This study demonstrates that apical exposure of RAECM to PEGylated quantum dots with amine-, carboxylate-, or non-modified surfaces for 24 hours does not affect alveolar epithelial barrier properties. Quantum dot translocation across RAECM via transcellular pathways does not appear to require caveolin-, clathrin-, or dynamin-dependent pathways, although quantum dot traffic via paracellular pathways may account (at least in part) for the observed fluxes.
We have previously reported that cationic polystyrene nanoparticles (amidine-modified, zeta potential about 71 mV) translocate across RAECM 20–40 times faster than anionic polystyrene nanoparticles (carboxylate-modified, zeta potential of about −45 mV).30
Although the range of surface charge of the amine-, carboxylate-, and non-modified PEGylated quantum dots was much less, no effect of surface charge on quantum dot flux across RAECM was seen.
No detectable translocation of quantum dots with non-modified, amine-modified, or carboxylate-modified surfaces ([apical quantum dot] = 25 nM) across RAECM over 24 hours was reported by Geys et al,40
while significant increases in transmonolayer resistance were observed. The quantum dots used by Geys et al40
were not PEGylated, although their hydrodynamic size and surface charge were similar to our quantum dots. The major difference between the studies is that measurable fluxes were noted for PEGylated quantum dots (our study) versus none for non-PEGylated quantum dots,40
likely due to differences in interactions between RAECM and differently coated quantum dots. Greater internalization of carboxylate-coated quantum dots compared with that of PEG-coated or PEG-amine-coated quantum dots has been reported for human epidermal keratinocytes.41
Although treatment of epithelial cells with EGTA (a calcium chelator) increases paracellular permeability to small solutes,42
it is unknown if nanoparticles traverse normal and/or EGTA-disrupted tight junctions. In this study, trafficking rates across RAECM of all three quantum dots with different surface modifications increased in the presence of 2 mM EGTA, indicating that these quantum dots traverse RAECM via tight junctional pathways following EGTA treatment. In contrast, Yacobi et al30
concluded that translocation of amidinated (20 and 120 nm) or carboxylated (20 and 100 nm) polystyrene nanoparticles across RAECM does not take place via paracellular pathways. Trafficking of small hydrophilic solutes (up to 10 nm) across epithelial barriers can occur via paracellular pathways,46
consistent with the small core size of the quantum dots utilized in this study (about 5 nm).
Quantum dot trafficking increased across RAECM when temperature was lowered from 37°C to 4°C. At lower temperature, energy-dependent mechanism(s) that may be involved in translocation of quantum dots transcellularly across RAECM will decrease or stop. However, when temperature was decreased to 4°C, transmonolayer resistance of RAECM decreased by about 90%, suggesting that trafficking of quantum dots is taking place primarily via paracellular pathways at low temperature.
Glycolipid rafts are detergent-insoluble, low-density membrane fractions that are rich in cholesterol and sphingolipids. 48
Methyl-β-cyclodextrin extracts cholesterol from plasma membranes and thereby inhibits lipid raft-mediated endocytosis (including caveolin-mediated endocytosis, CLIC/GEEC endocytosis, arf 6-mediated endocytosis, flotillin-mediated endocytosis, and macropinocytosis).37
We previously reported that 200 μM methyl-β-cyclodextrin markedly decreases flux of cholera toxin subunit B, a positive control for lipid raft-mediated transcytosis across RAECM.30
In the present study, flux of quantum dots observed in the presence of 200 μM methyl-β-cyclodextrin did not decrease, suggesting that translocation of quantum dots across RAECM is not taking place via lipid raft-mediated endocytosis. We have reported that translocation of polystyrene nanoparticles across RAECM and Madin Darby canine kidney type II cell monolayers (MDCK-II) was not decreased in the presence of 10–200 μM methyl-β-cyclodextrin.30
Clathrin is composed of three light and three heavy chains that form a triskelion. Assembly of the triskelion leads to formation of a net-like basket (clathrin-coated pit) at the cell plasma membrane. Involvement of clathrin-mediated endocytosis in uptake/translocation of various nanoparticles in cells and tissues, eg, amidine-modified polystyrene nanoparticles in MDCK-II,32
PEGylated D,L-polylactide (PLA) (PEG-PLA) nanoparticles into HeLa cells49
and fullerenic nanoparticles into rat fibroblasts and rat hepatoma cells,50
has been reported. Chlorpromazine and other inhibitors of clathrin- mediated endocytosis cause clathrin and AP-2 adaptor protein to relocate to multivesicular bodies and as a result inhibit clathrin-mediated endocytosis.51
We reported previously that transferrin utilizes clathrin-mediated endocytosis across RAECM which was inhibited by 28 μm chlorpromazine. 30
However, treatment of RAECM with 28 μM chlorpromazine in our current study did not decrease trafficking rates of any quantum dots utilized herein. It has been reported that a charge-dependent clathrin-mediated mechanism appears to be responsible for uptake of positively (but not negatively) charged PEG-PLA nanoparticles into HeLa and MDCK-II cells.49
In this regard, we recently reported that trafficking of positively (but not negatively) charged polystyrene nanoparticles across MDCK-II is clathrin-mediated.32
Dynamin, the membrane scission protein, is a large GTPase which forms a helical polymer around the neck of newly-formed cell plasma membrane invaginations and, upon GTP hydrolysis, mediates the fission of the vesicle from the plasma membrane. Dynamin is essential for caveolin-coated and clathrin-coated vesicle formation and appears to play a role in lipid raft-mediated processes as well. Dynasore, a small, cell-permeable molecule, reversibly inhibits the GTPase activity of dynamin-1 or dynamin-2 at the cell plasma membrane and rapidly blocks vehicle formation.53
Our data showing that trafficking rates of quantum dots did not decrease in the presence of 80 μM dynasore across RAECM are consistent with the lack of effect of inhibition of endocytosis involving clathrin or lipid rafts (including caveolin). Increased trafficking of quantum dots could be due to upregulation of other translocation pathways (although unlikely to include paracellular pathways with increased transmonolayer resistance) in the presence of dynasore. We reported recently that dynasore has no effect on trafficking rates of polystyrene nanoparticles (20 or 100/120 nm; amidine- modified or carboxylate-modified) across RAECM.30
On the other hand, 80 μM dynasore inhibited uptake of positively charged 100 nm polystyrene nanoparticles in HeLa cells by two-fold compared with that of negatively charged polystyrene nanoparticles of comparable size.54
Phagocytosis is prevalent in macrophages and dendritic cells, although it can be induced in most cells by expression of requisite receptors.55
Phagocytosis has also been reported for ATI and ATII cells.56
Internalization of large particles and microorganisms, typically greater than 0.5 μm, is known to occur via phagocytosis,37
but it is unlikely that PEGylated quantum dots (core size 5.3 nm, hydrodynamic size 25 nm) are significantly phagocytosed.
In summary, we have shown that translocation of amine-, carboxylate-, and non-modified PEGylated quantum dots across RAECM occurs both transcellularly (not involving major endocytic pathways) and paracellularly. Compared with trafficking of polystyrene nanoparticles across RAECM and MDCK-II, nanoparticle interactions with epithelial barriers appear to be both cell type-specific and dependent on nanoparticle physicochemical properties (eg, size, surface charge, and composition).