Amy Mihalchik

Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Yon Rojanasakul

Committee Co-Chair

John Barnett

Committee Member

Vincent Castranova

Committee Member

Dale Porter

Committee Member

Yong Qian

Abstract

Multi-Walled Carbon Nanotubes (MWCNT) were first described by Iijima in 1991 as "needle-like tubes" made up of concentric sheets of graphene lattice composed of hexagonal carbon units. MWCNT have been reported to be extremely strong, light-weight, and durable, thus making them a highly valued nanomaterial commercially. In order to improve their electrical conductivity and dispersibility for industrial and biomedical uses, materials scientists began functionalizing CNT in the mid-1990s. One type of functionalized MWCNT, nitrogen doped-MWCNT (ND-MWCNT), have nitrogen directly incorporated into the carbon lattice, disrupting sp2 bonding and decreasing the crystallinity of MWCNT. Nitrogen-doping significantly increases the brittleness, chemical reactivity, and n-type semiconductor activity of this material, and may also have interesting implications for its bioactivity. Alterations in physicochemical properties such as size, surface reactivity, agglomeration, and charge have been suggested to significantly impact the overall toxicity of MWCNT, but less in known on the effects of MWCNT functionalization. While the unique physicochemical properties of these materials generate exciting new possibilities for industrial and consumer products, the potential for unintended human exposure, especially and primarily through inhalation, has been of concern. Numerous animal studies have shown that MWCNT induce inflammation and pulmonary fibrosis at occupationally relevant exposure doses, but have yet to provide detailed information on the occupational risk of ND-MWCNT and other functionalized MWCNT. In recent years, the U.S. Environmental Protection Agency and National Toxicology Program have called for increased use and validation of predictive in vitro models to lessen the need for costly and time-consuming in vivo projects to determine general toxicity of nanomaterials. The studies presented here focused on developing and utilizing various in vitro models employing human Small Airway Epithelial Cells (SAEC), human bronchial epithelial cells (BEAS-2B), and normal human lung fibroblasts (WI-38) to study the bioactivities of ND-MWCNT and pristine Mitsui-7 MWCNT (MWCNT-7). However, it was found that the type of in vitro system utilized as well as the nanomaterial dosing schema could have serious and significant impact on the results and their subsequent interpretation and relevance to real-world exposures. Here, we provide an assessment of results on the inflammatory and fibrotic potential of ND-MWCNT and MWCNT-7 and the in vitro approaches we used to address these aims including monoculture and conditioned media, as well as concentration-based and surface area-based dosing. Results suggested that ND-MWCNT and MWCNT-7 induced an acute particle and dose-dependent inflammatory response in SAEC and BEAS-2B, suggesting that the physicochemical properties of these materials may impact their bioactivity. WI-38 directly exposed to ND-MWCNT and MWCNT-7 exhibited dose-dependent changes in collagen I production and gene expression of alpha-SMA, which is suggestive of pro-fibrogenic signaling. WI-38 exposed to biologically relevant concentrations of epithelial-derived IL-6 and IL-8 in the absence of CNT exhibited more subtle responses than those directly exposed to CNT, but still demonstrates that multiple approaches may be used in vitro to study fibroblast responses. Overall, the work suggests that while in vitro approaches may be useful for assessing acute cellular responses, the ability to accurately predict the fibrogenic potential of MWCNT with different physicochemical properties has yet to be fully elucidated. However, the conclusions drawn from this work and critical analysis of current literature provides clear direction for the future of in vitro nanoparticle risk assessment.

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