The most common liposomes protocols for encapsulating TX-TL mixtures are based on two strategies:
- hydration of dry lipids
- transformation of lipid-covered water-in-oil (w/o) droplets into liposomes (aka: droplet transfer / emulsion inversion)
The first strategy leads to samples composed only by lipids and the aqueous phase used for hydration. The second one leads to samples which can contain also traces of oil either as oil droplets, either as oil solubilized in the liposome membrane.
Note 1: by "oil" we mean an apolar solvent (generally a hydrocarbon, like dodecane or squalene, or a mixture of hydrocarbons, like the "mineral oil")
Hydration of dry lipids
We can distringuish three variants to this somehow "classical" preparation method.
- hydration of a dry lipid film stratified inside a glass vial
- hydration of a dry lipid film stratified on the surface of glass beads (diam. 0.2-2 mm)
- hydration of freeze-dried liposomes
The first two variants lead to liposome from multi-layer lipid lamellae deposited over the glass surface of a glass vial or of glass beads. To prepare the film, just let a chloroform solution of lipids evaporate. The third variant starts from freeze-dried liposomes. To prepare freeze-dried liposomes (sometimes referred to as "liposome cake"), liposomes must be firstly prepared by another method (e.g., hydration of lipid film), possibly extruded, then freeze-dried.
In these three variants, liposomes are formed just by adding the aqueous solution to the lipids. To have large vesicles (possibly giant one), minimal shear forces should be applied. After preparation, the inner and the outer solution are the same, so that there are no osmotic issues. If liposomes are manipulated after formation, the outer solution should be kept isotonic with the inner one.
Droplet transfer
This is a "novel" preparation method. Firstly, lipid-stabilized w/o droplets are formed by simple emulsification of a small amount of aqueous phase in a lipid-containing oil, next the droplets are allowed to pass through a flat oil/water interface, also stabilized by lipids, to acquire the second lipid layer, and form liposomes.
The method works well if the inner solution (that goes in the liposome lumen) and the outer solution are isotonic but have different densities. At this aim, sucrose and glucose are generally added to the inner and outer solution, respectively. Inner and outer solutions can have different buffer and different composition (e.g., TX-TL mix inside, PBS outside), provided that the two solutions are isotonic.
| Year | Reference | Size (diam.) | Comments |
|---|---|---|---|
| 1999 | [Oberholzer et al, Biochem Biophys Res Commun, 1999] | LUVs, 0.4 um | Hydration of lipid film in a vial |
| 2004 | [Ishikawa et al, FEBS Letters, 2004] | not given, probably few um | Rehydration of liposome "cake" |
| 2012 | [Danelon et al, Angew. Chem. Int. Ed, 2012] | <1-20 um | Hydration of lipid films on glass beads |
| 2014 | [Fujii et al, Nature Protocols, 2014] | 1-6 um | Droplet transfer |
| 2015 | [Ishiwata et al, Nature Protocols Exchange, 2015] | 1-10 um | Droplet transfer |
This is the very first report of ribosomal polypeptide synthesis inside liposomes. The liposome formation follows the hydration of lipid film stratified inside a vial, followed by freeze-thaw cycles, then extrusion Oberholzer et al, Biochem Biophys Res Commun, 1999 (note that the extrusion can be skipped). Additional work in this direction is descirbed in Yu et al, J Biosci Bioeng, 2001 (the very first report on GFP synthesis inside liposomes).
The preparation of liposomes is based on the hydration of freeze-dried extruded liposomes Ishikawa et al, FEBS Letters, 2004, based on a previous paper. The advantage of hydrating freeze-dried liposomes rather then lipid films lies in the higher entrapment rate.
A method for hydration of a lipid film stratified over 0.2 mm glass beads has been described by Danelon et al, Angew. Chem. Int. Ed, 2012; similar experiments (not referred to TX-TL reactions, and using 2 mm glass beads) can be found in an MDPI Life paper.
This paper presents an approach similar to the above-mentioned [Ishikawa et al, FEBS Letters, 2004], and monitoring of protein production. A JoVE paper and video is available, based on the paper by Sunami et al, Methods Mol. Biol., 2010.
This paper describes step-by-step, with explanatory pictures, the droplet transfer method. Yomo and collaborators have used this method in dozens of publications. The protocol is available in Fujii et al, 2014. In the following JoVE article a similar method is applied for encapsulating microspheres in liposomes. A detailed protocol is also available in the pdf companion article
This is another description of the droplet transfer method, for the in vitro reconstitution of biological processes in cellular compartments. Here the protocol.
A modified version of this protocol can be found at OpenWetWare The protocol was used by Malin Jonsson, Miroslav Gasparek (summer 2017 interns), and Zoila Jurado (PhD student at Caltech) to express the GFP in the liposomes in the summer of 2017 in the Murray Lab at Caltech.
To know more about general liposomes protocols:
- Szoka, F.; Papahadjopoulos, D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu. Rev. Biophys. Bioeng. 1980, 9, 467–508
- New, R. R. C. Liposomes. A practical approach.; 1st Edition.; IRL Press at Oxford University Press: Oxford, 1990
- Walde, P. Preparation of Vesicles (Liposomes) ASP Encyclopedia of Nanoscience and Nanotechnology (2003), Ed. H. S. Nalwa, Vol. 9, 43-79
- Walde, P.; Cosentino, K.; Hengel, H.; Stano, P. Giant Vesicles: Preparations and Applications. ChemBioChem 2010, 11, 848-865