A Novel Method of Measuring ENM Induced Lipid Disruption in Macrophages and Model Membranes Systems
Presentation Type
Poster Presentation
Abstract/Artist Statement
The expanded use of nanotechnology has led to increased production and use of engineered nanomaterials (ENM), resulting in an increased risk of human exposure. Human exposure to ENM has the potential to cause chronic inflammatory diseases. ENM generated occupationally can be airborne and are taken-up by immune cells (macrophages) in the lung, where the ENM accumulate in phagolysosome organelles. Some ENM have been shown to cause damage to phagolysosomes, resulting in phagolysosomal membrane permeability (LMP). LMP leads to the release of degradative enzymes into the cytosol, leading to release of inflammatory cytokines and cell death. This suggests that ENM may interact directly with the lipid membrane of the phagolysosomes, disrupting their normal state, resulting in LMP. The way in which various ENM disrupt lipid membranes, is not fully understood. Time-resolved fluorescence anisotropy measurements, using suitable lipid probes, can measure changes in membrane characteristics, such as lipid order (Lo) and disorder (Ld). Additionally, macrophage-like THP-1 cells, which stably express a YFP-ASC protein can be used to determine inflammasome formation, an immediate downstream event of LMP leading to inflammation. In this work, THP-1 cells and 100 nm liposomes made of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) were used as model systems determine interaction with ENM. Both models were exposed to 12.5 to 100 μg/ml of titanium dioxide (TiO2), zinc oxide (ZnO) nanospheres. Fluorescence membrane probe Di-4-ANEPPDHQ and a time-resolved fluorometer were used to determine the changes in lipid Lo/Ld of the liposomes. Inflammasome formation (measured as visable specks in THP-1 cells) were quantified using a laser scanning cytometer. THP-1 cells exposed to 100 μg/ml TiO2 for 4 hr had a significant increase (11%) in speck formation. There was no significant continued increase in speck formation at 100 μg/ml TiO2 between 4 and 24 hr. After 24 h the 50 μg/ml dose of TiO2 increased to nearly the same level as the 4 hr 100 μg/ml. ZnO (a more toxic ENM) exposed THP-1 had a significant increase in speck formation at 50 μg/ml after 4 hr (14%) and at 25 and 50 μg/ml after 24 hr, 25% and 45%, respectively. While both TiO2 and ZnO seemed to cause inflammasome formation, resulting from LMP, ZnO was confirmed to be more potent than TiO2. POPC liposomes exposed to 100 μg/ml TiO2 had a decrease in lipid order, but no significant change was observed using DOPS liposomes. ZnO exposure (100 μg/ml) to DOPS liposomes also showed a decrease in lipid order, but again there was no change using POPC liposomes. These results indicate that while both materials may be disrupting lipid membranes, the target lipids are different.
Mentor Name
Andrij Holian
A Novel Method of Measuring ENM Induced Lipid Disruption in Macrophages and Model Membranes Systems
UC North Ballroom
The expanded use of nanotechnology has led to increased production and use of engineered nanomaterials (ENM), resulting in an increased risk of human exposure. Human exposure to ENM has the potential to cause chronic inflammatory diseases. ENM generated occupationally can be airborne and are taken-up by immune cells (macrophages) in the lung, where the ENM accumulate in phagolysosome organelles. Some ENM have been shown to cause damage to phagolysosomes, resulting in phagolysosomal membrane permeability (LMP). LMP leads to the release of degradative enzymes into the cytosol, leading to release of inflammatory cytokines and cell death. This suggests that ENM may interact directly with the lipid membrane of the phagolysosomes, disrupting their normal state, resulting in LMP. The way in which various ENM disrupt lipid membranes, is not fully understood. Time-resolved fluorescence anisotropy measurements, using suitable lipid probes, can measure changes in membrane characteristics, such as lipid order (Lo) and disorder (Ld). Additionally, macrophage-like THP-1 cells, which stably express a YFP-ASC protein can be used to determine inflammasome formation, an immediate downstream event of LMP leading to inflammation. In this work, THP-1 cells and 100 nm liposomes made of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) were used as model systems determine interaction with ENM. Both models were exposed to 12.5 to 100 μg/ml of titanium dioxide (TiO2), zinc oxide (ZnO) nanospheres. Fluorescence membrane probe Di-4-ANEPPDHQ and a time-resolved fluorometer were used to determine the changes in lipid Lo/Ld of the liposomes. Inflammasome formation (measured as visable specks in THP-1 cells) were quantified using a laser scanning cytometer. THP-1 cells exposed to 100 μg/ml TiO2 for 4 hr had a significant increase (11%) in speck formation. There was no significant continued increase in speck formation at 100 μg/ml TiO2 between 4 and 24 hr. After 24 h the 50 μg/ml dose of TiO2 increased to nearly the same level as the 4 hr 100 μg/ml. ZnO (a more toxic ENM) exposed THP-1 had a significant increase in speck formation at 50 μg/ml after 4 hr (14%) and at 25 and 50 μg/ml after 24 hr, 25% and 45%, respectively. While both TiO2 and ZnO seemed to cause inflammasome formation, resulting from LMP, ZnO was confirmed to be more potent than TiO2. POPC liposomes exposed to 100 μg/ml TiO2 had a decrease in lipid order, but no significant change was observed using DOPS liposomes. ZnO exposure (100 μg/ml) to DOPS liposomes also showed a decrease in lipid order, but again there was no change using POPC liposomes. These results indicate that while both materials may be disrupting lipid membranes, the target lipids are different.