Characterization and application of fusogenic liposomes

Abstract

Conventional drug delivery strategies use the endocytic pathway to introduce biomolecules like proteins, DNA, or antibiotics into living cells. The main disadvantage of endocytic uptake is the quick intercellular degradation of the cargo. Compared to this, a more promising alternative for efficient molecular delivery is the induction of membrane fusion between liposomes and mammalian cells. Therefore special liposomes with extraordinary high fusion efficiency, so-called fusogenic liposomes (FLs), have been developed for such purposes. Due to the complete fusion of the liposomal membrane and the cellular plasma membrane, the cargo molecules can be effectively released into the cell cytoplasm, avoiding their degradation. In the last decade, applications relying on FLs became more and more relevant, however, the exact fusion mechanism is still to be elucidated. <br /> Therefore the aims of this work have been to investigate those liposomes and their fusogenicity with living mammalian cells dependent on lipid composition as well as environmental conditions to elucidate the most important factors inducing fusogenic structures within the liposomes. <br /> For structural characterization of the liposomes dynamic light and neutron scattering as well as solid state-NMR, freeze-fracture-STEM, Cryo-TEM, and differential scanning calorimetry were applied. Fusion efficiency was investigated by fluorescence microscopy and flow cytometry using Chinese hamster ovary (CHO) cells as an <em>in vitro</em> mammalian cell model. <br /> The first results showed that fusogenic liposomes (FLs) need cationic lipids with inverted conical molecular shapes and aromatic components at a distinct concentration as well as a neutral lipid for the best fusion induction. Neutral lipids with long and unsaturated chains and a small head group (e.g., PEs) do not change the liposomal fusion ability while those with saturated short chains and a big head group (e.g., PCs) do, and in most extreme cases revert the uptake mechanism to endocytosis. <br /> Additionally, a new application of fusogenic liposomes was established. For the first time, cationic liposomes with high fusion ability were successfully used as carrier particles for the delivery of the radionuclide ¹³¹I into mammalian breast cancer cells <em>in vitro</em>. The FLs reached the cancer cells with high efficiency and delivered their cargo into the cell cytoplasm. The control treatment of human red blood cells did not give positive results on fusion, and in this case, the delivery of the cargo was neglectable. These results considered FLs as an appropriate tool for applications in nuclear medicine. <br /> Further results showed that as the structural reorganization of the liposomal membrane supply the total required driving force to overcome the energy barrier of the different fusion intermediate steps, like in the case of FLs, changes of the fusion conditions such as temperature, osmolality or ionic concentration of the buffer did not influence the fusion success. In the case of the endocytic liposomes (ELs), buffer conditions played a crucial role in successful fusion, however, fusion efficiency remains infinitesimal under physiological conditions. <br /> To elucidate the correlation between efficient membrane fusion and liposomal characteristics, structural investigations of FLs with the best fusion efficiency were also carried out. Here, the simultaneous presence of lipid bilayers and small micelles of around 50 to 100 nm in diameter with high surface curvatures were found. Based on the obtained results, a theoretical mechanism of membrane fusion between FLs and cellular membranes could be proposed. The positively charged lipid is necessary for establishing contact between the two membranes. The micelles are formed by the neutral, phosphoethanolamine, lipids. The lipid bilayer enclosing inverted micelles has a high positive membrane curvature, which is especially favorable for the positively charged lipid molecules. Such curvature stress usually promotes the fusion-stalk formation and subsequent membrane fusion; therefore, the proposed fusion mechanism is called a modified stalk mechanism.<br /> Moreover, traces of other three-dimensional (3D) phases with high membrane curvature such us sponge-, inverted hexagonal-, and cubic phases could not be excluded. The present structures are probably metastable precursors, such as a rhombohedral phase, that reduce bilayer stability, which is leading to the pore formation occurring. In comparison to this, ELs formed only lamellar phases shown as non-fusogenic under physiological conditions. These results give rise to the hypothesis that the predominant presence of 3D-like and 3D phases with high membrane curvatures is the most important criterion for efficient membrane fusion induction.