Encapsulation Efficiency Measured on Single Small Unilamellar Vesicles

Abstract

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.

Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment

“…15). Recent experiments of this category are focused at interaction of peptides and proteins with vesicles, function of single membrane-active enzymes, [19][20][21] encapsulation efficiency, 22 and vesicle docking and fusion. 23 Alternatively, suspended vesicles may interact with various species attached to a supported lipid bilayer.…”

“…24 In all these experiments, the kinetics were simultaneously tracked on a large number of individual fluorescent dye-labelled vesicles by using total internal reflection fluorescence microscopy (TIRFM) 17,20,21,24 or confocal fluorescence microscopy (CFM). 14,15,19,22,23 (Historically, TIRFM was introduced to study various surface bioprocesses in the early 1980s; 25,26 for CFM and combination of TIRFM and CFM, see Refs. 27 and 28, respectively; the application of TIRFM and CFM to immobilized vesicles started in the early 2000s.…”

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates

“…The maximum fluorescence intensity of each vesicle was then measured (Figure 1 b (iii) and (vi), F ionomycin ) and the percentage of Ca 2+ influx was calculated. This approach corrected for the fact that the number of Cal‐520 dye molecules in a vesicle is not constant, owing to variations in the efficiency of dye encapsulation25 and in vesicle size. The fluorescence intensity change was quantitatively converted into a percentage of Ca 2+ influx for individual vesicles and then averaged over all the vesicles imaged (Figure 1, see Supporting Information for details).…”

Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer

“…Knowledge of the CA and the size of a vesicle allows us to infer both its shape and curvature of the bilayer. Nonspherical shape caused by adhesion can cause unwanted strain in the bilayer and increase the porosity of the bilayer, which is important to control in applications where vesicles are used as small reaction containers (18)(19)(20). Curvature in bilayers affects both molecular organization (21) and binding of membrane proteins (22).…”