One of the main problems of using a lipid that has the fluorescence probe on the head group is that it can interact with fusogens, proteins and ions and this interaction may alter the fluorescence intensity or the lateral diffusion of the probe (See this animation which explains various movements of lipids in liposome bilayer). If the fluorophores are only located in the inner monolayer of the liposomes then the fluorescence would no be affected by ions, proteins, peptides and other fusogens that bind to the surface of the liposomes. The fluorophores that are exposed to the outer monolayer can be reduced using dithionate and therefore eliminating the fluorescence of the outer monolayer. NBD-phosphatidylserine (NBD-PS) is is more suitable to be used for these formulations than NBD-phosphatidylethanolamine (NBD-PE) because PS is less prone to transbilayer movement following reduction by dithionate. This method was originally developed by The Liposome Company (Princeton, NJ) in 1999. See here:Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion.
Reduction of Outer Exposed Fluorphores/Inner Monolayer Mixing Assay
Liposomes containing 0.8 mole% of NBD-PS and 0.8 mole% Rhodamine-PE with total lipid concentration of 20 mM is prepared (Concentrated stock solution of labeled fluorescent liposomes)
Liposomes containing 0.08 mole% of NBD-PS and 0.08 mole% Rhodamine-PE with total lipid concentration of 20 mM is prepared (Concentrated stock solution of mock-fused liposomes)
Dithionate is added at a final concentration of 100 mM to 10 mM solution of labeled fluorescent liposomes and the solution is incubated for 1 hour at 4 C in refrigerator.
Dithionate is added at a final concentration of 100 mM to 10 mM solution of mock-fused liposomes and the solution is incubated for 1 hour at 4 C in refrigerator.
Dithonate is removed by either dialysis or spin column.
In order to perform fusion assay three batches of liposomes are needed: a) Inner monolayer labeled fluorescent liposomes (Containing 0.8 mol% of labeled lipid) b) Plain unlabeled liposomes c) Inner monolayer labeled mock-fused liposomes (Containing 0.08 mol% of labeled lipid).
On fluorometer set the excitation monochromator to 460 nm and emission monochromator to 535 nm.
For a fusion assay that utilizes 50 µM total lipid, take the appropriate amount of labeled liposomes and add it to the buffer inside the fluorometer cuvette to make a 5 µM of labeled fluorescent liposomes and also take the appropriate amount of plain liposomes and add it to the buffer inside the fluorometer cuvette to make a 45 µM of plain liposomes. The total lipid concentration will be 50 µM. The labeled and plain liposomes are mixed at 10:90 molar ratio.
Measure the fluorescence of the solution and adjust it to an arbitrary unit of 0%.
Take the appropriate amount of Mock-fused liposomes and add it to the buffer inside the fluorometer cuvette to make a liposome solution with total lipid concentration of 50 µM.
Measure the fluorescence of the solution and adjust it to an arbitrary unit of 100%. Mock-fused liposomes represent the theoretical fusion product.
The percentage of lipid mixing as a function of time is calculated using the following equation, where M(t) is the extent of lipid inter-mixing at time t, I (t) is the fluorescence intensity at time t, I(0) is the fluorescence intensity of the initial mixture of labeled liposome and plain liposomes (step 3 and 4). I(∞) is the fluorescence intensity of Mock-fused liposomes (steps 5 and 6).