Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

Abstract

Background: In this study, primary rat alveolar epithelial cell monolayers (RAECM) were used to investigate transalveolar epithelial quantum dot trafficking rates and underlying transport mechanisms. Methods: Trafficking rates of quantum dots (PEGylated CdSe/ZnS, core size 5.3 nm, hydrodynamic size 25 nm) in the apical-to-basolateral direction across RAECM were determined. Changes in bioelectric properties (ie, transmonolayer resistance and equivalent active ion transport rate) of RAECM in the presence or absence of quantum dots were measured. Involvement of endocytic pathways in quantum dot trafficking across RAECM was assessed using specific inhibitors (eg, methyl-β-cyclodextrin, chlorpromazine, and dynasore for caveolin-, clathrin-, and dynamin-mediated endocytosis, respectively). The effects of lowering tight junctional resistance on quantum dot trafficking were determined by depleting Ca 2+ in apical and basolateral bathing fluids of RAECM using 2 mM EGTA. Effects of temperature on quantum dot trafficking were studied by lowering temperature from 37°C to 4°C. Results: Apical exposure of RAECM to quantum dots did not elicit changes in transmonolayer resistance or ion transport rate for up to 24 hours; quantum dot trafficking rates were not surface charge-dependent; methyl-β-cyclodextrin, chlorpromazine, and dynasore did not decrease quantum dot trafficking rates; lowering of temperature decreased transmonolayer resistance by approximately 90% with a concomitant increase in quantum dot trafficking by about 80%; and 24 hours of treatment of RAECM with EGTA decreased transmonolayer resistance by about 95%, with increased quantum dot trafficking of up to approximately 130%. Conclusion: These data indicate that quantum dots do not injure RAECM and that quantum dot trafficking does not appear to take place via endocytic pathways involving caveolin, clathrin, or dynamin. We conclude that quantum dot translocation across RAECM takes place via both transcellular and paracellular pathways and, based on comparison with our prior studies, interactions of nanoparticles with RAECM are strongly dependent on nanoparticle composition and surface properties.

Nanoparticle translocation across mouse alveolar epithelial cell monolayers: Species-specific mechanisms

“…When Yacobi et al [23] applied the same experimental approach, it was found that translocation of both positively and negatively charged PNP across RAECM takes place predominantly via non-endocytic transcellular pathways. We also recently showed that PEGylated quantum dots (core size ~5 nm, hydrodynamic size ~25 nm, amine-, carboxylate- and non-modified) can traverse EGTA-disrupted tight junctions [29]. …”

“…The most abundant proteins found in clathrin-coated pits are clathrin and the heterotetrameric adaptor protein 2 (AP-2) [44]. Chlorpromazine causes clathrin and AP-2 to relocate to multivesicular bodies, causing inhibition of clathrin-mediated endocytosis [23, 29]. Treatment of MAECM with 28 µM chlorpromazine in this study led to ~60–80% decrease in trafficking of amidine-modified (positively charged, 20 and 120 nm) but not carboxylate-modified (negatively charged, 20 and 100 nm) PNP (Figure 3), consistent with the notion that positively (but not negatively) charged NP preferentially associate with clathrin-coated pits and are subsequently taken up into/translocated across certain (but not all) epithelial barriers.…”

“…To explore mechanisms of PNP uptake into and translocation across MAECM, effects of inhibition of lipid raft-mediated (including caveolin-mediated) endocytosis were evaluated using methyl-β-cyclodextrin (MBC) [23, 26–29], chlorpromazine was employed for inhibition of clathrin-mediated endocytosis [23, 29, 30] and dynasore was used to inhibit dynamin-dependent (including both clathrin- and caveolin-mediated) endocytosis [23, 27, 29]. MAECM were bathed on both sides with culture medium containing 200 µM MBC (Sigma), 28 µM chlorpromazine (Sigma) or 80 µM dynasore (Sigma) for 30 min prior to replacing apical fluid at t = 0 with fresh culture medium containing both 176 µg/mL of various PNP and respective specific endocytosis inhibitors.…”

Effect of modifying quantum dot surface charge on airway epithelial cell uptake in vitro

“…Therefore, uptake of charged particles can be cell specific, and our platform provides data about how the respiratory airway epithelium responds to inhaled nanoparticles. While there is data in the lower alveolar epithelium (Fazlollahi et al 2011; Fazlollahi et al, 2011; Kim et al 2010; Yacobi et al 2008, 2009, 2007), the airway epithelium has a higher paracellular permeability (Widdicombe J 1997), and transport could be substantially altered. The mechanisms dictating these routes are unknown.…”

“…Of note, it has been calculated that the airway epithelial barrier is more permeable than the alveolar barrier (Widdicombe J 1997), and therefore mechanisms of transport could be of great significance in airway epithelial cells. Studies have shown that charged particles can traverse the alveolar epithelium, primarily transcellularly (Fazlollahi et al 2011; Fazlollahi et al, 2011; Kim et al 2010; Yacobi et al 2008, 2009, 2007). Published studies, such as those by Yacobi et al(Yacobi et al 2009) have considered nanoparticle transport through the alveolar epithelium, and have shown that both positively and negatively charged particles traverse transcellularly, though at different rates.…”