The cell membranes of almost all organisms are formed of two layers of lipid molecules that prevent ions, proteins and other molecules from entering or leaving the cell. As phosphocholine (PC) phospholipids are the main component of these lipid bilayers, they are often used as simple models to study whether drug compounds are likely to be able to enter the cell. Phospholipids are non-toxic to cells and potentially useful for drug delivery and other medical applications. However, they are neither cheap nor easy to synthesise, unlike sulfobetaine (SB) based lipids. SB lipids are non-toxic and used in medical applications such as commercial eye drops, but their structure is less well studied. In work recently published in Physical Chemistry Chemical Physics, researchers from the University of Bath combined X-ray reflectometry (XRR) studies at Diamond with neutron reflectometry (NR) experiments at ISIS Neutron and Muon Source to investigate the structure of mixed monolayers of sulfobetaines and phosphocholine phospholipids. Their results confirm that the interactions between sulfobetaines and phospholipids are favourable and that these combinations have potential applications in future drug delivery methods.
Dr Naomi Elstone, lead author on the paper, was a PhD student at the University of Bath when we joined the team conducting this research. She says:
Our thought process when we started this experiment was that - as sulfobetaines have the opposite charge distribution in the head group to phospholipids - there might be some interesting interactions between them. And sulfobetaines have the advantage of being much cheaper and easier to synthesise than phospholipids, so we were hoping there might be the potential to use them in vesicles and other drug delivery mechanisms. To do that, we need to understand how the interactions of the two headgroups affect the properties of the mixed monolayers. So for these experiments, we created lipid mixtures with different ratios of sulfobetaines and phospholipids, and used neutron and X-ray reflectometry to investigate the structure of mixed monolayers.
The experiments used the sulfobetaine 3-(dimethylocta-decylammonio) propane-1-sulfonate (SB3-18), which has a single 18-carbon tail, and the phospholipid dimyristoylphosphatidylcholine (DMPC). DMPC has been well-characterised and has biological relevance. In addition, the two molecules have similar tail thicknesses, which makes it easier to see structural changes in the monolayer.
Dr Elstone explains;
By using a monolayer, a thin layer at the interface, we simplify the system and make it 2D rather than 3D. We're also able to manipulate the surface pressure and really understand the nature of the structure and how it changes with pressure. With the XRR experiments at Diamond, we get a lot of good information about the monolayer structure. But because there's a lot of hydrogen in the lipid tails, the contrast isn't as good compared to the headgroups. With the complementary NR experiments, we can selectively deuterate the molecules - add deuterium - to essentially make one of them invisible. And that gives us complementary information about the structure and adds to the overall picture.
The team found that adding the sulfobetaine to the mixture did not significantly affect the monolayer structure. However, the SB tails are longer than the PC tails, making the layer surface rougher. At higher SB concentrations, the data showed the phospholipids behaving normally, but that the sulfobetaine molecules were closer to perpendicular to the water surface. This suggests at these higher concentrations the two molecules have different configurations, making it harder to make vesicles, as they won’t pack well together. A very high concentration of sulfobetaines would probably also interact badly with cells, making the mixture toxic.
Dr Elstone concludes:
Our research team conducted some follow-up experiments at Diamond with different sulfobetaines, which we will publish in due course. We were interested in the potential for using sulfobetaines in vesicles specifically, but they are already widely used in medical applications, from contact lens solutions and eye drops to drug delivery. Mixtures of phospholipids and sulfobetaines are very interesting for drug delivery, and not just because they would be cheaper to produce. With their opposite charge distribution, sulfobetaines may interact differently with drug components, and facilitate the delivery of drugs that wouldn't be possible with phospholipids alone.
To find out more about the I07 beamline or discuss potential applications, please contact Principal Beamline Scientist Francesco Carlà: francesco.carla@diamond.ac.uk.
Elstone N et al. Structural investigation of sulfobetaines and phospholipid monolayers at the air-water interface. Physical Chemistry Chemical Physics 24.37: 22679-22690 (2022). DOI:10.1039/D2CP02695C.
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