Internalization and Fate of Individual Manufactured Nanomaterial within Living Cells
|Principal Investigator||Galya Orr|
|Relevance to Implications||High|
|Class of Nanomaterial||Engineered Nanomaterials|
|Impact Sector||Human Health|
|Broad Research Categories||
Generation, Dispersion, Transformation etc.|
|Anticipated Total Funding||$200,000.00|
|Anticipated End Year||2008|
Accumulating observations suggest that inhaled nanoscale particles (NSPs) exert harmful effects on human health to a greater extent than larger particles, and these effects have been linked to the surface properties of nanomaterial. Although large aggregates of NSPs have been found within cells, it is thought that such agglomerations occur as the result of experimental conditions. It is currently thought that NSPs in vivo are able to escape the alveolar macrophages and might directly enter the circulatory system through the epithelial wall. Our studies will therefore be guided by the working hypothesis that the initial interaction of NSPs with the living cell in vivo occurs at the level of individual or small NSP aggregates (<100 nm), and that the physical and chemical surface properties of the individual NSP dictate their mechanisms of interaction with the cell, and ultimately govern their level of toxicity. The specific chemical and physical surface properties that facilitate NSP interactions with macrophage and lung epithelial cells have not been fully characterized. Furthermore, little is known about the mechanisms that underlie the attachment, internalization or cellular fate of individual NSPs within these cells as they might be presented in vivo. To fill the gap in our understanding, two specific aims will be pursued, using cultured macrophage and lung epithelial cell-lines: 1) identify the internalization process and cellular fate of individual manufactured NSPs with specific surface properties, by characterizing the dynamic behavior of individual particles, the immediate response of membrane lipids at the encountered site, and the involved subcellular structures; and 2) determine the involvement of selected membrane receptors in the attachment and internalization of manufactured NSPs with specific properties, by exploring molecular interactions between the receptors and ligand-coated or naked particles.
Titania (TiO2) particles and single-walled carbon nanotubes will be used. The two manufactured NSPs differ in their physical and chemical properties, yet both have been shown to exert harmful effects. Nanoscale titania particles will be compared with larger particles and naked NSPs will be compared with surface modified particles. The first specific aim will be pursued using video-rate single-molecule fluorescence imaging techniques.Tracking individual NSPs with specific physical and chemical properties as they enter the cell, while staining or manipulating specific subcellular structures or membrane lipids, will identify the mechanisms of internalization and the involved cellular machinery. The second specific aim will be pursued using fluorescence resonance energy transfer (FRET).FRET will be used to identify interactions between ligand-coated or naked nanoparticles and specific receptors, and follow the dynamic changes in these interactions in the membrane and along the endocytic pathway.
The attachment, internalization and cellular fate of individual or small aggregates of manufactured NSPs, the immediate changes in membrane dynamics, and the involved subcellular structures will be identified, in relation to particle size, geometry, and surface chemistry. The role that specific membrane receptors play in the attachment and internalization of individual manufactured NSPs will be determined in respect to particle properties. Identifying the mechanisms that underlie the internalization and cellular fate of specific manufactured NSPs will enable the design of NSPs with desired chemical and physical surface properties that might reduce their toxicity, and ultimately the formulation of preventative approaches and exposure guidelines.