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 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.
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.
Table 1. Determination of the Test Dosage (mg/L)
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).