With the advent of powerful gene editing tools such as CRISPR/Cas9, advances in gene therapy have gained a second wind. Despite this, disease therapy still has not progressed beyond clinical trials due to limitations in current delivery methods. The work presented in this thesis studies the development of a non-viral gene delivery method which is the nanomagnetic transfection method, which is the delivery of genes to cells using magnetic nanoparticles (MNPs) with a cationic surface charge and an external magnetic _eld. The advantage of nanomagnetic transfection over other non-viral chemical methods is the low dosage required to transfect cells coupled with a short transfection time. The presence of an external magnet provides targeting functionality, whereby the MNPs carrying the gene of interest are pulled towards the cells, thus increasing the e_ciency of cell to MNP contact. The research looks at the synthesis of MNPs using thermal decomposition to obtain particles with a narrow size distribution and exhibiting a combination of Brownian and N_eel relaxation. The MNPs were coated with polyethyleneimine (PEI), which binds and condenses DNA to deliver into cells for protein expression. PEI is known to be toxic to cells at high concentrations, hence PEI not bound to MNPs were removed using dialysis. A unique study observing the gradual loading of PEI coating on MNPs using AC susceptometry (ACS) is described. ACS provided information on the MNP coating and aggregation process that was not accessible through dynamic light scattering (DLS) due to the additional presence of non-magnetic polymer particulates in the suspensions. In combination with complementary structural characterization techniques, a simple method was derived to obtain dense, uniform PEI coatings a_ording high-stability suspensions without excessive quantities of unbound PEI to reduce cytotoxic e_ects. This method can be used for improving coating and functionalization therefore advancing MNP-drug/gene delivery studies. The PEI-coated MNPs were subsequently studied for their transfection capabilities in HeLa cells and compared to commercial MNP transfection agents. It was found that nanomagnetic transfection had higher GFP reporter expression compared to Lipofectamine and PEI. The parameters a_ecting transfection activity were determined in order to improve transfection rates of synthesized MNPs. A trade-o_ between transfection e_ciency and cytotoxicity was observed, where the presence of unbound PEI improved transfection but a_ected cell viability. To overcome this, polymers and block-copolymers with a lower charge density should be developed. The proton-sponge e_ect, which is the mechanism of MNP-PEI escape from the endolysosome was studied by measuring the AC susceptibility of MNP-PEI in live cells. However, the low transfection e_ciency of MNP-PEI and low sensitivity of the AC susceptometer made it di_cult to obtain conclusive evidence. A novel study using Raman spectroscopy to obtain _ngerprint spectra of the MNP-PEI complexes and to determine their localization in cells is reported. Individual spectra of MNP and PEI were obtained, as well as the area map of the cell, however the localization of MNPs within the cell was not possible due to the limited sensitivity of the Raman spectrometer. Finally, the e_ect of the MNP-PEI transfection agents on cells were identi_ed. It was observed that MG-63 and HeLa cells expressed increased cell stress with the formation of actin stress _bres and increased cell adhesion. Between the two transfection components, PEI antagonized the cell adhesion e_ect compared to MNPs. In addition, the genes associated with actin _bres and cell adhesion were identi_ed, which were ACTA2, ACTN1, MVCL, VCL, P4HA2, PCDHB12, SVIL, and TGFBI, which showed increased expression to MNP-PEI treatment. Collectively, the study conducted reports the development of an MNP transfection agent, from synthesis to application.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:745340 |
| Date | January 2018 |
| Creators | Narayanasamy, Kaarjel Kauslya |
| Publisher | Keele University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.keele.ac.uk/5002/ |
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