We would like to add glucosylceramide and glucosylsphingosine to neuronal cells, (cell line later primary cells). I am interested in detailed protocols, specifically concerning resuspension, sonication times, storage. Also, how can I check if the lipids have entered the cells before continuing to the experiment itself? Anything will be helpful. Thank you.
At low concentrations, glycolipids tend to be soluble in aqueous medium and cell culture medium where they may complex with proteins. It has been far too many years for me to exactly recall concentration limits but if you were to do a Google search, the information should be available.
If the concentration of glycolipids in your medium exceeds critical micellar concentration, you may wish to consider sonication so everything becomes unilamellar. The best and most reliable way to do this is by dissolving the glycolipids in solvents of your choice (ethanol should work), dry in completely under vacuum (ideally in the absence of O2 and then add the aqueous medium of your choice and sonicate in short bursts (3-5) of
Thank you. Your answer helped me a lot. One very practical thing I haven't understood yet. When I can only resolve lipids for one experiment, I have to weigh every time I want to perform an experiment? Is this feasible?
Also I found a protocol where they generated micelles made of phosphatidylcholine and glucosylceramide for in vitro experiments and claimed that the mixture had to be done for stabilization? Is this also an issue for cell experiments or only for in vitro experiments?
What about adding BSA?
If you are going to dissolve a glycolipid in an organic solvent and dry it, the process can be made very simple. Weigh enough for many experiments, dissolve, and deliver aliquots to individual tubes, and dry the material. As long as tubes are properly sealed and stored, you need not be concerned for inconsistent amounts fron one to another experiment.
Inclusion of phosphatidyl choline or other phospholipids is useful if the purpose is to actually prepare liposomes of relatively uniform size - size, unilamelar or multi lamellar is determined by sonication time and composition of lipids (for example, including some cholesterol will change the fluidity of the bilayer). This type of system is often used for delivery of compounds that can be entrapped within the liposomes for intracellular delivery. These compounds are difficult to deliver within cells by simply adding them to the medium or if used in vivo, they may be too toxic if used systemically without entrapment. For in vivo delivery, one can design targeted delivery system, which can work preferentially if not selectively. For example, including a glycolipid that has galactose as the terminally exposed sugar residue, will help the liposomes go preferentially to the liver cells, courtesy of the galactose binding protein.
Of course, it goes without saying that if you use glycolipid containing liposomes, appropriate control will be liposomes without glycolipids.
Generally speaking, you can make glycolipid containing liposomes at levels of 2-10%. But as I mentioned, it has been a very long time since I used liposomes and for details you must review current literature. However basic chemical principles do tend to hold for a good long time... or not.
Thank you so much Tausif, you really helped a lot and you made things much clearer for me.
You should also realize that in vitro experiments (cell lines) in many cases would not be reproducible in vivo due to blood-brain barrier. The simplest way to avoid it is to use as smallest liposomes as possible.
So, the size of the liposomes is determined by the sonication process? Do I have to prepare liposomes if I only want to deliver the lipid itself into the cell? What about BSA? Is it for stabilization or for formation of the liposome?
If fact yes. But the final size is determined by the constrains in bilayer that is determined by the effective shape of lipid molecules present in the composition. In my opinion smallest stable liposomes would be in the range of 50-80 nm. As you are interested in liposome-cell interaction in vitro the BSA is not necessary but it can be used tp mimic future in vivo conditions. It would be even better to use whole serum.
Sanitation process will determine the size of liposomes to a substantial degree given all else being the same. If the aqueous suspension of lipids (after drying and creating a monolayer of dried lipids in a vessel) is sonicated for a minimum period of time, you will get mostly multlamellar (large) liposomes and as you sonicate the suspension for longer periods of time, more and more unilamellar liposomes will be formed in the range of 40-100nm diameter (depending on the lipid composition).
Including BSA or other proteins have no major effect on liposome preparation process; the only compelling reason to include a protein is that if your interest includes entrapping it so it can be delivered to the target cells. Since serum contains substantial amounts of lipids and lipoproteins, I would not use it to keep the composition of liposomes as intended.
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