Hello!
I am preparing liposomes using a membrane extruder.
The procedure is well-known and descibed extensively in the literature:
1. I start with a solution of the lipid in chloroform, then evaporate the chloroform. At this stage I know the mass of the lipid M.
2. I add buffer (volume V) and make several freeze-thaw cycles with vortexing.
3. The resulting mixture goes to extruder and becomes transparent upon several extrusion cycles.
What it the resulting concentration of the lipid? It should be lower than M/V due to adsorption of the lipid onto the membrane and the inner surface if the glass syringes. But what is the typical concentraion loss? Is it 10-15% of M/V?
Based on my experience using gas-tight syringes and a mini-extruder (Avanti Polar Lipids Extruder Set), lipid mass loss during the extrusion process is reduced and generally well below 10%. Lipid adsorption on the syringe surfaces is minimal, and on the polycarbonate membrane, it is significantly reduced due to its small surface area.
To accurately determine the final lipid concentration in your process, it is advisable to quantify the concentration after extrusion using analytical methods such as chromatography or spectrophotometry.
Hello,
A phosphate assay measures the concentration of phosphate ions in a sample. Since phospholipids contain phosphate groups, this type of assay can be used to indirectly quantify lipid levels.
Yes, it is common to experience some lipid and drug loss during liposome preparation due to adsorption onto the membrane filters and the internal surfaces of syringes or glassware. The exact amount of the initial lipid mass loss typically depends on the type of membrane, lipid composition, solubility of the lipids, and the handling process. I will not be able to tell you an exact estimate of how much lipid you are losing because there isn’t a fixed percentage for all situations. However, I will suggest that if you want to precisely quantify the loss in your case, you could measure the total lipid content before and after extrusion, in my case, I usually use freeze-drying to obtain a theoretical mass that I compared to the actual mass, so I recommend experimenting to determine the exact losses in your specific system. You can also pay attention while dissolving your lipids and drugs to make sure they completely dissolve which will facilitate easy pass through the filter. If you are using rubber tubing, you can consider using PBS and or your collecting solvent and running it through the rubber tubing after the lipid or drug solution to push through the residual lipid or drug solution remaining in the tubing. In my case, I use a microfluidics system, and this technique significantly reduced my drug and lipid loss.
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