Fluorescent Liposomes: Intermixing of lipids during liposome fusion (NBD/Rhodamine Assay)
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In order to perform fusion assay three batches of liposomes are needed: a) Labeled fluorescent liposomes (Containing 0.8 mol% of Rhodamine labeled lipid AND 0.8 mol% NBD labeled lipid- Please notice that BOTH labeled lipids should be present in the same liposomes ) b) Plain unlabeled liposomes c) Mock-fused liposomes (Containing 0.08 mol% of Rhodamine labeled lipid AND 0.08 mol% NBD 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).
In many papers, the maximal fluorescence was determined by lysing the labeled liposomes at the concentration to be used in the assay. The lysing is done by using a detergent. We have used “mock-fused liposomes” for measuring the maximal fluorescence. The reason is that using detergents can add a substantial error. As an example Triton X-100 has inhibitory effect on the fluorescence of NBD and affects the quantum yield of NBD. If Triton X-100 is used a correction factor of 1.4-1.5 should be used. Instead of Triton X-100, alternative detergents such as C12E8 adn C12E9 are recommended to be used.
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. To see a step by step instruction of this assay see here: Inner Monolayer Mixing Assay