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

Area of Science:

• Biotechnology
• Biophysics
• Materials Science

Background:

• Liposome fusion is crucial for delivering components into liposomes for biochemical reactions.
• Electrofusion offers a fusogen-free method for repetitive liposome fusion, preserving liposome dispersion.
• Microscopic observation limitations hinder understanding of liposome electrofusion dynamics.

Purpose of the Study:

• To elucidate the morphological changes during liposome electrofusion using fluorescent markers and flow cytometry.
• To analyze the impact of DC pulse, AC field, and lipid composition on electrofusion outcomes.
• To establish a high-throughput method for quantifying liposome fusion, fission, and destruction.

Main Methods:

• Utilized a calcein-Co2+-EDTA system for evaluating content mixing via fluorescence.
• Assessed liposome destruction by monitoring the reduction in total membrane volume.
• Quantified liposome fission by tracking the increase in giant liposome numbers.
• Employed high-throughput flow cytometry to analyze morphological changes under varying electrofusion parameters.

Main Results:

• Increased DC pulse number or voltage enhanced liposome fusion and destruction probability.
• Extended AC field application time increased the probability of liposome fusion, fission, and destruction.
• High concentrations of negatively charged POPG (≥19%) significantly inhibited liposome electrofusion.

Conclusions:

• Flow cytometry provides a robust platform for analyzing liposome electrofusion dynamics.
• Optimized DC pulse and AC field parameters can control liposome fusion, fission, and destruction.
• Lipid composition, particularly negative charge content, plays a critical role in modulating electrofusion efficiency.