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Quantitative analysis of the lamellarity of giant liposomes prepared by the inverted emulsion method.

ABSTRACT: The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin ?-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ?100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.

SUBMITTER: Chiba M

PROVIDER: S-EPMC4104052 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Quantitative analysis of the lamellarity of giant liposomes prepared by the inverted emulsion method.

Chiba Masataka M Miyazaki Makito M Ishiwata Shin'ichi S

Biophysical journal 20140701 2

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluores ...[more]

PMID: 25028876

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Project description:Increasing antibiotic resistance in pathogenic microorganisms has led to renewed interest in bacteriophage therapy in both humans and animals. A "Trojan Horse" approach utilizing liposome encapsulated phages may facilitate access to phagocytic cells infected with intracellular pathogens residing therein, e.g., to treat infections caused by Mycobacterium tuberculosis, Listeria, Salmonella, and Staphylococcus sp. Additionally, liposome encapsulated phages may adhere to and diffuse within mucosa harboring resistant bacteria which are challenges in treating respiratory and gastrointestinal infections. Orally delivered phages tend to have short residence times in the gastrointestinal tract due to clinical symptoms such as diarrhea; this may be addressed through mucoadhesion of liposomes. In the present study we have evaluated the use of a microfluidic based technique for the encapsulation of bacteriophages in liposomes having mean sizes between 100 and 300 nm. Encapsulation of two model phages was undertaken, an Escherichia coli T3 podovirus (size ~65 nm) and a myovirus Staphylococcus aureus phage K (capsid head ~80 nm and phage tail length ~200 nm). The yield of encapsulated T3 phages was 109 PFU/ml and for phage K was much lower at 105 PFU/ml. The encapsulation yield for E. coli T3 phages was affected by aggregation of T3 phages. S. aureus phage K was found to interact with the liposome lipid bilayer resulting in large numbers of phages bound to the outside of the formed liposomes instead of being trapped inside them. We were able to inactivate the liposome bound S. aureus K phages whilst retaining the activity of the encapsulated phages in order to estimate the yield of microfluidic encapsulation of large tailed phages. Previous published studies on phage encapsulation in liposomes may have overestimated the yield of encapsulated tailed phages. This overestimation may affect the efficacy of phage dose delivered at the site of infection. Externally bound phages would be inactivated in the stomach acid resulting in low doses of phages delivered at the site of infection further downstream in the gastrointestinal tract.

Dataset Information

Quantitative analysis of the lamellarity of giant liposomes prepared by the inverted emulsion method.

Publications

Quantitative analysis of the lamellarity of giant liposomes prepared by the inverted emulsion method.

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