Lipid Saturation and Cholesterol Drive the Mechanical Response of Lipid Bilayer to Ionic Liquid: An Atomic Force Microscopy Study

Ionic liquids (ILs) have recently emerged as promising amphiphilic electrolytes capable of modulating biomembrane properties, yet the mechanistic determinants underlying IL-lipid interactions remain insufficiently characterized. Here, we investigate the effect of the imidazolium IL [C6mim][Cl] on the structural and mechanical properties of phosphatidylcholine bilayers with controlled acyl-chain saturation (DPPC, POPC, DOPC) and cholesterol content. All measurements were performed at an IL concentration below the critical micellar concentration to avoid contributions from IL aggregation. Using atomic force microscopy, we show that even in the absence of topographical changes, the IL significantly affects membrane mechanics, as quantified by rupture force (RF) and Young's modulus (YM), exhibiting a strong dependence on lipid saturation and cholesterol content. For DPPC-Chol, incubation in [C6mim][Cl] leads to a pronounced decrease in RF (-26 ± 9%) and YM (-30 ± 10%), while no effect is observed without cholesterol. For POPC, a pronounced increase in RF is observed (+30 ± 9%), which reverts in the presence of cholesterol (-13 ± 7%), which also softens the bilayer (-19 ± 9%). For DOPC, a pronounced increase in YM is observed (+30 ± 14%), which reverts with the addition of cholesterol (-27 ± 5%) that, surprisingly, leads to a pronounced increase in RF (+34 ± 9%). Control measurements with NaCl further confirm that the observed effects are not due to ionic strength alone, highlighting the critical role of the IL organic cation in modulating bilayer mechanics. Overall, these findings establish lipid saturation and cholesterol content as key variables governing IL-membrane interactions, and offer a framework for rationally tuning ILs to modulate membrane mechanics─relevant for nanobiotechnology, drug delivery, and membrane engineering.