Membrane fusion of pH-sensitive liposomes – a quantitative study using giant unilamellar vesicles

Abstract

Drug delivery systems based on liposome carriers that are designed for induction of membrane fusion in response to the microenvironment have been investigated for more than two decades. However, most studies have focused on self-fusion among large unilamellar vesicles (LUVs) in solution, a system with limited biological relevance which suffers from averaging effects and non-trivial interpretation of results. Here we present a fusion assay capable of visualizing and quantifying heterogeneous fusion between fusogenic liposomes and stable giant unilamellar vesicles (GUVs), suitable for studying both lipid and content mixing. Fusion was visualized and quantified using fluorescence microscopy and a FRET-based lipid mixing assay and the number of fusion events with single GUVs was estimated. Fusogenic pH-sensitive oleic acid (OA):DOPE liposomes were used as a model system and fusion was quantified with giant vesicles containing 5 or 10 mol% positively charged lipids. It was shown that the number of fusion events with single GUVs after 30 min at room temperature is approximately 100 or 200 per 100 μm2 of GUV surface, for 5 or 10 mol%, respectively. Furthermore, the mixing of the aqueous content during fusion was visualized and quantified, which showed that the content of the fusogenic liposomes is transferred essentially without leakage. Fusion was confirmed to be pH-dependent and is enhanced by electrostatic attraction between the fusing vesicles, which was demonstrated by the absence of fusion in experiments performed with negatively charged GUVs.