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Differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type 1 epithelial cells.

Ruenraroengsak P, Tetley TD - Part Fibre Toxicol (2015)

Bottom Line: TT1 cells were the most resistant to the effects of UNP and CNP.MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo.The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Lung Cell Biology, Section of Airways Disease, National Heart & Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, UK. p.ruenraroengsak@imperial.ac.uk.

ABSTRACT

Background: Engineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo.

Methods: Amine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1-100 μg/ml (1.25-125 μg/cm(2); 0 μg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy).

Results: ANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP.

Conclusion: ANP induced marked oxidative damage and cell death via apoptosis in all cell types, while UNP and CNP exhibited low cytotoxicity via oxidative stress. MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo. The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells.

No MeSH data available.


Related in: MedlinePlus

Interaction and uptake of 100 nm polystyrene UNP. CNP and ANP by TT1 cells 4 h after exposure. Following exposure to 50 μg/ml NPs, compared to non-exposed TT1 cells (a), UNPs were taken up via endocytosis as an agglomerate (arrows in b) and as individual particles (arrows in c). UNPs were also observed paracellularly and were taken up individually (arrows in d-e). Similar observations were made following TT1 cell exposure to CNPs (f; arrows in g indicate endocytosis and macropinocytosis). The CNPs also travelled paracellularly (left arrow in h, right arrow indicates tight junction-tj). The ANPs were taken up via endocytosis as agglomerates (arrows in j) and individually (arrows in k), but few were observed paracellularly. The percent cell uptake of all NPs by all cell types is shown in Fig. 6m-o in comparison with the 50 nm NPs. Scale bars in a-k are 200 nm; a total of 90 cells were examined
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Fig7: Interaction and uptake of 100 nm polystyrene UNP. CNP and ANP by TT1 cells 4 h after exposure. Following exposure to 50 μg/ml NPs, compared to non-exposed TT1 cells (a), UNPs were taken up via endocytosis as an agglomerate (arrows in b) and as individual particles (arrows in c). UNPs were also observed paracellularly and were taken up individually (arrows in d-e). Similar observations were made following TT1 cell exposure to CNPs (f; arrows in g indicate endocytosis and macropinocytosis). The CNPs also travelled paracellularly (left arrow in h, right arrow indicates tight junction-tj). The ANPs were taken up via endocytosis as agglomerates (arrows in j) and individually (arrows in k), but few were observed paracellularly. The percent cell uptake of all NPs by all cell types is shown in Fig. 6m-o in comparison with the 50 nm NPs. Scale bars in a-k are 200 nm; a total of 90 cells were examined

Mentions: TEM and scanning electron microscopy (SEM) were employed to observe nanoparticle-cell interactions and particle uptake (Fig. 6, 7 and 8). We recently showed that the uptake and transport of the same set of latex nanoparticles (t = 4 h) through TT1 cells involved both passive and active transport depending on their size and surface chemistry [16]. The 50 nm NPs largely entered TT1 cells via passive transport, while the 100 nm NPs entered mainly via clathrin- and caveolin-mediated endocytosis; 50 nm ANPs were internalised more rapidly than the UNPs and CNPs [16]. 3−8 % of 50 nm UNP and CNP translocated across the TT1 monolayer, without interfering with TT1 monolayer integrity [16]. It was demonstrated that the NPs can traverse between TT1 cells, until they reach a tight junction [16], also shown here in Fig. 7h and which suggests that the integrity of the tight junction (white tj, arrow), and its location, controls the translocation of these NPs between epithelial cells. The aim of the study did not include the effect of NPs on cell monolayer integrity and their translocation; however, work on the TT1 cell in this respect is described elsewhere [16]. In the current study, the cells were exposed to 50 μg/ml NPs, as this concentration exhibited very low toxicity (viability was 92−95 %, Additional file 1: Figure S1, [31]) at 4 h exposure and was a critical concentration at which a change in mitochondrial structure was observed. 40−60 % of TT1 cells and 50−70 % of MACs internalised NPs, whereas only 7−22 % of AT2 cells contained NPs (Fig. 6m-o). Interestingly, there was little difference between NP-functionalisation and the number of NPs taken up by each cell type, despite marked differences in cell viability, where ANP were most cytotoxic. Neither was there any difference between the particle sizes. This indicates that surface charge is an important component of the cytotoxic effect of the ANP, with the exception of TT1 cells, where 100 nm ANP-functionalised NPs caused relatively little cytotoxicity. The uptake of NPs by AT2 cells was much lower than that of the TT1 cells and the number of cytosolic NPs in AT2 cells was also much lower than that of the TT1 cells (data not shown). TT1 cells internalised all types of NPs following 4 h exposure (Fig. 6b-d, m, Fig. 7). The 50 nm and 100 nm NPs were observed within TT1 cell vesicles, suggesting active uptake (Fig. 6c-d, Fig. 7). Particles in endo/lysosomal compartments of TT1 were in the form of agglomerates, possibly aggregates (Fig. 6b-d). Cytoplasmic NPs were present as individual particles (Fig. 6b, c, l and Fig. 7c-e and k), suggesting passive uptake of NPs or that NPs might escape from endo/lysosomes. Use of the LysoTracker® fluorescent probe indicated an effect of NPs on lysosomal membrane integrity. The decrease in mean fluorescence intensity (MFI) of the probe indicated a decrease in the number of intact lysosomes within the cells following NP exposure. ANPs, but neither UNPs nor CNPs, caused a significant reduction in the MFI of LysoTracker® (p < 0.001, n = 3, Fig. 6p) suggesting that the amine-surface modified NPs precipitated lysosomal membrane damage and, subsequently, escaped to the cytoplasm possibly via a ‘proton sponge’ mechanism [36]. We previously reported that ANPs caused pore formation in the cell membrane which may be one mechanism of passive uptake of 50 nm ANPs [31]. In addition, NPs (Fig. 7c and k) appeared to adhere to the TT1 cell membrane and penetrate into the cell cytoplasm. The uptake of individual particles was also observed to occur at the lateral, paracellular and cell-cell interface, where NPs had tracked between the cells, up to the tight junction, before translocation as individual NPs across the cell membrane (Fig. 7d-e and h-i) as we previously reported [16]. It is difficult to assess whether particles that appear to be within the cytosol are membrane-bound. We used a sample preparation and staining technique, with osmium, uranyl acetate and lead citrate post-stain, to specifically identify membranes and believe that any membranes, including vesicular membranes should have been apparent. Thus, we believe that some particles appear to be free within the cytosol. And, importantly, this latter, paracellular process was less obvious with ANP suggesting different uptake mechanisms (Fig. 7j-k). In contrast to TT1 cells, most of which internalised all types of 50 nm NPs, only a small proportion of AT2 cells (6−20 % from Fig. 6, [16, 29]) were found to contain NPs (Fig. 6f-g, n). Intracellular ANPs were only found in the endosomal compartment (Fig. 6h), indicating active uptake.Fig. 6


Differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type 1 epithelial cells.

Ruenraroengsak P, Tetley TD - Part Fibre Toxicol (2015)

Interaction and uptake of 100 nm polystyrene UNP. CNP and ANP by TT1 cells 4 h after exposure. Following exposure to 50 μg/ml NPs, compared to non-exposed TT1 cells (a), UNPs were taken up via endocytosis as an agglomerate (arrows in b) and as individual particles (arrows in c). UNPs were also observed paracellularly and were taken up individually (arrows in d-e). Similar observations were made following TT1 cell exposure to CNPs (f; arrows in g indicate endocytosis and macropinocytosis). The CNPs also travelled paracellularly (left arrow in h, right arrow indicates tight junction-tj). The ANPs were taken up via endocytosis as agglomerates (arrows in j) and individually (arrows in k), but few were observed paracellularly. The percent cell uptake of all NPs by all cell types is shown in Fig. 6m-o in comparison with the 50 nm NPs. Scale bars in a-k are 200 nm; a total of 90 cells were examined
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4489088&req=5

Fig7: Interaction and uptake of 100 nm polystyrene UNP. CNP and ANP by TT1 cells 4 h after exposure. Following exposure to 50 μg/ml NPs, compared to non-exposed TT1 cells (a), UNPs were taken up via endocytosis as an agglomerate (arrows in b) and as individual particles (arrows in c). UNPs were also observed paracellularly and were taken up individually (arrows in d-e). Similar observations were made following TT1 cell exposure to CNPs (f; arrows in g indicate endocytosis and macropinocytosis). The CNPs also travelled paracellularly (left arrow in h, right arrow indicates tight junction-tj). The ANPs were taken up via endocytosis as agglomerates (arrows in j) and individually (arrows in k), but few were observed paracellularly. The percent cell uptake of all NPs by all cell types is shown in Fig. 6m-o in comparison with the 50 nm NPs. Scale bars in a-k are 200 nm; a total of 90 cells were examined
Mentions: TEM and scanning electron microscopy (SEM) were employed to observe nanoparticle-cell interactions and particle uptake (Fig. 6, 7 and 8). We recently showed that the uptake and transport of the same set of latex nanoparticles (t = 4 h) through TT1 cells involved both passive and active transport depending on their size and surface chemistry [16]. The 50 nm NPs largely entered TT1 cells via passive transport, while the 100 nm NPs entered mainly via clathrin- and caveolin-mediated endocytosis; 50 nm ANPs were internalised more rapidly than the UNPs and CNPs [16]. 3−8 % of 50 nm UNP and CNP translocated across the TT1 monolayer, without interfering with TT1 monolayer integrity [16]. It was demonstrated that the NPs can traverse between TT1 cells, until they reach a tight junction [16], also shown here in Fig. 7h and which suggests that the integrity of the tight junction (white tj, arrow), and its location, controls the translocation of these NPs between epithelial cells. The aim of the study did not include the effect of NPs on cell monolayer integrity and their translocation; however, work on the TT1 cell in this respect is described elsewhere [16]. In the current study, the cells were exposed to 50 μg/ml NPs, as this concentration exhibited very low toxicity (viability was 92−95 %, Additional file 1: Figure S1, [31]) at 4 h exposure and was a critical concentration at which a change in mitochondrial structure was observed. 40−60 % of TT1 cells and 50−70 % of MACs internalised NPs, whereas only 7−22 % of AT2 cells contained NPs (Fig. 6m-o). Interestingly, there was little difference between NP-functionalisation and the number of NPs taken up by each cell type, despite marked differences in cell viability, where ANP were most cytotoxic. Neither was there any difference between the particle sizes. This indicates that surface charge is an important component of the cytotoxic effect of the ANP, with the exception of TT1 cells, where 100 nm ANP-functionalised NPs caused relatively little cytotoxicity. The uptake of NPs by AT2 cells was much lower than that of the TT1 cells and the number of cytosolic NPs in AT2 cells was also much lower than that of the TT1 cells (data not shown). TT1 cells internalised all types of NPs following 4 h exposure (Fig. 6b-d, m, Fig. 7). The 50 nm and 100 nm NPs were observed within TT1 cell vesicles, suggesting active uptake (Fig. 6c-d, Fig. 7). Particles in endo/lysosomal compartments of TT1 were in the form of agglomerates, possibly aggregates (Fig. 6b-d). Cytoplasmic NPs were present as individual particles (Fig. 6b, c, l and Fig. 7c-e and k), suggesting passive uptake of NPs or that NPs might escape from endo/lysosomes. Use of the LysoTracker® fluorescent probe indicated an effect of NPs on lysosomal membrane integrity. The decrease in mean fluorescence intensity (MFI) of the probe indicated a decrease in the number of intact lysosomes within the cells following NP exposure. ANPs, but neither UNPs nor CNPs, caused a significant reduction in the MFI of LysoTracker® (p < 0.001, n = 3, Fig. 6p) suggesting that the amine-surface modified NPs precipitated lysosomal membrane damage and, subsequently, escaped to the cytoplasm possibly via a ‘proton sponge’ mechanism [36]. We previously reported that ANPs caused pore formation in the cell membrane which may be one mechanism of passive uptake of 50 nm ANPs [31]. In addition, NPs (Fig. 7c and k) appeared to adhere to the TT1 cell membrane and penetrate into the cell cytoplasm. The uptake of individual particles was also observed to occur at the lateral, paracellular and cell-cell interface, where NPs had tracked between the cells, up to the tight junction, before translocation as individual NPs across the cell membrane (Fig. 7d-e and h-i) as we previously reported [16]. It is difficult to assess whether particles that appear to be within the cytosol are membrane-bound. We used a sample preparation and staining technique, with osmium, uranyl acetate and lead citrate post-stain, to specifically identify membranes and believe that any membranes, including vesicular membranes should have been apparent. Thus, we believe that some particles appear to be free within the cytosol. And, importantly, this latter, paracellular process was less obvious with ANP suggesting different uptake mechanisms (Fig. 7j-k). In contrast to TT1 cells, most of which internalised all types of 50 nm NPs, only a small proportion of AT2 cells (6−20 % from Fig. 6, [16, 29]) were found to contain NPs (Fig. 6f-g, n). Intracellular ANPs were only found in the endosomal compartment (Fig. 6h), indicating active uptake.Fig. 6

Bottom Line: TT1 cells were the most resistant to the effects of UNP and CNP.MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo.The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Lung Cell Biology, Section of Airways Disease, National Heart & Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, UK. p.ruenraroengsak@imperial.ac.uk.

ABSTRACT

Background: Engineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo.

Methods: Amine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1-100 μg/ml (1.25-125 μg/cm(2); 0 μg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy).

Results: ANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP.

Conclusion: ANP induced marked oxidative damage and cell death via apoptosis in all cell types, while UNP and CNP exhibited low cytotoxicity via oxidative stress. MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo. The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells.

No MeSH data available.


Related in: MedlinePlus