Asymmetric liposomes

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Contents

Protocol

Outer Bilayer Lipid Preparation:
  1. Measure out the lipid mixtures into sialinized glass vials. Evaporate the chloroform using nitrogen or argon. While drying, slowly turn the vial, to create a thin layer of lipid of even thickness around the bottom of the vial.
  2. Place the lipid-containing vial under a stream of nitrogen or argon for another 1-4 hours to ensure complete removal of all chloroform.
  3. Add a volume of 99:1 dodecane:silicone oil solution to make a final lipid concentration of 0.05mg/ml. Top the tube with nitrogen or argon, cap, and sonicate in a sonic cleaning bath for 30min. Incubate overnight at 25C to ensure complete suspension of the lipids.
  4. Add 3ml of aqueous buffer in to a 50ml concial tube (use whichever buffer the final liposomes will be suspended in). Layer above this 2ml of the dodecane:silicone oil:lipid mixture prepared above. Top the tube with nitrogen, cap, and let sit 3 hours. A lipid monolayer should form at the interface.
Inner Bilayer Lipid Preparation:
  1. Repeat steps 1-2 from outer bilayer lipid preparation stage.
  2. Suspend the lipids at 0.05mg/ml in >99% pure anhydrous dodecane ***without*** silicone oil. Top tube with nitrogen or argon, cap, and sonicate/incubate as in step 3 of the outer bilayer lipid preparation stage.
  3. Add 1/200th volume of the same aqueous buffer which will comprise the lumen of the liposomes to the solution prepared in step 2. Pass the solution several times through a 0.2um solvent-compatible syringe filter, or through a 100nm liposome extruder to emulsify the aqueous phase to a consistent size.
    • Note: the osmolarity of the luminal solution should be as close to the buffer the liposomes will be suspended in, in order to prevent shrinking or bursting of the liposomes.
    • A minimum of 100ul of emulsion will be needed per liposome preparation; due to loss in the filter and minimal volumes needed for emulsification, it is best to work with volumes >5ml.
  4. Cap the tube with nitrogen and let the emulsion sit for 3 hours. During this time a lipid monolayer will form around the emulsion.
Preparation of Asymmetric Liposomes:
  1. Layer 100ul of the inner bilayer preparation overtop the completed outer bilayer preparation.
  2. Centrifuge 120g for 10min; completed liposomes will appear in the lower aqueous solution.
  3. Fill a 5ml syringe with a 16-guage needle with buffer, and expel all but ~0.5ml of the buffer.
  4. Insert the needle through the solvent layer, into the aqueous layer. Expel the buffer to remove any oil or lipids that may have contaminated the syringe while passing through the solvent phase.
  5. Gently suck up, then expel, small volumes of the aqueous mixture. This will detach any liposomes stuck at the interface. After 5-10 recirculation’s, suck up as much buffer as possible without removing any of the interface or solvent layer.
  6. If required, liposomes can be concentrated using dialysis.
Checking liposome asymmetry:
  1. Prepare 2 samples of liposomes, using 0.5% NBD-PC to label the inner monolayer in one prep, and the outer monolayer in the second prep.
  2. Measure the fluorescence of NBD in the resulting liposomes (Ex=470nm, Em=540nm).
    • Adjust excitation/emission bandpass widths and PMT voltage to maximize signal.
  3.  Add 1:100th the reaction volume of quenching solution. Mix and incubate for 2 min, then re-measure NDB fluorescence.
  4. Add 1:10th the reaction volume of 10x Triton X-100 solution. Let sit 2min, then re-measure NDB fluorescence.
  5. The fraction of NDB in the outer verses inner membrane can be calculated by:
    • Outer Membrane = [(Em540 initial) – (Em540 post-quench)]/(ΔEm540)
    • ΔEm540 = (Em540 initial) - (Em540 post-X100)
    • Inner Membrane = [(Em540 post-quench) – (Em540 post-X100)]/( ΔEm540)
  6. Properly prepared liposomes should have an asymmetry of 90-95% or higher.

Recepies:

Normal aqueous buffer:
  • 0.584428g (100mM) NaCl
  • 0.07878g (5mM) Tris-HCl
    Bring upto 100ml in ddH2O, and pH to 7.4. Is good for 5 days if refrigerated.
100x Quenching Buffer:
  • 17.4097g (1M) sodium dithionate (Na2S2O4)
  • 0.114625g (5mM) TES
    Bring upto 100ml in ddH2O, pH to 9.0. Make fresh daily.
10x Triton Buffer:
  • 1.25ml Triton X-100
  • 8.75ml ddH2O
    Mix completely, makes a 10x solution for lysing liposomes. Is good for 1 week if refrigerated.
Other useful buffers:
  • 5-50mM Tris-HCL, pH 7.0-7.5 (ideal for experiments using salt addition)
  • PBS
  • Any culture media
  • Water
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