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About one in three of us will break a bone in our lifetime. Although painful, usually the bone will heal naturally. However, in about 1/20 cases the bone heals poorly or not at all. These are called delayed union or non-union bone fractures. They can be terrible for the person affected, sometimes taking many years of major surgery and rehabilitation to fix. They also cost a lot as well - about £40-50,000/patient, with the total cost in the UK at ~£350m every year. These fractures may be treated by implantation of bone harvested from other parts of the body or from donors, or with surgery and fixation of the bone using metal plates. Many research groups are investigating the use of drugs, materials and cells implanted at the bone fracture site to help speed up healing, but there is no drug that you can take to speed up or improve bone healing. Development of such an approach would improve the lives of thousands of patients each year. We think we can achieve this by using 'ultrasound responsive agents', including microbubbles and nanodroplets. Microbubbles have been used for a long time to help doctors see inside our bodies more clearly. They are filled with a gas and, because they are smaller than the smallest of our blood vessels, they can be safely injected into the bloodstream. Ultrasound waves are reflected by them much more than by surrounding tissues, and this makes it possible to use them to build up an image of organs and tissues much more clearly than without them. However, microbubbles can also be 'activated' by the right frequency of ultrasound from outside the body. This is somewhat similar to the way in which an opera singer might induce vibrations in a wine glass. By this method, energy can be transferred into the body to a site where microbubbles are present, a process that promotes drug uptake and physical stimulation. This has been used in cancer medicine to enhance delivery of chemotherapy to kill cancers. In this project we want to try to develop this method to see if we can deliver drugs to bone. Our vision is that in future a patient might visit a clinic, receive an injection of an ultrasound responsive agent, and subsequently receive ultrasound stimulation in their bone fracture to speed up bone healing. In recent work, we have found that we can detect microbubbles in human bone fractures and that we can make them resonate close to the bones of mice. This, combined with the work done in cancer medicine, gives us the confidence this idea might work. In the project we plan to find out when during human and mouse bone fractures that ultrasound responsive agents can be measured. To achieve this, we will do a small pilot study in patients who have had a bone fracture, and a controlled study in mice that have either a healing or non-healing bone defect. To do this we will inject and image or detect contrast agents at various stages using ultrasound imaging and detection. In parallel we will develop new formulations of ultrasound responsive agents, including microbubbles and their smaller cousins, nanodroplets, and do experiments in small 'acoustofluidic' devices containing mock bone fractures, or fractures created in real pieces of bone tissue to work out the right ultrasound and formulations to use. Finally, we will use information we learn from these 'in vitro' and 'ex vivo' models to test the idea that we can induce local delivery of molecules in real bone defects in experimental mice. Only by doing this work we will work out the right formulations and ultrasound methods to enable us to test this method as a way of delivering drugs in patients to help their bones heal faster and better. Our project involves close interaction with colleagues in the NHS, who are helping us run the clinical pilot study, and with a big healthcare device manufacturer, GE Healthcare, which will help us get this idea to the clinic as fast as possible.

Bone fractures are a major societal problem costing the UK economy more than £2 billion/year. This figure is predicted to increase markedly in the future as the average age of the population increases. A significant portion of this cost can be attributed to the 5-10% of bone fractures that fail to heal appropriately with current clinical interventions, leading to patients requiring major surgery and extensive rehabilitation. Hence there is an urgent need for new, minimally invasive and cost-effective treatments to be developed. The aim of the proposed research is to address this need by investigating the potential for targeted delivery of drugs that promote bone healing. This will be achieved using a combination of focused ultrasound applied externally to the body and drug-loaded nanodroplets (NDs) delivered by intravenous injection. NDs consist of particles (~200nm in diameter) of a volatile liquid that can be used to encapsulate a range of different types of drug. In preliminary work in a mouse model we have shown that upon exposure to ultrasound they undergo rapid expansion to form gas microbubbles, simultaneously releasing their drug payload and stimulating cell uptake. We have also demonstrated that NDs can be engineered to accumulate at bone fracture sites. These observations now provides the exciting possibility of controlling remotely the delivery of ND-loaded drugs at fracture sites. Our approach has the advantage of delivering molecules selectively to the injury site at the correct phase of healing and - importantly - also preserves the granulation and hematoma tissue, which are strong positive regulators of good fracture healing outcomes. Many molecules can have both positive and negative effects on fracture healing depending on the time and site of action, and so correct timing is fundamental to treatment efficacy. In this project, we plan firstly to build on our established ND chemistries to enable the delivery of proteins and small molecules known to be positive regulators of fracture healing in different temporal context, for example bone morphogenetic protein (BMP) and WNT protein. Building on our preliminary data, we will concurrently test what ultrasound parameters result in the optimal release, payload uptake and intracellular pathway activation, before assessing their osteogenic effects in cell culture, bioreactor culture and ex vivo systems of cell culture. In parallel, we will determine which ultrasound parameters are optimal to ensure molecule release and activation in vivo. Finally we will test whether optimised ND preparations can promote fracture healing in vivo using a combination of high resolution computed tomography, molecular and histological techniques. We have assembled a world-leading interdisciplinary team to conduct this research, comprising experts in ultrasound and drug release, bone repair, stem cell biology and nanoparticle chemistry. In addition, our research proposal has been developed in close collaboration with clinicians specialising in bone fracture treatment. We will also work closely with non-RCUK public sector stakeholders, Dstl, who have a strong interest in our technology as a means of better treatment of injured service personnel, and with commercial partners who will provide us with clinically approved materials and equipment. It is our aim that through these interactions, the outcomes of the work will have direct impact upon clinical practice and commercial uptake. Finally our results will also be of wide academic and applied relevance to other medical conditions for which control over timing and location of treatment delivery is important, for example, stroke and cardiovascular disease.

High blood pressure causes an increase in the size of the heart (cardiac hypertrophy) and is a major risk factor for the development of heart failure. One in five people die from this condition. Angiotensin II is a hormone that stimulates cardiac hypertrophy and it functions by binding to the Angiotensin II type I (ATI) receptor. A complex programme of intracellular signalling is initiated to stimulate hypertrophy and a new protein called ATRAP has been recently identified that protects against the effects of Angiotensin II. ATRAP was discovered because it binds to the ATI receptor but how ATRAP suppresses cardiac hypertrophy is not known. We have made an unexpected connection between ATRAP and a lipid binding protein, RdgB-beta. We propose to define the connection between the two proteins, ATRAP and RdgB-beta in the context of lipid signalling via enzymes called phospholipases that produce the 'signalling lipid', phosphatidic acid (PA). We will establish how this protein-lipid network operates during Angiotensin II signalling. The activity of phospholipases is stimulated when Angiotensin II binds to the receptor. RdgB-beta is uncharacterised and we have discovered that it has unusual lipid binding properties - it binds PA. Our concept is that RdgB-beta sequesters the 'PA' signal and therefore restrains the signalling cascade resulting in inhibition of cardiac hypertrophy. We will examine how RdgB-beta binds 'PA' and disposes of it. Because ATRAP binds RdgB-beta we think that a 'bridge' between two membranes is formed. This allows the 'PA' to be removed from the plasma membrane where signalling occurs and sent to the compartment where lipids are re-used for making new lipids. To form the bridge, RdgB-beta has to interact with ATRAP on one membrane and other proteins on the opposite membrane. We will therefore identify these proteins by using RdgB-beta as bait to fish for new proteins. We will also study the importance of RdgB-beta and ATRAP by increasing or decreasing the protein levels in the cells. This will inform us on how Angiotensin II signalling is affected. If RdgB-beta reinforces the restraint put by ATRAP on Angiotensin II signalling, this will provide strong evidence that the molecular mechanism used by ATRAP is to participate in the removal of the signalling lipid, PA. To further test the model, we will delete the gene for RdgB-beta in a model organism (Drosophila) and examine the phenotype in collaboration with our project partner in Bangalore, India. To determine the importance of PA binding to RdgB-beta, we will make mutant proteins that cannot bind PA. These mutants will be examined for rescue of the fly defect. The interaction between RdgB-beta and ATRAP together with the binding of PA to RdgB-beta could provide the molecular explanation of how ATRAP is able to suppress the function of Angiotensin II signalling and could therefore offer a novel therapeutic target for intervention in cardiovascular diseases. In the clinic, inhibition of Angiotensin II signalling by ACE inhibitors that prevents the production of Angiotensin II or drugs that prevent binding of Angiotensin II to its receptor are used for treatment for hypertension. Since most drugs have side-effects, drug combination that targets different systems are often used. Therefore the proposed research could well lead to a different molecular target which could provide a more effective treatment. Understanding how the endogenous inhibitor of Angiotensin II signalling, ATRAP, functions, may provide new strategies for drug targeting. Because ATRAP interacts with RdgB-beta, the possibility that targeting RdgB-beta may provide a unique opportunity to generate a new class of drugs that could be based on binding small hydrophobic molecules in the lipid binding pocket of RdgB-beta. The benefit derived from such drugs is huge as high blood pressure is one of the most common diseases that afflict humans.

The idea for this project came from a very simple observation. When healthy people have an infection, they feel ill. Almost everybody has experienced feeling terrible during a bout of flu, with symptoms such as tiredness and foggy thinking. This temporary effect on the brain is called sickness behaviour. However, when people with a brain disease have an infection, the symptoms of their brain disease flare up, sometimes dramatically. This is a quote from a person with multiple sclerosis (MS), a common brain disease: 'My water infection really knocked me out. My vision was affected for 16 hours, thankfully it has returned to normal now.' Why should a water infection - which is nothing directly to do with the brain - affect the brain in such a dramatic way? These flare-ups of symptoms can have a big impact on daily life, and do not have any specific treatment. This problem is 'hiding in plain sight' - it is very common and all doctors have seen it, but we do not know why it happens or what we can do to stop it. My theory is that the problem is inflammation. Inflammation is the body's response to infections. The immune system is activated to try and deal with the harm. However, sometimes inflammation itself can be harmful. There is a structure called the blood-brain barrier (BBB), which controls what substances from the blood can get into the brain. My theory is that inflammation in the body ('systemic inflammation') causes the BBB to become leaky. If the BBB is leaky, the brain's environment can change, leading to symptoms of the brain not working. I will be looking at a particular brain disease, MS. This is common, affecting over 100,000 people in the UK. MS causes symptoms such as fatigue, weakness, and problems with vision and balance. I will be looking at a particular infection - urinary tract infection (UTI or 'water infection'). This is very common in people with MS, and frequently leads to flare-ups, admission to hospital, and even death. I have an advanced brain scan which measures how leaky the BBB is. By scanning a person during a UTI, and scanning them when they are well, I can work out exactly how UTI affects the BBB. I will record the symptoms that people develop, to see if this relates to leaking of the BBB. I will do this in people with MS and also people with healthy brains, to see why it is that people with MS are more vulnerable. I have done a lot of work to prepare all of these techniques so I know I can finish this project within three years. This project is original and important for human health. We will know more about how the brain produces symptoms and how it responds to systemic inflammation. The lessons that we learn in MS will be relevant for other common brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease. We will also learn how the brain responds to inflammation in otherwise healthy people. For people with MS, understanding this is an important step in developing treatments to stop symptoms. If we discover that the BBB plays an important role, we can start to create treatments that strengthen the BBB. This project will have a number of other benefits. I will be testing a number of methods for detecting UTI, which can sometimes be missed. This will help work out the best strategy for detecting UTI. I will be improving the brain scan technique, which will be useful for other studies in MS and other diseases. I will be collecting blood samples, so that eventually we can find a blood test that checks on the BBB, without needing to do a scan at all.