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.