Nanoneedle-based gene transfer for cardiac regeneration & biophysical regulation of cellular state
File(s)
Author(s)
Leonardo, Vincent
Type
Thesis
Abstract
In recent years the advances in biomaterial science have made possible to use micro- and nano-fabrication techniques such as electro-spinning, soft- and photo-lithography to create numerous biomaterials with increasing sophistication in the ability to tune, mimic and manipulate complex physical and biological properties related to the cell niche. Modern biomaterials have a wide array of applications including but not limited to their use in surgical implants, medical devices, advanced applications in tissue engineering, biosensing and drug delivery, and as aids to understandthe interaction between cells and their niche to enablethe manipulation of cellular behaviour and identity.
This thesis aims to show how the combination of advanced biomaterials and biological tools can lead to the development of novel therapeutic tools and provides insight into cellular mechanosensing and cellular identity.
Firstly, the thesis aims at developing a proof-of-concept in vivo therapeutic tool targeted at rejuvenating the damaged myocardium post-myocardial infarction (MI) by nanoinjection of cardiac reprogramming factors to the epicardial layer of the heart. For this purpose, this study used a previously developed nanodelivery platform based on porous silicon nanoneedles (nN) that are capable of delivering a cargo in an efficient, safe and spatially resolved manner. An effective strategy was established through this study to uniformly load the nN with high concentrations of pDNA that are capable of rapid release under physiological conditions. A single polycistronic transposon-based plasmid containing the three cardiac transcription factors, MEF2C, GATA4 and TBX5 (MGT), was developed and used to initiate the direct conversion of the epicardial cells towards cardiomyocyte lineage. An in vitro study successfully demonstrated the ability of the nN to load and deliver the MGT containing transposon and the transposase to the epicardial derived cells (EPDC) and the transgenes were successfully integrated into the genome showing sustained prolong expression which in turn directed the EPDC to differentiate to cardiomyocytes. The in vivo proof of principle study further demonstrates the mostly successful nN-mediated intracellular gene delivery to the heart epicardium.
The purpose of the second study is to provide further insight into the cellular response to external biophysical cues from a global perspective. External biophysical cues can be
4
transferred via the cytoskeletal networks to alter the nuclear matrix mechanically. The
mechanisms that underlie this phenomenon have just started to be elucidated however there
are still substantial processes that are not entirely understood. To address this issue, mouse
embryonic fibroblasts (MEFs) were cultured on a high aspect ratio silicon-based nanomaterial
with defined geometry and physical properties, namely nanoneedles. The close interaction
with the nN induced substantial alteration on the nuclear shape and structure.
Furthermore, the positioning of the nucleosomes was also altered and the nanoneedles also
induced significant changes in the histone modifications specifically the methylation patterns.
RNA sequencing performed on MEFs grown on nN revealed enrichment in genes associated
with cytoskeletal organisation, chromosomes organisation, response to oxidative stress and
nuclear division and cell cycle. These results were mirrored by the label-free mass
spectrometry proteomic analysis performed on cells cultured under the same conditions. The
changes observed through the different analysis methodologies indicate that the cells were
in a different state as oppose to cells cultured on conventional tissue culture plastic (TCP)
potentially adopting a more responsive and open epigenetic state.
In conclusion, the work in this thesis demonstrates how biomaterials could be used in
conjunction with robust biology to further advanced regenerative medicine and the
understanding of complex biological problems.
This thesis aims to show how the combination of advanced biomaterials and biological tools can lead to the development of novel therapeutic tools and provides insight into cellular mechanosensing and cellular identity.
Firstly, the thesis aims at developing a proof-of-concept in vivo therapeutic tool targeted at rejuvenating the damaged myocardium post-myocardial infarction (MI) by nanoinjection of cardiac reprogramming factors to the epicardial layer of the heart. For this purpose, this study used a previously developed nanodelivery platform based on porous silicon nanoneedles (nN) that are capable of delivering a cargo in an efficient, safe and spatially resolved manner. An effective strategy was established through this study to uniformly load the nN with high concentrations of pDNA that are capable of rapid release under physiological conditions. A single polycistronic transposon-based plasmid containing the three cardiac transcription factors, MEF2C, GATA4 and TBX5 (MGT), was developed and used to initiate the direct conversion of the epicardial cells towards cardiomyocyte lineage. An in vitro study successfully demonstrated the ability of the nN to load and deliver the MGT containing transposon and the transposase to the epicardial derived cells (EPDC) and the transgenes were successfully integrated into the genome showing sustained prolong expression which in turn directed the EPDC to differentiate to cardiomyocytes. The in vivo proof of principle study further demonstrates the mostly successful nN-mediated intracellular gene delivery to the heart epicardium.
The purpose of the second study is to provide further insight into the cellular response to external biophysical cues from a global perspective. External biophysical cues can be
4
transferred via the cytoskeletal networks to alter the nuclear matrix mechanically. The
mechanisms that underlie this phenomenon have just started to be elucidated however there
are still substantial processes that are not entirely understood. To address this issue, mouse
embryonic fibroblasts (MEFs) were cultured on a high aspect ratio silicon-based nanomaterial
with defined geometry and physical properties, namely nanoneedles. The close interaction
with the nN induced substantial alteration on the nuclear shape and structure.
Furthermore, the positioning of the nucleosomes was also altered and the nanoneedles also
induced significant changes in the histone modifications specifically the methylation patterns.
RNA sequencing performed on MEFs grown on nN revealed enrichment in genes associated
with cytoskeletal organisation, chromosomes organisation, response to oxidative stress and
nuclear division and cell cycle. These results were mirrored by the label-free mass
spectrometry proteomic analysis performed on cells cultured under the same conditions. The
changes observed through the different analysis methodologies indicate that the cells were
in a different state as oppose to cells cultured on conventional tissue culture plastic (TCP)
potentially adopting a more responsive and open epigenetic state.
In conclusion, the work in this thesis demonstrates how biomaterials could be used in
conjunction with robust biology to further advanced regenerative medicine and the
understanding of complex biological problems.
Version
Open Access
Date Issued
2018-10
Date Awarded
2019-02
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Stevens, Molly
Payne, David
Publisher Department
Materials
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)