Carbon Nanotube - Intestinal Barrier Cell Interactions and Absorption
EMSL Project ID
40008
Abstract
Carbon nanotubes (CNT) are important components of the rapidly expanding nanotechnology field. We hypothesize that engineered, water dispersible CNTs, when exposed to gastrointestinal (GI) tract cells, will be absorbed by GI epithelia, interact with the barrier cells, partition between GI epithelia and the hepatic portal circulation, and exert effects that alter function in the gut-liver axis and beyond. The nature of these effects likely depends on the unique physico-chemical properties of the nanotubes. Particle translocation across the intestinal epithelial barrier relies on diffusion and accessibility through mucus, contact with enterocytes and lymphatic tissue, cellular trafficking, and post-translocation events, but the mechanism of CNT uptake and fate is unknown. We hypothesize that CNTs traverse this barrier and, in doing so, cellular interactions alter barrier cell function. We have recent evidence that carboxylated MWNT exposure to T84 cells (human intestinal epithelial cells) reduce transepithelial electrical resistance (TEER) and alter the expression of proteins associated with barrier cell function, suggesting that MWNT-mediated paracellular and intracellular effects are exerted. Tools that gain nano-scale insights into living cells are for a better understanding of the mechanisms whereby MWNTs exert these effects. Furthermore, tools that gain nano-scale structural and chemical characterization of the MWNTs within the cellular environment are needed to identify the specific physical and chemical properties of the particles that are responsible for the cellular response. Our proposed research will investigate cellular interactions, internalization pathways, and absorptive fate of individual or small agglomerates (~100 nm) of carboxylated MWNT labeled with Alexa Fluor (registered trademark) dyes, with well-defined surface properties and physico-chemical characterization, and correlate these processes with cellular responses. Labeled MWNTs will be exposed to cells in a co-culture system of the human colorectal adenocarcinoma epithelial cell lines. Caco-2 cells form polarized monolayers that are similar morphologically and functionally to intact intestinal tissues. HT29 cells are mucus-secreting goblet cells. Together, these particular cell types, with their highly developed mucosa in vivo, are particularly relevant for this study by providing a robust, in vitro model of the functionally absorptive barrier. High sensitivity time-lapse fluorescence imaging will be used to identify and follow individual CNTs as they enter the cells. Multiple lasers and emission channels will enable simultaneous imaging and tracking of fluorescent CNTs and endocytic vesicles or other cellular compartments, tagged with organelle specific fluorescent probes. This approach also will enable detection of CNT that cross paracellular pathways, such as tight-junctions. High resolution structural and chemical imaging will be used to determine CNT properties within the cell. Comprehensive physical and chemical characterization of the fluorescent CNTs will be done at New Jersey Institute of Technology. However, additional measurements of particle properties will be done in EMSL before and after cell exposures to determine changes that might occur in the cellular environment. The impact of the CNTs on the cells will be evaluated at Indiana University by assessing the functional integrity of the epithelia using TEER, and the oxidative stress response and survival of the cells using quantitative fluorescence assays. Large scale proteomic profiling of cells exposed to CNTs has been conducted at Indiana University with the over-reaching goal of identifying altered cellular networks. These studies, together with the studies conducted in EMSL, will gain new understanding of the intestinal barrier response mechanisms to CNTs.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2010-10-12
End Date
2011-10-16
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members