(16e) Membrane Outer Leaflet Is the Primary Regulator of Membrane Damage Induced By Silica Nanoparticles in Vesicles and Red Blood Cells

Plasma membrane damage is one of the primary mechanisms through which engineered nanomaterials cause cytotoxicity. Despite a number of studies, the role of the structural features of the plasma membrane, in particular membrane phospholipid asymmetry in regulating nanoparticle-membrane interactions has remained obscure. In the current study, the role of membrane phospholipid asymmetry, differences in the lipid composition of exofacial and inner leaflet of the plasma membrane in regulating nanoparticle-membrane interactions was investigated. Vesicles mimicking the outer (V_exo) and inner leaflets (V_cyto) of the plasma membrane of red blood cells (RBCs) were exposed to silica nanoparticles (50 nm), with different surface functional groups, plain, or carboxyl-, amine-, and polyethylene glycol (PEG)-modified and the effect of particles on vesicle integrity was examined. Symmetric and asymmetric vesicles with similar outer leaflet, but different inner leaflet lipid compositions, as well as live RBCs, were also used to elucidate the role of membrane asymmetry and individual leaflets in regulating nanoparticle-membrane interaction.

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 nanoparticles at 37 °C. Confocal fluorescence microscopy performed on giant unilamellar vesicles (GUVs) was used to monitor nanoparticle effects on vesicle morphology. Förster Resonance Energy Transfer (FRET) experiments were employed to examine particle localization at the membrane. Finally, nanoparticle-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.

V_exo and V_cyto vesicles showed significant difference in their interactions with nanoparticles. V_exo vesicles showed drastic leakage of intravesicular content after exposure to plain and amine-modified particles while none of the particles caused significant leakage in V_cyto vesicles. FRET experiments revealed that nanoparticle-induced disruption by plain and amine-modified particles occurs via pore formation. In agreement, FRET assay and GUV images revealed significant localization of plain and amine-modified particles at the surface of the V_exo vesicles. However, no localization was observed with the V_cyto vesicles. Interestingly, symmetric and asymmetric vesicles were disrupted similarly by plain and amine-modified particles. Importantly, hemolysis was observed after the incubation of RBCs with plain and amine-modified particles, consistent with leakage assays using V_exo vesicles. In conclusion, these results suggest that the outer leaflet of the plasma membrane is the primary regulator of nanoparticle-induced membrane damage in RBCs. Future studies focused on the understanding the role of outer leaflet lipids in regulating ENM-membrane interactions in live cells.