I'm trying to prepare liposomes (100-400nm) loaded with FITC-dextran for dye-release studies. I separate unincorporated dye from liposomes by gel filtration. When I perform my analysis (fluorimetric measure of FITC de-queching where addition of 2% triton-x to liposomes is my positive control), I see a lot of background fluorescence and very little de-quenching after addition of tx-100. I see nice-looking liposomes by EM and seemingly permeabilized ones after addition of tx-100, so I'm thinking I don't have very FITC loaded to begin with. I'm using freeze-thawing, sonication and extrusion to prepare my liposomes. Does anyone have any suggestions or general feedback?
Are you using the method described in this paper?
https://www.jstage.jst.go.jp/article/bbb1961/50/2/50_2_399/_pdf/-char/en
Hi Donald!
I'm not using their exact method to prepare the liposomes (Reverse-phase evaporation), but as far as I understand, my method of sonication, freeze and reuspending the dried lipid film in the presence of dye before final extrusion should work just as well to incorporate FTIC-dextran. I've also seen that reported in other studied. I know for a fact that my methods produced nice liposomes, and that's why I'm guessing incorporation is the problem...
A couple of thoughts...
1) "...I see a lot of background fluorescence"... This makes me wonder how effective your gel filtration chromatography is. Have you monitored the fluorescence of the fractions coming off the column?
2) Freeze-thaw followed by extrusion will give you lovely liposomes but I don't know how effective it will be at entrapping the FITC-dextran. I can imagine that entrapment would be sensitive to both the method used to make the liposomes and the lipid composition.
Good points,
First off, my liposomes come off in the void volume, clearly visible by 'cloudiness' and confirmed by EM. I see the dye traveling through the resin and eluting much later. These fractions are bright yellow, so I think most 'un-loaded' dye is separated effective.
Second, I totally agree, but I've seen most protocols using this methods so I'm confused. Do you have experience with RPE? It seems so much more complicated.
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