-
The synthesized MNPs were visible fine powder. Based on the position and relative intensity of diffraction peaks in the XRD patterns, the composite materials were determined as CuO, Al2O3, ZnO, and PbS. In addition, the TEM images revealed that nano-CuO was nearly spherical, the two types of nano-Al2O3 were sheet and short-rod-shaped, and nano-ZnO and nano-PbS were irregularly shaped (Figures 2, 3). Through the calculation and classification of the directional diameters of 200 particles in the TEM images, the dominant particle sizes of three batches of CuO-NPs were determined to be approximately 40, 80, and 100 nm, respectively (Figure 2). For convenience in the present study, they were named as CuO-40, CuO-80, and CuO-100. The average crystallite sizes of CuO-40, CuO-80, and CuO-100 were calculated by Scherrer's formula[24] using the full width at half maximum data of XRD peaks to be 19.61, 27.69, and 24.50 nm in sequence. The corresponding BET specific surface areas by Micromeritics ASAP 2020 surface area analyzer were 8.6044 ± 0.6017, 5.8084 ± 0.5629, and 1.4979 ± 0.1129 m2/g respectively. Because of their small sizes, nanomaterials aggregate easily, which considerably limits their toxic effects. To prevent agglomeration and facilitate observation in the present study, nano-samples were dispersed in 0.5 % HPMC solution. A pre-test called 'a standing test of nanomaterials in dispersant solution', aimed to test the stability of CuO-NPs including CuO-40, CuO-80, and CuO-200, revealed that the CuO-NPs (500 mg/L)-HPMC (0.5% and 1.0%) suspensions had a relatively uniform concentrations at different times (0, 3, 24, and 48 h) and at different depths below the surfaces of the suspensions (0, -10, and -20 cm). The observations indicated that the 0.5% HPMC was an optional dispersant solution for the test nanomaterials. In view of this, the 0.5% HPMC was selected for the preparation of the MNPs administration suspension preparation in the present study.
Figure 2. TEM images (A, B, and C) and the granulometric distribution (a, b, and c) of CuO-NPs [CuO-40 (A, a), CuO-80 (B, b), and CuO-100 (C, c)]. A. scale bar, 100 nm; B. scale bar, 100 nm; C. scale bar, 200 nm. The deeper coloring in image was because of the overlap of particles with each other. The particle size distribution graphs showed the dominant particle sizes of CuO-40 (a), CuO-80 (b), and CuO-100 (c) were approximately 40, 80, and 100 nm, respectively. For convenience, they were named as CuO-40, CuO-80, and CuO-100. TNP here meant total number of particle.
-
Two days after seeding, cells began to migrate from the edges of the pulmonary tissue (Figure 4A). The first cell appeared spindle-shaped. Cells proliferated vigorously in the following days and grew to form a monolayer to the 5th day (Figure 4C). Immunocytochemical detection of factor Ⅷ-related antigen in cells presented a positive result. Passaged PCECs grew rapidly and appeared in compact spindle and polygonal shapes with ovate nuclei (Figure 4D). Cell viability by resazurin method showed the subcultured PCECs exhibited logarithmic proliferation on the 2nd day and a peak viability value could be observed up to the 5th day. A549 cells grew in polygonal forms with a flower cluster arrangement. In model preparation activity, PCECs came to confluence at 6 days after seeding, and they were block-like and arranged compactly (Figure 4E). A549 growing in the internal surface of insert presented polygonal and closely aligned (Figure 4F). Some models, which exhibited incomplete confluence due to luminal growing, and which exhibited mechanical injury, were eliminated. The selected model had a considerable TEER value of 183.6 ± 7.4 Ω·cm2 up to the 13th day, which lasted for over 3 days (Figure 5). The LY leakage test revealed that the model had a satisfactory P value as 0.85 ± 0.19 × 10-3 cm/min.
Figure 4. PCECs primary culture and cells growth in model. Spindle-shaped cells began to migrate from lung tissue edge 64 h after initial seeding (A, 40×). Two days later, cells proliferated and appeared slimly spindle-shaped (B, 40×). Cells became confluent and formed a monolayer at day 5 (C, 40×). Subcultured cells grew compactly and appeared polyangular with clear boundaries (D, 40×). PCECs (E, 100×) and A549 (F, 60×) growing on the outside and internal surface of the cell insert membrane separately.
-
The 1-hour IC-50 values of the MNPs against the A549 cells and the PCECs were obtained and compared (Table 1). The lower IC-50s of each of the test objects were selected and their sqrt were used as the exposure doses in the subsequent permeability impact experiment.
Table 1. Determination of the Test Dosage (mg/L)
Test Samples IC-50 (95% CI) Dose Sqrt (IC-50)a A549 PCECs CuSO4 61.8 (45.1-85.2) 57.2 (37.1-87.1) 7.6 Al2(SO4)3 63.3 (35.4-116.4) 55.5 (44.9-68.4) 7.4 Zn(CH3COO)2 95.7 (69.4-145.8) 79.3 (56.3-120.0) 8.9 Pb(NO3)2 73.2 (60.5-90.0) 62.5 (50.6-77.5) 7.9 CuO-40 79.1 (48.9-179.6) 63.4 (51.3-81.2) 8.0 CuO-80 105.5 (86.0-137.3) 77.1 (62.2-100.3) 8.8 CuO-100 144.3 (108.5-220.0) 143.4 (94.7-298.4) 12.0 nano-Al2O3 (sheet) 43.7 (38.6-49.6) 34.5 (14.5-70.9) 5.9 nano-Al2O3 (short rod-shaped) 56.2 (34.2-109.3) 51.7 (43.1-62.8) 7.2 nano-ZnO 88.4 (65.0-137.3) 63.7 (53.7-77.3) 8.0 nano-PbS 51.1 (24.7-137.9) 60.1 (47.5-79.2) 7.1 HPMC > 2, 500b > 2, 500b 50.0 Note. aThe smaller one of the two IC-50 values of A549 and PCECs was adopted. bUnder 2, 500 mg/L, no obvious cell viability change was observed. When treated with CuO-40, CuO-80, sheet and short-rod-shaped nano-Al2O3, Al2(SO4)3, and Pb(NO3)2, the P values increased to (1.30 ± 0.21) × 10-3, (1.25 ± 0.25) × 10-3, (1.18 ± 0.28) × 10-3, (1.06 ± 0.23) × 10-3, (1.15 ± 0.16) × 10-3, and (1.19 ± 0.14) × 10-3 cm/min respectively, compared with non- treatment group [(0.86 ± 0.31) × 10-3 cm/min, P < 0.05, n = 5]. The results indicated the six materials above could penetrate the ABB. The effects observed among Cu-related materials, including CuO-40, CuO-80, CuO-100, and CuSO4 were somewhat different. CuO-40 and CuO-80 exhibited similar ABB permeability effects, and both effects were stronger than the effects observed in CuO-100 and CuSO4. No difference was observed between Al2(SO4)3 and sheet and short-rod-shaped nano-Al2O3, which all increased ABB permeability. In addition, Pb(NO3)2 increased ABB permeability more than nano-PbS. Both nano-ZnO and Zn(CH3COO)2 had no influence on ABB permeability. HPMC, which was the quality control, exhibited no significant difference from the control, which indicated that the introduction of 0.5% HPMC had no adverse effect on the model. Based on all the results above, some but not all nanomaterials tested had a stronger effect on ABB permeability than their bulk chemical counterparts. We also observed that the effects of CuO-NPs were associated with the sizes. Small CuO-40 and CuO-80 displayed greater impact on ABB permeability than CuO-100. In nano-ZnO, sheet and short-rod-shaped nano-Al2O3, no differences were observed between them and their chemical counterparts in their effects on ABB permeability. In the present study, Pb(NO3)2 particularly exhibited a greater effect compared to nano-PbS (Figure 6).
doi: 10.3967/bes2019.078
Comparative Toxicity of Nanomaterials to Air-blood Barrier Permeability Using an In Vitro Model
-
Abstract:
Objective To comparatively study the toxicity of four metal-containing nanoparticles (MNPs) and their chemical counterparts to the air-blood barrier (ABB) permeability using an in vitro model. Methods ABB model, which was developed via the co-culturing of A549 and pulmonary capillary endothelium, was exposed to spherical CuO-NPs (divided into CuO-40, CuO-80, and CuO-100 based on particle size), nano-Al2O3 (sheet and short-rod-shaped), nano-ZnO, nano-PbS, CuSO4, Al2(SO4)3, Zn(CH3COO)2, and Pb(NO3)2 for 60 min. Every 10 min following exposure, the cumulative cleared volume (ΔTCL) of Lucifer yellow by the model was calculated. A clearance curve was established using linear regression analysis of ΔTCL versus time. Permeability coefficient (P) was calculated based on the slope of the curve to represent the degree of change in the ABB permeability. Results The results found the increased P values of CuO-40, CuO-80, sheet, and short-rod-shaped nano-Al2O3, Al2(SO4)3, and Pb(NO3)2. Among them, small CuO-40 and CuO-80 were stronger than CuO-100 and CuSO4; no difference was observed between Al2(SO4)3 and sheet and short-rod-shaped nano-Al2O3; and nano-PbS was slightly weaker than Pb(NO3)2. So clearly the MNPs possess diverse toxicity. Conclusion ABB permeability abnormality means pulmonary toxicity potential. More studies are warranted to understand MNPs toxicity and ultimately control the health hazards. -
Figure 2. TEM images (A, B, and C) and the granulometric distribution (a, b, and c) of CuO-NPs [CuO-40 (A, a), CuO-80 (B, b), and CuO-100 (C, c)]. A. scale bar, 100 nm; B. scale bar, 100 nm; C. scale bar, 200 nm. The deeper coloring in image was because of the overlap of particles with each other. The particle size distribution graphs showed the dominant particle sizes of CuO-40 (a), CuO-80 (b), and CuO-100 (c) were approximately 40, 80, and 100 nm, respectively. For convenience, they were named as CuO-40, CuO-80, and CuO-100. TNP here meant total number of particle.
Figure 4. PCECs primary culture and cells growth in model. Spindle-shaped cells began to migrate from lung tissue edge 64 h after initial seeding (A, 40×). Two days later, cells proliferated and appeared slimly spindle-shaped (B, 40×). Cells became confluent and formed a monolayer at day 5 (C, 40×). Subcultured cells grew compactly and appeared polyangular with clear boundaries (D, 40×). PCECs (E, 100×) and A549 (F, 60×) growing on the outside and internal surface of the cell insert membrane separately.
Table 1. Determination of the Test Dosage (mg/L)
Test Samples IC-50 (95% CI) Dose Sqrt (IC-50)a A549 PCECs CuSO4 61.8 (45.1-85.2) 57.2 (37.1-87.1) 7.6 Al2(SO4)3 63.3 (35.4-116.4) 55.5 (44.9-68.4) 7.4 Zn(CH3COO)2 95.7 (69.4-145.8) 79.3 (56.3-120.0) 8.9 Pb(NO3)2 73.2 (60.5-90.0) 62.5 (50.6-77.5) 7.9 CuO-40 79.1 (48.9-179.6) 63.4 (51.3-81.2) 8.0 CuO-80 105.5 (86.0-137.3) 77.1 (62.2-100.3) 8.8 CuO-100 144.3 (108.5-220.0) 143.4 (94.7-298.4) 12.0 nano-Al2O3 (sheet) 43.7 (38.6-49.6) 34.5 (14.5-70.9) 5.9 nano-Al2O3 (short rod-shaped) 56.2 (34.2-109.3) 51.7 (43.1-62.8) 7.2 nano-ZnO 88.4 (65.0-137.3) 63.7 (53.7-77.3) 8.0 nano-PbS 51.1 (24.7-137.9) 60.1 (47.5-79.2) 7.1 HPMC > 2, 500b > 2, 500b 50.0 Note. aThe smaller one of the two IC-50 values of A549 and PCECs was adopted. bUnder 2, 500 mg/L, no obvious cell viability change was observed. -
[1] Wang ZL. Splendid one-dimensional nanostructures of zinc oxide: a new nanomaterial family for nanotechnology. ACS Nano, 2008; 2, 1987-92. doi: 10.1021/nn800631r [2] Osmond MJ, McCall MJ. Zinc oxide nanoparticles inmodern sunscreens: an analysis of potential exposure and hazard. Nanotoxicology, 2010; 4, 15-41. doi: 10.3109/17435390903502028 [3] Laha D, Pramanik A, Laskar A, et al. Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage. Mater Res Bull, 2014; 59, 185-91. doi: 10.1016/j.materresbull.2014.06.024 [4] Goh YF, Alshemary AZ, Akram M, et al. Bioactive glass: an in-vitro comparative study of doping with nanoscale copper and silver particles. Int J Appl Glass Sci, 2014; 5, 255-66. doi: 10.1111/ijag.12061 [5] Versavel MY, Haber JA. Lead antimony sulfides as potential solar absorbers for thin film solar cells. Thin Solid Films, 2007; 515, 5767-70. doi: 10.1016/j.tsf.2006.12.077 [6] Maximous N, Nakhla G, Wong K, et al. Optimization of Al2O3/PES membranes for wastewater filtration. Sep Purif Technol, 2010; 73, 294-301. doi: 10.1016/j.seppur.2010.04.016 [7] Rani VS, Kumar AK, Kumar P, et al. Pulmonary toxicity of copper oxide (CuO) nanoparticles in rats. J Med Sci, 2013; 13, 571-77. doi: 10.3923/jms.2013.571.577 [8] Lai XF, Zhao H, Zhang Y, et al. Intranasal delivery of copper oxide nanoparticles induces pulmonary toxicity and fibrosis in C57BL/6 mice. Sci Rep, 2018; 8, 4499. doi: 10.1038/s41598-018-22556-7 [9] Chuang HC, Chuang KJ, Chen JK, et al. Pulmonary pathobiology induced by zinc oxide nanoparticles in mice: a 24-hour and 28-day follow-up study. Toxicol Appl Pharm, 2017; 327, 13-22. doi: 10.1016/j.taap.2017.04.018 [10] Lin W, Stayton I, Huang YW, et al. Cytotoxicity and cell membrane depolarization induced by aluminum oxide nanoparticles in human lung epithelial cells A549. Toxico Enviro Chem, 2008; 90, 983-96. doi: 10.1080/02772240701802559 [11] Li Q, Hu X, Bai Y, et al. The oxidative damage and inflammatory response induced by lead sulfide nanoparticles in rat lung. Food Chem Toxicol, 2013; 60, 213-17. doi: 10.1016/j.fct.2013.07.046 [12] Yasuo M, Hiroto I, Yukiko Y, et al. Evaluation of pulmonary toxicity of zinc oxide nanoparticles following inhalation and intratracheal instillation. Int J Mol Sci, 2016; 17, 1241. doi: 10.3390/ijms17081241 [13] Jing X, Park JH, Peters TM, et al. Toxicity of copper oxide nanoparticles in lung epithelial cells exposed at the air–liquid interface compared with in vivo assessment. Toxicol in vitro, 2015; 29, 502-11. doi: 10.1016/j.tiv.2014.12.023 [14] Thomas L, Francoise R, Bénédicte T, et al. Predicting the in vivo pulmonary toxicity induced by acute exposure to poorly soluble nanomaterials by using advanced in vitro methods. Part Fibre Toxicol, 2018; 15, 25. doi: 10.1186/s12989-018-0260-6 [15] Legendre A, Froment P, Desmots S, et al. An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential. Biomaterials, 2010, 31, 4492-505. doi: 10.1016/j.biomaterials.2010.02.029 [16] Qosa H, Mohamed LA, Al Rihani SB, et al. High-throughput screening for identification of blood-brain barrier integrity enhancers: a drug repurposing opportunity to rectify vascular amyloid toxicity. J Alzheimers Dis, 2016; 53, 1499-516. doi: 10.3233/JAD-151179 [17] Adriani G, Ma D, Pavesi A, et al. Modeling the blood-brain barrier in a 3D triple co-culture microfluidic system. IEEE 2015 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) - Milan (2015.8.25-2015.8.29). 2015; 338-341. [18] Zhen C, Huan M, Gengmei X, et al. Acute toxicological effects of copper nanoparticles in vivo. Toxicol lett, 2006; 163, 109-20. doi: 10.1016/j.toxlet.2005.10.003 [19] Gao RD, Cao J, Shan LU, et al. Primary culture, identification and in vitro angiogenesis of mouse pulmonary microvascular endothelial cells. Chin J Pathophysiol, 2012; 28, 186-8. (In Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgblslzz201201037 [20] Hermanns MI, Unger RE, Kai K, et al. Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro. Lab Invest, 2004; 84, 736-52. doi: 10.1038/labinvest.3700081 [21] Srinivasan B, Kolli AR, Esch MB, et al. TEER measurement techniques for in vitro barrier model systems. J Lab Autom, 2015; 20, 107. doi: 10.1177/2211068214561025 [22] Culot M, Lundquist S, Vanuxeem D, et al. An in vitro blood-brain barrier model for high throughput (HTS) toxicological screening. Toxicol in Vitro, 2008; 22, 799-811. [23] Siflinger-Birnboim A, Vecchio PJ, Del, Cooper JA, et al. Molecular sieving characteristics of the cultured endothelial monolayer. J Cell Physiol, 2010; 132, 111-17. doi: 10.1002-jcp.1041320115/ [24] Khawal HA, Gawai UP, Dole BN. Substitutional effect of Ni on different properties of ZnO nanocrystals. AIP Conference Proceedings [AIP Publishing LLC NANOFORUM 2014 - Rome, Italy (22–25 September 2014)], - Substitutional effect of Ni on different properties of ZnO nanocrystals. 2015, 1665; 050140. [25] Nel A, Xia T, Lutz Mädler, et al. Toxic potential of materials at the nano level. Science, 311. [26] Girigoswami K. Toxicity of metal oxide nanoparticles. Adv Exp Med Biol, 2018; 1048, 99-122. doi: 10.1007/978-3-319-72041-8_7 [27] Koivisto AJ, Aromaa M, Koponen IK, et al. Workplace performance of a loose-fitting powered air purifying respirator during nanoparticle synthesis. J Nanopart Res, 2015; 17, 177. doi: 10.1007/s11051-015-2990-9 [28] Ghio AJ, Gilbey JG, Roggli VL, et al. Diffuse alveolar damage after exposure to an oil fly ash. Am J Resp Crit Care, 2001; 164, 1514-18. doi: 10.1164/ajrccm.164.8.2102063 [29] Foster KA, Oster CG, Mayer MM, et al. Characterization of the A549 cell line as a type Ⅱ pulmonary epithelial cell model for drug metabolism. Exp Cell Res, 1998; 243, 359-66. doi: 10.1006/excr.1998.4172 [30] Ahamed M, Siddiqui MA, Akhtar MJ, et al. Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells. Biochem Bioph Res Co, 2010; 396, 578-83. doi: 10.1016/j.bbrc.2010.04.156 [31] Moschini E, Gualtieri M, Colombo M, et al. The modality of cell–particle interactions drives the toxicity of nanosized CuO and TiO2 in human alveolar epithelial cells. Toxicol Lett, 2013; 222, 102-16. doi: 10.1016/j.toxlet.2013.07.019 [32] Gosens I, Cassee FR, Zanella M, et al. Organ burden and pulmonary toxicity of nano-sized copper (Ⅱ) oxide particles after short-term inhalation exposure. Nanotoxicology, 2016; 10, 1084-95. doi: 10.3109/17435390.2016.1172678 [33] Park EJ, Lee GH, Shim JH, et al. Comparison of the toxicity of aluminum oxide nanorods with different aspect ratio. Arch Toxicol, 2015; 89, 1771-82. doi: 10.1007/s00204-014-1332-5 [34] Pauluhn J. Pulmonary toxicity and fate of agglomerated 10 and 40 nm aluminum oxyhydroxides following 4-week inhalation exposure of rats: toxic effects are determined by agglomerated, not primary particle size. Toxicol Sci, 2009; 109, 152-67. doi: 10.1093/toxsci/kfp046 [35] Warheit DB, Sayes CM, Reed KL. Nanoscale and fine zinc oxide particles: can in vitro assays accurately forecast lung hazards following inhalation exposures? Environ Sci Technol, 2009; 43, 7939-45. doi: 10.1021/es901453p [36] Cho WS, Duffin R, Poland CA, et al. Metal oxide nanoparticles induce unique inflammatory footprints in the lung: important implications for nanoparticle testing. Environ Health Persp, 2010; 118, 1699-706. doi: 10.1289/ehp.1002201 [37] Sharma V, Singh P, Pandey AK, et al. Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles. Mutat Res-Gen Tox En, 2012; 745, 84-91. doi: 10.1016/j.mrgentox.2011.12.009 [38] Chusuei CC, Wu CH, Mallavarapu S, et al. Cytotoxicity in the age of nano: the role of fourth period transition metal oxide nanoparticle physicochemical properties. Chem Biol Interact, 2013; 206, 319-26. doi: 10.1016/j.cbi.2013.09.020 [39] Hanagata N, Zhuang F, Connolly S, et al. Molecular responses of human lung epithelial cells to the toxicity of copper oxide nanoparticles inferred from whole genome expression analysis. ACS Nano, 2011; 5, 9326-38. doi: 10.1021/nn202966t