| dc.description.abstract | Extracellular vesicles (EVs) have recently attracted much interest as natural therapeutic agents and promising drug delivery systems. They are important cellular mediators for the transport of various cargo and exhibit a homing ability to specific recipient cells. In addition, they have demonstrated immunomodulatory capabilities. While these findings present exciting opportunities for EV therapeutic applications, technical challenges, specifically low production yield of EVs and inefficient cargo loading, are considerable barriers to successful clinical translation. Manipulating EVs using synthetic biomaterials and surface modification techniques can provide strategies to enhance their therapeutic applicability. In this dissertation, research efforts focused on the development of (1) Semi-artificial, lipid-hybridized EVs for gene-modulating siRNA delivery, (2) a Multi-functional EV platform capable of selective co-delivery of siRNA and doxorubicin (DOX) to cancer cells, and (3) Azide-functionalized EVs enabling surface conjugation via bio-orthogonal click chemistry.
To enable mass production of the delivery vesicles, cancer cell-derived EVs were incorporated with various lipids using a sonication and extrusion technique to generate engineered EVs (eEV). Particle number characterization revealed this method produced a 6- to 43-fold increase in numbers of vesicles. Lipid to protein ratio and surface protein expression were evaluated to assess the membrane fusion efficiency. Exogenous siRNA was successfully loaded into eEVs via electroporation and effective gene silencing was demonstrated in cancer cells.
Next, a polymer layer-by-layer coated EV complex (LbL-eEV) was developed. Successful co-delivery of siRNA and DOX using LbL-eEV was verified by flow cytometry analysis. Cellular internalization efficiency was investigated and the inherent preferential delivery of LbL-EVs to cancer cells was demonstrated.
In addition to engineering EVs for drug delivery, modification of mesenchymal stem cell-derived EVs with bio-orthogonal functional groups (e.g., azide) to enable surface conjugation via click chemistry reaction was also studied. Successful modification of EV producer cells with azide and tetrazine reactive groups through metabolic engineering was demonstrated by fluorescent labelling. In summary, these studies demonstrate the surface composition and functionality of EVs can be tuned by multiple approaches, including membrane fusion with lipid-based materials, polymer coating, and natural biosynthetic pathways in cells. This work provided the proof-of-concept that EVs can be engineered to fill the needs of different therapeutic applications. | en |
