Threshold Rigidity Values for the Asbestos-like Pathogenicity. of High-Aspect-Ratio Carbon Nanotubes in a Mouse Pleural. Inflammation Model

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1 Supporting information for 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 and Merchandise Testing Institute, 8, Gaetbeol-ro 145 beon-gil, Yeonsugu, Incheon 21999, 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 *Correspondence and requests for materials should be addressed to Y.S.H. ( yunsuk.huh@inha.ac.kr) or W.-S.C. ( wcho@dau.ac.kr).

2 Figure S1. The fitting curves for fiber diameter against (a) Db and (b) SBPL using the Gompertz equation in non-linear regression analysis, leading to r 2 values of 0.76 for Db and 0.74 for SBPL.

3 Figure S2. The levels of total protein in the pleural lavage fluid at 24 h after intrapleural injection of test materials to ICR mice. n = 4 for each group. Significance vs. vehicle control (VEH) * p < 0.05, ** p < 0.01, *** p <

4 Figure S3. Boltzmann fitting curves for the correlation plots of (a) bending ratio (Db) and (b) SBPL against IL-1β levels in the high dosage group (5 μg/mouse) in Figs. 4d and e. Boltzmann fitting yielded values of 0.82 Db (Boltzmann fit, r 2 =0.79) and 1.01 SBPL (Boltzmann fit, r 2 =0.84) for the half-maximal concentration of IL-1β.

5 Figure S4. The levels of total protein in the pleural lavage fluid at 4 wks after intrapleural injection of test materials at 5 μg/mouse to ICR mice. n = 4 for each group. Significance vs. vehicle control (VEH) * p < 0.05, ** p < 0.01, *** p <

6 Figure S5. The levels of (a) IL-1β and (b) IL-6 in pleural lavage fluid at 4 wks after intrapleural injection of test materials at 5 μg/mouse to ICR mice. n = 4 for each group.

7 Figure S6. The cell viability of test materials to differentiated THP-1 cells. Cell viability was measured by MTS assay. n = 4 for each group. Significance vs. vehicle control (VEH) * p < 0.05, ** p < 0.01, *** p <

8 Figure S7. The levels of pro-inflammatory cytokines stimulated by test materials in the differentiated THP-1 cells. The levels of (a) IL-1, (b) IL-6, and (c) TNF- were measured in the supernatant of differentiated THP-1 cells treated with test materials at 24 h post-treatment. n = 4 for each group. Significance vs. vehicle control (VEH) * p < 0.05, ** p < 0.01, *** p <

9 Figure S8. Correlation plot of (a) bending ratio or (b) SBPL against the levels of IL-1 released from differentiated THP-1 cells treated with test materials at 50 μg/ml.

10 Figure S9. Dispersion of test materials for in vivo and in vitro assay. Evaluation of dispersion was performed by light microscopy and CNFtang1, CNTtang1, and CNTlong1 were presented as representative images. Dispersion of test materials in PBS with 3% rat serum showed better dispersity compared to in DW or in PBS only. Bar scale = 200 μm.

11 Table S1. Physicochemical properties of test materials. Test materials Diameter (nm) Length (µm) a Raman (IG/ID) BET (m 2 /g) Purity (%) b Endotoxin (EU/mL) CNF tang ± <0.1 CNT tang ± >90 <0.1 CNT tang ± <0.1 CNT tang ± <0.1 CNT tang ± <0.1 CNT long ± ± >99 <0.1 Amosite 270 ± ± 1.0 ND ND ND <0.1 Crocidolite ± ± 0.9 ND ND ND <0.1 a The length of tangled fibers could not be determined due to the severe curliness. b The data of purity was provided by the manufacturer. ND, not determined.

12 Table S2. Pearson s correlation coefficients between parameters of rigidity and toxicity in vitro toxicity endpoints of test materials. Toxicity endpoints a Db SBPL IL-1β IL TNF-α a Toxicity endpoints were selected from high dose group (50 μg/ml).