Lipid oxidation alters membrane mechanics favoring permeabilization

Ferroptosis is a form of regulated necrosis characterized by the iron-dependent accumulation of lipid peroxides in cell membranes. However, how lipid oxidation affects biophysical properties of cellular membranes and how these changes contribute to the opening of membrane pores are major questions in the field. Therefore, understanding the interplay between oxidized phospholipids and alterations in membrane biophysical properties is essential to unravel the underlying mechanisms of ferroptosis. Here, we characterized membrane alterations upon lipid oxidation in in vitro model membrane systems using lipid vesicles and supported lipid bilayers (SLBs) in which lipid oxidation was induced by Fenton reactions. We find that vesicle permeabilization kinetically correlates with the appearance of malondialdehyde (MDA), a product of lipid oxidation. Lipid oxidation also alters the lateral organization of SLBs exhibiting liquid-ordered (Lo)/liquid-disordered (Ld) phase coexistence, reducing the lipid mismatch between domains and decreasing the circularity of Lo domains, indicating a decrease in line tension at the phase boundaries. Further characterization of SLBs by force spectroscopy reveals a significant reduction in the average membrane breakthrough force upon oxidation, indicating changes in lipid bilayer organization that make it more susceptible to permeabilization. Our findings suggest that lipid oxidation induces strong changes in membrane lipid interactions and in the mechanical properties of membranes leading to reduced tension in the permeabilized state of the bilayer, which promotes membrane pore formation.