Unraveling Cholesterol-Dependent Interactions of Alkylphospholipids with Supported Lipid Bilayers

Alkylphospholipids are single-chain lipid amphiphiles that possess clinically relevant biological activities driven by membrane-destabilizing interactions. Subtle variations in alkylphospholipid structure can lead to significant differences in their biological effects, yet corresponding membrane interactions remain underexplored. Herein, we employed the quartz crystal microbalance-dissipation (QCM-D) technique to characterize the real-time membrane interactions of three alkylphospholipids–edelfosine, miltefosine, and perifosine–on supported lipid bilayers with varying cholesterol fractions. Our findings reveal that the tested alkylphospholipids had distinct membrane-interaction profiles: (1) edelfosine exhibited irreversible binding and caused weak membrane disruption; (2) miltefosine caused major disruption by affecting membrane packing; and (3) perifosine exhibited reversible binding while triggering structural rearrangements and modest disruption. Overall, alkylphospholipid micelles showed greater activity than monomers, and higher membrane cholesterol fractions resulted in more extensive disruption, highlighting the interplay between membrane stiffness and responsiveness. These results provide biophysical evidence that different alkylphospholipids have distinct membrane-interaction behaviors that align well with reported biological activities. Our supported lipid bilayer approach offers a valuable platform for designing and assessing alkylphospholipids with tailored membrane-interaction profiles.