Periodic Reporting for period 4 - CRYOMAT (Antifreeze GlycoProtein Mimetic Polymers) Reporting period: 2019-12-01 to 2020-05-31 Summary of the context and overall objectives of the project The overall aim of this ambitious ERC funded project was to develop new synthetic materials which mimic Antifreeze (Glyco)proteins (AF(G)Ps) also known as ice-binding proteins. Antifreeze proteins are produced by extremophile organisms, including polar fish species, to help them survive in sub-zero habitats. The native antifreeze proteins are, however, not easy to synthesise on scale and hence we set out to develop synthetic polymers which can reproduce the function of antifreeze proteins, without needing to ‘look like them’. The ability to obtain large quantities of these polymers will help find new applications for antifreeze protein inspired materials from controlling ice growth in crucial infrastructure, or in transport, and in particular to help the cryopreservation (freezing) of donated cells and tissues which are used in regenerative medicine. Furthermore, by using synthetic mimics we aim to be able to try to understand how these fascinating proteins work – put simply they can ‘see the difference between ice and water’ which is an incredibly complex biomolecular recognition challenge. When one considers the huge impact cryopreservation has on society, this research is of upmost importance. Especially for emerging ‘biologics’; medicines based on proteins or cells which are transforming medicine. These must be delivered to the patient intact and hence new methods to protect biologics from cold stress (during frozen transport) will have long term impact . This project set out to develop macromolecular (polymer) cryoprotectants, understand their mode of action and show their potential in cryopreservation. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far All aims and objectives of the project were met with work in each Workpackage completed. This has included nearly 50 publications and 3 patents filing as well as a major review article introducing this new field, which was published in Nature Communications. We have made exceptional progress in Workpackages A and B which involved the design, synthesis and biophysical testing of antifreeze glycoprotein mimetic materials. In particular we have made huge strides into understanding our model polymer – poly(vinyl alcohol) (PVA) and its interaction with ice. We have developed new architectures of PVA including block copolymers and star-shaped polymers and demonstrated how the ice growth activity is not affected by these changes, with all activity being retained. We have developed new synthetic methods to allow us to access degradable polymers based on PVA, which being based entirely on a carbon backbone is normally stable in solution. Working with colleagues at Warwick, we have also employed new analytical tools including wide-angle X-ray scattering and solid-state NMR to advance our understanding of how antifreeze proteins, and polymer mimics, interact with ice. We were also able to validate our findings (biophysical and cell biology, below) against native antifreeze proteins in each case. We have also investigated a new class of ice growth inhibitors based on poly(ampholytes) – polymers with positive and negative charged groups. Using a synthetic strategy to give alternating polymers, we were able to identify the key structural motifs needed for activity, including the role of hydrophobic groups. As part of an unplanned collaboration with partners at Warwick, we have also developed a new class of ice growth inhibitors based on self-assembled optically pure metallo-helicies and used these to test theories about the importance of ice-binding, verses amphiphilicity, in inhibiting ice growth. Finally, we introduced polyproline as a moderately active ice growth inhibiting polymer and linked its secondary structure to the ice growth properties. Workpackage C aimed to evaluate cryopreservation methodologies. In this we have made excellent progress and by using various cell-based models we have shown how we can increase the number of recovery cells post-thaw by modulating ice growth with our polymers. A range of cell types covering mammalian cells, blood cells, suspension and monolayer as well as bacteria cells have all been investigated and the cryoprotective benefits determined. In the next period we will investigate this further, using the panel of new polymers which we have synthesised. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Before this work, nearly all cryoprotectants were ‘small molecule’ solvents. This ERC project has successfully established the concept of macromolecular cryoprotectants and is an emerging theme of research globally, with several other research groups engaging with and moving into this field. Therefore, we anticipate significant growth in this field in the coming years, bringing exciting new discoveries, which is a key output of this project. One important advance was our demonstration that hydroxyl groups (alcohols) are not essential motifs for an ice-inhibiting compound. It had been speculated that these were essential to hydrogen bond with a growing ice crystal plane. However, we have introduced two diverse sets of materials which have no hydroxyl groups at all, and in fact no obvious ice-binding face. However, these are still very active. Self-assembled metallo-helicies were shown to be remarkably potent and their ‘patchy’ surface was linked to their activity. Secondly, polyproline was shown to have moderate ice growth activity but to be very potent in enhancing the cryopreservation of nucleated cell monolayers. These two key results demonstrate the un-tapped chemical space for new ice growth inhibitors and their exciting potential to advance biomedicine through improved cell storage.The second major breakthrough, which has also raised more scientific questions, is how the extent of ice recrystallisation inhibition activity scales with observed cryopreservation outcomes. Whilst this property is clearly beneficial, the exact method of cryopreservation is important – cells in suspension respond differently to those in monolayers and the use of non-IRI active macromolecular cryoprotectants, which appear to stabilise membranes, can give exception enhancements in cryopreservation outcomes. The affect of IRI on bacterial and protein cryopreservation was also (perhaps not unexpectedly) very different to that of mammalian cells. As this is an emerging field there remains many more questions. We have written review articles to try to summarise recent advances in the field, covering the aspects of polymer mimics of antifreeze proteins, polymers as macromolecular cryoprotectants and a critical comparison of ‘how active’ a large range of new and existing materials are. We fully expect to update these in the coming years, building on our own and others results. Summary of polymer mimetic approach taken
