Liposomes and osmolality of the solution

A liposome formulator should pay a very close attention to the osmolality of the solution that is encapsulated inside the liposomes, the osmolality of the buffer outside of the liposomes and the osmolality of the medium that is used for in vitro studies. 

Liposome can be osmotically active or osmotically inactive.  In osmotically active liposomes the lipid membrane is permeable and the osmosis will occur. This is not the case when liposomes are not osmotically active. 

The osmotic behavior of liposomes depend on their size, lamellarity and lipid composition. 

Large unilamellar liposomes are osmotically active and therefore the osmolality of the solution that is encapsulated inside the large unilamellar liposomes should be equal to the buffer that is outside of the liposomes. If the liposomes are added to a medium for in vitro studies then the osmolality of the liposome solution should match the osmolality of the medium. If the liposomes are used for in vivo studies then the osmolality of liposomes should match the osmolality of blood.

Small unilamellar liposomes are NOT active osmotically and therefore there is not need to have iso-osmatic solutions inside and outside of liposomes. 

Multilamellar phosphatidylcholine based liposomes that contain a small amount of charged lipids are osmotically active. Multilamellar phosphatidylcholine based liposomes that do not contain charged lipids are  NOT osmotically active. 

Very large oligolamellar liposomes with or without charged lipids are osmotically active.

Many liposome based fluorescent assays used 100 nm liposomes. Most of the liposome based injectable drugs are 100 nm in size. These are LUVs and therefore they are osmotically active. 

Package insert/injection instruction sheet for Clodronate liposomes: Clodrosome

Clodrosome-Clodronate Encapsulated Liposomes_ Package Insert Encapsula Nanosciences_2012

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MSDS for Plain liposomes-Clodronate liposome Control: Encapsome-CL

MSDS Encapsome CL_Clodronate Liposome Control-Encapsula NanoSciences_2012

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For more info go to www.clodrosome.com

MSDS for Clodronate liposomes: Clodrosome

MSDS Clodrosome_Clodronate encapsulated liposome_ Encapsula Nanosciences_2012

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Fluorescent Liposomes: Inner Monolayer Mixing Assay

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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

1. 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)
2. 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)
3. 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.
4. 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.
5. Dithonate is removed by either dialysis or spin column.
6. 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).
7. On fluorometer set the excitation monochromator to 460 nm and emission monochromator to 535 nm.
8. 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.
9. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 0%.
10. 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.
11. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 100%.  Mock-fused liposomes represent the theoretical fusion product.
12. 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).

Fluorescent Liposomes: Intermixing of lipids during liposome fusion (NBD/Rhodamine Assay)

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1. 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).
2. On fluorometer set the excitation monochromator to 460 nm and emission monochromator to 535 nm.
3. 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.
4. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 0%.
5. 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.
6. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 100%.  Mock-fused liposomes represent the theoretical fusion product.
7. 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).

Notes:

1. 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.
2. 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

Fluorescent Liposomes: ANTS/DPX Fusion Assay

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1. In order to perform fusion assay three batches of liposomes are needed: a) ANTS encapsulated liposomes b) DPX encapsulated liposomes c)  ANTS/DPX co-encapsulated liposomes
2. On fluorometer set the excitation monochromator to 360 nm and emission monochromator to 530 nm.
3. For a fusion assay that utilizes 50 µM total lipid, take the appropriate amount of ANTS encapsulated liposomes and add it to the buffer inside the fluorometer cuvette to make a 25 µM of ANTS-liposomes and also  take the appropriate amount of DPX encapsulated liposomes and add it to the buffer inside the fluorometer cuvette to make a 25 µM of DPX-liposomes. The total lipid concentration will be 50 µM. Please keep in mind that we are talking about lipid concentration and not the concentration of encapsulated molecules.
4. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 100%.
5. Take the appropriate amount of ANTS/DPX co-encapsulated liposomes and add it to the buffer inside the fluorometer cuvette to make a liposome solution with total lipid concentration of 50 µM (twice the lipid concentration of ANTS encapsulated liposomes).
6. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 0%.  This represents the theoretical fusion product (fully quenched) of all ANTS encapsulated liposomes and DPX encapsulated liposomes.
7. Add the proper fusogen, such as protons, fusogenic peptides or divalent cations and measure the fluorescence. Make sure that the solution inside the cuvette is under constant stirring.
8. Liposome fusion results in the decrease of fluorescence due to quenching of ANTS by DPX. DPX is not a fluorescent molecule and it is the collisional quencher of ANTS. ANTS is a fluorescent molecule.
9. For a fluorometer with computerized data acquisition, the assay can be calibrated by using the following equation:

where F(t) is the extent of fusion at time t, I (t) is the fluorescence intensity at time t, I(0) is the fluorescence intensity of the initial mixture of liposome encapsulated ANTS and liposome encapsulated DPX before adding the fusogen (step 3 and 4). I(∞) is the fluorescence intensity of the  liposome co-encapsulated ANTS/DPX (step 6).