched 4.2 cm2 transwell inserts (3 µm pores) supported by a 6-well companion plate (Corning, Germany). TT1 cells were seeded on day one and left to proliferate for one week. At day five, THP-1 cells were differentiated into macrophages using PMA and left for 24 hours. On day six, d.THP-1 macrophages were lifted with Acutase and transferred to the TT1 monolayer and left to adhere for one day. The completed co-culture was then ready to be used on day seven, this was confirmed with confocal laser scanning microscopy (LSM (images not shown)).
Confirmation of cellular interaction/entry using Transmission Electron Microscopy (TEM). ENM cellular uptake was confirmed by TEM imaging. TT1 cells, the primary focus of this study were exposed to ENM were fixed, embedded, sectioned and imaged as previously described (Wills et al., 2016). The analysis was performed with a FEI Titan3 Themis G2 operating at 300 kV fitted with 4 EDX silicon drift detectors, and a Gatan One-View CCD. EDX spectroscopy and mapping to identify potential heavy metal contamination was undertaken using Bruker Esprit v1.9 software.
TEM analysis of FLG lattice planes. In conjunction with TEM uptake imaging, confirmation of graphitic materials was confirmed through fast Fourier transform (FFT) using ImageJ. To begin analysis, firstly an intracellular vesicle containing an FLG crystal oriented on its z-axis was located, the image was captured and saved for ImageJ analysis. Using ImageJ software, the captured image was uploaded, the scale bar was set, and the lattice plane focused upon and analysed with FFT to produce an image (Supplementary Fig. 1) which reveals the interlayer spacing of FLG.
Cytotoxicity by relative population doubling (RPD) & genotoxicity. TT1 cells were seeded at 1.0 × 105 cells/ml and allowed to adhere for 24 hours after which the cells were then treated with ENMs for 24 hours. Cell culture media was added as the negative control, mitomycin-C (MMC) at 0.01 µg/ml was used as the positive control, the assay was performed as previously detailed by (Evans et al., 2019). All experiments were performed in triplicate (N = 3) and 2000 binucleate (BN) cells per replicate were scored for the presence of micronuclei per concentration (6000 BN cells in total).
d.THP-1 cytotoxicity. Cytotoxicity of d.THP-1 macrophages (seeded at 1 × 105 cells/ml) was assessed by trypan blue exclusion following 24-hour exposure to ENMs as described by Evans and colleagues (Evans et al., 2019). Following exposure cells were lifted gently with Acutase and live cells scored with a haemocytometer (1:5 dilution). This was performed in triplicate.
(Pro)-inflammatory response. Supernatant from the CBMN assay was harvested following 24-hour exposure to ENMs and analysed using an IL-6 & IL-8 ELISA (DuoSet ELISA; R&D Systems Europe). ELISA’s were performed in triplicate following the manufactures instructions. The optical density (OD) was recorded at a wavelength set to 450 nm with an Omega Multimode microplate reader (BMG LABTECH Ltd, UK). This was performed in triplicate.
Mitochondrial stress. TT1 cells were seeded at 1 × 105 cells/ml (100 µl/well) into XFe24 tissue culture plates (Seahorse Bioscience). The following day, 150 µl of growth media was added and exposed to ENMs for 24 hours. The assay was performed as described in the work by Burgum et al (Burgum et al., 2020). Data was normalised through quantification of cellular protein (DC Assay (Bio-Rad, UK)) and a series of BSA (Bio-Rad, UK) prepared using RIPA buffer (Thermo Scientific, UK). This was performed in triplicate.
Co-culture in vitro CBMN assay. The standard in vitro CBMN assay was adapted for use with advanced co-culture models and performed as described in detail by Evans and colleagues (Evans et al., 2019). Following co-culture construction, cells were exposed to ENMs for 24 hours. Following exposure, a complete media change was performed with media containing 3 µg/ml of cytochalasin B before incubating for 1.5 cell cycles (24 hours). Cells were then removed with trypsin and fixed in 3% paraformaldehyde (PFA) for 24 hours at 4 °C followed by permeabilization at 4 °C with 0.2% Triton X100. Cells were stained with 1 µg/ml anti-human CD-324 (FITC fluorophore) suspended in x1 PBS and incubated at 4 °C in the dark for 45 minutes. Cells were subsequently washed in PBS before 100 µl of cells (per slide) were drop cast onto ethanol-cleaned glass slides which were left to air-dry and finally 30 µl of DAPI counterstain was applied. A total of 500BN TT1 cells were identified per replicate (1500BN total per concentration); TT1 cells were positively identified by their FITC fluorescence signal produced by e-cadherin, while d.THP-1 cells were selected against due to their absence of FITC fluorescence. Further, the present study exposures were performed with and without a two-hour incubation with 1.5 mM N-acetylcysteine (NAC) to investigate the potential role of oxidative stress. Following the NAC incubation, a complete media change was performed, and exposures were performed for 24 hours. Cellular proliferation was evaluated due to the inclusion of cytochalasin B whereby the cytokinesis block proliferation index (CBPI) was calculated by Eq. (1);
CBPI = (Number of mononucleate Cells + (2x Number of binucleate cells) + (3x Number of multinucleate cells)) / Total number of cells
Data analysis and statistics. All data is presented as the mean +/- the standard deviation (SD). Statistical analysis was performed in SPSS statistics software (v.20 IBM, UK) where all data sets were firstly analysed for normality (Shapiro-Wilk test, p ≤ 0.05) and for equal variance p ≤ 0.05)). A one-way analysis of variance (ANOVA) was performed with post hoc Dunnett’s multiple comparisons applied to evaluate pairwise statistical significance between control and concentrations; the alpha value was set to 0.05.