(338c) Membrane Lipid Asymmetry Regulates Nanoparticle-Induced Cell Membrane Damage in Red Blood Cells

Over the past decade, there has been increasing interest in understanding the mechanisms through which engineered nanomaterials (ENM) disrupt the plasma membrane of mammalian cells. However, despite a number of mechanistic studies, primarily using vesicles as cell membrane models, the mechanisms of ENM-induced plasma membrane damage have remained elusive. This lack of understanding is in part due to the structural complexities of the cell plasma membrane. In particular, membrane asymmetry, the fact that the cell plasma membrane has different lipids on the exofacial leaflet (i.e. looking away from the cytoplasm) compared to the cytofacial leaflet (i.e. looking toward the cytoplasm) has been completely overlooked in previous studies of ENM-membrane interactions. The present study focused on the role of individual plasma membrane leaflets in regulating nanoparticle-plasma membrane interactions. Specifically, the effects of plain, carboxyl-, amine-, and polyethylene glycol(PEG)-modified fluorescent silica nanoparticles (50 nm) on the integrity of vesicles mimicking the exofacial (V_exo) and cytofacial leaflets (V_cyto) of the plasma membrane of red blood cells was examined and compared with ENM-induced cell death in red blood cells (hemolysis).

Vesicle integrity was studied by encapsulating the self-quenching fluorescent probe, carboxyfluorescein, in vesicles and studying its leakage after exposure to 0.0001-0.01 g/L of ENMs at 37 °C. Confocal fluorescence microscopy performed on giant unilamellar vesicles (GUVs) was used to monitor nanoparticle effects on vesicle morphology. Forster Resonance Electron Transfer (FRET) experiments were employed to examine particle localization at the membrane. Finally, ENM-induced hemolysis was evaluated by measuring the absorbance of hemoglobin released from red blood cells after incubation with 0.01 g/L of nanoparticles at 37 °C.

Nanoparticle interactions with V_exo and V_cyto vesicles were drastically different. V_exo vesicles showed significant leakage after exposure to plain and amine-modified particles, but were not disrupted by carboxyl-modified and PEGylated particles. Conversely, none of the particles caused significant leakage in V_cyto vesicles as evidenced by a lack of significant leakage. In agreement, FRET experiments revealed significant localization of plain and amine-modified particles, but not carboxyl-modified and PEGylated particles, at the surface of the V_exo vesicles. Only minor localization with the V_cyto vesicles was observed for all particles. Similarly, GUV images indicated visual disruption of V_exo vesicles by plain and amine-modified ENMs. Importantly, hemolysis of red blood cells by ENMs was consistent with leakage assays using V_exo vesicles. Hemolysis was observed after the incubation of cells with plain and amine-modified particles, but not after incubation with carboxyl-modified and PEGylated particles. In conclusion, these results suggest that artificial vesicles mimicking the exofacial leaflet of the cell plasma membrane predict ENM-induced membrane damage in red blood cells and could have important implications in predicting the cytotoxicity of ENMs.