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Development of a Novel Shallow Liquid Interface Exposure System for MWCNT Toxicity Assessment Chi-Yu Tien, Jui-Ping Li, Ding Han, Ziyi Li, Pin-Kuei Fu, Jen-Kun Chen, and Chuen-Jinn Tsai Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00067 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 31, 2019

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Chemical Research in Toxicology

Development of a Novel Shallow Liquid Interface Exposure System for MWCNT Toxicity Assessment Chi-Yu Tien1, Jui-Ping Li2, Ding Han1, Ziyi Li3, Pin-Kuei Fu4,5,6, Jen-Kun Chen2*, Chuen-Jinn Tsai1* Authors’ E-mail address: 1Chi-Yu

Tien, Master: [email protected], ORCID: 0000-0001-6398-7816

2Jui-Ping 1Ding 3Ziyi

Li, Master: [email protected],

Han, Master: [email protected],

Li, Associate Professor: [email protected],

4,5,6Pin-Kuei

Fu, Attending Physician: [email protected], ORCID: 0000-0002-9416-4094

Corresponding Authors: 1*Chuen-Jinn

Tsai, Chair Professor: [email protected]; Tel.: +886-3-5731880; Fax: +886-35727835, ORCID: 0000-0002-0459-3232 2*Jen-Kun

Chen, Associate Principal Investigator: [email protected]; Tel.: +886-37-246166 ext. 38117; Fax: +886-37-586440, ORCID: 0000-0002-3433-5971

1Institute

of Environmental Engineering, National Chiao Tung University, 1001 University Road, Hsinchu 30010, Taiwan 2Institute

of Biomedical Engineering and Nanomedicine, National Health Research Institutes, 35 Keyan Road, Miaoli 35053, Taiwan 3School

of Energy and Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China 4Department

of Critical Care Medicine, Taichung Veterans General Hospital, Taichung 40705,

Taiwan 5College

of Human Science and Social Innovation, Hungkuang University, Taichung 43302, Taiwan

6College

of Science, Tunghai University, Taichung 40704, Taiwan 1 ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only

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Abstract Increasing applications of multi-walled carbon nanotube (MWCNT) lead to significant occupational exposure and potential health concern. Toxicity of MWCNT should be carefully elucidated since conventional (CON) method with fully immersed condition fails to mimic the airliquid interface (ALI) in airways. Additionally, quantification of MWCNT in cells was a real challenge. Currently available ALI exposure devices are costly, posing problems to conducting in vitro evaluations for emerging nanomaterials. A novel system, consisting of a shaker fluidized-bed atomizer (SFA) and electrostatic shallow liquid interface (ESLI) exposure chamber, has been developed for investigating nanotoxicity of well-dispersed pristine-MWCNT (pMWCNT) and carboxylizedMWCNT (cMWCNT). After 24-hr exposure, LDH, MCP-1, IL-1 , IL-6 and TNF- releases were determined, and cell uptakes were quantified according to the molybdenum content in cells. Biological responses triggered by SLI exposure are obviously more sensitive compared with those caused by CON exposure at equivalent doses. Exposure dose-dependent release of LDH and IL-6 was highlighted in A549 cells, indicating higher cytotoxicity and inflammatory responses of cMWCNT attributed to its shorter length, smaller size, and higher cell uptake. Cell-associated dose-dependent release of LDH and IL-6 was highlighted in RAW264.7 cells, revealing higher adverse health risk of pMWCNT due to frustrated phagocytosis and its much higher molybdenum content. These results suggest that inherent characteristics of cells and distinct physicochemical properties of pMWCNT and cMWCNT lead to either exposure dose-dependent or cell-associated dose-dependent responses. Notably, the SLI is superior to the CON exposure method and well suited for nanotoxicity assessment of different MWCNTs.

Keywords: pMWCNT; cMWCNT; shallow liquid interface (SLI); exposure dose-dependent; cellassociated dose-dependent.



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Introduction Carbon nanotubes (CNTs) have been widely used in industry due to their unique mechanical and electrical properties. Increasing applications of CNTs lead to potential occupational exposure,1-4 posing possible health risks to humans.5 Safety assessments are required to evaluate health risks due to long-term and/or high-level CNT exposure. Recommended exposure limits (RELs) to CNTs range widely from 0.7 to 50 g/m3 due to differences in physicochemical properties of tested CNTs and various methods.6 Among different CNTs evaluated, the multi-walled carbon nanotube-7 (MWCNT7; length, 1-19 m; diameter, 40-170 nm; MITSUI Hodogaya Chemical Co.) is categorized as “possibly carcinogenic to humans (group 2B)” by the International Agency for Research on Cancer, while other CNTs are deemed “not classifiable as to their carcinogenicity to humans (group 3)” due to lack of toxicity evidence.7 Moreover, size, surface area, agglomeration status, and impurities could influence judgment on the toxicity of CNTs evaluated.8-12 Chemical functionalization is considered a fundamental approach to enhancing stability and dispersibility of CNTs in various media, thus triggering different biological responses.13-16 MWCNTs with hydroxyl and carboxyl groups were shown to penetrate cell membranes more easily compared with pristine MWCNTs (pMWCNT).14,15 Some studies indicated higher toxic and inflammatory responses caused by functionalized MWCNTs rather than pMWCNT, whereas others reported lower toxicity in functionalized MWCNTs than in pMWCNT.12,17,18 Conflicting results might be attributable to poor accuracy of the conventional (CON) fully immersed method and variations of physicochemical properties of MWCNTs from different 4 ACS Paragon Plus Environment

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manufacturers. In CON methods, cells are completely covered by a thick liquid layer of culture medium for assessing nanotoxicity. When fully immersed, nanoparticles (NPs) aggregate or agglomerate to an unpredictable status; hence, CON methods fail to mimic real-life interactions between NPs in the respiratory system or between NPs and cells at the air-liquid interface (ALI).19 On the other hand, ALI exposure methods have been developed 19-22 to simulate a realistic exposure situation in the respiratory system with NPs deposited directly onto the cells through cloud settling, gravitational settling, diffusional deposition, and electrostatic precipitation.23,24 Among these deposition approaches, electrostatic precipitation delivers NPs uniformly with high efficiency,21,22,25 along with less stress arising from electric field and air flow conditions.26,27 However, currently available ALI exposure devices are costly and complicated for operation, which does not allow most laboratories to conduct in vitro tests on ALI. Therefore, it would of interest and need to develop a novel and user-friendly exposure device for reliable assessment. When investigating nanotoxicity of dispersed MWCNTs on human lung epithelial cells (BEAS2B), Mishra, et al. (2012) 28 found that well-dispersed MWCNTs were more toxic compared with nondispersed MWCNTs.28 Pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs) in mice demonstrated larger agglomerated SWCNTs deposited in proximal alveoli, inducing granulomas in lungs; and single fiber-like SWCNTs deposited in distal alveoli, inducing interstitial lung fibrosis.29,30 The above-mentioned in vitro and in vivo studies revealed the dispersibility/agglomeration status as a 5 ACS Paragon Plus Environment


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critical physical factor leading to pulmonary toxicity induced by CNT exposure.31 Both electrospray and wet-atomization have been employed to disperse agglomerated CNT aerosols generated from aqueous solution.32-34 Surfactants and impurities in aqueous solutions cause unstable bimodal number distributions, causing abnormal toxicity assessment results.35 Consequently, dry-dispersion methods were developed to prevent such problems.36-41 Baron et al. (2008) used a mill and an acoustic feeder, comprising a speaker and a hopper, to generate SWCNT aerosols with a mass median aerodynamic diameter (MMAD) of 6-7 m.36 Madl et al. (2012) employed a cannula-based feeder and diamondcoated wheel to generate SWCNT aerosols with a MMAD of 1.3-1.7 m.40 McKinney et al. (2009) and Chen et al. (2012) developed a computer control feedback system to generate MWCNT-7 aerosols (MMAD: 1.5 m) with stable concentrations for up to 6-hr operation time.38,39 All these studies demonstrated well-dispersed conditions, and the stability of dispersion system has influence on the accuracy of toxicity assessments. In most published studies, the exposure duration was 1 hour or less; hence, the short-term stability of aerosol concentration is also of particular importance in the aerosol deposition exposure system.24 This study developed an integrated device comprising a shaker-fluidized bed-atomizer (SFA) disperser and an electrostatic shallow liquid interface (ESLI) precipitator for nanotoxicity assessment of MWCNTs. Comparison of cytotoxic and inflammatory responses was also made, using A549 and RAW264.7 cells under SLI and CON conditions exposed to MWCNTs. Finally, biological responses caused by exposure to pMWCNT and carboxylated MWCNT (cMWCNT) were examined. In this 6 ACS Paragon Plus Environment

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work, dose-dependent effects, physicochemical property-dependent, and exposure method-dependent biological responses were observed in terms of cell uptake alterations, lactate dehydrogenase (LDH) release, along with monocyte chemoattractant protein 1 (MCP-1), interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) levels after 24-h exposure.



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Methods MWCNT and cells pMWCNT grown catalytically by chemical vapor deposition process using molybdenum (Mo) as a catalyst was obtained from XinNano Materials, Inc. (XNM-LP-560000, Taoyuan, Taiwan; purity, > 95 %; average length, > 10 μm; average diameter, 10 nm; surface area, 224.24 m2/g). cMWCNT was carboxylized from pMWCNT using nitric acid treatment according to the previously reported method42. In brief, pMWCNT was functionalized using nitric acid (9.6 M) and then heated at 90oC for 12 hr. During the process, a magnetic mixer was employed to ensure uniform suspension of pMWCNT in the solution. Functionalized pMWCNT was then flushed with double-deionized water until the pH value of water exceeded 5.6 to ensure complete removal of nitric acid. A549 and RAW264.7 cells, cultured in Ham’s F12K medium and Dulbecco’s modified Eagle’s medium (DMEM), respectively, were provided by Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Both media comprise 10 % fetal bovine serum (FBS).

Construction of SFA-ESLI system Figure 1A shows the setup of the SFA-ESLI system developed for MWCNT exposure. As can be seen, it comprises a home-made SFA disperser and an ESLI exposure chamber. Dispersed MWCNT aerosols generated from the SFA were first neutralized by an

85Kr

neutralizer (TSI 3077A) before being

introduced into the mixing chamber at the total flow rate of 5.8 L/min. Neutralized MWCNT aerosols were subsequently introduced into the ESLI chamber placed inside a biosafety cabinet to ensure that 8 ACS Paragon Plus Environment

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the exposure system was sterile. Stability of MWCNT aerosol concentration was monitored by a scanning mobility particle sizer (SMPS, TSI model 3936). Figure 1B shows the schematic diagram of SFA, which is made up of an atomizer, a suspension chamber, a supporting base, and an orbital shaker. In detail, a critical orifice with an inner diameter (ID) of 0.33 mm is placed in the atomizer to control the flow rate at 3 L/min. Tested MWCNT (1g) is uniformly placed on a perforated plate with 25 holes (1.0 mm ID), which is supported by a porous metal (37 mm ID) in the suspension chamber. The bottom casing of the chamber comprises an air-flow distributor (20 holes with 0.5 mm ID) and an inlet for introducing dispersion air (Qdis). The air-flow distributor enables suspension of MWCNTs inside the chamber. The SFA is fixed on an orbital shaker which rotates at 250 rpm. Then, suspended MWCNTs transfer through a suction tube, while agglomerated MWCNTs are energetically dispersed by the sonic jet from the critical orifice to generate fine MWCNT aerosols. Figure 1C shows the ESLI exposure chamber, comprising a charger, an insulator casing, and a metallic bottom casing. The operating flow rate of ESLI is controlled at 5.5 L/min. Inside the charger, a carbon fiber bundle (CFB, Fu-Feng Co. Ltd., Taiwan) is used as the charging electrode to reduce ozone release which may lead to adverse effects on cells during corona discharge 43. The metal mesh is fixed on the insulator and connected to a high voltage supplier. A 35-mm culture dish is placed on the grounded bottom casing in which the culture medium is connected to the ground. When charged MWCNT aerosols pass through the electric field between the dish and the mesh, MWCNTs are deposited onto the dish via electrostatic force. Particle penetrations and deposition rates of ESLI were 9 ACS Paragon Plus Environment


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determined using experiments shown in Supporting Information (SI) (Figures S1 and S2). The compressed air, make-up air, and dispersion air are all passed through HEPA filters. All tests were repeated for at least 5 times.

Physiochemical properties of MWCNTs Bulk MWCNTs were dispersed in the ultra-pure water and ultra-sonicated for 1 hr. Appropriate amounts of the above-mentioned solution were pipetted onto copper grids for morphology observation by transmission electron microscopy (TEM). Inductively coupled plasma-mass spectrometer (ICP-MS, 7500cx, Agilent Technologies Inc., Tokyo, Japan) was employed to quantify the amount of Mo in MWCNTs. Carbon structure and functional groups were analyzed using Raman spectroscopy (DXR, Thermo Scientific, US) and Fourier-transform infrared spectroscopy (FTIR, Spectrum 100 FT-IR Spectrometer, PerkinElmer), respectively. A Zetasizer (Nano-ZS, Malvern Instruments Ltd., Worcestershire, UK) was employed to measure hydrodynamic size distribution and zeta-potentials () for investigating homogeneity and dispersibility of MWCNT suspensions. For airborne MWCNTs, number concentration distributions and stability were measured using an SMPS. Stability of mass concentration was monitored in real-time by a Dusttrak DRX (Model 8533, TSI Inc., MN) for 10 min at every 90 min after the concentration was stabilized. The lung deposition surface area concentration was monitored by an Aerotrak 9000 (TSI, Shoreview, MN, USA) for 10 min at every 60 min. A ten-stage NCTU micro-orifice cascade impactor (NMCI model 10R-A, Jusun 10 ACS Paragon Plus Environment

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Instruments Co. Ltd., Taiwan) was employed to measure mass distributions from 56 nm to 18 m. Additionally, the 3rd to 8th stages of the Marple personal cascade impactor (Model 290 Thermo Fisher Scientific Inc., Taiwan) were combined with a nanoparticle personal sampler (PENS)44 as the MPENS sampler (cut-off aerodynamic diameter, dpa50: 9.8, 6.0, 3.5, 1.55, 0.93, 0.52, 0.1 and < 0.1 m) to collect MWCNTs deposited in each stage for morphology inspection by TEM.

Cell Exposure by SLI and CON methods For the SLI method, A549 and RAW264.7 cells (9000 cells/cm2) were cultured on 35-mm dishes (area: 9 cm2) with 1.5-mL culture media and then placed inside the ESLI exposure chamber. Thus, the liquid layer in the culture dish was 1.7 mm deep. After adjusting the SFA-ESLI system to optimal conditions (Qdis = 0.5 L/min for SFA; Vc = +9000V and Vm = +2000V for ESLI), cells were exposed to different MWCNT aerosols until the deposition mass achieved 10, 25, 50 and 100 g, corresponding to exposure concentrations of 1.1, 2.8, 5.6 and 11.1 g/cm2, respectively. During exposure, the stability of output concentrations, and the distribution of MWCNT aerosols were monitored using the SMPS. To clarify whether air flow and electrical field induce cell stress, tests of the control group were conducted using air flowing through a HEPA filter installed at the inlet of the ESLI exposure chamber without MWCNT aerosols. During all tests on both experimental and control groups, ambient conditions with relative humidity of 60-70% and temperature of 25oC were maintained. All cells were only taken out from the incubator for a maximum of 30 min; hence, there should be no adverse effects on cells. 11 ACS Paragon Plus Environment


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For the CON method, exposure was conducted using the 24-well (11000 cells/cm2) plate with 2 mL media in each well (area: 1.9 cm2), presenting a culture media of 101 mm deep. In comparison with the SLI method, the exposure concentration of the CON method, Ccon (g/mL), could be calculated using the following equation:

Ccon 

CSLI  A

(1)

V

where CSLI is the exposure dose of the SLI method in the unit of g/cm2, A is the cross-sectional area of each well (1.9 cm2) in the 24-well plate, and V is the volume (2 mL) of the media added to each well. Therefore, exposure doses of 1.1, 2.6, 5.3, and 10.6 g/mL in the CON method corresponded to 1.1, 2.6, 5.6, and 11.1 g/cm2 in the SLI method. Cells for exposure using CON and SLI methods were both placed inside the incubator at the condition of 37oC, 5% CO2, and 70% RH. The ESLI exposure chamber placed inside the biosafety cabinet was disassembled for sterilization by ultraviolet (UV) light after experiments had been repeated for 12 hr to make sure that the exposure condition in the SLI method is sterile.

Cell uptake and inflammatory marker analysis After 24-hr exposure, cell uptake of MWCNTs, cytotoxicity, and inflammatory markers were analyzed following the procedure shown in Figure S4. MWCNTs, cells, and supernatants were separated by centrifugation (13000g*10min). To avoid false-positive interference, supernatants were collected and then mixed uniformly for measuring cytotoxicity and inflammatory response. To make sure whether 12 ACS Paragon Plus Environment

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the separated MWCNTs may lead to false-negative effect, standardized amounts of cytokines spiked into cultural media containing MWCNTs were carried out. MWCNTs (100 g) were added into the media (for both A549 and RAW264.7 cells) that were spiked with different cytokine standards, and then incubated for 1 and 24 hr. Then, supernatants were collected and subjected to ELISA measurements for checking the false-negative effect (Figure S5). As for cell uptake of MWCNTs, the cells were washed twice by PBS to remove residual MWCNTs not associated with cells. For quantitative analysis, Mo concentrations in pMWCNT and cMWCNT could be the tracer for estimating the uptake of MWCNTs in different cells. Nitric acid was employed to lyse A549 and RAW264.7 cells, which were subsequently subjected to ICP-MS for determining Mo concentrations. Uptakes of pMWCNT and cMWCNT in A549 and RAW264.7 cells could be calculated according to different Mo concentrations in pMWCNT and cMWCNT, respectively, as shown in the SI. This study defines cell uptake as the presence of interaction between cells and MWCNTs, including internalized MWCNTs and absorbed MWCNTs on the cell membrane. The quantitative cell uptake is further taken as cell-associated dose response. To confirm the retention of MWCNT-containing Mo in the media during cultivation, Mo contents in culture media were measured before and after exposures (page S8 of SI). For qualitative analysis, cells after CON and SLI exposure to different MWCNTs were examined using cytospin followed by microscopic inspections to validate results observed by ICP-MS analysis. This approach enables inspection of uptake of MWCNTs cell by cell. Briefly, 20000 cells/300 L were centrifuged (2000 rpm, 15 min) onto glass slides and fixed using methanol, followed by ASK 13 ACS Paragon Plus Environment


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staining (TONYAR BIOTECH. INC., Taiwan) prior to microscopic observation. Cytotoxicity of MWCNTs was evaluated in terms of lactate dehydrogenase (LDH) release, which is directly correlated with the integrity of the cell membrane. Inflammatory markers including human monocyte chemotactic protein (MCP-1), tumor necrosis factor (TNF-), interleukins (IL-6 and IL1), and mouse MCP-1, TNF-, IL-6, IL-1 were measured using corresponding enzyme-linked immune sorbent assay (ELISA) kits. LDH release was determined using the LDH-Cytotoxicity Colorimetric Assay Kit (BioVision, Milpitas, CA) and inflammatory markers were all obtained from R&D Systems (Minneapolis, MN).

Statistical analysis Data of cell uptake of MWCNTs, LDH release, and inflammatory biomarkers were analyzed using IBM® SPSS Statistics® version 17 and were shown as mean ± SEM (standard error of the mean). The Mann-Whitney test was employed to determine the statistical significance with p-values < 0.05 being considered significant.


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Results SFA-ESLI system For the SFA disperser, the dispersion air flow rate (Qdis) is crucial for stabilizing aerosol concentrations. Figure 2 shows the output pMWCNT aerosol concentration stability at different Qdis of SFA within 8 hr. Qdis at 0.5 L/min is optimal to maintain stability in an 8-hr dispersing operation with a 15-min time lag to reach the stable output aerosol concentration. It took 40 min to establish a lower aerosol concentration with Qdis = 0.3 L/min. When Qdis was increased to 1.9 L/min, the output aerosol concentration of pMWCNT increased gradually in the first 100 min but dropped rapidly after achieving peak concentration. A similar result was observed at Qdis of 1.0 L/min. For an optimal operating SFA disperser, Qdis should be kept at 0.5 L/min, consuming merely 0.03 g MWCNT in an 8-hr dispersing period. For the operation of an ESLI exposure chamber, the optimal discharge voltage of CFB electrode and the field voltage of the mesh were determined to be +9000 and +2000 V, respectively (Figure S3, SI). The maximum deposition rates were 4.40 ± 0.33 g/min for pMWCNT and 3.75 ± 0.29 g/min for cMWCNT. The time for accumulating 10, 25, 50 and 100 g of deposited pMWCNT were 2.3, 5.7, 11.4, and 22.8 min, respectively; while those for the same amounts of deposited cMWCNT were 2.7, 6.7, 13.4, and 26.8 min, respectively. More importantly, the measured ozone release in the ESLI exposure chamber was 4.53 ± 1.24 ppb, which is close to the background ozone concentration (2.88 ± 0.80 ppb) without corona discharge and would not lead to bias in the nanotoxicity assessment.43 15 ACS Paragon Plus Environment


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Physicochemical properties of MWCNTs Metal impurities in MWCNTs determined by ICP-MS showed Mo being the major impurity in pMWCNT. The Mo contents were 5290 g Mo/g-MWCNTs in pMWCNT and 486.4 g Mo/g MWCNTs in cMWCNT, revealing removal of 91% Mo content through functionalization. Further, TEM micrograms of pMWCNT and cMWCNT, shown in Figure 3A, are consistent with these results, indicating abundant metals present in pMWCNT but absent in cMWCNT. In addition to metal analysis, Raman and IR spectra were also employed to clarify the differences in chemical properties. Raman spectra, as shown in Figure 3B, displayed characteristic signals at 1332.5 cm- (D band) and 1567.3 cm(G band) for both pMWCNT and cMWCNT, respectively, implying similar chemical bonds in these two types of MWCNTs. In terms of IR spectra (shown in Figure 3C), signals were observed at 30003500 cm-1 and 1628 cm-1 in cMWCNT, corresponding to stretching vibrations of –OH and C=O groups, respectively. No signal was observed in pMWCNT. Therefore, these functionalized MWCNTs could be identified as carboxylated MWCNT (cMWCNT). In terms of physical properties, the specific surface areas were 264.7 and 317.8 cm3/g for pMWCNT and cMWCNT, respectively, with cMWCNT showing 20% higher surface area compared with pMWCNT. Table 1 shows that the hydrodynamic size of cMWCNT is smaller than that of pMWCNT in PBS, double-deionized water, and culture media. The carboxyl groups on cMWCNT may facilitate better hydrophilicity, leading to improved dispersibility in different solutions. The zetapotential (ξ) of pMWCNT was 0.69 and 5.4 mV in PBS and double-deionized water, respectively; 16 ACS Paragon Plus Environment

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while the zeta-potential of cMWCNT was -34.2 and -69.5 mV in PBS and double-deionized water, respectively. These observations echo IR spectra and confirm that cMWCNT is facilitating superior dispersibility compared with pMWCNT. Additionally, both pMWCNT and cMWCNT showed abnormal zeta-potential and PDI measurements in culture media, indicating non-uniformity of MWCNT suspensions caused by aggregation. These results again evidence inaccuracy for the CON method.

Table 1 Hydrodynamic diameter, PDI value, and zeta-potential (ξ) of pMWCNT and cMWCNT in different aqueous solutions and media. Hydrodynamic diameters (nm)

PDI

ξ (mV)

+ 10% FBS + 10% FBS

3740 27590 519 772

0.68 0.65 0.89 1.00

0.69 5.4 NA NA

PBS ddH2O +F12K + 10% FBS+ ++DMEM + 10% FBS

1502 4645 447 520

0.57 0.71 1.00 0.87

-34.2 -69.5 NA NA

Materials

Solutions PBS ddH2O

pMWCNT

+F12K

++DMEM

cMWCNT

+: medium for A549 cells ++: medium for RAW264.7 cells NA: not available



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For airborne MWCNTs generated by SFA, Figure 4A and 4B display time-dependent stability of mass concentration and lung deposition surface area concentrations for both pMWCNT and cMWCNT. The mass concentrations were stable at 2.02 ± 0.02 and 1.56 ± 0.02 mg/m3, while the lung deposition surface area concentrations were stable at 407 ± 11 and 258 ± 8 m2/cm3 for pMWCNT and cMWCNT, respectively. These results confirm that concentrations of MWCNT aerosols are not only stable in number but also in mass and surface area. The mass concentrations for the total suspended particles (TSP), PMResp (respiration) and PM1.0 of pMWCNT were 1.29, 1.30, and 1.31 times higher than those of cMWCNT; while the lung deposition surface area concentration of pMWCNT was 1.58 times higher than that of cMWCNT. The number distribution of MWCNT aerosols, shown in Figure 4C, indicate that both pMWCNT and cMWCNT display bimodal distributions with mode sizes of 93, 453 nm and 165, 453 nm, respectively. The number concentrations of MWCNT aerosols were maintained at 12889 ± 546 and 10740 ± 411 #/cm3 for pMWCNT and cMWCNT in the 8-hr period, respectively. Figure 4D shows pMWCNT and cMWCNT are bimodal mass distributions. The MMADs were 0.26 and 3.05 m with geometric standard deviations (g) of 1.91 and 2.26 for pMWCNT; while the MMADs were 0.18 and 1.28 m with g of 1.63 and 2.47 for cMWCNT. For pMWCNT aerosol, the ratios of PM0.056, PM0.1, PM1.0, PM2.5 and PM10 to TSP were 0.6, 1.3, 22.6, 54.1 and 93.8 % compared with 2.3, 4.1, 37.3, 63.2 and 92.8 % for cMWCNT aerosol. The PM1.0/TSP ratio of cMWCNT is 37.3% higher than that of pMWCNT (22.6%), indicating better dispersion of cMWCNT than of pMWCNT. In Figure 5A-H, aerosolized MWCNT samples collected on the 4th, 5th, 8th, and micro-orifice (9th) stages of 18 ACS Paragon Plus Environment

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MPENS (dpa50 = 6.0, 3.5, 0.52 and 0.1 m) show that cMWCNT was better dispersed and had shorter lengths than pMWCNT.

Cytotoxicity and inflammatory markers on A549 cells A549 and RAW264.7 cells were employed to evaluate cell uptake, cytotoxicity, and secretion of inflammatory markers (MCP-1, IL-1β, IL-6, TNF-α) caused by exposure to pMWCNT and cMWCNT. Two methods, CON and SLI, were used for comparison. For the quantitative cell uptake of MWCNTs, the background concentrations of Mo in media/supernatants are listed in Table S2. (page s9, SI). As can be seen, Mo concentrations in different groups are at low ppb levels. In comparison, Mo concentrations in pMWCNT and cMWCNT range between 500 and 4000 ppm (g/g), which are much higher than background concentrations of Mo (< 5 ppb) in MWCNT (100 g)-containing media (1 mL), and consequently, the loss of Mo during cultivation could be excluded. In Figure 6A, the uptake of MWCNTs in A549 cells after SLI and CON exposure showed a dosedependent effect. At the same doses, SLI-exposed A549 cells had more MWCNTs uptaken after than CON-exposed A549 cells. Significant differences between pMWCNT and cMWCNT were found after CON exposure at various exposure doses. Furthermore, a marked difference between pMWCNT and cMWCNT after SLI exposure at 11.1 g/cm2. In Figure 6B, LDH assay for A549 cells showed a dose-dependent effect after SLI and CON exposure at 2.8, 5.6, and 11.1 g/cm2. LDH release for A549 cells after SLI exposure was higher than 19 ACS Paragon Plus Environment


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that after CON exposure to cMWCNT at 5.6 and 11.1 g/cm2. However, the opposite was true (i.e., CON > SLI) after exposure to pMWCNT at 1.1, 2.8, and 5.6 g/cm2. Significant differences were observed between pMWCNT and cMWCNT in the SLI method at 2.8, 5.6, and 11.1 g/cm2 but were not observed in the CON method. In Figure 6C, IL-6 secretion by SLI-exposed A549 cells was dose-dependent but not by CONexposed ones. At various doses of either pMWCNT or cMWCNT, IL-6 secretion by A549 cells was markedly higher after SLI exposure than after CON exposure without significant differences between pMWCNT and cMWCNT. IL-1β and TNF-α secretions were not detectable in the culture media of A549 cells. In Figure 6D, MCP-1 secretion by A549 cells showed no dose-dependent effect after either SLI or CON exposure. Similar MCP-1 concentrations were observed in the culture media of CON-exposed A549 cells and the control group. In contrast, results showed a substantial increase in MCP-1 secretion to 2345 ± 161 and 3983 ± 394 pg/mL after SLI exposure to pMWCNT and cMWCNT, respectively. Almost constant secretion was maintained for exposure from 1.1 g/cm2 up to 11.1 g/cm2. The secretion of MCP-1 by A549 cells after SLI exposure was higher than that after CON exposure to either pMWCNT or cMWCNT at various doses. Significant differences between pMWCNT and cMWCNT were found with SLI exposure at 1.1, 2.8, 5.6, and 11.1 g/cm2.


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Cytotoxicity and inflammatory markers on RAW264.7 cells In Figure 7A, MWCNT uptake of SLI-exposed RAW264.7 cells showed a dose-dependent effect. The uptake rose from 2 to 15 g (pMWCNT) and from 8 to 60 g (cMWCNT) with an increase in exposure dose from 1.1 to 11.1 g/cm2. At the maximum exposure dose of 11.1 μg/cm2, CON-exposed cells showed deficient uptake, only 0.37 g pMWCNT and 0.48 g cMWCNT, compared with their SLI counterparts. Significant differences between pMWCNT and cMWCNT were observed after SLI exposure at doses exceeding 2.8 g/cm2. In Figure 7B, LDH assay showed a dose-dependent effect after SLI but not CON exposure. LDH release after SLI exposure to cMWCNT was higher than that to pMWCNT but only at 2.8 g/cm2. Opposite results were observed after CON exposure with significant differences between exposure to pMWCNT and cMWCNT only at low doses of 1.1 and 2.8 g/cm2 but not at higher exposure doses. Dose-dependent IL-1 secretion was observed only after SLI exposure, with significant differences between exposure to pMWCNT and cMWCNT, as shown in Figure 7C. Higher IL-1 secretion was observed after CON exposure to pMWCNT than to cMWCNT at 1.1 g/cm2, but lower IL-1 secretion was observed when dose increased to 2.8 g/cm2. For higher doses, at 5.6 and 11.1 g/cm2, no differences between exposure to pMWCNT and cMWCNT were found. There were significant differences in IL-6 secretion after SLI exposure to pMWCNT and to cMWCNT at various doses. Secretion of IL-6 was not detectable in the culture media of RAW264.7 cells. Both SLI and CON exposure to either pMWCNT or cMWCNT had a dose-dependent effect on 21 ACS Paragon Plus Environment


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MCP-1 secretion, as shown in Figure 7D, with no significant differences between the two methods at various exposure doses. Neither were there significant differences between exposure to pMWCNT and cMWCNT after either exposure approach. In contrast, TNF- secretion after SLI and CON exposure showed no dose-dependent effect (Figure 7E), but with significant differences between the two methods for the two MWCNTs. Significant differences were found after SLI exposure to pMWCNT and cMWCNT at 2.8 g/cm2 but not at other doses.

Discussion Physicochemical properties of CNTs, in particular, agglomerated states and surface modifications, are key factors strongly correlated with their toxicity.13,45 Compared with animal studies, in vitro approaches to nanotoxicity assessment are usually low cost. The CON method for assessing nanotoxicity is limited in its failure to simulate an in vivo environment, whereas ALI exposure systems mimic realistic exposure conditions in airways.24 Nevertheless, the high cost of existing ALI devices poses problems for conducting in vitro studies. Hence, the development of an efficient and low-cost ALI-like system is of great interest and importance. The SFA-ESLI system developed in this study can evaluate pMWCNT and cMWCNT on the shallow liquid interface in contrast to the fully immersed condition of the CON method, and without the use of transwell as required in ALI devices.


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Integration of SFA-ESLI system The SFA is designed to generate respirable MWCNT aerosols, which are stable in number, mass, and lung deposition surface area concentrations in both short-term (30-min) and long-term (8-hr) operations. The output aerosol concentrations could almost be kept constant at 2.02 ± 0.02 g/m3 for pMWCNT and at 1.56 ± 0.02 g/m3 for cMWCNT at the optimal Qdis (0.5 L/min), while the loaded amount of bulk MWCNT in SFA was around 1.0 g. To obtain the desired dosages for studying cell exposure, simultaneously increasing the loaded amount of bulk MWCNTs and Qdis gives higher concentrations while diluting the output aerosols yields lower concentrations. MWCNT aerosols produced from SFA are well-dispersed, as shown in mass distributions and TEM images. cMWCNT aerosols have smaller MMADs and shorter lengths than pMWCNT ones. MMADs of MWCNT aerosols reported in previous studies were approximately 1.5 m,37-40 close to the main mode diameter (1.28 m) of cMWCNT aerosols shown in Figure 4D. Some studies showed long-term stable output concentrations but not in short-term operation.40,41 Fluctuating exposure dosages lead to inaccurate nanotoxicity assessment for either in vivo or in vitro exposure. Additionally, the PM1.0/TSP ratio of cMWCNT (37.3%) is about 50% higher than that of pMWCNT (22.6%). The increase in particles of the submicron size fraction may be attributed to metallic impurity removal and breakage of the agglomerated state of pMWCNT during functionalization, which improves the dispersibility of cMWCNT. Ozone release in the ESLI chamber approximates the background ozone concentration (4.53 ± 1.24 ppb); hence, it does not affect cell culture or the subsequent interpretation of 23 ACS Paragon Plus Environment


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nanotoxicity.43 To obtain the maximum deposition of 100 g, 26.8 ± 2.1 min is needed for generating cMWCNT aerosols in the SFA-ESLI system. During this time period, no adverse effects were observed on A549 and RAW264.7 cells according to cell responses of the negative control group (Figure 6, Figure 7).

Conventional fully immersed exposure approach vs. shallow liquid interface exposure approach Higher cell uptake and dose-dependent responses are often observed in both A549 and RAW264.7 cells after SLI exposure. Only LDH release showed a dose-dependent response after CON exposure. Previous research attributed the interaction of deposited MWCNTs with cells in the culture medium to Brownian motion or gravity settling.46 That is, MWCNTs could interact with cells more directly in a thin liquid layer by the SLI exposure approach than in a thick liquid layer by the CON exposure approach. Although the cell density in CON exposure (11000 cells/cm2) is 1.2 times higher than that in SLI exposure (9000 cells/cm2), biological responses induced by CON exposure were mostly much lower than those by SLI exposure. The cell uptake of MWCNTs and secretions of MCP-1, IL-6, TNF-α on RAW264.7 cells did not significantly increase under CON exposure (Figure 7). Similarly, MCP-1, IL-1β, IL-6, and TNF-α secretions in A549 cells did not display apparent changes after CON exposure (Figure 6). In terms of LDH release after CON exposure, pMWCNT seems more cytotoxic than cMWCNT regardless of cells used, which is consistent with previous findings.14,15,17,18 However, disagreement has been suggested in other studies showing that cMWCNT is more cytotoxic than 24 ACS Paragon Plus Environment

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pMWCNT.12,47 Conflicting conclusions in these works are not only caused by different physicochemical properties of MWCNTs tested but also by various assays used for cytotoxicity assessment.48 Interactions among cells, cultural media, and MWCNTs may also influence toxicology evaluation because plasma proteins could adsorb onto MWCNTs, forming a protein corona and further changing toxicity and inflammation capabilities.49-51 The abnormal zeta-potential () and abnormal high PDI values in the culture media demonstrated the instability of MWCNTs in complicated solutions (Table 1), which may subsequently influence the agglomeration tendency, especially by CON method.15,52 As for inflammatory responses, no differences were observed between pMWCNT and cMWCNT on A549 cells, whereas higher inflammatory response due to IL-1β secretion was observed only in RAW264.7 cells exposed to cMWCNT at 2.8 μg/cm2, but not at other doses. That is, differences in inflammatory level caused by CON exposure to pMWCNT and cMWCNT are unclear; while using the SLI exposure approach can eliminate the above-mentioned issues.

Significant biological responses in SLI exposure Cell uptakes of pMWCNT and cMWCNT RAW264.7 cells demonstrate higher cell uptake of MWCNTs than A549 cells after SLI exposure (Figure 6A, Figure 7A), because MWCNTs can be internalized by macrophages (e.g., RAW264.7 and J774) through phagocytosis.15,16 Furthermore, cMWCNT presents higher potency of cell uptake 25 ACS Paragon Plus Environment


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capability compared with pMWCNT after SLI exposure. These results agree with observations in previous studies, indicating more effective transport of CNTs inside cells by negatively charged – COOH group than pristine CNTs.14,15 cMWCNT prevents formation of protein corona to eliminate micro-sized agglomerates that are not easily internalized through phagocytosis or through channels/holes in the cell membrane.47 Zhu et al. (2016) reported that non-agglomerated cMWCNTs, like nano-needles, can penetrate directly into cells.53 Moreover, in the SLI method, the number of cMWCNTs per unit mass is 10% higher than that of pMWCNT (7028 #/mg versus 6355#/mg) according to the measurements of Dusttrak and SMPS (Figure 4A, C), resulting in more cMWCNT aerosols deposited on the cell membrane and, consequently, higher cell uptake of cMWCNT. Figure 8 demonstrates that cell uptakes after SLI exposure are apparently higher than those after CON exposure regardless of cells or MWCNTs used (Figure 8C-F vs. Figure 8G-J). Furthermore, the uptakes of cMWCNT are obviously higher than those of pMWCNT in both cells. Consequently, qualitative observations are all in line with quantification results by ICP-MS analysis.

Cytotoxicity and inflammatory responses Nanomaterials usually lead to false-positive and/or false-negative effects while using ELISA assays for measuring cytotoxicity and inflammatory responses. In our study, false-positive effect caused by optical interference of MWCNTs could be excluded using centrifugation procedure to remove MWCNT. In terms of probably false-negative effect, Table S1 presents that measured concentrations 26 ACS Paragon Plus Environment

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of cytokines are as close as the spiked concentrations. These results suggest that the false-negative interference could be excluded (page S5-S7 of SI). LDH release from A549 cells exposed to cMWCNT at doses of 5.6 and 11.1 g/cm2 amount to 17.05% and 59.88% relative to total cell lysate, respectively (Figure 6B); and those from RAW264.7 amount to 12.39% and 26.41% relative to total cell lysate, respectively (Figure 7B), indicating higher cytotoxicity of cMWCNT-treated A549 cells. LDH release from A549 cells exposed to pMWCNT at doses of 5.6 and 11.1 g/cm2 amount to 3.32% and 25.14% relative to total cell lysate, respectively; and those from RAW264.7 cells amount to 12.67% and 21.83% relative to total cell lysate, respectively. The A549 cells show greater sensitively for identifying cMWCNT cytotoxicity, 2 to 5-fold higher than for pMWCNT. The RAW264.7 cells fail to differentiate the cytotoxicity caused by exposure to pMWCNT and cMWCNT. This biological significance may be attributed to MWCNTs interacting predominantly with A549 cells on the cell surface,16 damaging cell membrane integrity and leading to subsequent LDH release. For inflammatory markers on A549 cells after SLI exposure, IL-6 secretion showed higher dosedependent responses when exposed to cMWCNT than to pMWCNT at the same doses. There were no significant differences between pMWCNT and cMWCNT at various doses due to the relatively large standard deviation for cMWCNT exposure (Figure 6C). On the other hand, MCP-1 secretion was significantly higher than after exposure to cMWCNT than to pMWCNT at doses of 1.1, 2.8, 5.6, and 11.1 g/cm2 (Figure 6D). Secretion levels of MCP-1 caused by pMWCNT and cMWCNT exposure 27 ACS Paragon Plus Environment


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peaked at doses of 5.6 and 1.1 g/cm2, respectively, showing slight dose-dependency with a more severe inflammatory response triggered by cMWCNT compared with pMWCNT exposure. MCP-1 and IL-1 secretions on RAW264.7 cells displayed dose-dependent responses for both pMWCNT and cMWCNT exposures (Figure 7C, D). MCP-1 and IL-1 secretions were higher after exposure to cMWCNT than to pMWCNT without statistical significance. Therefore, to further investigate dosedependent responses on cells, IL-6 and IL-1β are suggested as representative inflammatory markers for A549 and RAW264.7 cells, respectively.

Exposure dose-dependent and cell-associated dose-dependent effects in SLI exposure A549 and RAW264.7 cells displayed limited uptake of pMWCNT and cMWCNT (< 2.44 g) by CON method (Figure 6A, Figure 7A) compared to SLI method. Uptake by A549 and RAW264.7 cells when exposed to cMWCNT reached the maximum at the dose of 11.1 g/cm2. There were no significant differences in cell uptake on A549 and RAW264.7 cells after SLI exposure to pMWCNT. Hence, changes in cell membrane integrity (LDH release) and inflammation markers (IL-1β and IL-6) after SLI exposure to pMWCNT and cMWCNT should be discussed according to either exposure dose (Figure 9) or cell-associated dose (Figure 10). Exposure dose-dependent response (Figure 9) and cell-associated dose-dependent response (Figure 10) of A549 and RAW264.7 cells display distinct characteristics. As shown in Figure 9, strong and positive correlation (r > 0.7) of LDH release with exposure doses was observed in both A549 and 28 ACS Paragon Plus Environment

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RAW264.7 cells. For A549 cells, LDH release induced by cMWCNT was significantly higher than that induced by pMWCNT at exposure doses of 5.6 and 11.1 g/cm2. However, there were no significant differences in LDH release on RAW264.7 cells after exposure to pMWCNT and cMWCNT. Similarly, the fitted curves in Figure 10A, B show strong and positive correlation of LDH release with cell-associated doses for both A549 and RAW264.7 cells. No significant differences in responses between pMWCNT and cMWCNT at different cell-associated doses were observed in A549 cells (Figure 10A). In contrast, with increasing cell-associated doses, pMWCNT demonstrated more rapid responses (higher slope) of LDH release compared with cMWCNT. pMWCNT induced much more cell membrane damage than cMWCNT at equivalent cell-associated doses (Figure 10A), indicating higher cytotoxicity for pMWCNT. No distinctive differences in LDH release versus exposure dose (sum of cell-associated dose and extracellular dose) were found between pMWCNT and cMWCNT exposure (Figure 8B). Similar uptake of pMWCNT on A549 cells (6.82 μg) and RAW264.7 cells (10.93 μg) demonstrated similar LDH release (25.14–21.83% relative to whole cell lysate), implying possible correlation of cell-associated dose of pMWCNT with changes in cell membrane integrity and cytotoxicity. On the other hand, RAW264.7 cells internalized more cMWCNT than A549 did (55.88 vs. 29.70 g) but RAW264.7 cells released less LDH than A549 cells did (26.41 vs. 59.88%), indicating greater resistance of RAW264.7 cells than A549 cells to cytotoxicity caused by cMWCNT exposure. In terms of inflammatory markers, A549 and RAW264.7 cells show distinct characteristics. A549 29 ACS Paragon Plus Environment


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cells secreted IL-6 without IL-1β while RAW264.7 secreted IL-1β without IL-6 after SLI exposure (Figure 9C, Figure 10C). Furthermore, A549 cells secreted more MCP-1 than RAW264.7 cells at equivalent exposure doses (Figure 6D, Figure 7D). Compared with RAW264.7 cells, A549 cells presented higher inflammatory response after exposure to either pMWCNT or cMWCNT. A549 cells showed a positive correlation between IL-6 secretion and exposure doses (Figure 9C). cMWCNTinduced IL-6 secretion in A549 cells was significantly higher than that caused by pMWCNT at the maximum dose (11.1 g/cm2). For RAW264.7 cells, data presented strong positive correlation between IL-1β secretion and exposure doses, with no significant differences between pMWCNT and cMWCNT at various doses (Figure 9D). In Figure 10C, D, the fitted curves of IL-6 and IL-1β secretions versus cell-associated doses were strongly and positively correlated for both A549 and RAW264.7 cells, but there were no significant differences between pMWCNT and cMWCNT on A549 cells (Figure 10C). With the increase in cell-associated doses, pMWCNT demonstrated more rapid responses (higher slope) of IL-1β release compared with cMWCNT. pMWCNT leads to more IL-1β secretion than cMWCNT at equivalent cell-associated doses (Figure 10D), revealing higher inflammatory response caused by pMWCNT than by cMWCNT. Results obtained in this study indicate cytotoxicity and inflammation determined by the cellassociated dose-dependent responses (Figure 9) different from those determined by exposure dosedependent responses (Figure 8). At the equivalent cell-associated dose level, pMWCNT induces higher LDH release along with IL-1β secretion in RAW264.7 cells than cMWCNT (Figure 10B, D). This 30 ACS Paragon Plus Environment

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phenomenon might be attributed to a higher metal impurity content in pMWCNT. Moreover, pMWCNT is relatively less dispersed and hence has a “frustrated phagocytosis” effect, in which macrophages fail to fully engulf oversized pMWCNT and try to tear it apart continuously, resulting in higher cytotoxicity and inflammatory responses.54,55 Figure 10 shows similar maximum uptake of pMWCNT on A549 and RAW264.7 cells (6.82 vs. 10.93 μg), as would be expected of frustrated phagocytosis for less dispersed pMWCNT. cMWCNT was more easily internalized by RAW264.7 macrophage cells than by A549 human alveolar basal epithelial cells (maximum amounts: 55.88 vs. 29.70 μg). The A549 cell line is relatively weak for phagocytosis while the RAW264.7 cell line tends by its nature to capture materials for protection of the biological system against exogenous stimulants. According to our findings, A549 and RAW264.7 cells can represent exposure dose-dependent and cell-associated dose-dependent effects, respectively. Differences in cytotoxicity and inflammatory responses caused by pMWCNT and cMWCNT on A549 cells are highlighted in Figure 9 which shows higher cytotoxicity in cMWCNT than in pMWCNT at relative high exposure doses (5.6 and 11.1 μg/cm2). Figure 9C reveals higher inflammatory response (secretion of IL-6) in cMWCNT than in pMWCNT at the maximum exposure dose (11.1 μg/cm2). Similar differences in cytotoxicity and inflammatory responses caused by pMWCNT and cMWCNT on RAW264.7 cells are highlighted in Figure 10. Instead of using qualitative analysis by TEM micrographs which provide a limited field of view16,47, this study used cytospin followed optical microscopy (OM) inspections to obtain the representatively qualitative 31 ACS Paragon Plus Environment


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results of cell uptake. In general, OM are not able to observe nanomaterials because of the optimal spatial resolution around 0.2 m, which is indeed the limitation of optical microscopy to identify a single-fiber MWCNT. However, the MMAD and length of MWCNTs are in micrometer levels according to the measurements of mass distributions and TEM images for dispersed MWCNTs (Figure 4D and Figure 5). This technique remains adequate to observe interaction between MWCNTs and cells. In contrast, the TEM provides very high spatial resolution (nanometer levels); however, it presents very narrow field of view to display the interaction between MWCNTs and cells. Figure S6 demonstrates OM is able to detect not only agglomerates of MWCNTs but also the uptake of single MWCNT fiber in cells. Notably, OM and TEM are complementary methods for the study of nanomaterials and its biological fates. Moreover, the qualitative results were in good accordance with the quantitative results performed by ICP-MS. This study facilitated a quantitative analysis to define the cell-associated dose, presenting higher cytotoxic and inflammatory responses in pMWCNT than in cMWCNT at equivalent cell-associated dose.

Conclusions The SFA-ESLI system developed in this research generated well-dispersed MWCNT aerosols for in vitro studies on the shallow liquid interface, with advantages of low cost and user-friendly operations. To obtain the desired dosages for exposure, output aerosol concentrations are tunable by simultaneously adjusting the loaded MWCNTs and compatible Qdis for elevating concentrations. 32 ACS Paragon Plus Environment

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Interactions between MWCNTs and cells depend on both physicochemical properties of MWCNTs and cell types. Consequently, measurements of physicochemical properties for tested MWCNTs and the selection of cell lines are crucial for nanotoxicity assessment. We suggest at least two different cells, for instance, epithelial cells (A549) and macrophages (RAW264.7), should be used for nanotoxicity evaluation in order to forecast complicated biological responses in real living animals. Biological responses, including cell uptake of MWCNTs, cytotoxicity, and secretion of inflammation markers observed in cell culture after SLI exposure are higher than those after CON exposure at equivalent doses, indicating higher sensitivity of the SLI method in toxicity assessment. Exposure dose-dependent responses of LDH and IL-6 releases in A549 cells can differentiate nanotoxicity between cMWCNT and pMWCNT. On the other hand, cell-associated dose-dependent responses of LDH and IL-1β releases in RAW264.7 cells are significant for differentiating nanotoxicity between pMWCNT and cMWCNT.



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Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: The supporting information includes: (1) Penetration test and quantitative test for ESLI exposure chamber, (2) Preparation of cells and supernatants for cellular response measurement after exposure, (3) Evaluation of false-negative interference, (4) Quantitative analysis of cell-associated doses, and (5) Qualitative analysis using cytospin followed optical microscopy inspection.

Acknowledgments The authors gratefully acknowledged the financial support from the Institute of Labor, Occupational Safety and Health, Taiwan under Grant no. IOSH-1040023, the National Health Research Institutes, Taiwan under Grant no. NHRI-BN-105-PP-27, and the Ministry of Science and Technology, Taiwan under Grant nos. MOST 106-2113-M-400-006 and MOST 107-2622-8-009-004-TE5.


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Abbreviations ALI: Air-liquid interface; CFB: Carbon fiber bundle; CNT: Carbon nanotubes; CON: Conventional fully immersed condition; ESLI: Electrostatic shallow liquid interface; ICP-MS: Inductively coupled plasma mass spectrometry; IL-1β: Interleukin 1β; IL-6: Interleukin 6; LDH: Lactate dehydrogenase; MCP-1: Monocyte chemoattractant protein 1; MMAD: Mass mean aerodynamic diameter; Mo: Molybdenum; MPENS: Constructed cascade impactor with 3rd to 8th stages of Marple personal cascade impactor and personal nanoparticle sampler; MWCNT: Multi-walled carbon nanotube; NMCI: NCTU micro-orifice cascade impactor; NMD: Number median diameter; NPs: nanoparticles; PM: Particulate matter; RELs: Recommended exposure limits; SFA: Shaker-fluidized bed-atomizer; SLI: shallow-liquid interface; SFA-ESLI: Constructed system with SFA and ESLI exposure chamber; SWCNT: Single-walled carbon nanotubes; TEM: Transmission electron microscopy; TNF-α: Tumor necrosis factor α; TSP: Total suspended particle; cMWCNT: Carboxylized multi-walled carbon nanotube; pMWCNT: Pristine multi-walled carbon nanotube dpa50: cut-off aerodynamic diameter; Qdis: dispersion air flow rate; ξ: Zeta-potential; g: Geometric standard deviation



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S. (2012) Effect of MWCNT surface and chemical modification on in vitro cellular response. J. Nanopart. Res. 14, 1181. (46) Teeguarden, J. G.; Hinderliter, P. M.; Orr, G.; Thrall, B. D.; Pounds, J. G. (2007) Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments. Toxicol. Sci. 95, 30012. (47) Ursini, C. L.; Maiello, R.; Ciervo, A.; Fresegna, A. M.; Buresti, G.; Superti, F.; Marchetti, M.; Iavicoli, S.; Cavallo, D. (2015) Evaluation of uptake, cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and -OH and -COOH functionalized multi-wall carbon nanotubes. J. Appl. Toxicol. 36, 394-403. (48) Geys, J.; Nemery, B.; Hoet, P. H. (2010) Assay conditions can influence the outcome of cytotoxicity tests of nanomaterials: better assay characterization is needed to compare studies. Toxicol. In Vitro 24, 620-9. (49) Dutta, D.; Sundaram, S. K.; Teeguarden, J. G.; Riley, B. J.; Fifield, L. S.; Jacobs, J. M.; Addleman, S. R.; Kaysen, G. A.; Moudgil, B. M.; Weber, T. J. (2007) Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials. Toxicol. Sci. 100, 303-15. (50) Casey, A.; Herzog, E.; Lyng, F. M.; Byrne, H. J.; Chambers, G.; Davoren, M. (2008) Single walled carbon nanotubes induce indirect cytotoxicity by medium depletion in A549 lung cells. Toxicol. Lett. 179, 78-84. (51) Monteiro-Riviere, N. A.; Inman, A. O.; Zhang, L. W. (2009) Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. Toxicol. Appl. Pharmacol. 234, 222-35. (52) Bruinink, A.; Wang, J.; Wick, P. (2015) Effect of particle agglomeration in nanotoxicology. Arch. Toxicol. 89, 659-75. (53) Zhu, S.; Zhu, B.; Huang, A.; Hu, Y.; Wang, G.; Ling, F. (2016) Toxicological effects of multiwalled carbon nanotubes on Saccharomyces cerevisiae: The uptake kinetics and mechanisms and the toxic responses. J. Hazard. Mater. 318, 650-62. 41 ACS Paragon Plus Environment


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(54) Brown, D. M.; Kinloch, I. A.; Bangert, U.; Windle, A. H.; Walter, D. M.; Walker, G. S.; Scotchford, C. A.; Donaldson, K.; Stone, V. (2007) An in vitro study of the potential of carbon nanotubes and nanofibres to induce inflammatory mediators and frustrated phagocytosis. Carbon 45, 1743-1756. (55) Figarol, A.; Pourchez, J.; Boudard, D.; Forest, V.; Tulliani, J.-M.; Lecompte, J.-P.; Cottier, M.; Bernache-Assollant, D.; Grosseau, P. (2014) Biological response to purification and acid functionalization of carbon nanotubes. J. Nanopart. Res. 16.


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Figure 1. Experimental setup for cell-based exposure test on shallow-liquid-interface (SLI) for MWCNTs nanotoxicity assessment. (A) Cell culture media was placed inside the electrostatic shallow-liquid interface (ESLI) exposure chamber and exposed to well-dispersed MWCNT aerosols on air-liquid-interface, which were generated by the shaker-fluidized bed-atomizer (SFA). Schematic diagrams of (B) SFA and (C) ESLI exposure chamber. 203x203mm (300 x 300 DPI)

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Figure 2. Output pMWCNT aerosol concentration stability by different Qdis of the SFA within 8 hrs. The Qdis is dispersed air flow rate of SFA. Qdis= 0.5 L/min, the optimized dispersed air flow rate, with high and stable output pMWCNT aerosol concentration. Qdis=0.3 L/min, stable output pMWCNT aerosol concentration but the concentration is lower than that of 0.5 L/min. Qdis=1.0 and 1.9 L/min, high but unstable output pMWCNT aerosol concentration. 208x189mm (300 x 300 DPI)


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Figure 3. Physicochemical properties of bulk MWCNTs before SFA dispersion. (A) TEM micrographs: The pMWCNT is shown to have metal impurity, but the metal impurity is absent in cMWCNT. (B) Raman spectra: carbon structure of pMWCNT and cMWCNT are nearly identical. (C) IR spectra: signals of C=O and O-H were found in cMWCNT but free from pMWCNT. 88x40mm (300 x 300 DPI)



Figure 4. MWCNT aerosols concentration and distribution. (A) The real-time mass concentrations and (B) lung deposition surface area concentrations of pMWCNT were higher than that of cMWCNT. The mass and surface area concentrations of pMWCNT and cMWCNT are all stable during 8-hr dispersion period. (C) Number distributions showed that both pMWCNT and cMWCNT have stable number concentrations and distributions within 8-hr dispersion period. (D) Mass distributions showed that cMWCNT has smaller MMADs than pMWCNT, implying that the cMWCNT is better-dispersed than pMWCNT. Both MWCNT aerosols showed the bimodal distributions in number and mass. 73x95mm (300 x 300 DPI)


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Figure 5. TEM micrographs of pMWCNT and cMWCNT aerosols collected on the MPENS. The samples were collected by a cascade impactor which is constructed with a Marple personal cascade impactor and personal nanoparticle sampler (PENS). (A)(B) The pMWCNT and cMWCNT samples collected on 4th stage impactor (dpa50= 6 μm). (C)(D) The pMWCNT and cMWCNT samples on 5th stage impactor (dpa50=3.5 μm). (E)(F) The pMWCNT and cMWCNT samples collected on 8th stage impactor (dpa50= 0.52 μm). (G)(H) The pMWCNT and cMWCNT samples collected on the micro-orifice stage impactor (dpa50= 100 nm). The cMWCNT is better-dispersed than pMWCNT, and cMWCNT has shorter length than pMWCNT in all stages samples. 85x181mm (300 x 300 DPI)



Figure 6. Comparison of (A) cell uptake and levels of (B) LDH (C) IL-6 and (D) MCP-1 in culture media of A549 cells after CON and SLI exposure to different doses of pMWCNT and cMWCNT. * denotes p-value < 0.05 indicating a significant difference between pMWCNT and cMWCNT. # denotes p-value < 0.05 indicating a significant difference between CON and SLI exposure. Data were shown in mean ± SEM (n=3). 403x427mm (300 x 300 DPI)


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Figure 7. Comparison of (A) cell uptake and levels of (B) LDH (C) IL-1β (D) MCP-1 and (E) TNF-α in culture media of RAW264.7 cells after CON and SLI exposure to different doses of pMWCNT and cMWCNT. * denotes p-value < 0.05 indicating a significant difference between pMWCNT and cMWCNT. # denotes p-value < 0.05 indicating a significant difference between CON and SLI exposure. Data were shown in mean ± SEM (n=3). 203x320mm (300 x 300 DPI)



Figure 8. Microscopic inspections for cells after CON and SLI exposure to MWCNTs (100 μg). (A) Nonexposed A549 cells and (B) non-exposed RAW264.7 cells. A549 cells after CON exposure to (C) pMWCNT; and (D) cMWCNT. RAW264.7 cells after CON exposure to (E) pMWCNT and (F) cMWCNT. A549 cells after SLI exposure to (G) pMWCNT and (H) cMWCNT. RAW264.7 cells after SLI exposure to (I) pMWCNT and (J) cMWCNT. (scale bar: 10 μm) 150x94mm (300 x 300 DPI)


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Figure 9. Exposure dose-dependent responses on LDH release and IL-1β/IL-6 secretion in A549 and RAW264.7 cells. LDH release in (A) A549 and (B) RAW264.7 cells exposed to pMWCNT and cMWCNT with SLI method. (C) IL-6 secretion in A549 and (D) IL-1β secretion in RAW264.7 cells exposed to pMWCNT and cMWCNT with SLI method. Both LDH and IL-6/IL-1β showed the high correlation with exposure dose of pMWCNT and cMWCNT in A549 and RAW264.7 cells. The exposure dose-dependent responses represent that cMWCNT is more cytotoxic and inflammatory than pMWCNT, which is highlighted in A549 cells. The data was presented in mean ± SEM (n=3). 63x65mm (300 x 300 DPI)



Figure 10. Cell-associated dose-dependent responses on LDH release and IL-1β/IL-6 secretion in A549 and RAW264.7 cells. LDH release in (A) A549 and (B) RAW264.7 cells exposed to pMWCNT and cMWCNT with SLI method. (C) IL-6 secretion in A549 and (D) IL-1β secretion in RAW264.7 cells exposed to pMWCNT and cMWCNT with SLI method. Both LDH and IL-6/IL-1β showed the high correlation with uptake of pMWCNT and cMWCNT in A549 and RAW264.7 cells. According to cell-associated dose-dependent responses, A549 cells show nearly the same responses on LDH release and IL-6 secretion caused by either pMWCNT or cMWCNT exposure, yet the pMWCNT is more cytotoxic and inflammatory than cMWCNT in RAW264.7 cells. The data was presented in mean ± SEM (n=3). 62x65mm (300 x 300 DPI)


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Development of a Novel Shallow Liquid Interface ... - ACS Publications

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