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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: Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery.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.

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

Effect of polystyrene nanoparticles on total cellular glutathione and TT1 cellular oxidised glutathione: reduced glutathione ratio (GSSG: GSH) at 1 and 4 h exposure. GSSG:GSH ratio following exposure to 50 nm (a, b) and 100 nm (d, e) NPs for 1 (a, d) and 4 h (b, e) respectively. Total GSH following exposure to 50 nm (c) and 100 nm (f) for 4 h with and without antioxidant N-acetylcysteine (NAC, 10 mM). ANPs caused the most significant increases in GSSG:GSH ratio, regardless of particle size. All three NPs caused a reduction in total cellular GSH, though this was most marked following ANP exposure (c, f). NAC treatment significantly prevented this effect (c, f). *p < 0.05, p** < 0.001; n = 3 replicates
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Fig3: Effect of polystyrene nanoparticles on total cellular glutathione and TT1 cellular oxidised glutathione: reduced glutathione ratio (GSSG: GSH) at 1 and 4 h exposure. GSSG:GSH ratio following exposure to 50 nm (a, b) and 100 nm (d, e) NPs for 1 (a, d) and 4 h (b, e) respectively. Total GSH following exposure to 50 nm (c) and 100 nm (f) for 4 h with and without antioxidant N-acetylcysteine (NAC, 10 mM). ANPs caused the most significant increases in GSSG:GSH ratio, regardless of particle size. All three NPs caused a reduction in total cellular GSH, though this was most marked following ANP exposure (c, f). NAC treatment significantly prevented this effect (c, f). *p < 0.05, p** < 0.001; n = 3 replicates

Mentions: As we have limited numbers of primary AT2 and MAC cells, we used TT1 cells as a model to study the effect of oxidative stress on glutathione flux. Cellular glutathione levels (GSH and GSSG) were measured in TT1 cells at 1 and 4 h after 50 nm NP exposure (Fig. 3a, b). After 1 h, the GSSG/GSH ratio (indicating the ratio of oxidised GSSG to reduced GSH) was increased, 2-3-fold above non-treated control cells, for all types of NPs (at concentrations of 50 and 100 μg/ml) reflecting oxidative stress (Fig. 3a-b). By 4 h (Fig. 3b), control (baseline) TT1 cell GSSG/GSH ratio had increased above that observed at 1 h. There was a further, significant increase in the GSSG/GSH ratio following ANP exposure, even at the lowest concentration of 1 μg/ml (1.5-fold control; p < 0.05, n = 3), which increased in a concentration dependent manner, reaching >6-fold that of unexposed cells (p < 0.001, n = 3) at 50 μg/ml ANP; this effect was not observed with UNP or CNP. Co-incubation of NPs with NAC for 4 h prevented the reduction of total cellular glutathione (GSH and GSSG combined; Fig. 3c). The highly significant fall (down to ~10 % of control, p < 0.001, n = 3) in glutathione following exposure to 50 nm ANPs could be markedly prevented by NAC treatment (down to ~67 % of control). A similar trend was also observed in TT1 cells exposed to 100 nm NPs, although the effect of all three NPs on increased GSSG/GSH ratio at 1 h was more noticeable than that seen following exposure to the 50 nm NPs at the same time interval (Fig. 3d). Remarkably, this effect disappeared for UNP and CNP at 4 h, but remained at very similar levels for the ANP-exposed cells (Fig. 3e). NAC prevented the reduction of cellular glutathione activated by 100 nm NPs (Fig. 3f) to a very similar extent to that seen with 50 nm NPs.Fig. 3


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)

Effect of polystyrene nanoparticles on total cellular glutathione and TT1 cellular oxidised glutathione: reduced glutathione ratio (GSSG: GSH) at 1 and 4 h exposure. GSSG:GSH ratio following exposure to 50 nm (a, b) and 100 nm (d, e) NPs for 1 (a, d) and 4 h (b, e) respectively. Total GSH following exposure to 50 nm (c) and 100 nm (f) for 4 h with and without antioxidant N-acetylcysteine (NAC, 10 mM). ANPs caused the most significant increases in GSSG:GSH ratio, regardless of particle size. All three NPs caused a reduction in total cellular GSH, though this was most marked following ANP exposure (c, f). NAC treatment significantly prevented this effect (c, f). *p < 0.05, p** < 0.001; n = 3 replicates
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Effect of polystyrene nanoparticles on total cellular glutathione and TT1 cellular oxidised glutathione: reduced glutathione ratio (GSSG: GSH) at 1 and 4 h exposure. GSSG:GSH ratio following exposure to 50 nm (a, b) and 100 nm (d, e) NPs for 1 (a, d) and 4 h (b, e) respectively. Total GSH following exposure to 50 nm (c) and 100 nm (f) for 4 h with and without antioxidant N-acetylcysteine (NAC, 10 mM). ANPs caused the most significant increases in GSSG:GSH ratio, regardless of particle size. All three NPs caused a reduction in total cellular GSH, though this was most marked following ANP exposure (c, f). NAC treatment significantly prevented this effect (c, f). *p < 0.05, p** < 0.001; n = 3 replicates
Mentions: As we have limited numbers of primary AT2 and MAC cells, we used TT1 cells as a model to study the effect of oxidative stress on glutathione flux. Cellular glutathione levels (GSH and GSSG) were measured in TT1 cells at 1 and 4 h after 50 nm NP exposure (Fig. 3a, b). After 1 h, the GSSG/GSH ratio (indicating the ratio of oxidised GSSG to reduced GSH) was increased, 2-3-fold above non-treated control cells, for all types of NPs (at concentrations of 50 and 100 μg/ml) reflecting oxidative stress (Fig. 3a-b). By 4 h (Fig. 3b), control (baseline) TT1 cell GSSG/GSH ratio had increased above that observed at 1 h. There was a further, significant increase in the GSSG/GSH ratio following ANP exposure, even at the lowest concentration of 1 μg/ml (1.5-fold control; p < 0.05, n = 3), which increased in a concentration dependent manner, reaching >6-fold that of unexposed cells (p < 0.001, n = 3) at 50 μg/ml ANP; this effect was not observed with UNP or CNP. Co-incubation of NPs with NAC for 4 h prevented the reduction of total cellular glutathione (GSH and GSSG combined; Fig. 3c). The highly significant fall (down to ~10 % of control, p < 0.001, n = 3) in glutathione following exposure to 50 nm ANPs could be markedly prevented by NAC treatment (down to ~67 % of control). A similar trend was also observed in TT1 cells exposed to 100 nm NPs, although the effect of all three NPs on increased GSSG/GSH ratio at 1 h was more noticeable than that seen following exposure to the 50 nm NPs at the same time interval (Fig. 3d). Remarkably, this effect disappeared for UNP and CNP at 4 h, but remained at very similar levels for the ANP-exposed cells (Fig. 3e). NAC prevented the reduction of cellular glutathione activated by 100 nm NPs (Fig. 3f) to a very similar extent to that seen with 50 nm NPs.Fig. 3

Bottom Line: Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery.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.

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