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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

Effect of polystyrene nanoparticles on cytochrome C (Cyt C) release and the mitochondrial network (Mito) in TT1, AT2 and MAC. Exposure to 50 μg/ml 50 nm UNP and CNP had no effect on the release of Cyt C or the mitochondrial network. ANP caused disruption of the mitochondrial network (arrows indicate breakdown of Mitochondria in green) and initiated the release of Cyt C (arrows indicate the red area of Cyt C release) in all cell types). Cell nuclei, mitochondrial networks and cytochrome C are stained blue, green and red, respectively; n = 45 cells analysed/sample
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Fig5: Effect of polystyrene nanoparticles on cytochrome C (Cyt C) release and the mitochondrial network (Mito) in TT1, AT2 and MAC. Exposure to 50 μg/ml 50 nm UNP and CNP had no effect on the release of Cyt C or the mitochondrial network. ANP caused disruption of the mitochondrial network (arrows indicate breakdown of Mitochondria in green) and initiated the release of Cyt C (arrows indicate the red area of Cyt C release) in all cell types). Cell nuclei, mitochondrial networks and cytochrome C are stained blue, green and red, respectively; n = 45 cells analysed/sample

Mentions: Mitochondrial membrane potential and mitochondrial structure of TT1 cells following NP exposure (Fig. 4–5) were observed to compare with changes in ROS production, using MitoTracker® fluorescent probe, confocal microscopy and transmission electron microscopy (TEM). The MitoTracker® probe reflected mitochondrial membrane potential of the intact mitochondria; a significant decrease in mean fluorescence intensity (MFI), indicating reduction of mitochondrial membrane potential in all cell types exposed to 50 nm ANPs (t = 4 h, Fig. 4a-c; p < 0.001, n = 3 replicates TT1 and 6 subject samples AT2 and MAC). This was accompanied by mitochondrial swelling and disruption of the mitochondrial network in ANP-exposed cells (Fig. 4–5), as shown by TEM (Fig. 4, n = 60 observed cells) and confocal microscopy (Fig. 5, n = 45 observed cells). Mitochondrial swelling is a pathology of mitochondria indicated by an increase in volume of mitochondria due to the fluid influx as a result of altered mitochondrial membrane potential. The enlarged size of the mitochondria can be seen at the same magnification (same scale bar) as we show here in Fig. 4k, n and o, in comparison to the control cells in Fig. 4d, h and l. The structure of cristae collapse during the swelling process cannot be detected by the osmium contrast agent when using TEM. We also investigated changes in mitochondrial structure at the lower NP concentration range (1-25 μg/ml) using transmission electron microscopy (TEM) and did not see a difference compared to non-treated cells (data not shown). In the normal healthy cells the mitochondria form a network where each mitochondrion is linked to another as seen in control cells in Fig. 5 (the connected green fluorescent feature, control panel). Disconnection of the green fluorescent feature indicated the disconnected mitochondria within the network (change from green connect line to green dot), as seen following exposure to ANP (ANP, green fluorescence panel). Cytochrome C was stained with a red fluorescent signal and when co-localised with the mitochondrial fluorescent green signal, showed yellow. However, in ANP-exposed cells, cytochrome C is released from the mitochondria and this can be observed in a clear pure red fluorescent signal, indicating loss of mitochondrial integrity and apoptosis (arrows on the right ANP column in Fig. 5). Unlike TT1 cells, all NPs induced ROS production in AT2 cells, however only ANPs induced mitochondrial swelling and loss of mitochondrial membrane integrity, as seen by TEM (Fig. 4k), and breakdown of the mitochondrial network (Fig. 5). This was associated with the release of cytochrome C (Cyt C indicated with arrow) within the cells, though this was not as noticeable as that observed in TT1 cells (Fig. 5). Again, all types of NPs induced ROS in MACs; interestingly, unlike the epithelial cells, in MACs, both CNPs and ANPs initiated mitochondrial swelling (Fig. 4–5); however, breakdown of the mitochondrial network and release of Cyt C could only be observed in MACs exposed to ANPs (see arrows in Fig. 5).Fig. 4


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 cytochrome C (Cyt C) release and the mitochondrial network (Mito) in TT1, AT2 and MAC. Exposure to 50 μg/ml 50 nm UNP and CNP had no effect on the release of Cyt C or the mitochondrial network. ANP caused disruption of the mitochondrial network (arrows indicate breakdown of Mitochondria in green) and initiated the release of Cyt C (arrows indicate the red area of Cyt C release) in all cell types). Cell nuclei, mitochondrial networks and cytochrome C are stained blue, green and red, respectively; n = 45 cells analysed/sample
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4489088&req=5

Fig5: Effect of polystyrene nanoparticles on cytochrome C (Cyt C) release and the mitochondrial network (Mito) in TT1, AT2 and MAC. Exposure to 50 μg/ml 50 nm UNP and CNP had no effect on the release of Cyt C or the mitochondrial network. ANP caused disruption of the mitochondrial network (arrows indicate breakdown of Mitochondria in green) and initiated the release of Cyt C (arrows indicate the red area of Cyt C release) in all cell types). Cell nuclei, mitochondrial networks and cytochrome C are stained blue, green and red, respectively; n = 45 cells analysed/sample
Mentions: Mitochondrial membrane potential and mitochondrial structure of TT1 cells following NP exposure (Fig. 4–5) were observed to compare with changes in ROS production, using MitoTracker® fluorescent probe, confocal microscopy and transmission electron microscopy (TEM). The MitoTracker® probe reflected mitochondrial membrane potential of the intact mitochondria; a significant decrease in mean fluorescence intensity (MFI), indicating reduction of mitochondrial membrane potential in all cell types exposed to 50 nm ANPs (t = 4 h, Fig. 4a-c; p < 0.001, n = 3 replicates TT1 and 6 subject samples AT2 and MAC). This was accompanied by mitochondrial swelling and disruption of the mitochondrial network in ANP-exposed cells (Fig. 4–5), as shown by TEM (Fig. 4, n = 60 observed cells) and confocal microscopy (Fig. 5, n = 45 observed cells). Mitochondrial swelling is a pathology of mitochondria indicated by an increase in volume of mitochondria due to the fluid influx as a result of altered mitochondrial membrane potential. The enlarged size of the mitochondria can be seen at the same magnification (same scale bar) as we show here in Fig. 4k, n and o, in comparison to the control cells in Fig. 4d, h and l. The structure of cristae collapse during the swelling process cannot be detected by the osmium contrast agent when using TEM. We also investigated changes in mitochondrial structure at the lower NP concentration range (1-25 μg/ml) using transmission electron microscopy (TEM) and did not see a difference compared to non-treated cells (data not shown). In the normal healthy cells the mitochondria form a network where each mitochondrion is linked to another as seen in control cells in Fig. 5 (the connected green fluorescent feature, control panel). Disconnection of the green fluorescent feature indicated the disconnected mitochondria within the network (change from green connect line to green dot), as seen following exposure to ANP (ANP, green fluorescence panel). Cytochrome C was stained with a red fluorescent signal and when co-localised with the mitochondrial fluorescent green signal, showed yellow. However, in ANP-exposed cells, cytochrome C is released from the mitochondria and this can be observed in a clear pure red fluorescent signal, indicating loss of mitochondrial integrity and apoptosis (arrows on the right ANP column in Fig. 5). Unlike TT1 cells, all NPs induced ROS production in AT2 cells, however only ANPs induced mitochondrial swelling and loss of mitochondrial membrane integrity, as seen by TEM (Fig. 4k), and breakdown of the mitochondrial network (Fig. 5). This was associated with the release of cytochrome C (Cyt C indicated with arrow) within the cells, though this was not as noticeable as that observed in TT1 cells (Fig. 5). Again, all types of NPs induced ROS in MACs; interestingly, unlike the epithelial cells, in MACs, both CNPs and ANPs initiated mitochondrial swelling (Fig. 4–5); however, breakdown of the mitochondrial network and release of Cyt C could only be observed in MACs exposed to ANPs (see arrows in Fig. 5).Fig. 4

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