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Free keywords:
giant vesicles, fusion intermediate, confocal microscopy, vesicle electrodeformation, FRET, FLIM-FRET, microfluidics, POPG, DOTAP
Abstract:
Membrane fusion is a ubiquitous process in biology and is a prerequisite for many intracellular delivery protocols relying on the use of liposomes as drug carriers. Here, we investigate in detail the process of membrane fusion and the role of opposite charges in a protein-free lipid system based on cationic liposomes (LUVs) and anionic giant unilamellar vesicles (GUVs) composed of different palmitoyloleoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol (POPC:POPG) molar ratios. By using a set of optical microscopy- and microfluidics-based methods, we show that liposomes strongly dock to GUVs of pure POPC or low POPG fraction (up to 10 mol%), in a process mainly associated with hemifusion and membrane tension increase, commonly leading to GUV rupture. On the other hand, docked LUVs quickly and very efficiently fuse with negative GUVs of POPG fractions at or above 20 mol%, resulting in dramatic GUV area increase in a charged-dependent manner. Importantly, both hemifusion and full fusion are leakage-free. Fusion efficiency is quantified by the lipid transfer from liposomes to GUVs upon fusion using fluorescence resonance energy transfer (FRET), which leads to con-sistent results when compared to fluorescence lifetime-based FRET. We develop an approach to deduce the final composition of single GUVs after fusion based on the FRET efficiency. We can conclude that fusion is driven by membrane charge and appears to proceed up to charge-neutralization of the acceptor GUV.
