Periodic Reporting for period 1 - USOME (Advanced Analysis of Polymersome and Exosome Hybrids for Potential Application in Therapeutics and Diagnostics) Reporting period: 2020-10-15 to 2022-10-14 Summary of the context and overall objectives of the project Synthetic and biosynthetic cargo-carrying vesicles are emerging as promising platforms for targeted drug delivery and diagnostics. However, they present significant variations regarding size, composition, stability and structure. Bearing that in mind, the USOME action was tasked with researching and developing: (1) advanced engineering strategies for synthetic and biosynthetic vesicles, and (2) robust analytical fractionation techniques for elucidating information on the corresponding size, structure, stability and composition. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far WP1. Polymersome Fabrication: Non-immunogenic and biocompatible vesicles called polymersomes were fabricated from synthetic amphiphilic block copolymers (ABCs) to function as artificial organelles. The ABCs were composed of a PEGylated and water-soluble segment conjoined with a water insoluble segment consisting of pH- and photo-sensitive functionalities. The pH sensitivity of the ABC was engineered to promote solubility in water at low pH, and insolubility at higher pH. Therefore, at higher pH the ABC chains undergoes a reversible phase separation and self-assembles into polymersomes. Subjecting the polymersomes to UV irradiation prompts the crosslinking of the photo-sensitive segment, which makes the self-assembly irreversible when subjected to any further pH changes, thus stabilizing the polymersomes. WP2. Biomolecule Release: Polymersomes were designed and fabricated to function as artificial organelles for performing the encapsulation and programmable release of enzymes and proteins under specific physiological conditions. To this end, it was necessary to determine the optimum protein/ enzyme size for sufficient and efficient encapsulation and release processes under various physiological conditions. At optimum protein/ enzyme size, it was necessary to define the permeability of the polymersomes membrane as a function of the membrane's thickness and composition for both deuterated and non-deuterated analogues. Overall, polymersomes as synthetic vesicles are attributed to better mechanical and chemical stability relative to biological alternatives. WP3. Fabrication of Exosome-DNA-Polymer Hybrid: As complimentary alternatives to artificial organelles, biologically produced vesicles called exosomes were surface-functionalized and stabilized with polymers to circumvent two major shortfalls associated with biological vesicles, namely, (1) naturally occurring biodegradation, which causes stability problems and (2) high rates of metabolic processing by the body, which results in short blood circulation life-time that is insufficient for adequate targeting of therapeutic sites. Overall, the two highlighted shortfalls have hindered the successful application exosomes as drug delivery nanocarriers, even though exosomes have shown better specificity in functionality relative to polymersomes. WP4. Advanced Characterization: Subject to analysis were the size, aggregation number, encapsulation quantification, degree of hybridization, morphology, shape, scaling properties, compactness, and stability of the novel synthetic and biosynthetic vesicles. These synthetic and biosynthetic vesicles have complex heterogeneity in their molecular properties, and therefore, separation techniques are prerequisite for accurate and reliable characterizations of their molecular properties and the distributions thereof. Moreso, these vesicles are fragile and delicate, and any one of the stipulated molecular parameter is only measurable with channel-based separation techniques such as multidetector FFF, as employed in the USOME action. For visual analysis of microstructure cryo-TEM was utilized. The functionalization and modification to the nanocarriers induced subtle changes in microstructure, which are not detectible with FFF. As such, SANS was employed as a robust and complementary analytical strategy, owing to its better sensitivity to size dynamics. 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) Understanding research excellence: Through the CMU partnership and internal collaborations at IPF, Dr. Muza was trained on the synthesis of complex polymers. Dr. Muza travelled to the USA for hands-on training at CMU, under the stewardship of world-renowned synthesis expert, Prof Krzysztof Matyjaszewski. Dr. Dietmar Appelhans and Dr. Silvia Moreno were the experts at IPF responsible for training Dr. Muza on the engineering and fabrication of artificial organelles using stimuli responsive polymers. Knowledge on advanced synthesis was necessary to proffer a balanced technical understanding of polymer chemistry for Dr. Muza. At ILL Dr. Muza was trained to perform his own SANS experiments and data interpretations. Extend network and new collaboration skills: Notably, the universal applicability of the analytical methods researched and developed in the USOME action allowed for Dr. Muza to initiate and foster new collaborations at IPF. Thus far, one article has been published, and a manuscript has been submitted in collaboration with the research group of Dr. Pospiech at IPF. Dr. Muza was actively involved in the design of the official FFF website and the FFF2022 announcements, and in organizing the inaugural FFF Virtual Symposium 2021, and Dr. Muza, has been supporting Prof. Lederer with setting up a vibrate multimedia department responsible for ensuring effective interactions during ISPAC 2023. Improved leadership and mentoring skills: Dr. Muza spearheaded the inaugural FFF Outreach Program in Africa. In addition, Dr. Muza spent about two months as a visiting academic at Stellenbosch University for mentoring and training students in FFF. Dr. Muza is also part of the pioneering group from IPF who are spearheading a Science Outreach Program for mentoring high school students. Subsequently, Dr. Muza intends to setup a similar science outreach projects for communities in Africa. Improved Science Communication: Dr. Muza successfully applied for a grant for SANS measurements at ILL, and is currently working on a M-ERA.NET 2023 research proposal as a coordinator. New management skills: Dr. Muza was responsible for the main aspects of managing the USOME project, such as budget allocation and prioritization. Furthermore, in the Polymer Separation Group at IPF, Dr. Muza successfully supervised a Master’s student from 2021-2022. Moreso, Dr. Muza was also responsible for supervising the research internship two Master’s students. Overall, being a Postdoctoral allowed Dr. Muza to mentor and support seven Masters and PhD students. Enhanced teaching skills: From 2020 to date, Dr. Muza has been a lecturer in Polymer Physics at TU-Dresden, thus gaining two years’ experience working at a world-class university. New decision-making and problem-solving skills: Dr. Muza successfully managed the USOME project, and a lot of critical decisions such as prioritizing tasks and allocating time and resources and strategies went into play. 4 Published articles; 2 Manuscripts; 1 Press release; Outreach activities; Website and Social media channels; Conferences and Conference awards; Near future conferences: SCM10; ISPAC2023. Related documents LogoUsome_fin.jpg
