Using a Model Lysosome Membrane to Study Nanomaterial-Membrane Interactions

Abstract

Some engineered nanomaterials (ENM), phagocytosed by cells and transported to lysosomes can cause the release of hydrolytic enzymes into the cytosol of the cell, resulting in an inflammatory response and cytotoxicity. We designed model lipid systems to study interactions between ENM and lipid membranes to assess ENM-lipid interaction. Liposomes (100 nm) were generated from lipids found in lysosomes: bis(monooleoylglycero)phosphate (BMP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), in a ratio of 2:3:3. Tris-buffered saline (TBS, pH 7.4) and acetate-buffered saline (ABS, pH 4.5) was used to mimic the pH of early and late lysosomes. Liposome structure was evaluated using transmission electron microscopy; all preparations were ∼100 nm in diameter and unilamellar. Liposomes were exposure to two types of titanium dioxide (TiO2) ENM, nanospheres (TNS) and nanobelts (TNB), or zinc oxide (ZnO) nanospheres for 2 hours at 25, 100 or 200 µg/ml. Time-resolved fluorescence anisotropy was performed using the fluorescence probe di-4-ANEPPDHQ and a custom-built time-resolved fluorometer to measure changes in membrane fluidity (reported in cone angle). Liposomes (BMP:DOPC:DOPE) exposed to TNS (100 µg/ml) showed no difference in cone angle compared to control in either buffer. However, TNB exposure produced significant decreases in cone angle in TBS at 200 µg/ml (36.42±0.31° to 32.84±0.33°) and in ABS at both 100 and 200 µg/ml (34.92±0.52° to 34.30±0.83° and 30.88±0.12°). Even though the surface area of TNS was greater than TNB the lack of effect supported that shape was important. ZnO ENM exposed liposomes composed of BMP:DOPC:DOPE showed no significant change in cone angle (100 µg/ml). However, BMP:DOPC:DOPS liposome had a significant decrease in cone angle (37.60±0.35° to 34.12±0.30°), indicating lipid headgroup can influence interactions. Funding: NIH R01ES023209, 1F32ES027324, P20GM103546, P30GM103338 and J Murdock Charitable Trust.