Periodic Reporting for period 1 - ProDelivery (High Throughput Synthesis of Polymeric Vesicles for Protein Delivery) Reporting period: 2019-09-01 to 2021-08-31 Summary of the context and overall objectives of the project Although protein-based therapeutics have become increasingly prevalent in the clinic due to their high selectivity, their overall potency has been hampered by their instability in vivo. As a result, conventional drugs such as chemotherapeutics though generally possessing lower selectivity (and hence, more significant side effects), are still commonly employed as first line therapeutics. To improve the stability and hence potency of protein therapeutics, one approach is to develop nanoformulations encapsulating and protecting the sensitive therapeutics during delivery to the target site. The overall goal of ProDelivery has been to develop a chemical synthetic platform enabling the efficient synthesis of proteins encapsulated within polymeric nanoparticles known as polymersomes. A key aspect of the developed platform has been the optimisation of ultralow volume chemistry in order to allow for cost-efficient nanoformulation of expensive protein therapeutics. This enables greater chemical space to be explored for screening formulations and also allows a broader range of protein therapeutics to be readily accessed in the academic environment. Using the developed protein-loaded polymersome formulations, the proteins retain their activity after encapsulation, the polymersomes themselves are not cytotoxic over a broad range of concentrations and the polymersome membrane can protect the encapsulated cargo from thermal, proteolytic and intracellular stresses over prolonged periods of time. Overall, these results establish the developed chemical platform as a highly promising proof-of-concept for the stabilisation, screening and delivery of clinically relevant protein therapeutics. The multidisciplinary nature of this highly ambitious project was strongly supported by its localisation within the world-renowned Stevens Group at Imperial College London. The diverse nature of the project required input from a number of personnel within the Group with research backgrounds across chemistry, materials science, spectroscopy and cell biology and was crucial to meeting project outcomes. Ongoing collaborations which have been established as a result of this fellowship will continue to drive this work towards future applications. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The following summarizes the main research tasks and results from this fellowship so far:1. A novel strategy to downscale polymersome synthesis to the microlitre scale was optimised and developed. This overcomes some limitations of traditional polymersome forming strategies such as thin film rehydration which typically requires millilitre scale volumes. As a result, when loading proteins into the lumen of polymersomes, significantly lower quantities of expensive proteins are required and higher therapeutic loadings become economically feasible. We have also developed a range a complementary tools to characterise enzyme loaded polymersomes based on single-particle fluorescence and Raman spectroscopic techniques. 2. When loading a luminescence enzyme as a model protein therapeutic to form enzyme-loaded polymersomes, we have demonstrated significantly enhanced enzymatic resistance to physical and chemical stresses. This demonstrates the importance of nanoformulation of protein therapeutics in enhancing not only in vivo half-life (and hence, potency) but, also in improving the overall shelf-life and reducing the economic impact of cold-chain transportation for drug delivery applications. 3. We’ve demonstrated that optimised polymersome formulations loaded with model enzymes have no detectable cytotoxicity across a range of concentrations and can be efficiently taken up into a model cell line. Further, the enzymatic activity inside uptaken polymersomes can be retained for at least a week in culture demonstrating the excellent stability of the nanoformulation. These optimised polymersomes show excellent promise for the delivery of therapeutic enzymes whereby the mode of action is primarily catalytic. 4. For the release of protein-based payloads, we have also developed mechanisms to release encapsulated protein therapeutics. For example, we have developed polymersome formulations that can be disassembled in response to lowered pH conditions such as those commonly encountered in diseased tissue microenvironments. This disassembly of the nanoparticle results in release of the encapsulated therapeutics. We are currently exploring suitable in vitro models to continue this work beyond the end of this Marie Curie fellowship.The key concepts from this fellowship were presented at the Recent Appointees in Polymer Science (RAPS) conference in Leeds with a larger overview of the project to be presented at the upcoming ACS Spring 2022 meeting. Two publications are currently under preparation for submission to high impact publications which will report the crucial findings of this project. These publications will acknowledge all European Commission funding and will comply with EU open access policies. I have also participated in broader outreach activities such as Imperial College’s Great Exhibition Road Festival, to disseminate my research findings and general research interests to the general public. Although significant disruption to planned dissemination activities was incurred due to the COVID-19 pandemic, these will be greatly pursued as further opportunities become more available. Finally, the fundamental knowledge gained from this work has facilitated the training and project development of several research students and will result in further outcomes from this fellowship. 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) This fellowship has advanced the state-of-the-art across several areas in polymer chemistry and (bio)materials. One of the most significant advances has been in miniaturisation of polymersome synthesis which is a significant limitation of conventional methods based on thin film rehydration or solvent exchange. This has several benefits: i) lower quantities of expensive protein therapeutics are required for screening formulations allowing for more extensive optimisation, ii) a greater number of experiments can be performed allowing for structure-activity correlations and (iii) the amount of nanoformulated therapeutic can be tuned to more closely match the scale needed for basic biological screening, reducing waste of costly therapeutics. This advance therefore establishes a fundamental and versatile platform for greatly advancing the potency and clinical relevance of nanoformulated protein therapeutics. Secondly, I have used optimised formulations to demonstrate the long-term stability of these protein-loaded polymersomes which retain significant activity even when in the intracellular environment for more than a week. This is a promising finding for long-term enzyme replacement therapies but also for the storage and transportation of formulated protein therapeutics which are normally susceptible to degradation under ambient conditions reducing their effective shelf-life. This work therefore also has significant socio-economic implications for reducing the reliance of protein therapeutics on cold-chain transportation. Finally, we have developed chemical mechanisms to release encapsulated protein therapeutics allowing for therapeutics to be delivered to diseased tissues. This process will be combined with miniaturisation and high throughput approaches in the near future leading to a sophisticated screening platform for optimising the delivery of protein therapeutics.