The Institut Laue-Langevin (ILL) facility in Grenoble, France, has for the past 40 years been using neutron science to collect important data on fundamental questions in physics, such as what is dark matter and do parallel universes exist? More recently it has investigated industrial topics such as magnetism, superconductivity, materials engineering, biological substances and nanoparticle behaviour.
For example, recent projects have used neutron diffraction to understand how segments of DNA move from one place to another, or to characterise biomaterials formed on titanium dioxide implants. Another project has revealed important information about how potential drug delivery agents such as gold nanoparticles react with biological cells. The results could have implications for many of the companies – including AstraZeneca1 – that are looking at nanoparticles as drug delivery agents.
So how is it, that these subatomic particles, which normally reside in the nucleus of an atom, can tell us so much?
It is largely due to the fact that they are neutral and have a wavelength typically comparable to inter-atomic spacing of matter. This means that – once freed by neutron fission – the neutrons can penetrate deep into solid matter and can reveal the atomic structure of material crystal structures simply by rebounding off the nucleus of atoms inside a sample. They also have a magnetic moment, which means they can interact with internal magnetic fields. These strange properties make them useful for non-destructive ways of looking at nanomaterials and advanced multilayer composites.
Neutrons can also provide a powerful tool in spectroscopy, for looking at materials that are normally opaque to infrared and for Raman spectroscopy. Working catalysts, for example, are typically opaque to infrared and Raman spectroscopy but by putting chemical systems into a beam of neutrons, their spectroscopic properties can reveal the changes in chemicals as they undergo a chemical process.
But first the neutrons have to be freed, and a quick tour of the ILL reactor facility reveals the neutron reactor housed within the facility’s thick concrete walls and submerged in a deep pool of water. Dr Peter Geltenbort, a physicist at ILL, explains that within an internal chamber (only about twice the size of a basketball) the enriched uranium is initiated in a chain reaction such that, at a certain point, the excited neutrons bounce off in all directions, at great speeds. Thankfully, dense materials – such as the aluminium casings, concrete walls and water – prevent the neutrons from escaping beyond a predetermined point, but the strange blue glow, due to a phenomenon known as Cherenkov radiation, is clearly visible through the water.
Some of the neutrons bounce their way into the sampling tubes or guides of varying lengths, placed around the reactor and at the end of these tubes, scientists from universities and institutions around the world are able to set up experiments of all kinds, using up to 40 different instruments. It is these experiments that are revolutionising our understanding of the universe, matter, genetics, nanoparticles and potential new drug delivery methods.
The reactor provides beams of neutrons that vary in energy over a wide range, from ‘hot’ (high energy or fast), thermal (medium energy), cold (slow), to ultra cold (very slow) neutrons.
Scientists working at ILL have used some of these neutrons to show how the charge of gold nanoparticles can affect gold’s interaction with the protective outer membrane of biological cells. These insights, published in Langmuir2, provide a first step in the effective design of safe nanoparticles for biomedical applications and in safe handling practices for their use in consumer products.
Gold nanoparticles are considered to be promising delivery agents because they are easy to load with other molecules such as cancer drugs; easy to produce; chemically stable in the body; and have optical, electronic and thermal properties that allow them to be switched on once they arrive at the right location in the body. However, at present, the interactions between nanoparticles and the cell membrane (a cell’s outer defences), are not well understood – beyond the fact that shape, size, composition and charge can all have an effect.
The technique being used to look at biomembranes and how well certain drug delivery mechanisms work, is described as ‘neutron reflection’. Using the most simplistic of terms, ILL Instrument Scientist Dr Rob Barker explains that by bouncing neutrons off surfaces, the scientists are able to look at variations in density of different layers. ‘Neutron reflectometry is very powerful for measuring layered structures. If nanoparticles enter the lipid bilayers the density of the layers will rise and we can quantify this rise,’ he says.
Cationic nanoparticles with their positive charge were found to penetrate the membrane when at low concentration, but at higher concentration they disrupted the membrane
However, while real biological membranes are made of a very complex mixture of lipids and proteins, the work carried out by Dr Marco Maccarini looking at the structural changes due to the interaction between nanoparticles and membranes has been carried out on simpler systems built using a double lipid layer on a substrate to mimic the structure of a real biological membrane. The initial studies looked simply at charged nanoparticles. A nanoparticle that is ‘functionalised’ with a cationic group obtains a positive charge. These cationic nanoparticles with their positive charge were found to penetrate the membrane when at low concentration, but at higher concentration they disrupted the membrane. When similar experiments were carried out with nanoparticles functionalised to have a negative charge, it was found that these nanoparticles did not penetrate or stabilise the membrane.
Predictive modelling
According to Barker, this model is just a simple starting point and making more complex layers takes a lot of lab time and optimisation. ‘But we now have a framework to look at different nanoparticle parameters that can be changed – size, shape and composition. In the next few years we will look at what effects these different nanoparticle parameters have on membranes. We can even start to look at how drug actives work with the membranes,’ he says.
‘The idea of functionalising the surface of gold nanoparticles is well known. For targeting cancer cells, this reaction could be desirable but cationic particles have been found to be much more toxic than positive ones. We are trying to see what they actually do. We want to know why at low concentration they penetrate and stay there but if you raise the concentration they disrupt the membrane.’ He adds: ‘It is not the gold itself that is important, but what is on the surface. Which is OK if you can control that chemistry – you can have one particle that is interacting with the membrane and, depending on what you want to do with it, one that is destroying the membrane.
‘Neutron scattering can help us to see the mechanism by which this happens, so that when thinking about designing these for drug delivery we can start to see what reacts with a cell very well and what can deliver into the cell.’ He adds: ‘You can even start to see ways of functionalising the membrane surface to get things across quicker or you can look at ways of better designing molecules.’
The aim is that in the future, rather than trial and error, there will be a system in place that is predictive
The aim is that in the future, rather than trial and error, there will be a system in place that is predictive, so that researchers can reduce the temperature, slow things down, use different lipids to see how the nanoparticle inserts itself, and change components.
In addition to drug delivery, ILL researchers have used the technique to investigate titanium dioxide, to try and understand the surface interaction on surgical implants. Barker says: ‘While titanium is biocompatible, we can penetrate deep into the surfaces of membranes and look at how our bodies interact with it on the surface.’ For example, both titanium and calcium have strong surface charges – so adding calcium ions to the mix can lead to dynamic processes of exchange.’
They have found, for example, that a very stable membrane bi-layer can be formed on the surface of the titanium dioxide – but calcium ions, if added, will go to the membrane surface and destroy it. This work has implications for surgeons who have tried various means to improve the rate of implant acceptance. For example, some researchers have coated the surface of implants with antibiotics to see if it would help take-up by the body and make the recovery process quicker.
‘Until now scientists have been unable to really know how these antibiotics are interacting,’ says Barker. ‘We can now see what is happening on these surfaces. For example, we can see if we put a cell on it, or plasma from the body, how these things are absorbed and how to improve these interactions. Such experiments could be used to improve the process of recovery or even stop the build-up of cholesterol on stents.’
Figure 2: A schematic comparison of how negatively and positively charged nanoparticles react with the outer membrane of a cell
There are also interesting ways of using the neutrons in genomic research. For example, the fact that neutrons are sensitive to hydrogen atoms means that they can be used to look at natural hydrogen-containing organic and biological samples, such as membranes, bound water and DNA.
Professor Andrew Harrison, one of ILL’s directors, explains that the scattering power of different nuclei can vary dramatically between isotopes of the same element. In particular, hydrogen scatters very differently from its heavier isotope deuterium. This enables structural components to be highlighted by substituting atoms of one isotope for another. Isotopic substitution provides information about the orientation and interactions of atoms and molecules. It can be used to make the scattering strength in one part of a structure the same as in the surrounding medium, rendering it ‘invisible’. This allows another part of the structure to stand out in contrast (a technique known as contrast matching).
Harrison explains: ‘Scientists at the institute can tune the scattering contrast selectively by duteriorating parts of genes, allowing them to monitor in real time what one gene is doing.’
Researchers from the universities of Edinburgh and Keele used ILL Life Sciences group’s deuterium labelling techniques to show for the first time how the protein enzymes first bind to the DNA to become a single molecule complex, then separate out the DNA and carry it away. The next stage of this project is to look at the ‘pasting’ process, which is what happens when the protein moves the DNA elsewhere. According to researcher Dr Max Cuypers, the findings will provide increased genetic understanding for creating useful genetic tools and increasing gene efficiency.
Isotopes can be used to target specific cells or tissues, enabling the cells that are labelled to be irradiated, thereby delivering controlled doses of radiation to cancerous tissue
Also important for the field of drug therapy is ILL’s ability to produce radioactive isotopes. The Institute is currently involved in work that will lead to the generation of specific isotopes with specific radio therapeutic and diagnostic applications. These can be used in cancer therapy, to target specific cells or tissues, enabling the cells that are labelled to be irradiated, thereby delivering controlled doses of radiation to cancerous tissue. They can also deliver the isotope to particular parts of the body.
These discoveries are a long way from the initial ideas of the facility’s founders – Louis Néel and Heinz Maier-Leibnitz – who initiated it back in 1967, but increasingly new partnerships with ILL are making neutrons accessible to new communities. Harrison explains that in the early days the institute’s users were mainly solid state physicists, experts who understood the technique; but increasingly the users are not experts but rather chemists wanting to look at the structure and biologists wanting to look at membranes and the way things pass through them. As a result, over the years the institute has built up a much needed infrastructure and array of support labs to help the researchers prepare, or ‘dutorate’, samples and that also allows them to do other light scattering research work at nearby facilities, at the same time.
These changes have been carried out at the same time as modernistation to the infrastructure and installing new instruments, such as FIGARO, a horizontal-surface reflectometer, which will allow the study of fluid interfaces and extend the scope of studies of biomembranes, and which could be key in developing better drug delivery processes.
On 9 August 2013, the reactor was shut down for a planned modernisation programme that is scheduled to finish in June 2014.
References
1. http://www.astrazeneca.com/Research/news/Article/27122012--cytimmune-and-astrazeneca-to-research-potential, last accessed 13 Aug 2013.
2. Sabina Tatur, Marco Maccarini, Robert Barker, Andrew Nelson, and Giovanna Fragneto. Langmuir, 2013, 29 (22), pp 6606–6614 DOI: 10.1021/la401074y
