A panel of five SiO2 NPs particles was produced by FSP as previously reported in the literature [18, 42]. Briefly, a precursor solution of hexamethyldisiloxane (HMDSO, puriss, #98.5%) in ethanol (puriss, #98.5%) at various Si concentration was prepared. The solution was fed with a syringe pump in a metal capillary with a predetermined rate (x ml/min) where it was dispersed by an oxygen flow (y l/min) that had a 1.5&#;bar pressure drop at the nozzle tip. The aerosolized precursor was then ignited with a supporting flame of premixed 1.5&#;L/min CH4/3.2&#;L/min O2. The produced heat converts the precursor to the metal oxide. The ratio x/y determines physicochemical properties such as the primary particle size and silanol content.
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In addition commercially available fumed SiO2 NPs (Aerosil® 200) and WetChem SiO2 NPs (porous SiO2 NPs mostly synthesized by wet-chemistry approaches) were obtained as benchmark materials from Evonik Industries and Sigma Aldrich (product # ), respectively, and were hereafter denoted as Commercial fumed SiO2 and WetChem SiO2.
For each flame the ratio of the combustion enthalpy introduced by precursor, solvent and methane into the flame (\( \dot{\mathrm{H}} \) C in MJ/min) over the total (liquid and gas) inlet mass flow rate, mtot,in (kg/min) was calculated, based on Spyrogianni et al. [18] as follows:
$$ \frac{{\dot{\mathrm{H}}}_{\mathrm{C}}}{{\dot{\mathrm{m}}}_{\mathrm{tot},\mathrm{in}}}=-\frac{\left({\dot{\mathrm{n}}}_{\mathrm{precursor}}\Delta {\mathrm{H}}_{\mathrm{C}}^{\mathrm{precursor}}+{\dot{\mathrm{n}}}_{\mathrm{solvent}}\Delta {\mathrm{H}}_{\mathrm{C}}^{\mathrm{solvent}}+{\dot{\mathrm{n}}}_{\mathrm{C}{\mathrm{H}}_4}\Delta {\mathrm{H}}_{\mathrm{C}}^{\mathrm{C}{\mathrm{H}}_4}\right)}{{\dot{\mathrm{m}}}_{\mathrm{precursor}-\mathrm{solution}+}{\dot{\mathrm{m}}}_{\mathrm{dispersion}-{\mathrm{O}}_2+}{\dot{\mathrm{m}}}_{\mathrm{flame}-{\mathrm{C}\mathrm{H}}_4+}{\dot{\mathrm{m}}}_{\mathrm{flame}-{\mathrm{O}}_2}} $$
where \( \dot{\mathrm{n}} \) and \( \dot{\mathrm{m}} \) are the inlet flow rates in mol/min and kg/min, respectively, and ΔHC (MJ/mol) is the combustion enthalpy of each compound. ΔHC was calculated assuming complete combustion using the reactant and product enthalpies of formation (at 25&#;°C) from Iseard et al. [43] for HMDSO and from Haynes [44] for ethanol, CH4, CO2 and H2O. SiO2 NPs from low enthalpy flames (<&#;11&#;MJ/kg) are referred to as &#;cold SiO2 NPs&#; (FSP SiO2 5/5, FSP SiO2 3/5, FSP SiO2 4/5) while high enthalpy (>&#;15&#;MJ/kg) SiO2 NPs are reported as &#;hot SiO2 NPs&#; (FSP SiO2 9/3, FSP SiO2 11/3, commercial fumed SiO2). The commercial WetChem SiO2 NPs is considered as cold silica since there is no heat production in the synthesis process.
Transmission electron microscopy (TEM, FEI Tecnai F30 ST microscope operated at 300&#;kV) was used to ascertain the morphology of different types of SiO2 NPs. The powders were dispersed in ethanol at 100&#;μg/ml with cup-horn sonication at 100&#;kJ/L energy (95% amplitude, 30s on, 1&#;s off) [18] and deposited onto a perforated carbon foil supported by a copper grid. A similar sample preparation procedure was used to analyze the morphology of FSP SiO2 5/5 via a TEM, JEOL [42].
Brunauer-Emmett-Teller (BET) N2-adsorption at 77&#;K was conducted to determine the specific surface area (SSA) of SiO2 NPs using a five-point BET isotherm ((TriStar II Plus, Micromeritics) after degassing the samples for &#;1&#;h at 140&#;°C) [10]. Similar protocols were used to determine the SSA of FSP SiO2 5/5 via a high-throughput surface area and pore-size analyzer (Quantachrome Instruments, NOVAtouch LX4). The equivalent BET particle size (dBET) was calculated assuming that the particles are spherical and of equal size, and following dBET&#;=&#;/(SSA·ρ), where the SSA is the specific surface in m2/g, ρ is the material density in g/cm3, and dBET is in nm [42].
SiO2 NPs density in powder form was measured using a pycnometer (Quantachrome Instruments, ULTRAPYC e). SiO2 NPs in powder form were used without any preparation. The sample volume was measured 15 times and the average value was used as the value of the density.
The total silanol content (surface and internal) of all samples but the FSP 5/5 SiO2 has been previously reported [18]. For the FSP 5/5 SiO2 NPs, the total silanol content was quantified by thermogravimetric analysis (TGA) with a thermobalance (TGA/SDTA 851e, Mettler Toledo), modifying a previously described procedure [18]. Briefly, slightly compacted SiO2 powder was filled into 900&#;μL alumina crucibles and a TGA method consisting of two steps was employed. In step 1, the samples were heated in 40&#;ml/min Ar from 40 to 140&#;°C at 5&#;°C/min and held at this temperature for 180&#;min. In step 2, the gas flow was changed to 40&#;ml/min O2 to allow for the oxidation of possible carbon-containing residues on the particle surface. The temperature was increased to 800&#;°C at 10&#;°C/min and held constant for 60&#;min. The mass loss during step 2 was used to calculate the number of silanol groups per surface area according to:
$$ \mathrm{OH}/{\mathrm{nm}}^2=2\left[\left({\mathrm{m}}_{140{}^{\circ}\mathrm{C}}-{\mathrm{m}}_{800{}^{\circ}\mathrm{C}}\right)\ {\mathrm{N}}_{\mathrm{A}}/{\mathrm{M}}_{\mathrm{H}2\mathrm{O}}\right]/\left[{10}^{18}\ \mathrm{SSA}\ {\mathrm{m}}_{140{}^{\circ}\mathrm{C}}\ \right]+1 $$
where m140 °C and m800 °C are the sample mass in grams at the beginning and at the end of step 2, SSA is in m2/g, MH2O (g/mol) is the molar mass of H2O and NA (#/mol) is Avogadro&#;s constant [45&#;48]. Furthermore, it is assumed that the silica surface is still covered with one hydroxyl group per nm2 at 800&#;°C [49].
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XPS was used to analyze the surface chemistry of the SiO2 NPs (quantification of the &#;Si&#;O&#;H, &#;O&#;Si&#;O&#;, and organic carbon/oxygen) and its stoichiometry. The NPs powders were used without any treatment. A double-sided carbon tape was used to fix the powders on the XPS plate. Approximately 100&#;mg of the ENM were pressed to create a small pellet that was placed on the carbon tape and pressed to adhere on the carbon tape. The Thermo Scientific K-Alpha XPS was used to perform XPS analysis. The survey range was set from &#;&#;10&#;eV to &#;eV with 200&#;eV pass energy and 400&#;μm spot size. Three different spots of the pellet were used for the survey. Once all the elements were identified, high-resolution elemental scans were performed for each element to minimize the Noise to Signal ratio (N2S) without saturating the detector. For the data analysis, the calculation software Avantage&#; Software (Thermo Scientific, Waltham, MA) was used. The XPS spectra were calibrated in respect to the Carbon 284.6&#;eV peak.
The dispersion preparation, colloidal characterization and dosimetric analysis were performed as described in great detail by the authors in previous publications [40, 42, 50, 51]. The cup horn sonicator (Branson Sonifier S-450D, 400&#;W, with Branson 3-in. cup horn) was calibrated according to the protocol by Taurozzi et al. [51] and found to deliver 2.59&#;W/ml. A stock solution of ENMs in distilled water (Invitrogen) was prepared at a concentration of 0.5&#;mg/ml and was used to determine the critical delivered sonication energy (DSEcr). One milliter of the stock solution was used to measure the hydrodynamic diameter (dH) with DLS (Malvern Nanosizer, Worcestershire, UK). The solution was sonicated for 1&#;min, vortexed for 30&#;s, and measured again. The process continued until the dH and polydispersity index (PDI) were not changing significantly (± 5%). The DSEcr of an ENM is defined as the DSE (in J/ml) required to achieve the lowest particle agglomeration state in DI H2O and is ENM-specific. Once the DSEcrt was determined, a fresh suspension was prepared, and it was diluted with RPMI+&#;10% (vol/vol) FBS to a final concentration of 0.1&#;mg/ml and its dH was measured with DLS at 0&#;h and 24&#;h to assess stability overtime. Further, the effective density (ρeff) was determined using the volumetric centrifugation method (VCM) as described previously [42].
The distorted grid (DG) model was utilized to calculate the concentration profiles across the well of a 96-well plate, the concentration at the bottom of the well (bottom concentration) and the fraction of deposited particles to the cell surface as a function of the exposure time (fD) for the SiO2 NP suspensions [51]. The developed code was executed on MATLAB (MathWorks, Massachusetts, USA). Inputs for the model were the agglomerate volume-weighted hydrodynamic diameter (dH) and ρ eff of SiO2 NPs agglomerates suspended in RPMI+&#;10% (vol/vol) FBS.
Acellular measurement of ROS generated by various silica NPs is based on the oxidation of Trolox (a water-soluble variant of Vitamin E) to Trolox quinone (TQ), followed by TQ quantitation with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The principle of the method is described in Zhao et al. [52]. The method quantifies the highly reactive radicals (such as hydroxyl radicals, superoxide anions, and singlet oxygen), as a group (fast-reacting ROS) as well as the stable hydrogen peroxide (H2O2) [53]. Notable advantages of this method over traditional fluorescin-based assays such as the DCFH assay are high sensitivity, interference-free measurements, and simultaneous quantitation of fast-reacting ROS and H2O2.
Different volumes of 1&#;mg/ml stock of SiO2 NPs (10, 25, 50, 100&#;μL) were added to 0.5&#;ml solution (containing 100&#;nmol) Trolox in 7&#;ml amber vials, and the volume was adjusted to a final 1&#;ml with phosphate buffer (50&#;μM, pH&#;7.4), to obtain a range of SiO2 NPs concentrations (10&#;μg/ml &#; 100&#;μg/ml) in 0.1&#;mM Trolox solution in pH&#;7.4 phosphate buffer. The above vials were placed in a Thermo Forma 420 shaker (Thermo Fisher Scientific, Waltham, MA, USA) at 37&#;°C and 50&#;rpm for 30&#;min. Then the samples were filtered through a 20&#;nm pore PTFE membrane filter (Whatman, 10&#;mm diameter) to remove SiO2 NPs. NP removal efficiency was confirmed via DLS and Tunable Resistive Pulse Sensing (TRPS) measurements of the filtrate showing no particles present. Two 0.4&#;ml aliquots of the filtrate Trolox solution were transferred into two separate 1.8&#;ml amber liquid chromatography vials. One unit of horseradish peroxidase (HRP) was added into one of the vials used for H2O2 quantification. The other vial was not modified. Subsequently, both vials were incubated at 37&#;°C for 30&#;min and then subjected to LC-ESI&#;MS/MS analysis for Trolox Quinone (TQ) quantitation.
Trolox and TQ, were analyzed by LC&#;ESI-MS/MS as described earlier [52, 54]. Electrospray ionization (ESI) was performed in the positive ion mode (ion spray voltage &#;V) with nitrogen as the nebulizing, heater, curtain, and collision gas. Gas flow parameters were optimized (nebulizer 65&#;psi, heater 50&#;psi, and curtain gas 30&#;psi) by making successive flow injections while introducing mobile phase into the ionization source at 600&#;μL/min. The turbo ion spray temperature was set to 500 ̊C. Quantitative analysis was performed in the multiple reaction monitoring (MRM) mode by monitoring the transition 267&#;221, with a dwell time of 500&#;ms. The following compound-specific parameters used were: Declustering potential DP, 48&#;V; collision energy, 21 (eV); and collision energy exit potential, 5. Chromatographic separation was achieved on a Kinetex C18 column, (4.6&#;×&#;100&#;mm, 2.6&#;μm particle size) (Phenomenex, Torrance, CA) at a flow rate of 600&#;μL/min and column temperature set at 40 ̊C. The isocratic separation was accomplished with 60% solvent A (0.1% ammonium acetate in water) and 40% solvent B (0.1% formic acid in methanol). Injection volume was 10&#;μL.
Fast reacting ROS species (such as hydroxyl and superoxide radicals), which have a short half-life in the milliseconds&#; range, were measured as the amount of TQ formed in the first vial [52]. H2O2, a stable product, does not react appreciably with Trolox under the current experimental conditions (verified experimentally), but in the presence of HRP it is converted to hydroxyl radical (1H2O2:1OH&#;) which oxidizes Trolox to TQ. The amount of TQ in vial #2 (with HRP) is the sum of fast-reacting ROS species and H2O2. H2O2 was calculated from the difference between TQ in the HRP containing vial (#2, total ROS) and TQ in vial #1 (fast-reacting ROS). This approach was validated independently by treating H2O2 containing samples and standards with catalase, an enzyme specialized in converting H2O2 into water, and then re-quantifying ROS. In the presence of catalase, the amount of H2O2 was reduced to zero.
RAW 264.7 cells, purchased from ATCC (ATCC, Rockville, MD), were grown as a monolayer using DMEM medium (Gibco-Life Technologies) supplemented with 10% heat-inactivated FBS, 100&#;IU/ml Penicillin, 100&#;μg/ml Streptomycin (Gibco-Life Technologies) and 1&#;mM HEPES (Gibco-Life Technologies). Normal small airway epithelial cells (SAEC) were purchased from Lonza (Walkersville, Maryland) and maintained in serum-free SABM with the following supplemental growth factors (Bovine Pituitary Extract, Hydrocortisone, Human Epidermal Growth Factor, Epinephrine, Transferrin, Insulin, Retinoic, Triiodothyronine, Gentamicin Amphotericin-B, and BSA-fatty acid free) provided by the manufacturer (Lonza Inc., Allendale, New Jersey). Both cell lines were cultured at 37&#;°C in a humidified 5% CO2 incubator and subcultured at 80% confluence.
For each experiment, cells were plated at 50,000 cells/well in a 96-well plate and allowed to fully attach for 24&#;h. After that, the medium was changed to RPMI 10% FBS and cells were treated with the different SiO2 NPs. Based on the dosimetry data obtained via the DG model [40, 51], the administered doses were chosen to yield the delivered-to-cell dose in terms of mass per surface (μg/cm2). The delivered-to-cell doses for all the materials at 24&#;h were 0.026, 0.052 and 0.104&#;μg/cm2. After 24&#;h of treatment, cells were analyzed for different toxicological endpoints.
After being exposed to the test particles for 24&#;h, cells were evaluated for cytotoxicity using the Pierce&#; LDH Cytotoxicity Assay Kit (Thermo Scientific, Waltham, MA, USA). LDH release, used as an indicator of cell membrane damage, was measured in the culture medium according to the manufacturer&#;s instruction. Briefly, 80&#;μl cell-free supernatants from the culture treatments were collected, and centrifuged at &#;rpm for 10&#;min; 50&#;μl of the media supernatant was then added to a fresh 96-well plate along with LDH assay mix reagent. After incubating for 30&#;min, the absorbance values were recorded at both 490&#;nm and 680&#;nm using SpectraMax M5/M5e (Molecular Devices, Sunnyvale, California). Maximum cellular LDH activity was measured in cell lysates obtained by treatment with Lysis buffer 1X solution. Data from control and treated cells were calculated as percent LDH leakage (100&#;×&#;LDH activity in medium/maximum LDH activity) and expressed as the mean, using triplicate wells per concentration. The same protocol was performed in parallel without seeding cells for checking possible interaction between NPs and the reagent.
PrestoBlue® (Thermo Fisher, USA) was used for cell viability measurements. This resazurin-based solution was used to quantify the reducing power of living cells as a cell health indicator. After 24&#;h treatment cells were washed twice with PBS 1X. Fresh media containing PrestoBlue 1X reagent was added to the cells and incubated at 37&#;°C for 10&#;min. Fluorescence was detected using excitation and emission pair of 560/590&#;nm using SpectraMax M5/M5e. Possible interaction of NPs with the reagent was evaluated as mentioned before.
The induction of oxidative stress was measured by using both CellRox Green (Invitrogen) and CM-H2DCFDA (Invitrogen) in separate experiments. After 24&#;h of treatment and followed by two washes with 1X PBS, CellRox Green was diluted to 10&#;μM in media without FBS, added to the cells and incubated at 37&#;°C for 30&#;mins. Two washes with 1X PBS were done before measuring fluorescence by SpectraMax M5/M5e, using excitation and emission pair of 485/520&#;nm. Again, media only and media with NPs were assessed to ensure interference-free measurements. For CM-H2DCFDA, after 24&#;h of cell seeding, the probe was diluted to 10&#;μM in RPMI media without FBS and added to the cells for 40 mins at 37&#;°C. After the incubation cells were washed twice with PBS and nanoparticle treatment was applied. Cell imaging was done at 24 and 72&#;h using InCell analyzer (GE Healthcare LifeSciences) in epifluorescence mode. Four different fields were acquired for each well. CM-H2DCFDA fluorescence (488/510&#;nm excitation/emission) was acquired at a laser power of 100% and exposure of 400&#;ms. Images were processed using FIJI software.
Results were expressed as mean&#;±&#;SD of three independent experiments. Data were analyzed using two-way analysis of variance (ANOVA) with Tukey&#;s multiple comparison test to determine statistical significance among treatments. In all cases p&#;<&#;0.05 was considered significant.