Published ahead of print on July 2, 2009 Am. J. Respir. Cell Mol. Biol. 2009, doi:10.1165/rcmb.2009-0138OC
Submitted on April 23, 2009 Mechanisms of alveolar epithelial translocation of a defined population of nanoparticlesNazanin R Yacobi1,1 Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California, United States; Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California, United States, 2 Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California, United States, 3 Department of Medicine, University of Southern California, Los Angeles, California, United States; Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California, United States, 4 Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California, United States, 5 Department of Medicine, University of Southern California, Los Angeles, California, United States; Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California, United States; Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California, United States, 6 Department of Medicine, University of Southern California, Los Angeles, California, United States; Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California, United States; Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California, United States, 7 Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California, United States; Department of Medicine, University of Southern California, Los Angeles, California, United States; Department of Pathology, University of Southern California, Los Angeles, California, United States; Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California, United States * To whom correspondence should be addressed. E-mail: ecrandal{at}usc.edu.
To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECM) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNP, 20 and 100 nm, carboxylate (negatively charged)- or amidine (positively charged)- modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy (CLSM), we investigated PNP colocalization with early endosome marker EEA1, caveolin-1, clathrin heavy chain (CHC), cholera toxin B (CTB) and wheat germ agglutinin (WGA). Leakage of 5-carboxyfluorescein diacetate (5-CFDA) from RAECM, and trafficking of 22Na and 14C-mannitol across ALBF, were measured in the presence and absence of PNP. Results showed that trafficking of positively charged PNP was 20-40 times that of negatively charged PNP across both RAECM and ALBF, while translocation of PNP across RAECM was 2-3 times faster than that across ALBF. Trafficking rates of PNP across RAECM did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. CLSM revealed no intracellular colocalization of PNP with EEA1, caveolin-1, CHC, CTB or WGA. Leakage of 5-CFDA from alveolar epithelial cells, and sodium ion and mannitol fluxes across ALBF, were not different in the presence or absence of PNP. These data indicate that PNP translocate primarily transcellularly across RAECM but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNP through the lipid bilayer of cell plasma membranes. Key words: epithelial transport • lipid bilayers • cell monolayers • particle trafficking • pneumocytes
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