Mar 04, 2017
Version 2
Part 2: Extrusion and suspension of phospholipid liposomes from lipid fims V.2
- James R. Collins1,
- Krista Longnecker1,
- Helen F. Fredricks1,
- Benjamin A. S. Van Mooy1
- 1Woods Hole Oceanographic Institution
- Van Mooy Lab @ WHOI
Protocol Citation: James R. Collins, Krista Longnecker, Helen F. Fredricks, Benjamin A. S. Van Mooy 2017. Part 2: Extrusion and suspension of phospholipid liposomes from lipid fims. protocols.io https://dx.doi.org/10.17504/protocols.io.hatb2en
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 04, 2017
Last Modified: March 22, 2018
Protocol Integer ID: 5171
Abstract
The protocol can be used to create and extrude phospholipid liposomes. This extrusion protocol roughly follows some guidelines published by the manufacturer. However, we found that the manufacturer's directions were insufficient in several areas. In particular, the manufacturer guidance on initial preparation of lipids was incomplete; 'good' liposomes cannot be prepared directly from dry lipid mixtures. In addition, our protocol provides supplementary guidance for extrusion and suspension of different classes of polar lipids; efficacy of the various matrixes we recommend was largely determined through trial and error. The manufacturer directions (downloaded March 4, 2017) are attached to this protocol as a .pdf document.
This protocol was original created by Krista Longnecker and Jamie Collins for creating lipsomes to be used in lipid photo-oxidation experiments. The results of these experiments are detailed in Chapter 4 of:
Collins, J. R. 2017. The remineralization of marine organic matter by diverse biological and abiotic processes. Ph.D. thesis. Cambridge, Massachusetts: Massachusetts Institute of Technology, 300 pp; doi:10.1575/1912/8721
A manuscript of the chapter is forthcoming.
Attachments
Materials
STEP MATERIALS
MilliQ water
Sodium ChlorideCatalog #PubChem CID: 5234
Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
Sodium sulfateCatalog #PubChem CID: 24436
0.2 µm polycarbonate membranesAvanti Polar Lipids, Inc.Catalog #610006
10 mm filter supports for Avanti mini-extruderAvanti Polar Lipids, Inc.Catalog #610014
MilliQ water
Sodium ChlorideCatalog #PubChem CID: 5234
Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
Sodium sulfateCatalog #PubChem CID: 24436
0.2 µm polycarbonate membranesAvanti Polar Lipids, Inc.Catalog #610006
10 mm filter supports for Avanti mini-extruderAvanti Polar Lipids, Inc.Catalog #610014
Protocol materials
Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
Sodium sulfateCatalog #PubChem CID: 24436
10 mm filter supports for Avanti mini-extruderAvanti Polar Lipids, Inc.Catalog #610014
MilliQ water
Sodium ChlorideCatalog #PubChem CID: 5234
Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
0.2 µm polycarbonate membranesAvanti Polar Lipids, Inc.Catalog #610006
Sodium sulfateCatalog #PubChem CID: 24436
MilliQ water
Sodium ChlorideCatalog #PubChem CID: 5234
0.2 µm polycarbonate membranesAvanti Polar Lipids, Inc.Catalog #610006
10 mm filter supports for Avanti mini-extruderAvanti Polar Lipids, Inc.Catalog #610014
MilliQ water
Sodium ChlorideCatalog #PubChem CID: 5234
Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
Sodium sulfateCatalog #PubChem CID: 24436
0.2 µm polycarbonate membranesAvanti Polar Lipids, Inc.Catalog #610006
10 mm filter supports for Avanti mini-extruderAvanti Polar Lipids, Inc.Catalog #610014
Before start
Note: Plan to use the liposomes you are making within a day or two because they are not stable for longer periods of time. If necessary, store the extuded liposome solutions at 4ºC.
This protocol assumes you have an Avanti mini-extruder with heating block, available at https://avantilipids.com/product/610000/
Obtain lipid films
Obtain lipid films
Obtain lipid films for the desired liposomes. The companion lipid film preparation protocol contains instructions for producing the necessary films.
Protocol
NAME
Part 1: Preparation of lipid films for phospholipid liposomesCREATED BY
James R Collins
For this work, we used a variety of phosopholipids obtained from Avanti Polar Lipids, Inc. Where available, we obtained lipids as a powder. When the desired lipid was not available from the supplier as a powder (e.g., 22:6, 22:6 PC), we ordered the product dissolved in chloroform. The lipids given in the reagents list for this step were those used for the lipid photo-oxidation experiments, but this protocol can be followed for preparation of films of any phospholipid.
1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1, 18:1 PC)Avanti Polar Lipids, Inc.Catalog #850375P
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16:0, 16:0 PC)Avanti Polar Lipids, Inc.Catalog #850355P
1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16:1, 16:1 PC)Avanti Polar Lipids, Inc.Catalog #850358P
1,2-distearoyl-sn-glycero-3-phosphocholine (18:0, 18:0 PC)Avanti Polar Lipids, Inc.Catalog #850365P
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (18:2, 18:2 PC)Avanti Polar Lipids, Inc.Catalog #850385P
1,2-dibehenoyl-sn-glycero-3-phosphocholine (22:0, 22:0 PC)Avanti Polar Lipids, Inc.Catalog #850371P
1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6, 22:6 PC)Avanti Polar Lipids, Inc.Catalog #850400C
Weigh out desired quantity of lipid(s) into a glass test tube. Use a round-bottom tube, such a disposable culture tube or small (i.e., 20 mL) round-bottom vial from an HPLC fraction collector. In applying this protocol, we aimed for approx. 1.65 mg of each lipid per tube. If making up multiple tubes of lipid fims for each lipid, weigh out the total quantity of lipid needed (1.65 × no. of tubes) into a single tube; you can then aliquot the solution into separate tubes once the chloroform has been added (see step below) using a solvent-rinsed glass syringe.
In a fume hood, add ~ 200 μL of chloroform to each glass test tube to dissolve the lipids. Just add enough chloroform that the lipids dissolve. The actual volume of chloroform does not matter because it will be removed in a later step.
Note that this step does not apply if lipid was obtained from vendor already in chloroform.
If making multiple films of each lipid: Add more chloroform to this first tube — 200 μL × no. of desired films — and then aliquot the solution into separate tubes in volumes of 200 μL.
Safety information
Chloroform is a hazardous solvent. Conduct all work in a fume hood, while wearing proper PPE.
Chloroform
Disposable borosilicate glass culture tubes, with rounded bottom Fisher ScientificCatalog #14-961-26
If you want to know the exact concentration of lipids in any of these initial chloroform solutions, remove an aliquot of the lipid/chloroform solution at this point, and keep track of the volume. The lipid solution will need to be diluted further with chloroform before it can be analyzed. Helen Fredricks (HPLC-MS guru in the Van Mooy Lab) has suggested a final concentration of 0.01 mg mL-1 for analysis.
If you made up more than 200 μL of lipid solution in your initial tube, now is the time to dispense ~ 200 μL aliquots into separate round-bottom tubes. A volumetric, solvent-washed glass syringe can be used for this purpose.
The object here is to create a lipid 'film' that evenly coats the inside of the lower side walls and bottom of each tube once the solvent has been evaporated. This requires some concentration and attention when evaporating the solvent.
You cannot just stick the vial under a needle with N2 flowing out of it and walk away, because that will not form the lipid film needed for this method to work.
Best practices:
- Connect a disposable glass pasteur pipette (use one with a longer length) to the nitrogen supply by way of some flexible non-reactive (e.g., Tygon) tubing. Use some lab tape or other means to secure the pipet at about eye height inside the fume hood. You will want the end of the pipette to point slightly downward. The precise orientation of the pipette doesn't matter all that much; you want it oriented in such a way to make the next few steps convenient and easy!
- Hold a tube containing ~ 200 μL of the lipid solution so that the end of the pipette protrudes just into the mouth of the tube.
- While contantly rolling the tube in your fingers, gradually let a weak stream of the N2 blow down the chloroform. Remember: You don't want to concentrate all the lipid at the very bottom of the tube. The goal is to create a film that evenly and continuously coats the lower third of the side wall and bottom of the tube.
- At the end, you should have an even film of the lipid coating the lower side walls and bottom of the tube. If you end up with any chunks or visible crystals, add chloroform and repeat the film-coating and blowdown process again.
Glass pasteur pipettes
Nitrogen gas, UHP
Once the vial appears dry, remove any residual chloroform by putting the tubes in a Speed Vac (or Vacufuge) for ~ one hour.
Safety information
Note: Make sure you use a vacuum evaporator and tubing suitable for work with organic solvents.
Top the dry lipid film with argon (as a stabilizer), cap, and then and store at −20ºC until needed.
We have stored the films for up to 4 months when argonized and capped.
Argon gas, UHP
Prepare matrixes for lipid suspension
Prepare matrixes for lipid suspension
In this protocol, you will resuspend lipids from lipid films in a buffered solution prior to beginning the extruding process. By trial and error, we determined that the following matrixes were appropriate for various classes of polar lipid:
| Class(es) of lipid | Solution(s) appropriate for suspending lipid films prior to extrusion | |
| PC | Milli-Q water 100 mM NaCl 100 mM NaCl / 50 mM Tris 10 mM Na2SO4 260 nM NaCl / 50 mM Tris† | |
| PC:SQDG | 100 mM NaCl / 50 mM Tris | |
| PC:MGDG | Milli-Q water | |
| PC:DGDG | Milli-Q water | |
| PC:PG | 100 mM NaCl | |
| PC:Soy CRB | 10 mM Na2SO4 | |
| PC:SPG | Milli-Q water | |
| PC:PCE | Milli-Q water | |
| PC:Di-O-P | Milli-Q water | |
| PC:Di-O-H | Milli-Q water |
† This was the chosen solution for preparation of PC liposomes for our photo-oxidation experiments.
Once the appropriate matrix solution has been selected, prepare a suitable volume for the suspension and extrusion process; this will depend on how many vials of lipid film(s) you will be making into liposomes.
MilliQ water
Sodium ChlorideCatalog #PubChem CID: 5234
Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
Sodium sulfateCatalog #PubChem CID: 24436
Warm ThermoMixer
Warm ThermoMixer
Power on ThermoMixer and warm to 60°C. Use the ThermoMixer tube block appropriate to the size of tube in which you prepared the lipid films (and will be resuspending them). An incubator/shaker warmed to the same temperature could be used in lieu of a ThermoMixer.
Warm liposome mini-extruder block
Warm liposome mini-extruder block
Place the Avanti mini-extruder heating block (heavy black metal component) on a heating block or warming plate. Set temperature to 60°C. Monitor temperature of the extruder block using a thermometer inserted into the provided hole. Do not let the extruder block get hotter than 70ºC, even if you increased the set temperature of the ThermoMixer to a higher point during the suspension process. Temperatures above 80°C can damage the syringes.
Assemble the mini-extruder
Assemble the mini-extruder
Assemble the mini-extruder according to the following figure, linked from the Avanti web site:
- Place the two Internal Membrane Supports o-ring side up on the counter.
- Pre-wet two of the small (~ 5 mm) filter supports with Milli-Q water, and place over the holes in each of the Internal Membrane Supports.
- Insert one of the Internal Membrane Supports with small filter support into the Extruder Outer Casing with the o-ring side facing up.
- Place a 0.2 μm polycarbonate membrane‡ over filter support and o-ring.
- Carefully place the second Internal Membrane Support (o-ring facing down) over the polycarbonate membrane. Do not twist the Membrane Supports.
- Place the Teflon bearing into the Retainer Nut.
- Put the Retainer Nut onto the Extruder Outer Casing
- Tighten the nut enough to allow it to fit into the spot on the extruder heating block (do not use a wrench, but this will need to be quite tight).
- Wet the extruder parts by passing a syringe filled with the chosen buffer solution (see step above) through the extruder. Discard this volume of buffer.
‡ A different size membrane could be used. We found the 0.2 μm pore size suitable for our needs.
0.2 µm polycarbonate membranesAvanti Polar Lipids, Inc.Catalog #610006
10 mm filter supports for Avanti mini-extruderAvanti Polar Lipids, Inc.Catalog #610014
Hydrate lipid films
Hydrate lipid films
Hydrate the lipid film(s) by adding some volume of the appropriate buffer/suspension matrix to the glass test tube(s). 200-500 µL was suitable for our purposes, but the volume will depend largely on the capacity of your glass syringes.
Suspend lipids
Suspend lipids
Insert the tube(s) containing the lipid films and suspension solution(s) into the ThermoMixer. Mix for 30 minutes at 60ºC.
After 30 minutes, take the tube out of the ThermoMixer and shake/swirl the solution. At this stage, you should see a milky/cloudy solution. If you see chunky bits of lipids floating around in the solution, something is wrong. Try again, using a new film, new buffer, and a higher or lower suspension temperature.
In this image, the top vial contains a liposome suspension which has been properly hydrated. The bottom vial has been improperly hydrated and should be discarded.
Note: The 60ºC temperature should be sufficient for most IP-DAG composed of short- and medium-chain fatty acids with at least some degree of unsaturation. However, you may need to increase the temperature for certain lipids containing saturated, long-chain fatty acids; these can have very high phase transition (i.e., melting) temperatures. A useful table of phase transition temperatures is provided by the manufacturer at https://avantilipids.com/tech-support/physical-properties/phase-transition-temps/
00:30:00
Transfer suspension to syringe and allow temperature to equilibrate
Transfer suspension to syringe and allow temperature to equilibrate
- Immediately transfer the solution to a glass syringe; ensure this syringe has been solvent-rinsed and is compatible with the extruder. Insert the syringe into the Teflon pieces of the mini-extruder as shown in the figure below (from manufacturer site):
- Then put the assembled extruder (with syringes on both ends) into the extruder block. Use the yellow clips to hold each syringe in place. Insert the hex nut so that the apex of the hex nut points straight up:
- Allow the temperature of the lipid suspension to equilibrate with the temperature of the extruder block (~ 5 minutes).
Further directions and guidance on proper extruder assembly are provided on the manufacturer web site: https://avantilipids.com/divisions/equipment/mini-extruder-extrusion-technique/
00:05:00
Extrude lipsomes from the lipid suspension(s)
Extrude lipsomes from the lipid suspension(s)
- Gently push the plunger of the filled syringe until the lipid solution is completely transferred to the alternate syringe.
- Then, push the plunger of the alternate syringe to transfer the solution back to the original syringe.
- Repeat the above steps multiple times in order to have at least ten passes through the membrane inside the extruder. The final pass through the extruder should fill the alternate syringe.
- Remove the extruder from the block, and then remove the syringe from the extruder by pulling the syringe straight out of the extruder.
- Inject the lipid solution into a clean vial.
Other directions and guidance are provided on the manufacturer web site: https://avantilipids.com/divisions/equipment/mini-extruder-extrusion-technique/
Clean up
Clean up
Clean the extruder apparatus with Milli-Q water and thoroughly dry it before storing. Solvent-rinse syringes before storing. If extruding additional liposomes, disassemble and clean all parts of extruder and replace the membrane and filter supports.