Chapter 5 The Single Guv Method to Reveal Elementary Processes of Leakage of Internal Contents from Liposomes Induced by Antimicrobial Substances

Abstract

Abstract So far almost all studies of biomembrane function have been done using a suspension of many small-size vesicles such as large unilamellar vesicles (LUVs) (the conventional LUV suspension method). In these studies, the average values of the physical parameters of vesicles have been obtained from a large number of vesicles, and thereby much information has been lost. Recently we have proposed a novel method, the single GUV method. In this method, we observe and measure physical properties of single giant unilamellar vesicles (GUVs) with a diameter of ≥10 μm, and analyze these results over many GUVs statistically, which will provide a great deal of new information that cannot be obtained by the conventional LUV suspension method. In this review, we describe an application of the single GUV method to investigations of leakage of internal contents such as small fluorescent probes from liposomes. Such investigations using the conventional LUV suspension method have been extensively used to obtain information on interactions of various kinds of substances (e.g., antimicrobial substances, peptides/proteins, and drugs) with biomembranes/lipid membranes. However, this method could not reveal main cause of the leakage and its elementary steps. Here, we show two examples of the single GUV method studies on the leakage of internal contents from liposomes induced by antimicrobial peptide, magainin 2, and antibacterial substance, tea catechin. We have succeeded, for the first time, in observing detailed elementary processes of the substance-induced leakage and also obtaining a direct evidence for the cause of the leakage. The statistical analysis of individual events in single GUVs over many single GUVs gave us important information on the rate constants of elementary processes, such as the rate constant of the magainin 2-induced pore formation and that of the EGCg-induced bursting of GUVs. These results clearly show that the single GUV method is much more useful than the conventional LUV method in studies of leakage of internal contents from liposomes. We also discuss the advantage of the single GUV method in biomembrane research.