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. 2011 Dec 27;5(12):9772-87.
doi: 10.1021/nn2033055. Epub 2011 Nov 22.

Dispersal state of multiwalled carbon nanotubes elicits profibrogenic cellular responses that correlate with fibrogenesis biomarkers and fibrosis in the murine lung

Xiang Wang  1 , Tian Xia, Susana Addo Ntim, Zhaoxia Ji, Sijie Lin, Huan Meng, Choong-Heui Chung, Saji George, Haiyuan Zhang, Meiying Wang, Ning Li, Yang Yang, Vincent Castranova, Somenath Mitra, James C Bonner, André E Nel
Affiliations

Dispersal state of multiwalled carbon nanotubes elicits profibrogenic cellular responses that correlate with fibrogenesis biomarkers and fibrosis in the murine lung

Xiang Wang et al. ACS Nano. .

Abstract

We developed a dispersal method for multiwalled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on profibrogenic responses in tissue culture cells and in mouse lung. We demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA, and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells, and alveolar macrophages (AM) and induced more prominent TGF-β1 and IL-1β production in vitro and TGF-β1, IL-1β, and PDGF-AA production in vivo than nondispersed tubes. Moreover, there was good agreement between the profibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bioprocessed by AM and induced less prominent collagen deposition at sites of nongranulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects profibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

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Figures

Figure 1
Figure 1. Comparison of the biological effects and uptake of dispersed (D) and non-dispersed (ND) MWCNTs in BEAS-2B cells
(A) Cells were treated for 24 hr with the indicated concentrations of the AP, PD and COOH-MWCNTs, which were prepared in the presence or absence of BSA (0.6 mg/mL) plus DPPC (0.01 mg/mL) as dispesants. The supernatants were collected to measure the TGF-β1 levels by ELISA as described in Materials and Methods. No significant TGF-β1 production was seen with ND tubes, except at the highest COOH-MWCNT concentration. * denotes a p-value < 0.05, comparing control to CNT-exposed cells; # defines a p-value < 0.05, comparing D to ND tubes. (B) Representative TEM images to show the subcellular uptake of D vs ND tubes in cells treated with 50 μg/mL AP-MWCNTs for 24 hr. The red arrows point to tubes localized in membrane-lined vesicles. The MWCNT identity was confirmed by the Raman spectroscopy data obtained in a Renishaw inVia Raman microscope system. MWCNTs exhibit characteristic peaks at 1355 nm and 1597 nm as shown in the middle panel. Identical results were obtained for PD-MWCNTs (Fig. S2A). The right side panel shows that ND tubes are not taken up by the cells. (C) Confocal Raman analysis of BEAS-2B cells viewed under a light optic microscope to show quantitative differences in the uptake of well-dispersed AP, PD, and COOH-MWCNTs in BEGM over 24 hr. Raman microscopy was performed at different cellular planes (top, middle and bottom) to compare the signal intensities when the beam is focused above at or below the cellular localization level of the tubes.
Figure 1
Figure 1. Comparison of the biological effects and uptake of dispersed (D) and non-dispersed (ND) MWCNTs in BEAS-2B cells
(A) Cells were treated for 24 hr with the indicated concentrations of the AP, PD and COOH-MWCNTs, which were prepared in the presence or absence of BSA (0.6 mg/mL) plus DPPC (0.01 mg/mL) as dispesants. The supernatants were collected to measure the TGF-β1 levels by ELISA as described in Materials and Methods. No significant TGF-β1 production was seen with ND tubes, except at the highest COOH-MWCNT concentration. * denotes a p-value < 0.05, comparing control to CNT-exposed cells; # defines a p-value < 0.05, comparing D to ND tubes. (B) Representative TEM images to show the subcellular uptake of D vs ND tubes in cells treated with 50 μg/mL AP-MWCNTs for 24 hr. The red arrows point to tubes localized in membrane-lined vesicles. The MWCNT identity was confirmed by the Raman spectroscopy data obtained in a Renishaw inVia Raman microscope system. MWCNTs exhibit characteristic peaks at 1355 nm and 1597 nm as shown in the middle panel. Identical results were obtained for PD-MWCNTs (Fig. S2A). The right side panel shows that ND tubes are not taken up by the cells. (C) Confocal Raman analysis of BEAS-2B cells viewed under a light optic microscope to show quantitative differences in the uptake of well-dispersed AP, PD, and COOH-MWCNTs in BEGM over 24 hr. Raman microscopy was performed at different cellular planes (top, middle and bottom) to compare the signal intensities when the beam is focused above at or below the cellular localization level of the tubes.
Figure 1
Figure 1. Comparison of the biological effects and uptake of dispersed (D) and non-dispersed (ND) MWCNTs in BEAS-2B cells
(A) Cells were treated for 24 hr with the indicated concentrations of the AP, PD and COOH-MWCNTs, which were prepared in the presence or absence of BSA (0.6 mg/mL) plus DPPC (0.01 mg/mL) as dispesants. The supernatants were collected to measure the TGF-β1 levels by ELISA as described in Materials and Methods. No significant TGF-β1 production was seen with ND tubes, except at the highest COOH-MWCNT concentration. * denotes a p-value < 0.05, comparing control to CNT-exposed cells; # defines a p-value < 0.05, comparing D to ND tubes. (B) Representative TEM images to show the subcellular uptake of D vs ND tubes in cells treated with 50 μg/mL AP-MWCNTs for 24 hr. The red arrows point to tubes localized in membrane-lined vesicles. The MWCNT identity was confirmed by the Raman spectroscopy data obtained in a Renishaw inVia Raman microscope system. MWCNTs exhibit characteristic peaks at 1355 nm and 1597 nm as shown in the middle panel. Identical results were obtained for PD-MWCNTs (Fig. S2A). The right side panel shows that ND tubes are not taken up by the cells. (C) Confocal Raman analysis of BEAS-2B cells viewed under a light optic microscope to show quantitative differences in the uptake of well-dispersed AP, PD, and COOH-MWCNTs in BEGM over 24 hr. Raman microscopy was performed at different cellular planes (top, middle and bottom) to compare the signal intensities when the beam is focused above at or below the cellular localization level of the tubes.
Figure 2
Figure 2. Comparison of the biological effects and uptake of dispersed (D) and non-dispersed (ND) MWCNTs in THP-1 cells
(A) Cells were treated for 24 hr with the indicated concentrations of the different MWCNTs in their D and ND states as described above. The supernatants were collected to measure the IL-1β production by ELISA as described in Materials and Methods. The * and # symbols denote statistical significance at p < 0.05 as described in Fig. 1. (B) Representative light optical images looking at cellular uptake of dispersed and non-dispersed AP, PD and COOH-MWCNTs. THP-1 cells were treated with D and ND tubes for 24 hr and then observed by a phase contrast microscopy (Carl Zeiss, Inc. Peabody, MA, USA).
Figure 2
Figure 2. Comparison of the biological effects and uptake of dispersed (D) and non-dispersed (ND) MWCNTs in THP-1 cells
(A) Cells were treated for 24 hr with the indicated concentrations of the different MWCNTs in their D and ND states as described above. The supernatants were collected to measure the IL-1β production by ELISA as described in Materials and Methods. The * and # symbols denote statistical significance at p < 0.05 as described in Fig. 1. (B) Representative light optical images looking at cellular uptake of dispersed and non-dispersed AP, PD and COOH-MWCNTs. THP-1 cells were treated with D and ND tubes for 24 hr and then observed by a phase contrast microscopy (Carl Zeiss, Inc. Peabody, MA, USA).
Figure 3
Figure 3. Dose-dependent pulmonary affects of well-dispersed AP-MWCNTs in mice
(A) Assessment of the suspension stability index of the different tube types after their addition at a concentration of 50 μg/mL in PBS, followed by sonication in the absence or presence of 0.6 mg/mL BSA, 0.01 mg/mL DPPC, or a combination of both. The stability index was determined by comparing the initial MWCNT absorbance at t = 0 to the absorbance at 1, 2, 3, and 20 hr. The absorbance measurements were carried out at a λ=550 nm in a SpectroMax M5e (Molecular Devices Corp., Sunnyvale, CA) as described in Materials and Methods. (B and C) Anesthetized C57BL/6 mice were exposed one time to well-dispersed AP-MWCNTs at 0.5, 1, 2 and 4 mg/kg by oropharyngeal aspiration. There were 6 animals per group. Animals were euthanized after 21 days and BAL fluid collected to determine TGF-β1 (B) and PDGF-AA levels (C). Six mice treated with Min-U-Sil at 5 mg/kg by oropharyngeal exposure served as positive control. TGF-β1 levels were determined by an Emax ImmunoAssay System (Promega, Madison, WI) while PDGF-AA levels were assessed by the Quantikine ELISA kit from R&D Company (R&D Systems, Minneapolis, MN) as described in Materials and Methods. Absorbance was measured at 450 nm using a plate reader (SpectroMax M5e, Molecular Devices Corp., Sunnyvale, CA). The experiment was reproduced a second time. * p < 0.05 compared to control, # p < 0.05 for pair-wise comparisons as shown.
Figure 3
Figure 3. Dose-dependent pulmonary affects of well-dispersed AP-MWCNTs in mice
(A) Assessment of the suspension stability index of the different tube types after their addition at a concentration of 50 μg/mL in PBS, followed by sonication in the absence or presence of 0.6 mg/mL BSA, 0.01 mg/mL DPPC, or a combination of both. The stability index was determined by comparing the initial MWCNT absorbance at t = 0 to the absorbance at 1, 2, 3, and 20 hr. The absorbance measurements were carried out at a λ=550 nm in a SpectroMax M5e (Molecular Devices Corp., Sunnyvale, CA) as described in Materials and Methods. (B and C) Anesthetized C57BL/6 mice were exposed one time to well-dispersed AP-MWCNTs at 0.5, 1, 2 and 4 mg/kg by oropharyngeal aspiration. There were 6 animals per group. Animals were euthanized after 21 days and BAL fluid collected to determine TGF-β1 (B) and PDGF-AA levels (C). Six mice treated with Min-U-Sil at 5 mg/kg by oropharyngeal exposure served as positive control. TGF-β1 levels were determined by an Emax ImmunoAssay System (Promega, Madison, WI) while PDGF-AA levels were assessed by the Quantikine ELISA kit from R&D Company (R&D Systems, Minneapolis, MN) as described in Materials and Methods. Absorbance was measured at 450 nm using a plate reader (SpectroMax M5e, Molecular Devices Corp., Sunnyvale, CA). The experiment was reproduced a second time. * p < 0.05 compared to control, # p < 0.05 for pair-wise comparisons as shown.
Figure 3
Figure 3. Dose-dependent pulmonary affects of well-dispersed AP-MWCNTs in mice
(A) Assessment of the suspension stability index of the different tube types after their addition at a concentration of 50 μg/mL in PBS, followed by sonication in the absence or presence of 0.6 mg/mL BSA, 0.01 mg/mL DPPC, or a combination of both. The stability index was determined by comparing the initial MWCNT absorbance at t = 0 to the absorbance at 1, 2, 3, and 20 hr. The absorbance measurements were carried out at a λ=550 nm in a SpectroMax M5e (Molecular Devices Corp., Sunnyvale, CA) as described in Materials and Methods. (B and C) Anesthetized C57BL/6 mice were exposed one time to well-dispersed AP-MWCNTs at 0.5, 1, 2 and 4 mg/kg by oropharyngeal aspiration. There were 6 animals per group. Animals were euthanized after 21 days and BAL fluid collected to determine TGF-β1 (B) and PDGF-AA levels (C). Six mice treated with Min-U-Sil at 5 mg/kg by oropharyngeal exposure served as positive control. TGF-β1 levels were determined by an Emax ImmunoAssay System (Promega, Madison, WI) while PDGF-AA levels were assessed by the Quantikine ELISA kit from R&D Company (R&D Systems, Minneapolis, MN) as described in Materials and Methods. Absorbance was measured at 450 nm using a plate reader (SpectroMax M5e, Molecular Devices Corp., Sunnyvale, CA). The experiment was reproduced a second time. * p < 0.05 compared to control, # p < 0.05 for pair-wise comparisons as shown.
Figure 4
Figure 4. Dose-dependent increase in lung fibrosis, using the organs collected in Fig. 3
(A) Total lung collagen content from the same animals as in Fig. 3, using a Sircol Collagen Assay. The experiment was reproduced a second time. * p < 0.05 compared with control, # p < 0.05 compared among different doses of well-dispersed AP-MWCNTs and Min-U-Sil (positive control). (B) Trichrome staining depicting collagen deposition in the lungs of representative animals treated with dispersed AP-MWCNTs. Lungs were embedded, sectioned and stained by the Masson’s Trichrome stain. The red color represents staining of muscle tissue while collagen is stained blue. Lungs from Min-U-Sil exposed animals served as positive control.
Figure 4
Figure 4. Dose-dependent increase in lung fibrosis, using the organs collected in Fig. 3
(A) Total lung collagen content from the same animals as in Fig. 3, using a Sircol Collagen Assay. The experiment was reproduced a second time. * p < 0.05 compared with control, # p < 0.05 compared among different doses of well-dispersed AP-MWCNTs and Min-U-Sil (positive control). (B) Trichrome staining depicting collagen deposition in the lungs of representative animals treated with dispersed AP-MWCNTs. Lungs were embedded, sectioned and stained by the Masson’s Trichrome stain. The red color represents staining of muscle tissue while collagen is stained blue. Lungs from Min-U-Sil exposed animals served as positive control.
Figure 5
Figure 5. Comparison of the fibrotic effect of AP-, PD- and COOH-MWCNTs in their dispersed (D) as compared to their non-dispersed (ND) states
This experiment was performed identical to the experiment in Fig. 3, using 6 mice in each group. Oropharyngeal aspiration of a dose of 2 mg/kg AP, PD and COOH-MWCNTs was performed using dispersed (BSA and DPPC) and non-dispersed (ND) tube preparations. 21 days after the one-time exposure, the animals were euthanized and BAL fluid collected to determine TGF-β1 levels (A) and PDGF-AA activity (B). The experiment was repeated once, * p < 0.05 compared to control, # p < 0.05 when comparing D to ND tubes. Mice treated with Min-U-Sil at 5 mg/kg by oropharyngeal exposure served as positive control.
Figure 5
Figure 5. Comparison of the fibrotic effect of AP-, PD- and COOH-MWCNTs in their dispersed (D) as compared to their non-dispersed (ND) states
This experiment was performed identical to the experiment in Fig. 3, using 6 mice in each group. Oropharyngeal aspiration of a dose of 2 mg/kg AP, PD and COOH-MWCNTs was performed using dispersed (BSA and DPPC) and non-dispersed (ND) tube preparations. 21 days after the one-time exposure, the animals were euthanized and BAL fluid collected to determine TGF-β1 levels (A) and PDGF-AA activity (B). The experiment was repeated once, * p < 0.05 compared to control, # p < 0.05 when comparing D to ND tubes. Mice treated with Min-U-Sil at 5 mg/kg by oropharyngeal exposure served as positive control.
Figure 6
Figure 6. Differential collagen deposition in the lungs of mice for the same experiment shown in Fig. 5
(A) Total collagen content of the lung tissue was determined using the Sircol Soluble Collagen Assay kit (Biocolor Ltd., Carrickfergus, UK) as described in the Materials and Methods. * p < 0.05 compared with control, # p < 0.05 when comparing D to ND tubes. (B) Masson’s Trichrome staining to assess the tissue distribution of collagen in the lungs as described in Fig. 4. These lung images obtained at 100 × magnification are representative of the animals in each group. More prominent collagen deposition is seen in animals exposed to D vs ND tubes.
Figure 6
Figure 6. Differential collagen deposition in the lungs of mice for the same experiment shown in Fig. 5
(A) Total collagen content of the lung tissue was determined using the Sircol Soluble Collagen Assay kit (Biocolor Ltd., Carrickfergus, UK) as described in the Materials and Methods. * p < 0.05 compared with control, # p < 0.05 when comparing D to ND tubes. (B) Masson’s Trichrome staining to assess the tissue distribution of collagen in the lungs as described in Fig. 4. These lung images obtained at 100 × magnification are representative of the animals in each group. More prominent collagen deposition is seen in animals exposed to D vs ND tubes.
Figure 7
Figure 7. Confocal Raman analysis of the dispersed MWCNTs in the lung
The lung sections were obtained from the same experiment as in Fig. 5 and stained with hematoxylin and eosin. Lower (100 ×) and higher (400 ×) magnifications are shown of representative regions in the lungs of AP and COOH-MWCNT exposed animals. PD-MWCNTs had similar effects than AP-MWCNTs. The same lung sections were analyzed in a Renishaw inVia Raman microscope system. Two representative spots in each high magnification image were chosen for Raman scanning, namely a spot in an area of granulomatous inflammation for AP-MWCNTs and an alveolar impact site for COOH-MWCNTs. Arrow A is a representative cell not engulfing the MWCNTs, while arrow B denotes a representative cell that loaded with MWCNT. The right panel shows the corresponding Raman spectra identifying the specific peaks at 1355 nm and 1597 nm.
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