Anurag Mishra

Date of Graduation

2012

Document Type

Dissertation/Thesis

Abstract

Multi-walled carbon nanotubes (MWCNT) have many applications due to their unique mechanical and electrical properties. However, these fiber shaped materials have been shown to possess unusual fibrogenic activity in vivo and are currently the focus of intense toxicological investigations. To date very few detailed mechanistic studies on the fibrogenic activity of CNT have been reported. The aim of this series of studies is to determine fibrogenic potential of well dispersed MWCNT in human lung cell culture models and develop a novel platform for understanding the cellular mechanisms of MWCNT-induced lung fibrosis. Dispersion of large agglomerates of MWCNT in suspension to fine structures similar to aerosolized particles was achieved by using Survanta® , a natural non-toxic lung surfactant. Human lung epithelial BEAS-2B and fibroblast CRL-1490 cells were exposed to physiologically relevant low doses (0.02-0.2 µg/cm2) of non-dispersed (ND)- or Survanta®-dispersed (SD)-MWCNT which is equivalent to the in vivo doses of 10-80 ug in mice. In the first study, our results showed that MWCNT exhibited a dose-dependent anti-proliferative effect on the lung epithelial cells which is more pronounced in the dispersed form as compared to non-dispersed form. The well characterized SD-MWCNTs were used in further evaluation for in vitro studies. Significantly elevated levels of fibrogenic mediators such as transforming growth factor-β1 (TGF-β1) and matrix metalloprotienases-9 (MMP-9) were observed in the SD-MWCNT treated lung epithelial cells. Upon direct exposure to human lung fibroblasts, SD-MWCNT showed a dose-dependent dual-effect: induced proliferation and upregulation of collagen and fibroblast growth factor-2 (FGF-2) expression at low dose (0.002-0.06 ug/cm2), and cytotoxic effect at high dose (0.2-0.6 ug/cm2). These results indicate that the dispersion status of MWCNT determines their fibrogenic activity which is consistent with in vivo findings. The observations of the direct stimulation of lung fibroblast cell proliferation and collagen expression suggest novel mechanisms of MWCNT-induced lung fibrosis. TGFβ is a well-established central mediator in the development of lung fibrosis, but its role in CNT-induced lung fibrosis has not been systemically investigated. The results of the second study showed that MWCNT induced collagen I via the activation of TGF-β receptor 1 (TGF BR1) and SMAD pathway in human lung fibroblasts at low exposure doses (0.02-0.6 µg/cm2). Significant reduction in MWCNT-induced collagen was observed in the cells where TGF BR1 and Smad were knocked down (shRNA) or chemically inhibited. These results indicate that the TGF BR1-SMAD signaling pathway plays a crucial role in the pathogenesis of MWCNT-induced lung fibrosis. In the third study, the impact of certain physicochemical characteristics (e.g. surface functionalization and metal ion content) of MWCNT on their biological activities was evaluated. Raw (high metal ion content), acid purified (low metal ion content) and COOH functionalized (hydrophilic) MWCNT were evaluated for their cytotoxic and fibrogenic effects in vitro in human lung bronchial epithelial (Beas-2B) and fibroblast (CRL-1490) cells. Higher cytotoxicity was observed in raw MWCNT as compared to purified or COOH-functionalized MWCNT in the two cell systems. The COOH-functionalized MWCNT was more potent in stimulating collagen production in fibroblasts as compared to raw and purified MWCNT on an equal mass basis. Our findings suggest that surface modification can affect the fibrogenic activity of MWCNT, which is important in designing safe nanomaterials. The rapid increase in the number of newly synthesized nanoparticles has made in vivo animal studies impractical (i.e. due to the amount of time and cost required); therefore, there is a need for rapid in vitro models for risk assessment of nanomaterials. The development of cytotoxic and fibrogenic lung cell models reported in this study provide the experimental tools for rapid assessment of the fibrogenic potential of nanomaterials and allow mechanistic investigations of the disease process.

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