Jayachandran Kizhakkedathu

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Patients with thalassemia, myelodysplastic syndromes, sickle cell disease and other acquired anemia require life sustaining and often repeated red blood cell transfusions. Since humans lack an iron excretion pathway, excess iron results in systemic iron overload either due to an underlying genetic component or acquired through disease pathogenesis or repeated transfusions. Toxicity arises from the generation of reactive oxygen species and elicits considerable damage. The resulting iron toxicity accounts for a majority of premature deaths, primarily from liver and heart dysfunction and failures. The current standard of care for the treatment of transfusion-dependent iron overload is iron chelation therapy, which effectively reduces the toxicity associated with labile iron by lowering the iron burden. However, the toxicity, shorter circulation time and non-specificity of the current iron FDA approved iron chelators proved challenging and patient compliance are poor for this life-long therapy.Recently, Dr. Kizhakkedathu’s team developed a macromolecular iron chelating system with decreased toxicity, increased half-life and iron excretion profiles compared to the current standard iron chelator, deferoxamine in mice. Further, since the discovery of the asialoglycoprotein receptor and its specificity for N-acetyl galactosamine, there has been significant research pertaining to liver targeting and delivery of various drugs. Thus, we hypothesize that a macromolecular iron chelating system conjugated with liver targeting groups would enhance the iron removal from liver thereby preventing complications due to iron overload.In this thesis, a novel class of liver targeted macromolecular iron chelators were developed for the treatment of iron overload. The macromolecular scaffold was optimized for hepatocyte uptake in vitro and HPG-GalNAc₅₀ and HPG-TAG₂ were selected for iron chelation. The tolerability, biodistribution, and excretion of liver targeted iron chelating systems, HPG-DFO-GalNAc and HPG-DFO-TAG, were investigated in vivo. Remarkably, HPG-DFO-GalNAc and HPG-DFO-TAG exhibited significant hepatocyte accumulation with immediate lysosomal localization and subsequent rapid excretion with over 70% eliminated within 24 h. Liver targeted iron chelating systems with higher DFO units translated into superior systemic iron removal in a mice iron overload model. This thesis demonstrates the utility of liver targeted iron chelator for the removal of excess iron.

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Catheter-associated urinary tract infection is one of the most common medical device-associated complications that has caused significant morbidity, mortality and costs. There is a significant need for new technologies to prevent such catheter-related infections. Despite advancements in the development of antimicrobial and antibiofilm coatings in recent years, current coating technologies to prevent biofilm formation fail to address all factors, including prevention of biological deposition, inhibition of bacterial colonization, adaptation to diverse materials, easy application to devices of various sizes and shapes, and stability of the coating. This thesis addresses my attempts to explore new knowledge and develop novel technologies to address this important medical need. In Chapter 2, by using a high throughput screening method, we identified a highly durable thin hydrophilic coating which prevents biofilm formation over a long-term period (>4 weeks) in the presence of high concentration of bacteria. Furthermore, this coating can easily be applied to diverse substrates of varying shapes and material properties via a dip coating process and demonstrates a broad spectrum of bacterial adhesion resistance. When the coating was applied to commercial catheters, biofilm formation was consistently less with coated catheter than with uncoated catheters both in vitro and in vivo.In Chapter 3, we propose a new mechanism for the stable coating formation between polycatecholamines and hydrophilic polymers. The hydrophilic polymers have an active role in the co-assembly and co-deposition process, which is influenced by the molecular weight and chemistry of the hydrophilic polymer. We determined that the self-assembly of different polycatecholamines is influenced by different polymers but the nature of polycatecholamine is not the major factor that influences the final characteristics of the coating. In Chapter 4, a facile layer-by-layer assembly process of a hydrophilic polymer with a natural polyphenol tannic acid was used to fabricate stable bacteria-resistant multilayers with controlled thickness. We demonstrated that the main driving force in this layer-by-layer assembly process is the hydrogen bonding between the polymers and tannic acid. This work demonstrates the fabrication of novel bacteria-resistant coatings and provides a potential platform incorporate antimicrobial agents for the development of multifunctional coatings.

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Residing at the interface between circulating blood and the vessel wall, the endothelial glycocalyx is a prominent regulator of the immune response. Existing as a highly complex, glycoprotein-rich brush-like structure on the surface of the endothelium, it dynamically regulates endothelial behavior by changing its architecture and chemical composition. As such, it is an interesting target for cell surface engineering (CSE), a methodology in which the cell surface is chemically or genetically tailored to modulate cellular behavior. Glycocalyx engineering can also be explored as a therapeutic iteration of CSE, as the breakdown of the glycocalyx plays a pivotal role in the immune regulated pathways that initiate organ damage and rejection at the blood vessel surface. Though a variety of preventative strategies have been developed to attenuate immune-mediated organ damage, none exist which actively reverse glycocalyx damage to re-initiate the native immunoprotective structure on the cell surface. In this body of work, we present new techniques and tools for engineering the glycocalyx surface with glycocalyx mimicking polymers. Architecturally defined, biocompatible polymers like hyperbranched polyglycerol (HPG), linear polyglycerol (LPG) and polyethylene glycol (PEG) were explored as promising candidates for recapitulating glycocalyx function as they are non-immunogenic and can be easily functionalized post-polymerization. Utilizing the enzyme tTGase in concert with Q-tagged polymers, we developed a CSE technique that mediates extensive and gentle attachment onto lysine substrates on the endothelial glycocalyx. Once attached to the cell surface, the polymers were able to re-establish the immunosuppressive barrier functions of the glycocalyx following breakdown. Next, we built upon the therapeutic efficacy of our strategy by introducing immunosuppressive sialic acids onto the LPG-Q scaffold. Endothelial cell surfaces engineered with α2,3 sialic acid-decorated glycopolymers provided a potent localized therapy which combined sterically driven immunocamouflage against leukocyte binding with sialic acid-mediated CD8+ T-cell and NK-cell inhibition. The immunosuppressive nature of this CSE strategy was also recapitulated in vivo using an arterial transplant model.Finally, we presented two new tools designed to improve the control over and assessment of glycocalyx engineering strategies in cell culture by re-directing CSE in the longitudinal direction and developing physiological relevant glycocalyx structures in vitro.

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With the advent of antibiotic resistance and crisis, it is crucial to find substitutes to conventional antibiotics. Antimicrobial peptides (AMPs) are considered to be viable alternatives, because they are broad spectrum and bacteria develop little or no resistance towards AMPs. Interestingly, only few AMPs are used as therapeutics, due to problems such as host toxicity, protease cleavage and short half-life. Therefore, there is a need to improve the efficacy of AMPs by the use of D-peptides and/or delivery vehicles. The introduction of the thesis describes the diversity and various mechanisms of action (MOA) of AMPs. The issues and ways to improve the efficacy of AMPs, which forms the foundation of this thesis, are also discussed.Recently, hyperbranched polyglycerol (HPG) has gained attention due to its excellent biocompatibility, multifunctionality and long blood circulation time. The body of the thesis describes a methodology to covalently attach aurein 2.2 and its mutants to HPG and study the influence of the molecular weight on the antimicrobial activity. A peptide array was used to design tryptophan and arginine mutants of aurein 2.2. Mutant peptide 77 had significantly superior antimicrobial and antibiofilm activity compared to aurein 2.2 but was more toxic. We found that HPG can be used as a general scaffold to alleviate the toxicity of the peptides. The conjugates/peptides were tested in an in vivo mice skin infection (abscess) model. Surprisingly, peptide 73 and aurein 2.2 has similar efficacy in vivo indicating both the antimicrobial activity and toxicity, i.e. therapeutic index, are important. The conjugates (HPG-73c) were not active in mice abscess model, whereas 73c and D-73 encapsulated in micelles composed of DSPE-PEG2000 had excellent activity suggesting the release of the peptide from the delivery vehicle is necessary for in vivo activity. Without encapsulation D-73 was too toxic. A bacterial expression system was used to produce isotopically (¹⁵N) labeled aurein 2.2 and its interaction with whole bacterial cells was examined by nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM) confirming the MOA. Finally, the results presented will be discussed in the broad context of designing AMPs for therapeutics and understanding their MOA.

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Heparins exert anticoagulation by potentiating anti-factor (F)Xa and anti-thrombin activity of antithrombin (AT), whereas oral anticoagulants (DOACs) directly target FXa or thrombin. FXa and thrombin are the key proteases required for blood clotting. Anticoagulants are therefore used for the prophylaxis and treatment of thrombosis and during surgeries. However, anticoagulation associated haemorrhage is a concern. The only approved antidote for unfractionated heparin (UFH), protamine do have limitations including cardiovascular complications. No approved antidotes are available for low molecular weight heparins, fondaparinux and direct FXa inhibitors. Therefore, there is a need for antidotes that are nontoxic and efficient. In this thesis, we reveal the mechanism of action, hemocompatibility and efficiency of three antidote molecules that are under development. UHRA: UHRA is a synthetic antidote developed by the Kizhakkedathu laboratory at the UBC. Thermodynamics based on isothermal titration calorimetry (ITC) and fluorescence studies revealed the molecular design of UHRA, the importance of steric shield produced by PEG brush, the selectivity of UHRA against heparins and its mechanism of action. Clotting studies confirm the antidote activity of UHRA. Studies also show that UHRA even in the absence of heparins, do not interact with fibrinogen, alter fibrin polymerization or abrogate blood clotting. Unlike protamine, UHRA does not incorporate into blood clots, form clots with normal morphology, and lysis profile. Studies in mice reveal that UHRA reverses UFH anticoagulant activity without the lung injury as seen with protamine. Studies confirm the superiority of UHRA compared to protamine.Andexanet Alfa (AnXa) and PER977: AnXa is a truncated FXa recombinant protein developed by Portola Pharmaceuticals and PER977 is a small cationic molecule developed by Perosphere Pharmaceuticals. ITC confirms high-affinity binding of AnXa to heparin/AT complex and to DOACs studied. PER977 shows weak binding to heparins and no binding to DOACs tested. Both antidotes do not influence fibrin polymerization, fibrin and blood clot architecture even in the absence of anticoagulants. Electron micrographs of blood clots containing edoxaban treated with AnXa or PER977 reveal restoration of impaired fibrin formation. However, in clotting assays, PER977 failed to show antidote activity, whereas AnXa neutralized the anticoagulation activity of all tested anticoagulants.

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Traumatic brain injury (TBI) has been proven as an established risk factor of Alzheimer’s disease (AD). Historically, progesterone (Pro) has been found to promote recovery from moderate TBI. However, the utility of this drug as a TBI treatment is severely hampered by its near total insolubility in water due to its hydrophobicity, which contributes to an inability to rapidly administer the drug after injury. The present work describes the synthesis, characterization, development and in vitro evaluation of nanoparticulate formulations of Pro for treatment of TBI. The nanoparticles developed for Pro consist of a library of hyperbranched polyglycerols (HPGs), which were hydrophobically modified with alkyl chains (C₆,₈,₁₀,₁₂,₁₄,₁₈) to enable loading the hydrophobic drug, and were further modified with MPEG chains to increase the solubility and stability of the formulations. Hydrophobically derivatized HPGs (HPG-Cn-MPEG), also known as dHPG(Cn), were characterized by GPC and NMR methods. Pro encapsulation by and release from the drug-binding pocket was determined through a reverse-phase UPLC method. Combination of binding, release and kinetic studies of the dHPG(Cn)/Pro library presented a relatively high number of drug molecules encapsulated, slow release and stable formulations. In vitro assays, including blood biocompatibility, cytotoxicity and cellular uptake, were performed on dHPG(Cn)/Pro. Blood biocompatibility studies demonstrated that the polymer-drug formulations do not cause significant changes in blood coagulation time (APTT assay), nor have they significant effects on red blood cell aggregation, lysis or platelet aggregation. There was no platelet activation observed in this study. Study of viability of human cortical microvascular endothelial cells and human astrocytoma cells in the presence of dHPG(Cn)/Pro demonstrated no toxicity. Studies on the same cells presented significant uptake with relatively even distribution of the formulation inside the cells. Further investigations indicated no degradation pathway for dHPG(Cn) over short periods of time (~ 8 h). Overall, the in vitro studies suggest that dHPG(Cn) are compatible and harmless to cells, suitable for carrying hydrophobic drugs and molecules, such as Pro, to the target tissues.

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Desferrioxamine (Desferal®, DFO), deferiprone (Ferriprox®, L1) and desferasirox (Exjade®, ICL-670) are clinically approved iron chelators used to treat transfusion associated iron overload, a common condition in patients with severe hemoglobin disorders like β-thalassemia, sickle-cell disease and the myelodysplastic syndromes. The poor pharmacokinetics and inefficacy of iron chelators necessitate administration of almost maximum tolerated doses to achieve adequate iron removal. This causes toxicity ranging from neurological dysfunction in DFO users, agranulocytosis and neutropenia in L1 users, and severe kidney toxicity in ICL-670 treated patients. This also hinders the use of iron chelators during gestation. Thus, developing iron chelators with improved long-term efficacy and reduced toxicity is essential. All currently approved iron chelators are of low molecular weight (MW) (
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