Jayachandran Kizhakkedathu

Faculty of Medicine

Research Classification

Medical and biomedical engineering

Pathology (except oral pathology)

Research Interests

Biomaterials

Blood Coagulation

Cell-surface Engineering

Implants and Medical Devices

Iron Chelators

Macromolecular Therapeutics

Polymers

Proteomics Tools

Thrombosis

Relevant Thesis-Based Degree Programs

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Affiliations to Research Centres, Institutes & Clusters

Life Sciences Institute

Centre for Blood Research

Bionics Network

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Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Development of a liver targeted macromolecular iron chelator for the treatment of iron overload (2021)

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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Design and development of universal antibiofilm coatings for urinary catheters (2020)

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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Glycocalyx engineering: development of biomaterials, ligation strategies and in vitro systems to modulate inflammation at the endothelial surface (2019)

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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The development of novel antimicrobial peptides and various strategies to improve their activity and biocompatibility (2018)

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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Novel anticoagulant neutralizing agents for the management of bleeding: studies on the design, mechanism of action and their influence on blood coagulation (2017)

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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Progesterone-binding Modified Hyperbranched Polyglycerols: Synthesis, Characterization and Biological Assessment (2016)

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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Innovative polymeric iron chelators with iron binding affinity and biocompatibility for the treatment of transfusional iron overload (2015)

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) ( 600 Da) and the objectives reported for the “ideal” chelator of low MW is yet to be realized in practice. However, the limited attempts towards developing higher MW, long circulating iron chelators has shown tremendous promise. This thesis assesses the role of a new polymer, hyperbranched polyglycerol (HPG) in improving the properties of iron chelators. High MW iron chelators were developed by conjugating DFO to HPG of various MWs, forming a library of HPG-DFO conjugates. Iron binding affinity of HPG-DFO was investigated using isothermal titration calorimetry, UV-visible spectroscopy and studying iron removal from ferritin. Biocompatibility and toxicity were investigated using coagulation assays in human blood and cell culture. Since iron chelator toxicity during development remains an under-explored area, the second goal of this thesis was to expand knowledge of chelator toxicity during development. The toxicity of FDA-approved and HPG-DFO in developing embryos was investigated using zebrafish. Studies indicate that HPG-DFOs are biocompatible, efficient chelators, capable of binding ferritin iron and preventing harmful redox reactions. Moreover, combining a low MW iron chelator with HPG-DFO enhances chelation. In vivo chemical screening indicated that while low MW chelators L1 and ICL-670 may interact with zebrafish embryos and cause toxicity, DFO and HPG-DFO did not have this effect. Results indicate that HPG-DFO is a new class of efficient, biocompatible iron chelator, potentially useful for development into clinical agents for the prevention of transfusion associated iron overload.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

The development of an anti-thrombotic and anti-inflammatory coating for medical devices (2024)

Blood-contacting biomaterials are commonly and increasingly used in medical devices including for example, vascular grafts, coronary stents, prosthetic heart valves, and extracorporeal pumps. A major and persistent problem of exposing blood to a foreign biomaterial surface is that a thromboinflammatory response is triggered that may cause device failure and/or systemic inflammation and clotting. Several biomaterial coatings, particularly heparin, are currently used to prevent thromboinflammation, but these do not reliably work. The goal of my thesis was to develop a novel blood compatible surface coating that simultaneously prevents thrombosis and inflammation by immobilizing anticoagulant and anti-inflammatory molecules in a more functional manner, onto a universal surface coating. This thesis is divided into two parts. In the first part, I chemically modified unfractionated heparin, a potent anticoagulant, on one end such that it could be conjugated to the universal antifouling surface coating in an orientation that optimally and uniformly exposed its anticoagulant antithrombin binding site. The functional properties of this heparin coating were tested under static and dynamic conditions, and these verified that this novel heparin coating was protective against thrombosis. In the second part, I modified the cDNA encoding the naturally occurring anti-inflammatory lectin-like domain of thrombomodulin (TM-LLD), such that it could be expressed by cultured mammalian cells, purified by affinity chromatography, and then conjugated to the antifouling surface coating in an accessible orientation. The purified soluble TM-LLD exhibited anti-inflammatory properties, in that it dampened leukocyte adhesion and complement activation. However, its activity when bound to the surface coating has yet to be confirmed. While further studies are required to optimize the function and to test the stability of these immobilized molecules, this thesis has provided proof-of-concept that the potent anti-coagulant, heparin, and the anti-inflammatory TM-LLD, can be chemically modified for conjugation to the antifouling surface in an orientation to optimize function. Future work will include experiments to upscale production of the modified proteins, to verify their functions and stability under different conditions, and finally, to apply combinatorial approaches to simultaneously bind the molecules to the coating to yield a truly biocompatible, biomaterial surface that prevents both thrombosis and inflammation.

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A novel non-glucose based osmotic agent with liver uptake potential for peritoneal dialysis solutions (2021)

Peritoneal Dialysis (PD), a treatment modality for end-stage renal disease (ESRD) uses high concentrations of glucose in its dialysis solutions. Since, 40% ESRD patients are diabetic, use of glucose-based PD solutions is non-rational because of the local and systemic adverse effects of glucose. There is a need for a biocompatible, non-glucose based osmotic agent that can replace glucose in peritoneal dialysis solutions. Recent studies on low molecular weight hyperbranched polyglycerol (HPG) as an osmotic agent show good ultrafiltration and reduction of peritoneal membrane injury but elimination through the kidneys. Our studies show that HPG is excreted via kidneys, and in ESRD patients, kidney function is impaired which could potentially result in poor excretion of this osmotic agent. N-acetylgalactosamine (GalNAc) carrying proteins and macromolecules has shown to be effectively up taken by the liver through asialoglycoprotein receptor (ASGPR). Thus, we hypothesize that HPG modified by GalNAc conjugation can be recognized by ASGPR in the liver, taken up and excreted through feces. GalNAc epoxide was synthesized by standard organic modifications and conjugated with HPG (3K) at two different densities (one or three GalNAc molecules per HPG) (denoted as HPG+1S or HPG+3S). The polymers were labeled with alexa647 and screened for effective internalization in a panel of hepatocyte cell lines with different levels of ASGPR expression using flow cytometry. Results of dose-dependent uptake of polymers and ASGPR staining of hepatocytes suggested receptor mediated internalization of HPG+3S in HepG2 and HuH7.5.1 cells. Kₘ values were assessed to determine the efficacious HPG conjugate followed by competitive inhibition with natural ligands of ASGPR in HepG2 cells. HEK293 cells ectopically expressing ASGPR1 and ASGPR1&2 genes were used to confirm HPG+3S uptake through ASGPR specifically. Mice tissue distribution studies of radiolabeled HPG and HPG+3S showed better uptake of HPG+3S by the liver than HPG. Percentage of HPG+3S excreted via feces was significantly more than HPG in mice with normal kidney function, which support the enhanced binding of HPG+3S to ASGPR in hepatocytes. In conclusion, both our in vitro and in vivo results substantiate our claim that GalNAc conjugated HPG can be rerouted to be excreted via feces.

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Developing releasable antimicrobial peptide-polyethylene glycol conjugates by targeting infection site-associated host matrix metalloproteinases (2021)

The rapid generation of multidrug-resistant (MDR) bacteria has caused bacterial infections to become a global health concern. Antimicrobial peptides (AMPs), or host defence peptides (HDPs), offer a viable solution to these pathogens due to their broad-spectrum activity and low generation of resistance. In addition, many AMPs possess immunomodulatory properties (e.g., anti-inflammatory activity) that may provide a more robust treatment of infection. However, high toxicity and short biological half-lives have greatly limited the production of clinically available AMP therapeutics. Conjugation of the peptides to delivery vehicles such as polyethylene glycol (PEG) has significantly improved these properties but has also been associated with large reductions in antimicrobial activity, making formulation challenging. In this thesis, an enzymatically releasable PEG-AMP delivery system was developed by incorporating a cleavage sequence susceptible to matrix metalloproteinases (MMPs), enzymes released by the host during the inflammatory response to infection, onto an aurein 2.2-derived AMP. N- vs. C-terminal addition of the sequence found the former to best maintain the activity of the AMP after MMP cleavage, likely due to the maintenance of its amidated C-terminus and higher positive charge. Subsequent conjugation of the cleavable AMP to 2 kDa PEG significantly improved the AMP’s blood biocompatibility in vitro but also eliminated its activity until cleaved by isolated human MMP. This activity was mimicked in an in vivo abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, where both free peptide and conjugate displayed strong activity confirmed to be dependent on the accumulation of MMPs at the infection site, as non-cleavable D-isomeric counterparts of the compounds showed no activity. Following this, the system was expanded to larger PEG molecules by incorporating a tetraglycine spacer between carrier and MMP cleavage sequence. This spacer enabled cleavage of the AMP when bound to 5, 10, and 22 kDa PEG, not possible for the initial peptide, allowing for further improvements in biocompatibility compared to the 2 kDa PEG-AMP conjugate. Altogether, the enzyme-releasable delivery system developed here may provide a suitable platform for the development of infection site-targeting AMP therapeutics where both high biocompatibility and activity can be achieved.

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Development of a cardiac-targeted macromolecular chelator for the treatment of iron overload (2020)

Disorders of hemoglobin, such as sickle cell anemia and thalassemia, are an increasing global health concern for which patients must regularly receive blood transfusions. Unfortunately, chronic blood transfusion can lead to a condition known as transfusion-associated iron overload wherein iron becomes available for catalytic reactions that cause oxidative damage to cells. Further, because the human body lacks an iron excretion pathway, excess iron accumulates in vital organs such as the liver, heart and endocrine organs leading to organ damage and failure. The current standard of care is chelation therapy using small molecule Fe³⁺ chelators such as deferoxamine (DFO), deferiprone (L1) and deferasirox (DFX) which suffer from short circulation time, low iron excretion efficiency, and adverse side effects. To date, no methods are available to remove iron from specific organs to prevent organ toxicity; consequently, iron overload remains associated with significant morbidity and mortality. Recently, the Kizhakkedathu group demonstrated conjugation of DFO onto hyperbranched polyglycerol (HPG) improves the circulation profile of the drug (Hamilton, 2017). In addition, EP4 receptors, which prostaglandin E₂ (PGE₂) has high affinity for, has been shown to be abundant in the heart tissue of several species including humans (Regan, 1994; Castleberry, 2001; Sugimoto, 2007). Thus, I hypothesize that the use of PGE₂ as a cardiomyocyte targeting moiety in a macromolecular chelation approach would greatly enhance iron chelation efficacy of DFO in cardiomyocytes and protect heart from iron mediated injury.In this thesis, a novel macromolecular cardiac-targeted macromolecular iron chelator, termed CTMC, has been developed and assessment of its targeting ability, circulation and biodistribution, toxicity and iron removal efficiency have been conducted. CTMC was selectively taken up by cardiomyocytes in an in vitro co-culture model. CTMC was taken up 11.5-fold higher than non-targeted controls in the hearts of female C57BL/6 mice and shows long circulation. Studies conducted in an in vitro iron overload model of HL-1 murine cardiomyocytes demonstrated that after 2 days of treatment, CTMC was able to reduce intracellular iron levels and reactive oxygen species generation to those of baseline. Overall, the presented work suggests that CTMC is a promising candidate for organ-specific iron removal.

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Investigation of complement activity induced by hyperbranched polyglycerol grafting to red blood cell membranes (2015)

Repeated transfusion of red blood cells (RBCs) is the only treatment modality currently available for certain blood related genetic disorders such as thalassemia and sickle cell anemia. Due to chronic transfusion of RBCs in these patients, clinical problems surrounding alloimmunization develops in approximately 30% of patients. The pathology arises from adverse immune reactions to minor antigens that are either not routinely typed for, or cannot be readily matched. Hence, the development of donor RBCs that reduces the risk of alloimmunization would be highly beneficial. An innovative approach to address this problem involves the use of polymers to mask the immunogenic blood group antigens on RBC membranes. Given potential applications of polymer grafted RBCs, non-toxic and non-immunogenic materials are desired. In this research, we have investigated the covalent attachment of hyperbranched polyglycerols (HPG), a highly biocompatible polymer, to red blood cell surfaces. The aim is not only to shield immunogenic blood group antigens, but also to prevent the degradation of biomaterial modified cells by the immune system, particularly by the proteolytic convertases of the complement system. We investigated the mechanism of complement activation on HPG modified cells, and the influence of various polymer properties, including: grafting concentration, molecular weight, and degree of HPG functionalization in an effort to optimize the grafting process on cells. Traditional assays using antibody sensitized sheep erythrocytes and rabbit erythrocytes were used to assess the overall complement activation. Complement activation products C4a, C3a, Bb, and SC5b – 9 were quantified by ELISAs to determine the specific pathway of complement activation by HPG modified RBCs. Flow cytometry was also performed to demonstrate the effectiveness of antigen protection by the different graft properties.HPGs with a molecular weight greater than 28 KDa at grafting concentrations greater than 1.0 mM, as well as a high degree of HPG functionalization result in the activation of complement via the alternative pathway. No activation was observed when these threshold levels were not exceeded. These insights may have an impact on devising key strategies in developing novel therapeutics, especially in the fields of both transfusion and transplantation medicine.

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Bioactive Polymers: A Comparative Study on the Antithrombotic Properties of Soluble Polymers and Surface Grafted Polymers (2010)

Use of synthetic materials in medical applications is one of the most common practices in modern medicine. Yet occurrence of surface-induced thrombus formation on these materials, especially those associated with cardiovascular applications, generates a need for surface modifications. Limiting thrombus formation on a biomaterial surface represents the ultimate success for blood contacting devices. One interesting approach is to enhance fibrinolysis before the blood clot becomes stabilized. Herein, two synthetic polymers, poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide (PDMDOMA) and poly- (N-isopropylacrylamide) (PNIPAm), were tested for this particular antithrombotic property. Surface-grafted PNIPAm samples, brush-PNIPAm and star-PNIPAm, were also tested for the biological activity.We evaluated the influence of these synthetic polymers on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties of blood clot such as its clot strength, clot elasticity and clot fibrinolysis. Both linear PDMDOMA and PNIPAm altered the normal fibrin polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of these synthetic polymers exhibited thinner fibers with less branching and resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The structural changes in these clots led to significant difference to their mechanical properties. Lower clot strength and clot elasticity were recorded from the thromboelastography study. More interestingly, enhanced clot lysis was measured by thromboelastography when whole blood was clotted in presence of PDMDOMA or PNIPAm. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease in D-dimer levels measured from degraded plasma clot. Similar results were obtained when star-form of PNIPAm was used but not for brush-form PNIPAm.The antithrombotic activity of soluble PDMDOMA and PNIPAm could potentially lead to the development of novel antithrombotic agents that could enhance endogenous fibrinolytic activity by modulating the fibrin clot structure. In the exploratory analysis of surface grafted PNIPAm (brush-PINPAm), brush-PNIPAm showed that the biological activity of attached chains is quite different from soluble polymers and several parameters need to be optimized to generate an antithrombotic coating for biomaterials.

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