Janus Liposomes: Exploring Liquid–Liquid Phase-Separating Lipid Systems Alternative to DOPC/DPPC/Cholesterol

Existing work on Janus liposomes almost exclusively employs the ternary lipid system based on 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC), and cholesterol (Chol), offering limited material choice, stability, and operating temperature window. In this work, we have systematically investigated >30 binary/ternary lipid combinations as potential alternatives to DOPC/DPPC/Chol to produce microsized Janus liposomes. A variety of structural/chemical differentiators are explored to induce phase separation in these systems, such as chain length (C10 to C18), unsaturation level (1 to 3 double bonds per acyl chain) and headgroup identity (phosphatidylcholine, phosphatidylglycerol and sphingomyelin). Using confocal fluorescence microscopy, we have identified eight new Chol-containing systems that produce Janus liposomes in decent to high yields. Furthermore, we also discover that global liquid-liquid phase separation can occur within individual Chol-free, binary liposomes, despite at much lower yields compared to Chol-containing systems. Evaluating these successful cases together with other tested systems, we also attempt to provide a set of guidelines for designing and preparing phase-separated liposomes. These findings may be of value to workers in membrane biophysics, particularly related to lipid liquid-liquid phase separation, as well as those interested in exploring Janus liposomes as an anisotropic colloidal material, for example, in liposomal drug delivery and active matter.