For the last several years (more than I actually want to think about, I think we started around 2007) I have been looking into what happens when we mix a phospholipid and a triglyceride. As we now have published the fourth paper on this subject, it might be time to give a short overview.
The project started many years ago when we, a visiting Phd student and I, where looking at lysosomes. She had isolated the lipids from them and now she wanted to make GUV (giant unilamellar vesciles)
to get pretty pictures to study the lateral structure of these. But they just didn’t want to form, so we went on to do a lot of different experiments and among them was TLC, where we found a large amount of triglycerides. Okay the might prevent the formation of liposomes, we thought, but we where so wrong. The GUV could be formed much faster and easier from a mixture of POPC and triolein (TO) than from pure POPC. We then started to do all the thing we normally do with lipids mixture like DSC, EPR and fluorescence spectroscopy.
During one of the EPR experiments I discovered that a mixture of 10% TO and 90% POPC separated into to different phases when they where centrifugated. One went to the bottom of the tube, the other to the air-water interface, like this:
The lower phase we labelled HF, for heavy fraction and the upper LF for light fraction. We also found that there spectroscopic properties where different, so we ended up writing a paper on this, which went into chemistry and physics of lipids.
In this we had TLC’s which showed that both fractions had the same amount of TO (which is why we where talking of fractions instead of phases originally) which made it hard to explain what was going on, but we where bold (stupid) enough to suggest the following model:
The “driving force” here would be that TG are not that happy with having all three FA chains parallel, like in B. But we never figured out how this could explain the difference in density.
After this got out we managed to get our resident MD simulation guy to run MD simulations on a system with 2 and 5 mol% TO and he found a different picture:
In A is the results for 5 mol% and in B and C the result for 2 mol%. The figure is from this paper. Now this starts to make a bit more sense, once we accept that the TO molecules can go into the centre of the membrane, which was a bit hard for me. And the biological relevance also went up. This is highly relevant for the formation of lipids droplets and or for the so-called mobile lipids in cancer cells.
In the next paper in we focused on microscopy and mechanics, showing confocal picture and a video on the dynamics. We also showed that TG doesn’t make the liposomes more prone to fusion, as we had speculated as mechanism for the fast and efficient formation of liposomes. And we showed that the presence of TO lowered the bending rigidity significantly, which could be relevant for cancer cells as they also have reduce bending rigidity.
And now the forth and probably last on only POPC TO is now online. Here is the abstract:
We have in this study investigated the composition, structure and spectroscopical properties of multilamellar vesicles composed of a phospolipid, POPC, and up to 10 mol% of triolein (TO), a triglyceride. We found in agreement with previous results that the mixtures with 10 mol% TO spontaneously separate into two distinct phases, heavy (HF) and light (LF), with different densities and found this also to be the case for 2 and 5 mol% TO. The compositions of the two phases were investigated by quantitative lipid mass spectrometric analysis, and with this method we found that TO had a solubility maximum of about 4 mol% in the HF, whereas it was markedly up-concentrated in the LF. Electron paramagnetic resonance spectroscopy indicated POPC membranes of all tested concentrations of TO in both phases to be almost unperturbed by the presence of TO and to exist as vesicular structures containing entrapped water. Bilayer structure of the membranes was supported by small angle X-ray scattering that showed the membranes to form a lamellar phase. Fluorescence spectroscopy with the polarity sensitive dye Nile red revealed, that the LF samples with more than 5 mol% TO contained pure TO domains. These observations are consistent withan earlier MD simulation study by us and co-workers suggesting triglycerides to be located in lens shaped, blister-like domains between the two lipid bilayer leaflets (Khandelia et al. (2010)).
And the ToC picture:
Seen here are the Nile red spectra of o, 2, 5 and 10 mol% TO samples. Nile red is used for detecting lipid droplets in cells as is changes it emission wavelength depending on the polarity of the surrounding medium. On the right are the spectra from the LF fraction shown and it shows pretty clearly that it contains TG domains, based on the increased intensity at 571 nm. This combined with the SAXS measurements, where we show the presence of lamellar structures is al in all experimental evidence for the model suggested from the MD simulations.
And the pretty pictures I posted some time ago were made for that paper.