Traumatic spinal cord injury (SCI) is a global health problem involving complex pathophysiological cascade and afflicts both developing and developed countries. Transplantation of Mesenchymal stem cell population such as dental stem cells (DSC) have demonstrated preclinical potential for central nervous system (CNS) repair. The work presented in this thesis has evaluated the potential of dental stem cells from apical papillae (SCAP) in combination with different biomaterials for SCI repair. ECM scaffolds were produced from different mammalian tissues including spinal cord, bone and dental hard tissue using different decellularisation processes. Scaffolds were then digested with pepsin to allow solubilisation and hydrogel formation. The ECM hydrogels were characterised and embedded with SCAP to investigate the effect of morphological and biochemical properties upon cell characteristics. All the hydrogels maintained high cell viability and an increase in the cell number with a satisfactory metabolic activity. However, only ECM hydrogels from decellularised spinal cord and bone tissue supported the expression of neural lineage and pro angiogenic markers with stronger responses observed with spinal cord ECM hydrogels. Biodegradable PLGA-Triblock (PLGA-TB) microparticles were fabricated to provide controlled release of glial cell derived neurotrophic factor (GDNF) and may facilitate SCAP attachment. An optimal PLGA-TB microparticle formulation was selected based on the size, surface morphology and release profile achieved. All commercial preparation of GDNF being stabilised in salt, a modified protocol was required to prepare microparticles. The formulation was modified with 10mM sodium acetate which led to a successful encapsulation and sustained release of bioactive GDNF. To support SCAP attachment and survival, PLGA-TB microparticles surfaces were coated with different ECM pre-gel solutions (spinal cord and bone tissue ECM) and laminin. Assessment of surface coating with ToF-SIMS showed protein adsorption on all the coated microparticles, with a higher adsorption on ECM pre-gel coated microparticles. All the surface modified PLGA-TB microparticles supported prolonged SCAP attachment and survival. Laminin and bone ECM pre-gel coated microparticles promoted a significant increase in SCAP number after 7 days. Over all, the result in this thesis have shown that SCAP combined with decellularised mammalian tissue derived ECM hydrogels or GDNF loaded PLGA-TB microparticles may facilitate delivery of autologous stem cells to promote spinal cord repair.

Chronic liver diseases represent a primary worldwide health concern. Liver transplantation is the only practical cure for severe and chronic diseases, but due to the lack of human livers donor an insufficient number. The alternative is the cell-based transplantation of hepatocytes or hepatocyte-like cells derived from human stem cells. We aim to produce a physiologically relevant human liver model from human stem cells and to scale-up their production to be utilised in disease modelling and drug testing. Wnt/ β-catenin signalling pathway involved in hepatoblast proliferation and differentiation during liver development and regeneration. Here we showed that inhibition of GSK3β to stabilise β-catenin during hESC- derived hepatoblast differentiation resulted in high level of β-catenin with the early appearance of truncated form, improvement of HLCs morphology and upregulation of HNF4α and albumin expression. Although hESC can differentiate into almost any cell type including hepatocytes, their genetic instability in cultures limited their utilisation in regeneration medicine and cell-based transplantation therapy. Adult liver stem/ hepatic progenitor cells (HPCs) on the other hand upon activation following severe liver injury can differentiate into both hepatocytes and biliary epithelial cells. EpCAM+ progenitor cells isolated from human livers presented their expandability with the maintenance of progenitor characteristics and capacity to differentiate into hepatocytes in 3D culture (organoids). Differentiation of organoids- resulted in a loss of hepatic progenitor cell markers expression while the expression of differentiated hepatocyte-specific genes increased. Differentiated organoids showed low albumin expression compared to primary human hepatocytes; suggesting that the differentiation may have arisen from a limited population of cells or the organoids- derived hepatocytes were not fully mature. FACS sorting using a combination of specific markers (EPCAM, CD133 and CD24) produced a more homogeneous cell population that differentiated more effectively into hepatocytes compared to EpCAM+ cells. The acellular rat liver scaffolds obtained by decellularisation using detergents was naturally directed the cultured HPCs and NPCs to their in localisation and improved the albumin expression in the differentiated HPC providing an applicable model for drug testing and bio-engineered liver transplantation. Generating functional hepatocytes from human liver progenitor cells could provide an unlimited supply of hepatocytes; replacing the animal use in drug screening, testing new chemical entities and modelling human liver diseases.

The HER family of receptors plays a major role in a variety of cancers including breast cancer. Several researchers have shown that HER family overexpression in breast cancer is a significant prognostic factor, especially for survival and relapse. Therefore, many therapeutics are being developed to test the impact of HER family blockade in breast cancer. Although numerous therapies have been developed, many have not been very successful in the clinic. This is often a consequence of cancer cells developing new mechanisms to activate HER family signalling indirectly through cross talk with compensatory pathways. Thus, it is vital to consider the biology of the HER signalling network to a greater extent, which includes RAS/MAPK, PI3K/AKT, mTOR, JAK/STAT, ER and AhR pathways and, also identify breast cancer patient populations that will benefit from specific targeted therapies that target these pathways. In the current study, 6 breast cancer cell lines (MCF7, T47D and ZR-75-1, SKBR3, MDA-MB 468 and MDA-MB 231) representing distinct molecular subtypes of breast cancer have been used to investigate anti-cancer effects of a variety of agents. These agents include clinical as well as currently experimental and entirely novel pharmacological agents alone or in combination. Among the clinical agents studied, it was found that EGF and Gefitinib were significantly potent against the HER2 overexpressing SKBR3 cell line, out of the panel of cell lines studied. EGF and Gefitinib showed a slightly different spectrum of activity from each other against the SKBR3 cell line. However, more research is needed to determine whether EGF could be used as a therapy for HER2 overexpressing breast cancer. Even though Gefitinib is currently used as a treatment in the clinic, the therapeutic window of this agent is drastically narrowed by its poor bioavailability, acquired resistance and systemic toxicity. Thus, in the current study, encapsulation of Gefitinib within the cavity of human heavy chain (H) apoferritin (AFt), provided a route for sustained release of Gefitinib from the H-AFt cavity, which demonstrated enhanced anti-tumour activity, at a longer duration against the SKBR3 cell line compared to Gefitinib alone. Overexpression of HER2 is considered to confer a more aggressive phenotype in breast cancer. Many patients have shown resistance to existing clinical agents such as Trastuzumab, demonstrating the need for novel therapies. Hence, 2 novel HER2 targeting human H and light chain (L)-AFt-fusion proteins were tested, and it was found that the nanoagent - H-AFt-fusion protein was very potent against the SKBR3 cell line compared to the L-AFt-fusion protein. This novel H-AFt-fusion protein abolished SKBR3 colony formation completely, caused a G1 arrest and a reduction in the orchestration of S and G2/M cell cycle events and also induced a large SKBR3 apoptotic population demonstrating its potent cytotoxic effects. Furthermore, this agent down-regulated the HER2 protein remarkably which resulted in significant down-regulation of the RAS/MAPK, PI3K/AKT and JAK/STAT signal transduction pathways in SKBR3 cells. Previous research has shown that a combination of pharmacological agents are more effective against cancer than individual agents due to up-regulation of compensatory signalling pathways which cancer cells use to thrive and acquire resistance to agents. Thus, several agents were tested in combination. Out of the agents tested it was found that 3 dual PI3K/mTOR inhibitors were potent against the triple negative breast cancer cell line - MDA-MB 468 and the HER2 overexpressing SKBR3 cell line. Further, Gefitinib in combination with an experimental AhR ligand - 5F 203, showed synergistic growth inhibition against the SKBR3 cell line by inducing CYP1A1, thereby resulting in a large apoptotic population. It was observed that the effect of Gefitinib was mainly potentiated by the effect of 5F 203 within the agent combination. There is a momentous unmet medical need for the development of effective therapies that can stabilise or slow the progression of breast cancer, therefore, these results may contribute to existing knowledge or enhance further understanding of the HER signalling network and therapies targeting this network. It may also guide potential treatment options which might lead to significant improvements in breast cancer therapy in the clinic thereby personalising therapy for patients with breast cancer.

The field of nanomedicine has progressed to a stage where a diverse set of materials are available for controlling how a drug is delivered in the body. Although these materials can be engineered to overcome many of the obstacles associated with drug delivery, the complexity of cellular trafficking mechanisms means controlling intracellular delivery remains a major challenge. The primary portal for the cellular internalisation of nanomedicines is endocytosis, which involves transport through a network of highly complex intracellular compartments undergoing a dynamic process of acidification. As a result, nanoparticle-based pH sensors offer a new perspective from which to investigate this process. In this study, ratiometric polyacrylamide pH nanosensors were utilised to probe fundamental aspects of intracellular trafficking with the view of developing biological insights to aid the rational design of nanomedicines. Nanosensors were fabricated with a dynamic range covering the entire range of the endocytic pathway (4.0 – 7.5), with sizes between 50 and 100 nm. Endocytic uptake of nanosensors was induced in four different cell types (HeLa, 3T3, MRC-5 and JAWS II) by increasing the surface charge on the nanosensor. Dynamic pH measurements were found to be highly sensitive to experimental methodology for performing ratiometric measurements, particularly image analysis. Consequently an optimised procedure for performing ratiometric measurements was developed, and subsequently validated by correlating pH measurements with intracellular location using 3D structured illumination microscopy (3D-SIM). Application of pH nanosensors in studies investigating fundamental aspects of intracellular trafficking resulted in three key findings: 1) HeLa, 3T3 and JAWS II cells process material in different ways with respect to the extent and rate of acidification in endocytic organelles, 2) surface charge does not affect the final intracellular location of polyacrylamide nanoparticles internalised by endocytosis, and 3) lipid-mediated transfection of siRNA is associated with a greater degree of lysosomal disruption compared to cationic polymer-mediated transfection, with the former observed to show increased toxicity. These findings represent biological insights, which can be utilised to provide a rational basis for tailoring the response of pH-sensitive nanomedicines to a specific cell type, tuning the physicochemical properties of a material for more efficient intracellular trafficking and optimising siRNA formulations for endo-lysosomal release.