Unraveling phospholipid binding selectivity of miltefosine at membrane interfaces: Factors beyond lipid net charge.

Miltefosine (MIL) is a clinically relevant alkylphospholipid that selectively targets cancer cell membranes and has attracted interest for therapeutic and diagnostic applications. Although MIL is known to preferentially bind anionic phosphatidylserine (PS) lipids that are exposed on cancer cell membranes, the molecular basis of its interactions with PS and other membrane lipids remains unclear. Herein, we employed a two-phase polymerized liposome platform, which is sensitive to binding events and resulting membrane perturbations, to investigate MIL interactions with membranes containing dioleoyl PS (DOPS), anionic phosphatidylglycerol (DOPG), or zwitterionic phosphatidylcholine (DOPC) lipids. UV-vis and fluorescence spectroscopy showed that MIL interactions with polymerized liposomes, which contained one of those three lipids, occurred in the order of DOPS > DOPG > DOPC. In marked contrast, there were negligible MIL interactions with polymerized liposomes that did not contain the dioleoyl phospholipid component. Zeta potential measurements indicated that MIL binding generally reduced the membrane surface charge of polymerized liposomes incorporating DOPS, DOPG, or DOPC. ATR-FTIR spectroscopy further revealed that MIL interactions occurred mainly at the membrane interface while the specific binding profile was markedly influenced by the headgroup features of the dioleoyl phospholipid component in the liposomal membrane. Strong binding to DOPS-containing membranes involved both electrostatic attraction and hydrogen bonding whereas MIL binding to DOPG-containing membranes was driven mainly by electrostatics and interactions with DOPC-containing membranes were less favorable. These findings demonstrate that MIL membrane selectivity arises from specific molecular interactions with distinct lipid headgroups that go beyond membrane charge effects and provide a mechanistic framework for selective membrane targeting by alkylphospholipids.