Fluorescein chemiluminescence method for estimation of membrane permeability of liposomes

Abstract

Unilamellar liposomes have a compartment enclosed by a single lipid bilayer. Encapsulation of compounds, such as drugs, genes, and enzymes, into liposomes has been applied successfully to drug delivery systems and cosmetics [1,2]. For these applications of liposomes, control, and characterization of release and leakage of compounds are very important. Release and leakage are attributable to the membrane permeability, and in some cases to instability, of the liposomes. The assay of membrane permeability is generally performed by monitoring the release of fluorescein derivatives, such as 5(6)-carboxyfluorescein and calcein (bis[N,N 0-di(carboxymethyl)-aminoethyl] fluorescein), encapsulated in liposomes [3,4]. In these assays, the dyes are encapsulated in liposomes at concentrations greater than 50–100 mM, where more than 95% of fluorescence of the dyes is self-quenched. Release of the dyes inside liposomes leads to an increase in fluorescence intensity due to the reduction of self-quenching. The percentage of released dye over a certain period is used as an index of membrane permeability. The dyes exhibit low permeability to bilayer because the dyes have multiple negative charges. Release of the dyes, therefore, requires several hours or days. It is generally difficult to use fluorescein for dye-release assay because encapsulated fluorescein leaks rapidly from the inner phase of liposomes during separation of the free dye. On the other hand, we previously reported a chemiluminescence (CL) reaction inside liposomes encapsulating horseradish peroxidase