Successful gene therapy depends upon specific gene delivery into the cells and tissues of interest. Nanomagnetic gene transfection is a relatively new gene delivery technique that has attempted to meet that need and has been effectively used with both viral and non-viralvector systems. It uses magnetic nanoparticles (MNPs) in assisting and directing specific delivery of reporter or therapeutic genes on a single cell basis, in the presence of an externally introduced oscillating magnet. The novelty of the lateral oscillation further stimulates endocytosis of MNP:plasmid complexes with improved in vitro transfection efficiency compared to the static magnet application and other non-viral gene delivery approaches. This work’s purpose was to contribute in the optimisation of this tool for safe and efficient gene delivery, and to investigate the applicability of the method in a wider range of cell types used for regenerative medicine purposes, improving transfection efficiency and duration. Novel transfection experiments using commercially available MNPs coupled to a reporter gene, demonstrated higher levels of transfection efficiency (differing among cell types) and cell viability (80-94%), at the lowest reagent concentrations across all posttransfected cell types, compared with the most widely used cationic lipids (Lipofectamine) and/or electroporation. In particular, using human lung mucoepidermoid carcinoma cells (NCI-H292), the magnetic field requirements for transfection were evaluated; using human osteosarcoma fibroblasts (MG-63), a nanomagnetic transfection protocol at shorter transfection times (30 min) was established for increased transfection efficiency (53% oscillating and 49% static magnet, 7% at 30 min and 24% at 6 hr Lipofectamine, and 21% electroporation); using mouse embryonic fibroblasts (NIH-3T3), the 30 min-protocol was applied further (25% oscillating and 22% static magnet, 2% at 30 min and 22% at 6 hr Lipofectamine); using human mesenchymal stem cells (hMSCs), the ability of the method to transfect primary cells and to retain key markers for multipotency was demonstrated; using human embryonic stem cells (hESCs), the transfection capability in additional types of primary cells was shown, along with indications of retention of key markers for pluripotency and differentiation, although further work is required to confirm this finding. Nanomagnetic gene transfection shows promising results for in vitro and in vivo non-viral gene delivery and biomedical engineering applications. Data from this study could to be used for MNP drug delivery strategies, ultimately for clinical translation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:733261 |
| Date | January 2017 |
| Creators | Fouriki, Angeliki |
| Publisher | Keele University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.keele.ac.uk/4280/ |
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