Synthesis and Advanced Characterisation of Cancer-Derived Exosome Mimetics: Novel Vectors for Targeted Cancer Therapy

Abstract

Biotechnology has reshaped modern medicine through the development of biopharmaceuticals, including monoclonal antibodies, viral vectors, and extracellular vesicles. Despite this progress, viral vectors and vesicle-based systems are still in the early stages of clinical use, with limited analytical methods and underdeveloped quality control. This project addresses these challenges by applying fluorescence-based tools to study stability and dynamics, while also developing practical approaches for fabricating and characterising drug delivery vesicles.
The first part of this work applies the Red Edge Excitation Shift (REES) method, a Tryptophan-based fluorescence technique, to viral vectors. This novel approach can track conformational changes in protein structure using Tryptophan as the fluorophore. REES was previously used for antibodies but is extended here to intact viruses, including adeno-associated viruses and bacteriophages. REES is highly sensitive to conformational changes induced by thermal stress, enabling early detection of destabilisation events and providing a new approach for viral quality control.
The second part focuses on extracellular vesicle mimetics, specifically cancer-derived exosome mimetics (CDEMs) generated from ovarian cancer cells. Two fabrication methods, extrusion and spin cup filtration, were compared, with spin cup filtration showing better reproducibility, scalability, and ease of use. Characterisation using NTA, DLS, and SEM confirmed expected size distributions (50–200 nm) and morphologies, while storage tests identified −80 °C as the most effective condition for long-term preservation, and 4 °C as the most effective for short-term stability. Finally, applying REES provided novel insights into CDEM membrane dynamics, revealing subtle, batch-specific differences in flexibility and rigidity, and highlighting its potential as a complementary quality-control tool.

The final part examines the biological behaviour of CDEMs in vitro. Using fluorescent dye labelling (DiD), uptake of SKOV3-derived CDEMs was studied in both parental SKOV3 ovarian cancer cells and non-parental MRC5 fibroblasts, focusing on uptake mechanisms, cell selectivity, and the effects of pathway inhibition. Both SKOV3 and MRC5 cells internalised CDEMs efficiently, but uptake was significantly higher in the parental SKOV3 cells, suggesting selective tropism. Inhibition studies showed that CDEMs rely on several endocytic pathways, with clathrin-mediated and Arf6-dependent entry being the most prominent, and caveolae-mediated uptake playing a smaller role. These results shed light on how CDEMs are internalised and provide a basis for designing more effective mimetic-based delivery systems, as well as for comparing their uptake with that of natural extracellular vesicles in future therapeutic studies.
Together, this work advances both methodological and translational aspects of biotechnology. It shows that REES is a sensitive and practical approach for studying the stability of viral vectors and nanovesicles. It also establishes strategies for producing and characterising CDEMs. By addressing analytical and quality control gaps that limit viral and vesicle-based therapies, this project helps develop more stable, reliable, and clinically viable biopharmaceutical platforms.

Date of Award	24 Jun 2026
Original language	English
Awarding Institution	University of Bath
Supervisor	Christopher Pudney (Supervisor), Jody Mason (Supervisor) & Steve Conlan (Supervisor)