Medicine & Health

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  • (1998) Wool, R; Kusefoglu, S; Khot, S; Zhao, R; Palmese, Gaetano; Boyd, Andrew; Fisher, Keith; Bandyopadhyay, Srikanta; Williams, J; Wang, Chaoyuan
    Conference Paper

  • (2022) Forest, Chelsea
    In the last 40 years there have been many strides taken towards better and more selective cancer treatment using nanoparticles. Nanoparticles can have inherent passive accumulation in tumour cells, known as the enhanced permeability and retention effect (EPR) which makes them a strong therapy candidate; however this effect is not as well defined or effective as once thought. There is a large variance of efficacy between different patients due to the heterogeneity of tumours, therefore a more targeted nanoparticle systems needs to be designed to increase selectivity and efficacy. This thesis describes the design, synthesis, and characterisation of 20 novel ellipsoidal polymersomes decorated with peptide ligands for selective targeting of medulloblastoma, a childhood brain cancer. These ligands were FSRPAFL 1 a medulloblastoma cell targeting peptide and T7 26 a transferrin targeting peptide designed to aid in crossing the blood brain barrier (BBB). A new synthetic method was designed to attach the peptide ligands post self-assembly, so the peptides were attached to the hydrophilic corona rather than the hydrophobic membrane of the polymersomes. Analysis of these polymersomes showed more ligand available for binding but this did not translate to increased cell association due to an over saturation of ligand. The ratio and density of the targeting peptide 1 and BBB peptide 26 was altered on the surface of the polymersomes and it was found that the polymersomes with 100% T7 ligand showed rapid and high cell association with two different subtypes of SHH medulloblastoma (DAOY and UW228) as well as high association with brain endothelial cells that make up the BBB (HBEC-5i) making it a promising candidate as a drug delivery system for SSH medulloblastoma. Finally, linearly conjugated dual peptides made up of both targeting peptide 1 and T7 peptide 26 sequence, were synthesised and attached to the polymersome hydrophilic corona and analysed against the non-conjugated dual-functionalised peptide polymersomes. There was no significant difference between the two ligand conjugation method analysed but further research should be conducted to confirm this. The work described in this Thesis has shed light on the multitude of nuances that make up the composition of mono and dual functionalised peptide nanoparticle systems and how these can influence biological function. Future work will allow for a better understanding of fundamental questions about targeted nanoparticles therapies and how ligand characteristics directly impact biological function, selectivity and efficacy.

  • (2022) Khosravanihaghighi, Ayda
    The two leading causes of failure of orthopaedic implants are aseptic loosening and periprosthetic joint infection. Since the numbers of primary and revision joint replacement surgeries are increasing, strategies to mitigate these failure modes have become increasingly important. However, most recent work has focused on the design of coatings to prevent infection or to enhance bone mineralisation. However, long-term success of the implants is contingent on addressing both of these issues. Consequently, the present work focussed on multifunctional orthopaedic coatings that inhibit microbial cells while still promoting osseointegration. Nanoceria has considerable potential to be used in biomedical applications owing to its unique bio-responsive redox switching and its capacity to be doped with different therapeutic ions of varying functionalities. Therefore, the effect of different cations incorporated in ceria on cellular behaviour in vitro as well as the anti-bacterial performance were investigated. The two main foci were: (1) characterisation of the bioceramic materials and (2) biological response to undoped and doped ceria ceramics in vitro using bacteria colonies forming unit (CFU) and cytotoxicity Ceria (CeO2) thin films (~820 nm thickness) doped with 0-9 mol% Ga or Mn were fabricated by spin coating on 3D-printed Ti6Al4V followed by heat treatment at 650°C for 2 h, and these were characterised by transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) (microstructure), 3D laser scanning confocal microscopy (topography), glancing angle X-ray diffraction (GAXRD) (structure and mineralogy), and X-ray photoelectron spectroscopy (XPS) (surface chemistry). In vitro testing was conducted, including inhibition of bacterial growth, simulated body fluid (SBF) testing, and cell attachment and proliferation studies. The data are interpreted in terms of the following: (1) The roles of the sol-gel precursor viscosity, which affected pore filling and surface coverage, (2) Lattice contraction, which contradicted the XPS data, (3) Intervalence charge transfer, which increased the Ce3+ concentration but was a minor effect, (4) Substitutional solid solubility, which is consistent with Hume-Rothery’s rules and the GAXRD data, (5) Redox charge compensation, where the defect equilibria highlight the key role of this mechanism, which decreased the Ce3+ concentration and provided the majority effect, (6) Electronegativity, which plays a small, if any, role in affecting the ion valences but is important in initiating intervalence charge transfer, (7) Multivalence charge transfer, which combined the electron exchanges between film matrix, dopants, and Ti substrate. The most significant outcome was that the bioactivity of ceria derives directly from the Ce3+ concentration, which itself results from solid solubility (substitutional and interstitial) and charge compensation and redox. This challenges the common assumption of the dominance of oxygen vacancies in the performance of ceria. The antibacterial activity was dependent on the type, amount, and valence of the dopant, where opposite trends were observed for gram-positive S. aureus and gram-negative E. coli bacteria. All of the doped samples resulted in enhanced cell proliferation, although this was greatest at the lowest dopant concentration. Surface hydroxyapatite formation on the samples was achieved by soaking in SBF at 2 weeks and 1 month.

  • (2023) Seyedzadeh, Hadi
    Cytotoxic T lymphocytes (CTLs) can target and induce apoptosis in cancer cells during the anti-tumour immune response. However, the cytotoxicity (or killing) function of CTLs can be perturbed directly by cancer cells or via the tumour microenvironment (TME). Among the various factors in TME that can influence T cell function, the effect that mechanical properties of the extracellular matrix (ECM) have on CTL responses is unclear. Research into CTL-mediated cytotoxicity is typically performed in either two-dimensional (2D) matrix-free culture or in complex in vivo animal models. In vitro, 2D studies are limited in recapitulating the CTL response in vivo, whereas it is very difficult to manipulate the TME and perform high-throughput experiments using in vivo models. Recently 3D culture models have been introduced to fill the gap between 2D and in vivo studies. In this study, we used an automated 3D bioprinter to incorporate OT-I T cells and cognate and non-cognate target cells in a polyethylene glycol (PEG)-based hydrogel and studied the killing efficiency in comparison with 2D culture and manually-prepared gels. Here, we showed that the 3D bioprinter embeds both CTLs and target cells in the hydrogel and enables control over the dimensions of the embedding matrix as well as the number and spatial organisation of cells. Moreover, the ability to digest the gel and release the cells allowed us to perform killing efficiency comparisons and downstream high-throughput CTL functional analyses using flow cytometry. This novel 3D cell culture system allowed us to investigate the effects of tunable ECM mechanical properties in a reproducible cytotoxicity model of matrix-embedded CTL and target cells. Our results demonstrate that in matrices with higher density, CTL killing efficacy was compromised. This demonstrates that matrix stiffness, independent of matrix porosity or other variable characteristics, has a large impact on CTL function. From another perspective, cancer cells can directly induce dysfunctional programming in CTLs. Repeated stimulation of the T cell receptor (TCR) on CTLs with the tumour-associated antigen leads to overexpression of inhibitory receptors such as programmed cell death (PD)-1 on the surface of T cells, leading to aberrant response and eventually tumour escape. TCR signalling machinery can be affected by the expression of inhibitory receptors, but it is not clear whether inhibitory receptors alone are responsible for the dysfunction of exhausted T cells or to what degree other mechanisms contribute. To address this, we used a mouse model of T cell dysfunction, finding that T cells could exhibit a dysfunctional phenotype with minimal upregulation of inhibitory receptors and without downregulation of TCR. Instead, we found a decrease in the proximal signalling kinases Lck and ZAP70, specifically in dysfunctional cells. To confirm these results, we developed a human primary in vitro CD8+ T cell dysfunction model, which allowed us to study the effect of repeated antigen stimulation on the inhibitory receptors expression and expression of Lck and ZAP70 in human T cells. In this model, we again found that dysfunctional T cells had lower expression of Lck and ZAP70, confirming the results from the mouse model. Future experiments could be performed in which Lck and/or ZAP70 expression is enhanced in dysfunctional T cells. If this restores the functional phenotype, it may confirm that a low level of Lck and ZAP70 protein expression is a cause of T cell dysfunction. This research sheds light on how the external (matrix stiffness) and internal factors (TCR stimulation) affect the CTL response.

  • (2022) Joshi, Nidhi
    RNA interference (RNAi) has emerged as a promising tool to silence any kind of gene expression from viral infection to genetic disease, especially in the field of cancer treatment. siRNA-based therapeutics offer an efficient and specific targeting of disease-causing genes. An aberrant expression of Wnt pathways and ROR receptors which are the transmembrane protein of tyrosine kinase family, have been reported in many cancers, including ovarian cancer. The upregulation of Wnt pathways and ROR receptors are known to be potential contributors in ovarian cancer progression and metastasis. Therefore, targeting these receptors could be a powerful approach towards designing and developing new therapeutic materials. In that regard, siRNA-based therapeutics offer an efficient and specific targeting of overexpressed disease-causing genes. However, the challenge remains for the effective and safe delivery of siRNA therapeutics while maintaining its efficacy and therapeutic integrity. Polymeric nanostructures hold great promise towards designing a compatible delivery vector for nucleic acid therapeutics, especially for siRNA drugs. Among polymeric systems, polycationic carriers based on PDMAEMA are widely explored as nucleic acid delivery vectors. The low toxicity and high transfection efficiency make them an excellent candidate for targeted siRNA therapy. There is an immense scope to study PDMAEMA based vectors as cancer therapeutic carriers for siRNA drugs in platinum-resistant and high-grade ovarian cancer. Therefore, this study aims to develop well-defined biocompatible polymeric nanocarriers for targeted siRNA (ROR2 siRNA) therapy in cisplatin resistance (A2780) and high grade serous ovarian cancer (HGSOC) cells. Polymeric systems containing PDMAEMA as siRNA condensing core have been employed with various structural modifications to investigate the delivery efficiency and therapeutic potential of siRNA drugs. The ability of polymeric nano-vector either generated from PEGylation or BSA modification of PDMAEMA to efficiently bind and release ROR2 siRNA in both 2D and 3D ovarian cancer cell model have been investigated. It was observed that the inhibitory effect of ROR2- siRNA encapsulated in the core of polyion complex (PIC) is strongly dependent on the polyPEGMEMA block length. Reduction of ROR2 expression in both transcription and translation levels was observed in A2780 cells. Alternatively, the BSA modified PDMAEMA nanoparticles present a biocompatible approach for targeted siRNA delivery in HGSOC cells. BSA decorated nanoparticles have shown potential to deliver ROR2-siRNA efficiently in the cytoplasm and hence displayed a significant reduction in migration and invasive features of HGSOC cells.

  • (2022) Srivastava, Pallavi
    Embryogenesis is a complex process orchestrated through local morphogen gradients and physical constraints that give rise to the three germ layers. In vitro models of embryogenesis have been demonstrated by treating pluripotent stem cells in adherent or suspension culture with soluble morphogens and small molecules, which leads to tri-lineage differentiation. However, treatment with exogenous agents override the subtle spatiotemporal changes observed in vivo that ultimately underly the human body plan. In this thesis, we demonstrate how deformable hydrogels for pluripotent stem cell culture catalyse gastrulation-like events using materials alone. Micro-confinement through soft lithography enhances cell-cell adhesion and proliferation within the colony, where stress at the interface leads to mechanosensing mediated nuclear shape changes, epithelial to mesenchymal transition, and emergence of defined patterns of primitive streak containing SOX17+ T/BRACHYURY+ cells. Immunofluorescence staining, transcript analysis, and the use of pharmacological modulators reveal a role for mechanotransduction-coupled non-canonical wingless-type (Wnt) signalling and YAP1 signalling dynamics in promoting epithelial to mesenchymal transition at the interface, and multilayered organization within the colonies. These microscale gastruloids were removed from the substrate and grown in suspension culture. Rather than uniform growth as observed using traditional embryoid body culture, the gastruloids undergo multi-lobed outgrowth with evidence of further differentiation both in vitro and in vivo. Encapsulating the gastruloids into several hydrogel biomaterials indicates that materials properties will influence the interfacial positioning of primitive streak, which may prove a useful strategy to further direct differentiation. Together, this thesis demonstrates how materials alone can nurture embryonic gastrulation, with defined microenvironmental cues that mimic pre-streak events, thereby providing an in vitro model of early development. This work provides a new tool to link the biophysical and biochemical parameters of the local microenvironment to cell and tissue morphogenesis for fundamental studies. Using materials to guide morphogenesis in the laboratory provides an approach for directing differentiation, towards new materials-directed organoid models for tissue engineering and regenerative medicine.

  • (2022) Yu, Tsz Tin
    The rapid emergence and development of antibacterial resistance is a major global threat to public health. There is an urgent need for the development of antibacterial agents with novel therapeutic strategy to tackle the increasing incidence of antibacterial resistance. In recent years, antimicrobial peptides (AMPs) and their synthetic mimics have been under the spotlight of the development of a novel class of antibiotics to combat antibiotic resistance. This research project focused on the utilisation of phenylglyoxamide and benzothiazole scaffolds in the development of antimicrobial peptidomimetics. The synthesis of phenylglyoxamide-based peptidomimetics was achieved via the ring-opening reactions of N-sulfonylisatins with primary amines followed by salt formation. Minimum inhibitory concentrations (MIC) of the peptidomimetics against different bacterial strains were determined to assess their antibacterial activity. Structure-activity relationship (SAR) studies revealed the inverse relationship between the alkylsulfonyl chain length and the bulkiness of the phenyl ring system for high antibacterial activity. The most active peptidomimetics exhibited high antibacterial activity with the lowest MIC to be 4, 16 and 63 μM against S. aureus, E. coli and P. aeruginosa, respectively. These peptidomimetics also showed significant biofilm disruption (up to 50%) and inhibition (up to 70%) against S. aureus at 2–4× MIC. In addition, terphenylglyoxamide-based peptidomimetics synthesised by the ring-opening reaction of N-acylisatins with amines and amino acid esters were evaluated for their quorum sensing inhibition (QSI) activity against P. aeruginosa MH602. The most potent peptidomimetic possessed high QSI activity of 82%, 65% and 53% at 250, 125 and 62.5 μM, respectively, with no bacterial growth inhibition. On the other hand, benzothiazole-based peptidomimetics were synthesised via the Jacobson method of cyclisation of phenylthioamides, followed by the installation of cationic groups. 2-Naphthyl and guanidinium hydrochloride as the hydrophobic and cationic groups, respectively, were important for high antibacterial activity of the peptidomimetics against both Gram-positive and Gram-negative bacteria. The most potent peptidomimetics against S. aureus, E. coli and P. aeruginosa possessed MIC values of 2, 16 and 32 μM, respectively. These active peptidomimetics inhibited 39% of S. aureus biofilm formation and disrupted 42% of preformed S. aureus biofilms at sub-MIC.

  • (2022) Gadde, Satyanarayana
    High-risk neuroblastoma is one of the most aggressive and treatment-refractory childhood malignancies. MYCN (v-myc avian myelocytomatosis viral related oncogene, neuroblastoma derived) is a major oncogenic driver for neuroblastoma (NB) tumorigenesis. Developing direct inhibitors of MYCN has been challenging due to several limitations. Hence, targeting MYCN-binding proteins which regulate the stability of MYCN protein is a promising alternative approach. This study is aimed at developing novel inhibitors of ubiquitin specific protease 5 (USP5), a deubiquitinating enzyme, which is known to prevent MYCN protein degradation by deubiquitination. The first results chapter describes the synthesis of novel pyrido[1,2-a]benzimidazole compounds and their cytotoxicity against MYCN amplified NB cells with high expression of USP5 protein (SK-N-BE(2)-C and Kelly cells). However, none of the tested compounds displayed better cytotoxicity than the parental compound, SE486-11. The second results chapter describes a one-pot synthesis of novel γ-carbolinone, γ-carboline and spiro[pyrrolidinone-3,3′]indoles. One of the γ-carboline compounds (42d) displayed promising cytotoxicity against NB cells (SK-N-BE(2)-C (IC50 = 1.21 μM) and Kelly (IC50 = 3.09 μM)) but showed little therapeutic selectivity when compared to normal human fibroblasts, MRC-5 cells (IC50 = 3.75μM). The synthesis and cytotoxicity of novel pyrimido[1,2-a]benzimidazoles is described in the third results chapter. The active compound, 65a displayed promising cytotoxicity against SK-N-BE(2)-C (IC50 = 0.78 μM) and Kelly (IC50 = 2.00 μM) cells with a reasonable therapeutic window compared to MRC-5 cells (IC50 = 15.0 μM). 65a bound to USP5 protein by microscale thermophoresis assay (Kd = 0.47 µM). USP5 and MYCN protein levels were decreased in NB cells by treatment with 65a. Moreover, the cytotoxicity of 65a was dependant on the expression of USP5 and MYCN proteins. 65a showed synergy in combination with HDAC inhibitors, SAHA and panobinostat. In the fourth results chapter, the synthesis of more potent pyrimido[1,2-a]benzimidazoles with di- and tri- substitutions on the pendant phenyl ring (86b (SK-N-BE(2)-C IC50 = 0.31 μM; Kelly IC50 = 0.65 μM) and 91 (SK-N-BE(2)-C IC50 = 0.03 μM; Kelly IC50 = 0.07 μM)) are described. Importantly, 86b displayed significant in vivo efficacy in TH-MYCN homozygous NB mice when treated with 60 mg/kg for three weeks. The last results chapter describes the synthesis and cytotoxicity of novel benzo[4,5]imidazo[2,1-b]thiazole and pyrido[2,3-b]indole compounds. Collectively, this thesis identifies promising novel scaffolds with great potential for further development.

  • (2022) Amaldoss, Maria John Newton
    Nanocatalytic tumor therapies involve established strategies to increase the concentration of endogenous oxygen species (ROS) H2O2 to cytotoxic levels. These strategies are based on increasing the ROS levels through stimuli from drugs, the action of ROS-producing agents, and nanoparticulate catalysis. However, these techniques frequently are indiscriminatory, being cytotoxic to diseased cells and normal cells alike, leading to significant unwanted side-effects. The present work reports a new paradigm strategy based upon the catalytic action of a cell-discriminative, ROS-mediating, autophagy-suppressive nanoparticle, which is CePO4·H2O (rhabdophane). CePO4·H2O nanoparticles were synthesised using CeNO3·6H2O precipitated in an aqueous solution of sodium tripolyphosphate (STPP) at room temperature. The nanoparticles were well crystallised, equiaxed (~10-35 nm), of positive surface charge, and of general valence ratio 〖"Ce" 〗_"0.8" ^"3+" 〖"Ce" 〗_"0.2" ^"4+" 〖"PO" 〗_"4.1" . Materials characterisation involved particuological (hydrodynamic particle size, surface area, zeta potential), mineralogical (X-ray diffraction, laser Raman microspectroscopy), chemical (X-ray photoelectron spectroscopy), structural (Fourier transform infrared spectroscopy), and microstructural (transmission electron microscopy) analyses. Biological characterisation involved examination of the effects on HT-1080 fibrosarcoma cells and MRC-5 normal fibroblasts in terms of cellular interactions (cell viability by MTT assay), cellular uptake and trafficking (confocal laser scanning microscopy, biological transmission electron microscopy, flow cytometry), ROS generation (confocal laser scanning microscopy, flow cytometry), apoptosis (annexin V-FITC assay), gene expression (q-RT-PRC), and protein expression (western blot analyses). The key observations and conclusions from the biological evaluation are as follows: Discriminative Cytotoxicity: CePO4·H2O nanoparticles are the first to exhibit discriminative cytotoxicity: At 24 h, fibrosarcoma HT-1080 cell viability is ~10% but MRC-5 normal cell viability is ~45%. Discriminative Uptake: CePO4·H2O nanoparticles are the first, without the use of a targeting ligand, to be internalized readily by cancer cells but scarcely by normal cells. Self-Targeting: CePO4·H2O nanoparticles are trafficked toward the mitochondrial environment and possibly the converse trafficking. Mitochondrial Starvation: The preceding proximity between CePO4·H2O nanoparticles and cancer cell leads to increased phosphate concentration in the cellular environment, the concentration gradient of which effectively starves the mitochondria, leading to mitochondrial stress and dysfunction. Discriminative ROS Generation: CePO4·H2O nanoparticles are the first to demonstrate elevated cellular ROS in cancer cells by multiple mechanisms while normal cells exhibit only a low level of such elevation. Autophagy Suppression: CePO4·H2O nanoparticles suppress autophagy, thereby increasing cellular stress and suppressing cancer cell survival, thus offering a complement to mitochondrial starvation. Redox Switching: CePO4·H2O nanoparticles are the first nonmetallic nanoparticles to balance redox switching through simple electronic charge compensation rather than more complex ionic charge compensation. Biocompatibility: As hydrated phosphates, CePO4·H2O nanoparticles are more biocompatible than metals or oxides, suggesting greater feasibility of renal clearance. These advantages derive from the key role of the redox and defect equilibria arising from the oxidation reaction Ce3+ → Ce4+ + e′, which is induced by the acidic pH environment of the cancer call versus the stability of the Ce3+ valence in the basic pH environment of the normal cell. The former both elevates the ROS level and disrupts the electron transfer chain. Ultimately, the suppression of the proliferation of cancer cells derives from the cross-talk involving cellular ROS elevation, autophagy suppression, and their mitochondrial control.

  • (2023) Browne, Kate
    Healthcare-associated infections are the most common adverse event affecting hospital patients. With the emergence of antibiotic-resistant bacterial strains, there is an increasing number of untreatable infections. Therefore, there is a clear need for the development of novel antibacterial strategies to both prevent, and treat, healthcare-associated infections. This thesis explores three key approaches to treat and prevent the transmission of healthcare-associated infections. First of all, a library of novel antibacterial compounds were assessed in vitro for their clinical potential. The compounds with the greatest clinical potential were then tested in various biomaterial applications (biocompatibility in an animal model, and their activity when covalently attached to biomaterial surfaces). Finally, this thesis explores antibacterial strategies to prevent the transmission of bacteria from clinical surfaces. The lead antimicrobial candidates, melimine and RK758, are peptidomimetics that address the drawbacks of conventional antibiotic therapy. They showed broad-spectrum antibacterial activity (including clinical antibiotic-resistant isolates), anti-biofilm activity and thermal stability. When peptidomimetics were used in combination with traditional antibiotics, bacteria were unable to develop resistance towards antibiotics or peptidomimetics. This combination antibiotic-peptidomimetic therapy also showed synergistic activity, which further increases the potential for these compounds to treat multidrug-resistant infections. When assessed in two clinical applications, peptidomimetics showed promise for their continued clinical development. Both melimine and RK758 were biocompatible in animal models. When peptidomimetics were loaded into bone allograft, there was no inhibition of bone healing in a critical-sized distal femur defect model. Moreover, when these peptidomimetics were covalently attached using a new polydopamine coating method to biomaterial surfaces, they retained their broad-spectrum antibacterial activity and prevented biofilm formation in Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. This thesis also explores the transmission of pathogens from clinical surfaces, and how ultraviolet-C disinfection could be used in healthcare-facilities to reduce the bacterial bioburden on various clinical surfaces. Overall, adjunct ultraviolet disinfection significantly reduced the bioburden on surfaces in operating theatres and clinical rooms. Overall, these three strategies work together to prevent healthcare-associated infections. Preventing the transmission of pathogens from the environment to patients, is known to reduce healthcare-associated infection rates. Furthermore, the development and application of novel-acting antibiotics offers a solution to the antibiotic-resistance crisis and addresses the urgent need for antimicrobials with broad-spectrum antibacterial activity. If commercialised, the strategies proposed in this thesis have the potential to increase activity of ineffective antibiotics and prolong their use in the clinic.