Crystalline silica samples
Two synthetic α-quartz samples with as-grown (gQ) or fractured (gQ-f) faces were used in this study. A commercial fractured α-quartz dust, largely used in studies of experimental silicosis and lung cancer (Min-U-Sil 5 quartz, US Silica Co., Berkeley Springs, WV, USA, lot number 15062696, in this work named cQ-f), was used as positive control.
Synthetic quartz crystals with as-grown regular faces (gQ) were obtained following the procedure developed by Pastero and coworkers . Briefly, a 25% w/w Na-metasilicate (Na-MTS) solution was polymerized into a gel by the addition of 1 M HNO3. The gel was stabilized at pH ≈ 8. Growth runs were performed in PTFE liner steel autoclaves at 210 °C, for 168 h. The finest fraction (< 30 mm), obtained through 30 minutes of sieving in 100 and 30 mm sieves, was used in all the experiments.
To obtain crystals with fractured faces (gQ-f), a further synthesis was carried out, where the Na-MTS solution was polymerized by bubbling CO2 until gel formation, at pH ca. 8. The largest fraction (> 30 mm), obtained as above, was mechanically fractured by milling in a MM 200 mixer mill (Retsch, Haan, Germany) in agate jars (27 Hz, two spheres, 500 mg of dust/jar) for 6 hours, in order to induce surface alterations.
Particle size distribution
Particle size distribution was determined by Flow Particles Image Analyzer (FPIA), using a Sysmex FPIA 3000 particle size and shape analyser (Malvern Panalytical, Malvern, UK) and by dynamic light scattering (DLS), using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). For FPIA analysis, a sample dispersion of 0.5 mg/ml in MilliQ water was prepared and probe sonicated on ice for 3 minutes at 30% amplitude, power 25 W, using a Sonopuls HD3100 homogeniser (Bandelin, Berlin, Germany). For DLS analysis, a sample dispersion in RPMI medium (0.5 mg/ml), with the addition of 10% FBS, was prepared and sonicated as described.
Surface area determination
Specific surface area (SSA) was evaluated by the Brunauer-Emmett-Teller (BET) method, based on Kr adsorption. Quartz samples were firstly outgassed for two hours, at 150 °C. The analysis was then performed at -196 °C using an ASAP 2020 physisorption analyser (Micromeritics, Norcross, USA).
The ζ potential of the quartz samples was evaluated by means of electrophoretic light scattering (ELS) with a Zetasizer Nano–ZS (Malvern-Panalytical, Malvern, UK). In this technique, the velocity of a particle in an oscillating electric field, which is proportional to its ζ potential, is measured by light scattering. The ζ potential was measured after suspending quartz (0.5 mg/ml) in serum free RPMI medium or RPMI + 10% FBS, to evaluate the surface charge at exposure conditions (pH ca. 8) and the modulation given by serum proteins. Investigation of zeta potential at lysosomal pH (pH 4.5) was measured suspending quartz particles (0.5 mg/ml) in 0.01 M NaCl, and adjusting the pH of the suspension to the experimental value with 0.1 M HCl or 0.1 M NaOH.
Cell culture and THP-1 differentiation
RPMI 1640, FBS and DPBS were purchased from Gibco (Thermo Fischer Scientific, Waltham, MA, USA). Experiments were performed on human THP-1, a human monocyte-like cell line derived from a patient with leukaemia (ATCC#TIB-202) . Cells suspensions were cultured in RPMI 1640 supplemented with 10% FBS (complete medium, cRPMI), at 37 °C and 5% CO2. Just before seeding, (200000 or 100000 cells/well in a Cellstar 24-well plate for LysoTracker staining and PI staining, respectively, or 50000/well in a Cellstar transparent 96-well plate for MTT assay) cells were differentiated into macrophages by incubation with 100 nM PMA (phorbol 12-myristate 13-acetate) in cell culture medium for 48 h, at 37 °C and 5% CO2 . Transparent 24-well and 96-well plates were purchased from Greiner Bio-One (Kremsmunster, Austria).
Particle uptake by THP-1 macrophages was investigated by means of transmission electron microscopy on cross sections of fixed cells, upon 24 h incubation with 50 µg/ml of synthetic or commercial quartz samples. After exposure, the cells were fixed with 0.2% glutaraldehyde and 2% PFA in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h. Then, cells were rinsed twice for 5 min in 0.1 M cacodylate buffer at room temperature followed by post-fixation in 1% osmium tetroxide/1.5% potassium ferrocyanide in 0.1 M sodium cacodylate at 4 °C for 30 min. The cells were then washed with Milli-Q water, dehydrated through serial incubation in a graded ethanol series (30, 50, 70 and 100%), and lastly embedded in EPON resin and polymerized at 37 °C for 16 h followed by 58 °C for 24 h. Given the size of the particles and the fact that they are hard to cut with a standard diamond knife, sections were cut at a thickness of 200 nm instead of the standard 80 nm using an UC7 ultramicrotome (Leica, Vienna, Austria). This allowed to partially reduce the presence of holes in the section, corresponding to areas were particles accumulate and that cannot be sectioned, while still being able to determine whether particle uptake was present. Sections were then contrasted using 5% uranyl acetate for 20 min, followed by Reynolds lead citrate for 2 min. Images were recorded with a CM100 Biotwin transmission electron microscope (FEI, Eindhoven, The Netherlands) operated at 80 KV using a Morada digital camera.
Cell metabolic activity was assessed through a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay . After differentiation, THP-1 macrophages, previously plated at a density of 50000 cells/well in a transparent 96-well plate, were exposed for 24 h to quartz particles at increasing concentrations (10, 25, 50, 100, 250 μg/ml) in complete medium (cRPMI, RPMI medium + 10% FBS). Before the assay, each well was washed with Dulbecco’s Phosphate-Buffered Saline (DPBS) to remove the extracellular particles and eventual cell debris and treated with MTT (0.5 mg/ml) in cRPMI for 20 minutes at 37 °C. Then, MTT reagent was discarded and DMSO (200 μl) was added to each well, to solubilise formazan crystals. Each well was pipetted again to mix, and absorbance at 550 nm was measured using a THERMOmax microplate reader (Molecular Devices, San Josè, CA, USA). All values were normalized to the results obtained in untreated cells.
MTT assay was also carried out after 4 h of exposure at 4 °C, in order to block active cellular processes [65,66]. Plates with cells were pre-incubated at 4 °C for 30 minutes, just before the exposure to quartz particles (25, 50, 100, 250 μg/ml) in cRPMI. After 4 h of incubation, wells were washed three times with cRPMI (100 μl), and further incubated in fresh medium without particles for 20 h at 37 °C and 5% CO2. The same protocol was applied for the 4-hours exposure, at 37 °C.
To assess protein modulation on quartz cytotoxicity, the MTT assay was carried out as previously explained, exposing PMA-differentiated THP-1 cells (50000 cells/well) for 4 or 24 h to quartz samples (100 mg/ml), in serum-free RPMI or RPMI supplemented with 10% FBS. The analysis was carried out as described above.
Lyso Tracker staining and flow cytometry
The impact on the lysosome after interaction and internalization of particles was investigated by staining cells with Lyso Tracker Red DND-99 (Thermo Fisher Scientific, Waltham, MA, USA), a fluorescent acidotropic probe for labelling and tracking acidic organelles in live cells. Increase in Lyso Tracker intensity from basal condition can be due to an increase in lysosomal volume, lysosomal number, or lysosomal acidity. THP-1 cells differentiated into macrophages were exposed to particle dispersions (25, 50, 100 mg/ml) prepared by diluting a stock dispersion (1 mg/ml) in cRPMI. Cells were grown on transparent 24-well plates (200000 cells/well) and exposed to particles 48 h after seeding. After 24 h exposure, at 37 °C and 5% CO2, wells were washed (1x500 ml with complete medium) to remove quartz particles, then cells were incubated 15 minutes at 37 °C, 5% CO2, with a solution (25 nmol, 500 ml) of Lyso Tracker Red DND-99 (Thermo Fisher Scientific, Waltham, MA, USA), in cRPMI. Then, the dye solution was discarded and wells were washed again with cRPMI (1x500 ml) and DPBS (2x500 ml). Cells were detached with 0.05% trypsin/EDTA (300 ml) at 37 °C for 5 minutes, followed by a treatment with EDTA solution (5 mmol) in PBS (pH 7.4), at 37 °C. Then, cells were harvested in FACS tubes and pelleted by centrifugation at 250g for 3 minutes. Cells were resuspended in DPBS (100 ml) and analysed using a CytoFlex flow cytometer (Beckman-Coulter, Brea, CA, USA). LysoTracker Red fluorescence intensity was analyzed in FL3 channel. Quantitative analysis of flow cytometry data was carried out using the FlowJo software (Tree Star, USA). Gates were set in order to discriminate cell debris and cell doublets from the analysis, according to their forward and side scattering. 20000 cells were acquired, unless specified in the case of samples for which strong cytotoxicity was detected (in these cases a variable number of cells, ranging from 20000 to at least ca. 7000 was acquired, as specified in figure captions). The same experiment was performed after 4 hours exposure at 4 and 37 °C, for cells exposed to 50 and 100 µg/ml of particles. The exposure at 4° C was preceded by a 30-minutes pre-incubation step of cells, at the same temperature.
Propidium iodide (PI) assay
THP-1 cells differentiated into macrophages were seeded on transparent 24-well plates (100000 cells/well) and, 48 h after seeding, cells were exposed to particles (final concentration: 100 mg/ml) in serum free (- FBS) or complete RPMI (+ FBS). After 4 h exposure at 37 °C and 5% CO2, quartz dispersions were discarded and wells were washed with cRPMI (1x500 ml). Then, cells were harvested as described, collected into FACS tubes and stained with a PI solution (5 μg/ml) at room temperature. After incubation with the dye (15 minutes, 37° C), samples were measured as described above. A variable number of cells, ranging from 12000 to ca. 7000, were acquired for each sample (as specified in figure caption). Data were analyzed using the FlowJo software (Tree Star, USA).
The hemolytic activity of quartz particles refers to a method described by Lu & coworkers , with minor modifications given by Pavan & coworkers . RBCs were separated from fresh human blood of healthy volunteer donors, not receiving any pharmacological treatment. To assess the effect of FBS proteins on quartz hemolytic activity, particles were incubated with increasing concentration of FBS (0.03%, 0.06%, 0.3%) in the exposure medium (DPBS). Briefly, quartz suspensions (75 μl/well,) at a concentration of 0.37 mg/ml were dispensed in a transparent 96-well Cellstar microplate (Greiner Bio-One, Kremsmunster, Austria), and FBS (75 μl/well) was added, to each well. After 30 minutes of incubation at room temperature (25 °C), red blood cells suspension (75 μl/well) was added, and the plate, gently shaken on an orbital plate shaker, was incubated for 30 minutes at 25 °C.
The plate was centrifuged for 5 minutes at 1200 RPM, using a Centrifuge Rotina 420R centrifuge (Hettic Instruments, Beverly, MA, USA). The supernatant (75 μl) was removed and transferred to a clean transparent microplate. The amount of haemoglobin released into the supernatant was spectrophotometrically determined at a wavelength of 540 nm with an Infinite 200 UV/Vis spectrophotometer (Tecan, Grödig, Austria). Absorbance values were converted into percentages of haemolysis according to the formula (Equation 1):
where OD is the optical density or absorbance.
DPBS and 0.1% Triton-X100 were used as negative and positive controls, respectively. Released hemoglobyn did not adsorbed on quartz samples (data not shown).
Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, as appropriate. Differences with p value < 0.05 were considered statistically significant.