High-Density Inverted Micellar Intermediates Promote Membrane Fusion of Cationic Liposomes in Drug Delivery

Liposomes have become increasingly popular as carriers for pharmaceutically relevant molecules such as nucleic acids, proteins, or anticancer drugs. The bottleneck in delivering such vehicles is their inefficient endosomal uptake by target cells. To bypass endosomal degradation and enhance delivery efficiency, fusogenic liposomes have been developed. They fuse with extraordinary efficiency with the plasma membrane of mammalian cells and deliver their cargo directly into the cell cytoplasm. Here, we set out to decipher the key to membrane fusion and optimize the liposomal composition accordingly. Special focus has been placed on identifying the intrinsic phase properties of these liposomes. Therefore, giant and small cationic liposomes with outstandingly high membrane fusion efficiency were prepared, and their thermal phase behavior was investigated using fluorescence microscopy, solid-state NMR, small-angle neutron scattering (SANS), and cryo-electron microscopy techniques. Our experiments revealed a temperature-dependent phase behavior of those liposomes. At 25 °C and below, mainly a lamellar phase formed without elevated membrane fusion capacity. At the physiological temperature of 37 °C and above, we found high concentrations of inverted micellar intermediates and interlamellar attachments, presumably as precursors of a high-temperature 3D phase, embedded in a lamellar phase. Their structures were resolved by cryo-electron tomography. We believe that the presence of these metastable fusion intermediate structures enables highly efficient fusion with complex biological membranes under physiological conditions, as is necessary in biomedical applications.