The phase behavior of phospholipids dictates various membrane properties, such as permeability, shape, and stiffness. Some phospholipids exhibit a polymorphic gel phase and are capable of undergoing membrane interdigitation under specific conditions, such as in the presence of short-chain alcohols (e.g., ethanol). Ethanol-induced interdigitation of diacyl–phosphatidylcholine bilayers has been leveraged to formulate large unilamellar vesicles with high entrapped aqueous volumes via the so called interdigitation-fusion vesicle method. In this case, ethanol is added to a small unilamellar vesicle (SUV) suspension below the lipid transition temperature (T_m), which induces vesicle fusion and rupture, together with bilayer interdigitation. A sol–gel transition of the vesicle suspension typically occurs as a result. Upon a temperature increase above the lipid T_m, the interdigitated bilayers refold into large unilamellar vesicles.

In order to induce bilayer interdigitation, water molecules at the lipid–water interface need to be replaced by ethanol, which alters the magnitude of hydration forces responsible for interbilayer repulsion. Binding of cations to the polar headgroups of phospholipid molecules can also modify the water layer coordinated to the lipid bilayer. However, most of the studies on the interactions between ethanol and PC bilayers in the context of bilayer interdigitation have been carried out in water and little is known on the role of cations, in particular Ca²⁺ and Na⁺. Here, we employed a combination of small- and wide-angle X-ray scattering (SAXS and WAXS), small-angle neutron scattering (SANS), and all-atom MD simulations to elucidate the nature of complex interactions between calcium and sodium ions, ethanol, and PC membranes. While bulk phase DPPC exhibited membrane interdigitation at all of the NaCl and CaCl₂ concentrations explored, DPPC vesicles underwent membrane interdigitation and vesicle fusion in interdigitated lamellae only for a specific CaCl₂ concentration. Large, multilamellar vesicles formed in the other conditions. These findings highlight the important role of cations in ethanol-induced membrane interdigitation and have implications for cell biology and biochemistry, as well as being directly applicable for, i.e., the fabrication of interdigitation-fusion vesicles with high entrapped volumes.