Threshold Rigidity Values for the Asbestos-Like Pathogenicity of High

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Threshold Rigidity Values for the Asbestos-Like Pathogenicity of High Aspect Ratio Carbon Nanotubes in a Mouse Pleural Inflammation Model Dong-Keun Lee, Soyeon Jeon, Youngju Han, Sung-Hyun Kim, Seonghan Lee, Il Je Yu, Kyung Seuk Song, Aeyeon Kang, Wan Soo Yun, Sung-Min Kang, Yun Suk Huh, and Wan-Seob Cho ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b03604 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Three representative electron microscopic images of HARNs used in this study show the degree of piercing to phagocytes and frustrated phagocytosis in the cells according to the rigidity values of HARNs (orange colour is HARNs and green is cells). Based on the value of rigidity (Db & SBPL), the biological outcome can be classified into three stages: tier 1 is no effect or normal, tier 2 is acute & chronic inflammation, and tier 3 is the carcinogenic. 276x197mm (149 x 149 DPI)

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Threshold Rigidity Values for the Asbestos-Like Pathogenicity of High Aspect Ratio Carbon Nanotubes in a Mouse Pleural Inflammation Model Dong-Keun Lee†, Soyeon Jeon†, Youngju Han†, Sung-Hyun Kim†, Seonghan Lee†, Il Je Yu‡, Kyung Seuk Song§, Aeyeon Kang‖, Wan Soo Yun‖, Sung-Min Kang¶, Yun Suk Huh¶,*, Wan-Seob Cho†,*

†Lab

of Toxicology, Department of Medicinal Biotechnology, College of Health Sciences, Dong-

A University, 37, Nakdong-daero, 550 beon-gil, Busan, 49315, Republic of Korea ‡HCTm

Co., LTD., 74, Seoicheon-ro 578 beon-gil Majang-myeon, Icheon-si, Gyeonggi-do,

17383, Republic of Korea §Korea

Environment & Merchandise Testing Institute, 8, Gaetbeol-ro 145 beon-gil, Yeonsu-gu,

21999, Incheon, Republic of Korea ‖Department

of Chemistry, Sungkyunkwan University, 2066, Seobu-ro, Jangan-gu, Suwon-si,

Gyeonggi-do, 16419, Republic of Korea ¶Department

of Biological Engineering, Inha University, 100, Inharo, Nam-gu, Incheon, 22212,

Republic of Korea

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*Correspondence and requests for materials should be addressed to Y.S.H. (email: [email protected]) or W.-S.C. (email: [email protected]).

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Abstract The qualitative and quantitative evaluation of the physicochemical parameters associated with the pathogenicity of high-aspect-ratio nanomaterials is important for comprehensive regulation efforts and safety-by-design approaches. Here, we report quantitative data on the correlations between the rigidity of these nanomaterials and toxicity endpoints in vitro and in vivo. As measured by new ISO standards published in 2017, rigidity shows a strong positive correlation with inflammogenic potential, as indicated by inflammatory cell counts and IL-1β (a biomarker for frustrated phagocytosis) levels in both the acute and chronic phases. In vitro experiments using differentiated THP-1 cells find that only highly rigid multi-walled carbon nanotubes (MWCNTs) and asbestos fibers lead to piercing and frustrated phagocytosis. Thus, this study suggests a bending ratio of 0.97 and a static bending persistence length of 1.08 as threshold rigidity values for asbestos-like pathogenicity. However, additional research using MWCNTs with rigidity values that lie between those of non-inflammogenic (Db=0.66 and SBPL=0.87) and inflammogenic fibers (Db=0.97 and SBPL=1.09) is required to identify more accurate threshold values, which would be useful for comprehensive regulation and safety-by-design approaches based on MWCNTs.

Keywords: high aspect ratio nanomaterial, carbon nanofiber, multi-walled carbon nanotube, rigidity, frustrated phagocytosis, inflammation, carcinogen

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Nanomaterials exhibit novel properties, such as a high tensile strength, low weight, and high electrical and thermal conductivity, which distinguish them from their bulk material counterparts.1,2 These properties mean that nanomaterials are indispensable in various industrial applications, with multi-walled carbon nanotubes (MWCNTs) particularly common owing to their advantageous thermodynamic and electronic properties.3-5 However, safety concerns associated with the use of nanomaterials can hinder the development and commercialization of nanotechnology. Because high-aspect-ratio nanomaterials (HARNs) such as MWCNTs pose a threat to public health and their role in industry is rapidly increasing, it is important to establish rational quantitative criteria for safety-by-design and proper regulation of HARNs based on an in-depth analysis of the correlation between key physicochemical and toxicity parameters. The physicochemical properties of nanomaterials, such as size, shape, composition, and surface reactivity, have been known to be closely related to toxicity. Slight differences in or modifications to these physicochemical properties can significantly influence the toxicity of nanomaterials, even if they have identical constituent elements.6 Therefore, the assessment of toxicity-related physicochemical factors is extremely important in the regulation of nanomaterial use due to the costs and ethical issues involved in in vivo studies. In 2014, the International Agency for Research on Cancer (IARC) classified only one type of MWCNT, manufactured by Mitsui & Co., as a Group 2B human carcinogen based on animal studies.7 Other types of MWCNT were classified as Group 3 carcinogens owing to a lack of data. However, the IARC and other national and international regulatory bodies are currently attempting to regulate all types of HARN based on the qualitative and quantitative evaluation of pathogenic parameters related to toxicity.6,7 Pulmonary exposure to some types of HARN has been shown to induce 4


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asbestos-like lesions because of their unique physicochemical properties, such as aspect ratio, length, and biopersistence.8-12 Further studies are needed to determine whether other physicochemical factors (i.e., diameter, biopersistence, and rigidity) are related to the pathogenicity of HARNs, thus strengthening any regulatory approach. Rigidity, which represents the ability of a material to resist deformation, can be highly variable among MWCNTs due to differences in the manufacturing process and defects.13 In recent studies, it has been suggested that rigidity is a key factor responsible for frustrated phagocytosis because rigid and long MWCNTs can pierce phagocytic cells, which is also a pathognomonic feature of asbestos fibers.14,15 However, previous research has been limited to small sets of MWCNTs and has not conducted quantitative correlation analysis on rigidity and toxicity parameters. In this study, we carried out a number of experiments using carbon nanofibers (CNFs), MWCNTs, and asbestos fibers to evaluate their toxicity potential in vivo and in vitro and to determine which physicochemical parameters are most strongly related to the toxicity of HARNs. MWCNTs have been known to transmigrate into the pleural space after inhalation and cause mesothelioma.16 However, it is difficult to evaluate the dose response for pleural damage via pulmonary exposure because the concentration of MWCNTs in the pleural space varies depending on the type of MWCNT.16 Therefore, we selected a mouse intrapleural injection model for in vivo experiments because this model is a reliable way to understand the behavior of MWCNTs in the pleural space.17-19 The quantitative evaluation of the association between physicochemical parameters, including rigidity, and toxicity parameters in HARNs will serve as a powerful tool in the risk assessment of these nanomaterials. 5


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Results Physicochemical characterization of CNFs, MWCNTs, and asbestos fibers. Our strategy for establishing the criteria for the toxicity assessment of HARNs is schematically presented in Figure 1a. In this study, we focused on identifying the physicochemical parameters considered to be highly related to systemic toxicity while controlling other properties (e.g., length, aspect ratio, and biopersistence). To evaluate the effect of CNF and MWCNT rigidity on pleural inflammation, one type of long tangled CNF (designated as CNFtang1), four types of long tangled MWCNT (designated as CNTtang1-CNTtang4), one type of long rigid MWCNT (designated as CNTlong1), and two types of long rigid asbestos fiber (amosite and crocidolite) were injected into the pleural space of the mouse model. The rigidity of each material was correlated with inflammogenic potential, which was classified into Tier 1 (minimal-to-mild inflammation), Tier 2 (moderate inflammation), and Tier 3 (severe inflammation) based on inflammatory cell counts and IL-1β expression levels in the acute and chronic phases. The physicochemical properties of the nanomaterials investigated in this study are presented in Supporting Information (Table S1). All samples were long fibers with a mean length greater than 10 m. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images confirmed that the CNFs (CNFtang1) and four types of MWCNT (CNTtang1-CNTtang4) were composed of tangled individual fibers, while the long rigid MWCNTs (CNTlong1) and asbestos fibers (amosite and crocidolite) were straight (Figure 1b). The diameter of the tangled fibers (CNFtang1 and the four tangled MWCNTs) was smaller than that of the straight individual fibers (CNTlong1, amosite, and crocidolite). None of the samples exhibited endotoxin contamination. 7


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Figure 1. The study design and morphology of the test materials. (a) A schematic depiction of the study design used to investigate the effect of rigidity in CNFs and MWCNTs. Eight types of HARN were injected into the pleural space of ICR mice and the rigidity of each material was correlated with inflammogenic potential, which was classified into Tier 1 (minimal-to-mild inflammation), Tier 2 (moderate inflammation), and Tier 3 (severe inflammation) based on inflammatory cell counts and IL-1β expression levels in the acute and chronic phases. (b) SEM (the large square images) and TEM (the small square inserts) images of the test materials.

The rigidity of the test materials and correlation with diameter. The rigidity of nanomaterials is an emerging concern in nanotoxicology, and recent efforts to establish international standards 8


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for the measurement of rigidity have made it possible to quantitatively evaluate its role in toxicity. We measured the rigidity of the samples in terms of their bending ratio (Db) and static bending persistence length (SBPL) in accordance with the methods published by the International Standards Organization (ISO/TS 11888:2017).15,20 The Db of CNFtang1 and the four tangled MWCNTs was less than 0.66, while that of CNTlong1, amosite, and crocidolite was greater than 0.97 (Figure 2a). The SBPL of the tangled fibers was less than 0.87, while that of the straight individual fibers was greater than 1.09 (Figure 2b). In the experiment to determine which structural parameters were most strongly related to rigidity, the diameter of the test materials demonstrated an excellent positive correlation with both Db and SBPL (Figures 2c & d). The Gompertz non-linear regression curve fit also produced excellent correlations (Figure S1). However, other physicochemical parameters such as purity, length, and the IG/ID ratio of Raman data had no clear correlation with either rigidity parameter (data not shown).

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Figure 2. Rigidity of the test materials and the correlation with fiber diameter: (a) bending ratio (Db) and (b) static bending persistence length (SBPL). Plotting fiber diameter against (c) Db and (d) SBPL generated high Pearson’s correlation coefficients (0.6813 for Db and 0.6404 for SBPL). The Gompertz non-linear curve fit for Figs. 2c and d is presented in Figure S1.

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Pleural inflammation due to the test materials in the acute phase and the correlation with rigidity. The number of total cells, macrophages, and granulocytes in the pleural lavage fluid (PLF) all demonstrated a dose-dependent increase, while the rigid HARNs (CNTlong1, amosite, and crocidolite) had a higher inflammogenic potential than did the tangled HARNs (Figures 3ac). However, the number of lymphocytes was not significantly affected in a dose-dependent manner (Figure 3d). Both Db and SBPL exhibited a strong correlation with the cytological PLF data, including the number of total cells, macrophages, and granulocytes (Figures 3e & f, and Table 1). Total protein levels significantly increased for all types of rigid HARN compared to the vehicle control, while the tangled HARNs demonstrated only marginal increases (Figure S2). In order to identify the pro-inflammatory cytokines related to the pathogenesis of HARNs, IL-1β and IL-6 levels were measured in the PLF supernatant (Figure 4a). The levels of IL-1β and IL-6 increased for all types of rigid HARN in a dose-dependent manner, while only marginal increases were observed for all types of tangled HARN (Figures 4b & c). Db and SBPL demonstrated a high correlation with IL-1β and IL-6 levels when analyzed using Pearson's linear regression (Figures 4d & e, and Table 1) and Boltzmann non-linear regression curves (Figure S3).

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Figure 3. Pleural inflammation due to the test materials in the acute phase and the correlation with rigidity. Cytological analysis of PLF was performed 24 h after the intrapleural injection of the test materials into ICR mice. The number of (a) total cells, (b) macrophages, (c) granulocytes, 12


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and (d) lymphocytes. Correlation plots of the (e) bending ratio (Db) and (f) SBPL against the number of granulocytes at a high dosage (5 μg/mouse). The Pearson’s correlation coefficients were 0.9296 and 0.8551 for Db and SBPL, respectively, against granulocyte number. n = 4 for each group. Significance vs. vehicle control (VEH): *p < 0.05, ** p < 0.01, *** p < 0.001.

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Figure 4. The levels of pro-inflammatory cytokines in PLF and the correlation with rigidity. (a) The hypothetical depiction of the association between rigidity and pro-inflammatory cytokine production for CNFs and MWCNTs. The levels of (b) IL-1β and (c) IL-6 in PLF were evaluated 24 h after intrapleural injection into ICR mice. Correlation plots of the (d) bending ratio (Db) and 14


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(e) SBPL against IL-1β levels at a high dosage (5 μg/mouse). The Pearson’s correlation coefficients were 0.9113 and 0.8965 for Db and SBPL, respectively, against IL-1β levels. The Boltzmann non-linear curve fit for Figs. 4d and e is presented in Figure S3. n = 4 for each group. Significance vs. vehicle control (VEH): *p < 0.05, ** p < 0.01, *** p < 0.001.

Table 1. Pearson’s correlation coefficients for the relationship between the rigidity parameters and toxicity endpoints in the in vivo toxicity study of the test materials. Toxicity

At 24 h after injection

At 4 weeks after injection

endpointsa

Db

SBPL

Db

SBPL

Total cell

0.9244

0.8430

0.4226

0.3065

Macrophages

0.9072

0.8192

-0.1156

-0.2486

Granulocytes

0.9296

0.8551

0.8514

0.8552

Lymphocytes

-0.0599

-0.2678

0.5919

0.4318

Total protein

0.9052

0.8936

0.7969

0.6587

IL-1β

0.9113

0.8965

0.2903

0.4323

IL-6

0.8193

0.8608

-0.1744

-0.2137

aToxicity

endpoints were selected from the high dosage group (5 μg/mouse).

Pleural inflammation due to the test materials in the chronic phase and the correlation with rigidity. The induction of pleural inflammation by the test materials was evaluated using cytological PLF data four weeks after injection. Among inflammatory cells, the number of granulocytes increased for all rigid HARNs (CNTlong1, amosite, and crocidolite) (Figure 5). The 15


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rigidity parameters exhibited a strong positive correlation with the number of granulocytes in the chronic phase. The threshold values of Db and SBPL at which granulocytes were recruited into the pleural space were determined to be 0.97 and 1.08, respectively (Figures 5e & f). The levels of total protein, which is a marker for vascular permeability, increased for rigid fibers and exhibited a high correlation with the rigidity measures (Figure S4 and Table 1). In contrast, IL1β and IL-6 levels were comparable to those of the vehicle control (Figure S5).

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Figure 5. Pleural inflammation caused by the test materials in the chronic phase and the correlation with rigidity. Pleural inflammation was evaluated four weeks after the intrapleural injection of the test materials at 5 μg/mouse into ICR mice. The number of (a) total cells, (b) 17


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macrophages, (c) granulocytes, and (d) lymphocytes. Correlation plots of the (e) bending ratio (Db) and (f) SBPL against the number of granulocytes at a high dosage (5 μg/mouse). The Pearson’s correlation coefficients were 0.8514 and 0.8552 for Db and SBPL, respectively, against granulocyte number. n = 4 for each group. Significance vs. vehicle control (VEH): *p < 0.05, ** p < 0.01, *** p < 0.001.

Histological analysis of the visceral and parietal pleura. In order to assess histological changes in the visceral and parietal pleura, hematoxylin and eosin staining and picrosirius red (PSR) staining were conducted for lung and chest wall tissue collected from non-lavaged mice 24 h and four weeks after the injection of the test materials. Twenty-four hours after injection, all of the tested HARNs produced small inflammatory cell aggregations without collagen deposition on the surface of lungs (visceral pleura) and on the inner surface of the chest wall (parietal pleura; Figure 6). Four weeks after injection, only the rigid HARNs (CNTlong1 and the asbestos fibers) produced large granulomas with significant collagen deposition on the surface of the lungs and the inner surface of the chest wall (Figure 6). Rigid HARNs were distributed widely in the areas of cell proliferation, while most of the tangled CNTs were unattached to the surface of the pleura and did not stimulate the pleural cells.

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Figure 6. Representative histological lesions on the visceral and parietal pleura 24 h and 4 weeks after a single intrapleural injection of HARNs (5 μg/mouse). Four weeks after the injection, rigid MWCNTs (CNTlong1) induced significant collagen deposition and large granulomas containing MWCNTs (indicated by the arrowhead) in both the visceral and parietal pleura, while CNTtang1 produced only minimal tissue reaction. The sections were stained with hematoxylin and eosin (upper panel) or picrosirius red, which stains the collagen bright red (lower panel). All pictures were taken at a magnification of × 200. L = lung; CW = chest wall.

SEM observations of the surface of the parietal pleura. The surface of the parietal pleura was investigated using SEM four weeks after the intrapleural injection of the test materials. The 19


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vehicle control and tangled HARNs exhibited a normal mesothelial layer with no inflammatory cell aggregations, while the rigid HARNs (CNTlong1, amosite, and crocidolite) produced aggregations of inflammatory cells of various sizes. Some rigid fibers were found within the aggregated inflammatory cells demonstrating frustrated phagocytosis (Figure 7).

Pro-inflammatory cytokines in differentiated THP-1 cells and the correlation with rigidity. High doses of the eight test materials led to a cell viability of more than 70%, and no cytotoxicity was observed below the medium dose (Figure S6). All of the test materials increased IL-1β, IL-6, and TNF- levels in a dose-dependent manner, with the IL-1β levels slightly higher for the rigid HARNs than for the tangled ones (Figure S7). The rigidity measures exhibited a strong positive correlation with IL-1β levels (Figure S8 and Table S2).

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Figure 7. SEM images of the parietal pleura of ICR mice four weeks after the pleural injection of test materials at a dosage of 5 µg/mouse. The insert figures in a-f are high magnification images of the white rectangles. High magnification images for g-i are presented as j-l. The white arrows indicate fibers.

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SEM observations of the interactions between the test materials and differentiated THP-1 cells. SEM observations of differentiated THP-1 cells treated with HARNs (Figure 8) found that minimum of the tangled HARNs entered or pierced the differentiated THP-1 cells. However, the presence of all types of rigid HARN (CNTlong1, amosite, and crocidolite) led to frustrated phagocytosis and the piercing of cells.

Figure 8. SEM images of differentiated THP-1 cells 24 h after treatment with the test materials at a dosage of 25 µg/mL. Note that CNFtang1 and CNTtang1-CNTtang4 did not pierce the 22


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macrophages but CNTlong1, amosite, and crocidolite fibers did and induced frustrated phagocytosis. Fibers are indicated with white arrows. Scale bar = 2 μm.

Discussion In this study, we aimed to determine the threshold MWCNT rigidity values for the occurrence of asbestos-like pathogenicity by analyzing the relationship between physicochemical parameters and toxicity endpoints. HARNs can produce several forms of lung damage, such as granulomatous inflammation, fibrosis, and lung cancer, due to their asbestos-like physicochemical properties.21,22 Of these physicochemical properties, aspect ratio, length, and biopersistence have previously been suggested as key parameters related to the pathogenicity of HARNs, with an aspect ratio greater than 3:1 and a length greater than 5 μm reported as critical values.17,21,23 However, there is a need for the further qualitative and quantitative assessment of physicochemical parameters in relation to HARN toxicity as a part of risk assessment and safetyby-design approaches. The next stage in the IARC’s carcinogen classification system for MWCNTs plans to cover various types of HARN (including SWCNTs, MWCNTs, and CNFs) based on the association between their physiochemical properties and toxicity endpoints.6,24,25 In this study, the rigidity and length of HARNs were proven to be major physicochemical determinants of toxicity. MWCNTs with a Db and SBPL greater than 0.97 and 1.09, respectively, demonstrated similar toxicity to asbestos fibers, while CNFs and MWCNTs with a Db and SBPL lower than 0.66 and 0.87 did not exhibit any asbestos-like pathogenicity, although they would be classified as hazardous if judged by the length and aspect ratio (Figure 9). 23


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Figure 9. A schematic depiction of the quantitative correlations found in our study between the rigidity of HARNs and their inflammogenic potential using intraperitoneal injection models. The tangled HARNs produced only minor cell damage, while the rigid HARNs produced high cytokine levels by piercing the phagocytes and inducing frustrated phagocytosis, which mimics the pathogenesis of hazardous asbestos fibers. The threshold rigidity for asbestos-like pathogenicity was a Db of 0.97 and an SBPL of 1.08.

Rigidity is an indicator of fiber curliness. Recent studies suggested Db and SBPL as quantitative parameters that can be calculated based on the mesoscopic shape of fibers.13,26 The rigidity of MWCNTs is a complex issue because the more rigid MWCNTs are, the better their thermal and electrical properties,26,27 but they may also demonstrate toxic properties similar to those of asbestos. In this study, both Db and SBPL demonstrated significant positive correlations with the 24


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diameter of the test HARNs. A previous study that investigated 34 types of SWCNT reported that diameter of SWCNTs was closely related with their persistence length.28 Thus, our results indicate that the diameter of HARNs can be useful in the calculation of the rigidity of fibers, although more research is needed on the quantitative correlation between physicochemical parameters and rigidity. This study demonstrated that the rigidity of MWCNTs is the most suitable indicator of asbestoslike pathogenicity in terms of inflammogenic potential and IL-1β expression levels. Two recent studies have suggested that the rigidity of MWCNTs can be a critical factor in asbestos-like lung damage, involving the activation of mesothelial cells, macrophages, and fibroblasts.14,15 However, these studies were unable to provide quantitative proof for this hypothesis, either because rigidity was not measured or because only a small range of MWCNT types was tested. In light of this, the results of the present study indicate that MWCNTs with a rigidity similar to CNTlong1 (Mitsui & Co.) can induce asbestos-like pathogenicity because the rigidity of HARNs has a strong association with frustrated phagocytosis and cell piercing.11,29-32 Past asbestos data also suggests that more rigid crocidolite fibers have a higher potential for inducing cell proliferation and fibrosis in the lungs and pleural cells than less rigid chrysotile.33 The most important mechanism underlying the toxicity of rigid HARNs is frustrated phagocytosis in macrophages. Macrophages subject to frustrated phagocytosis exhibited a slow clearance of HARNs and continuously produce pro-inflammatory cytokines and reactive oxygen species.18,29,34,35 In this way, HARNs can provoke pathological changes similar to those induced by asbestos fibers, such as granulomatous inflammation, pulmonary fibrosis, and neoplasia.22,36 25


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Therefore, high aspect ratios (i.e., greater than 3:1), smaller fiber diameters (thinner than 3 μm), and longer fibers (longer than 5 μm) have been suggested as the major pathogenic factors of HARNs.17,37,38 In addition to these factors, the present study clearly shows that HARN rigidity is another important factor determining asbestos-like pathogenicity because rigidity has a strong relationship with frustrated phagocytosis. The molecular mechanisms of toxicity via frustrated phagocytosis include NLRP3 inflammasome activation and IL-1β production.39-43 All types of HARN can be phagocytosed to form phagolysosomes but only rigid MWCNTs and asbestos fibers can produce severe lysosomal damage, which in turn leads to NLRP3 inflammasome activation and inflammatory responses.39,44 Thus, IL-1β production through this pathway has been shown to be an indicator of HARN toxicity.42,45,46 Therefore, the strong correlation between rigidity and IL-1β production in macrophages observed in this study confirms that the rigidity of fibers determines whether they can induce frustrated phagocytosis and IL-1β production. In this study, we quantitatively evaluated the role of rigidity in the toxicity of MWCNTs, and found that the rigidity determines shape (i.e., straight length) and shape determines clearance/frustrated phagocytosis. Thus, the pathogenic factor for MWCNTs is the straight length as surface area is the key modifier in poorly soluble low toxicity nanoparticles such as TiO2 and polystyrene latex beads.47,48 However, our study was limited to pleural inflammation and carbon-based HARNs, so further quantitative correlation studies on lung inflammation models involving a wider variety of HARNs may provide more useful information for future HARN risk assessment. 26


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Conclusion The results of this study show that the rigidity of fibers is highly correlated with both acute and chronic inflammation. We also determined the threshold rigidity values for the generation of inflammation in the pleural space. In vitro experiments using THP-1 macrophages also suggested that rigidity is an important factor in frustrated phagocytosis and IL-1β production, which are indicators of HARN toxicity. Therefore, MWCNTs with a similar or higher rigidity than CNTlong1 cause frustrated phagocytosis, while MWCNTs with a lower rigidity do not. This study suggests that a Db of 0.97 and an SBPL of 1.08 are the threshold values for asbestos-like pathogenicity. However, additional research using MWCNTs with rigidity values that fall between the non-inflammogenic (Db=0.66 and SBPL=0.87) and inflammogenic fibers (Db=0.97 and SBPL=1.09) in the present study is required to determine more accurate threshold values.

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Methods Panel of test materials. CNFtang1 (Carbon Nano-material Technology Co., Pohang, Korea), CNTtang1 (Kumho Petrochemical Co., Daejeon, Korea), CNTtang2 (Neone Co., Seoul, Korea), CNTtang3 (JEIO Co., Incheon, Korea), CNTtang4 (Hanwha Nanotech Co., Seoul, Korea), and CNTlong1 (Mitsui & Co., Tokyo, Japan) were purchased from commercial sources. Amosite and crocidolite fibers were purchased from General Science Corporation (Tokyo, Japan) as Union for International Cancer Control (UICC) standard reference asbestos samples. Physicochemical characterization of the test materials. The shape and size of the HARNs were characterized using SEM and TEM under the same conditions as those used in this animal experiments. The powdered form of the fibers (1 mg/mL) was dispersed in heat-inactivated fetal bovine serum (FBS) and sonicated for 1 h in a bath sonicator (Saehan-Sonic, Seoul, Korea). This solution was then diluted 10-fold (100 μg/mL) with the addition of distilled water (DW) followed by 30 min of sonication in the bath sonicator. This working solution was washed twice using centrifugation at 15,000 ×g for 15 min to remove any unbound serum components. Following this, 100 μL of the solution was placed on an SEM filter (Merck Millipore Ltd., Cork, Ireland) and dried overnight at room temperature, and 10 μL of the solution was dried at room temperature on a copper grid (Electron Microscopy Sciences, Hatfield, PA, USA) for TEM analysis. The morphology of the test materials was observed using SEM (JSM-6700F; JEOL, Tokyo, Japan) and TEM (JEM-1200EX II, JEOL, Tokyo, Japan). The mean length and diameter of the HARNs were measured for 300 fibers using a built-in analysis program. Raman spectroscopy was used on the CNFs and MWCNTs to evaluate defects using a WITec alpha300 28


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system (WITec GmbH, Ulm, Germany) with incident laser light at a wavelength of 532 nm. The surface area of the HARNs was measured with the Brunauer-Emmett-Teller method using a BELSORP-mini II (BEL Japan Inc.). To confirm the absence of endotoxin contamination of the test materials, Endpoint Chromogenic Limulus Amebocyte Lysate assays (Cambrex, Walkersville, MD, USA) were conducted according to the instruction manual at a concentration of 600 μg/mL in DW, which was the highest dose in the animal study. Evaluation of HARN rigidity. The bending force measurement by atomic force microscopy (AFM) can be more accurate method for the measurement of rigidity, but no ISO standard method is exist for this technique. While, there is an ISO standard method (ISO/TS 11888:2017) for the measurement of rigidity by SEM.15,20 Thus, we used this ISO standard method for the evaluation of rigidity of HARNs. SEM images of the test materials were taken at a magnification of ×10,000 and the length from end to end (R) and along the axis (L) of the individual strands were measured using Gatan DigitalMicrograph 3.11.0 software (Gatan, Pleasanton, CA, USA). More than 10 strands that were clearly separated from the others were measured for each test material. Db and SBPL were calculated using the following equations: 𝐷𝑏 = 𝑅2/𝐿2 𝑆𝐵𝑃𝐿 = (𝐷𝑏 × 𝐿) 2 Preparation of the samples for in vivo and in vitro experiments. To develop an optimal dispersion protocol for the test materials in phosphate-buffered saline (PBS) or RPMI-1640 culture medium for the in vivo and in vitro experiments, a previously described method was employed with slight modifications.18 The stock solution was prepared by dispersing 1 mg/mL of 29


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the test materials in either 30% heat-inactivated mouse serum for the in vivo experiments or FBS (Corning Life Sciences, Corning, NY, USA) for in vitro experiments and sonicated for 1 h in a bath sonicator to break up agglomerations. PBS or RPMI-1640 medium (Gibco Laboratories, Grand Island, NY, USA) was then added to obtain the final treatment doses (10, 25, and 50 μg/mL for the in vivo experiments and 12.5, 25, and 50 μg/mL for the in vitro experiments). The concentration of the mouse serum for the in vivo research was lower than 3%. To evaluate the dispersity of the final samples, glycerin (Sigma-Aldrich, Saint Louis, MI, USA) and 100 μg/mL of the samples were added at a ratio of 1:1 to reduce the flow of the fibers. These changes were observed using light microscopy (Nikon Inc., Tokyo, Japan). To demonstrate the efficacy of the dispersion protocol, test materials in DW without the dispersion medium and in PBS without the dispersion medium were also compared (Figure S9). Intrapleural injection of the test materials. Six-week-old female ICR mice (Samtako, Gyeonggi-Do, Korea) were purchased and acclimatized for one week before the experiment began. The mice were maintained and handled according to guidelines approved by the Institutional Animal Care and Use Committee at Dong-A University. The mice were housed in a micro-ventilation cage system (MVCS; Three Shine Inc., Daejeon, Korea) under controlled conditions (a temperature of 22 ± 1 ºC, humidity of 50 ± 10%, and a 12 h light/dark cycle). Food and water were supplied ad libitum. The intrapleural injection of the HARNs was conducted according to a previously described method.17 The mice were anesthetized with isoflurane (Piramal Critical Care, Bethlehem, PA, USA) using a VetEquip rodent anesthesia system (Pleasanton, CA, USA). Suspensions of the test materials (100 μL) were then injected into the pleural space. The doses for the acute experiments (24 h after injection) were 1, 2.5, and 5 30


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μg/mouse, while 5 μg/mouse was used for the chronic experiments (four weeks after a single injection). PBS with 3% heat-inactivated mouse serum was used as a vehicle control. Four mice per group were used for the evaluation of inflammation in the pleural space, three mice per group were used for SEM analysis of ultrastructural changes in the chest wall, and three mice per group were used for histological analysis of the visceral and parietal pleura.

Cytological assessment of the inflammogenic potential of HARNs in the pleura. The mice were sacrificed after 24 h and four weeks by removing blood from the inferior vena cava under deep isoflurane (Primal Critical Care) anesthesia. The pleural space was lavaged three times with 1 mL of PBS. The supernatant of the first lavage was stored separately for the measurement of cytokine levels and for biochemical analysis. The cellular fractions of the three lavages were pooled in 1 mL of PBS with 10% FBS (Corning Life Sciences). The number of nucleated cells was then counted using a NucleoCounter (Chemometec, Allerod, Denmark). For differential cell counting, the cytospin method was employed by attaching 4 × 104 cells to a glass slide using a cytocentrifuge (Hanil Cellspin, Incheon, Korea). At least 300 nucleated cells were then counted after staining with Diff-Quik (Baxter, McGaw Park, IL, USA). Biochemical and pro-inflammatory cytokine analysis of the PLF. The levels of total protein in the PLF were measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The levels of pro-inflammatory cytokines, including IL-1β and IL-6, were measured in the PLF using a Duoset ELISA kit (R&D Systems, Minneapolis, MN, USA).

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Histological analysis and collagen staining of the visceral and parietal pleura. After the sacrifice of the mice, lung and chest wall tissue was fixed with 10% neutral buffered formaldehyde for 24 h. Decalcification was then conducted with ethylenediaminetetracetic acid (EDTA) for the histological analysis of the bone-containing chest wall. EDTA (10% in DW, pH 7.4) was applied for two weeks, with the solution replaced once every four days. The tissue was then trimmed, processed, and waxed according to routine histological methodological practice. Paraffin blocks were sectioned at thicknesses of 3 µm and stained with hematoxylin and eosin. To analyze collagen deposition, PSR staining was employed according to a previously described method.18 SEM analysis of ultrastructural changes in the chest wall. To assess the ultrastructural changes in the chest wall, non-lavaged parietal pleura tissue was dissected and washed with icecold 0.1 M PBS (pH 7.4), followed by fixation under incubation with 2% glutaraldehyde (SigmaAldrich) in 0.1 M PBS overnight. The tissue was then washed three times with 0.1 M of PBS for 10 min. A second fixation step was conducted by incubating the tissue with 1% osmium tetroxide (Ted Pella, Redding, CA, USA) for 1 h, followed by washing with DW for three times (10 min for each wash). The tissue was dehydrated using an ethanol/water solution (30–100%) and submerged in isoamyl acetate (Sigma-Aldrich). The tissue was then immediately dried using liquid CO2 replacement in a Polaron E3000 critical-point dryer (Quorum Technologies, East Sussex, UK). The surface of the specimens was sputter-coated with a platinum layer and observed using SEM.

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In vitro treatment of differentiated THP-1 cells with the test HARNs. To determine the effect of the HARNs’ structural factors on phagocytosis by macrophages, differentiated THP-1 cells were incubated with the HARNs, and the pro-inflammatory cytokine levels and ultrastructural changes were monitored. THP-1 cells (ATCC, Manassas, VA, USA) were cultured in RPMI1640 medium (Gibco Laboratories) supplemented with 10% FBS (Corning Life Sciences), 1% penicillin/streptomycin, and 1% L-glutamine at 37 ºC with 5% CO2. Before treatment with the HARNs, the THP-1 cells (5 × 105 cells/mL) were incubated with 10 ng/mL of phorbol-12myristate-13-acetate (PMA; Sigma-Aldrich) in 96-well plates for two days to allow time for them to differentiate into macrophage-like cells. The cells were then washed three times with pre-warmed sterile PBS and treated with HARNs at various doses (12.5, 25, and 50 μg/mL). To evaluate the cytotoxicity of the HARNs 24 h after treatment, the cells were washed three times with pre-warmed PBS, and a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-((4sulfophenyl)-2H-tetrazolium) chloride (MTS; Promega, Madison, WI, USA) assay kit was used according to the instruction manual. The nanomaterial-free supernatant was collected using centrifugation twice at 15,000 ×g for 20 min, and the levels of pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α, were measured using a Duoset ELISA kit (R&D Systems). Ultrastructural analysis of the interaction between the test materials and cells. THP-1 cells treated with PMA (10 ng/mL) were seeded on coverslips (Nunc, Roskilde, Denmark) in 6-well plates at a density of 5 × 105 cells/mL and incubated for two days. The cells were then washed three times with 0.1 M PBS for 10 min, and 2% glutaraldehyde (Sigma-Aldrich) in 0.1 M PBS was applied for 1 h. Following this, the cells were washed three times with 0.1 M PBS for 10 min and incubated with 1% osmium tetroxide (Ted Pella) for 20 min. The cells were then 33


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washed three times with DW for 10 min and dehydrated with an ethanol/water solution (30– 100%). The cells were dried via the evaporation of hexamethyldisilazane (Sigma-Aldrich), and their surface was sputter-coated with a thin platinum layer and observed using SEM. Statistical analysis. All of the figures are presented as the mean ± standard error of the mean. The data was analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). A one-way analysis of variance (ANOVA) with post hoc Tukey’s pairwise comparisons was employed to compare the groups. The correlations between the physicochemical parameters and toxicity endpoints were calculated based on the fit of a linear regression curve using the nonparametric Pearson’s correlation test and the fit of a non-linear regression curve using Gompertz or Boltzmann curves. A p < 0.05 was considered statistically significant. Fig. 2, which displays similar behavior to the growth curves for bacteria and cancer cells, was analyzed with the Gompertz equation, while Fig. 4 was analyzed with Boltzmann’s sigmoidal equation.49,50 For the threshold value derived from the fit of a non-linear regression curve in Fig. 4, we used the half-maximal concentration in a similar approach to the results of previous research.51,52

Additional Content Supporting Information The following files are available free of charge on the ACS Publications website at DOI:xxx. The fitting curve of diameter of fibers against Db or SBPL using non-linear Gompertz equation.

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The total protein levels in the PLF 24 h after the intrapleural injection of the test materials into ICR mice. Boltzmann fitting curve for the correlation plot of bending ratio or SBPL against the levels of IL1β of high dose group (5 μg/mouse).

The total protein, IL-1β, and IL-6 levels in the PLF four weeks after the intrapleural injection of the test materials into ICR mice. The cell viability and levels of pro-inflammatory cytokines after treatment with the test materials of differentiated THP-1 cells. Correlation plots of the bending ratio and SBPL against IL-1 levels in differentiated THP-1 cells treated with the test materials. Dispersion data for the test materials for the in vivo and in vitro assays. Physicochemical property data for the test materials. Pearson’s correlation coefficients for the association between the parameters of rigidity and the in vitro toxicity endpoints of the test materials (PDF).

Corresponding Authors Correspondence and requests for materials should be addressed to Y.S.H. (email: [email protected]) or W.-S.C. (email: [email protected]). 35


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Authors Contributions IJU, KSS, YSH, and W.-S.C. conceived the idea including the development and design of methodology. D.-K.L., Y.H., S.J., S.-H.K., S.L., and S.-M.K. performed in vitro and in vivo studies. A.K. and W.S.Y. evaluated the rigidity of test materials. D.-K.L., YSH, and W.-S.C. wrote the manuscript with input from other co-authors.

Acknowledgements This research was supported by the BB21+ Project in 2018 and the Industrial Strategic Technology Development Program (10059132, Promotion and Utilization of Safety Evaluation Based Technology Development for Nanomaterials and Nanoproducts) through the Korea Evaluation Institute of Industrial Technology, Korean Ministry of Trade, Industry & Energy.

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Threshold Rigidity Values for the Asbestos-Like Pathogenicity of High

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