Date of Award

1-1-2010

Document Type

Campus Access Thesis

Department

Environmental Health Sciences

First Advisor

Tara Sabo-Attwood

Abstract

The same properties that make engineered nanoparticles (ENPs) appealing for use in novel pharmaceuticals, biomedical and product enhancement applications (small size, large surface area and increased solubility) may also influence their ability to modulate immune defense and inflammatory responses in humans. Specifically, recognition of ENPs by Toll-Like Receptors (TLRs) may trigger an innate immune response through activation of the Nuclear Factor-kappa B (NF- κB) transcription factor. To test the capacity of ENPs to stimulate this transcription factor in vitro, two Human Embryonic Kidney cell lines (HEK293) were employed; one stably expressing TLR 2 (HEK293-TLR2) and the other devoid of TLRs (HEK293-WT). A select group of ENPs; silver nanoparticles (75 nm), gold nanorods (48 nm + 4 nm), and carboxylated, and pristine semi-conducting (0.8 nm) single-walled carbon nanotubes (SWNT) was used to assess the capacity of ENPs to transcriptionally activate a NF- κB reporter gene in these cell lines. All the ENPs were characterized to determine their morphology, surface charge, and degree of aggregate formation using Transmission Electron Microscopy (TEM), Zeta PALS, and Dynamic Light Scattering (DLS) respectively. Prior to assessing NF-κB activation in both cell lines, a dye-based viability assay was used to determine non-toxic exposure doses for each ENP. Results show that all ENPs were relatively non-toxic to the cells after a single acute dose in the range of concentrations assessed (0-100 μg/ml). Interestingly, gold nanorods and functionalized carboxylated SWNT did not stimulate NF- κB. However, pristine unmodified SWNT (SG65) enhanced reporter gene activity resulting in a 14- and 17-fold increase in NF- κBactivation at the 50 μg /ml and 75 μg /ml doses respectively in HEK-TLR2 cells. Silver nanoparticles also activated NF- κBin HEK293-TLR2 cells 2-fold over control unexposed cells at the highest dose tested. Overall, these results suggest stimulation of TLR2 is ENP-specific. Preliminary characterization data indicate that TLR2 activation by pristine SWNT is not likely due to charge. All of the ENPs had similar negative surface charges in the presence of cell culture media. However, other morphological characteristics that may afford these SWNT the ability to stimulate TLRs are still being explored. Results from this work highlight the differential biological response of ENPs, particularly activation of TLRs in human cell systems. These findings lay the groundwork for further investigations into the physiochemical properties and characteristics of ENPs that afford them the ability to trigger innate immunity by acting as non-traditional ligands for TLRs.

Rights

© 2010, Leslyn Brusch-Richardson

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