Flow Cytometric Analysis To Evaluate Morphological Changes in Giant Liposomes As Observed in Electrofusion Experiments.

Abstract

Liposome fusion is a way of supplying additional components for in-liposome biochemical reactions. Electrofusion is a method that does not require the addition of fusogens, which often alter the liposome dispersion, and is therefore useful for repetitive liposome fusion. However, the details of electrofusion have not been elucidated because of the limitations surrounding observing liposomes using a microscope. Therefore, we introduced fluorescent markers and high-throughput flow cytometry to analyze the morphological changes that occur in liposome electrofusion. (i) The content mixing was evaluated by a calcein-Co2+-EDTA system, in which green fluorescence from dequenched free calcein is detected when the quenched calcein-Co2+ complex and EDTA are mixed together. (ii) Liposome destruction was evaluated from the decrease in the total membrane volume of giant liposomes. (iii) Liposome fission was evaluated from the increase in the number of giant liposomes. By applying the flow cytometric analysis, we investigated the effect of three parameters (DC pulse, AC field, and lipid composition) on liposome electrofusion. The larger numbers or higher voltages of DC pulses induced liposome fusion and destruction with higher probability. The longer application time of the AC field induced liposome fusion, fission, and destruction with higher probability. Higher content of negatively charged POPG (≥19%) strongly inhibited liposome electrofusion.