DOI: 10.25549/usctheses-c127-666554
title: Diverse interactions of epithelial cells and nanoparticles: search for underlying mechanisms

description: Nanoparticles have at least one dimension between 1 and 100 nm. Inhaled nanoparticles have been associated with cardiac arrhythmias, atherogenesis, clotting disorders and other health effects and have been found in liver, blood vessels, heart and other organs. The most likely route by which inhaled nanoparticles enter the systematic circulation is across alveolar epithelium with its large surface area and thin barrier thickness. Potential mechanisms for the pathophysiologic effects of inhaled nanoparticles include lung inflammation and/or direct interactions of nanoparticles with end organs. Concerns about the health risks of nanoparticles, and interest in the use of nanoparticles for therapeutic applications, require increased understanding of interactions of inhaled nanoparticles with alveolar epithelium with regard to injury, uptake and trafficking. ❧ Our laboratory has reported transalveolar epithelial trafficking characteristics of polystyrene nanoparticles (PNP) in vitro across primary rat alveolar epithelial cell monolayers (RAECM) We showed that trafficking rates of PNP across RAECM are dependent on both surface charge density and size, in that we found (a) ~20-40 times greater flux for positively (versus negatively) charged PNP of similar size and (b) larger PNP (~100 nm) cross RAECM ~3-4 times slower than smaller PNP (20 nm) of similar surface charge properties (126). PNP cross RAECM via non-endocytic transcellular pathways, possibly involving diffusion across the lipid bilayer of cell plasma membranes. Based on our recent preliminary data, we hypothesize that interactions of nanoparticles with epithelia is dependent both on the physicochemical characteristics of the nanoparticles and the specific epithelial cell type. ❧ To evaluate epithelial cell type-specificity of PNP trafficking, we studied PNP flux across Madin Darby canine kidney cell II monolayers (MDCK-II). Effects of calcium chelation (ethylene glycol-bis(β-aminoethyl ether) N,N,N’,N’-tetraacetate (EGTA, 2 mM)), energy depletion (sodium azide (NaN₃, 10 mM) or decreased temperature), and endocytosis inhibitors like, methyl-β-cyclodextrin (MBC, 200 µM), monodansylcadaverine (200 µM) and dynasore (80 µM) (for caveolin-, clathrin-, or dynamin-mediated endocytosis, respectively) were determined. Amidine-modified PNP cross MDCK-II 500 times faster than carboxylate-modified PNP. PNP flux did not increase in the presence of EGTA. PNP flux at 4°C and after treatment with NaN3 decreased 75% and 80%, respectively. MBC exposure did not decrease PNP flux, whereas dansylcadaverine- or dynasore-treated MDCK-II exhibited ~80% decreases in PNP flux. Confocal laser scanning microscopy revealed intracellular colocalization of PNP with clathrin heavy chain. These data indicate that PNP translocation across MDCK-II (1) occurs via clathrin-mediated endocytosis and (2) is dependent upon PNP physicochemical properties. ❧ In Order to further investigate if interactions of nanoparticles with epithelia depend on the specific epithelial cell type, we studied PNP trafficking across primary cultured mouse alveolar epithelial cell monolayers (MAECM). Effects of calcium chelation (EGTA, 2 mM) and various endocytosis inhibitors (e.g., MBC (200 µM), chlorpromazine (28 µM) and dynasore (80 µM) for caveolin-, clathrin-, or dynamin-mediated endocytosis, respectively) were determined. Results show that flux of 20 and 120 nm amidine-modified PNP is ~65 times faster than that of 20 and 100 nm carboxylate-modified PNP, respectively. Calcium chelation has little effect on flux of amidine-modified PNP, while flux of carboxylate-modified PNP increases ~60-fold and ~30-fold for 20 and 100 nm negatively charged PNP, respectively. PNP flux is unaffected by MBC, while ~80% decrease in amidine- (but not carboxylate-) modified PNP flux is observed in chlorpromazine- and dynasore-treated MAECM. Confocal laser scanning microscopy revealed intracellular presence of both amidine- and carboxylate-modified PNP with colocalization of amidine- (but not carboxylate-) modified PNP with clathrin heavy chain. Localization of carboxylate-modified PNP in disrupted junctions in EGTA-treated MAECM is noted. These data indicate that (1) positively charged (amidine-modified) PNP translocation across MAECM occurs primarily via clathrin-mediated endocytosis and (2) physicochemical properties (e.g., surface charge) determine interactions with mouse alveolar epithelium. ❧ To study effects of physicochemical characteristics of the nanoparticles on their translocation mechanism(s), we utilized RAECM to study transalveolar epithelial trafficking rates of quantum dots (QD) and underlying transport mechanisms. Trafficking rates of QD (comprised of PEGylated CdSe/ZnS with core size of 5.3 nm and hydrodynamic size of 25 nm) in the apical-to-basolateral direction across RAECM were determined. Changes in bioelectrical properties (i.e., transmonolayer resistance (Rt) and equivalent active ion transport rate (Ieq)) of RAECM in the presence or absence of QD were measured. Involvement of endocytic pathways in QD trafficking across RAECM was assessed using specific inhibitors (e.g., MBC (200 µM), chlorpromazine (28 µM) or dynasore (80 µM)). Effects of lowering tight junctional resistance on QD trafficking were determined by depleting Ca⁺⁺ in apical and basolateral bathing fluids of RAECM using EGTA (2 mM). Effects of temperature on QD trafficking were studied by lowering temperature from 37 to 4ºC. Results showed that: i) apical exposure of RAECM to QD did not elicit changes in Rt or Ieq for up to 24 h, ii) QD trafficking rates were not surface charge-dependent, iii) methyl-β-cyclodextrin, chlorpromazine or dynasore did not decrease QD trafficking rates, iv) lowered temperature decreased Rt by ~90% with concomitant increase in QD trafficking by ~80%, and v) 24 h treatment of RAECM with EGTA decreased Rt by ~95% with increased QD trafficking up to ~130%. These data indicate that QD do not injure RAECM and that QD trafficking does not appear to take place via endocytic pathways involving caveolin, clathrin or dynamin and that QD translocation across RAECM takes place via both transcellular and paracellular pathways. ❧ Finally, we conclude that uptake/trafficking of nanoparticles into/across epithelial barriers are dependent on both nanoparticle physicochemical properties (like, composition, surface charge, and size) and specific epithelial cell type (based on comparison with our prior results).