Nanoparticle interactions with alveolar epithelium: Barrier and trafficking properties

We investigated the effects of ultrafine ambient particulate suspensions (UAPS), polystyrene nanoparticles (PNP; positively and negatively charged; 20, 100, 120 nm), quantum dots (QD; positively and negatively charged) and single-wall carbon nanotubes (SWCNT) on alveolar epithelial barrier properties. We also investigated trafficking characteristics and mechanisms of PNP transport across primary rat alveolar epithelial cell monolayers (RAECM). Transmonolayer resistance (Rt) and equivalent short-circuit current (Ieq) of RAECM were measured in the presence of varying apical nanoparticles. We measured fluxes of PNP, in the apical-to-basolateral direction under various experimental conditions. Using confocal laser scanning microscopy (CLSM), we investigated PNP co-localization with cell-cell junction, an early endosome marker (EEA1), caveolin-1 and clathrin heavy chain (CHC). Fluxes of PNP across an artificial lipid bilayer model (ALBM) were measured. Results revealed that exposure to UAPS decreased Rt and Ieq significantly. Positively charged QD and SWCNT decreased Rt significantly (with subsequent recovery). PNP exposure had no effects on Rt or Ieq. PNP fluxes across RAECM and ALBM increased as surface charge density increased, with positively charged PNP trafficking 20-40 times faster than highly negatively charged PNP of comparable size. Fluxes decreased with increasing PNP diameter, although surface charge seems to be a stronger determinant of PNP fluxes. PNP fluxes at 4°C were significantly lower than those measured at 37°C. PNP fluxes did not change in the presence of EGTA or various inhibitors of endocytosis. Co-incubation of PNP with cationic polypeptides decreased the positively charged PNP fluxes significantly. CLSM revealed no evidence for co-localization of PNP with cell-cell junction, caveolin-1, CHC or EEA1. These data indicate that disruption of alveolar epithelial barrier properties due to nanoparticle exposure is composition, shape and/or surface charge dependent. PNP trafficking across RAECM is strongly influenced by surface charge density, occurs transcellularly but not via known major endocytic pathways, and PNP likely translocate across RAECM by direct penetration through cell plasma membranes.