Grantee Research Project Results
2008 Progress 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 Period Covered by this Report: 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 physicochemical properties of materials at the nano-scale. However, a great deal of confusion still exists about the properties that lead to nanoparticle toxic or biocompatible. Since the cellular interactions and fate of the particles dictate the cellular response and ultimately determine the impact on human health, two specific aims have been pursued in cultured alveolar epithelial cells and macrophages: 1) determine the internalization process and cellular fate of individual manufactured nanoparticles with well defined surface properties; and 2) identify membrane receptors that mediate the attachment and internalization of the particles. This information will gain a mechanistic understanding of particle toxicity or biocompatibility, and guide the design of preventative approaches and safer nanomaterials.Progress Summary:
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 materials at the nano-scale. 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 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 flowcytometry, 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. 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 distinct pro-inflammatory responses in alveolar type II epithelial cells and macrophages. Our studies therefore have begun to draw new relationships between distinct particle properties, defined cellular responses, and underlying mechanisms delineated from cellular interactions and intracellular fate of the particles. These connections will eventually help to predict toxic or biocompatible properties of nanomaterials and guide their design and exposure.Future Activities:
Our data show that the interaction of syndecan with the particles facilitates the clustering of the molecules, which is required for their coupling with actin and their subsequent internalization. These observations indicate that the particles can induce their own internalization by triggering syndecan clustering. The possibility that particles smaller than the tested 100 nm might not be able to efficiently induce cross-linking or clustering of syndecan molecules, and therefore their subsequent internalization via this pathway, is currently being tested. Our future studies will focus on additional nanomaterials to delineate the roles of particle chemistry and shape in cellular response and identify the underlying cellular and molecular mechanisms.Journal Articles on this Report : 2 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 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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Relevant Websites:
1. Publication (Orr et al., 2009) was featured on the cover of Toxicology and Applied Pharmacology: http://www.sciencedirect.com/science/journal/0041008X
, Volume 236, Issue 2 (15 April 2009).
2. Publication (Orr et al., 2009) was featured on Pacific Northwest National Laboratory - Science Highlights: http://www.pnl.gov/science/highlights/highlight.asp?id=593
3. Publication (Orr et al., 2007) was highlighted by a special Perspective article written by Hess H and Tseng Y: “Active Intracellular Transport of Nanoparticles: Opportunity or Threat?” ACS Nano. 2007, 1(5), 390–392. http://pubs.acs.org/doi/full/10.1021/nn700407v
4. Publication (Orr et al., 2007) was highlighted in a podcast interview of Orr. Posted at the ACS Nano site: http://pubs.acs.org/page/ancac3/audio/index.html Episode 5.
.5. Presentation (Orr et al., at the American Chemical Society 236th National Meeting) was highlighted by an article in Pesticide and Toxic Chemical News: Nanotech materials' safety confronted at American Chemical Society meeting , in Sept 01, 2008 issue.
, 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.