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INTERNALIZATION AND FATE OF INDIVIDUAL MANUFACTURED NANOMATERIAL WITHIN LIVING CELLS
Impact/Purpose:
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.
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
Description:
Using quantitative fluorescence imaging with single molecule sensitivity, combined with molecular biology techniques, we have been investigating the cellular interactions and fate of one nanoparticle or nanoscale aggregate at a time, identifying molecular interactions and cellular processes that are relevant to the properties of nanomaterials. Our work has been focused on positively or negatively charged synthetic amorphous silica particles, which are used extensively in a wide range of industrial applications, and are explored for drug delivery and medical imaging and sensing. By studying alveolar type II epithelial cells (C10), a target cell type for inhaled particles, we find that positively charged particles can take advantage of the actin turnover machinery within the microvilli to advance their way into the cell body. This pathway is strictly dependent on the positive surface charge of the particles and on the integrity of the actin filaments, unraveling charge-dependent coupling of the particles with the intracellular environment across the cell membrane. To identify the molecules that capture the particles at the cell surface we therefore searched for a negatively charged, transmembrane molecule that could mediate the coupling of the particles with the actin filaments. Using flow cytometry, time lapse fluorescence, and laser confocal microscopy we find that syndecan I, a transmembrane heparan sulfate proteoglycan, mediates the initial interactions of the particles at the cell surface, their coupling with the intracellular environment, and their internalization pathway.