Student Research: Tao Lin

PhD, , 2010
Faculty Advisor: Terrance J. Kavanagh

Glutathione Modulates Amphilic Polymer-Coated CdSe/ZnS Quantum Dot-induced Pro-inflammatory Cytokine/Chemokine Expression


Abstract

The unique characteristics of engineered nanomaterials have received enormous attention for their potential applications in biology and medicine. Quantum dots (QDs), in particular, with their bright, photostable and size tunable fluorescence have shown great promise as alternatives to organic dyes for biological imaging. A key issue in evaluating their utility is assessing their potential toxicity, which however is not fully understood. In order to address problems associated with QDs manufacturing and to assess their immunotoxicity, we evaluated recently synthesized CdSe/ZnS core/shell QDs with a tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coating. In this study, we investigated the potential pro-inflammatory response that these nanoparticles might elicit and the signaling pathways that regulate such events. We measured the gene expression of pro-inflammatory cytokines/chemokines in mouse macrophages. The mRNA transcription of a list of pro-inflammatory cytokines and chemokines were induced by QDs in a time- and dose-dependent manner as assessed by quantitative real-time PCR. Considering the importance of glutathione (GSH) in modulating the disposition of Cd, Se and Hg in biological systems and in regulating redox-mediated gene expression of cytokines, we studied the effect of GSH depletion on gene expression of these cytokines/chemokines in mouse macrophage-like cell line RAW264.7 and in primary peritoneal macrophages isolated from wild type and mice with compromised GSH synthesis. In RAW264.7 cells, pretreatment with BSO, a specific and irreversible inhibitor of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, significantly suppressed the gene expression of most of the cytokines/chemokines that were induced by QDs. On the other hand, only IL6 and MCP 1 gene expression was significantly reduced in primary peritoneal macrophages. Finally, the protein expression and subcellular translocation of nuclear transcription factor NF-κB, which regulates the expression of various inflammatory genes, was found to be augmented by BSO as measured by Western blot analysis and immunocytochemistry. Although BSO suppressed the gene expression of pro-inflammatory cytokines/chemokines despite of the enhanced expression and nuclear translocation of NF-kB subunits, studies have shown that highly oxidized environment actually prohibits the DNA-binding ability of NF-kB. Therefore, current efforts are focused on studying the DNA-binding activity of NF-kB and exploring alternative signaling pathways that might account for the down-regulation of these cytokines/chemokines while GSH is depleted. Along with the preliminary data from our ongoing in vivo experiments, these results collectively suggested that the TOPO-PMAT-coated QD is capable of inducing pro-inflammatory cytokine/chemokine gene expression of macrophages both in vitro and in vivo.