(16g) Loss of Membrane Asymmetry Alters the Interactions of Red Blood Cells with Nanoparticles | AIChE

(16g) Loss of Membrane Asymmetry Alters the Interactions of Red Blood Cells with Nanoparticles


Bigdelou, P. - Presenter, Ohio University
Farnoud, A. M., Ohio University
Silica nanoparticles have been shown to induce cytotoxicity in red blood cells by disrupting the cell plasma membrane. Mechanistic studies on nanoparticle-induced membrane damage have used membrane models, such as lipid vesicles, to examine the molecular basis of nanoparticle toxicity. While membrane models have provided significant valuable insight, the role of membrane phospholipid asymmetry had been previously ignored in studies with membrane models. Studies from our group, using lipid vesicles, have shown that membrane lipid asymmetry plays a significant role in regulating nanoparticle-membrane interactions. However, it is unclear whether such results can be translated to cells. In the current study, the role of the plasma membrane asymmetry in regulating nanoparticle- membrane interactions has been investigated using healthy red blood cells, and red blood cells in which membrane asymmetry has been abolished.

Silica nanoparticles (30 nm) with different surface chemistries, plain, amine-modified, and carboxyl-modified, were purchased and characterized for their size and charge using dynamic light scattering and laser Doppler anemometry. De-identified human blood was commercially procured in acid citrate dextrose as anti-coagulant. Red blood cells were isolated and their membrane asymmetry was abolished by ionomycin in the presence of Ca2+ at 37ºC. Loss of membrane asymmetry was confirmed by monitoring the translocation of the cytofacial lipid, phosphatidylserine (PS), from the cytofacial to the exofacial leaflet of the cell membrane. PS externalization was examined by monitoring the binding of annexin V-Alexa Fluor 488 to PS in the exofacial leaflet of cells. Annexin V binding was monitored by confocal microscopy and flow cytometry and was used to calculate the population of cells with scrambled (symmetric) membranes. To study the hemolysis of treated and untreated cells by nanoparticles, 0.01 mg/ml of plain-, amine-, and carboxyl-modified nanoparticles of silica were added to the cells for 2 hours of incubation, and the supernatant was collected. Absorbance was measured at 541 nm and hemolysis was calculated.

To induce loss of membrane asymmetry, erythrocytes were treated with 1 to 10 µM of ionomycin for 2 hours at 37ºC. A maximum of 48% eryptosis was observed using this method as confirmed by flow cytometry. Nanoparticle interactions with asymmetric and symmetric cells were different, but depended on particle surface properties. In normal (asymmetric) cells, plain nanoparticles induced the highest degree of hemolysis (28%), followed by amine-modified particles. Carboxyl-modified particles did not show hemolytic activity in cells with asymmetric membrane. However, loss of membrane asymmetry led to a dose-dependent reduction in the hemolytic activity of plain silica nanoparticles, likely due to the presence of PS in the cell exofacial leaflet. In addition, the hemolytic activity of both amine- and carboxyl-modified nanoparticles increased in cells with symmetric membranes. Together, these results suggest that membrane asymmetry is an important factor in regulating nanoparticle-membrane interactions. Future work is focused on studies of nanoparticle binding to cells with symmetric and asymmetric membranes using electron microscopy and fluorescence spectroscopy.