Extracellular vesicle-inspired asymmetric vesicles for nucleic acid delivery
| datacite.contributor.Supervisor | Holme, Margaret | |
| dc.contributor.author | Nicdao, Deborah | |
| dc.contributor.other | Nicdao, Deborah | |
| dc.date.accessioned | 2025-05-21T14:48:39Z | |
| dc.date.available | 2025-05-21T14:48:39Z | |
| dc.date.issued | 2023-06-22 | |
| dc.degree | masterThesis | |
| dc.description.abstract | As natural carriers of biomolecules, extracellular vesicles (EVs) have great potential as drug delivery systems due to their biocompatibility, enhanced in vivo stability, weak immunogenicity, and ability to cross biological barriers. However, their heterogeneity and the challenges associated with their isolation, formulation, and scale-up pose limitations to their clinical translation. Hence, EV-inspired synthetic vesicles with asymmetric lipid distributions across the membrane bilayer were formulated in this study to mimic cell membranes and deliver nucleic acid for potential therapeutic applications. Asymmetric vesicles (aLUVs) composed of DOPC on the inner leaflet and DOPA or SM:Chol on the outer leaflet were successfully prepared using two methods – 1) mβCD-catalyzed lipid exchange and 2) enzymatic reaction with phospholipase D. Membrane asymmetry was then confirmed through zeta potential, NMR spectroscopy, and Amplex Red PLD assay, with 44.25-98% exchange efficiency. Afterwards, a splice-switching oligonucleotide (SSO) was encapsulated into the aLUVs and the loaded aLUVs were purified using size-exclusion chromatography. The lipid vesicles were found to be non-toxic to HeLa_Luc705 cells through Alamar Blue assay. Through fluorescence imaging, DOPC:SM:Chol aLUVs at 1:1 and 1:10 dilutions were successfully internalized by the cells and reached the nuclei after 4 hours, with increased uptake as compared to symmetric LUVs. These results were further supported by luciferase assay data, in which enzyme activity was observed in cells treated with 1:1 aLUVs after 4 and 24 hours, indicating the functional delivery of SSO cargo. Overall, EV-inspired aLUVs offer great potential as nanocarriers for nucleic acid delivery, leading towards diagnostic and therapeutic applications. Further studies involving more complex lipid compositions and protein receptors, other preparation methods (e.g., microfluidics), and a combination of quantitative techniques (e.g., flow cytometry) and high-resolution imaging (e.g., confocal microscopy) to better understand cellular uptake and intracellular localization can also be explored. | |
| dc.identifier.uri | https://hdl.handle.net/10889/29497 | |
| dc.language.iso | en | |
| dc.rights | Attribution-NonCommercial-ShareAlike 4.0 International | en |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-sa/4.0/ | |
| dc.subject | Asymmetric vesicles | |
| dc.subject | mβCD-catalyzed lipid exchange | |
| dc.subject | Phospholipase D | |
| dc.subject | Nucleic acid delivery | |
| dc.subject | Splice-switching oligonucleotide | |
| dc.title | Extracellular vesicle-inspired asymmetric vesicles for nucleic acid delivery | |
| dc.title.alternative | Ασύμμετρα κυστίδια εμπνευσμένα από εξωκυτταρικά κυστίδια για χορήγηση νουκλεϊκών οξέων |