I am trying to deliver a 20kDa protein to cancer mono-layer cells using L4395 Sigma kit ( cholesterol; L-α-phosphatidylcholine and stearylamine). We just have to dissolve this lipid powder in the protein of interest and it is expected to encapsulate it. Any idea once I have it in liposome form, how I have to treat the cancer mono-layer cells with it such that I get a good delivery of this protein in the cells?
Any precaution/modification in this protocol would be highly appreciated.
It is unlikely that all of the protein will be encapsulated. If you simply mix the protein with the lipids, forming multilamellar liposomes, some of the protein will be internal and some will be external. If you need to remove unencapsulated protein, you may be able to do so by ultracentrifugation to pellet the liposomes.
The pH may affect the amount of protein that associates with the cationic liposomes electrostatically, so you may want to try a range of pHs. The ionic strength (saltiness) of the solution will also have an effect, with lower ionic strength favoring greater electrostatic protein association.
You can extrude the liposomes through a porous polycarbonate membrane to convert the multilamellar vesicles into unilamellar ones of a defined size, which may have an effect on delivery. If you do this, do it before removing unincorporated protein. Unincorporated protein can then be removed quickly and easily by passing the unilamellar vesicles through a suitable gel filtration medium, such as Sepharose A5m.
Thanks for a comprehensive answer. I don't have Sepharose A5m. I have Avanti extruder-100 nm membrane. I can do extrusion to get unilamellar vesicles. Can I spin after this (what would be speed?).
Also, once I have liposomes encapsulated protein, how to deliver it on the cells. Do you have any protocol?
I have found it to be difficult to pellet SUVs quantitatively, even with an ultracentrifuge, so it is a good idea to preprae them in as high a concentration as possible from the start.
To deliver the liposomes to cells in culture, you may want to replace the usual medium with serum-free medium, using the smallest practical volume of medium so that the liposome concentration is maximized. For reference, see this information about liposomal transfection of cells.
https://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_2000_Reag_protocol.pdf
If you extrude your liposome in a raffinose buffer then you can spin the liposomes down at 21,000xg - 16000xg. Only thing you have to note is once you have raffinose in your system then all the work needs to be carried out at room temperature since raffionose precipitates at low temperatures.
You can check the protocol in this paper Article Phosphatidic acid binding proteins display differential bind...
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