Detecting liposome fusion via fluorescence self-quenching: The fusion efficiency and repeatability of cationic dye-labeled liposomes

Abstract

Liposomes have proven to be suitable drug delivery systems across many medical applications. In order to deliver the drug to a desired place, the liposomes are met with an essential task of penetrating the cell membrane. Currently, the fusion of a liposome into a membrane is hard to detect and little is known about the fusion mechanisms. However, the liposomal fusion can be studied with fluorescent dye-labeled liposomes and by utilizing fluorescence spectroscopy methods.

In this Master’s thesis, the aim was to construct a simple measurement system to detect successfully fusion between liposomes. A self-quenching Octadecyl Rhodamine B dye was used to label liposomes and detect liposomal fusion between dye-labeled and unlabeled liposomes. Two distinct liposome compositions, neutral DPPC and cationic DOTAP liposomes, were labeled with six different amounts of the dye. The fluorescence intensity and lifetime of prepared liposome batches were compared to the respective dye concentrations and further constructed into a calibration curves used during the fusion measurements analysis. The fusion measurements were done by monitoring the kinetics of fluorescence intensity when dye-labeled liposomes were mixed with unlabeled liposomes. In addition, the effect of temperature change was tested during fusion. Finally, the fluorescence lifetime of fusion sample was measured as well as the size distribution and concentration of liposomes.

Analysis of the results showed clear dequenching of the dye signal in both fluorescence intensity and lifetime with cationic liposome mixture that indicates successful fusion. The neutral liposomes didn’t show signs of fusion when mixed with each other, however temperature induced fusion was observed upon mixing neutral and cationic liposomes. It was established that the cationic lipid composition and higher amount of PEG enhanced fusion efficiency. Therefore, the repeatability of cationic DOTAP liposomes upon fusion was studied further. The fusion measurements showed repeatability in both fluorescence intensity kinetics as well as fluorescence lifetime results. The fusion efficiency of replicate samples was estimated to be 100 % based on fluorescence lifetime while 54 % efficiency was calculated based on fluorescence intensity measurements. The fusion was further attempted to confirm by using DLS and NTA measurements. However, change in liposome size was not observed and the NTA results fell within the error limit of the method. Different experimental parameters would have been required for these measurements. It was determined that steady-state and time-resolved fluorescence spectroscopy results as well as the liposome composition endorse detection of fusion. Furthermore, additional measurements could have fully confirmed detection of fusion and ruled out possibility of lipid-mixing happening during hemifusion stage. Nevertheless, this simple system based on self-quenching could be useful in detecting fusion of more complicated membranes, for example between liposomes and extra-cellular vesicles.