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- PublicationSuperparamagnetic Nanoparticles for the Assessment of Intracellular Nanoparticle-Cell Interactions(University College Dublin. School of Chemistry, 2022)Since their discovery, nanomaterials have been employed in an increasing number of products and applications. Thanks to their unique properties they enabled the advancement of many technologies and improved our lives under several aspects. Because of their nanometric dimensions, nanomaterials interact with the biological matter in a completely different way compared to their smaller (molecular scale) or larger (macro scale) counterparts. Despite the efforts of the scientific community in unravelling the network of machineries involved in the interaction of nanomaterials with the cells and human body, our understanding of the bio-nano interactions is still limited. The lack of knowledge around the dynamics that regulate the nanoparticles’ (NPs) trafficking in the human body is limiting the development of nanomedicine and, at the same time, is rising concerns in the regulatory bodies for the safe commercialisation of nanomaterial-based products. One of the main issues related to this critical gap in the knowledge is the lack of methodologies and tools that prevents unravelling the complexity of the bio-nano interactions. Techniques commonly used for the study of the intracellular dynamics result limiting for the study of the NPs intracellular trafficking for which the combination of different analysis and methodologies is required to obtain reliable and robust results. However, the correlation of data from different techniques is difficult when different NPs systems are used for the experiments. In this thesis, we develop new tools and methodologies for the study of the bio-nano interactions, exploiting one of the unique properties of iron oxide NPs, the superparamagnetism. In this context, superparamagnetic NPs are extremely useful tools because allow to label the machineries involved in the intracellular trafficking of NPs and enable their isolation from the biological matrix, which in order allows a more in-depth downstream analysis. To label the machineries involved in the intracellular trafficking with superparamagnetic NPs, two strategies are possible: exploiting the natural uptaking mechanisms of the cells or targeting specific compartments with NPs designed ad hoc. To exploit the first strategy, we designed multifunctional core-shell NPs with iron oxide multicores that provided the superparamagnetic properties and a silica shell doped with an organic fluorophore. Combining the magnetic and fluorescent properties in one nanoconstruct enabled to resolve the NPs intracellular trafficking by optical microscopy and to isolate the vesicles loaded with the NPs by magnetic separation, at different stages of their voyage inside the cell. The vesicles were then analysed with a set of technique to evaluate their integrity and functionality. To exploit the second strategy, we designed antibody grafted iron oxide NPs for the targeting of specific biological species. For this purpose, we adopted a thoughtful strategy of NPs surface modification that enabled the grafting of the antibody through bio-orthogonal chemistries, stabilise the particles in biological conditions, and limit the adsorption of undesired biomolecules typically responsible for NPs off-targeting. Although this second nanoconstruct is still under development, the preliminary results showed excellent targeting ability and specificity. Overall, the work presented in this thesis provides a solid base for the isolation, by magnetic separation, of biological species involved in the NPs intracellular trafficking and for the development of methodologies for the investigation of the machineries involved in the process.
- PublicationThe Physiochemical and Biological Characterization of Nanostructured Materials Derived from Natural Processes(University College Dublin. School of Chemistry, 2022)The thesis focuses on the study of nanostructured materials obtained from natural sources. The work presented in this thesis was designed to 1) develop protocols for a complete Physico-chemical characterization of micro-and nanomaterials derived from calcified seaweed. Such protocols were achieved by combining several different characterization techniques and were able to unravel unexpected material features at the nanoscale; 2) obtain nanostructured metallic particles from the calcified seaweeds through a multistep extraction protocol and investigate their interactions with cells. The extensive characterization work carried out along this project, especially using electron microscopy, also allowed to develop methods for the visualization of endogenous biological material associated with nanoparticles after their interaction with biomolecules and the intracellular environment.
- PublicationComplex Nanostructures and Bio-Nanoscale Interactions: Well Defined Synthesis, Identification and Biological Effects(University College Dublin. School of Chemistry, 2022)In this thesis, a framework was proposed in chapter II, aiming to identify distinct shape populations and build a quantitative linkage of well-defined nanoscale shapes to biological impacts. This inductive nanoscale shape discovery and evaluation framework is biologically relevant, and we believe by utilizing machine learning it could benefit the field of shape dependent therapy. In chapter III, the shape dependent histone modifications were reported. As histone modifications are one of the crucial epigenetic regulators that control chromatin structure and gene transcription, shape dependent histone modifications indicate that some important cellular phenotypes differences induced by nanoscale shapes may be related to the histone modifications, which opens a new window for the investigation of nanoscale shape effects and nano therapy. In chapter IV, we proposed a method to modify the surface of the nanostructures by endogenous cellular processes and studies found that this re-engineered particle complex was able to transfer the loading genes to recipient cells, which indicates their potential to work as an efficient nucleic acid delivery machine.
- PublicationInvestigation of Nanostructures generated in cells(University College Dublin. School of Chemistry, 2022)Nanoparticles have established themselves firmly within the biomedical field due to their fine tuneability. Recent work explores NP behaviour in complex biological media lending itself to a growing body of research investigating the biological identity of NPs. Biomolecules have been found to readily adsorb to NPs upon interaction in complex biological media forming a protein shell. Previously viewed as an impediment to NP application in vivo, the protein shell, now referred to as the ‘biological corona’ displays potential for beneficial and independent applications. This has highlighted the need for a sounder understanding of the way in which cells receive, process, and clear nanoparticles and the pathways implicated. This research investigates the ability of magnetic magnetite multicore silica fluorescent nanoparticles to adsorb biomolecules to form a biological corona and further enrich with target EGFP fusion proteins upon administration to and interaction with stable EGFP-transfected, adherent cells in culture. The cell – engineered, biosynthetic particle recovered following internalisation, trafficking and subsequent recycling will be referred to here as a bio nanostructure (BNS). This research builds upon a large body of data encompassing all proteins comprising the BNS generated in HEK-293T cells. Here I will explore several highly abundant RNA binding proteins that are incorporated onto the BNS during cellular processing of the nanoparticle-protein corona. The application of high-resolution confocal laser scanning microscopy, along with a ‘masking’ technique will facilitate observation of EGFP fusion proteins on the BNS coat. The results presented herein support the establishment of a reliable and reproducible method for the fluorescent mapping proteins of interest on cell – generated bio nanostructures. It provides a platform for further investigation of potential subpopulations of cell generated bio nanostructures and the intricate processing networks underlying the export of these bio nanostructures.
- PublicationThe Preparation and Application of Novel Planar Chiral Ferrocenyl Ligands in Asymmetric Catalysis(University College Dublin. School of Chemistry, 2022)Asymmetric catalysis, employing both transition-metal complexes of chiral ligands and organocatalysts has become one of the most popular methods for the preparation of enantioenriched and enantiopure compounds. This is important for the synthesis of chiral biologically active compounds or drug targets. The design of efficient and novel chiral ligands and catalysts has become a prominent area of chemistry in itself. In this PhD thesis, the concept of chirality and the importance of chiral synthesis in chemistry is introduced while highlighting the interesting properties of ferrocene and the development of chiral ferrocenyl scaffolds in this expanding area of chiral ligand synthesis. The aim of this project was to exploit the reactivity and selectivity of ferrocenyl mono- and diketone scaffolds optimised previously in the group to develop a range of interesting chiral catalysts and ligands. The first of these novel structures was an extended diol structure which would serve as a second generation to diols produced previously in the group which possessed a quaternary centre at the alpha-ferrocenyl position. Three chiral diols extended by two methylene units, were synthesised in six high yielding steps with minimum purification required. These catalysts were applied in an asymmetric hetero Diels-Alder reaction affording the product in low yield and low enantioselectivity of up to 33% ee. The presence of a tertiary stereocentre at the alpha-ferrocenyl position caused instability issues compared to the previous library. Following on from this, five novel ferrocenyl aniline-containing amino alcohol ligands were developed diastereoselectively in as few as three high yielding steps. These ligands were very successful in asymmetric diethylzinc addition to aldehydes, achieving ees of up to 99% and in asymmetric phenyl transfer reactions with moderate yields and enantioselectivities of up to 88%. With the assistance of X-ray crystallographic studies, transition states for the novel gamma-amino-alcohol system were proposed to explain the stereochemical outcome of the reactions. Finally, a fascinating rearrangement was observed when attempting to synthesise a ferrocenyl phosphoramidite, resulting in the formation of novel chiral phosphonamidate structures. Compounds that possess this moiety have been shown to evoke therapeutic effects such as anti-cancer and anti-viral properties. By utilising both X-ray crystallography and 31P NMR spectroscopy, a mechanistic understanding of phosphonamidate formation was reached, with four diastereomers theoretically possible in their synthesis. By employing a series of secondary amines, six chiral phosphonamidate analogues were synthesised with both a major and minor diastereomers isolated and their structures successfully elucidated.