Hello,
Im trying to make a nanoliposome to encapsulate plasmid DNA. I am using the standard thin film method followed by resuspension in sodium acetate 25mM pH4 with my DNA (Im using the ionizible cationic lipid DODMA) for an hour or two at 70degrees (Higher than the Tm of DSPC) and then sonication followed by manual extrusion.
My lipid formulation is DODMA/DSPC/Cholesterol/DSG-PEG2000 at molar ratios 50/10/38/2 at a total lipid concentration of 2.5mM. I am in the process of optimizing the formulation and protocol.
When making the thin film I dilute all lipids in chloroform, add appropriate amounts to the rotary evaporator flask (pear shaped 25ml capacity) and evaporate at 100rpm in a 45 degree water bath, I place in vacuum seal overnight. However, my thin film looks very white (attached picture), and it does not resuspend properly, even at longer times and with constant magnetic stirring. Eventually it just peels off and forms these relatively big film-like lamps of lipids which I doubt could make nanoliposomes.
Any ideas on how to optimize? I thought of using a round bottom flask to increase surface area and lowering total lipid? However I am already at the low end of all protocols I have seen.
Thanks!
Dear Bruno Salomone Gonzalez de Castejon
I see two problems:
-First it might be that the cholesterol content is too high according to Article Nanoliposomes: Preparation and Analysis
they state and I quote” Anionic vesicles containing 10% Chol were also able to incorporate DNA molecules in the presence of calcium ions. Increasing cholesterol content of these vesicles from 10 to 40%, however, caused them to be unable to interact with model membranes and also unable to incorporate DNA molecules.”-Second I think your dispersion procedure requires a sonication step (see step 7 page 36)
Indeed, a round-bottom flask is preferred for reasons you already indicated.
Hope this helps you any further.
Best regards.
Dear Rob,
Thank you for your advice, I will optimize accordingly and update.
Kind regards,
The purpose of creating a lipid film after organic solvent evaporation is to increase the surface are upon contacting the film with an aqueous solution, you are not achieving this. Despite this, I normally use a tip sonicator (high power) to help dispersing the lipids on the aqueous solution. But you have to produce a thin film.
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