In this study, human airway epithelial cells grown at an air–liquid interface were used to investigate the effect of physicochemical properties on nanoparticle transport. These cells are grown on a porous support with their upper (apical) surface exposed to air and the lower (basolateral) surface to medium, allowing them to differentiate into ciliated, goblet and basal cells, forming a pseudostratified columnar epithelium that secretes an airway surface liquid (ASL) on their apical surface, similar to airway epithelial cells in vivo
() (Sidhaye et al. 2008
; Sidhaye et al. 2011
; de Jong et al. 1994
). This ASL is composed of water, salts, osmolytes and macromolecules such as mucins (Tarran 2004
; Thornton & Sheehan 2004
). Analysis of this type of platform has been over the past two decades and several studies support this to be a good model of in vivo
airway epithelial cells, in terms of its ability to recapitulate the cell populations, the barrier integrity and the apico-adherens junctions found in human airways (de Jong et al. 1994
), (Lin et al. 2007
; Lam et al. 2011
). The kinetics of transport was determined from the increase in fluorescence of the medium in the lower chamber due to transport of QDs (). The biochemical response of the epithelium was determined using immunofluorescence microscopy, fluorescence measurements of QD transport, transepithelial electrical resistance (TER) measurements and measurements of permeability to fluorescently labelled dextran. In order to traverse the epithelium, the QDs have to first penetrate the ASL above the cells, which includes the mucus layer. Thus, this evaluation will provide insight into the ability of QDs to traverse the mucus layer, and enter and potentially cross the epithelial cells' layer as well.
Figure 1. Schematic illustration of the platform used to study the response of the human airway epithelium to nanoparticles. (A) Human airway epithelial cells are grown on an insert such that the apical (top) surface is exposed to air and the basolateral (bottom) (more ...)
CdSe/ZnS QDs were synthesised as reported previously (Park et al. 2008
; Galloway et al. 2009
). The QDs were water solubilised by first replacing the surfactants from synthesis with dodecanethiol (DDT) and then incubating with lipids in chloroform to form a hydrophilic outer leaflet (Galloway et al. 2011
). The composition of the outer leaflet included a zwitterionic single C14 acyl chain lipid (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine, MHPC) along with 30 mol% of Octadecylamine (ODA) and stearic acid (SA) (). The ODA introduces positive charge resulting in QDs with an average zeta potential of 49 mV, whereas SA introduces negative charge, resulting in QDs with an average zeta potential of –50 mV ().
Figure 2. (A) Schematic illustration of lipid functionalised quantum dots. The outer leaflet contains 20 mol% Octadecylamine (positive charge) or stearic acid (negative charge). (B) The zeta potential was 49 mV for ODA-modified QDs and -50 mV for SA-modified QDs. (more ...)
The CdSe/ZnS core/shell QDs are 8 nm in diameter. With the DDT inner layer (≈ 1 nm) and the lipid outer leaflet (≈ 2 nm), the overall size of the QDs is expected to be about 14 nm. From dynamic light-scattering measurements, the average diameters of the ODA- and SA-modified QDs were 13.4 and 14.5 nm, respectively, in excellent agreement with the expected values (). The stated particle sizes are obtained from the maximum of the number density distributions. There are no additional peaks, either in number, volume or intensity distributions. Qualitatively, excessive aggregation of QDs in suspension can be seen under illumination by a black lamp, and quantitatively by a decrease in the absorbance and the appearance of a second peak in the DLS distributions. The ODA- and SA-functionalised QDs used here were selected for their stability in media. The QD suspensions were stable in media for at least 24 h (the maximum length of experiment that we have performed). From DLS measurements, there is no change in particle size after 12 h in media (). In addition, there is no change in the absorbance after 12 h in media indicating no aggregation or sedimentation (). The curve surrounding the first exciton peak at 580 nm remains unchanged after the transfer to serum indicating no change in the electronic properties of QD in serum.
In summary, the ODA- and SA-modified QDs are both about 14 nm in diameter and present an outer lipid layer with either positive or negative charge.
Workplace concentrations of nanoparticles (NP) (Davenpeck et al. 1995
) on the order of 104
and as high as 107
have been reported in the literature (Demou et al. 2008
). The ventilation volume for the lungs is about 6000 cm3
per min, and the number of airway epithelial cells for an average adult is about 1010
(Mercer et al. 1994
). An ambient concentration of 107
corresponds to a dose of 3.6 NP per cell for an exposure of 1 h. Therefore, we chose to study doses on the order of 1, 10 or 100 NP per cell. A dose of 1 NP/cell corresponds to a high workplace exposure (107
) for 17 min or a low workplace exposure (104
) for seven working days. A dose of 100 NP/cell corresponds to a high workplace exposure for 3.5 working days or a low workplace exposure for 87 working weeks.
Particles can cross the epithelium by either transcellular or paracellular transport (). To determine the mode of entry of the QDs into the epithelial monolayer, the membranes of the airway epithelial cells were labelled with Image-IT (wheat germ agglutinin conjugated to Alexa Fluor 488, Molecular Probes). After exposure of the apical surface to QDs, live-cell confocal microscope imaging was performed to determine whether charge affected the localisation of QDs in the epithelium. Within 5 min of exposure, fluorescence imaging reveals that the QDs penetrated the epithelium (). For negatively charged particles, the QDs are co-localised with the membrane marker indicating that they are located between cells. In contrast, for positively charged particles, there is increased intracellular fluorescence indicating more QDs both at the apical surface of the cell, near the cilia and within the cell instead of between cells. This suggests that particle charge affects the route of transit. In EM images, in cells exposed to positively charged particles, there were several found within cells of different sizes (15 nm–50 nm), suggesting variable levels of aggregation. However, there were very few particles identified in cell monolayers exposed to negatively charged particles. Afew identified did in fact seem to be between cells, and were of smaller sizes (15 nm) suggesting less particle aggregation (). It is possible that during the monolayer processing, many of the particles between cells were washed, and therefore so few were found. Of note, in response to exposure of epithelial cells to QDs, there was no significant trypan blue staining of the monolayer, suggesting minimal cell death at the time points studies (not shown).
Figure 3. (A) After 5 min exposure, QDs penetrate the epithelium. Fluorescence images for airway epithelial cells 5 min after incubating with a dose of 10 NP/cell. (Green) Cell membrane, (red) QDs. After 5 min exposure, the majority of the negatively charged QDs (more ...)
While other models have indicated charge-based differences in nanoparticle transit, the differences are specific to the cell studied. It has been suggested in Hydra vulgaris
that positively charged quantum rods are endocytosed by cells (Tortiglione et al. 2009
). While Dausend et al. did not find an effect of charge on nanoparticle uptake in HeLa cells (Dausend et al. 2008
), their studies indicated that endocytosis of positively and negatively charged particles occurred by different mechanisms, and thereby can influence uptake in cells depending on which mechanisms predominate. 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
), the airway epithelium has a higher paracellular permeability (Widdicombe J 1997), and transport could be substantially altered. The mechanisms dictating these routes are unknown. In addition, it is not clear whether negatively charged QDs are primarily transported via the paracellular pathway due to exclusion from a transcellular pathway, or due to selectivity by proteins mediating cell–cell contacts. Unlike the transcellular route, which has both active and passive processes regulating transport, paracellular transport is believed to have only passive processes driven by the gradients created by transcellular transport mechanisms (Anderson & Van Itallie 1995
). The major barrier in the paracellular route is the tight junction, which varies in ion selectivity. At physiological pH, most tight junctions are thought to be slightly cation selective (Colegio et al. 2003
), potentially contributing to the increase in negative QDs between cells.
Since the QDs are photoluminescent, we can determine whether they cross the epithelial barrier by sampling the fluorescence in the basolateral medium (). Within 30 min of exposure to QDs there was evidence of increased fluorescence in the basolateral medium of cells incubated with both positively and negatively charged particles, indicating that both crossed the epithelial barrier. After 30 min incubation with QDs, the amount of negatively charged particles in the basolateral medium was relatively independent of dose. In contrast, the amount of positively charged particles was significantly higher at doses of 10 and 100 NP/cell compared to negatively charged particles. Since the negatively charged particles are preferentially localised at the cell–cell junctions, this suggests that either transcellular transport in faster than paracellular transport or, more likely, that the capacity for storing QDs in the paracellular regions is high resulting in a delay in transport into the basolateral media. The fluorescence does not increase linearly with dose, implying that there is a significant fraction of QDs within the epithelial layer. After 4 h incubation, the amount of QDs increased approximately linearly with dose, and was independent of charge, suggesting that the amount of QDs in the epithelial layer has saturated and that the global transport kinetics have reached a steady state, independent of charge.
One of the fundamental features of the airway epithelium is its barrier properties. The epithelial barrier is the first line of defence preventing access of inhaled particles to subepithelial tissues (Gabrielson et al. 1994
; Yu et al. 1994
; Yu et al. 1994
; Rothen-Rutishauser et al. 2008
). However, the epithelium also serves to segregate the apical and basal compartments (Humlicek et al. 2007
), (Sidhaye et al. 2011
; Winter et al. 2006
) so that regulation of epithelial barrier properties can influence cell signalling (Humlicek et al. 2007
; Sidhaye et al. 2011
). In order to determine if QD charge could affect airway epithelial barrier function, we measured TER using two probe electrodes inserted on either side of the barrier (). The TER decreased for both positively and negatively charged particles in a dose-responsive fashion, indicating that exposure to QDs compromises barrier function. The largest change in TER occurred between 1 and 5 h of exposure, consistent with the fluorescence results shown in . In particular, even a very low exposure of QD, at a concentration of approximately 1 NP/cell was sufficient to cause a significant decrease in TER after 5 h exposure. The change in resistance after 24 h exposure increases exponentially with dose (), independent of charge.
Figure 4. Nanoparticle exposure results in altered airway epithelial barrier function. (A) TER versus time for airway epithelial cells incubated with 1, 10 or 100 NP/cell. Both positively and negatively charged QDs decrease TER in a dose-responsive fashion. The (more ...)
Previous studies have concluded that there is no correlation between change in TER and solute flux,(McCarthy et al. 1996
) and hence we assessed the effects of QDs on paracellular permeability by measuring the barrier permeability to 4 kDa FITC-dextran (). The TER measurements described above are sensitive to ionic transport and show that QD exposure decreases the resistance corresponding to an increase in ionic transport. The hydrodynamic diameter of dextran is about 2.5 nm (Armstrong et al. 2004
), an order of magnitude larger than Na+
, and hence the dextran permeability experiments probe larger length scales.
After incubation with QDs for different times, the fluorescently labelled dextran was injected into the ASL. Dextran transport across the airway epithelial cells was determined from fluorescence measurements of the medium from the basolateral chamber. After 5 min of exposure, there is no change in permeability compared to a barrier that had not been previously exposed to QDs. After 30 min of exposure, however, the permeability decreased with decreasing dose. At this larger length scale, exposure to QDs at low dose decreases dextran permeability after 30 min, whereas exposure to large doses has no effect. While this result is unexpected, one potential explanation is that the epithelial response to low doses resulting in enhanced barrier function is overwhelmed at higher doses. This is in contrast to previous work where we showed that initial exposure to micron-sized particles can result in decreases in permeability without dose dependence (Sidhaye et al. 2011