Liposome and Lipid tubule Production

To make lipid tubes instead of vesicles, substitute in galactocerebrosides (Sigma C 1516) for the PE, up to 40% GalCer: e.g., 10% Chol, 40 % GalCer, 40% PC 10% PIP2. These lipid tubes will have a diameter of approximately 40 nm. To make Folch liposomes, simply use folch extract straight (10 mg/ml in 19:1 ChCl3/MeOH) instead of mixing lipids.

See also advice on making Liposomes from Avanti Polar Lipids.

1. Rinse 1.8 ml glass vials (with teflon caps, Wheaton #224740) in chloroform to wash.

1. Mix 100l of chloroform (special stuff) and 30l of methanol. Dry chloroform, stored with dessicant, is recommended (e.g. Sigma C-2432)

2. Add lipids (most important last) eg. 10% cholesterol (stock: 10mg/ml), 35% PC (stock: 10mg/ml), 35% PE (stock: 10mg/ml), 10% PS (stock: 20mg/ml), 10% PIP2 (stock: 1mg/ml). Lipids are already in chloroform, keep them on ice. The concentration of the final mixture is 1mg/ml. Gently swirl in-between additions. Be sure to use protonated PIP2 (see protocol, or use the pre-protonated ones from Avanti Polar Lipids), mix them first or sonicate in the bath sonicator.

3. Evaporate chloroform + methanol using slow-flow Argon (0.1 units) to produce a film on the glass. When this film becomes white, turn up the flow and spread out last drops. Avoid leaving thick lumps of precipitated lipid.

4. Put in a dessicator, pump out the air, leave for 5-15 minutes.

5. Slowly release the valve.

6. Add 1ml of filtered buffer. Leave to hydrate for 5 minutes at room temp. Gently agitate occasionally.

7. Put in a bath sonicator for about 30 seconds to 2 minutes to resuspend the lipids and form liposomes/tubes, until solution just begins to clear.

8. Apply two to five brief pulses with a small (2 mm) probe sonicator. Solution should be only slightly cloudy.

9. Filter using 0.1, 0.2, or 0.4m polycarbonate filters, e.g., Whatman Cyclopore Cat # 7060 2501, with 1 ml syringes and Avanti mini-extruder. Push 3-11 times through the filter (odd number so that the material trapped in the filter does not get washed back into the final vial) and deposit into a buffer-rinsed glass vial. NB: the filtration process usually leads to loss of around 100-200 ul of liquid, and 10-20% of the lipid in the reamining volume.

NB. Galactocerebrosides and PIP2 may need to be left on the bench for a few minutes to warm up since they are insoluble at low temperatures.

PIP2 = PtdIns(4,5)P2

PS= Phosphatidylserine

PC= Phosphatidylcholine

PE= Phosphatidylethanolamine

Folch extract= Total brain lipids from Sigma (B-1502), approx. 10% phosphoinositides.


Link: http://endocytosis.org/techniqs/Liposome.htm

As a specialist for ultrasonic systems, I have a serious remark on your step 8:
Why are you using such a small microtip when having such a "big" sample. When using such a samll surface tip, the power density delierec into the sample is much to low and the risk problems like splashing, local overheating etc. is much too high.
If you want some help for improving just let me know.
Best regards
Andreas