Cancer is one of the most feared diseases. Radiotherapy is commonly used in about 50% of all cancer patients. Although the radiotherapeutic protocols have been improved significantly, only a limited number of patients are entirely cured and severe side effects are often induced. Hadrontherapy and protontherapy are approaches superior to conventional photon radiotherapy because of the maximal energy they deposit at the end of the track (the Bragg peak) and the absence of damage induced behind the tumor. However, the technique is limited by the damage caused along the beam path in healthy tissue located in front of the tumour. Dr. Lacombe's work focuses on improving the hadrontherapy performances using metal nanoparticles (NPs) as radiosensitizers. This is a highly innovative strategy aiming at increasing efficiency and tumour targeting of the treatments. So far, the group focused on the physical properties of the nanoparticles. The objective of my project is to characterize fully the biological effects induced by NPs in DNA and living cells, submitted to medical ion radiations. During the project I will address the cellular toxicity, localization, uptake and quantification of NPs, and I will specifically dissect the cellular response to the combined treatment of NPs and fast ions/protons down to the molecular pathways. To answer these questions, I will make use of the highly advanced experimental instruments and methods available at the host institution and collaborating laboratories. The results obtained during this project will be highly relevant not only for the research community, but also for the industrial sector that synthetizes the NPs and for the medical community who will be closely involved in the project. This multidisciplinary research at the interface of physics, chemistry and biology is unique in Europe and promises novel propositions for future cancer treatments.

Among the numerous classes of drug delivery systems, drug-loaded polymer nanoparticles have attracted much attention. Although this approach has led to numerous encouraging results and proofs of concept in vitro, important limitations still remain, which may explain the lower number of successful in vivo studies and the limited number of marketed nanomedicines. In this view, the current project aims at developing an innovative and versatile nanoparticulate platform to prepare well-defined amphiphilic polymer-drug 'prodrug' nanoassemblies by means of controlled/living radical polymerization (CLRP) techniques, such as nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP), or reversible addition-fragmentation chain transfer (RAFT). The new, facile and general strategy we propose relies on the controlled growth of biorelevant polymers (hydrophobic or hydrophilic) from functionalized drugs of the opposite solubility under CLRP conditions. This will result in well-defined amphiphilic species, bearing one drug molecule at the extremity of each polymer chain, able to self-assemble into nanoassemblies of various morphologies, exhibiting high drug payloads and thus high biological activities. This new methodology tends to be: (i) universal as it is virtually applicable to multiple drugs and consequently to multiple pathologies and (ii) versatile as various polymer prodrugs can be produced with high degree of control and flexibility over their structures (e.g., nature of the drug/polymer couple, nature of the linker, polymer chain length, etc.). This will be illustrated by the synthesis of a broad range of polymer prodrugs from a wide selection of drugs having demonstrated activities against cancer. Comprehensive characterization of the resulting nanoassemblies will be performed as well as their pharmacological evaluation in vitro (cell culture) and in vivo on relevant models, according to standardized protocols.

Angiogenesis a major contributor to tumor development and metastases. Formation of new blood vessels supports tumor proliferation and disease progression and is often associated with poor clinical prognosis. Recent advances targeting angiogenesis and inhibition of tumor neovascularization has led to the approval of several new antiangiogenic drugs for clinical use in many types of cancers. Yet despite promising potential, the efficacy of these treatments has been relatively limited. Additionally, as with chemotherapeutics, over time these therapies are associated with tumor resistance and escape. Antiangiogenic therapy (target-specific or broad spectrum) aims to eradicate the tumor by damaging its blood supply; as a result tumor tissue becomes hypoxic and, consequently, necrotic. By current clinical measure, this tissue death is considered a positive outcome; however it is recently discovered that the necrotic tissue in turn releases signals contributing to inflammation and angiogenesis, eventually initiating aggressive revascularization, overriding the foreseen beneficial effects of the antiangiogenic drug. For this grant, we propose to design a novel drug-delivery system based on multifunctional polymer nanomicelles which combine two small molecule drugs: one a potent antiangiogenic drug, and second which is an antagonist of these necrotic signals, combating the feed-back loop which can undermine the positive effects of therapy. Using this innovative approach, we intend to significantly improve cancer treatment by minimizing resistance to antiangiogenic drugs. This technology is based on our previous development of the PEG-PLA polymer conjugate of a small molecule angiostatic compound, TNP-470, which form nanomicelle with improved pharmacological properties compared with the free drug. By combining a second drug with distinct mechanism of action we will provide an important, clinically relevant tool, and a future platform for antiangiogenic or other drugs.

Our ability to tailor individual proteins is now sophisticated, but our ability to assemble such proteins into larger structures is still primitive. Proteins are typically joined by reversible or non-specific linkages. We have designed a unique way to connect protein building blocks irreversibly and precisely, via spontaneous isopeptide bond formation. This involves modifying proteins with a short peptide tag (SpyTag) that is based upon remarkable chemistry used by pathogenic Gram-positive bacteria. Here we will develop this novel approach to address major challenges in synthetic biology. We will engineer SpyTag capture towards infinite affinity (defined as diffusion-limited on-rate and no off-rate), to transform the sensitivity of peptide detection in living systems. We will also apply SpyTag to create a new generation of protein polymers, irreversibly assembled with molecular precision and tailored branching. In parallel we will harness SpyTag to enhance circulating tumor cell (CTC) capture, one of the most promising ways to achieve early cancer diagnosis. In capturing CTCs and other rare cells from blood, the high forces mean that even the strongest non-covalent linkages fail. SpyTag covalent bridging, in concert with super-resolution live cell fluorescence microscopy, will give us the opportunity to answer key questions about the forces and membrane dynamics at the magnetic bead:cell synapse. We will exploit these insights and SpyTag-assembled antibody polymers to dramatically reduce the threshold of antigen expression for CTC capture. This comprehensive program of research will explore novel concepts in protein recognition and cellular response to force, while creating conceptually new tools, making it possible for biologists in a wide range of areas to step beyond existing barriers.

The main goal of this proposal is to induce and detect in real time and at single-cell level the differentiation of human mesenchymal stem cells using mechanical loads in 2D and 3D conditions. This proposal integrates disciplines from nanotechnology, bioengineering and cell and molecular biology. First we propose to develop a method to track the differentiation of stem cells in real time and at the single cell level using cytoskeletal organization of actin, microtubules and intermediate filaments as a suitable cell biomarker. We will then establish mechanical loading protocols to induce, via direct force application onto cells, the first stages of stem cell differentiation towards specific cell lineages. We will apply cyclic tensile strain and compression to stem cells in 2D and 3D conditions, and track their differentiation status in real time using the cytoskeletal biomarkers that we will have identified before. The results of this proposal will have implications for the field of stem cell mechanobiology in particular, and some of the techniques developed will also contribute to the wider field of directed stem cell differentiation.

Recent advances made in the field of crystallization-driven self-assembly (CDSA) of block copolymers (BCPs) with a crystallisable core-forming block in selective solvents have opened up exciting opportunities in the creation of well-defined nanostructures such as monodisperse cylinders with precisely controlled length. Herein, we propose to study linear-dendritic BCPs and to obtain new, well-defined materials with the dimensional precision provided by CDSA and also higher orders of complexity arising from surface functionalization with dendrimers. The overall objectives of this proposal are two-fold. First, to combine well-defined dendrons with crystallizable linear blocks such as metal-containing polyferrocenylsilane (PFS) and crystalline biodegradable organic blocks such as polycaprolactone (PCL) and polylactide (PLA) to yield linear-dendritic BCPs to further advance fundamental knowledge by studying their self-assembly behavior. Second, by combining CDSA and dendrimer science we intend to take a significant step toward the creation of precisely surface-engineered materials for potential applications in nanomedicine. The proposed research objectives will be accomplished by bringing a highly talented researcher, Dr. Nazemi, from Canada with his extensive experience in dendrimer synthesis and bionanomaterials to stay for 2 years in one of Europe's highest ranked research laboratories, that of Professor Ian Manners at Bristol. This group is recognised as being among the world leaders in the fields of metallopolymers, BCP self-assembly, and in particular the use of CDSA. At the end of the 2 year stay Dr. Nazemi wishes to return to Canada to take up an academic position at a research-intensive University.

The aim is to produce a platform technology that combines the price-point and ease-of–use of lateral flow immunoassays with high-sensitivity, quantitative measurements currently requiring capital intensive equipment. This could ultimately allow its use as a consumer-technology, to diagnose and monitor health. e-Gnosis is an interdisciplinary project combining the current state-of-the-art in recessed ring-disk nanoelectrode arrays with a novel signal amplification strategy in the form of ferrocene loaded pH sensitive polymer nanobeads. e-Gnosis will combine these technologies to produce a multiplexed biosensor chip capable of running several ultra-high sensitivity immunoassays without the need for complex or expensive read-out equipment. The use of nanostructured electrode arrays allows the local pH to be changed electrochemically, such that the pH sensitive beads bound to the analyte dissolve and release ferrocene, leading to a large, proportional signal amplification of each binding event. Amplification can be triggered without external reagent addition and, importantly, without necessitating a wash step to remove unbound labels (the rapid initial current peak is bound to originate from within the nanostructures). Performance characteristics will include low sample volume requirement, sensitivity, limit of detection, robustness to interferrents, dynamic range and speed to result. Fabrication is based on highly parallel semiconductor fabrication technology and standard methods for antibody immobilisation, allowing low cost production. The e-Gnosis platform can also be used for other affinity based recognition events. Apart from the potential commercial and societal benefits of such a sensing technology, the project will also produce two enabling methods within nanofabrication and surface functionalisation. The project feeds into key areas identified by EU and industry stakeholders within ETP Nanomedicine and Horizon 2020, amongst others.

Drug delivery transport across the cell membrane involves many biological processes. It is complex and dynamic in nature. In this regard, model lipid membranes which mimic many aspects of cell membrane lipids, are a very useful membrane model. It has been shown that physical characteristics of drug delivery nanoparticles such as polarity and surface charge can significantly influence their interactions with lipids. A clear understanding of the interactions of lipids with drug delivery systems and their transport mechanisms is required to enable the design of an efficient and biologically compatible drug delivery vehicle. The aim of this project is to focus on the physico-chemical understanding of the relationships between the structure of the nanoparticles and their penetration and efficiency as a drug delivery vehicle. In this research, we wish to clarify the mechanisms involved in the interactions between nanogels and dermal membranes and find out how these are influenced by physico-chemical conditions and the morphology of the nanogels. The outcome of this research would aid the design of the next generation of dermal delivery systems and also contribute to the risk assessment of skin exposure to nanoparticles. The scientific challenges to be investigated in this project will focus on the understanding of the molecular mechanisms of interaction between organic nanomaterials and biological barriers, in particular skin. The project will benefit from the combination of high level experimental facilities available in the Host Institute and the longstanding expertise of the group in the development of novel functional nanomaterials for drug delivery applications as well as experience in the area of understanding the biological processes and potential drug targets (by means of e.g. neutron scattering and reflectivity) with the knowledge and experience acquired by the applicant on nanoparticle synthesis, their physico-chemical characterization and interactions.

Supramolecular assemblies -formed by the self-assembly of hundreds of protein subunits -are part of bacterial nanomachines involved in key cellular processes. Important examples in pathogenic bacteria are pili and type 3 secretion systems (T3SS) that mediate adhesion to host cells and injection of virulence proteins. Structure determination at atomic resolution of such assemblies by standard techniques such as X-ray crystallography or solution NMR is severely limited: Intact T3SSs or pili cannot be crystallized and are also inherently insoluble. Cryo-electron microscopy techniques have recently made it possible to obtain low- and medium-resolution models, but atomic details have not been accessible at the resolution obtained in these studies, leading sometimes to inaccurate models. I propose to use solid-state NMR (ssNMR) to fill this knowledge-gap. I could recently show that ssNMR on in vitro preparations of Salmonella T3SS needles constitutes a powerful approach to study the structure of this virulence factor. Our integrated approach also included results from electron microscopy and modeling as well as in vivo assays (Loquet et al., Nature 2012). This is the foundation of this application. I propose to extend ssNMR methodology to tackle the structures of even larger or more complex homo-oligomeric assemblies with up to 200 residues per monomeric subunit. We will apply such techniques to address the currently unknown 3D structures of type I pili and cytoskeletal bactofilin filaments. Furthermore, I want to develop strategies to directly study assemblies in a native-like setting. As a first application, I will study the 3D structure of T3SS needles when they are complemented with intact T3SSs purified from Salmonella or Shigella. The ultimate goal of this proposal is to establish ssNMR as a generally applicable method that allows solving the currently unknown structures of bacterial supramolecular assemblies at atomic resolution.

One-atom thin two-dimensional nanomaterials possess unique properties different from their bulk counterparts. Initiated by the discovery of graphene, many stable one atom-thick layers such as boron nitride, molybdenum disulphide, tungsten disulphide etc., have been isolated and characterized. However, the individual properties of such 2D-atomic crystals (except graphene) were modest. The combination of isolated single atomic layers into designer structures, named as 2D-heterostrcutures, is predicted to give synergetic properties. In order to harness the interesting properties the combination of various 2D-atomic crystals have to offer, a method to assemble them in a simple and scalable way is required. Currently, the only method known is manual placing of the 2D-atomic crystal layers sequentially which limits the scope of the study of such structures. The objective of the proposal is to assemble layered (each layer is one atom thick) stacks of graphene superlattices and heterostructures with other 2D-atomic crystals such as BN, MoS2, WS2 etc., by deoxyribonucleic acid (DNA)-mediated assembly. DNA mediated assembly is highly programmable by chemically specific interaction between nucleotides, length of the DNA, strength of the interactions in addition to the symmetry control of the assembled structures. Top-down lithography will be combined with bottom-up DNA assembly to fabricate seed layers of DNA for the guided assembly which lead to patterned heterostructures. This approach is targeted toward combinatorial screening of exotic properties of varied architectures of heterostructures with control over the composition of 2D-atomic crystals and spacing between the layers (controlled by DNA). The anticipated structures would be vertical atomic scale Legos of 2D-atomic crystal layers with DNA spacers.