Narayanasamy, Kaarjel Kauslya (2018) Preparation and Evaluation of Polymer Coated Magnetic Nanoparticles for Applications in Gene Delivery. Doctoral thesis, Keele University.

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Abstract

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.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Medicine and Health Sciences > Institute for Science and Technology in Medicine
Depositing User: Lisa Bailey
Date Deposited: 08 Jun 2018 13:14
Last Modified: 08 Jun 2018 13:14
URI: http://eprints.keele.ac.uk/id/eprint/5002

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