The objective of this project is to establish an integrated Infrastructure that will cater to a starting community of multi-disciplinary chemistry users in need of cutting-edge instrumentation and experimental expertise in solid-state nuclear magnetic resonance (NMR). We aim at applying to the call INFRAIA-02-2020: Integrating Activities for Starting Communities. Chemistry is at the heart of the European economy and the wider Chemical Industry is one of the largest manufacturing sectors. As an ‘enabling industry’, Chemistry plays therefore a pivotal role in providing innovative materials and technological solutions to support Europe's industrial competitiveness. The chemical industry alone, including basic chemistry and pharmaceuticals, contributes to more than 15% of European industrial production (http://ec.europa.eu/growth/sectors/index_en.htm). Development of modern chemistry relies on the capacity for atomic level investigation of increasingly complex solid substrates in frontier research areas crossing disciplines from catalysis and energy materials through polymers to pharmaceutical formulations and medical implants. Among the range of physical and analytical techniques used for atomic level characterisation, breakthroughs have been made since the beginning of the century in the development and use of solid-state NMR spectroscopy that now do indeed enable the characterisation of structure and dynamics at the atomic-level, and can reveal morphology in solids. Such state-of-the-art methods rely on the use of sophisticated and costly solid-state NMR instrumentation that is only available in a handful of national facilities. The rarity of the instrumentation and associated operational know-how has restricted the uptake of these enabling methods by the broader base. This project aims at providing the European chemistry community, both academic and industrial, effective and convenient access to the best research infrastructures (RIs) in solid-state NMR available worldwide, ensuring their optimal use and joint development. The French partner, who will coordinate the project is composed by two leading groups in NMR spectroscopy, the CRMN Lyon (Centre RMN à Très Haut Champs) and the CEMHTI Orléans (Conditions Extrêmes et Matériaux : Haute Température et Irradiation) that will operate jointly as a single-entry-point facility (named LOFT). These two sites are part of the French Research Infrastructure for high-field NMR (IR-RMN). The other partners are spread across Europe (Switzerland, United Kingdom, the Netherlands, Denmark and Italy), and one is located in Florida, USA. The seven RIs will provide users with access more than 30 NMR spectrometers with proton frequencies ranging from 100 to 1500 MHz. This will advance knowledge and foster innovation by helping to structure a new scientific community of chemistry users in need of cutting-edge instrumentation and experiments in solid-state magnetic resonance. The constitution of this user community around the infrastructures will allow us to educate a new generation of cross disciplinary chemists able to optimally exploit advanced NMR methods for their research. The partner infrastructures currently have only a limited degree of coordination and networking, and there is no integration at the European level. A key aim is to achieve integration by substantially developing networking in order to implement standard operating procedures and to establish a common single-entry procedure for trans-national access provision, thereby harmonizing, optimizing and improving access procedures and interfaces. Joint research activities will improve the quality and quantity of the access to the infrastructures, and facilitate the use of modern solid-state NMR by non-expert users, widening the opportunities for novel application areas in chemistry.

Many devices of interest to sustainable chemistry and climate change have functions that depend on the underlying materials, ranging from the average long-range structure, down to more local environments of specific atoms and ions, whether the structure is ordered/disordered, and whether it is dynamic. Of particular interest are paramagnetic materials, since these lend many unique properties due to the unpaired electrons of their paramagnetic metal ions. The characterization of the structural environments of these metal ions is key to understanding the functions and limitations of these materials. Whilst some structural information is provided by X-ray, and neutron diffraction, and electron microscopy techniques, these methods often fail to appreciate the complexity of the all-important local structure, how this local structure varies throughout the material, and thus how these important features affect performance of the corresponding devices. Solid-state paramagnetic nuclear magnetic resonance (pNMR) is a key method for understanding this atomic-level structure, but the unpaired electrons result in broad, low intensity signals that are very difficult to excite, resolve, and interpret using standard NMR methods. We will develop new pNMR and computational methods for paramagnetic materials, on three themes. Firstly, we will develop new NMR methods for the broadband excitation and resolution of NMR signals from quadrupolar nuclei close to paramagnetic metal ions. We will also test new density-functional theory (DFT) protocols to enable unambiguous assignment. Secondly, we will push the boundaries of dynamic nuclear polarization (DNP) to the intrinsic metal ions to enhance the pNMR sensitivity in the immediate vicinity of the metal ions, and in parallel reduce shift dispersion by perturbing the measured paramagnetic shifts. Thirdly, we will take pNMR from the atomic to the nano-scale and develop a protocol based on bulk magnetostatics to characterize the distributions of shapes/sizes of paramagnetic nano/micro particles, and lengthscales of the surface layers deposited on these particles. The methods developed around these three themes will then be put into action enabling us to fourthly, solve the complete local and global structure of three materials with important applications in sustainable energy, and to link these structures to the performances of the corresponding devices.

In this project I will develop new spectroscopy approaches that will allow one to obtain unprecedented structural information of drug molecules throughout the whole pharmaceutical process. High resolution structure determination ideally of unmodified drugs (i) in their free form, but also (ii) in complex dosage formulations, as well as (iii) during their delivery and target engagement in cells represents one of today’s major challenges in pharmaceutical industry, key to ensure productive drug uptake and improved efficacy. Current routine characterisation methods (such as detection of drugs in cells) often require sample modification (e.g. tagging with fluorescent labels) which may alter the behaviour of the drug. Here I will make use of the fact that a increasingly growing percentage (currently about 30%) of Active Pharmaceutical Ingredients (API) contain at least one fluorine atom, while hardly any excipients and no endogenous biomolecules in human cells do. By implementing innovative 19F solid state Nuclear Magnetic Resonance (NMR) approaches under fast Magic-Angle Spinning (MAS) and Dynamic Nuclear Polarisation (DNP) techniques, I will develop a new analytical tool with enhanced resolution and sensitivity, which will allow one to overcome the above mentioned challenges and to obtain structural information of unmodified drug molecules in complex and diverse formulation, in vitro and in cellular environments, as well as their interactions with various substrates (excipients, biological targets). The 19F NMR observables will be correlated with those of other nuclei (1H, 13C and 15N at natural abundance), using multidimensional 19F detected methods to distinguish between the drug molecule and excipients or cell background. The methods will be benchmarked on a variety of pharmaceutically relevant molecules and will concern both their pure constituents and their delivery systems.

Lipid nanoparticles (LNPs) are a newly emerging type of drug formulation that encapsulates biological molecules such as nucleic acids and proteins. They have recently emerged as effective vehicles for mRNA vaccines. The performance, stability and delivery properties of these particles crucially depend on their architecture. However, due their complexity, their molecular-scale organization has so far escaped from full characterization. Several important structural features, such as the interactions between their many components, the environment of the nucleic acid cargo, its hydration or the lipid distribution, remain elusive, preventing a rational design of improved formulations. Atomic force microscopy (AFM) imaging, cryogenic transmission electron microscopy (cryo-TEM) as well as small-angle X-ray scattering (SAXS) can in principle be applied to image the individual LNPs, their size and shape, and internal fine structure. Yet, none of these techniques provides molecular-scale insight into the LNP structure nor quantitative compound-specific information. Our project aims at developing innovative analytical approaches to examine the internal atomic, molecular and nanoscale structure of mRNA-loaded LNPs. Leveraging recent instrumental and methodological breakthroughs in DNP (Dynamic Nuclear Polarisation) and ultra-fast Magic Angle Spinning (MAS) NMR (Nuclear MAgnetic Resonance), the first objective of the LNP-HiRes project is to implement high-sensitivity solid-state NMR approaches to probe the internal architecture of LNPs. Notably we aim at identifying the location of the mRNA cargo, probing its hydration and secondary structural elements as well as its interactions with other LNP components. The development of advanced fluorescence spectroscopy and imaging techniques specifically tailored to LNPs is the second objective. This will include the design of fluorescent probes that will locate in specific LNP environments as well as the development of single particle imaging techniques. The methods will be benchmarked on LNPs of pharmaceutically relevant compositions as well as on innovative formulations, not yet in use in industry, prepared by appropriate microfluidic protocols. The preparation of customized mRNA strands that will incorporate isotopically (15N,13C or 2H) labelled or chemically-modified fluorescent nucleotides will be essential to reach these two first objectives. Improving the current understanding of LNP structure and refining existing models by analyzing jointly the NMR and fluorescence data is the final objective of the project. Links between LNP composition and structural features will be established and put in perspective, whenever possible, with differential biological readouts. Our project is expected to create new basic knowledge and provide advances beyond current state-of-the-art with the implementation of innovative biochemical strategies for the preparation of customized NMR- and fluorescence-active mRNA strands as well as the introduction of pioneering solid-state NMR and fluorescence methodologies, tailored for the in-depth investigation of multicomponent nano-objects. Unique structural insights into LNPs at the atomic, molecular and nanometer scales will be provided. LNPs represent one of the most widely used and promising platforms for the mRNA therapeutics. The project results are expected to lead to an improved understanding of their organization, stability and (physicochemical and biological) properties, fostering further innovation in the field of mRNA vaccines.

Aluminum-hydroxide-based adjuvants can absorb protein antigens from an aqueous solution and it is these adjuvants, as well as those based on aluminum phosphate, that are now widely used in vaccine formulation because of their immunostimulant behavior. The limited data in the literature show that the immune response of a vaccine formulation is related to (i) the structure of the adjuvant, (ii) the surface properties and (iii) the nature of the interaction with antigens and highlight the lack of thorough understanding of the molecular forces driving absorption/release. The objective of this project is to characterize protein/adjuvant interactions at a fundamental level using a model antigen and implementing state-of-the-art solid-state NMR approaches to describe the protein-adjuvant interface with atomic resolution.