Mechanisms of Alveolar Epithelial Translocation of a Defined Population of Nanoparticles

Abstract

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 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, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Lung vascular targeting through inhalation delivery: Insight from filamentous viruses and other shapes

“…Yacobi et al studied the mechanisms of nanoparticle interactions with lung alveolar epithelium during translocation utilizing rat alveolar epithelial cell monolayers (RAECM). Polystyrene nanoparticles of size 20-100 nm appeared to translocate across RAECM in the apical to basolateral direction transcellularly, primarily via nonendocytic pathways with direct involvement of the lipid in the cell's plasma membrane (51). The results also suggested that 20 nm particles transport twofold to threefold faster than 100-120 nm particles of similar charge while positively charged particles translocated 20-to 40-fold faster than negatively charged particles.…”

Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

“…To explore if quantum dot translocation across RAECM involves endocytotic mechanisms, methyl-β-cyclodextrin, chlorpromazine, and dynasore were used to disrupt lipid raft-mediated endocytosis, 30,32,36,37 clathrin-mediated endocytosis, 30,38 and dynamin-dependent endocytosis (including clathrin-mediated and caveolin-mediated endocytosis), 30,32,39 respectively. Briefly, RAECM were pretreated for 30 minutes with methyl-β-cyclodextrin (200 µM, Sigma), chlorpromazine (28 µM, Sigma), or dynasore (80 µM, Sigma) in both apical and basolateral fluids.…”

“…[24][25][26][27][28][29] It is currently thought that both the size and surface properties of nanoparticles affect the cytotoxicity and intracellular fate(s) of nanoparticles. [30][31][32][33] In this regard, we recently reported the trafficking properties of polystyrene nanoparticles with variable sizes and surface charges across RAECM. 30,31 In brief, trafficking rates of polystyrene nanoparticles across RAECM are dependent on both surface charge density and size, in that we found an approximately 20-40 times greater flux for positively (versus negatively) charged polystyrene nanoparticles of similar size and that larger polystyrene nanoparticles (about 100 nm) cross RAECM about 3-4 times slower than smaller polystyrene nanoparticles (20 nm) of similar surface charge.…”

“…[30][31][32][33] In this regard, we recently reported the trafficking properties of polystyrene nanoparticles with variable sizes and surface charges across RAECM. 30,31 In brief, trafficking rates of polystyrene nanoparticles across RAECM are dependent on both surface charge density and size, in that we found an approximately 20-40 times greater flux for positively (versus negatively) charged polystyrene nanoparticles of similar size and that larger polystyrene nanoparticles (about 100 nm) cross RAECM about 3-4 times slower than smaller polystyrene nanoparticles (20 nm) of similar surface charge. 31 Polystyrene nanoparticles appear to be translocated across RAECM via nonendocytic transcellular pathways, possibly involving diffusion across the lipid bilayer of cell plasma membranes.…”

“…31 Polystyrene nanoparticles appear to be translocated across RAECM via nonendocytic transcellular pathways, possibly involving diffusion across the lipid bilayer of cell plasma membranes. 30 In this study, we used PEGylated quantum dots with three surface modifications (amine-, carboxylate-, and nonmodified) with which to characterize nanoparticle uptake/ trafficking into/across RAECM. We found that these quantum dots do not affect the barrier properties of alveolar epithelium.…”