Atomic layer deposition coating of carbon nanotubes with aluminum oxide alters pro-fibrogenic cytokine expression by human mononuclear phagocytes in vitro and reduces lung fibrosis in mice in vivo

Abstract

Background: Multi-walled carbon nanotubes (MWCNTs) pose a possible human health risk for lung disease as a result of inhalation exposure. Mice exposed to MWCNTs develop pulmonary fibrosis. Lung macrophages engulf MWCNTs and produce pro-fibrogenic cytokines including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and osteopontin (OPN). Atomic layer deposition (ALD) is a novel process used to enhance functional properties of MWCNTs, yet the consequence of ALD-modified MWCNTs on macrophage biology and fibrosis is unknown.

Methods: The purpose of this study was to determine whether ALD coating with aluminum oxide (Al2O3) would alter the fibrogenic response to MWCNTs and whether cytokine expression in human macrophage/monocytes exposed to MWCNTs in vitro would predict the severity of lung fibrosis in mice. Uncoated (U)-MWCNTs or ALD-coated (A)-MWCNTs were incubated with THP-1 macrophages or human peripheral blood mononuclear cells (PBMC) and cell supernatants assayed for cytokines by ELISA. C57BL6 mice were exposed to a single dose of A- or U-MWCNTs by oropharyngeal aspiration (4 mg/kg) followed by evaluation of histopathology, lung inflammatory cell counts, and cytokine levels at day 1 and 28 post-exposure.

Results: ALD coating of MWCNTs with Al2O3 enhanced IL-1β secretion by THP-1 and PBMC in vitro, yet reduced protein levels of IL-6, TNF-α, and OPN production by THP-1 cells. Moreover, Al2O3 nanoparticles, but not carbon black NPs, increased IL-1β but decreased OPN and IL-6 in THP-1 and PBMC. Mice exposed to U-MWCNT had increased levels of all four cytokines assayed and developed pulmonary fibrosis by 28 days, whereas ALD-coating significantly reduced fibrosis and cytokine levels at the mRNA or protein level.

Conclusion: These findings indicate that ALD thin film coating of MWCNTs with Al2O3 reduces fibrosis in mice and that in vitro phagocyte expression of IL-6, TNF-α, and OPN, but not IL-1β, predict MWCNT-induced fibrosis in the lungs of mice in vivo.

Figure 1. Atomic Layer Deposition Functionalization of MWCNTs.

A) Transmission electron microscopy (TEM) image of MWCNTs from Helix that went through the ALD process with no coating added. B) TEM image of a semi-uniform ALD-coating of Al₂O₃ on MWCNT; 10 cycles of ALD resulted in a 3 nm thick coating. C) TEM image of a uniform ALD-coating of Al₂O₃ on MWCNT; 50 cycles of ALD resulted in a 9 nm thick coating. D) TEM image of a uniform ALD-coating of Al₂O₃ on MWCNT; 100 cycles of ALD resulted in a 20 nm thick coating. E) Al₂O₃ coating thickness (nm) on MWCNTs as a function of ALD cycle. Data represent mean values (±SEM) with r² = 0.9618. F) Percent mass gain with increasing ALD cycles. Data represent mean values (±SEM) with r² = 0.9471.

Figure 2. Transmission electron microscopy of human macrophages engulfing MWCNTs.

A) Lower magnification (x18000) of uncoated, sonicated MWCNTs contained in vesicles within a THP-1 macrophage. B) Higher magnification (x56000) of uncoated, sonicated MWCNTs within a vesicle of a THP-1 macrophage. C) High magnification (x56000) of a single (unsonicated) MWCNT within the cytoplasm of a THP-1 macrophage. D) Lower magnification (x7100) of a THP-1 macrophage with sonicated Al₂O₃ coated MWCNTs (50 ALD cycles) in the cytoplasm. E) Higher magnification (x56000) of a cluster of sonicated Al₂O₃-coated MWCNT fragments (50 ALD cycles within the cytoplasm of a THP-1 macrophage. F) High magnification (x36000) of a THP-1 macrophage with Al₂O₃ coated MWCNTs (50 ALD cycles –unsonicated) in the cytoplasm in close proximity to the nucleus. G) Low magnification (x7100) of a macrophage with Al₂O₃-coated (100ALD cycles) sonicated MWCNTs in the cytoplasm. H) Inset showing a higher magnification (x56000) of a cluster of sonicated Al₂O₃-coated MWCNTs (100 ALD cycles) within the cytoplasm of a macrophage. I) High magnification (x56000) showing unsonicated Al₂O₃-coated (100 ALD cycles) MWCNTs in the cytoplasm of a THP-1 macrophage. J) High magnification inverted image of ALD-coated MWCNTs from panel H showing fracture points of breakage (arrows). K) Al₂O₃ coated MWCNT length (nm) as a function of ALD cycle post sonication. Data represent mean values (±SEM) of 20 measurements of nanotubes thickness and length.

Figure 3. Cytokine protein levels secreted by THP-1 cells 24 hr after exposure to MWCNTs coated with Al₂O₃ by ALD.

THP-1 cells were exposed to low (10 µg/ml) and high (100 µg/ml) doses of uncoated MWCNTs (U-MWCNT) or atomic layer deposition (ALD)-coated MWCNTs (A-MWCNT) functionalized with increasing layers of Al₂O₃ achieved by 10, 50, or 100 ALD cycles (A-MWCNT). ELISAs were performed on cell supernatants for A) IL-1β, B) OPN, C) IL-6 and D) TNF-α. Data are representative graphs of three separate experiments and are expressed as means ± SEM. P values are indicated for each graph. *Significant compared to control. ∧Significant effect between A-MWCNT and U-MWCNT at the same dose.

Figure 4. Cytokine protein levels secreted by THP-1 cells 24 hr after exposure to Al₂O₃ or carbon black nanoparticles (CB NP).

THP-1 cells were exposed to low (10 µg/ml) and high (100 µg/ml) doses of Al₂O₃ or CB NP. ELISAs were performed on cell supernatants for ELISAs were performed on cell supernatants for A) IL-1β, B) OPN, C) IL-6 and D) TNF-α. Data are representative graphs of three separate experiments and are expressed as means ± SEM. Asterisk directly above bar indicate comparison to control, whereas asterisk over connecting bar indicate comparison between CB NP and Al₂O₃ NP. *P<0.05, **P<0.01, ***P<0.001. Data are representative of three experiments and expressed as means ± SEM.

Figure 5. Effect of Al₂O₃-coated MWCNTs or Al₂O₃ nanoparticles on the secretion of cytokines by primary human blood monocytes (PBMCs).

Human PBMCs were treated with or without LPS for 2 hrs then exposed to nanoparticles for 24 hrs prior to collecting cell supernatants for ELISA. A single nanoparticle dose of 100 µg/ml was used and corrected for estimated particle number to account for the difference in mass between uncoated ‘U-MWCNT’ and 10 ALD cycle Al₂O₃-coated ‘A-MWCNT’. ELISAs were performed on cell supernatants for A) IL-1β and B) OPN. Data are expressed as the mean +/− SEM of quadruplicate cultures of PBMCs. *P<0.05, **P<0.01, ***P<0.001 compared to corresponding controls (−LPS or +LPS).

Figure 6. Effect of Al₂O₃-coated MWCNTs on Lung Inflammation and Fibrosis in C57BL6 mice.

Uncoated (U)-MWCNT or MWCNT coated with Al₂O₃ by 50 ALD cycles (A-MWCNT) were delivered to the lungs of mice by oropharyngeal aspiration (4 mg/kg) and necropsy performed at 1 or 28 days post-exposure to collect bronchoalveolar lavage fluid (BALF) and lung tissues for histopathology. A) Inflammatory cell differential counts in 1 day BALF demonstrated that U-MWCNTs caused primarily a neutrophilic inflammatory response that was not significantly different in A-MWCNT-treated mice. Data are the mean +/− SEM from 4 mice per group where 500 cells were counted from each animal randomly as described in Methods . B) Quantification of macrophages containing MWCNTs shows that approximately 50% of macrophages engulfed U-MWCNT or A-MWCNT after 1 day. 100 cells were counted in each BALF cytospin slides from U- or A-MWCNT-exposed mice. Data are mean +/− SEM of 4 animals in each group. Photomicrographs above each bar show representative inflammatory cells in BALF cytospin slides from U- or A-MWCNT-exposed mice where ‘Pos’ indicates MWCNT positive and ‘Neg’ indicates MWCNT-negative. Red arrows indicate neutrophils. C) Hematoxylin & eosin-stained lung sections at 1 day showing alveolar macrophages containing U-MWCNT or A-MWCNT (arrows). D) Representative microscopic images of trichrome-stained slides (20X) and semi-quantitative morphometry at 28 days following exposure to 0.1% pluronic/PBS vehicle (Control), U-MWCNT, or A-MWCNT.

Figure 7. Effect of Al₂O₃-coated MWCNTs on cytokine protein levels in bronchoalveolar lavage fluid (BALF) in C57BL6 mice at 1 day or 28 days.

Uncoated (U)-MWCNT or MWCNT coated with Al₂O₃ by 50 ALD cycles (A-MWCNT) were delivered to the lungs of mice by oropharyngeal aspiration (4 mg/kg) and necropsy performed at 1 or 28 days post-exposure to collect bronchoalveolar lavage fluid (BALF) and lung mRNA for measurement of A) IL-1β, B) OPN, C) IL-6 and D) TNF-α by ELISA and Taqman real-time RT-PCR, respectively.

Figure 8. Illustration showing Al₂O₃ thin film coating of MWCNT by ALD and the effect on mononuclear cell cytokine production in vitro and pro-fibrogenic responses in the lungs of mice in vivo.

Al₂O₃-coated MWCNTs (A-MWCNT) increased IL-1β secreted by THP-1 cells and PBMCs in vitro, but decreased IL-6, OPN, and TNF-α protein levels compared to uncoated (U)-MWCNTs. In vivo, ALD coating reduced lung mRNA levels of OPN and IL-6 but had no effect (NE) on lung mRNA levels of IL-1β or TNF-α at either 1 or 28 days post-exposure and reduced protein levels of IL-1β, OPN, and TNF-α measured in BALF at 1 or 28 days. ALD coating significantly reduced MWCNT-induced lung fibrosis in mice. Also, A-MWCNTs undergo breakage along the radial axis, which could potentially increase lung clearance of MWCNTs and reduce the potential for fibrosis.