Silica nanoparticles
Rhodamine-labeled SiO2 NPs, “Sicastar“, of 30 nm in diameter, functionalized with carboxy (Cat. #40-02-301), amino group (Cat. #40-01-301) or non-functionalized (Cat. #40-00-301) were purchased from Micromod Partikeltechnologie, Rostock, Germany. All NPs were dispersed in water at 25 mg/mL. The size of the NPs in water or in complete cell culture medium was characterized by DLS, Zeta potential analysis and TEM. Zeta-potential was measured with Photal LEZA-600 (Otsuka Denshi Co., Osaka). The fluorescence intensities of the three types of silica NPs were measured at different concentrations using ARVOTMMX 1420 Multilabel Counter (Perkin Elmer, Waltham, MA). The slopes of the regression lines for independent variable of concentration and dependent variable of fluorescence intensity were calculated to obtain the relative fluorescence intensity between different types of silica NPs labeled with rhodamine (Supplementary file 1).
Animals
Forty-two male C57BL/6JJcl mice of 7-week-old were purchased from CLEA Japan Inc (Tokyo, Japan). All mice were housed and acclimatized to the new environment for one week in a pathogen-free animal room controlled at 23–25 °C and 55–60% humidity. Light was set within a 12-h light-dark cycle (on at 09:00 and off at 21:00), and food and water were provided ad libitum. This study was conducted according to the Japanese law on the protection and control of animals and the Animal Experimental Guidelines of Tokyo University of Science. The experimental protocol was also approved by the Animal Ethics Committee of Tokyo University of Science prior to the experiment.
Mice were randomly divided into seven groups of six each and exposed to plain SiO2 NPs, carboxyl SiO2 NPs or amino SiO2 NPs at 2 or 10 mg/kg bw, which were equivalent to 40 or 200 µg per mouse if body weight was 20 g, or endotoxin-free water. These exposure levels were half of those adopted in a previous study, which demonstrated that exposure to SiO2 NPs by intratracheal instillation increased the number of macrophages in BALF at 0.5 mg silica/mouse (20 mg/kg bw) but not at 0.1 mg silica/mouse (4 mg/kg bw) (Morris 2016). With regard to the relation of the above exposure levels by intratracheal instillation to those by inhalation, one study showed that 690 µg of titanium dioxide deposited in the rat lung after exposure to ultrafine titanium dioxide at 125 mg/m3 for 2 hours (Osier 1997). Since the alveolar surface areas of rat and mice are 2970 [30] and 82.2 cm2 [31], respectively, the estimated lung deposition of titanium dioxide in mice was 690 × 82.2/2970 = 19 µg when exposed to ultrafine titanium dioxide at 125 mg/m3 for 2 hours. The higher dose of 200 µg in the present study is comparable to ultrafine particles deposited in the lungs after inhalation exposure to ultrafine particles at 125 mg/m3 for 21 hours. SiO2 NPs dispersed in water at 25 mg/mL were vortexed and then further diluted with endotoxin-free water to obtain the NP solution at 1 and 5 mg/mL.
Mice were anesthetized with pentobarbital and then exposed to 40-µl aliquot of samples of SiO2 NPs by pharyngeal aspiration, as described previously (Gabazza et al. 2004, Wu et al. 2015). The technique of pharyngeal aspiration involved placement of the nanoparticle suspension on the back of the tongue followed by pulling of the tongue to induce a reflex gasp with resultant aspiration of the droplets. At 24 h after administration, the mice were euthanized by intraperitoneal injection of pentobarbital. BAL was performed by cannulation of the trachea with 18-gauge needle, and infusion and collection of 5/6 mL of saline was repeated six times.
Total and differential cell counts in BALF
The recovered BALF was centrifuged (1,500 rpm, 5 min, 4 °C), and the cell pellet was mixed with 1 mL of ACK lysing buffer (Gibco-Thermo Fischer Scientific, Waltham, MA) for hemolysis. Ten mL of Dulbecco’s phosphate buffer saline (DPBS) was added and centrifuged at 1,500 rpm and 4 °C. The resultant pellet was re-suspended in DPBS for total and differential cell counts. The total cell count was measured using hematocell counter. Aliquots of 5 × 104 cells in 400 µl DPBS per slide were prepared for cytospins. The cell mixture was added to EZ Single Cytofunnel®(Thermo, UK and centrifuged for 10 min at 1,000 rpm with Cytospin, using cytoslides. The slides were dried overnight at room temperature and then stained with the Differential Quik Stain Kit (Modified Giemsa, Sysmex Co., Kobe, Japan) for differential cell count in 10 fields (20 x magnification) of each slide.
Fluorescence immunocytology
A slide obtained by Cytospin was washed in DPBS three times and incubated with blocking agent (1% BSA) for one hour. The slide was further incubated with Biotin anti-Ly6G and Ly6C (Gr-1) (BD, Franklin Lakes, NJ), which was diluted 400 folds in 1% BSA, for one hour at room temperature, washed in DPBS three times and then incubated with 200-fold diluted Cy5 streptavisin (BioLegend, San Diego, CA) for 30 min at room temperature to stain the neutrophils. Ly6G and Ly6C (Gr-1) were used as markers of neutrophils, as their expression levels are known to correlate with differentiation and maturation of granulocytes [32, 33] and are only expressed transiently on bone marrow cells in the monocyte lineage [33]. The slides were counterstained with Hochest33342 for 10 min at room temperature and enclosed with Fluorescent Mounting Media (Dako, Agilent, Santa Clara, CA). Cells and SiO2 NPs were observed with confocal microscope Fv10i (Olympus, Tokyo)
Cell culture
Murine macrophages RAW 264.7 cell were kindly provided by Prof. Kenneth Dawson, University of College Dublin and grown in Dulbecco’s Modified Eagle Medium (DMEM, high glucose) with L-glutamine and phenol red (Wako, Cat. #044-29765) supplemented with 1 mM sodium pyruvate (100 mM, Gibco, Cat. #11360-070), 100 U/mL penicillin, 100 mg/mL streptomycin and 250 ng/mL amphotericin B (anti-anti, Gibco, Cat #15240-062), 2 mM glutamine and 10% FBS. All experiments were performed with cells from passages 4 to 15. Cells were grown in T25-flasks (Violamo, AS ONE Co., Osaka) in monolayers. Exponentially growing cells were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C and were passaged once every two days using a cell scraper (NEST 100071).
NP treatment
Depending on the experiment and after reaching 70–80% confluence, the cells were seeded onto appropriate cell culture plates and treated with silica nanoparticles of different surface modification. Before exposure to NP, the cells were rinsed with PBS to eliminate trace amounts of FBS. Treatments were performed under FBS-free condition for two reasons: 1) serum is reported to modulate NP uptake [34], and 2) to mimic in vivo condition whereby bronchial cells are not directly exposed to serum proteins. Stock of 30 nm rhodamine-labeled silica nanoparticles (25 mg/mL in water) was vortexed shortly before the preparation of the final dilution for the treatment.
MTS assay
MTS assay was conducted using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as described previously [35] and the instructions provided by the manufacturer. Briefly, RAW264.7 cells were seeded at 1.5 × 104 cells/well onto 96-well plates and incubated at 37 ºC in a humidified atmosphere of 5% CO2 and 95% air for 24 hours. After incubation, the cell culture medium was removed from each well with a multichannel pipette, and the cells were washed three times with DPBS to remove FBS. The cells were incubated for 4 or 24 hours with the three types of silica nanoparticles dispersed in FBS-free cell culture medium at a final concentration ranging from 0.3 to 30 µg/cm2. After incubation with the silica nanoparticles, the cells were washed twice with 1xDPBS, and incubated with MTS reagent (1.4 mL CellTiter 96® AQueous One Solution Reagent and 7.1 mL of complete phenol red-free cell culture medium with FBS). Cellular viability was determined by measuring absorbance at 490 nm, which reflected the reduction of {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium} (MTS) to formazan by mitochondria in viable cells.
Confocal microscopy
Cells were seeded in 8 well Lab-TekIITM chambered cover glasses (Nunc, Thermo Scientific, Dominique Dutscher, Brumath, France) at 16,590 cells/well in complete cell culture medium, and then incubated for 24 h. Upon 70–80% confluence, the cells were washed three times with DPBS to remove FBS and treated with 0.3 mL/well of NPs at pre-selected concentrations for 24 h at 37 °C and 5% CO2. After removal of medium, the cells were washed one time and fixed in 4% PFA for 20 min at 25 °C, rinsed twice with DPBS and then incubated with Cell Mask Green Plasma Membrane Stain (C37608 Thermo Fisher Science, Waltham, MA) and Hoechst for 10 min. The cells were washed with DPBS twice. After embedding the cells in Mounting Medium, they were observed by confocal microscopy (model FV.10, Olympus, Tokyo). The maximum concentration of silica nanoparticles 35.2 × 105 µg/cell (8.40 µg/cm2) was determined to be equivalent to exposure level of 40 µg/mouse in vivo, given that the average number of macrophages collected in BALF was 1.14 × 105 cells/mouse.
Flow cytometry
RAW264.7 cells were seeded onto 6-well plates at 2.37 × 104 cells/cm2 in complete cell culture medium and incubated for 24 h before treatment. After treatment with 3.0 mL/well of silica NPs at the preselected concentrations for 1 or 4 h in dark, the medium was removed, cultures were thoroughly washed with PBS three times and treated with 0.1% trypan blue for 1 min to quench the fluorescence of rhodamine on the cell surface. The concentration of trypan blue for quenching the fluorescence of rhodamine was determined beforehand by plot of trypan blue and rhodamine intensity in RAW264.7 cells exposed to bare rhodamine-labeled SiO2 NPs (Supplementary Fig. 2). The cells were washed with PBS, mixed with 500 µL of FACS buffer (PBS containing 0.5% FBS and 0.1% NaN3) and harvested by cell scraper. Cell-associated fluorescence was detected using FACSCalibur™ and results were analyzed with FlowJo software (BD, Franklin Lakes, NJ). The Mean Fluorescence Intensity (MFI) of rhodamine (excitation 488 nm, Filter range 564–606 nm) from three different size fractions indicated by the forward scatter (FS) was shown by the flow cytometer. The results are reported as the median of the distribution of cell fluorescence intensity obtained by analyzing cells in the gate. To adjust the differences in the fluorescence intensity relative to weight, the intensity of rhodamine fluorescence was measured at different concentrations of three types of SiO2 NPs using ARVOMx-fla system (485 nm/535 nm 1.0 s).
LDH cytotoxicity assay
LDH cytotoxicity assay was conducted using Pierce LDH cytotoxicity assay kit following the instructions provided by the manufacturer (Thermo Fisher Scientific). Briefly, RAW264.1 cells were plated at 104 cells/well in 100 µl of medium in a 96-well tissue culture plate. To minimize the cytotoxic effect of lack of FBS, DMEM was replaced by Opti-MEM for LDH cytotoxicity assay and further investigation of the effect of pan-caspase or necroptosis inhibitor. After incubation at 37 ºC under 5% CO2 for 24 hours, the cells were exposed to bare, carboxyl-functionalized, amino-functionalized rhodamine-labeled SiO2 NPs at 5.85 µg /cm2 (19.5 µg/mL). After the exposure for 1, 4, 12, 24, 36 and 48 hours, the supernatant of the culture medium was collected by centrifugation, and dispensed at 50 µl/well into another 96-well plate. Each cell was incubated in the presence of 50 µl of LDH reaction mixture at room temperature for 30 min in dark. The reaction was stopped by adding 50 µl of stop solution, and absorbance at 490 and 680 nm was read by Plate reader Gen5 (BioBik, Osaka, Japan).
Evaluation of effects of caspase inhibitor Z-VAD-FMK
Z-VAD-FMK, {Benzyloxycarbonyl-L-valyl-L-alanyl-[(2S)-2-amino-3-(methoxycarbonyl)propionyl]}fluoromethane, cell-permeable pan-caspase inhibitor was dissolved at 20 mM in DMSO and diluted to 20 or 40 µM by culture medium Opti-MEM® | Reduced Serum Media (Thermo Fisher Scientific). RAW264.1 cells were preincubated with Z-VAD-FMK at 20 or 40 µM or vehicle for one hour, and exposed to plain rhodamine-labeled silica nanoparticles at 28 µg/mL for 18 hours in Opti-MEM®. Cytotoxicity was evaluated using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as mentioned above.
Evaluation of effects of necroptosis inhibitor necrostatin-1
Necrostatin-1 or necrostatin-1 inactive control (Cayman Chemical, Ann Arbor, MI) was dissolved in 20 mM in DMSO and diluted to 10 or 20 µM by culture medium Opti-MEM® | Reduced Serum Media (Thermo Fisher Scientific). Raw264.1 cells were preincubated with Z-VAD-FMK at 10 or 20 µM or vehicle for one hour, and exposed to plain rhodamine-labeled silica nanoparticles at 8.4 µg/cm2 (28 µg/mL) for 18 hours in Opti-MEM®. Cytotoxicity was evaluated using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as mentioned above.
Quantitative real time PCR
RAW264.1 cells were seeded onto 6-well plates at 23,700 cells/cm2 and exposed to silica NPs at the concentration of 0.33, 1.67 and 8.37 µg/cm2 (1.06, 5.30, and 26.50 µg/mL, respectively). The cells were collected by centrifugation at 1,000 rpm for 5 min at 4ºC. Total RNA from the cells was isolated by using ReliaPrepTM RNA Cell Miniprep System (Promega, Fitchburg, WI). The concentration of total RNA was quantified by spectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE). RNA was reverse transcribed to single-strand cDNA using SuperScript III First-Strand Synthesis System for RT-PCXR (Life Technologies). The cDNA was subjected to quantitative PCR analysis with Thunderbird cyber green master mix (TOYOBO, Osaka, Japan) and primers designed by TAKARA (Kusatsu, Japan). The primers are 5’-GTCCCTCAACGGAAGAACCAA-3’ (forward) and 5’-TCTCAGACAGCGAGGCACAT-3’ (reverse) for MIP-2, 5’-GATCGGTCCCCAAAGGGATG-3’ (forward) and 5’-GTGGTTTGTGAGTGTGAGGGT-3’ (reverse) for TNFα and 5’-GATCATTGCTCCTCCTGAGC-3’ (forward) and 5’-ACTCCTGCTTGCTGATCCA-3’ (reverse) for β-actin.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). Differences with the control or between groups were analyzed respectively by Dunnett’s or Tukey’s multiple comparison following one-way ANOVA. A probability (p) of < 0.05 denoted the presence of a statistically significant difference. For the analysis of relative fluorescence intensity among different types of particles, regression lines were obtained by forcing the intercept to zero using Excel 2016 (Microsoft, Redmond, WA). All statistical analyses were performed using JMP (version 14, SAS Institute, Cary, NC).