Oxidative and Toxicological Evolution of Engineered Nanoparticles

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Environmental Processes

Oxidative and Toxicological Evolution of Engineered Nanoparticles with Atmospherically Relevant Coatings Qifan Liu, John Liggio, Dalibor Breznan, Errol M. Thomson, Premkumari Kumarathasan, Renaud Vincent, Kun Li, and Shao-Meng Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06879 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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Environmental Science & Technology

Oxidative and Toxicological Evolution of Engineered Nanoparticles with Atmospherically Relevant Coatings Qifan Liu,† John Liggio,*,† Dalibor Breznan,‡ Errol M. Thomson,‡ Premkumari Kumarathasan,*,§ Renaud Vincent,‡ Kun Li,† and Shao-Meng Li† †

Atmospheric Science and Technology Directorate, Science and Technology Branch,

Environment Canada, 4905 Dufferin Street, Toronto, Ontario M3H 5T4, Canada ‡

Inhalation Toxicology Laboratory, Healthy Environments and Consumer Safety Branch, Health Canada, 0803C Tunney's Pasture, Ottawa, Ontario K1A 0K9, Canada §

Analytical Biochemistry and Proteomics Laboratory, Healthy Environments and

Consumer Safety Branch, Health Canada, 0803C Tunney's Pasture, Ottawa, Ontario K1A 0K9, Canada *Corresponding

authors.

e-mail: [email protected] [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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The health impacts associated with engineered nanoparticles (ENPs) released into the atmosphere have not been adequately assessed. Such impacts could potentially arise from the toxicity associated with condensable atmospheric secondary organic material (SOM), or changes in the SOM composition induced by ENPs. Here, these possibilities are evaluated by investigating the oxidative and toxicological evolution of TiO2 and SiO2 nanoparticles which have been coated with SOM from the O3 or .OH initiated oxidation of α-pinene. It was found that pristine SiO2 particles were significantly more cytotoxic compared to pristine TiO2 particles. TiO2 in the dark or under UV irradiation catalytically reacted with the SOM, increasing its O/C by up to 55% over photochemically inert SiO2 while having negligible effects on the overall cytotoxicity. Conversely, the cytotoxicity associated with SiO2 coated with SOM was markedly suppressed (by a factor of 9, at the highest exposure dose) with both increased SOM coating thickness and increased photo-chemical aging. These suppressing effects (organic coating and photo-oxidation of organics) were attributed to a physical hindrance of SiO2-cell interactions by the SOM and enhanced SOM viscosity and hydrophilicity with continued photo-oxidation, respectively. These findings highlight the importance of atmospheric processes in altering the cytotoxicity of ENPs.

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INTRODUCTION

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Engineered nanoparticles (ENPs) are used extensively in a vast range of consumer goods and industrial processes due to their novel physical, chemical, and biological properties.1 In particular, nano-sized titanium dioxide and nano-sized silicon dioxide (denoted TiO2 and SiO2 respectively) are the two most commonly manufactured and frequently used ENPs globally.2 TiO2 is well-known as a semiconductor material used in products such as solar cells,3 coatings,4 sunscreens,5 and water purification agents.6 SiO2 is widely used in chemical-mechanical polishing7 and as an additive to cosmetics,1 pharmaceuticals,8 and toothpaste.9 The estimated global annual production of nanoTiO2 and SiO2 is 83,500–88,000 and 82,500–95,000 tons in 2010 respectively,10 and is expected to increase into the foreseeable future.11

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ENPs are likely to be released into the environment during their manufacture, packaging, from accidental spills, and from the degradation of products in which they are contained.2 Given the global widespread use of TiO2 and SiO2, their release into the environment could pose a significant risk to humans, particularly since a number of studies have demonstrated their toxicity.12-15 For example, subacute exposure of mice to 2–5 nm TiO2 nanoparticles caused a significant lung inflammatory response within


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the first two weeks of exposure.13 In addition, a significant decrease in cell viability was observed for A549 human lung cancer cells exposed to 46 nm SiO2 nanoparticles.14 Instillation of amorphous SiO2 nanoparticles caused significant inflammatory changes in rat lungs.15 Together, these results suggest that TiO2 and SiO2 are capable of generating adverse health outcomes on their own, due to their pro-oxidative and proinflammatory properties. However, ENPs including TiO2 and SiO2 are not likely to remain physically or chemically unchanged upon entering various environmental media. Indeed, changes in the surface characteristics of ENP have been observed in soil and aquatic environments.16,17 In the atmosphere, the role of ENPs may be two-fold; they (TiO2) can potentially serve as a photo-initiator and/or reactive site for free-radical mediated reactions of gaseous species,18 converting gases to particulate products, or they (TiO2 and SiO2) may act as seeds for the condensation of low volatility products originating from the gaseous oxidation of various volatile organic compounds (VOCs) present in the atmosphere.19 Ultimately, both processes are likely to result in ENPs that are coated and/or internally mixed with secondary organic material (SOM). With continued atmospheric aging, the interaction between the underlying nanoparticle and the condensed SOM may profoundly alter the physical and chemical properties of both the nanoparticle core and/or the overlying SOM. The association between nanoparticle physiochemical properties and various biological endpoints20 as well as the observation that secondary organic aerosols (SOA) alone can induce a cellular response21,22 suggests that the interaction between nanoparticle and SOM coatings has the potential to alter the toxicological effects associated with particles containing both. While limited evidence of this effect exists within soil/aquatic ecosystems,23,24 no information exists on the impact of atmospheric aging and transformations of TiO2 and SiO2, despite inhalation from ambient air being recognized as an important future ENP exposure pathway.25 In a previous study, the adsorption of organic gases onto single wall carbon nanotubes (SWCNTs) during ambient air exposures was suggested as the cause of an observed reduction in the overall SWCNTs cytotoxicity.26 However, the exact chemical composition, precursor source, atmospheric age, and the amount of adsorbed materials onto those SWCNTs was not determined. Such findings highlight a need to systematically study the relationship between the amount and nature of atmospherically relevant organic coatings on more common ENPs and their potential for corresponding air quality and health impacts. In the current study, TiO2 and SiO2 nanoparticles were systematically coated by SOM from the reactions of α-pinene with O3 or .OH radicals, simulating an exposure equivalent to photo-chemical aging times of 0.8–8.0 days in the atmosphere.



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Monitoring of the SOM oxidative evolution in these experiments was combined with simultaneous in vitro cytotoxicity analysis, across a range of SOM coating thickness and photo-chemical age. The results here provide a first insight into the impact of TiO2 and SiO2 exposed to the atmosphere, on both the oxidative evolution of the overlying SOM, and the corresponding effect on cellular toxicity endpoints. The present findings can advance the understanding on atmospherically transformed nanoparticles of all types, in support of associated risk assessment efforts.

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EXPERIMENTAL METHODS

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O3 Oxidation Experiments

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The O3 oxidation of α-pinene was performed in a dark flow tube reactor in the presence of pre-existing TiO2 or SiO2 using an experimental approach described previously26 and in the Supporting Information (SI; Figure S1A). TiO2 (Sigma-Aldrich, #677469) and SiO2 (US Research Nanomaterials Inc., #3438) nanoparticles were suspended in ultrapure water at a concentration of 1.5 g L−1, followed by 20−40 minutes of sonication. The particles used in the experiments were generated by atomization of the above solution (TSI, model 3076), dried through a diffusion dryer (TSI, model 3062), and exposed to α-pinene and O3 (Ozone Solutions, model TG-10). The relatively monodisperse TiO2 and SiO2 seed particles were generated with similar size distributions (Figure S2), having mode mobility diameters of approximately 120 nm and 150 nm, respectively. Gaseous α-pinene (70 ppb) was produced by passing a gas flow of 20 mL min−1 over a permeation tube in a temperature-controlled oven. The O3 exposure was converted to an equivalent atmospheric exposure representing 0.5−8.0 days by assuming an average tropospheric O3 concentration of 30 ppb.27 Gas-phase αpinene and O3 concentrations were monitored using a high-resolution time-of-flight proton transfer reaction mass spectrometer (HR-ToF-PTR-MS, Ionicon Analytik) and an O3 analyzer (2B Technologies, model 202), respectively. Particle size distributions were measured using a scanning mobility particle sizer (SMPS). An Aerodyne HRToF-AMS28 was used to determine the bulk chemical composition of the organic fraction of the coated ENPs, including the elemental ratios of H/C and O/C as described previously.29 A shift in the unimodal particle size distribution upon the addition of SOM demonstrated that the nanoparticles were approximately evenly coated with negligible new particle formation (Figure S3). Assuming the nanoparticles were evenly coated resulted in a calculated coating thickness ranging from 4 to 20 nm (achieved by varying the α-pinene concentration). SOM coated ENPs from each experiment were collected on 47 mm Teflon filters at the exit of the reactor for subsequent cytotoxicity analysis (Table S2). The specific types of ENP used in this work are provided in Table S1.

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.OH

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The TiO2 and SiO2 particles generated above were also exposed to SOM produced from the photo-oxidation of α-pinene by .OH radicals in a manner described previously26,30 and in Figure S1B. .OH radicals were generated by the UV photolysis of O3 at 254 nm in the presence of water vapor. Upon achieving a steady-state concentration of O3 and ENPs, gaseous α-pinene (18 ppb) was introduced into the photo-chemical reactor. In offline calibrations, the .OH radical concentration was systematically varied by changing the voltages applied to the UV lamp (Jelight) between 0 and 120 V, and was determined using CO as a tracer compound (see SI). The .OH exposures, quantified by measuring the loss of CO via its reaction with .OH (1.54×10−13 cm3 molecules–1 s–1 at 298 K),31 were in the range of 1.0×1011–1.0×1012 molecules cm–3 s. Assuming an average atmospheric .OH concentration of 1.5×106 molecules cm−3,32 this experimental exposure is equivalent to 0.8–8.0 days of atmospheric photo-chemical oxidation.33 The resultant ENPs coated with SOM (1.5–7.5 nm coating thickness) were also analyzed for their cytotoxicity (Table S2).

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Cell Culture and Cytotoxicity Assays

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J774A.1 murine macrophage cells (ATCC) were maintained as previously described.34 For experiments, J774A.1 cells were seeded in 96-well plates (~0.32 cm2 surface area) at 20,000 cells well–1, in 100 µL DMEM (phenol red-free (Hyclone)), with 10% heatinactivated FBS (Hyclone), 50 µg mL–1 gentamicin (Sigma-Aldrich) and incubated for 24 h prior to particle exposure. Stock suspensions of ENPs were prepared at 1 mg mL–1 in serum-free DMEM (containing 50 µg mL–1 gentamicin), briefly vortexed, and sonicated for 5 min using a Branson 2510 water bath sonicator with ice present. The particle stocks were prepared newly for each experimental repeat. Particle standards EHC6802 (Ottawa urban dust), Si12 (Sigma-Aldrich, 16±3.1 nm SiO2) and Aeroxide P25 (Evonik Industries, 21±5 nm TiO2) were included in the assays.

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Radical Oxidation Experiments

For cell dosing, working ENP suspensions were diluted in DMEM as required, sonicated for an additional 2 min, briefly vortexed, and dosed to cell monolayers at 10, 30 and 100 µg cm–2 of well surface area (96-well plate) in 100 µL of DMEM +10% FBS. The equivalent exposure concentration was 16, 50 and 160 µg mL–1 of DMEM +5% FBS (final conc.). The cells were incubated (37 °C, 5% CO2, 95% RH) for 24 h prior to the integrated cytotoxicity assay. “No-cells” wells containing particle suspensions only were included in the experiments to test for potential nanoparticle interference with the assays. Furthermore, supernatants and cell lysates utilized for the cytotoxicity analysis were clarified by centrifugation to remove any traces of ENPs.



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The integrated cytotoxicity assay was conducted to obtain the cytotoxicity profile of the J774A.1 cells exposed to the ENPs. The assay tested for cell membrane integrity (LDH release, CytoTox 96, Promega), energy metabolism (cellular ATP, ViaLight Plus, Lonza), and redox state (resazurin reduction, Alamar Blue, Invitrogen), from the same exposure experiment. The integrated cytotoxicity assay was performed as previously described.35 All experiments were conducted three times (n=3) with duplicate samples per experiment. The cytotoxicity data were normalized within an experiment for all doses (including zero dose control), to the grand mean value of all zero dose controls, to obtain fold effect (FE) for each particle dose. These data were also analyzed using an independent sample t-test and a two-way ANOVA (SPSS Statistics) with dose and coating thickness or dose and photo-chemical age as factors, as given in Table S2 and S3.

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RESULTS AND DISCUSSION

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Impact of ENP seed on SOM: O3 Oxidation of α-pinene

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While the HR-ToF-AMS is not able to detect or quantify the underlying ENP core, its utility is in the ability to characterize the oxidation state of the SOM in real-time as a measure of the oxidative evolution. This is typically achieved through the analysis of various fragment ratios and the direct determination of the O/C ratios.36,37 For example, the measured fraction of the total AMS organic signal contributed by C2H3O+ (fC2H3O+) and CO2+ (fCO2+) ion is often used as a measure of the oxidation state of SOA.37 These metrics are similarly used here to provide insight into the effect of the underlying ENP seed on the oxidation state of α-pinene SOM condensed onto the surface of TiO2 (denoted SOMTi) and SiO2 (denoted SOMSi). It is important to evaluate the ability of an ENP core to alter the oxidation state of SOM during atmospheric aging processes, as increased organic oxygenation has been linked to changes in the organic aerosol toxicity,38 and has a significant impact on climate and air quality by strongly affecting the phase state, optical properties, hygroscopic growth, and cloud condensation nucleus (CCN) activity of aerosol particles.39-41

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The evolution of fC2H3O+ and fCO2+ for SOMTi and SOMSi as a function of O3 exposure is shown in Figure 1A. For both types of ENP, increased O3 exposure, equivalent to increased aging time in the atmosphere, results in a decrease in fC2H3O+ and increase in fCO2+ respectively, reaching a constant fraction at an O3 exposure of 2.8×1017 molecules cm–3 s (approximately equivalent to 4 days of ambient exposure). The C2H3O+ ion is linked to non-acid containing oxygenates (alcohols, aldehydes, ketones), while CO2+ ion is predominantly due to the presence of acidic groups.36 The decrease in fC2H3O+ and corresponding increase in fCO2+ at low O3 exposures (0.5–2.0 days) is consistent with a


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conversion of oxidation products containing carbonyl functional groups (higher fC2H3O+) to products containing carboxylic acid groups (higher fCO2+ and lower fC2H3O+) and thus a higher overall oxygen content of the SOM. For example, campholenic aldehyde, a known α-pinene ozonolysis product which contains a double bond, can undergo further reaction with O3 to form terpenylic aldehyde, which can be further oxidized in the particle phase to form terpenylic acid.42 This is also reflected in the O/C evolution for these experiments shown in Figure 1B which varies from (0.39±0.01) to (0.42±0.01) depending upon the exposure time and seed type and is consistent with literature values (0.33–0.52) of SOA generated from the dark ozonolysis of α-pinene at an O3 exposure of 1.4 days.43 Given the absence of NOx in these experiments which serves to propagate radical production and enhance secondary chemistry, once all α-pinene and oxidation products containing double bonds are consumed, it is expected that the O/C should remain relatively constant at high O3 exposures (4–8 days; Figure 1B).

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With the exception of low photo-chemical age experiments (0.8 days .OH exposure), the type of seed particle has a negligible effect on overall SOM formation yields from O3 or .OH oxidation (see Figure S4 & SI). However, there are observable differences in the degree of SOM oxidation between experiments of different particle core types. Figure 1B indicates that the presence of TiO2 (in O3 experiments) slightly increases the oxygen content of the SOM compared to the SOM formed in the presence of photochemically inactive SiO2. This is attributed to dark reactions which occur on the surface of TiO2. Although the majority of previous studies have focused on the photo-activity of TiO2,3-6,18,44 limited data suggests that organic oxidation induced by TiO2 in the dark is also possible, particularly for carbonyl containing species.45-47 For example, exposure of gaseous methacrolein (MAC) to TiO2 under dark conditions resulted in the oxidation of MAC to carboxylate on the TiO2 surface,47 while MAC was only physically adsorbed onto SiO2.48 It was hypothesized that the reactivity of TiO2 towards MAC was caused by oxygen vacancies on the TiO2 surface, which were often involved in the catalytic reactivity of TiO2.44 Similar dark reactions were also observed during the heterogeneous interaction between gaseous aldehydes (HCHO and CH3CHO) and TiO2, where the oxidative adsorption converted aldehyde to carboxylate.45,46 The propensity of carbonyl oxidation to occur on the surface of TiO2 described above is consistent with the slight increase in O/C for the SOM studied here. This is particularly relevant for αpinene SOM formed through O3 oxidation, as it is known to contain significant carbonyl functionality.49

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Impact of ENP seed on SOM: .OH Oxidation of α-pinene

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The oxidative evolution of TiO2 and SiO2 seeded SOM formed from the .OH initiated photo-oxidation of α-pinene is shown in Figure 2A, and B. Similar to the results for

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SOM formed from O3 oxidation (Figure 1A), the fCO2+ generally increases and fC2H3O+ decreases as a result of increased photo-chemical aging (see Figure 2A). The relative change of fCO2+ in .OH experiments (101–276%) is significantly higher compared to the O3 experiments (10–12%), since .OH can easily induce further oxidation by converting first-generation α-pinene product to later-generation product; while O3 has a much lower reactivity, it can only react with limited products which contain a double bond (e.g. campholenic aldehyde42). During the initial .OH exposure, the measured photochemical age profile for fC2H3O+ of SOMSi and SOMTi follow different trends, with the fC2H3O+ of SOMSi initially increasing whereas the fC2H3O+ of SOMTi begins at a higher relative fraction and continually decreases with photo-oxidation. This suggests a progression from less oxidized species in SOMSi with a higher contribution to fC2H3O+, to more oxidized products with a lower contribution to fC2H3O+ as has been noted during the formation of α-pinene derived SOA.33 A similar overall oxidative evolution to that of O3 experiments (i.e. O/C, Figure 1B) is shown in Figure 2B for .OH initiated SOM formation. In this case, the O/C of the SOM are in the range of 0.43–1.20 at .OH exposures of 1.0×1011–1.0×1012 molecules cm–3 s (0.8–8.0 days), with an O/C at 0.8 days of .OH exposure similar to that of SOA from α-pinene photo-oxidation in smog chambers.50 The higher range of O/C for .OH initiated experiments (for both particle seed types) compared to O3 experiments is consistent with the formation of latergeneration SOM products which can be easily achieved in the presence of .OH but not O3, as mentioned above. The difference in oxygen content between SOMTi and SOMSi from .OH initiated oxidation is significant. Under otherwise identical conditions, the O/C for SOMTi at a given .OH exposure is consistently higher (11–55 %) than that of SOMSi (Figure 2C), which is also reflected in the fCO2+ curvature of Figure 2A. A higher oxygenation level for SOMTi can be attributed to the unique photo-chemical surface reactivity of TiO2, which is known to participate in interfacial photo-catalytic reactions,44 while a SiO2 surface is chemically inert. More specifically, TiO2 irradiated by UV light (λ

Oxidative and Toxicological Evolution of Engineered Nanoparticles

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