Fluorescence anisotropy based single liposome assay to measure molecule-membrane interactions

Nanometer-scaled liposomes are used frequently for research, therapeutic, and analytical applications as carriers for water-soluble molecules. Recent technical advances allow the monitoring of single liposomes, which provides information on heterogeneous properties that were otherwise hidden due to ensemble averaging. Recent observations demonstrated that the efficiency of entrapping water-soluble molecules increases with decreasing vesicle size. The molecular mechanism behind this observation is not clear, but enhanced molecule-membrane interactions due to the increase of the surface area-to-volume ratio could play an important role. To investigate this hypothesis, we extended our single liposome assay based on confocal fluorescence imaging by implementation of fluorescence anisotropy. This combination has not been widely exploited, and confocal fluorescence anisotropy imaging in particular has seldom been used. We investigated different small dye molecules and were able to determine if these molecules interact or not with the liposome membrane. We confirm the liposome size-dependent entrapment of molecules whereas the molecule-membrane interactions appear to be independent of liposome size. Our fluorescence anisotropy assay can be used as a general method to investigate molecule-membrane interactions or molecule-molecule interactions in a high-throughput manner in nanometer-scaled containers like liposomes.