Date of this Version
Weerakkody, J.S. (2023) Characterizing Extracellular Vesicles and their Isolation Methods towards Engineering Therapeutic Vesicles [Unpublished Master's Thesis]. University of Nebraska-Lincoln.
Extracellular vesicles (EVs) and exosome-like vesicles (ELVs) are nanoscale lipid particles secreted by cells that mediate intercellular communication by delivering bioactive molecules. Isolation of pure EVs and ELVs are important for downstream analyses and potential therapeutic applications. Isolation of pure actively-loaded engineered ELVs are also important as potential delivery vehicles for future biotherapeutics. Nano-flow cytometry (nFCM) is a recently developed technology that can measure nanoparticle concentration, size, and phenotype individual nanoparticles using suitable antibodies and membrane stains. The objective of this thesis was to use nFCM in combination with other techniques such as nanoparticle tracking analysis (NTA) to characterize EVs secreted by HEK293T cells, which were isolated using various methods including differential ultracentrifugation, polymer-based precipitation, ultrafiltration, and size exclusion chromatography, in order to compare these techniques by quantifying parameters such as EV/ELV yield, size, purity, and concentration. HEK293T cells were transfected with plasmid DNA encoding CD63-GFP to actively load GFP inside ELVs to assess loading efficiency. Using the nFCM, isolation using ultracentrifugation resulted in the highest EV and ELV yield and purity, compared to all other isolation techniques. ELV-enrichment using a commercial CD63-specific immunocapture kit was attempted but was unsuccessful. It was also determined that compared to nFCM, nanoparticle concentrations and sizes were overestimated using NTA highlighting a challenge in the field, as discrepancies can affect downstream applications. We demonstrated that transfection of cells with CD63-GFP can enable high loading of ELVs with GFP. Insights from this thesis will inform future work towards engineering ELVs to deliver biotherapeutics.
Advisor: Angela K. Pannier