Design of nanofibre based adsorbent for extracellular vesicle isolation

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BackgroundExtracellular vesicles (EVs) are nature's vectors to shuttle information in multicellular environments. Relatively recently attention has focused on the therapeutic potential of these entities. This has been driven by increased understanding of their biological importance and the success of other vector strategies such as viral and non-viral vectors (most notably lipid nanoparticles (LNPs)).EVs (also referred to as exosomes) are under active development in native and engineered forms. In both cases understanding of the product characteristics is challenging. EVs are complex cellular products consisting of a lipid envelope with a range of embedded proteins and carrying a cargo within. A single cell can make vesicles by multiple pathways, the one of greatest interest is the endosomal pathway (which gives rise to exosomes), but even from in one cell a population of vesicles will be produced. Understanding this heterogeneity is an unmet need.Therefore characterisation methods have to deal with a huge range of diversity alongside the complexity and labile nature of EVs.AimIn this project we will investigate the use of nanofibre based adsorption as a means to separate EV populations for use in the characterisation and manufacture of these nascent products. Current approaches used to isolate and enrich for exosomes do not produce high functional yields of EVs. They also do not differentiate the different sizes of EVs and the different types as well, e.g., exosomes, exomeres, ectosomes, microvesicles, microparticles, apoptotic bodies. This programme of research involves development of a novel method for isolation of exosomes that can address those issues by applying a specialised, high porosity material modified with affinity ligands at a high, medium, and low density in an ion exchange chromatography system. The material causes low shear so issues with high shear from other separation methods, e.g., ultracentrifugation, that damage exosomes and compromise their function will be avoided.Objectives1) Establish a model EV upstream process and associated analytics such that product mass and quality using a relevant bioassay can be reliably determined.2) Determine mechanisms of EV yield loss during bind elute IEX chromatography by understanding:a. The kinetics of loss during processing.b. The manipulation of EV structure / population to determine the EV components associated with irreversible binding.3) Determine mechanisms of product quality loss during bind elute chromatography by:a. Correlating differing purification methodologies with product loss.b. Correlating differing feed materials / EV population with product loss.c. Rational manipulation of purification conditions based on the above.