Figures & data
Figure 1 Characterization of c-SWNTs.
Notes: TEM image (A). Length and diameter distribution by TEM (B). Raman spectrum (C). IG/ID by Raman was 5.50. Zeta potential was −44.1 mV. Metal impurities of Al, Fe, Ti, Cr, Co, and Ni were 0.89, 0.43, 0.30, 0.08, 0.08, 0.03 wt%, respectively.
Abbreviations: TEM, transmission electron microscopy; IG, Raman intensity of G band; ID, Raman intensity of D band; c-SWNTs, carboxylated single-walled carbon nanotubes.
Figure 2 The dissected lungs from rats after long-term intravenous injection of c-SWNTs.
Notes: The rat lungs were dissected at the end of the setting periods (30, 60, 90, and 120 days) in the two groups: the control group (A1–A4, 5.00 wt% glucose solution, 1 mL per rat) and the c-SWNTs group (B1–B4, 2.0 mg of c-SWNTs/kg body weight per injection per rat).
Abbreviation: c-SWNTs, carboxylated single-walled carbon nanotubes.
Figure 3 Long-term intravenous exposure to c-SWNTs induces capillary embolization, persistent inflammation, and fibrogensis in rat lungs.
Notes: Representative histopathology of H&E-stained lung sections from the rats at the end of the setting periods (30, 60, 90, and 120 days) after persistent intravenous injection with 5.00 wt% glucose solution (control) or 2.0 mg of c-SWNTs/kg body weight per injection (c-SWNTs). (I) The control group (A1–A4); The c-SWNTs group (B1–B4). Agglomerates of c-SWNTs accumulate in the pulmonary capillary lumen, leading to embolization (magnification: 100×). (II) No fibrosis in the control group (A1–A4); Colllagen deposition and fibrogenesis in the c-SWNTs group (B1–B4); Infiltration of inflammatory cells within the embolized capillaries, inducing granuloma formation in the c-SWNTs groups (C1–C4) (magnification: 400×).
Abbreviations: c-SWNTs, carboxylated single-walled carbon nanotubes; H&E, hematoxylin and eosin.
Figure 4 c-SWNTs traverse alveolar interstitium and induce ultrastructural alteration in AECs-II.
Notes: Representative TEM images of the lung sections from the rats after 30-day intravenous injection with 5.00 wt% glucose solution (control) or 2.0 mg of c-SWNTs/kg body weight per injection (c-SWNTs). Control lungs (A) and (B), wherein (B) is an enlarged image of the boxed region in (A), present normal ultrastructure of AECs-II. Exposed lungs (C)–(G), wherein (D) and (G) are enlarged images of the boxed regions in (C) and (F), respectively, show increasing lamellar bodies with decreasing sizes in AECs-II and c-SWNTs (red arrows) incorporated into the alveolar interstitium and trapped inside the vacuoles or the phagolysosomes, as well as fibril formation (E), (white arrows).
Abbreviations: c-SWNTs, carboxylated single-walled carbon nanotubes; AECs-II, alveolar epithelial cells type-II; TEM, transmission electron microscopy.
Figure 5 Long-term intravenous exposure to c-SWNTs promotes deposition of collagens and ECM remodeling by upregulating both MMP-2 and TIMP-2.
Notes: Immunohistochemical examination of Col I and Col III, MMP-2, and TIMP-2 (A) in the rat lungs after intravenous injection at indicated periods (positive signal: brown; c-SWNT aggregations: red arrows; magnification: 100×). Mean optical density of Col I, Col III, MMP-2, and TIMP-2 (B) based on immunohistochemical assay. Data represent mean ± SEM. *P<0.05, **P<0.01, and ***P<0.001 vs control.
Abbreviations: c-SWNTs, carboxylated single-walled carbon nanotubes; ECM, extracellular matrix; MMP-2, matrix metalloproteinase-2; TIMP-2, tissue inhibitor of metalloproteinase-2; Col I, type-I collagen; Col III, type-III collagen; SEM, standard error of the mean.
Figure 6 SWNTs stimulate the synthesis of collagen and activate NF-κB signaling in rat lungs.
Notes: Western blotting analysis of total level of Col III (A) and cytoplasmic levels of NF-κB/p65 and IκBα (B) after intravenous injection for 1, 7, and 30 days.
Abbreviations: SWNTs, single-walled carbon nanotubes; NF-κB, nuclear factor-kappa B; Col III, type-III collagen; IκBα, inhibitor of kappa B alpha; c-SWNTs, carboxylated SWNTs.
Figure 7 SWNTs stimulate production of pro-inflammatory and pro-fibrotic cytokines in rat lungs after intravenous exposure.
Notes: ELISA determination of TNFα and IL-1β levels (A) in the lung tissue of the rats after intravenous injection at indicated periods. Immunohistochemical examination of TGF-β1 and α-SMA, as well as the corresponding mean optical density (B) in the rat lungs after intravenous injection at indicated periods (positive signal: brown; c-SWNT aggregations: red arrows; magnification: 100×). Data represent mean ± SEM. *P<0.05, **P<0.01, and ***P<0.001 vs control.
Abbreviations: SWNTs, single-walled carbon nanotubes; ELISA, enzyme-linked immunosorbent assay; TNFα, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; TGF-β1, transforming growth factor-beta 1; α-SMA, alpha smooth muscle actin; c-SWNTs, carboxylated SWNTs; SEM, standard error of the mean.
Figure 8 Schematic representation of mechanisms of pulmonary fibrosis induced by long-term intravenous exposure to c-SWNTs.
Notes: Long-term intravenous injection induces persistent accumulation of c-SWNTs in rat lung, embolization in alveolar capillary, and inflammation regulated by NF-κB pathway, resulting in EMT of AECs-II and ECM deposition.
Abbreviations: c-SWNTs, carboxylated single-walled carbon nanotubes; NF-κB, nuclear factor-kappa B; EMT, epithelial-to-mesenchymal transition; AEC-II, alveolar epithelial cell type-II; ECM, extracellular matrix; AEC-I, alveolar epithelial cell type-I; RBC, red blood cell; EC, endothelial cell; IL-1β, interleukin-1 beta; TNFα, tumor necrosis factor alpha; TGF-β1, transforming growth factor-beta 1; IκBα, inhibitor of kappa B alpha; p-p65, phosphorylated p65.
