Grantee Research Project Results
Final Report: Internalization and Fate of Individual Manufactured Nanomaterial within Living Cells
EPA Grant Number: R833338Title: Internalization and Fate of Individual Manufactured Nanomaterial within Living Cells
Investigators: Orr, Galya , Tarasevich, Barbara J.
Institution: Pacific Northwest National Laboratory
EPA Project Officer: Hahn, Intaek
Project Period: May 1, 2007 through December 30, 2009
Project Amount: $200,000
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: a Joint Research Solicitation-EPA, NSF, NIOSH, NIEHS (2006) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
Accumulating observations suggest that inhaled nanomaterials exert harmful effects on human health and these effects have been linked, in part, to the surface properties of materials at the nano-scale. Although large aggregates of nanomaterials have been found within cells, it is thought that such agglomeration occurs as the result of experimental conditions. It currently is thought that nanomaterials are able to escape the alveolar macrophages in vivo and might directly enter the circulatory system through the epithelial wall. Our studies therefore have been guided by the working hypothesis that the initial interaction of nanomaterials with the living cell in vivo occurs at the level of individual or small particle aggregates (<100 nm), and that the physical and chemical surface properties of the individual particle dictate its mechanisms of interaction with the cell, and ultimately govern the level of toxicity. However, a great deal of confusion still exists about the properties that determine nanoparticle toxicity or biocompatiblity.
To fill the gap in our understanding, two specific aims have been pursued, using cultured alveolar epithelial cells and macrophages:
1) Determine the internalization process and cellular fate of individual manufactured nanoparticles with well defined surface properties.
2) Identify membrane receptors that mediate the attachment and internalization of the particles.
Identifying the mechanisms that underlie the internalization and cellular fate of specific manufactured nanoparticles will aid in understanding particle properties that govern the cellular response and ultimately the impact on the pulmonary system. This information will enable the design of nanoparticles with desired chemical and physical surface properties that might increase their biocompatibility, and the formulation of preventative approaches and exposure guidelines.
Summary/Accomplishments (Outputs/Outcomes):
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.Conclusions:
Together, our findings reveal a new mechanism by which positive surface charge supports particle recruitment by polarized epithelial cells bearing microvilli, and identify a critical role for syndecan I in the cellular interactions and subsequent cellular response to these particles. By studying a model macrophage cell-line (RAW 264.7), we find that scavenger receptor A (SR-A), expressed predominantly by macrophages, mediates the cell surface interactions and subsequent internalization of negatively charged amorphous silica nanoparticles, via clathrin mediated endocytosis. Both positively and negatively charged amorphous silica nanoparticles have been shown to induce pro-inflammatory responses. Our observations identify a new role for syndecan-1 in alveolar type II epithelial cells and SR-A in macrophages as mediators of the initial interactions and subsequent internalization, and therefore cellular fate and response of positively and negatively charged inorganic particles, respectively. Our studies have begun to draw new connections between distinct particle properties, defined cellular responses, and underlying mechanisms delineated from cellular interactions and intracellular fate of the particles. These connections eventually will help to predict toxic or biocompatible properties of nanomaterials and guide their design and exposure.Journal Articles on this Report : 3 Displayed | Download in RIS Format
| Other project views: | All 12 publications | 4 publications in selected types | All 3 journal articles |
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Orr GA, Chrisler WB, Cassens KJ, Tan R, Tarasevich BJ, Markillie LM, Zangar RC, Thrall BD. Cellular recognition and trafficking of amorphous silica nanoparticles by macrophage scavenger receptor A. Nanotoxicology 2011;5(3):296-311. |
R833338 (Final) |
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Orr G, Panther DJ, Phillips JL, Tarasevich BJ, Dohnalkova A, Hu D, Teeguarden JG, Pounds JG. Submicrometer and nanoscale inorganic particles exploit the actin machinery to be propelled along microvilli-like structures into alveolar cells. ACS Nano 2007;1(5):463-475. |
R833338 (2008) R833338 (Final) |
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Orr G, Panther DJ, Cassens KJ, Phillips JL, Tarasevich BJ, Pounds JG. Syndecan-1 mediates the coupling of positively charged submicrometer amorphous silica particles with actin filaments across the alveolar epithelial cell membrane. Toxicology and Applied Pharmacology 2009;236(2):210-220. |
R833338 (2008) R833338 (Final) |
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Supplemental Keywords:
airways, respiratory tract, inflammatory, manufactured, nanoparticle, ultrafine, particulates, endocytosis, phagocytosisRelevant Websites:
, Volume 236, Issue 2 (15 April 2009). , in Sept 01, 2008 issue. Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.