Encapsulation efficiency measured on single small unilamellar vesicles

In this communication we present a fluorescent based method to measure the encapsulation efficiency in single small unilamellar vesicles. The single small unilamellar vesicles are loaded with a dye in the membrane and a dye in the lumen. They are immobilized on a surface and then imaged with a fluorescent microscope. The dye in the membrane is used to determine the vesicle size, and the lumen dye is used to determine the absolute amount of encapsulant. The correlation of the two signals allows us to calculate the encapsulation efficiency in a single vesicle as a function of size. We discovered that the encapsulation efficiency is inversely proportional to the vesicle radius and that a significant number of vesicles are empty. Both observations would be averaged out in bulk experiments. They pertain for vesicles prepared through the rehydration technique but may be relevant for other formulations as well.