Water is vital to life on planet Earth. We see it every day, we drink and bathe in it; we use it to clean, cook, grow crops, provide energy, and we complain when it falls from the skies. It makes up around two thirds of a healthy human, and covers 70% of the Earth’s surface. Yet, despite its importance in everyday life, water has managed to retain some of its mystery.
Comprised of just three atoms, two hydrogens flanking an oxygen atom, water is deceptively simple. It interacts with other molecules, including other water molecules, using hydrogen bonds – the same forces that hold two DNA strands together in our cells or help proteins fold into 3D shapes. The ability to form these bonds gives water some unusual and useful properties. Almost anything will dissolve in water to some extent; even a few molecules of a mug or glass will dissolve when water is poured into it, and this property is exploited in our bodies to absorb nutrients from food.
Neutrons, such as those produced at the ISIS Neutron and Muon Source, are an ideal tool for investigating water because of how clearly they can see hydrogen atoms. Using a technique called ‘contrast variation’, specific molecules can be made more visible by deuteration of their hydrogen atoms, making them heavier. This allows researchers to ‘tune’ the neutron beam to the heavier hydrogen atoms and focus on areas of interest, minimising background noise.
From creating ‘designer saliva’ to understanding our weather systems, scientists from all over the world come to ISIS to harness the power of neutrons. While one research group may be looking at how to overcome drug insolubility, another is inspired by a frog’s natural ability to freeze and thaw.
Man-made lubricants used in the moving parts of vehicles and machinery are oil-based. However, biological lubricants, such as those on the surface of the eyes, mouth and small intestine must be based on water. As water is a poor lubricant by itself, nature must recruit additional biomolecules to overcome this limitation. For example, saliva is accompanied by up to 2000 proteins and peptides that promote lubrication. These proteins include mucins, long glycoproteins with the ability to form gels that retain water on surfaces, which is essential for the hydration of internal body surfaces.
Dr Javier Sotres, from Malmö University in Sweden, is part of a team researching mucins using the INTER reflectometer at ISIS. Although it is well known that mucins are required for effective lubrication, their exact role in the stability of salivary films is still unclear. Using a recently developed confinement cell system, Dr Sotres and his team can apply pressure to mucin films to mimic the movement of the eye against the eyelid, or the tongue over the teeth. By focussing neutrons on the system, Dr Sotres is able to see how much water is retained at differing pressures, shedding light on the interactions between mucins and water.
Previous experiments by Dr Sotres have characterised the mechanical stability of salivary films on different surfaces, and the protective role of mucins. Early data from the experiments at ISIS appear to show that mucin films retain significant amounts of water to a higher pressure than other hydrophilic polymers. It is hoped that these findings could eventually lead to the development of better artificial saliva for cancer patients undergoing radiotherapy and elderly people on multi-medication, who suffer with “dry mouth” and are at risk of tooth decay.
The Future of Pharma
It is estimated that up to 90% of active pharmaceutical ingredients (APIs) being discovered today are poorly soluble in water. If drugs don’t dissolve efficiently in water, they cannot be absorbed through the intestinal wall to the bloodstream, and cannot be taken by patients orally. Research groups are addressing this problem by devising drug delivery systems, such as micelles and microemulsions, which contain surfactants to lower the surface tension between the drug and the water, and increase drug solubility in the gut.
Microemulsions are already being used clinically to deliver drugs, for example in Neoral, a formulation of the immunosuppressant, cyclosporine, which is given to organ transplant recipients. Professor Jayne Lawrence, a research scientist from King’s College London studying drug delivery, explains, “A micelle only contains surfactant, but you can convert a micelle to a microemulsion by adding oil. The oil sits in the hydrocarbon chains and swells those chains, and ultimately forms a drop of oil in the centre of the emulsion.” The drug compound can then be dissolved in the oil, which in theory will assist dissolution in water later on.
However, each drug is structurally different, requiring a different combination of surfactant and oil to become sufficiently soluble. By understanding how the surfactants and oils interact with the drug, and how this affects solubility, better delivery systems can be designed.
One particular class of drugs Professor Lawrence is investigating is steroids, which are often injected directly into the bloodstream because of their poor water solubility. In her studies with the steroid drug, testosterone propionate (TP), a surfactant called SDS was found to significantly increase solubility. However, SDS is toxic in the body, so the group must now try to select the important part of the compound, a sulfate group, and link it to something known not to be toxic in order to achieve the same therapeutic effect. This could pave the way for drugs such as TP to be taken orally for the first time.
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Researchers are also using ISIS to ‘put the chemistry into climate models’. Professor Martin King’s group from Royal Holloway University are using the INTER reflectometer at ISIS to understand how the Earth’s climate are affected by chemical reactions in clouds. A cloud is formed by air rising, cooling and the water vapour condensing on atmospheric aerosol. Atmospheric aerosols are produced by man burning fossil fuels but also naturally from forest fires, dust storms, sea spray and volcanoes. Every cloud droplet starts as an aerosol particle in the atmosphere. These droplets scatter sunlight to give their characteristic white appearance.
In the atmosphere, cloud droplets can be ‘shrink-wrapped’ by organic films, affecting growth and therefore the amount of water that will nucleate. The size of the aerosol dictates how long it survives in the atmosphere and how much light the cloud scatters. Professor King is using neutrons and real atmospheric samples from Antarctica and London to contribute to more realistic climate models, by investigating the effect of organic films on droplet size and finding out how this may impact life on Earth.
Reaction may allow the droplets to shrink, and the smaller droplets will scatter more sunlight back into space, with a climatic cooling effect. If the droplets grow larger due to these films, less sunlight will be reflected back into space and the Earth may grow warmer. Chemical reactions that affect the size of cloud droplets may make clouds form, evaporate or even rain, although it’s good to know that only 1 in 10 clouds rain.
The Eastern Wood frog is the only North American frog to be found in the colder territories of Canada and Alaska. This cold-blooded amphibian belongs to a group of organisms called psychrophiles, and can survive weeks in temperatures as low as -8oC. This ability to thrive in low temperature environments is due the frog’s use of nature’s very own antifreeze, glycerol.
Cryoprotectant molecules like glycerol protect cells and tissues from the formation of destructive ice crystals. However, the molecular mechanism behind cryopreservation is still relatively unclear, prompting a group of physicists from the University of Leeds to investigate.
In studies on the SANDALS neutron diffractometer at ISIS, paired with computer simulations, the team mimicked the freezing conditions the frog faces in North America to look at the impact of glycerol on the structure of water. They found that when a water-glycerol mixture is cooled to -35oC, the water molecules form clusters, and the formation of a larger ice crystal network is prevented by a cage of glycerol molecules.
It is hoped these findings could offer a new way to study water’s fundamental properties at low temperatures. They may also be used to improve cryopreservation techniques in the freeze storage of sperm and eggs for fertility treatments, and embryos for the conservation of endangered species.
Water’s Hidden Mysteries
In a 20 year debate, researchers are still arguing about some of water’s more unusual properties. Water has a freezing point of zero degrees Celsius, although it is possible to supercool it down to -40oC under the right conditions. This is where it begins to puzzle scientists, including Professor Alan Soper, world expert in the molecular structure of water and Senior Research Fellow at ISIS. “Normally when you cool things down they shrink and they get smaller… The specific heat [the amount of heat needed to raise the temperature of something by one degree] gets smaller as well,” he explains. “When you supercool water, first of all it gets bigger, it expands… and secondly, the specific heat gets bigger. So what’s going on?”
Some scientists believe they know. If water is cooled below a certain temperature, the so-called ‘critical point’, it will split into two distinct but coexisting forms: liquid and vapour. However, an elusive ‘second critical point’ of water has also been suggested. The idea behind the second critical point is that below this point, water separates into low density liquid and high density liquid, and the presence of a second critical point affects the behaviour of the overall stable liquid.
Although mixtures of different liquids can have several critical points, this has never been seen in a single liquid. Despite computer models showing this theory to fit the available data for supercooled water, studies on the NIMROD and SANDALS instruments at ISIS are yet to find any experimental evidence of a second critical point in real water samples. Thus the debate continues…
When you consider the diverse range of investigations involving water at ISIS, it reflects the prevalence water has in our lives. Whether scientists are studying climate, nature or human health, understanding of the fundamental properties of water is vital. However, just as there is more to discover about clouds and cryopreservation, there are still many fundamental questions that remain unanswered about the liquid of life.
Research date: ---- 2016
Sotres, J., Madsen, J., Arnebrant, T. and Lee, S. (2014). Adsorption and nanowear properties of bovine submaxillary mucin films on solid surfaces: Influence of solution pH and substrate hydrophobicity.Journal of Colloid and Interface Science, 428, pp.242-250.
Znamenskaya, Y., Sotres, J., Engblom, J., Arnebrant, T. and Kocherbitov, V. (2012). Effect of Hydration on Structural and Thermodynamic Properties of Pig Gastric and Bovine Submaxillary Gland Mucins. The Journal of Physical Chemistry B, 116(16), pp.5047-5055.
Znamenskaya, Y., Sotres, J., Gavryushov, S., Engblom, J., Arnebrant, T. and Kocherbitov, V. (2013). Water Sorption and Glass Transition of Pig Gastric Mucin Studied by QCM-D. The Journal of Physical Chemistry B, 117(8), pp.2554-2563.
A paper on the technique used in these experiments can be found here.
Abbott, S., de Vos, W., Mears, L., Cattoz, B., Skoda, M., Barker, R., Richardson, R. and Prescott, S. (2015). Is Osmotic Pressure Relevant in the Mechanical Confinement of a Polymer Brush?. Macromolecules, 48(7), pp.2224-2234.
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Jones, S., King, M., Rennie, A.R., Ward, A.D., Campbell, R.A. and Hughes, A.V. (2016). Aqueous radical initiated oxidation of an organic monolayer at the air-water interface as a proxy for thin films on atmospheric aerosol studied by neutron reflectivity. Langmuir (in preparation)
The lead author on the paper, Dr James Towey, who completed the experiments during his PhD with Dr Lorna Dougan, was funded by an EPSRC DTA studentship. (Now known as a Doctoral Training Partnership: https://www.epsrc.ac.uk/skills/students/dta/)
Lorna Dougan is funded by a European Research Council grant on 'Extreme Biophysics'.
Towey, J.J, Soper, A.K and Dougan, L. (2016) Low-Density water structure observed in nanosegregated cryoprotectants solution at low temperatures from 285 to 238 K. Journal of Physical Chemistry B, 120(19), pp. 4439-4448.