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Innovative polymeric iron chelators with iron binding affinity and biocompatibility for the treatment… Hamilton, Jasmine La Juanie 2015

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  INNOVATIVE POLYMERIC IRON CHELATORS WITH HIGH IRON BINDING AFFINITY AND BIOCOMPATIBILITY FOR THE TREATMENT OF TRANSFUSIONAL IRON OVERLOAD  by   Jasmine La Juanie Hamilton   BSc. (Honors), Trent University, 2009    A thesis submitted in partial fulfillment of the requirements for the degree of    DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES   (Pathology and Laboratory Medicine)     THE UNIVERSITY OF BRITISH COLUMBIA   (Vancouver)    April 2015   © Jasmine La Juanie Hamilton, 2015   ii  ABSTRACT  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. iii  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.  iv  PREFACE  Ethics approval was received from UBC for studies conducted at the Centre for Blood Research (UBC Ethics approval no: H07-02198). All ethics approval for zebrafish experiments was obtained from the University of Saskatchewan and complied with the Canadian Council of Animal Care guidelines and the animal care protocol (2012-0082). This thesis was conducted under the supervision of Jayachandran N. Kizhakkedathu at the Centre for Blood Research UBC, Vancouver. Chapter 1 is published as a first authored, invited review paper in the journal of Molecular and Cellular Therapies. I was responsible for conducting the literature review, conceptualizing and writing the review.  Chapter 2 was conducted in collaboration with the department of Chemical and Biological Engineering at UBC and has been submitted for peer review. I was responsible for synthesizing some of the conjugates tested, designing all experiments, conducting all ITC and ferritin experiments and preparing the initial draft of the manuscript. Imran ul-Haq synthesized some of the conjugates tested, B. Lai and F. Hsu assisted in taking UV readings.  Chapter 3 is based on work published in the journal ACS Nano and describes the blood and biocompatibility, in vivo efficacy and pharmacokinetics of the novel polymeric chelators described herein. I was responsible for conducting in vitro analyses of blood compatibility and toxicity, analyzing data and editing the manuscript, for which I am second author.  Chapter 4 is based on work conducted at the Veterinary Biomedical Sciences Facility at the University of Saskatchewan in Saskatoon during my term as a visiting scholar at the University of Saskatchewan and in collaboration with Dr. Suraj Unniappan who provided supervision during my visit. Imran ul-haq, M synthesized the ICL-670. I was responsible for characterizing the samples that were tested, conducting and optimizing all zebrafish v  experiments with the help of Hatef, A, data collection, analysis and preparing the initial draft of the manuscript, which is published in the journal PLOS ONE. Nair, N provided assistance when conducting the ultrasound study. Some of the rationale for the remarks on biodegradability in Chapter 5 is supported by a third authored study published in the Journal of the American Chemical Society which demonstrated that HPG can be engineerd to degrade under certain conditions. I was responsible for the cell culture  study and confocal imaging in this paper.  Publications Hamilton JL, Imran ul-haq M, Lai BF, Creagh AL and Kizhakkedathu JN. Influence of Molecular Characteristics of Macromolecular Iron Chelators on Fe (III) Binding Thermodynamics and Iron Removal from Ferittin. Submitted 2015.  Hamilton JL and Kizhakkedathu JN. Polymeric Nanocarriers for the Treatment of Systemic Iron Overload. Molecular and Cellular Therapies. 2015, 3:3, DOI: 10.1186/s40591-015-0039-1.  Hamilton JL, Hatef A, Imran ul-haq M, Nair N, Unniappan S and Kizhakkedathu JN. Clinically Approved Iron Chelators Influence Zebrafish Mortality, Hatching Morphology and Cardiac Function.  Plos One 2014; 9 (10):e109880.  Imran ul-haq M, Hamilton JL, Lai BF, Shenoi RA, Horte S, Constantinescu I, Leitch HA, Kizhakkedathu JN. Design of Long Circulating Nontoxic Dendritic Polymers for the Removal of Iron in Vivo. ACS Nano 2013; 7(12):10704-16.  Shenoi RA, Narayanannair JK, Hamilton JL, Lai BFL, Horte S, Kainthan RK, Varghese JP, Kallanthottathil RG, Manoharan M, Kizhakkedathu JN. Branched multifunctional polyether polyketals: variation of ketal group structure enables unprecedented control over polymer degradation in solution and within cells. J. Am. Chem. Soc. 2012; 134 (36):14945-57. vi  TABLE OF CONTENTS  ABSTRACT .............................................................................................................................. ii PREFACE ................................................................................................................................ iv TABLE OF CONTENTS ......................................................................................................... vi LIST OF TABLES .................................................................................................................... x LIST OF FIGURES ................................................................................................................. xi LIST OF SYMBOLS AND ABBREVIATIONS .................................................................. xiv ACKNOWLEDGEMENTS ................................................................................................... xvi DEDICATION ..................................................................................................................... xviii Chapter 1: Introduction ............................................................................................................. 1 1.1: Transfusion Associated Iron Overload .......................................................................... 4 1.2: Iron Chelation Therapy: Treatment of Transfusion Associated Iron Overload ............. 8 1.2.1: Desferrioxamine...................................................................................................... 8 1.2.2: Deferiprone ........................................................................................................... 11 1.2.3: Desferasirox .......................................................................................................... 13 1.3: Continuous Advances Towards Improved Iron Chelators ........................................... 16 1.3.1: HBED.................................................................................................................... 16 1.3.2: Pyridoxal Isonicotinoyl Hydrazone (PIH) ............................................................ 17 1.3.3: FBS0701 ............................................................................................................... 17 1.3.4: CM1 ...................................................................................................................... 18 1.3.5: Thiosemicarbazones .............................................................................................. 19 1.3.6: The Development of High Molecular Weight Polymeric Iron Chelators ............. 21 1.4: Advancing Iron Chelation Therapy ............................................................................. 26 vii  1.4.1: Investigating the Effects of Iron Chelation Therapy During Development .......... 26 1.4.2: Monotherapy Is Inadequate to Ensure Negative Iron Balance ............................. 29 1.5: Theis Rationale, Hypothesis, Specific Aims ................................................................... 32 1.5.1: The Development and Characterization of Novel, High Molecular Weight Polymeric Iron Chelators ................................................................................................ 32 1.5.3: Significance and Novel Contributions of Thesis .................................................. 37 Chapter 2: Polymer Size and the Degree of Functionalization with Desferrioxamine Modulate Chelator Iron Binding Thermodynamics and Efficacy ........................................... 39 2.1: Overview ...................................................................................................................... 39 2.2: Background .................................................................................................................. 40 2.3: Materials and Methods................................................................................................. 43 2.4: Results .......................................................................................................................... 48 2.4.1: Synthesis and Characterization of HPG-DFO ...................................................... 48 2.4.2: Iron(III) Binding Studied by Isothermal Titration Calorimetry............................ 53 2.4.3: Iron Removal from Ferritin: Influence of Molecular Weight and Concentration of HPG-DFO on Iron Removal ........................................................................................... 57 2.4.4: Combination of Macromolecular Chelators and Small Molecular Weight Chelators on Iron Removal ............................................................................................. 61 2.4.5: Influence of Molecular Weight on Inhibition of Iron Mediated Oxidation of Hemoglobin..................................................................................................................... 63 2.5: Discussion .................................................................................................................... 66 2.6: Conclusions .................................................................................................................. 70 Chapter 3:  Biocompatibility of Novel Macromomolecular Iron Chelators ........................... 71 viii  3.2: Background .................................................................................................................. 71 3.1: Overview ...................................................................................................................... 72 3.3: Materials and Methods................................................................................................. 73 3.4: Results .......................................................................................................................... 79 3.4.1: The Effect of Polymer Conjugation on the Blood Compatibility of DFO ............ 79 3.4.2: Cytotoxicity of DFO and HPG-DFO .................................................................... 86 3.4.3: Cellular Uptake of DFO Differs with Conjugation to HPG ................................. 87 3.5: Discussion .................................................................................................................... 91 3.6: Conclusions .................................................................................................................. 92 Chapter 4: The Effect of Clinically Approved and Novel High Molecular Weight Iron Chelators on Zebrafish Embryo Mortality, Hatching and Morphology ................................. 93 4.1: Overview ...................................................................................................................... 93 4.2: Background .................................................................................................................. 94 4.3: Materials and Methods................................................................................................. 97 4.4: Results ........................................................................................................................ 101 4.4.1: Effects of Carrier Solvent on the Mortality, Hatching Success and Morphology of Zebrafish Embryos ........................................................................................................ 105 4.4.2: Effects of Clinically Approved Iron Chelators on Mortality, Hatching Success and Morphology of Zebrafish Embryos .............................................................................. 105 4.4.3: Acute Exposure to Iron Chelators on Cardiac Output and Heart Rate in Adult Zebrafish ....................................................................................................................... 112 4.4.4: Acute Exposure to Iron Chelators on the Expression of Hepcidin, Ferroportin and DMT-1 Genes in Adult Zebrafish ................................................................................. 114 ix  4.4.5: HPG Conjugation to DFO does not Enhance the Toxicity of DFO in Zebrafish 116 4.4.6 Positive Control HPG Increases Mortality and Reduces Hatching Success of Zebrafish Embryos ........................................................................................................ 119 4.5: Discussion .................................................................................................................. 121 4.6: Conclusions ................................................................................................................ 126 Chapter 5: Conclusions and Future Directions ..................................................................... 128 5.1: Conclusions ................................................................................................................ 128 5.2 Significance of Thesis ................................................................................................. 130 5.3: Implications of Increasing Chelator Half-life ............................................................ 135 5.4: Future Directions ....................................................................................................... 138 5.5: Polymeric Iron Chelation: Beyond Transfusional Iron Overload .............................. 142 References ............................................................................................................................. 144 Appendix A ........................................................................................................................... 170 Appendix B ........................................................................................................................... 171 Appendix C ........................................................................................................................... 173 x  LIST OF TABLES Table 1: Summary of the properties of iron chelators in clinical use ..................................... 15 Table 2: Contrasting features of ideal and currently approved iron chelators ........................ 31 Table 3: The reported benefits of polymer conjugation to iron chelators............................... 33 Table 4: Characteristics of iron chelators used in this study. ................................................. 51 Table 5: Apparent Fe(III) binding thermodynamic paramaters for polymeric chelators measured using Isothermal Titration Calorimetry. ................................................................. 57 Table 6: TEG values for HPG-DFO conjugates ..................................................................... 82 Table 7: Characteristics of HPG-DFO used in cellular uptake study ..................................... 87 Table 8: The forward and reverse primers used for real-time qPCR. ................................... 100 Table 9: The anticipated reduction in drug needed with increased half-life of DFO through polymer conjugation. ............................................................................................................ 134 Table 10: The properties of HPG-DFO molecules tested for Biocompatibility in Chapters 2-4............................................................................................................................................. 170 Table 11: Species present in ITC titration of Fe(NTS) and DFO, HPG-DFO and 40SDO2. 171 xi  LIST OF FIGURES Figure 1: Iron recycling and distribution in the body. .............................................................. 2 Figure 2: Harmful redox cycling of iron. .................................................................................. 4 Figure 3: Excess labile iron causes damage to the body’s organ systems. ............................... 7 Figure 4: The structure of clinically approved iron chelators and their complexes. ................. 9 Figure 5: The structures of previously reported and potential iron chelators for treating transfusional iron overload. .................................................................................................... 20 Figure 6: The structures of polymer components used in DFO modification. ....................... 22 Figure 7: Synthesis scheme for HPG-DFO. ............................................................................ 48 Figure 8: Iron chelation by HPG-DFO depends on the number of DFO conjugated per polymer. .................................................................................................................................. 49 Figure 9: Conjugation of DFO to HPG does not influence DFO chelating properties. .......... 50 Figure 10: HPG-DFOs are high affinity Fe(III) binding chelators. ........................................ 55 Figure 11: Influence of MW of conjugates on ferritin iron binding. ...................................... 59 Figure 12: The effect of reaction temperature on iron extraction by chelators....................... 60 Figure 13: Small MW chelator deferiprone (L1) enhances iron removal by larger MW chelators. ................................................................................................................................. 62 Figure 14: HPG-DFO can prevent the Fe(III) mediated oxidation of hemoglobin. ............... 64 Figure 15: HPG-DFO prevent the Fe(III) mediated oxidation of hemoglobin. ...................... 65 Figure 16: Conjugation of DFO to HPG did not affect the intrinsic or extrinsic pathways of coagulation. ............................................................................................................................. 80 Figure 17: Influence of HPG-DFO on blood coagulation as analyzed by thromboelastograpy (TEG). Shown are TEG traces of HPG-DFO incubated with human whole blood. ............... 81 xii  Figure 18: Polymer Conjugation did not affect the activation of complement (A) or platelets (B). .......................................................................................................................................... 83 Figure 19: Influence of HPG-DFO on Red Blood Cell Aggregation and Hemolysis ............ 85 Figure 20: Cytotoxicity of HPG-DFO conjugates againist HUVECs .................................... 86 Figure 21: Fluroscence profile of  fluorescein conjugated DFO and different HPG-DFOs. .. 88 Figure 22: Cellular uptake of iron chelators in HUVECs. ...................................................... 89 Figure 23: Cellular uptake of iron chelators in CHO cells. .................................................... 90 Figure 24: Developmental changes in zebrafish embryos with time after fertilization. ......... 95 Figure 25: The effect of DMSO on chelator exposure. ........................................................ 101 Figure 26: DMSO does not interfere with the development of embryos. ............................. 102 Figure 27: Influence of DMSO on the development of zebrafish embryos. Data show that DMSO does not interfere with the development of embryos in the concentration range studied (0-1%). ...................................................................................................................... 103 Figure 28: DMSO does not influence the morphology of zebrafish embryos. ..................... 104 Figure 29: Mortality of zebrafish embryos exposed to iron chelators. ................................. 107 Figure 30: Hatching success and morphological alterations of zebrafish embryos. ............. 108 Figure 31: Zebrafish larvae morphology after 72 h of exposure to iron chelators. .............. 110 Figure 32: Zebrafish embryo morphology after 72 h of exposure to iron chelators. ............ 111 Figure 33: Iron chelator exposure significantly influences heart rate and cardiac output in adult zebrafish. ...................................................................................................................... 113 Figure 34: Effect of chelator exposure on gene expression. ................................................. 115 Figure 35: Toxicity of HPG-DFO conjugates in zebrafish embryos. ................................... 117 xiii  Figure 36: HPG conjugation to DFO does not influence the hatching success of zebrafish embryos. ................................................................................................................................ 118 Figure 37: The effect HPG-NH2 on zebrafish embryo mortality and hatching. ................... 119 Figure 38: Zebrafish larvae morphology after 72 h of exposure to polymeric iron chelators................................................................................................................................................ 120 Figure 39: Polymeric iron chelators result in a higher unsaturated iron binding capacity. .. 137 Figure 40: The proposed intracellular degradation of liver-specific HPG-DFO. ................. 142 Figure 41: Speciation plot of Fe-NTA complexes existing in the syringe. .......................... 172 Figure 42: Tolerance studies of 44K-85 chelator in Balb/C mice. ....................................... 173 Figure 43: Tolerance studies of 44K-85 chelator in Balb/C mice. ....................................... 174  xiv  LIST OF SYMBOLS AND ABBREVIATIONS ANOVA  Analysis of Variance β-TM  Beta thalassemia major DFO  Desferrioxamine DMSO  Dimethyl sulfoxide DMT-1 Divalent Metal Transporter 1 dw  Dry Weight EDTA  Ethylenediamine Tetraacetic Acid FDA   Food and Drug Administration FPN  Ferroportin FTN  Ferritin HES  Hydroxyethyl Starch HMW   High Molecular Weight HPG  Hyperbranched polyglycerol IC  Iron Chelators ICL-670 Desferasirox ICT   Iron Chelation Therapy i.p.  Intraperitoneal  i.v.  Intravenous  ITC  Isothermal Titration Calorimetry  kDa   Kilo Dalton L1   Deferiprone LIP  Labile Iron Pool xv  LPI  Labile Plasma Iron mRNA  Messenger RNA MDS  Myelodysplastic syndromes MEM   Minimum Essential Medium MW   Molecular Weight NTBI  Non transferrin Bound Iron OD   Optical Density PPP   Platelet Poor Plasma PRP   Platelet Rich Plasma P-DFO  Polyethylene Glycol Conjugated to DFO PEG   Polyethylene Glycol RAFT   Reversible Addition-Fragmentation Chain-Transfer RBC   Red Blood Cell RES  Reticuloendothelial System Rh  Hydrodynamic radius ROS  Reactive Oxygen Species RT   Room Temperature SCD  Sickle Cell Disease SD   Standard Deviation s.c.  Subcutaneous 40SDO2  Starch Conjugated to DFO Tf  Transferrin TAIO  Transfusion Associated Iron Overload  xvi   ACKNOWLEDGEMENTS  I thank God for every challenge, blessing and opportunity that arose during this journey. I am especially thankful for the caring, dedicated and generous people that have contributed to my personal and professional growth throughout this journey. I am thankful for the research fellowships/scholarships that I received from the Canadian Blood Services, the Centre for Blood Research, the International BioIron Society, CIHR’s Canadian Student Health Research Forum, Albert B and Mary Steiner Summer Research Award and UBC.  I am thankful to my supervisor Dr. Jayachandran Kizhakkedathu for providing guidance and direction throughout this project. Thank you for being approachable, for helping me during difficult trials, challenging me when necessary and especially for encouraging my unconventional professional development. I am grateful Dr. Suraj Unniapann for welcoming me into his lab and for leadership at the University of Saskatchewan, thank you for your guidance. Thanks to the members of my supervisory committee; Drs. Don Brooks, Helene Cote, Marcel Bally and Mark Scott for continual guidance. Special thanks to Dr. Mark Scott for providing starch DFO (40SDO2) samples. Thanks to Drs. Imran ul-haq, Azadeh Hatef and Louise Creagh for training me during various aspects of this project; Benjamin Lai, Neelima Nair, Iren Constantinescu, Irina Chafeeva, and Sonja Horte for training and supporting me in various ways. Special thanks to Ben who always demonstrated vast knowledge and willingness to share lab protocols and other important information.   Thanks to Dr. Heather Leitch (St. Paul’s Hospital), who has provided invaluable guidance with interpretation of research findings through our collaboration. I am indebted to xvii  Dr. Haydn Pritchard for constant guidance and mentorship and Aleya Abdullah for her consistent help in solving difficult problems.  My sincerest gratitude to my CBR colleagues and friends Dana Price, Benjamin Lai, Erika Das, Narges Hadjesfandiari, Marie Weinhart, Johan Janzen, Vincent Leung, Yan Mei, Anil Kumar, Srinivas Abbina, Kai Yu, Xeifei Yu and Prashant Kumar for providing a supporting and positive atmosphere to work. Vince, thank you very much for your constant encouragement throughout the years. Thanks for being a good listener; I am happy for every interesting and honest conversation that we had. Narges, Mahsa, Anil and Srinivas, thank you for answering my myriad questions about conjugation Chemistry and for your patient explanations of concepts and structures.   I am most grateful to my mother, siblings and the rest of my family for constant support, encouragement and love. I am equally grateful to my friends Brant and Susan Levert, Tony and Judy Vick, Yves and Carole Perron, Roger and Wendy Schmidt, Ute and Gordon Carkner, Rolinda Carter and the Kwakyes; thank you for your continued prayers, spiritual guidance, support, generosity and friendship. Thanks to Len and Pat Cuthill, and Elizabeth McTaggart for providing warm, welcoming spaces for me throughout this journey.    xviii  DEDICATION  1.) To my mother, Donna Ederle Chalmers; for every sacrifice that you made for me.   2.) To my sister, Jonne-l Melissa Hendrickson, for being the biggest inspiration in my life and the reason why I pursued a PhD in this area.    3.) To Mr. Peter Chesham; for recognizing my ability, investing in me and enabling me to pursue this passion. I am so grateful for your generosity.  4.) To my grandmother, Urla Roslin Chalmers (1927-2013), for convincing me that I could achieve anything. 1  Chapter 1: Introduction Iron is essential for oxygen transport, DNA synthesis and energy metabolism (1). Thus, it is life sustaining in virtually all living organisms. The usefulness of iron results from its ability to cycle between its ferrous (Fe2+) and ferric (Fe3+) forms in oxidation and reduction reactions (1-2). Although iron is abundant in the earth’s crust, Fe2+ is highly toxic, while Fe3+ is insoluble in aqueous solution at physiological pH, rendering it inaccessible. As a result, obtaining bioavailable iron is a continual challenge which living organisms have overcome by evolving to conserve iron (1-3). Organisms have acquired highly organized mechanisms of iron acquisition, transport and storage; microorganisms use low molecular weight (MW) high affinity iron ligands or siderophores, while more complex life forms like mammals use specialized storage and transport proteins (3).  Under normal physiological conditions, the human body contains 3.5-5g of iron, with the majority (over 70%) existing in hemoglobin (3). The remainder is found in myoglobin, intracellular storage iron in the hepatocytes of the liver, spleen and bone marrow macrophages, and in proteins and enzymes that are involved in cellular respiration (1-3). Iron metabolism is highly conservative in man, with the efficient recycling of hemoglobin iron and limited external exchange forming the major component of iron regulation. Intestinal iron uptake also plays a key role in maintaining human iron homeostasis; 1-2 mg of dietary iron is absorbed daily and approximately 1-2 mg of iron is lost daily due to the sloughing off of epithelial cells, secretions from the skin and gut, and small losses of blood from the gastrointestinal tract (2-3). This indicates the conservative nature of iron metabolism and recycling. The process of iron recycling and metabolism is schematically demonstrated in Figure 1. 2      Figure 1: Iron recycling and distribution in the body.  Body iron is primarily located in erythrocytes (>70%) which are efficiently recycled by macrophages of the liver and spleen. Enterocytes obtain iron from the diet. Macrophages, which obtain iron from the phagocytosis of senescent RBCs release iron into the circulation where it binds to plasma transferrin, the iron transport protein. Transferrin delivers iron to the erythron of the bone marrow and to other sites like hepatocytes of the liver, the main iron storage site in the body. There is no physiological excretion pathway for iron.    3   Under normal physiological conditions, iron is complexed with proteins like transferrin (Tf) or small organic molecules like citrate and acetate which ensure that it is unable to cause free radical production (4-5). In the plasma, iron is transported by Tf and is unavailable for redox activity. Tf has a high iron binding capacity which prohibits the accumulation of toxic unshielded or non-transferrin bound iron (NTBI). Tf contains 2-3 mg of iron and is hyposaturated at ~30% under normal physiological conditions. Tf delivers iron to hepatocytes and specific binding sites on red cell precursors of the bone marrow involved in the synthesis of hemoglobin. Tf also captures iron released into the plasma from intestinal enterocytes or cells which catabolize senescent RBCs (4-5).  Within cells, ferritin is the major storage molecule for reusable iron and accounts for ~27% (1g) of the total body iron in normal individuals (6). Ferritin has an iron storage capacity of 4500 atoms per ferritin molecule and iron storage in ferritin ensures that iron is stored within cells in a safe redox inactive form. Therefore, ferritin reduces the toxicity from free radical generation while ensuring that iron is also available for mobilization for metabolic processes. Ferritin also helps to re-establish normal redox conditions during oxidative stress by removing ferrous ions and oxygen from the cytoplasm. Under pathological conditions in which ‘iron overload’ occur, excess iron is deposited as insoluble ‘iron cores’ of partially degraded ferritin or hemosiderin, primarily in liver, spleen, endocrine organs and myocardium of the heart (1-2, 6). Although electron shuttling is vital in metabolic processes, under conditions of excess, iron may catalyze harmful reactions that generate free radicals which amplify the development of reactive oxygen species (Figure 2) (7-8). This may occur via the Haber-Weiss reaction in which hydrogen peroxide (H2O2) reacts with the superoxide radical (O2·-) to produce the hydroxyl radical (OH·), the most reactive radical in the body (8). Although this reaction occurs at minimal levels 4  under normal physiological conditions, it can be catalyzed by iron, leading to accumulation of free radicals, which can interact with cellular components and disturb metabolic functions (8). It has been shown that the increased generation of free radicals can oxidize lipids, proteins, and DNA in major organs with the heart being most susceptible. Thus, the disruption of normal cellular redox equilibrium is possible with very small amounts of misplaced iron, and the magnitude of the body iron burden is the most important determinant of the ensuing organ damage.   Figure 2: Harmful redox cycling of iron.  Iron (Fe) can participate in one electron oxidation and reduction reactions. This leads to the generation of harmful free radicals in the presence of oxygen. The hydroxide radical and hydroxide anion (OH-) are produced when hydrogen peroxide reacts with ferrous iron. Ferric iron is in turn reduced by the superoxide radical (O2·-). Redox active or “labile” iron reacts with cellular hydrogen peroxide (H2O2) producing the hydroxyl radical (OH·), which perpetuates free radical production, ultimately increasing cellular ROS generation. The resulting oxidative stress is associated with damage to cellular components and organs (8).  1.1: Transfusion Associated Iron Overload In contrast to the highly evolved methods of iron acquisition, storage and transport, the ability to offload excess iron remains challenging as there is no known physiological pathway to actively excrete iron. This is the major challenge in disease states like β-thalassemia (β-TM), sickle cell disease (SCD) and myelodysplastic syndromes (MDS) that invariably lead to iron overload (1-3, 9-11). 5  Red blood cell (RBC) transfusions are used to ameliorate anemia in patients with β-TM and MDS and can prevent vaso-occlusive events in SCD (9-11). Patients with these disorders develop severe anemia due to ineffective erythropoiesis and hemolysis, which causes large numbers of marrow erythrocyte precursors to undergo apoptosis before undergoing maturity into erythrocytes. In the case of SCD, there is the added risk of stroke due to the lack of deformability and enhanced stickiness of RBCs which may cause the obstructive adhesion of sickled cells to each other and the vasculature (11).  Further, ineffective erythropoiesis results in a drastic increase in plasma iron turnover, with the turnover of plasma iron occurring at a rate that is 10-15 times greater than in patients with normal erythropoiesis (12). As a result, patients can accumulate over 2.5 g of iron annually from this process, which results in a “primary” iron overload state. In addition to this inherent iron accumulation, patients receive red blood cell transfusions frequently which, although highly beneficial in suppressing erythropoiesis and anemia, put patients at risk of developing “secondary” or transfusion associated iron overload.  Each unit of RBC contains approximately 250 mg of iron (10). Since humans lack an iron excretion pathway, chronically transfused patients accumulate iron at a rate of 0.2-0.4 mg/kg/day if transfused more than twice per year (10, 12). This excess iron accumulation causes a saturation of the body’s iron regulatory mechanisms and a subsequent disruption of normal iron regulation.  As the iron loading from transfusions increase, transferrin in the plasma becomes saturated and NTBI appears in the serum (13). This toxic pool of partially ligated iron accumulates in plasma and is subsequently, and in some cases, preferentially taken up by cells. For example, the rate of NTBI uptake by cultured rat heart cells is greater than 300 times that of transferrin bound iron (12). As this toxic pool of NTBI accumulates, an intracellular labile iron pool (LIP) is formed and 6  ultimately facilitates harmful redox damage to tissues through the formation of the free hydroxyl radical.  The excess iron is accumulated primarily in the liver, spleen, endocrine organs and myocardium where toxic “labile” iron pools develop. The cytosolic LIP mirrors the cellular iron content and its fluctuations are considered to trigger homeostatic adaptive responses. Once homeostatic mechanisms become saturated, excess iron can ultimately lead to organ dysfunction and death if left untreated (12, 14-16). Figure 3 shows some of the potential effects of iron overload on major organs.     7    Figure 3: Excess labile iron causes damage to the body’s organ systems.  Chronic transfusion therapy results in the saturation of serum transferrin and the development of toxic iron pools in cells and tissues. NTBI in the plasma and labile cellular iron (LCI) react with cellular membranes and organelles, causing peroxidation, DNA damage and protein dysfunction. The liver, heart, pancreas and other endocrine organs are most commonly damaged. These events ultimately lead to organ dysfunction, failure and death if left untreated (10,12, 14-16). 8  1.2: Iron Chelation Therapy: Treatment of Transfusion Associated Iron Overload Iron chelation therapy (ICT) is used for the treatment of transfusion associated iron overload. ICT is achieved when iron binding molecules (iron chelators) bind iron forming a “chelate” which is subsequently excreted via the feces and/ urine, enabling safer body iron levels (9-10, 12, 14-17). ICT protects cells against oxidative damage by reducing the pool of reactive iron in the plasma and cytosolic LIP in cells and is used to manage iron overload in transfusion dependent patients with β-TM, SCD and MDS (8-10, 12, 15-17). ICT inhibits the lipid peroxidation, protein oxidation and cellular damage that accompanies iron overload (18-19). ICT is recommended after receiving 10-20 transfusions of erythrocytes in order to prevent severe iron loading and damage in major organs (10). Currently, three iron chelators are approved for treating transfusion associated iron overload (Figure 4).  1.2.1: Desferrioxamine  The most thoroughly characterized iron chelating drug is desferrioxamine (Desferal®, DFO) which has been the standard of therapy for over 40 years. Used since the 1960s, DFO has demonstrated efficacy in prolonging life and improving quality of life for transfusion dependent thalassemic patients (9, 10, 12, 15, 16). DFO has demonstrated efficacy at preventing lipid peroxidation which leads to organ damage, promoting iron excretion, arresting fibrosis, significantly decreasing deaths by cardiac disease, reducing hepatic iron concentrations and extending lifespan in iron overloaded patients (9, 15-16, 18-20).    9    Figure 4: The structure of clinically approved iron chelators and their complexes.  10    DFO is a high affinity iron(III) chelator with a overall stability constant  (log K) of 30.6 for the Fe(III) complex and a molecular weight (MW) of 560 Da. Due to its hexadentate nature, DFO binds iron in a 1:1 ratio producing a stable complex that prevents iron from catalyzing the formation of harmful free radicals (20-21). DFO can access iron by two methods; directly interacting with hepatocellular iron and subsequent biliary excretion, as well as from the destruction of RBCs in the reticuloendothelial system (RES), directly or following its release into plasma as NTBI (10, 12). DFO enters the liver via active transport and interacts with liver and extracellular iron which leads to excretion primarily by urine as well as some biliary iron excretion (22-23). The DFO–Fe(III) complex does not redox-cycle and this reduces the chances of iron redistribution and toxicity within the body (21).  DFO therapy improves lifespan and quality of life (9,12,15-17,20). Borgna-Pignatti et al. showed that mortality at 20 years of age had fallen significantly after the advent of DFO chelation; diabetes had fallen from 15.5% in those born between 1970-74 when DFO therapy was relatively new, to 0.8% in those born after 1980, when DFO was more frequently prescribed (20). This strongly supports the idea that the age at which transfusion dependent patients begins DFO therapy, as well as adherence to therapy may modulate risk of heart disease and other complications. Similar studies in the UK have shown the benefit of iron chelation with DFO in prolonging life, significantly reducing the incidence of cardiac disease, liver failure and other endocrine disorders in compliant patients (15). Despite these advantages, DFO is hardly the “ideal” chelator. Due to its low lipophilicity and high MW (560 Da), DFO is not readily absorbed by gastrointestinal cells. In addition, it has a very short circulation half-life of ~20 minutes in humans and must be subcutaneously infused at doses of 40-60 mg/kg for 8-12 h a day, 5-7 days per week. Additionally, DFO at high doses has 11  been associated with severe neurotoxicity, causing sensorineural hearing loss, visual electroretinographic disturbances, and impaired growth and bone development (23-25). Thus, the use of DFO has been hindered by its shortcomings and attempts toward generating more efficient iron chelators have continued. 1.2.2: Deferiprone  Deferiprone (Ferriprox®, Cipla, L1) is the second chelator to receive approval for the treatment of iron overload. It was first reported as a potential orally active iron chelator and efficient at in vivo iron removal in 1987 and was subsequently licensed for use in India in 1994 and Europe in 1999 with special conditions (17, 26). Due to questions regarding safety and chelation efficiency, L1 only received full marketing authorization in Europe in 2002 and by the FDA in 2011. L1 is a bidentate chelator thus, 3 L1 molecules are needed to chelate one atom of iron (10, 21, 26, 27). As a result, the efficacy of L1 as an iron chelator is highly dependent on the concentration ratio of chelator and iron in the environment. At low L1 to iron concentrations, L1 may bind to iron incompletely. These partially bound forms of iron with unoccupied coordination sites may accumulate and remain reactive. Furthermore, these partially chelated forms of iron are able to catalyze the formation of harmful radicals and other reactive oxygen species (27).  In the first study reporting its efficacy, L1 was shown to cause iron excretion at a rate proportional to the iron load of the patients and the dose given in the 4 MDS and 4 β- thalassemia major patients participating in the study (26). Further, the iron excretion levels in urine were found to be similar to that obtained with therapeutic doses of DFO. Rombos et al. reported that L1 was safe and caused a reduction in iron overload in Greek thalassemics without causing considerable side effects (28). However, several subsequent studies have shown that L1 therapy alone may be ineffective in ensuring negative iron balance in many patients, especially in patients with less 12  severe iron loading (29-32). Hoffbrand et al. found no significant reduction in urinary iron excretion in any of the patients enrolled in the study and no significant change in the serum ferritin levels of more than half of the patients who received L1 treatment for more than 3 years (32). While Cohen et al. found that L1 can reduce and maintain body iron in some but not all patients; L1 did not reduce body iron overload to a level below that achieved by DFO in those patients that had lower baseline iron levels (30). This demonstrates that the daily L1 dose of 75 mg/kg body weight/day induces less iron excretion than the standard daily dose of DFO 50 mg/kg body weight/day.  Other studies show that L1 reduces serum FTN levels in some but not all patients and that the effects of prolonged therapy were not sustained (29-32). In addition, although L1 can mobilize iron intracellularly and has been shown to reduce cardiac iron it is unable to promote adequate iron removal and prevent death by cardiac disease in some patients. This was well described in a review by Hoffbrand et al. For example, it was reported that 9 out of 532 thalassemic patients undergoing consistent L1 treatment for 3 years died of heart failure (29). In addition, Hoffbrand et al. found that out of 51 L1-treated patients, 4 out of the 5 patient deaths were caused by cardiac dysfunction (32). This indicates that in some patients with myocardial iron overload and continuing need for blood transfusions, L1 was not reliable at preventing further iron loading.  One of the major reasons for the limited efficacy of L1 in clinical use is its rapid metabolism in the liver. The 3-hydroxyl functional group that is found on the L1 molecule is required for effective iron chelation. However, this is also the site of rapid metabolism by glucoronidation in liver cells (21, 29). Studies which measured L1 recovery in the urine found that over 85% of the L1 dose given to patients may be recovered in the urine as the inactive 3-O-glucuronide conjugate (21, 29).  In addition to the challenging metabolism of L1 described above, 13  severe side effects of L1 can also be limiting without adequate monitoring of patients. Agranulocytosis is considered to be the most serious side effect of L1 use (10, 30). Milder neutropenia is also common, occurring in up to 4.8% of patients in some studies (29). Thus, it is necessary to carefully monitor blood counts, especially in patients that are given higher doses. Arthralgia, nausea, gastrointestinal symptoms, zinc deficiency and fluctuating liver enzymes have also been reported (29-30, 32). 1.2.3: Desferasirox  Desferasirox (Exjade®, ICL-670) is the second orally active iron chelator and the most recent to become approved for the treatment of transfusion associated iron overload (33-35). It has a MW of 373 Da. Although tridentate, that is, requiring 2 molecules to bind each iron atom, ICL-670 has been shown to be highly selective for iron without promoting the excretion of other metals like zinc and copper (34). Studies in rats and humans demonstrate that ICL-670 possesses a half-life of 8-16 h, which allows ICL-670 plasma levels to be sustained at a therapeutic range for longer than either DFO or L1. Subsequent clinical studies have confirmed the iron chelation efficacy of ICL-670.  ICL-670 has been reported to be significantly more efficient than DFO and L1 at promoting iron excretion. At equal molar concentrations ICL-670 is reported to be five times more efficient than DFO and ten times more effective than L1 (34). Several studies show a linear dose-dependent increase in the amount of iron excretion by iron overloaded patients and the doses of ICL-670 given. ICL-670 was reported to induce iron excretion in a manner that would likely prevent iron accumulation in most patients requiring standard transfusion therapy for iron overload (33-35).  14  Like L1, ICL-670 is highly cell permeable. Moreover, it is absorbed by some cells more rapidly than L1 (37). The active molecule is highly lipophilic and cell permeable in vivo and feces is the main route of excretion for ICL-670 and its metabolites. Renal excretion accounts for approximately 8% (35-36). Unlike L1, which is absorbed but rapidly inactivated through metabolism, ICL670 rapidly increases in concentration in the plasma of patients and persists at detectable levels for several hours.  Although the long half-life and iron removal efficacy of ICL-670 allows once-daily dosing and offers significant improvement in convenience for patients when compared to DFO and L1, the toxicities reported to accompany prolonged ICL-670 use warrant deep consideration (38-43). In early studies, changes to the renal tubular epithelium were observed as side effects of ICL-670 use (39-40). Subsequent studies have confirmed that renal toxicity, hepatic dysfunction and thrombocytopenia are the main concerns for patients undergoing iron chelation therapy with ICL-670. Reports indicate that prolonged use can cause Fanconi syndrome (39-42). Additionally, a mild, dose-dependent increase in serum creatinine occurs in some patients. Thus, ICL-670 use requires meticulous monitoring of kidney, liver, and hematopoietic function.  In a recent report by Kontoghiorghes, the fatalities associated with ICL-670 use are shown to be the highest among the clinically approved chelators. When compared to DFO and L1 which have been in use for much longer periods, the toxicity due to chelation with ICL-670 is high (43). More importantly, according to this report, ICL-670 was listed as the drug associated with the second highest number of deaths in 2009. Kontoghiorghes, reported that there is a steady increase in the ICL-670 induced deaths in patients per year and that most are caused in elderly patients with MDS. Although the evidence presented in this report was questioned by Riva, the potential seriousness of ICL-670 induced toxicity should not be overlooked (44). 15  A comparison of the major characteristics of the 3 clinically approved iron chelators is given in the Table 1 below.   Table 1: Summary of the properties of iron chelators in clinical use  Property of Chelator Desferrioxamine (DFO) Deferiprone (L1) Desferasirox (ICL-670) Date approved for clinical use 1960s 1999 in Europe and Asia  2011 in USA 2005 Usual dose 20-50 mg/kg/day 75-100 mg/kg/day 20-40mg/kg/day Molecular weight 560 139 373 Fe binding log stability constant 30.6 35 38 Chelator: Iron 1:1 (Hexadentate) 3:1 (Bidentate) 2:1 (Tridentate) Potential Toxicities Reactions at the infusion site, neurotoxicity, bone abnormalities Neutropenia, agranulocytosis, arthralgia, elevation of liver enzyme Gastrointestinal, rash, renal and liver   16  1.3: Continuous Advances Towards Improved Iron Chelators   The shortcomings of DFO, L1 and ICL-670 highlight the need for improved chelation therapy. Challenges such as the inefficiency of DFO and necessity for continuous subcutaneous infusion; the toxicities of L1 and its inability to adequately control body iron levels with prolonged use; and the severe toxicities associated with ICL-670, have sustained the interest among researchers to develop better options for iron chelation.  Fe(III) selective chelators are the most fitting for biological applications because they are less likely to deplete other essential metals, which are commonly divalent. However, because the size of a drug influences intestinal absorption, creating orally active hexadentate chelators has proven difficult (21, 45). Instead, the greatest emphases have been placed on generating novel bi and tridentate chelators or modifying the properties of existing ligands. Over the years, several promising agents which vary in denticity, metal selectivity for Fe(III), toxicity, stability of the Fe-chelator complex and lipophilicity have been proposed and tested in several in vivo models including rodents, marmosets, dogs, and primates, with promising agents progressing to clinical trials (46-63). Figure 5 shows the structures of a few previously reported iron chelators with potential clinical utility. 1.3.1: HBED N, N’-bis (2-hydroxybenzyl) ethylenediamine-N-N’-diacetic acid (HBED) is a hexadentate, phenolic aminocarboxylate (MW 388) which has been tested for utility as an iron chelating agent (Figure 5). HBED has a high affinity and specificity for Fe(III) and like DFO, renders it virtually inert and incapable of forming harmful radicals which damage cellular components and organs (46-48). HBED has been thoroughly characterized for iron chelation efficiency and toxicity. Initial studies showed that HBED is ineffective at promoting iron excretion when given orally. However, 17  when given subcutaneously or intravenously, HBED was more than twice as effective as DFO at mobilizing iron from rats and cebus apella. Importantly, HBED is a powerful antioxidant and was not associated with any major toxicity in the models tested. Compared to DFO, HBED showed potential as a therapeutic for treating transfusional iron overload with the potential for dosing every other day. HBED has been used in man but has not been further developed for use in treating transfusional iron overload (46-48).  1.3.2: Pyridoxal Isonicotinoyl Hydrazone (PIH) Pyridoxal isonicotinoyl hydrazone (PIH) is tridentate iron chelator (MW 287) effective at scavenging and mobilizing iron (Figure 5). Ponka et al first reported the potential of this class of chelator, and have also shown its efficacy as anti-proliferative agents, preventing free-radical mediated injury, and as anti-malarias (49-54).  PIH has been shown to chelate both forms of iron, and like other chelators it caused depletion in zinc levels at physiological pH. At neutral pH, the neutral charge of PIH ensures oral absorption and allows access to the cytosol, where labile iron can be chelated. PIH showed efficacy at removing iron from rat reticulocytes which contained labile non-heme iron and mobilizing iron from Chang cells. PIH also reduced iron levels in major organs in mice and was tested in man but did not promote adequate iron excretion. PIH has been reported to be highly toxic in cebus monkeys and although analogues of PIH have been created none has been further developed for use in treating iron overload in transfusion dependent patients (49-54).  1.3.3: FBS0701 FBS0701 (SPD-602) is a novel tridentate chelator of the desazadesferriothiocin (DADFT) class and has been tested in phase II clinical trials (Figure 5). FBS0701 has a MW of 400 (salt form 440), binds iron tightly and has a higher affinity for Fe(III) than other divalent metals. It can 18  enter cells and has demonstrated efficacy in iron chelation and a comparable safety profile to currently approved chelators (55-59).  A one-week, dose escalation, phase Ib study demonstrated its potential clinical utility and efficacy. While a phase II multicenter trial, which dosed patients with 50-375 mg of the FBS0701-salt, showed a statistical significant reduction in liver iron, confirming the potential benefit of this agent to reduce iron burden from transfusions. Although adverse events were reported, they did not appear to be dose related and occurred at low frequency. Future studies with larger sample sizes will provide more information on the potential of this chelator for treating transfusion associated iron overload. FBS0701 is currently undergoing development and represents a promising agent for future treatment (55-59). 1.3.4: CM1 CM1, 1-(N-Acetyl-6-Aminohexyl)-3 Hydroxy-2-Methylpyridin-4-One), is an orally active, bidentate L1 analogue, possessing a MW of 256 Da and is currently being developed for the treatment of iron overload (Figure 5). CMI has shown higher lipophilicity than L1 and can bind both Fe(II) and Fe(III). CM1 is effective at mobilizing cytosolic labile iron in primary mouse hepatocytes hepatocytes and HepG2 cells, and plasma NTBI. It has been studied in transgenic β-thalassemic mice, and has demonstrated efficacy and low toxicity in the liver and peripheral blood of iron overloaded mice. Importantly, CM1 showed efficacy at preventing lipid peroxidation, one of the underlying causes of cellular damage. Further studies are needed to determine clinical utility of this agent (60-63).   19  1.3.5: Thiosemicarbazones  Thiosemicarbazones represent a versatile group of chelators previously described and tested widely for efficiency and utility in iron chelation and for the treatment of a wide range of iron mediated pathological conditions (64-67).  Thiosemicarbazone ligands have been derived from condensing aliphatic, aromatic or heterocyclic aldehydes or ketones with thiosemicarbazide compounds and have demonstrated the ability to complex a wide range of transition metals. In some cases, this class of chelator complexes the metal in the cis configuration as a bidentate ligand, forming complexes using the pyridyl and imine nitrogen and sulfur as donor atoms. Studies conducted in cell culture and mice showed that these ligands have high chelator efficiency and diverse utility for treating a diverse set of iron related conditions (66-67). Alternatively, thiosemicarbazone ligands can stabilize the ferric iron state when using donor ligands such as oxygen. In the latter case, it can be useful for treatment of iron overload disorders and reducing iron mediated damage (65-67).      20                           Figure 5: The structures of previously reported and potential iron chelators for treating transfusional iron overload.  21  1.3.6: The Development of High Molecular Weight Polymeric Iron Chelators Although all currently approved chelators are of low MW, reports of polymeric iron chelators have demonstrated that the development of high MW chelators can be a viable alternative to improve the pharmacokinetics and systemic toxicity of small MW chelators. The approach toward developing improved, polymeric chelators has varied from using iron binding dendrimers, hydrogels, the covalent attachment of DFO to a wide range of biocompatible materials and the use of amino acid amide derivatives. Due to its hexadentate nature, efforts have focused on DFO modification. Figure 6 shows the structures of polymeric components previously used to modify DFO toxicity and systemic circulation. The major motivations for producing polymeric iron chelators are to overcome the challenges of rapid plasma elimination and degradation, prolonged infusions and widespread DFO toxicity that has hindered the achievement of safe iron levels in many iron overloaded patients undergoing iron chelation therapy. Indeed, several studies have indicated that polymeric iron chelators possess unique advantages over their low MW counterparts.     In a 1989 report, Hallaway et al described the advantages associated with attaching DFO covalently to dextran and hydroxyethyl starch (HES) (68). This resulted in significant increase in the plasma half-life and reduction in toxicity with no apparent loss in the iron chelating properties. The increase in size of starch conjugated DFO (S-DFO) resulted in improved plasma half-life from 5 min for DFO to 87 min for S-DFO in mice. The LD50 in mice increased from 250 mg/kg to 4000 mg/kg for dextran-DFO and there was an absence of pulmonary hypotension when intravenously administered in dogs. 22    Figure 6: The structures of polymer components used in DFO modification.    23  This is a major improvement as both the iron free and iron saturated forms of DFO caused rapid and significant hypotension in dogs at a dose of 100 mg/kg. Moreover, once hypotensive, the blood pressure did not return to normal during the 60 minutes of the experiment.  In contrast, neither HES nor dextran conjugated DFO caused any significant change in blood pressure. Additionally, the half-life was > 10 times greater after conjugation. In 2005 Polomoscanik et al reported the generation of a non-toxic formulation of DFO hydroxamic acid based iron chelating hydrogels and evaluated utility to prevent iron absorption in the gut (69). These gels were effective in preventing gastric iron absorption and did not cause any change in hemoglobin and hematocrit. They also conducted a study to determine whether other divalent metals compete with iron for binding to the polymeric chelator and found that Zn and Cu did compete. However, the overall binding strength of the polymeric chelators for iron was affected only modestly. These agents prevented the rise of hematocrit and hemoglobin in treated mice and suggest that arresting the intestinal uptake of dietary iron is a viable option for depleting iron levels. The hydrophilic polymeric hydroxamic acid gel was non-toxic and suggests the feasibility of using non-absorbed iron binding polymers as oral agents to sequester dietary iron in the GI tract. In 2009, our group reported the development of well-defined, blood compatible and degradable PEG based copolymers conjugated with DFO (P-DFO) for application in iron chelation therapy (70). PEG methacrylate was copolymerized by the RAFT method with a functional monomer for the conjugation of DFO to the polymer backbone via degradable or non-degradable linkage. The presence of PEG increased the biocompatibility of these nanoconjugates. The presence of hydrolysable ester linkages was anticipated to result in slow degradation of the conjugate via the ester linkages between the PEG side chains and copolymer backbone. P-DFO had MWs ranging from 27-127 kDa with between 5-26 DFO units per polymer chain and 24  demonstrated improved biocompatibility and toxicity profile as compared to unconjugated, small MW DFO. Like dextran and HES-DFO, P-DFO had a drastically improved toxicity profile; while unconjugated DFO exposure in HUVECs resulted in death at 3 μM, P-DFO was associated with ~90% cell viability up to 700 μM.  The most significant evidence existing for the potential clinical utility of polymeric iron chelators was published by Harmatz et al (71). In this first reported human clinical trial of polymeric chelators, S-DFO (40SDO2) caused clinically significant iron excretion after single dose infusion of S-DFO. The highest plasma chelator level of 6 mmol/L was achieved by 40SDO2 after 4 h intravenous infusion, an order of magnitude higher than that which occurs with DFO treatment. More importantly, the unsaturated iron binding capacity of S-DFO remained for one week and the dose given was not associated with any toxicity while resulting in significant urinary iron excretion (71).  Apart from the previously described polymeric structures with specific chelators attached to the polymer backbone, other polymeric chelators have also been generated. Winston et al prepared polymeric chelators with hydroxamic acid terminated side chains (72). These polymeric chelators were composed of amino acid amide derivatives of acrylic and methacrylic acid with the terminal carboxyl group converted to the hydroxamic acid. Polymeric chelators demonstrated a high affinity for iron(III) and were able to remove iron from iron overloaded mice when administered via i.p. injection.  Zhou et al report the synthesis of 3-hydroxypyridin-4-one hexadentate ligand-containing copolymers by copolymerization of a 3-hydroxypyridin-4-one hexadentate ligand with N,N-dimethylacrylamide (DMAA), and N,N’-ethylene-bis-acrylamide (EBAA) using (NH4)2S2O8 as the initiator (73). This class of chelator has demonstrated high selectivity and affinity for iron(III), 25  and has demonstrated potential clinical utility for the treatment of iron overload diseases associated with the hyper-absorption of iron.            Since iron accumulates as a result of transfusions as well as dietary absorption, it has been suggested that blocking the intestinal absorption of iron may also significantly reduce iron levels in patients. This has been attempted by administering high affinity, high MW chelators that are not absorbed by intestinal cells, which bind iron and promote its removal from the body. Zhou et al designed hydroxypyridinone-containing polymers which significantly reduced intestinal iron uptake. In their in vitro intestinal perfusion study, the accumulated absorbed iron was significantly reduced compared with the control groups in the presence of polymeric iron chelator.  Dendrimers have also demonstrated suitability for the generation of polymeric iron chelators (74). Zhou et al designed novel dendrimeric iron chelators by terminating dendrimers with hexadentate ligands formed from hydroxypyridinone, hydroxypyranone, and catechol moieties and have demonstrated that these novel conjugates can reduce iron absorption efficiently. This supports the idea that polymeric and dendrimetric iron chelators may be able to uniquely diminish iron absorption through the intestine and may have potential clinical utility in reducing dietary iron absorption due to their high MW (74).   Although the encapsulation of DFO into liposomes has also been attempted as a means of improving its therapeutic index, it has been unsuccessful (75). This is likely because DFO encapsulation does not mean that DFO is continuously sequestered and protected from rapid excretion or degradation once released. More importantly, once released from the liposome, DFO can cause toxicity in cells. Liposome encapsulation of DFO can be valuable but maybe more useful in treating cancers or tumors that have a high iron requirement but it also has to be targeted to ensure a very specific release location. 26  1.4: Advancing Iron Chelation Therapy 1.4.1: Investigating the Effects of Iron Chelation Therapy During Development  In addition to the side effects and failures of individual chelators, there are additional questions and uncertainties concerning the use of chelation in some populations of iron overloaded β-TM patients. To date, there has been very little research conducted on the safety and mechanisms of iron chelators in early stages of development and all chelation therapy is regarded as potentially teratogenic in the first trimester of pregnancy (76-79).  A few studies have been conducted on the topic (78-81). Bosque et al reported that DFO may cause high maternal toxicity and may also cause developmental toxicity (78). DFO was reported to have some effects on bone formation in mice when large doses up to 5 times the maximum daily human dose were administered (78). Similar results were found in rabbits while there was no effect in rats. It has been shown that DFO at high doses during organogenesis can cause reduced body weight gain in Swiss mice, at and above doses of 88 mg/kg/day. There was also a significant decrease in the number of live births at doses above 300 mg/kg/day. DFO caused early deliveries in a dose dependent manner with 32% of those treated with 88 mg/mL delivering early. This number increased with increasing does to 22.2% and 29.4% in those given 176 and 352 mg DFO/kg/day. Developmental toxicity occurred secondary to maternal toxicity and only at the dose of 352 mg/kg/ day of DFO. Although there has been reference to a case of abortion in humans due to early exposure to DFO, DFO is the only chelating drug that has been successfully used to treat women in the 2nd and 3rd trimester of pregnancy (76-77). Although data on DFO use later in pregnancy is promising, 27  DFO safety is not fully established and the drug label only recommends use when “benefits outweigh risks” (76-77).  Chelation with L1 and ICL-670 has been reported to be teratogenic in mice (80, 82). The range and mechanisms of developmental toxicity of L1 has also been scarcely reported. Berdoukas et al reported that LI may exert teratogenicity in rats, cynomolgus monkeys and rabbits (80). Organ atrophy was observed in rats that were treated with 100 mg/kg/day or more and L1 was thought to exert significant effects on the bone marrow which depleted the number of red and white blood cells.  Studies of ICL-670 induced toxicity during early development have been scarce. However, studies conducted in rats have demonstrated that ICL-670 can cross the placental barrier, albeit to a low extent. Moreover, Bruin et al also reported the accumulation of ICL-670 (at a level of 3% of the dose) in breast milk of rats (36).   As a result, patients are generally withheld chelation therapy once pregnancy is confirmed. For example, in a Greek study reported by Aessopos et al, in which pregnancies in this population were being planned, chelation therapy was interrupted one month before patients conceived and in any cases that resulted in unplanned pregnancies chelation therapy was ceased as soon as the pregnancy was confirmed (76-77). In other countries like Australia, all chelation therapy is withheld in this population, while in other countries like the USA and Greece, chelation therapy with DFO may be used after the first trimester if necessary (82-83). It is well documented that iron overload during pregnancy increases the number of complications in this population and may greatly increase the risk of cardiomyopathy in the mother and may also lead to a reduction in fetal growth (76-77, 85). For example, Farmakis et al describe a pregnant patient whose iron levels drastically increased after 12 months without iron chelation. 28  The patient’s ferritin level went from 67 μg/L to 1583 μg/L. Additionally, this patient’s heart function rapidly deteriorated and her liver iron concentration went from 1 mg/g dry weight to 11.3 mg/g. This indicates that in a short period of ceasing chelation therapy moderate to severe iron loading may develop in pregnant patients (85). During her pregnancy she accumulated 10.3 mg/g of Fe, which is approximately 0.85 mg/g dry weight per month. This demonstrates that stopping chelation in these patients is associated with significant risks and may worsen iron overload status, increase morbidity and the likelihood of cardiac failure.   Singer and Vichinsky also reported the increased risk of iron overload with pregnancy (79). They report the case of a 20 year old female who was chelated during pregnancy after having a liver iron concentration of 22 µg/g Fe dry weight.  DFO was restarted at 18 weeks of gestation both as infusions and intravenously. The dose was increased from 40-50 mg/kg at 26 weeks as her FTN rose to 7000 ng/mL. Chelation with DFO caused a reduction in her ferritin levels.  Assepos et al report that 9 months or more with no chelation could cause new endocrinopathies like hypothyroidism and diabetes mellitus to develop due to the increased iron burden. Although some longitudinal studies have demonstrated that patients can be aggressively chelated to combat this problem before pregnancy and that these patients are less likely to suffer from endocrinopathies or cardiac problems, this is not possible for some patients (76-77). Due to the greatly improved quality of life and life expectancy of patients with β-TM, it is likely that pregnancies in this population will continue to increase. Given this and the evidence that suggests increased iron overload can pose significant risks to mothers, it is important to conduct studies which define the effects of chelation in early gestation. This may advance our knowledge and enable the development of guidelines and more efficient, safer chelators for this population in the future. Moreover, there are no current recommendations for chelation therapy in 29  pregnant women that are based on well controlled studies. Therefore, investigating chelator exposure at early developmental stages is vital, and will become increasingly important with the increased likelihood of transfusion dependent women reaching reproductive age.  1.4.2: Monotherapy Is Inadequate to Ensure Negative Iron Balance  Despite the availability of three iron chelators, some patients fail to achieve adequate iron excretion with a single chelator. Furthermore, it has been estimated that 20% of patients undergoing iron chelation therapy will be inadequately chelated and remain susceptible to organ damage (10, 12). This is due to factors such as poor compliance in DFO-treated patients, inefficacy in some L1-treated patients and a wide range of toxic side effects like kidney failure among ICL-670 users. For example, in some patients that are treated with DFO infusions for at least 5 times a week, serum ferritin levels may still exceed 1000 ng/mL and many remain susceptible to cardiac iron overload and failure. Additionally, 80% of patients undergoing DFO therapy will experience reactions at the infusion site (10). Oral L1 therapy alone will ensure a negative iron balance in some but not all patients and ICL-670 may cause severe kidney failure in some patients leading to death (29-30, 32, 39-43).  In order to achieve effective iron chelation therapy, chelators must be efficient at removing equal or greater amounts of iron than that which accumulates from chronic red cell transfusions. This requires chelators to be present in the body for prolonged periods in order to reach the target sites at relevant concentrations. Since there are several iron pools that develop in iron overload, the LIP in plasma and labile cell iron in the cytosol, chelators which are effective at mobilizing iron from all labile iron pools would be more advantageous. Secondly, the cardioprotective effect of iron chelation therapy is a critical feature for measuring the efficacy of iron chelators as cardiac disease caused by iron overload is the leading cause of death in those that are insufficiently 30  chelated (9-10, 12, 15-16). These desirable properties depend in part on the ability of chelators to bind NTBI over sustained periods of time, thereby decreasing tissue uptake and iron-catalyzed toxic reactions. In addition, iron chelators that are clinically effective must be able to selectively bind to iron in the presence of other physiologically important metal ions. Table 2 describes some of the preferred features of an “ideal” iron chelator and contrasts it with the features of currently approved iron chelators DFO, L1 and ICL-670.   31  Table 2: Contrasting features of ideal and currently approved iron chelators     Chelator Property Ideal Chelator DFO L1  ICL-670 Cost Affordable for patients in low income countries where β-TM, SCD and MDS are common Moderate  Moderate Unaffordable and unavailable  for most Route of Administration Oral i.v. injection or s.c. infusion Oral Oral Circulation t1/2 Long enough to allow once-daily dosing and effective iron removal Short (~20 min) requires all-day (8-12h) delivery   Moderate; requires at least 3 times per day dosing Ideal; 8-16 hours, requiring once-daily dosing Therapeutic index High High at high doses in patients with high burden Unpredictable High  Toxicity  None Neurotoxic, swelling at infusion sites, bone deformities Agranulocytosis and mild neutropenia are common Reversible kidney failure Unsaturated Iron Binding Capacity  (UIBC) High: Long enough to prevent drastic fluctuations in LIP None Moderate High Ability to remove iron from heart, liver etc. High Low High  High  32  1.5: Theis Rationale, Hypothesis, Specific Aims 1.5.1: The Development and Characterization of Novel, High Molecular Weight Polymeric Iron Chelators DFO is currently on the World Health Organization’s Model List of Essential Medicines (2013) and is recommended as the front-line choice for both acute and chronic iron poisoning. DFO is also the most thoroughly understood iron chelator due to its many years of use in patients. Several studies have demonstrated the potential benefits associated with modifying DFO properties with polymers (68-75). Therefore, we approached this study aware of the previous reports of DFO modification, our expertise developing biocompatible polymers for use in clinical applications and our experience engineering low MW drugs to enhance their properties. Our goal was to develop a new class of polymeric iron chelator.   Global Thesis Aim: To develop a novel class of high molecular weight, polymeric chelator and define its iron binding affinity, biocompatibility and toxicity profile compared to small molecular weight DFO.  Global Hypothesis: Conjugating DFO to high molecular weight, biocompatible polymers (HPG) generates high affinity, efficient, iron chelators capable of stably binding iron under physiological conditions and preventing iron mediated oxidative damage. High molecular weight chelators will also exhibit diminished toxicity profiles and enhanced biocompatibility compared to unconjugated DFO. Specific Aim I: The first aim of this thesis was to develop a library of high MW iron chelators through conjugation of DFO to biocompatible polymers. This was based on previous 33  studies conducted in our laboratory and elsewhere, which showed that polymer conjugation can increase the half-life and diminish the toxicity associated with iron chelators. Table 3 shows a summary of the reported benefits of DFO conjugation to polymers.  Table 3: The reported benefits of polymer conjugation to iron chelators.   Conjugated forms of DFO are associated with reduced toxicity both in vitro and in vivo. Dextran-DFO, HES-DFO and HPG-DFO demonstrated significantly higher plasma half-lives than unconjugated DFO.   † Novel Polymeric Iron Chelator Developed Through this Thesis  To achieve this, hyperbranched polyglycerol (HPG) was used as the polymeric component for engineering DFO into a high MW chelator. Our lab has been a pioneer in the synthesis and characterization of HPG for use in biomedical applications (86-93). HPG represents a class of versatile, biocompatible, inert, dendritic polymers that can be synthesized in a controlled one-step Polymeric-DFO Conjugate Pharmacodynamic effect Pharmacokinetic effect Dextran-DFO  LD50 increased from 250 mg/kg to 4000 mg/kg    Reduction of pulmonary hypotension in dogs Increased circulation half-life  Starch-DFO (40SDO2)  Reduced retinal toxicity in albino rats   Reduction of pulmonary hypotension in dogs, excess free iron binding capacity in healthy males Increased circulation half-life   Increased in vivo iron excretion efficiency  PEG-Methacrylate-DFO  Reduced endothelial cell toxicity  --  HPG-DFO† Increased LD50  Increased circulation half-life in mice, decreased clearance  Increased in vivo iron excretion efficiency in mice 34  reaction with narrow polydispersity. Detailed testing of these polymers conducted in vitro and in vivo has demonstrated the potential unique advantages that HPG may have in nanomedicine (86-88). Our group has previously developed HPGs as a synthetic substitute for serum albumin that closely mimics the binding and transport properties of natural albumin and is considered to hold advantages over the current clinically used plasma expanders (89-93). We have also coated HPG on red blood cells to mask different antigens towards the development of universal blood cells, developed DNA delivery agents, and developed anticoagulant neutralizing agents for use as heparin antidotes (92-93). Due to the high biocompatibility, multi-functionality and long circulating nature of high MW HPG, we anticipated that it would be a promising candidate for the development of new generation non-toxic macromolecular chelators for the removal of toxic iron in vivo.  Study hypothesis: Conjugating varying numbers of DFO molecules to high molecular weight polymers could generate a class of macromolecular iron chelator with high iron chelation efficacy, capable of binding iron and preventing harmful iron mediated reactions to an extent that varies with the degree of functionalization with DFO. We conjugated HPG, the polymeric component to DFO forming HPG-DFO and investigated whether there was any change in the iron chelating properties of DFO after conjugation to the polymer using isothermal titration calorimetry, UV-Visible Spectroscopy and ferritin iron binding studies. The results of this study are described in the Chapter 2 of this thesis. After developing and characterizing the iron binding properties of novel, high molecular weight DFO conjugates, studies aimed at defining the biocompatibility and toxic effects of the polymeric component were conducted in vitro (Chapter 3). Since DFO is administered by s.c. 35  infusion or i.v. administration, emphasis was placed on defining the blood compatibility of novel HPG-DFO conjugates with the human blood components.  Specific Aim II: The goal of this study was to investigate the blood and biocompatibility, and toxicity of HPG-DFO in human blood components. We hypothesized that the HPG component used to develop HPG-DFO does not enhance DFO toxicity but improves the biocompatibility of DFO. The results of this study are described in the third data chapter of this thesis (Chapter 3). 1.5.2: Comprehensive Investigation of Iron Chelator Toxicity in Zebrafish Embryos The use of chelating agents during gestation has been a controversial topic for decades. In general, LI and ICL-670 are contraindicated. DFO use during pregnancy is also highly discouraged unless benefits out weight the risks. Exposure to chelators during pregnancy varies widely from abstention of chelation, accidental exposure followed by abstention in the case of one ICL-670 case report, and treatment in the second and third trimesters for many DFO patients (76-85). Thus, understanding the effects and potential toxicities of iron chelators in models of early vertebrate development is of special interest. Specific Aim III: The first part of the study reported in Chapter 4 was exploratory and aimed to investigate the potential toxicities caused by FDA-approved DFO, L1 and ICL-670 in developing vertebrate embryos. In addition, high molecular weight HPG-DFO was compared to DFO to determine whether any major changes to DFO toxicity would occur upon conjugation. Owing to the ability to observe effects in the embryo independent of maternal factors, the 36  transparency of zebrafish embryos and their rapid development, this study used zebrafish as a model for assessing chemical toxicity.  Prior to this thesis, there have been a few studies conducted on the effects of iron chelation in early developmental stages. Thus, we approached this work with the awareness of current deficits in knowledge regarding iron chelator-induced toxicity in early development and the challenges associated with interpreting the data that does exist on the subject. Further, we considered it important to use a simple model that could provide unambiguous data about direct chelator effects in embryos.  Zebrafish (Danio rerio) are increasingly used as a cost-effective in vivo model and have been shown to be useful in evaluating chemical toxicity (94-99). The toxicity data obtained in zebrafish correlates well with developmental toxicity data from rat in vivo studies and previous studies have demonstrated that the prediction success rate for some drugs can be as high as 100% in zebrafish (96).  Further advantages of zebrafish include the rapid rate of organogenesis and the large number of embryos obtained from each spawn which allows throughput screening (97-99). Most importantly, the optical transparency and ex-utero development of zebrafish embryos allow the direct assessment of functional, morphological and behavioral effects of test compounds on embryos in the absence of interference from maternal factors.  It is anticipated that the findings of this study would begin to improve our knowledge and understanding of chelator induced toxicities in early stages of development. Additionally, it is our hope that this study will help to direct the focus of future studies on chelator-induced toxicity in early development. Moreover, it is a significant contribution to the field as such knowledge is sparse for iron chelators.  37  We exposed developing zebrafish embryos to various concentrations of DFO, L1 and ICL-670 and assessed their effects on mortality, hatching and morphology in embryos. To determine whether the effects were specific to embryos and to probe possible underlying mechanisms of the observed toxicities, we investigated the effects of chelator exposure on the cardiac function and gene expression in adults. This study improves our understanding of the differences in potential effects of clinically approved iron chelators at the earlier stages of embryonic development. Results will guide our approach in further understanding the effect of iron chelation in vertebrates during development, and will provide clues on the possible organ systems perturbed by iron depletion in developing embryos. In addition, to further define the properties of HPG-DFO and determine whether the polymeric component of the conjugates was associated with any toxicity, HPG-DFO were also screened for toxicity in zebrafish. These results are presented in Chapter 4.  1.5.3: Significance and Novel Contributions of Thesis There are several significant contributions arising from this thesis. First, this thesis develops and describes the properties of a novel class of high molecular weight iron chelator, which was generated by using HPG and DFO, the most extensively used and oldest form of iron chelation therapy. DFO modification with polymers generated a new class of larger or “macromolecular” chelator which was subsequently characterized for iron chelation efficacy in vitro, efficacy of preventing iron mediated damage, biocompatibility and toxicity in vitro and in vivo. Secondly, this thesis investigates the toxicity of iron chelators in embryos. An important feature of these studies was the use of a model that precluded the interference of maternal factors. Furthermore, attempts were made to probe the organ systems most susceptible to iron depletion in 38  early development. Given the increased lifespan and reduced morbidity in thalassemic patients, it is conceivable that there will be significant increases in pregnant women in need of iron chelation in the near future. Given the reported potential dangers associated with withholding chelation therapy in pregnant women and the controversy surrounding optimal therapy during pregnancy there is an urgent need to understand the potential toxicities and mechanisms of FDA licensed chelators and develop new, safer iron chelators with improved toxicity profiles, and iron binding efficiency. We have investigated the toxicity in zebrafish to begin to build a knowledge base on the mechanisms by which iron chelators may exert toxicity in developing vertebrates. Further, we have probed the potential mechanism of toxicity by measuring gene expression and changes in heart function.      39  Chapter 2: Polymer Size and the Degree of Functionalization with Desferrioxamine Modulate Chelator Iron Binding Thermodynamics and Efficacy 2.1: Overview  Desferrioxamine (DFO) is a clinically approved, high affinity iron(III) chelator used for the treatment of transfusion-associated iron overload. Due to poor oral availability and short half-life, DFO is administered by subcutaneous infusion for 8-12 hours per day, 5-7 days per week. These prolonged administrations, as well as high doses of DFO cause irritation at the infusion site in 80% of patients, and may cause neuro toxicity and bone deformities. DFO was conjugated to soluble, non-interactive hyperbranched polyglycerol (HPG) of molecular weights (MWs) 25 kDa to 500 kDa to enhance its pharmacokinetics. This study investigates the thermodynamic and metal-binding properties of this new macromolecular iron chelator. Isothermal titration calorimetry (ITC) was used to measure the binding enthalpy (ΔH), the stability of the chelator-iron complex after polymer conjugation (ΔG), and the stoichiometry of binding (n). The influence of polymer size on iron removal efficiency from ferritin, the primary iron storage protein in humans, is also reported. Results demonstrate that HPG-DFO conjugates are high-affinity iron(III) chelators. Additionally, HPG-DFO conjugates remove iron from ferritin efficiently, with an iron-removal rate that varies with polymer MW. The addition of small molecular weight chelator deferiprone and increasing the reaction temperature resulted in increased iron extraction from ferritin by all chelators, regardless of MW. Although polymer MW and the extent of modification with chelator molecules are important determinants of chelator efficiency at room temperature, chelation properties of higher MW polymer conjugates were more similar to that of smaller MW conjugates when the iron binding reactions proceeded at physiological temperature. These results therefore help to 40  define the iron binding thermodynamics of polymeric chelators and their dependence on molecular weight, and can be extended to improve our general understanding of polymeric chelator-iron interactions in situ. 2.2: Background  Desferrioxamine (DFO) is a high affinity, hexadentate iron chelator (21, 100). It has been used clinically since the 1960s for the treatment of iron overload in transfusion dependent patients with β-thalassemias, SCD and MDS and for metal poisoning (10-12, 101). DFO binds ferric iron stably in a 1:1 ratio, with a stability constant, log K=~30, which renders iron metabolically inert. Thus, DFO can prevent the dangerous redox cycling and radical production that causes oxidative damage to cells and tissues (18-19, 21).   While DFO therapy has improved the quality of life and lifespan for transfusion dependent patients, its use has been limited by its poor pharmacokinetic properties and the toxic side effects that are associated with higher doses. Due to its relatively large size and hydrophilicity, DFO is not orally absorbed and must be administered by subcutaneous infusion for 8-12 hours daily 5-7 days per week (10-12, 21-25). In addition, DFO is associated with sensorineural toxicity when given at high doses and pain, itching, erythema, swelling and discomfort at the site of infusion is reported to occur in approximately 80% of patients. Due to these challenges, compliance to DFO therapy among some patients, especially in young children and teenagers is difficult to achieve (21-25, 102).  Patients that comply with DFO therapy have a longer lifespan, higher quality of life and are less likely to develop organ damage from transfusional iron loading than non-compliant patients. Non-compliance has been shown to cause dramatic reduction of life expectancy in patients performing less than 225 DFO infusions per year as compared to those with a more regular 41  treatment (102). Thus, the direct influence of poor compliance to DFO therapy on the clinical outcome of patients is significant.    In order to improve the properties of DFO, many efforts have been made towards engineering it into a high molecular weight (MW) chelator (68-75). It has been demonstrated that the toxicity profile, the circulation half-life, number of doses needed to achieve iron removal, and efficacy of DFO can be redefined by conjugating it to PEG based polymers, dextran, hydroxyethyl starch (HES), triazene dendrimers and hyperbranched polyglycerol (HPG) without any major changes in iron (III) chelating properties (68, 70-72, 103). Through the development of 40SDO2, a starch-based polymeric chelator, Harmatz et al have demonstrated the potentially high clinical utility that can be associated with high MW DFO. Moreover, their study demonstrated that polymer conjugation can increase the unsaturated iron binding capacity of DFO; a property that could potentially reduce the number of injections required by transfusion-dependent patients undergoing iron chelation therapy (71). Gehlbach et al showed that polymer conjugation completely prevented the retinal damage in an albino rat model, indicating that polymer conjugation may strongly influence cellular distribution and toxicity (103).  Therefore, we hypothesized that: Conjugating various numbers of DFO molecules to high molecular weight polymers produces a class of macromolecular iron chelator with high iron chelation efficacy, capable of binding iron and preventing harmful iron mediated reactions to an extent that varies with the degree of functionalization with DFO. We have developed a library of high MW DFO conjugates that vary in MW and the number of DFO attached to the polymer (DFO density), subsequently referred to herein as HPG-DFO. In this study, we define the properties of this novel class of polymeric iron chelator by evaluating their binding thermodynamics by ITC. To date, the study of iron binding to polymeric, DFO based 42  iron chelators, has been conducted primarily by spectroscopic techniques, while there is significantly less information available about the thermodynamics of ferric iron binding to polymeric chelators. Therefore, we compare the binding thermodynamics and iron chelating properties of HPG-DFO and DFO using ITC. We were especially interested in whether the size and functionality of the conjugates exert any “penalty” on the iron binding affinity DFO.   In addition to ITC studies, we characterize the influence of polymer size on the iron binding ability of the chelator by studying iron removal from ferritin and the prevention of hemoglobin oxidation (HbOx). Ferritin is an intracellular multimeric protein which specializes in storing large amounts of iron (1, 6, 104). Ferritin forms an important component of the body’s antioxidant strategy and the access of chelators to its iron core has been shown to be dependent on chelator size (1, 6, 104).  Under normal physiological conditions, excess iron in the body is stored in the liver, spleen, and bone marrow in the form of ferritin molecules. However, under pathological conditions such as the thalassemias, SCD, and MDS, iron overload occur and the excess iron is deposited as insoluble ‘iron cores’ of degraded or partially degraded or denatured ferritin (described as hemosiderin) primarily in liver, spleen, endocrine organs and myocardium (10, 12). Although low MW, cell permeable chelators are more likely to have continued access to the intracellular ferritin core, higher MW, DFO-based chelators with extended half-lives may also access this iron pool due to two important reasons. It has been shown that DFO targets cellular ferritin to the lysosome. It has also been shown that polymers and nanomaterials may be targeted to the lysosome, thus extened exposure to higher MW chelators may allow access to this pool, making this an important comparative study.  43  Thus, we considered that the interaction of polymeric chelators with ferritin would provide important information about the possible mechanisms of iron removal by macromolecular chelators as well as the influence of polymer size on iron chelation ability. Moreover, since previous studies have shown that iron removal from ferritin is possible by DFO, comparing the iron removal from ferritin by polymer conjugated DFO would allow the direct determination of deviations imposed by polymer conjugation, polymer size and DFO density of polymer conjugates.  2.3: Materials and Methods  Chemicals: Ferric ammonium sulfate dodecahydrate (FAS), sodium periodate, sodium cyanoborohydride, iron(II) sulfate heptahydrate (>99 %), ferric(III) chloride, nitrilotriacetic acid (NTA), HEPES, sodium acetate buffer, imidazole buffer, horse spleen ferritin (F4503), desferrioxamine mesylate, deferiprone (3-hydroxy-1,2-dimethylpyridin-4(1H)-one) and 1, 10-phenanthroline were purchased from Sigma-Aldrich Canada, Oakville, ON. Starch conjugated DFO (40SD02) was received as a gift from Dr. Mark D. Scott, the Centre for Blood Research,  University of British Columbia.  Synthesis and characterization of HPG-DFO: Different molecular weight of HPG were synthesized by a ring opening anionic polymerization of glycidol. The absolute molecular weights of the polymers were determined by gel permeation chromatography (GPC) on a Waters 2695 separation module fitted with a DAWN EOS multiangle laser light scattering (MALLS) detector coupled to an Optilab DSP refractive index detector, both from Wyatt Technology Inc., Santa Barbara CA; the details have been described previously (87). DFO was then conjugated to HPG using Schiff-base chemistry as previously reported (105). In a typical reaction, 100-200 mg of HPG (25, 44, 500 kDa) were dissolved in MilliQ water (2-4 mL). To oxidize the 1, 2-diol groups on the HPG, 84 µL of a 0.5 M solution of NaIO4 was slowly added to the solution over a period of 44  1-3 minutes and the solution was left to stir for 24 h. The solution was then dialysed against water to remove the unreacted NaIO4. Dialysis was carried out for 24 h using a Spectra/Por dialysis membrane (MWCO 1000). DFO was then added in 1.2 molar excess of the number of aldehydes generated. After 8 h, NaCNBH3 was added at 1.2 molar excess of the number of aldehydes generated and this solution was left to stir for 24 h. The remaining product was put to dialyze against water for 3 days with frequent changes of water. Excess, unreacted aldehyde groups were quenched using glycine. Using similar protocols, HPG-DFO conjugates with different MWs and DFO density were prepared. Appendix A shows the properties of an array of HPG-DFO conjugates synthesized by this method. The analysis of some selected compounds is reported here.  UV-Visible Spectroscopy was used to determine the functionality of HPG-DFO conjugates. A thermally controlled Varian Cary 400 UV-Vis Spectrophotometer was used to determine the amount of DFO attached to the polymer. Conversion of chelators to the DFO-iron complex was achieved by incubating HPG-DFO conjugates with 10 mM iron(II) sulphate heptahydrate and left for 24 h at room temperature (22°C) as previously reported. After 24 h, the absorbance of the DFO-Fe chelates were measured at 429 nm and the concentration of DFO in the polymer conjugate was determined by using the molar absorptivity of ferrioxamine (2300 M-1 cm-1) (70, 105). Iron(III) binding thermodynamics by isothermal titration calorimetry:  ITC studies were carried out on a MicroCal VP-ITC (MicroCal Inc., Northampton, MA). Chelating agents studied by ITC included DFO, HPG-DFOs of different molecular weight and DFO density, and 40SDO2. All data analysis was performed using the Origin software supplied by MicroCal. All experiments were carried out at 25 °C in 100 mM HEPES buffer, pH 7.0 + 0.2. Reactions were carried out by injecting 25 consecutive 10 μL aliquots of 0.1 mM FeCl3-NTA (2-fold molar excess of NTA) into the sample cell which contained 1.424 mL of 10 μM of chelator (DFO, HPG-DFO 45  or 40SDO2). The time between each injection was 5 minutes. Control experiments were performed by titrating FeCl3-NTA solution into chelator-free buffer and unmodified HPG. All samples were degassed before loading into the sample cell and injection syringe. All titrations were carried out 2-3 times to ensure consistency of the data and stability of the solutions.  The experimental data were fitted to a one-site binding model and the association constant (K), stoichiometry (n) and enthalpy change (ΔH) were recorded for each reaction. The entropy and Gibbs free energy of binding were then determined from the relationship ΔG°=-RTlnK=ΔH°-TΔS°. Preparation of Ferritin: Horse spleen ferritin was used without further purification. Apoferritin was prepared according to previously established protocols by Hoy et al (106). Briefly, apoferritin was obtained by dialyzing ferritin solution in 1 M sodium acetate buffer , pH 5.4 containing 3% sodium dithionite. The protein concentration was determined according to the Lowry method (107). Ferric ammonium sulfate, potassium iodate (0.07 M) and sodium dithionite (0.1M) were used for iron loading of apoferritin (106, 108). The protein concentration was 3.0 mg/mL and was loaded with 5 mM iron. The amount of ferrous iron incorporated into ferritin was determined using 1,10-phenanthroline and an extinction coefficient ε535 nm of 22100 mol iron L-1 cm-1 as previously reported (109).  Evaluation of molecular characteristics of iron chelators on iron removal from ferritin: The iron removal from ferritin by macromolecular iron chelators was investigated using UV-Vis spectroscopy either alone or in combination with small molecular weight chelator deferiprone (L1).  Different molecular weights HPG-DFO (44K and 500K) and DFO were used for the study. Different incubation times and temperatures were used to determine the efficacy of HPG-DFOs and the factors that influence chelation. In a typical experiment,  chelators were dissolved in 20 mM imidazole buffer, pH 7.00 and were added to ferritin solutions to produce final 46  chelator concentrations of 50-500 µM. Solutions were allowed to incubate for 1 h, 24 h and 48 h before conducting spectroscopic readings at room temperature (22°C) or 37 °C. A broad absorption peak around 429 nm was indicative of Fe(III) complexes of DFO or HPG-DFO as reported (70, 105). Both DFO and HPG-DFO samples without Fe(III) showed no maximum absorption at 429 nm. Using a molar absorptivity coefficient of 2300 M-1 cm-1, the Fe(III) content in the conjugate was determined. This value was taken as the amount of iron removed from the ferritin. To determine whether small molecular weight chelators enhance the efficacy of polymeric chelators in removing the iron from ferritin, L1 (250 µM) was added along with DFO and polymeric chelators (250 µM) and monitored for 1, 24 and 48 h. Readings were obtained at 22°C and 37°C.  Inhibition of iron mediated oxidation of hemoglobin: Hemoglobin (Hb) oxidation was used to determine the ability of HPG-DFO to protect proteins against iron-mediated damage.  Hb concentration (g/L) was determined spectrophotometrically at a wavelength of 540 nm by the cyanmethemoglobin method based on Drabkin’s assay with cyanide–ferric cyanide solution and as previously described (70, 105, 110). Hb solutions prepared in 5 mM tris buffer at pH 7.4 were adjusted to a final concentration of approximately 22 μM heme at a final pH of between 7.1 and 7.4 at 25 °C. Fe3+ GSH driven oxidation of Hb was determined by spectrophotometric analysis between 500 and 700 nm (111-112). The scans were performed with 30s cycle times for 5 minutes and 200-400 uM of Fe 3+ was used for experiments. The mixture of Fe3+ and HPG-DFO was incubated for 5 minutes before measurement was taken. The test reactions were carried out in disposable polystyrene cuvettes, with a mixture of Hb solution with either pre-chelated DFO or HPG-DFO Fe3+ and reduced glutathione (GSH). The percentage of oxyhemoglobin was calculated as a function of the ratio of the oxyhemoglobin absorbance peaks (500–600 nm) and the 47  methemoglobin absorbance peak (630 nm)  (111-112). DFO or HPG-DFO (200-500 µM) were used to examine whether chelation of free iron could inhibit hemoglobin oxidation. Statistics: The paired t-test was used to compare differences between chelation at room temperature (22 °C) or 37 °C and with the addition of deferiprone (L1). P values < 0.05 were considered to be significant.    48  2.4: Results 2.4.1: Synthesis and Characterization of HPG-DFO  Different MWs of HPG-DFO were synthesized and characterized. The method for conjugation is illustrated in Figure 7 below.   Figure 7: Synthesis scheme for HPG-DFO.  DFO is conjugated to HPG using the reaction scheme shown above. HPG is oxidized using sodium periodate (NaIO4) and DFO is subsequently added in the presence of sodium cyanoborohydride (NaCNBH3). HPG possessing MWs of 25, 44 and 500 KDa were conjugated to various numbers of DFO molecules.  Schiff-base chemistry allowed for the variation of the number of DFO molecules conjugated per HPG molecule. Based on the absorption maximum of the HPG-DFO chelate at 429 nm, the number of DFO molecules conjugated per polymer was determined (Figures 8-9). This result also confirmed that the general spectroscopic characteristics of the DFO-iron complex were not changed upon conjugation to HPG and the molecular weight of the conjugate did not influence iron complex formation. The characteristics of the HPG-DFO prepared are given in Table 4 and 49  Appendix A. Molecular weights of the conjugates ranged from 25 kDa to 500 kDa and the DFO density was varied for each MW class. For instance, in the case of 44 kDa conjugate, the number of DFO molecules was varied from 10 to 85. Similarly for 500 kDa conjugates, the number of DFO per polymer varied  from 55 to 129 DFO molecules per HPG for this study (Table 4).    Figure 8: Iron chelation by HPG-DFO depends on the number of DFO conjugated per polymer.  500 kDa HPG-DFOs (1mg/mL) with increasing DFO density were incubated with 10 mM iron(II) sulphate at room temperature and left to complex overnight. HPG-DFO with higher numbers of DFO per polymer complexed more iron than HPG-DFO with lower DFO density. This is demonstrated by the deepening color of the solution with increasing DFO density. The identity of all chelators is given in Appendix A.    50   Figure 9: Conjugation of DFO to HPG does not influence DFO chelating properties.   HPG-DFO (1mg/mL) was incubated with 10 mM iron(II) sulphate at room temperature and left to complex overnight. HPG conjugated to varying densities of DFO produce a maximum absorbance in proportion to DFO density. Fe-free DFO, DFO-free Fe, nor unmodified, oxidized, HPG did not produce the maximum absorbance peak at 429 nm.     00.10.20.30.40.50.60.70.80.91300 400 500 600AbsorbanceWavelength (nm)DFO-FeFe44K-1-Fe44K-3-Fe44K-4-FeDFO51  Table 4: Characteristics of iron chelators used in this study.  Macromolecule Ligand Molecular  weight of the  conjugate (kDa) @ # of DFO &Rh (nm) DFO Fe (III) -- -- -- 25K-65 ≠ Fe (III) 67.6 65 2.7 44K-1 ┼ Fe (III) 50.5 10 4.2 44K-3 ┼ Fe (III) 73.5 45 4.5 44K-4 ┼ Fe (III) 99.8 85 --- 500K-2 ┼┼ Fe (III) 536.1 55 7.3 500K-4 ┼┼ Fe (III) 584.7 129 7.7 40SDO2 a Fe (III) 26 *40 mmol > 3.0   $HPG of various MWs between 25kDa and 500kDa were conjugated to various numbers of DFO molecules. DFO was used as a control in all experiments. Starch DFO (40SDO2), a previously reported polymeric chelator was also included in some analyses for comparison. Conjugates were prepared from HPG of MW 25kDa≠, 44kDa┼ and 500 kDa┼┼. &Rh- hydrodynamic radius determined by quasy elastic light scattering (QELS) (Wyatt Technology. Inc., Santa Barbara CA ) analysis in aqueous 0.5 N NaNO3 (pH-8) solution.   a40SDOS was prepared from a 26 kDa hydroxyl-ethyl starch polymer.  * The batch of 40SDO2 utilized in this study has a 40 mMol iron binding capacity. Conjugates were prepared from HPG of MW 25kDa≠ (Mn= 25, 000, Mw/Mn=1.1), 44kDa┼ (Mn = 44, 000, Mw/Mn=1.2) and 500 kDa┼┼ (Mn =500, 000, Mw/Mn=1.1).   52  @The molecular weight of the conjugates were calculated from the number average molecular weight of the parent polymer (determined using GPC-MALLS) and the number of DFO molecules per polymer.  53  2.4.2: Iron(III) Binding Studied by Isothermal Titration Calorimetry  ITC is the ideal technique to determine the thermodynamics of binding interactions between polymeric iron chelators and Fe(III). From a single titration experiment, the binding constant (K), binding enthalpy (ΔH) and reaction stoichiometry (n) can be obtained (113-115). As a result, ITC has been used to characterize the thermodynamics of metals binding to polymeric scaffolds (116). However, the technique has not been applied to Fe(III) binding to DFO or DFO-HPG conjugates. DFO or HPG-DFO binding to Fe(III) was monitored by titrating with 0.1 mM Fe:NTA in a 1:2 ratio at 25°C and pH 7.0 ± 0.2(HEPES buffer). Under these conditions, the concentration of free ferric ions is vanishingly small. Instead, iron is present in solution in the following speciation: 0.2% Fe·NTA, 9.0% Fe·NTA2, 18.9% Fe·NTA·OH2, and 71.9% Fe·NTA·OH (Appendix B) (117). Here, to simplify discussion, we collectively refer to the equilibrium set of species present at pH 7.0 as Fe(III)-NTA and report apparent binding thermodynamics for interaction of the Fe(III)-NTA complex with various chelating agents. Figure 10 reports representative raw ITC-generated titration curves and integrated heats fit to an independent-site binding model. For each system studied, the titration of Fe(III)-NTA into DFO or HPG-DFO shows that the titrant Fe(III) binds to DFO or HPG-DFO (the titrand) with a higher average affinity relative to the apparent Fe(III)-NTA complex.  A stoichiometry of one ferric-ion bound to each DFO (chelator) moiety present on the titrand molecule is observed. Titrations with pure buffer or the DFO-free control polymer as titrand were similar, indicating that the ferric ion does not non-specifically adsorb to the HPG scaffold (Appendix B). Binding isotherms indicate that like DFO, HPG-DFOs of all MWs have an affinity for ferric ions that is greater than that of NTA for Fe(III) (Figure 10). 40SD2 is also seen by ITC to bind iron efficiently and in a manner similar to DFO. Values for the Fe(III)-54  binding stoichiometry (n), apparent free energy (Gapp) and enthalpy (Happ) for HPG-DFOs, 40SDO2 and DFO are provided in Table 5. In all cases the binding is enthalpically driven. The binding stoichiometry reported is calculated based on the number of Fe(III) ions bound to HPG-DFO, DFO or 40SDO2 at saturation. For HPG-DFO conjugates 25K-65 and 44K-4, the polymeric chelators form complexes with iron in a 1:1 saturation ratio (Figure 10), as reflected in the n values near unity (Table 5). Thus, the polymeric scaffold does not change the intrinsic binding properties of DFO in these constructs. Other HPG-DFOs exhibit a saturated Fe(III) binding stoichiometry less than unity, which may reflect (steric) inaccessibility of some DFO moieties within those architectures, at least at lower thermal energies (low kT). This conclusion is supported by the fact that, on average, n decreases with increasing size of the polymer scaffold (Table 5).   55   Figure 10: HPG-DFOs are high affinity Fe(III) binding chelators.  Raw ITC curves showing the integrated heats and best fit for the binding interaction between chelators and Fe(III). FeCl3 (0.1 mM: 0.2 mM NTA) was titrated into 10 µM DFO, HPG DFO and S-DFO of various MWs at 25 °C. Solutions were prepared in 100 mM HEPES buffer at pH 7.0 (+ 0.02). The upper panel demonstrates the heat change due to the heat of dilution during each injection. The lower trace shows the integrated areas corresponding to each injection normalized as a function of molar ratio (metal/chelator). The best least-squares fit of the data to a one site model is given by the solid red line.  56  The values obtained for the apparent binding constants (different from the overall stability constant of Fe (III) complex) are in the order of 107 mol-1 from the current ITC measurements. The binding stoichiometry reported was calculated based on the number of bound Fe(III) to HPG-DFO, DFO or 40SDO2. Except for few cases (500K), the polymeric chelators formed complexes with iron similar to DFO, in a 1:1 ratio as values of N are near unity. This was verified by the sigmoidal shape of the curve demonstrating the binding of a single ligand (Table 5, Figure 10).  ITC data revealed that HPG-DFO exhibits similar binding in comparison to DFO (G & H) except for the case of 500K conjugates. In the case of 500K-4 for example, the values are notably lower compared to DFO. The binding stoichiometry (N) of this molecule was also smaller compared to DFO or other HPG-DFOs.  It is most likely that the binding stoichiometry is influenced by the ratio of DFO: polymer and the distribution of the DFO on the polymer.        There is a clear dependence on the degree of functionality of DFO per polymer on binding stoichiometry for the lower MW series. In the case of 44K conjugates, increasing the DFO per polymer increased the binding stoichiometry. However, an opposite trend was observed in the case of 500K conjugates where the stoichiometry decreased with increasing molecular weight (Table 5). This data suggest the iron (III) binding to this conjugate is different from 44K or 25K conjugates. The lower molecular weight HPG-DFO conjugates behave in a manner that is similar to DFO.     57  Table 5: Apparent Fe(III) binding thermodynamic paramaters for polymeric chelators measured using Isothermal Titration Calorimetry.  Chelator ΔG°app (kcal/mol) -ΔHapp (kcal/mol) TΔSapp (kcal/mol K) n DFO  -9.752 -19.2 + 0.5 -9.59 + 0.21 0.85+ 0.07 25K-65  -9.829 -18.4 + 1.1 -8.55 + 0.55 1.05 + 0.14 44K-1  -9.331 -20.1 + 0.2 -10.74 + 0.19 0.45+ 0.02 44K-3  -10.100 -18.8 + 0.7 -8.70 + 0.76 0.70+ 0.12 44K-4  -9.587 -17. 9 + 0.0 -8.38 + 0 1.09 + 0.17 500K-2  -9.349 -19.1 + 0.2 -8.31 + 2.52 0.58+ 0.10 500K-4  -9.399 -19.8 + 1.2 -9.64 + 1.08 0.36 + 0.04 40SDO2 -9.071 -17.7 +  0.8 -10.67 + 0.63 0.63 + 0.00  $ITC experiments were conducted at 25˚C and pH7.0+0.2 (HEPES buffer). Results were fit to an independent-site binding model to regress the apparent association constant (Kapp), binding stoichiometry (n) and apparent enthalpy change (ΔHapp) per mole of Fe(III) for the Fe(III):chelating agent interaction. The apparent binding entropy ∆Sapp and Gibbs free energy of binding were then determined from the relationship ΔG =- RTlnK = ΔH-TΔS. Reported error values are the standard deviation of 2-3 independent experiments.  2.4.3: Iron Removal from Ferritin: Influence of Molecular Weight and Concentration of HPG-DFO on Iron Removal   We next investigated whether polymeric chelators could remove iron from ferritin, the cytosolic iron storage protein (6, 104). Initially we tested the influence of molecular weight and concentration of chelator on iron removal from ferritin at physiological pH at 22°C. The 58  concentration of chelators incubated with ferritin varied from 50-500 µM and the ferritin was reloaded to have a ferric iron concentration of 5 mM. Different incubation times (1 h to 48 h) were used.  Results are shown in Figure 11. The pattern of iron removal by HPG-DFO from ferritin was similar to iron removal by DFO, increasing with chelator concentration and time of incubation, reaching a maximum after 48 h incubation. Additionally, the amounts of iron removed after 1 h, 24 h and 48 h varied with the molecular weight of the polymer; the amounts of iron removed decreased with increase in MW of the polymer which suggest that high molecular weight polymer conjugates may have less accessibility to iron rich core of ferritin. This data agrees with previously reported studies showing that DFO is capable of removing iron from ferritin, albeit slowly (118). Moreover, when compared to the polymeric DFO-based iron chelators tested in this study, DFO was able to remove more iron at each time-point at identical concentration.  We next investigated the influence of temperature on the iron removal; 22°C and 37°C were studied. The concentration of the chelators (500 µM) and ferritin iron (5 mM) was fixed. Increasing the temperature from 22°C to 37°C resulted in a significant increase in iron removal at 37°C after 1 h, 24 h and 48 h (Figure 12). The temperature effect was greatest for higher MW chelators (500K) whose rate of iron removal from ferritin approached that of the smaller MW, 44K chelator with longer incubation time. This suggests that for high molecular weight chelators longer incubation time is needed to achieve saturation of the chelator.   59                                      Figure 11:  Influence of MW of conjugates on ferritin iron binding.  Chelators were incubated with iron loaded ferritin for 1, 24 and 48 h at 22°C at pH 7.4 in imidazole buffer. The concentration of iron bound by each chelator was determined by UV-Vis Spectroscopy. Chelators removed iron in a time and concentration dependent manner and the iron removal efficiency was inversely proportional to polymer MW for all concentrations tested.  60   Figure 12: The effect of reaction temperature on iron extraction by chelators.  HPG-DFO and DFO (500 μM) were incubated with ferritin at room temperature (22°C) and at 37°C for 1, 24 and 48 h in imidazole buffer at pH 7.4. Increasing the reaction temperature significantly increased the amount of iron removed from ferritin by each chelator tested in a time-dependent manner. The asterix (*) denotes values that are significantly different (p < 0.05). 61  2.4.4: Combination of Macromolecular Chelators and Small Molecular Weight Chelators on Iron Removal  The influence of scaffold-mediated steric factors on Fe(III) removal from the clustered-metal core of ferritin was explored further through the use of the low-MW chelator deferiprone (DFP) as a Fe(III) ion shuttle (119-120). Results described in Figures 12 and 13 show that the high molecular weight chelator conjugates have limited accessibility to ferritin iron as unconjugated DFO gave higher iron extraction than polymer conjugates in all the cases. We anticipated that a combination of small molecular weight iron chelator with polymer conjugates may increase iron removal via a previously reported “shuttle” mechanism (119-120). We used a combination of deferiprone (L1) and HPG-DFO for this purpose. Iron removed by DFP may then be shuttled to either DFO or a HPG-DFO conjugate to achieve Fe(III) transfer to those larger chelators without requiring their direct interaction with ferritin. As shown in Figure 13, DFP alone is highly effective in extracting Fe(III) from ferritin.  Moreover, for DFO and each HPG-DFO conjugate tested, the co-presence of DFP significantly enhances the amount of iron removed from ferritin and shuttled into DFO or the polymeric chelator, providing strong supporting evidence that, at low kT, polymer-scaffold mediated steric effects moderate rates of Fe(III) uptake from ferritin by HPG-DFOs. One additional point of more modest importance is that observation that saturation of all DFO moieties in 500K-2 is not observed when Fe(III) is shuttled to the conjugate in a 2:1 NTA:Fe (dominant species predicted by Hyperquad software stoichiometry (Figure 10, Appendix B), but does occur when shuttled as part of the smaller complex formed with DFP (117). Thus, at room temperature, modest changes in the size of the transfer agent can impact Fe(III) loading into 500K-2, while 44K-4 and 500K-2 show similar loading rates at the more relevant thermal energy of the body.  62   Figure 13: Small MW chelator deferiprone (L1) enhances iron removal by larger MW chelators.  HPG-DFO and DFO (250 μM) were incubated with ferritin and 250 μM L1 at room temperature (22°C) for 1, 24 and 48 h in imidazole buffer at pH 7.4. L1 significantly increased the amount of iron extracted by each chelator in a time-dependent manner. Paired t-test showed a significant benefit for each chelator. The asterix (*) denotes values that are significantly different (p < 0.05). 63  2.4.5: Influence of Molecular Weight on Inhibition of Iron Mediated Oxidation of Hemoglobin DFO protects cells from iron overload by inhibiting Fe(III) mediated oxidation of proteins. To determine whether HPG-DFO conjugates could prevent iron mediated oxidation of hemoglobin in a manner comparable to DFO, the hemoglobin oxidation model was used (111-112). Figure 14-15 shows that the addition of 200 μM iron resulted in a rapid reduction in the percentage of oxyhemoglobin, indicating that Hb was oxidized. However, the addition of 200-500 μM of DFO was able to prevent this process from occurring. Similarly, HPG-DFO of various MWs and DFO density are shown to prevent hemoglobin oxidation. However, the ability of polymer conjugates to prevent iron mediated oxidation is dependent on the HPG MW. Similar to the ITC stoichiometry data, higher MWs of HPG are slower at reversing this process over the 5 minute period of data collection. This indicates that for a higher MW of polymer, iron binding may occur at a slower rate, depending on the density and location of conjugated DFO molecules.    64     Figure 14: HPG-DFO can prevent the Fe(III) mediated oxidation of hemoglobin.  Prevention of iron(III) mediated oxidation of hemoglobin by HPG-DFO. Hemglobin A produces a distinct spectrum that can be used to monitor changes in oxidation. This figure shows the UV–vis absorbance scans of hemoglobin after addition of different amounts of iron, DFO and HPG-DFO. Fe can cause oxidation of hemoglobin in a concentration dependent manner. DFO and HPG-DFO can prevent hemoglobin oxidation.  65    Figure 15: HPG-DFO prevent the Fe(III) mediated oxidation of hemoglobin.  UV–Vis absorbance scans of hemoglobin after the addition of Fe(III), DFO and the molar equivalent of HPG-DFO is shown. Like DFO, HPG-DFO of all molecular weights were able to prevent Fe(III) mediated oxidation of hemoglobin. Each sample, except for the HbA control, contains 200 µM Fe(III) and 1 mM of GSH.    66  2.5: Discussion Polymer conjugation to low MW iron chelators such as DFO can result in drastic improvement to pharmacokinetics and biodistribution without compromising their iron chelation efficiency. Several studies have demonstrated significant reduction in cellular and in vivo toxicity, as well as prolonged circulation half-life associated with polymer conjugation to DFO (70-71, 103). These results suggest the importance of further developing macromolecular iron chelators for the treatment of iron overload diseases.  In an effort to optimize and understand the iron binding properties of macromolecular iron chelators, we investigated the influence of polymer size on iron binding thermodynamics, efficiency in removing iron from ferritin and prevention of iron mediated oxidation of proteins using Hb as a model.   Our results indicate that HPG-DFO conjugates are high affinity iron(III) binding chelators with similar iron binding properties as that of unconjugated DFO. ITC binding thermodynamics data indicate that the binding of DFO to iron remains a favorable, enthalpy driven reaction after polymer conjugation. Since the enthalpy of bond formation is similar among all chelators, polymer conjugation of DFO leads to no enthalpic penalty (Figure 10 and Table 5). Not surprisingly, there appears to be a relationship between the binding stoichiometry (N), the polymer MW and the number of DFO molecules conjugated to the polymer.   While the high MW, deformability and globular shape of the HPG is anticipated to provide extended half-life for these conjugates in vivo, it may also influence the dynamics of the iron binding reaction. With increasing polymer size, there is increased availability of reactive groups available for conjugation to DFO. Therefore, during polymer conjugation, the number of possible arrangements of DFO on the polymer also increases with increasing MW. Presumably, the arrangement or distribution of DFO on the polymer surface or within the HPG structure, 67  determines the rate at which the iron becomes bound. Furthermore, the steric hindrance from the crowded dentritic structure may influence the approach of the Fe ions, resulting in the observed changes.  The data from the iron removal and inhibition of hemoglobin oxidation is also following a similar trend and supports this idea (Figure 14-15). In the case of iron removal, large molecular weight polymer conjugates needed more time to achieve saturation with iron (for example, Figure 12, data at 48 h in comparison to data at 1 h). High MW conjugates, as well as conjugates possessing a low degree of functionalization were not as effective in short term iron removal when compared to DFO or HPG-DFO conjugates with lower MW and higher degree of DFO functionalization/DFO density. These observations lend support to the idea that the amount of iron that is initially bound depends on the number and location of DFO molecules on the HPG, especially at higher MWs.  Despite the observed variation in the N values, this effect is not likely to influence the chelating ability of the polymer conjugates in vivo. This is clearly demonstrated when comparing HPG-DFO values to that of 40SDO2, the previously reported polymeric iron chelator. The value of N obtained for 40SDO2 was 0.63. However, this chelator has previously demonstrated a superior ability to remove iron in vivo when compared to unconjugated DFO (71). This is due to the long circulating ability of 40SDO2 and the high unsaturated iron binding capacity, which ensured that patients had chelator coverage for prolonged time periods. A similar observation holds true for HPG-DFO t1/2 in vivo (105). In contrast to high MW chelators, DFO rapidly disappears from the circulation (t1/2 of ~20 min), leaving patients unprotected outside of subcutaneous administration of the drug (10, 12, 15-17).  68  The inherent toxicity and reactivity of iron is precluded by its storage in the form of a ferrihydrite nanoparticle core inside of ferritin, the major iron storage protein in humans (6, 104). Ferritin reduces iron toxicity that can result from free radicals by storing iron in a redox inactive/“harmless” form and providing an easily mobilizable iron reserve that can be utilised for metabolic processes when needed. We investigated the ability of chelators to remove iron from ferritin over a 48 h period. Polymeric chelators were effective at removing iron from ferritin but at a slower rate than DFO; with larger polymers (500 kDa) removing less iron over time than smaller polymer-DFO conjugates (25kDa and 44kDa).  Ferritin is made of 24 subunits of H and L types. H and L subunits of ferritin co-assemble to form a shell-like structure surrounding a cavity which is approximately 8 nm in diameter. Fe(II) cations enter ferritin through its eight hydrophilic three-fold channels that are 4 Angstrom wide.  Fe(II) is oxidized rapidly upon entry at conserved di-iron centres and subsequently forms the ferrihydrite mineral core (6, 104). Thus the size of the chelator influences access to the iron mineral core of ferritin.   This interpretation was supported by the addition of deferiprone (L1), a smaller MW bidentate chelator which can freely enter ferritin pores (118). It has been demonstrated that combining a small MW chelator with a larger one may enhance iron chelation both in vitro and in vivo (119-121). We found that L1 significantly increased the amount of iron extracted by each chelator at all concentrations and time points investigated. In particular, for the highest MW conjugate (500kDa), the presence of L1 significantly enhanced both the rate as well as the amount of iron removed (~4-fold). In a similar study conducted with hydroxypyridinone chelators, Brady et al found that the size of chelators was a major factor which determined the effectiveness of iron 69  removal from ferritin (122). This is most likely due to restriction of entry through the ferritin pores, resulting in slower access to the ferritin iron nanoparticle core.  There are several mechanisms by which chelators may access iron from ferritin. For example, iron may passively dissolve, which can cause it to seep out into the surrounding solution on occasion (122). This would allow it to become complexed with available chelator. Chelation of metal irons near the protein surface may also account for some of the iron that is removed. Small bidentate iron(III) chelate ligands such as L1, catechols and hydroxamates are not restricted from entry into ferritin and therefore possess the ability to directly remove iron from the ferritin inorganic core. These small MW chelators can then diffuse out of the protein shell with iron(III) bound to it. In some cases, it is assumed that Fe equilibrates between sites on the core and protein shell that are accessible to the chelator (122). Thus, the size of the chelator as well as the activity occurring on the outer protein surface may influence the process of iron extraction from within the ferritin centre. The bulk of the polymeric component of the chelator may also increase the steric hindrance, leading to a reduction in the number of chelate molecules that can bind the core surfaces at the same time. As a result, it is not surprising that larger molecules may take longer to penetrate the protein shell or are excluded entirely. The protein shell must be flexible enough to permit the passage of chelators and their iron complexes to move in and out of the shell. Increasing the temperature to 37⁰C also increased the amount of iron removed from ferritin for all chelators tested in a time-dependent manner. Interestingly, the rate of iron removal from ferritin by higher MW chelators (500kDa) approached that of the 44 kDa chelator with longer incubation time at 37⁰C. This observation has important implications. First, the increased efficacy at 37⁰C temperature implies that chelators are more efficient iron scavengers under physiological conditions and that the effect of polymer size observed in vitro may be deleted in vivo. Additionally, 70  given that these HPG-DFO conjugates have demonstrated significantly increased circulation time compared to DFO, these findings indicate that these high molecular weight chelators will result in more efficient iron removal than DFO at equal concentrations, or when used in combination with low MW chelators such as L1 (105).  2.6: Conclusions  Toxicity and poor pharmacokinetics are major challenges associated with treating transfusion dependent patients with DFO.  We have previously demonstrated that polymeric iron chelators can be generated by the covalent attachment of DFO onto HPG, a biocompatible polymer. In this study, we investigated the iron binding thermodynamics and chelating abilities of HPG-based iron chelating conjugates. ITC studies show that polymeric chelators are efficient at binding iron and that the favourability of the reaction remains unchanged after polymer conjugation. Conjugates with relatively smaller sizes showed more DFO-like properties, likely due to a reduction in polymer-induced steric crowding in solutions, while high MW and low DFO density were associated with greater deviations and slower removal of iron from ferritin at 22⁰C. However, at physiological temperatures the iron chelated by 500kDa conjugates approaches that of the 50kDa, demonstrating that effect of MW is negligible at physiological temperature. The polymer properties and DFO density are also influencing the efficacy of conjugates in preventing the iron mediated oxidation of hemoglobin. Previous in vivo studies of iron removal demonstrated efficacy of HPG-DFO conjugates. Results from this study allow us to further optimize iron chelating properties of the conjugates to enhance iron excretion in vivo (105).  71  Chapter 3:  Biocompatibility of Novel Macromomolecular Iron Chelators 3.2: Background  Many clinically approved drugs have MWs of approximately 500 g/mol and are generally considered to be of the low MW class (125-126). Such drugs may exhibit a short plasma circulation half-life and as a result, are removed from the body rapidly after administration. Furthermore, low MW drugs may move more freely into healthy cells resulting in an equal distribution and drug accumulation in healthy and un-healthy cells and tissues. Consequently, smaller amounts of active drug may reach the target site and severe side effects may develop with prolonged use at high drug doses. This is the major challenge associated with treating iron overloaded, transfusion-dependent patients with DFO. DFO is too large and hydrophilic to be orally absorbed and must be subcutaneously administered. However, DFO has a very short circulation half-life of approximately 20 minutes in man and is rapidly filtered and degraded by enzymes when free in the circulation. As a result, DFO must be either intravenously or subcutaneously infused for 8-12 h daily to promote adequate iron excretion (10, 12, 15-17).  Polymers have been frequently used to improve the therapeutic efficiency, drug-delivery methods, modes of drug action, pharmacokinetic and pharmacodynamics properties of drugs (68-75, 86-93, 126). Polymer conjugation and encapsulation of chelators in liposomes are two approaches that have been used to optimize the properties of DFO. Polymer conjugation has been reported as a successful method owing to the ability of polymers to significantly influence the biophysical properties of DFO. Moreover, these polymeric chelating nanomedicines have highlighted the importance of polymers in advancing the treatment options for those suffering from transfusion associated iron overload (68-71). 72  In Chapter 2, we showed that engineering DFO into a high MW chelator through conjugation to HPG resulted in the formation HPG-DFO conjugates with high iron binding affinity. Several conjugates with different MW and degree of functionalization with DFO were developed and characterized. In this chapter, the blood and biocompatibility of HPG-DFO were investigated. We hypothesized that HPG-DFO are more biocompatible than DFO and that the HPG component does not enhance but attenuates DFO toxicity.  Since DFO is administered primarily by subcutaneous or intravenous infusion, we investigated the blood compatibility of HPG-DFO using the human blood components as our model. Coagulation assays including thromboelastography (TEG), activated partial thromboplastin time (aPTT) and prothrombin time (PT) were used to determine whether there were any deviations in coagulation when the polymer is conjugated to DFO. The influence of HPG-DFO on platelet and complement activation, red cell aggregation, and hemolysis were also studied. Cellular toxicity and cellular uptake of the high MW chelators were also investigated. The results from these biocompatibility studies contribute to our understanding of the biocompatibility of these novel high MW iron chelators and are an important first step in determining the suitability of these chelators for further testing in more advanced biological models.  3.1: Overview  The interactions that occur between biomaterials and biological components under physiological conditions must be investigated when developing novel nanomedicines. Chapter 2 of this thesis reports the development and characteristics of a novel, high MW, polymer based iron chelator, HPG-DFO. HPG-DFO was developed by conjugating DFO, a low MW chelator to hyperbranched polyglycerol. HPG-DFO has demonstrated high iron binding affinity and our studies have shown that polymer conjugation does not drastically alter the thermodynamics of 73  DFO. Herein, our goal was to determine whether conjugation of HPG to DFO resulted in any alterations in DFO biocompatibility. This was achieved using in vitro and in vivo models of blood compatibility and cytotoxicity. The influence of polymer conjugation on blood clot formation via the intrinsic and extrinsic pathways of coagulation; red blood cell aggregation and hemolysis; complement and platelet activation were investigated. The effect of conjugation on the cellular toxicity as well as the underlying mechanisms, were also investigated using human umbilical vein endothelial cells (HUVECs) and Chinese Hamster Ovary (CHO) cultures.  HPG-DFO did not cause any deviation in blood coagulation, complement activation nor platelet activation. Additionally, compared to low MW DFO, HPG-DFO was less toxic than DFO in endothelial cells and the cellular uptake of HPG-DFO conjugates appeared to be dependent on molecular weight. Taken together, the data obtained from this chapter, as well as previous and subsequent studies of HPG-DFO toxicity, iron binding efficiency, and tolerance studies conducted in mice, demonstrate that HPG-DFOs are highly efficient, biocompatible chelators with potential clinical utility.  3.3: Materials and Methods  Synthesis of HPG-DFO: HPG was conjugated to DFO using the protocol reported in Chapter 2. Many conjugates that vary in MW and DFO density were created initially. These conjugates were screened in a variety of tests, some of which are reported in this chapter. The properties of HPG-DFOs synthesized are given in Appendix A.   Blood Collection: Blood was drawn from healthy un-medicated consenting donors at the Centre for Blood Research at UBC (UBC Ethics approval no: H07-02198). Blood was collected in a 3.8% sodium citrated tube with a blood/anticoagulant ratio of 9:1 (BD Vacutainer Buffered Citrate Sodium, 0.105 M; 9:1) EDTA (Fisher Scientific, New Jersey), or in serum tubes. Platelet-74  rich plasma (PRP) was prepared by gently centrifuging citrated whole blood samples at 150 X g for 10 min in an Allegra X-22R centrifuge (Beckman Coulter, Canada). Platelet-poor plasma (PPP) was prepared by centrifuging citrated whole blood samples at 1200 X g for 20 min. Serum was prepared by centrifuging whole blood collected in a serum tube at 1200 X g for 30 min. Red blood cell (RBC) suspensions were prepared by washing packed red blood cells with phosphate buffered saline four times.  Blood compatibility was investigated using thromboelastography (TEG), activated partial thromboplastin time (aPTT), prothrombin time (PT), platelet activation, complement activation, and red blood cell aggregation and hemolysis. HEPES buffer and saline of pH 7.4 were used as controls for in vitro compatibility studies. All chelator solutions (DFO and HPG-DFO) were prepared in HEPES buffer, and the pH was monitored after the polymer dissolution to ensure pH stability. Thromboelastography (TEG) Analysis: The effect of different HPG-DFO conjugates on blood clotting was examined using the TEG technique (128-129). TEG measures the physical properties of the fibrin clot formed and produces values for four main parameters that relate to the kinetics and strength of clot formation: R, the time from the start of a run until the first signs of detectable clot formation; K, the time taken from the start of the run until substantial clot formation occurs; alpha angle, a function representing the kinetics of fibrin polymerization and cross-linking; and maximum amplitude (MA), a measurement of the maximum strength or stiffness of the formed clot. The effect of different HPG-DFO conjugates on blood clotting was examined by mixing citrate anticoagulated whole blood with the conjugate solution (9:1 ratio) for a final concentration of 1.0 mg/mL of HPG-DFO at 37 ⁰C. Stock solutions of HPG-DFO conjugates were prepared in HEPES buffer. In a typical experiment, 40 μL of chelator stock solution was mixed with 360 μL 75  of citrate anticoagulated whole blood. After thorough mixing of HPG-DFO with blood, 340 μL of the mixture was added to the TEG cups in the analyzer. Parameters and hemostasis profile were measured upon the addition of 20 μL of calcium chloride solution (0.2 M) to each of the HPG-DFO/blood mixtures in the TEG analyzer. The measurements of all parameters were made on freshly drawn whole blood (experiments began within 5-10 min of venipuncture). Each experiment was repeated at least three times with three different donors and the average with standard deviation of TEG parameters from different donors was reported. Prothrombin Time (PT): The effect of HPG-DFO on the extrinsic pathway of coagulation was determined by measuring the PT in sodium citrate anticoagulated PPP at 37 ⁰C. The detailed experimental procedure was published previously (130-131). The final concentration of conjugates in plasma was 1 and 5 mg/mL (9:1 v/v, PPP: conjugate). Stock solution of the conjugates was mixed with PPP and the coagulation cascade was initiated when recombinant human tissue factor with synthetic thromboplastin was added to the mixture. The experiment was repeated in triplicate on the STart4 coagulometer (Diagnostica Stago) using plasma from at least three different donors. HEPES buffer solution, saline and DFO were used as normal controls and values reported are mean from three measurements with standard deviation.  Activated Partial Thromboplastin Time (aPTT): The effect of HPG-DFO on the intrinsic pathway of coagulation was determined by measuring the aPTT. HPG-DFO conjugates dissolved in HEPES buffer were mixed with platelet poor plasma (PPP) (9:1 v/v PPP: conjugate) to obtain a final concentration of 1.0 and 5.0 mg/mL. Coagulation time was determined by the addition of partial thromboplastin reagent and calcium chloride (CaCl2) to sodium citrated anticoagulated PPP. The conjugate/ PPP mixture was warmed in cuvette-strips at 37 ⁰C for 3 min before addition of coagulation reagents. HEPES buffer solution, saline and DFO were used as 76  normal controls. Each experiment was repeated in triplicate. The mean values of three replicates with standard deviation from three different donors were reported.  Red Blood Cell Aggregation and Hemolysis: Red blood cell (RBC) aggregation and morphology were measured in EDTA anticoagulated whole blood. Whole blood (90 μL) was incubated at 37 ⁰C with HPG-DFO conjugates in HEPES buffer (10 μL) to obtain a final concentration of 0.5, 5.0, and 10 mg/mL of HPG-DFO in the blood (1:9 v/v HPG-DFO: blood). HEPES buffer incubated whole blood was used as a normal control. Polyethyleneimine (PEI) was used as a positive control. Following incubation, the suspension was gently centrifuged and resuspended in the supernatant plasma on microscopic slides before examination by light microscopy. Samples were examined and images were captured using a digital microscope camera.   Platelet Activation Analysis: The level of platelet activation in PRP upon incubation with HPG-DFO conjugates was measured by flow cytometry. Briefly, 90 μL of PRP was mixed with 10 μL of HPG-DFO solution (final concentration of the polymer was 1.0 mg/mL) and incubated at 37⁰C. After 1 h incubation, 5 μL of PRP-HPG-DFO mixture was incubated with 5 μL of anti-CD62P-PE (Immunotech) and 45 μL of HEPES buffer solution for 15 min in the dark. Finally the mixture was diluted with 300 μL of phosphate-buffered saline and the platelet activation level was analyzed in a BD FACS Canto II flow cytometer (Becton Dickinson) by gating platelet specific events based on their CD62P-PE fluorescence and side scattering profile. Activation of platelets was expressed as the percentage of the platelet activation marker CD62P detected in 10 000 total events counted. Bovine thrombin (1 U/mL, Sigma) was used as a positive control, and PE-conjugated goat anti-mouse IgG polyclonal antibody (Immunotech) was used as the nonspecific binding control. PRP incubated with HEPES buffer was used as normal control. Triplicate 77  measurements were performed for each donor, and three different donors were used. The mean values with standard deviation are reported. Complement Activation Analysis: Complement activation by the HPG-DFO conjugates was measured by antibody sensitized sheep erythrocytes (CH50 analysis, CompTech, Tyler, TX). Stock solutions of HPG-DFO were prepared in HEPES buffer (10 mg/mL and 50 mg/mL). A 10 mL portion of conjugate stock solution was mixed with 90 μL of GVB2þ-diluted fresh serum and incubated for 1 h at 37 ⁰C. The final concentration of HPG-DFO in diluted serum was 1 and 5 mg/mL. The serum incubated with heat aggregated human IgG (1 mg/mL) and 5 mM EDTA solution in saline was used as positive and negative controls, respectively. Following 1 h of incubation, 60 μL of this mixture was diluted with 120 μL of GVB2þ, and 75 μL of the diluted sample mixtures was then incubated with 75 μL of antibody-sensitized sheep erythrocytes for 1 h at 37 ⁰C. The incubation was stopped by the addition of 300 μL of cold GVB-EDTA to each sample. The samples were centrifuged, and the optical density of supernatants was measured in a spectrophotometer at 540 nm. Antibody-sensitized sheep erythrocytes incubated with distilled H2O were used as 100% lysis control for the calculation. Triplicate measurements were performed using serum and the mean plus standard deviation is reported.  In Vitro Cytotoxicity: DFO is known to be cytotoxic (132). Therefore, we explored whether HPG-conjugation enhances or attenuates the toxicity of DFO in cell culture. This was achieved by measuring the cell viability of human umbilical vein endothelial cells (HUVECs) following incubation with HPG-DFO at concentrations up to 600 µM DFO equivalent for 48 h and examining cell viability by MTS assay.  Fluorescent Labeling of Iron Chelators: To determine whether the reduction in toxicity is due to reduced cellular uptake, studies investigating cellular trafficking in HUVECs and Chinese 78  Hamster Ovary cells (CHO) were conducted. 5-(Aminoacetamido) Fluorescein (Fluoresceinyl Glycine Amide) (FGA) and Alexa 488-NHS ester were used to label HPG-DFO and DFO, respectively. In a typical reaction, 100-200 mg of HPG (25, 44, 500 kDa) was dissolved into in MilliQ water (2-4 mL). To oxidize the OH groups on the HPG, 84 µl of a 5M solution of NaIO4 was slowly added to the solution over a period of 1-3 minutes and the solution was left to stir for 24 h. The solution was then dialysed against water to remove the unreacted NaIO4. Dialysis was carried out using a Spectra/Por dialysis membrane (MWCO 1000). Approximately 0.1 mole percent of FGA were reacted with CHO groups. This was allowed to stir for 1 hr. DFO was then added in 1.2 molar excess of the number of aldehydes generated. After 8 h NaCNBH3 was added at 1.2 molar excess of the number of aldehydes generated and this solution was left to stir for 24 h. The remaining product was put to dialyze against water for 3 days with frequent changes of water. Excess, unreacted aldehyde groups were quenched using glycine. Using similar protocols, HPG-DFO conjugates that vary in MW and DFO density were prepared.   DFO was tagged with Alexa 488-NHS ester in methanol in 1:10 ratio w/w. All samples were dialysed for 2 days with frequent changes of water after conjugation. Fluorescent labeling was confirmed using UV-vis analysis. The presence of fluorescein and Alexa-488 are indicated by a maximum absorbance at 494 nm and the actual mass per molecule was determined using the extinction coefficient of 80 000 cm-1 M-1. Samples were acetone precipitated prior to taking absorbance readings to ensure that conjugation was successful.  Cellular Uptake Studies: CHO and HUVECs (ATCC) containing 10% FBS (v/v %) were seeded on 8 chamber polystyrene tissue culture treated slides (BD Falcon) 24 h prior to treatment. The media was removed prior to the addition of the treatment solution; which consisted of polymer labeled with FGA and alexa fluor 488-NHS-labelled DFO (Invitogen Canada Inc.) dissolved in 79  complete media to a final concentration of 10 mg/ml and filtered with 0.22 μM purogenic filters. Cells were incubated with fluorescently labeled polymers for 1 h, 24 h and 48 h. After incubation, cells were washed two times with PBS buffer (Invitrogen Canada Inc.), pH 7.4, to remove excess polymers at 37 °C. Cells were fixed using 4% paraformaldehyde, washed with PBS 3 times, and stained with wheat germ agglutinin, Alexa Fluor 633 conjugate membrane dye (Invitrogen) for 15 minutes at 37° C. Prolong gold anti-fade reagent containing DAPI was then added and samples were left to dry for 3 minutes prior to adding cover slips. Slides were analyzed using a confocal microscope (Olympus Fluoview FV1000, Japan). Slides of untreated cells were used as a negative control (no polymer). Images were taken from randomly selected areas on separate chambers of culture slides.  3.4: Results 3.4.1: The Effect of Polymer Conjugation on the Blood Compatibility of DFO  To determine whether high MW polymer conjugates of DFO (HPG-DFO) are compatible with the human blood components, studies of clot formation via the extrinsic and intrinsic pathways of coagulation were conducted in human plasma. Results demonstrate that HPG-DFO do not change the clot forming properties of human blood in vitro; there was no difference in the time taken for clots to form when PPP was incubated with HPG-DFO conjugates. The PT and aPTT values in human plasma at 1.0 and 5.0 mg/mL of HPG-DFO are shown in Figure 16. Values reported are the average of three different donors and triplicate measurements per donor. There was no statistically significant difference between the values obtained for PT and aPTT for HPG-DFO compared to buffer, saline or DFO only controls (p=ns for all, except aPTT for the 25K-65 and 44K-4 conjugate).  80                         Figure 16: Conjugation of DFO to HPG did not affect the intrinsic or extrinsic pathways of coagulation.  HPG-DFO conjugates were incubated with platelet poor plasma and the time taken for clots to form was recorded on a coagulation analyzer. Conjugation of DFO to HPG did not affect the coagulation cascade in vitro; polymeric chelators did not activate the intrinsic or extrinsic pathways of coagulation. Moreover, increasing the concentration to 5mg/mL did not significantly reduce biocompatibility of HPG-DFO. At 5 mg/mL all polymer conjugates of varied molecular weights had coagulation times that were comparable to the buffer, saline and DFO only controls, except the 25K-65 and 44K-4 conjugated which had significantly increased clotting times at higher concentration. * * 81  The TEG assay is the most comprehensive in vitro test of blood coagulation and allows the observation of coagulation in fresh human whole blood (128-129).  As demonstrated by the TEG trace in Figure 17 and Table 6, conjugation of DFO to HPG did not affect the rate or strength of clot formation as analyzed by the TEG assay. A representative TEG trace as well as the values obtained from TEG analysis in the presence of HPG-DFO is shown in Figure 17 and Table 6. It is shown that the time taken from the start of the run until substantial clot formation occurs, alpha angle (R), and the maximum amplitude (MA) or stiffness of the formed clot remained within the normal range after polymer conjugation to DFO. Moreover, neither the size of the polymer nor the number of DFO attached to it appeared to influence coagulation properties of whole blood.  Figure 17: Influence of HPG-DFO on blood coagulation as analyzed by thromboelastograpy (TEG). Shown are TEG traces of HPG-DFO incubated with human whole blood.     TEG traces for saline control, DFO and HPG-DFO did not differ. The time taken for clots to form and clot strength did not vary. TEG analysis shows that the HPG-DFO conjugates did not affect initial coagulation time (R-time), formation of clot or blood clot structure (alpha, K-value and MA value). Clot strength is indicated by MA. The values for R-time, alpha, K-value and MA value are 82  calculated from the TEG trace and standard deviations (n=4). The similarity of control (saline) and HPG-DFO profiles indicates that there was no effect on blood coagulation.   Table 6: TEG values for HPG-DFO conjugates    ¥ Scarpelini, S.G. et al Normal range values for thromboelastography  in healthy adult volunteers. Brazilian Journal of Medical and Biological Research (2009) 42: 1210-1217.   We next investigated complement activation in human serum in the presence of HPG-DFO using antibody sensitized sheep erythrocytes. Total complement consumption was measured in this assay and compared with serum incubated with buffer at a concentration up to 5 mg/mL.  HPG-DFO R(min) Alpha(Ѳ) K(min) MA (mm) Normal values ¥ 9-27 48-78 2-9 49-72 Buffer 13.4 52.85 2.8 57.75 25K-65 15.3 40.9 4.7 54.3 44K-1 12.3 31.2 3.2 58.2 44K-4 14.6 42.2 3.8 57.8 500K-7 9.6 53.1 2.8 56.7 500K-2 13.3 38.65 3.2 57.7 500K-500 13.4 55.05 2.65 59.2 500K-900 12.95 54.9 2.65 59.4 83                              Figure 18: Polymer Conjugation did not affect the activation of complement (A) or platelets (B).  The total consumption of complement proteins was measured at different conjugate concentrations in serum at 37⁰C at polymer to serum concentration (1:9 v/v). IgG was used as a positive control.  HPG-DFO conjugates do not activate the complement cascade.    A B 84  Data shown in Figure 18 demonstrates that none of the HPG-DFO conjugates activated the complement system compared to the positive control (IgG); the level of complement activation between negative control and polymer conjugates were comparable;  n=4 and p=ns (p > 0.05) for all.  Platelet activation was investigated in platelet-rich plasma (PRP) in the presence of HPG-DFO by measuring the expression of the activation marker CD62P using fluorescently labeled anti-CD62P antibody. When compared to the positive control of bovine thrombin, polymer conjugation to DFO did not enhance platelet activation and the activation level of platelets in the presence of HPG-DFO was similar to that of control platelets incubated with buffer solution, see Figure 18, p = ns for all. RBC aggregation was measured by incubating HPG-DFO in whole blood at 37 ⁰C. Under normal conditions, RBC form reversible aggregates called roleaux. However, irreversible RBC aggregation can damage RBCs and could potentially obstruct capillaries. Irreversible aggregates may be induced by the positive control polyethyleneimine (PEI). Photomicrographs of RBCs incubated with HPG-DFO at 0.5, 5.0 and 10 mg/mL are shown in Figure 19 and indicate that the incubation of HPG-DFO with human blood did not result in the formation of aggregates. The concentration-dependent, visible association of red cells in Figure 19 are indicative of roleaux formation, which is reversible. Neither MW nor DFO density appeared to influence aggregate formation, and the shape of the RBCs appeared normal. However, incubation of blood with PEI resulted in the formation of irreversible aggregates.    85        Figure 19: Influence of HPG-DFO on Red Blood Cell Aggregation and Hemolysis  HPG-DFO conjugates did not cause any unwanted RBC aggregation or lysis when incubated with RBC suspension at 1:9 v/v at 37 º C for 1 h.  HPG-DFO of all MWs showed similar levels of hemolysis to that of the buffer control.    86  3.4.2: Cytotoxicity of DFO and HPG-DFO To determine whether conjugation to HPG influenced DFO toxicity, HUVECs were exposed to DFO and HPG-DFO for 48 h. Cellular metabolic activity was measured using the MTS assay. At all concentrations tested, HPG-DFO and unconjugated HPG were considerably less toxic than DFO (Figure 20).  Moreover, cells exposed to HPG regardless of MW, were viable up to very high concentrations, indicating that HPG does not increase DFO toxicity in HUVECs after 48 h incubation. Other studies report similar findings (70, 105).  Figure 20: Cytotoxicity of HPG-DFO conjugates againist HUVECs  The toxicity profiles of DFO and HPG-DFO conjugate. Cell culture media were used as normal control. HUVECs were treated with up to 0.5 mM of HPG-DFO and DFO for 48h. Results show that the conjugation of DFO to high MW HPGs does not enhance but attenuates the toxicity of DFO; HUVECs treated with HPG-DFO remain viable at higher concentrations while DFO treated cells are showing lower viability at all concentrations.   87  3.4.3: Cellular Uptake of DFO Differs with Conjugation to HPG  To determine whether reduced toxicity of DFO upon polymer conjugation was due to reduced cellular uptake, HPG-DFO of various MWs and DFO were fluorescently labeled and their uptake into HUVEC and CHO cells was investigated for 48 h. Care was taken to ensure that the number of fluorescent moleculues present per chelator molecule was similar (Figure 21). Fluorescently labeled HPG-DFO and DFO accumulated within HUVECs and CHO cells with increasing incubation times (Figures 22 and 23).  Cellular accumulation increased with longer incubation times for all chelators tested with 25K-65 and 44K-85 showing the greatest accumulation. Accumulation in HUVECs was greater with longer incubation time for DFO and the 25K conjugate however; 44K-85 did not show any cellular accumulation at any time point in HUVECs. Surprisingly, there is a greater accumulation of larger MW chelators after 48 h compared to smaller MWs in CHO cells. Table 7: Characteristics of HPG-DFO used in cellular uptake study   $HPG of various MWs were conjugated to Fluorescein (Flu) and DFO. DFO was conjugated to Alexa-488-NHS ester.   Nomenclature Average MW of the chelator (kDa) DFO density/Polymer molecule Mean diameter (nm) Fluorophore DFO 0.560 -- -- Alexa-488 25K-65 65 65 2.7 Fluorescein 44K-85 77 85 5.5 Fluorescein 88              Figure 21: Fluroscence profile of  fluorescein conjugated DFO and different HPG-DFOs. HPG of various molecular weights were labelled with FGA and DFO was labelled with Alexa-488-NHS according to the scheme above (A). After conjugation, HPG-DFO samples were acetone precipitated and the specta were read to confirm fluorophore conjugation (B).    B A 89                                      Figure 22: Cellular uptake of iron chelators in HUVECs.  Confocal images of HUVECs incubated with 10 mg/mL of HPG-DFO for 1 h, 24 h and 48 h at 37°C. The DFO and HPG-DFO were stained with alexa-488-NHS and FGA respectively, and the nucleus was stained with DAPI. The green colour indicates cellular uptake of chelators. Cellular uptake was greatest for the 25K conjugate at all time-points while DFO and the 44K-4 conjugate did not accumulate inside the cell to the same degree as the 25K conjugate after 48 h.   10µM 90   Figure 23: Cellular uptake of iron chelators in CHO cells.  Confocal images of CHO cells incubated with 10 mg/mL of HPG-DFO for 1 h, 24 h and 48 h at 37°C. The DFO and HPG-DFO were stained with alexa-488-NHS, FGA and the nucleus was tained with DAPI. Cellular uptake increased with incubation time, with high MW chelator conjugates accumulating inside the cell more than DFO.  10 µM 91   3.5: Discussion  The biocompatibility of HPG was investigated in order to further define the properties of HPG-DFO. We have previously demonstrated that the conjugation of DFO to HPG polymers yields a library of effective high MW iron chelators with high iron binding affinity and similar thermodynamic properties as unconjugated DFO (105). Herein, we compare the biocompatibility and in vitro toxicity of unconjugated DFO to that of HPG-DFO. We also probed mechanisms underlying the reduced toxicity of DFO in cells upon polymer conjugation. Since the toxicity of DFO and small MW chelators have been a significant challenge to patient compliance, any prospective chelators with clinical utility must demonstrate suitable biocompatibility and toxicity profiles in order to positively impact patient treatment options.  In addition, because polymers are known to be agonists of complement activation and thrombosis, failure to design biocompatible materials may result in unwanted side effects. Thus, this study assessed blood compatibility of HPG-DFO using the human blood components. In vitro blood compatibility analyses of the intrinsic and extrinsic pathways of coagulation, whole blood coagulation, red cell aggregation, complement, platelet activation and cell toxicity were used to assay biocompatibility. In general, there were no deviations from normal values of blood coagulation, platelet activation or complement activation after conjugating HPG to DFO.  Conjugation of DFO to HPG reduced toxicity of DFO in HUVECs indicating that the HPG component is not likely to worsen DFO toxicity. This has been demonstrated by several studies previously (70, 105). Fluorescence studies confirmed that the uptake of DFO and HPG in HUVECs and CHO differs, although the trend is inconclusive. Previous studies reported similar findings which suggest that the uptake of HPG into CHO cells may vary depending on charge and HPG 92  MW (133). Reichert et al showed that the higher molecular weight HPGs which ranged from 40-870 kDa predominantly accumulated in the cytosol while their low-molecular-weight counterparts (2-20 kDa) accumulated to a lesser extent (133). The mechanism or reason for this trend is yet to be elucidated and will require further investigation. Further studies on the toxicity and efficacy or HPG-DFO were conducted in mice in addition to the studies mentioned in this chapter (105). Tolerance studies conducted in mice have also demonstrated that conjugating DFO to HPG does not cause toxicity at doses of more than twice the LD50 of DFO (1000 mg/Kg) HPG-DFO (105). Detailed biocompatibility testing of these polymers conducted in vitro and in vivo has demonstrated the unique advantages that HPG may have (105, 131). 3.6: Conclusions  The biocompatibility of polymer conjugates may be influenced by the MW of the polymer component. In addition, the large number of functional hydroxyl end groups that are present on the periphery of the HPG could potentially affect interactions with biological macromolecules or cells. Thus, we selected high MW HPGs with of various sizes and with various degrees of modification and studied their biocompatibility in detail.   These in vitro biocompatibility studies contribute to our understanding of HPG-DFO compatibility and toxicity. Furthermore, these studies have enabled the suitable development of subsequent in vivo experiments of tolerance, pharmacokinetics and efficacy studies in mice, which showed HPG-DFO to be superior to DFO in all areas tested. The efficient iron chelation properties and highly biocompatible nature of HPG-DFO are expected to reduce the iron mediated toxicity associated with repeated blood transfusions, and are thus promising candidates for further development. 93  Chapter 4: The Effect of Clinically Approved and Novel High Molecular Weight Iron Chelators on Zebrafish Embryo Mortality, Hatching and Morphology  4.1: Overview  Iron chelation therapy using iron (III) specific chelators such as desferrioxamine (DFO), deferiprone (L1) and deferasirox (ICL-670) is the current standard of care for the treatment of iron overload. Although each chelator is capable of promoting some degree of iron excretion, these chelators are also associated with a wide range of well documented toxicities with prolonged use and at high doses.  However, there is less information available on their effects in developing embryos. In this study, we took advantage of the rapid development and transparency of the zebrafish embryo, Danio rerio to assess and compare the toxicity of iron chelators that vary in molecular weight and chemical properties. Chelators were delivered to zebrafish embryos by direct soaking and their effects on mortality, hatching and developmental morphology were monitored for 96 hours post fertilization (hpf). To determine whether toxicity was specific to embryos, we examined the effects of chelator exposure via intra peritoneal injection on the cardiac function and gene expression in adult zebrafish. In addition to defining the range of toxicity of DFO, L1 and ICL-670 in embryos, this study sought to further define the properties of HPG-DFO, the novel class of polymeric chelators described in Chapters 2 and 3. The previously reported starch-DFO conjugate (40SDO2) was also tested for comparison. The effect of HPG-DFO and 40SDO2 on the survival, hatching success and morphology of zebrafish embryos was investigated. Chelators varied significantly in their effects on embryo mortality, hatching and morphology. While none of the embryos or adults exposed to DFO, HPG-DFO or 40SDO2 were negatively affected, ICL-treated embryos and adults differed significantly from controls, and L1 exerted toxic effects in 94  embryos alone. ICL-670 significantly increased the mortality of embryos treated with doses of 0.25 mM or higher and also affected embryo morphology, causing curvature of larvae treated with concentrations above 0.5 mM. ICL-670 exposure (10 μL of 0.1 mM i.p. injection) also significantly increased the heart rate and cardiac output of adult zebrafish. While L1 exposure did not cause toxicity in adults, it did cause morphological defects in embryos at 0.5 mM. Neither DFO nor higher molecular weight HPG-DFO and 40SDO2 were not associated with any toxic effects up to 1 mM. This study provides the first toxicological evidence of FDA-approved and high molecular weight, polymeric iron chelators in early development and will help to guide our approach on better understanding the mechanism of iron chelator toxicity in embryos.  4.2: Background  Iron chelation therapy is used to treat patients with transfusion associated iron overload (9-10, 14-17, 20, 26-34). Desferrioxamine (DFO) is the oldest form of therapy and has been used since the 1960s. Deferiprone (L1), the second iron chelator to be licensed and is recommended for use when chelation with DFO alone does not work well enough. Desferasirox (ICL-670) is the most recent iron chelator to become available for use (9-10, 15-17, 20, 26-34, 35-41).  Iron chelators can reduce complications such as cardiomyopathy, the major cause of death from iron overload. Further, iron chelation therapy can slow the progression of liver fibrosis and reduce glucose intolerance in transfusion dependent patients (10, 12, 14). However, a wide range of chelator-induced toxicities have also been reported. The use of DFO at high doses may cause neurological disturbances, growth retardation, peripheral neuropathies, vision changes, endocrine dysfunction and bone deformities (23-25). Severe toxicities associated with L1 include agranulocytosis and neutropenia (29-32). Gastrointestinal disturbances, arthropathy, increased 95  liver-enzyme levels and severe kidney failure, increased serum creatinine as well as gastrointestinal hemorrhage in some patients were also reported in ICL-670 treated (39-43).   While toxicities of iron chelators are well documented in adult patients, there is currently very limited data regarding the toxicity of DFO, L1 and ICL-670 during the earlier stages of development. A few studies have been conducted in mice which show that DFO caused developmental toxicity at high doses and only in the presence of maternal toxicity. L1 was shown to cause toxicity in rats, monkeys and rabbit embryos (76-77, 80-82). ICL-670 is reported to cross the placental barrier but no specific toxicity data is available regarding toxicity or teratogenicity in mammals (36).  Due to the challenges associated with differentiating maternal and embryo toxicity in mouse studies of DFO, the rapid development, optical transparency and ex-utero development of zebrafish embryos was ideal and allowed the direct assessment of the functional, morphological and behavioral effects of clinically approved iron chelators on embryos in the absence of interference from maternal factors.   Figure 24: Developmental changes in zebrafish embryos with time after fertilization.    96  Zebrafish (Danio rerio) are increasingly used as a cost-effective in vivo model and have been shown to be useful in evaluating chemical toxicity. Seventy per cent of the human genome is homologous with the zebrafish genome and 86 percent of disease causing genes in humans have homologs in zebrafish. In addition, the toxicity data obtained in zebrafish correlates well with developmental toxicity data from rat in vivo studies and previous studies have demonstrated that the prediction success rate for some drugs can be as high as 100% in zebrafish (96). Furthermore, zebrafish represented the most suitable model for this study due to the limitation of stem cell cultures in over-predicting toxicity and the short lifetime of whole embryonic cultures which can only capture in vivo embryonic development for 48 h (94-99, 134-135). The rapid rate of organogenesis, the reduced amount of test compound needed and the large number of embryos obtained per spawns which easily allows optimal study sizes provided additional advantages.   In this study, we exposed developing zebrafish embryos to various concentrations of chelators and assessed their effects on mortality, hatching and morphology in embryos. To determine whether the effects were specific to embryos and to probe possible underlying mechanisms of the observed toxicities, we investigated the effects DFO, L1 and ICL-670 exposure on the cardiac function and gene expression in adults. Since the toxicity of iron chelators can result from sequestration of iron required for biological processes, we investigated the change in expression of hepcidin, ferroportin (FPN) and DMT-1, proteins directly involved in iron transport and recycling (136-140).  In order to further define the properties of the HPG-DFO conjugates developed herein, 40SDO2 and HPG-DFO were also screened for effects on mortality, morphology and hatching success in zebrafish embryos. This study improves our understanding of the differences in 97  potential effects of clinically approved as well as novel polymeric iron chelators at the earlier stages of embryonic development. Results will guide our approach in further understanding the effect of iron chelation in vertebrates during development, and will provide clues on the possible organ systems perturbed by iron depletion in developing embryos.   4.3: Materials and Methods  Chemicals: Desferrioxamine (> 99%), deferiprone (1,2-dimethyl, 3-hydroxy, pyrid-4-one) and 100% cell culture grade DMSO were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON). Desferasirox (4-[3, 5-bis (2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid) was synthesized in house according to established protocols (141). Aquacalm was obtained from Western Chemical Inc.(Ferndale, WA, USA). Zebrafish Husbandry: All experiments complied with the Canadian Council of Animal Care guidelines and the animal care protocol (2012-0082), which was approved by the University of Saskatchewan Animal Care Committee. Zebrafish were obtained from (Aquatic Imports, Calgary) and were maintained in a recirculating, light and temperature controlled facility on a standard 14:10 h light: dark cycle in standard system fish water. Embryos were maintained at 28 °C throughout all experiments. Embryos were washed and healthy, non-coagulated embryos were selected for exposure to chelators.  Chelator Exposure to Zebrafish Embryos: Embryos were grown in 6-well plates (Corning, Life sciences) containing 4 mL of chelator solution in DMSO and serial dilutions were done using fish water. DMSO was used as control. Chelator solutions were replenished every day and five concentrations of chelator (0.015, 0.03, 0.06, 0.125, 0.25, 0.5 and 1 mM) were used. The maximum DMSO concentration for DFO and ICL-670 was 0.45%. The maximum concentration tested for L1 was 0.5 mM, with a DMSO concentration range of 0.02-1% as shown in Figure 25.  98  For studies conducted with high MW chelators, DFO, 40SDO2 and HPG-DFO were dissolved in fish water. A positive control (HPG-ethanolamine) was also generated by reaction of oxidized HPG with ethanolamine. The resulting HPG was used as a positive control to investigate the effect of HPG surface groups on zebrafish embryo morphology and hatching. All embryos were derived from the same spawn of eggs to allow statistical comparison. After fertilization, embryos were collected and transferred to 6-well plates; 15 embryos per well and 3 wells per concentration (n=45) for a treatment period of 96 h. This study was repeated twice after an initial optimization study.   Mortality, Hatching and Morphology Readings: Mortality, hatching and morphology were assessed and recorded every 24 h using a CCD digital camera (OLYMPUS DP70, Japan) mounted on a microscope (Olympus, BX51, Japan) every 24 h from 6 h to 96 h post fertilization (hpf).  Percent mortality, morphology, and hatching success were determined. Other defects such as swim behavior were also monitored. Percent hatching success was defined as: (the number of larvae/initial number of embryos) 100.  Chelator Exposure and Ultrasound Analysis in Adult Zebrafish: Adult fish were anesthetized with 28 mg/L aquacalm solution. Stock solutions were prepared in DMSO and serial dilutions were prepared before each experiment using fish water. The final concentration of DMSO present in each solution was noted and is given in Figure 25-27. Six adult zebrafish were injected i.p. with 100 µM of DFO, L1 and ICL-670 and subjected to ultrasound monitoring using a VEVO 660 high frequency ultrasound machine (VisualSonics, Markham, ON), 30 minutes post injection. Each zebrafish was removed from fish water and placed in 28 mg/L aquacalm (Syndel Laboratories, Canada) until they succumbed to the anesthetic. Once sufficiently anesthetized, they were positioned ventral side up and secured in a 3% agarose gel with minutien pins (Fine Science 99  Tools, Vancouver, BC). The probe (A RMV 708B), attached to the machine is kept at startup mode so that the probe is already moving. The probe is then lowered above the horizontally placed zebrafish and adjusted as the view is needed. The ultrasound monitoring was carried out using the VisualSonics software (Markham, ON). Three short axis views; A1 (towards the gills), A2 (slightly away from the gills) and A3 (towards the tail), were taken. One long axis view of the ventricle was also taken by placing the fish in a vertical position. All these readings were measured both at the systole and diastole using the VisualSonics Software. Calculations were conducted as follow: Stroke Volume (SV) = EDV (end diastolic volume) – ESV (end systolic volume). Heart Rate = heart rate/10 sec x 6 and cardiac output = stroke volume x heart rate. Quantitative PCR: Six adult zebrafish from each group were injected intraperitoneally with10 μL of DFO, LI and ICL-670 (100 µM) and sacrificed 24 h after injection. Liver, heart and gut were harvested for RNA extraction. Total RNA was extracted using the TRIzol RNA isolation reagent (Invitrogen, Canada).  The purity of extracted RNA was assessed by optical density absorption ratio (OD 260 nm/OD 280 nm) using the nanodrop (ND 100, NanoDrop Technologies Inc. Wilmington, DE, USA). One µg of RNA was used for iScript cDNA synthesis as directed by the manufacturer (BioRad, Canada). cDNAs were diluted 1:3 before qRT-PCR using a CFX connect (BioRad Laboratories Inc. Canada) with iQSYBR Green supermix (BioRad, Canada). The cDNAs were amplified using the forward and reverse primers shown in Table 8 (Sigma-Aldrich Canada Ltd). For each sample, qRT-PCR was run in duplicate to ensure consistency. Each reaction contained 2 µL (30ng) of cDNA, 10 µL SYBR® Select Master Mix (2X), 0.5 µL(10nM) of forward and reverse primers and 7 µL nuclease free water. The thermal profile for all reactions was 3 min at 95 °C and 40 cycles of 10 s at 95 °C, and 30 s at 60 °C. Fluorescence monitoring occurred at the end of each cycle. Relative expression levels were determined by normalizing to 100  the β-actin housekeeping gene and the relative change in gene expression was determined using the ∆∆Ct method of Livak and Schmittgen for relative quantification (142). The expression of hepcidin, ferroportin (FPN) and the divalent metal transporter (DMT-1) were investigated (137-140).  Table 8: The forward and reverse primers used for real-time qPCR.   Statistical Analysis: Statistical analysis was performed using SPSS 19.0 software. Data were expressed as means ± SEM. Comparisons between groups were made using one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis for multiple comparisons. Homogeneity of variance and normal distribution of data were tested for all data using Levene’s test. Quantitative PCR data were log transformed to meet assumptions of normality and homoscedasticity when data was non-homogeneous.  P values less than 0.05 were considered statistically significant.    Gene   Primer Accession No. GEN Bank  Amplicon size, bp Hamp1 F: 5'-CCGAGCAGAAGACAAGTAGAT-3' R: 5'-GCAGCCAGAAACACGTTAGA-3' NM_205583.1 104 DMT-1   F: 5'-ACCGCAGCAATAAGAAGGAG-3' R: 5'-TTGGTTTTCCCGTAGAAGGC-3' NM_001040370.1 136  Ferroportin F: 5'-ATTTACTTTGCCCGAGCCTT-3' R: 5'- CAGCGAGGTTTCTTTGATGC-3' NM_131629.1 104 β-Actin  F: 5'-TTCAAACGAACGACCAACCT-3' R: 5'-TTCCGCATCCTGAGTCAATG-3' NM_131031.1 93 101  4.4: Results  In order to ensure that the solvent, DMSO did not contribute to the observed effects of chelator exposure at the concentrations tested, control experiments were run with DMSO alone. Since DFO is highly soluble in both DMSO and fish water, a control experiment was conducted with DFO to determine the effects of solvent on embryos. Figure 25-27 demonstrate that DMSO neither induced toxicity, nor did it influence exposure to the embryo. Embryos exposed to DMSO at all concentrations used in the study, were normal and did not differ significantly from fish water controls in mortality, morphology nor hatching.   Figure 25: The effect of DMSO on chelator exposure.  To ensure that the amount of carrier solvent used did not influence the observed effects, DMSO was titrated and the effects on morphology were observed. The morphology of DMSO treated embryos after 96 hpf exposure and the concentration of DMSO present per concentration of each chelator. There were 3 replicates of 15 embryos (n=45) used in this study. 102   Figure 26: DMSO does not interfere with the development of embryos.  To determine whether DMSO influenced or enhanced the toxicity of chelators, embryos were exposed to DFO (0-1 mM) dissolved in either fish water or DMSO for 96 hours post fertilization (hpf). The number of dead embryos out of the number of total embryos was recorded after 24, 48, 72 and 96 h of exposure and are shown above. Embryos did not differ significantly in mortality rates throughout the duration of the study, 45 embryos were used for each concentration. Comparisons were made between fish water treated and DMSO treated embryos.          103     Figure 27: Influence of DMSO on the development of zebrafish embryos. Data show that DMSO does not interfere with the development of embryos in the concentration range studied (0-1%).  To determine whether DMSO influenced or enhanced the toxicity of chelators, embryos were exposed to DFO (0-1mM) dissolved in either fish water or DMSO for 96 hpf. The number of dead embryos out of the number of total embryos was recorded after 24, 48, 72 and 96 hpf of exposure and are shown above. Embryos did not differ significantly in mortality rates or hatching success throughout the duration of the study, 45 embryos were used for each concentration. Comparisons were made between fish water treated and DMSO treated embryos. 104    Figure 28: DMSO does not influence the morphology of zebrafish embryos.   To determine whether DMSO influenced or enhanced the toxicity of chelators, embryos were exposed to DFO (0-1mM) dissolved in either fish water or DMSO and their morphology was observed for 96 hpf. The morphology of zebrafish embryos exposed to DFO in fish water did not differ from the morphology of zebrafish embryos exposed to DFO dissolved in DMSO.   105  4.4.1: Effects of Carrier Solvent on the Mortality, Hatching Success and Morphology of Zebrafish Embryos  To determine whether DMSO can influence chelator exposure in zebrafish, embryos were exposed to various concentrations of DFO dissolved in 2 solvents; fish water and DMSO. The  difference in embryo mortality, and hatching success did not reach significance at any of the time-points tested compared to control. Additionally, embryos exposed to DFO in fish water and DFO in DMSO did not vary in morphology throughout the study. Figure 28 shows that the larvae of embryos treated in both solvents hatched normally. DFO was chosen for this study due to its high solubility in both water and DMSO.  4.4.2: Effects of Clinically Approved Iron Chelators on Mortality, Hatching Success and Morphology of Zebrafish Embryos  Iron chelators (DFO, L1, ICL-670) differed significantly in their effects in developing zebrafish embryos. The mortality of zebrafish embryos exposed to different iron chelators at different time points are shown in Figure 29. The mortality of DFO-treated embryos did not differ significantly (p=0.961) from the control regardless of exposure duration and drug concentration (Figure 29). In contrast, the mortality of ICL-670 varied significantly from controls (p <<0.05) in a dose and time-dependent manner (Figure 29); while the mortality of embryos exposed to L1 caused a slight but insignificant increase in mortality at 0.5 mM. The percentage mortality was highest in ICL-670 treated embryos; with more than 80% mortality occurring above 0.25 mM of ICL-670. The onset of mortality in embryos exposed to ICL-670 varied from 24 h for 1 mM treated embryos to 72 and 96 h in embryos treated with 0.5 mM and 0.25 mM, respectively. L1 treated embryos did not differ significantly from control although there was a slight increase in mortality 106  at 0.5 mM after 96 h exposure. Control experiments with different concentrations DMSO used for dissolving the different iron chelators suggest that there is no interference from DMSO in this study (Figures  25-27).   DFO, L1 and ICL-670 also differed significantly in their effect on zebrafish embryo morphology upon hatching. Similar to the mortality, DFO-treated embryos did not show any differences in hatching when compared to control, up to concentrations of 1 mM (Figure 30). While all the embryos treated with L1 hatched into larvae successfully, hatching success was reduced in ICL-670 treated embryos due to increased mortality; embryos treated with 1 mM of ICL-670 did not survive to hatch.    107                            Figure 29: Mortality of zebrafish embryos exposed to iron chelators.   The mortality of DFO and L1-treated embryos did not vary significantly from control after 96 h of exposure (A-D). The mortality of ICL-670-treated embryos increased in a dose and time-dependent manner and was significantly higher than control embryos; death occurred within 24 h of exposure to 1mM ICL-670 (A) and 96 h for 0.25 and 0.5 mM treated embryos (D). § denotes missing bar for L1 at 1 mM due to high DMSO content; the DMSO concentration would have been 2%. The concentration of chelator for which mortality was significantly different from control (p = <0.05) is denoted by *.108                   Figure 30: Hatching success and morphological alterations of zebrafish embryos.  DFO and L1-treated embryos hatched successfully, while ICL-670-treated embryos hatched less successfully than control due to high mortality (A). Bars missing at 1 mM are due to the death of embryos prior to hatching. § denotes the concentration of L1 that was excluded due to high DMSO content. There were no significant defects observed in DFO treated embryos. However, the percentage of embryos demonstrating behavioral and morphological defects increased in a time and concentration dependent manner in L1 and ICL-670-treated embryos (B). The time of onset of morphological and behavioral alterations is outlined in Figure 28. Swimming behavior was affected and bent bodies (Figure 29) were observed at concentrations above 0.25 mM for ICL 670-treated embryos and at 0.5 mM for L1 treated embryos. Chelator/Solvent Onset of morphological deformities   24 h  48 h  72 h DFO - - - L1 - - 0.5 mM ICL-670 - 0.5 mM 0.25 mM 0.5 mM  DMSO (0.1-1.25% v/v) - - - 109  We next looked at the morphological deformities of the hatched larvae at different concentrations after exposure to iron chelators (Figures 30-32). The percentage of deformities was highest for ICL-670. The deformities increased with increasing exposure time (Figure 30). Representative optical images of zebrafish embryos after exposure to iron chelators and control are shown in Figures 31-32. Bent bodies (BB) of larvae were the most commonly observed defects for L1 and ICL-670. However, DFO at the doses tested, did not cause any deformities compared to controls. ICL-670 samples showed deformities at much lower concentrations than L1. Signs of rapid breathing and change in the swimming behavior were also observed. The ICL-670-treated larvae exhibited lethargy and often moved only when a small stimulus was provided. Edema was also observed in ICL-670 treated larvae. Similar but less abundant morphological defects occurred in embryos exposed to 0.5 mM L1 (Figures 31-32). Chelator dose and duration of exposure were the two most important factors influencing the toxicity.   110                                         Figure 31: Zebrafish larvae morphology after 72 h of exposure to iron chelators.   Representative optical images of zebrafish morphology after 72 h exposure to DFO, L1 and ICL 670. Zebrafish kept in DMSO was used as control. Embryos treated with 1 mM ICL-670 did not survive to hatch (DNS); while those treated with 0.25 mM and 0.5 mM hatched abnormally and had bent bodies (shown with arrows). Images were captured at 2X magnification.   111                                      Figure 32: Zebrafish embryo morphology after 72 h of exposure to iron chelators.   Representative optical images of zebrafish morphology after 72 h exposure to DFO, L1 and ICL 670. Higher magnification (4X) shows edema in L1 and ICL-670 treated embryos. No abnormalities were observed in DFO treated embryos up to concentrations of 1 mM 4X magnification.   112  4.4.3: Acute Exposure to Iron Chelators on Cardiac Output and Heart Rate in Adult Zebrafish To enhance our understanding of iron chelator-induced toxicities in zebrafish, we investigated the effects of DFO, L1 and ICL-670 on the cardiac function of adult zebrafish. Adult fish were injected with 100 µM of each chelator and subjected to ultrasound 30 minutes after treatment. We chose a lower concentration of the chelators that was not lethal and would allow us to determine more subtle changes. The heart rate, cardiac output, stroke volume, end systolic volume and end diastolic volume were measured and are shown in Figure 33.  ICL-670 but not L1 nor DFO caused a significant increase in the heart rate and cardiac output of zebrafish (Figure 33 A and B). The average heart rate increased from 102 to 138 beats per minute and cardiac output increased from 12 μL/min in controls compared to 30 μL /min in ICL-670, (p<0.05; Pheart rate = 0.001; Pcardiac output = 0.01). Interestingly, there was a significant difference in heart rate between L1 treated and ICL-670 treated fish. The heart rate of DFO treated fish did not differ significantly from control. Chelators did not significantly affect the end-diastolic volume, end-systolic volume and stroke volume in adult zebrafish (Figure 33 C-E) (Pend diastolic volume = 0.3421; Pend systolic volume = 0.25; Pstroke volume = 0.1137).          113  Figure 33: Iron chelator exposure significantly influences heart rate and cardiac output in adult zebrafish. Two independent experiments were conducted and 3 fish were used per concentration per experiment; a total of 6 fish per concentration were used for analysis. Zebrafish were injected with 10 μl of 100 µM of each chelator and subjected to analysis by ultrasound 30 min after injection. The heart rate of fish treated with ICL-670 differed significantly from controls. There was also a significant difference in heart rate between LI and ICL-670 treated fish. Similarly, the cardiac output of fish exposed to ICL-670 differed significantly from control and L1-treated fish. However, DFO and L1 treated fish did not vary significantly from controls in any of the measurements obtained. DFO, L1 and ICL-670 did not significantly affect the end-diastolic volume, end-systolic volume or the stroke volume compared to the saline or 0.1% DMSO controls, n=6 adult zebrafish and values represent the mean + SD, * denotes values that are significant from control (p= <0.05). A B E C D 114  4.4.4: Acute Exposure to Iron Chelators on the Expression of Hepcidin, Ferroportin and DMT-1 Genes in Adult Zebrafish Hepcidin, FPN and DMT-1 are involved in iron regulation in zebrafish and changes in iron status have been shown to influence their expression levels (131-135, 138). Thus, we investigated the effect of different chelators on mRNA levels in adult fish in the gut, liver and heart tissue. Hepcidin is a multifunctional peptide and the chief iron regulatory hormone in vertebrates (137-138, 140). One of its functions is to bind to FPN, which is located on the basolateral surface of the enterocyte, and prevent iron absorption. Upon binding to FPN, hepcidin causes it to be broken down when the body's iron supplies are adequate. DMT-1 functions as an iron transporter in vertebrates. It can also transport other divalent metals (136, 140). Adult fish were injected intraperitoneally with 10 µl of 100 µM chelators and the gene expression at 24 h post-treatment was measured.  None of the iron chelators tested significantly altered the levels of hepcidin in the liver or heart. DFO and L1 caused a down-regulation of hepcidin expression in the gut (Figure 34). Chelators did not significantly alter FPN expression in the gut. L1and ICL-670 down-regulated, while DFO up-regulated FPN expression in the liver. All chelators caused a slight up-regulation of FPN in the heart with ICL-670 showing a significant difference from control. DFO, L1 and ICL-670 caused a down regulation of DMT-1 in the gut with ICL-670 causing the strongest change. However, none of the iron chelators significantly altered DMT-1 expression in the liver and heart.   115                             Figure 34: Effect of chelator exposure on gene expression.  Expression of hepcidin, ferroportin (FPN) and divalent metal transporter (DMT-1) in the gut, liver and heart tissues of adult Zebrafish after 24 h exposure to 100 μM iron chelators. Gene expression values were normalized to β-actin and are presented as means + SEM. Treatments that do not share a common letter are significantly different from each other (p = <0.05), n=6 for all treatments.  116  Hepcidin expression in the liver and heart tissue of adult zebrafish did not differ significantly from control. However, DFO and L1 significantly down-regulated hepcidin expression in the gut. Changes in FPN expression in the gut upon chelator exposure did not reach statistical significance. However, L1 and ICL-670 down-regulated, while DFO upregulated FPN in the liver and all chelators upregulated FPN in the heart.  Treatment with DFO, L1 and ICL-670 resulted in a down-regulation of gut DMT-1.  4.4.5: HPG Conjugation to DFO does not Enhance the Toxicity of DFO in Zebrafish  To determine whether HPG-DFO exerted toxic effects in vivo, we used zebrafish embryos as a model of toxicity. DFO and previously characterized high MW 40SDO2 based iron chelator 40SDO2 were used as controls. A positive control was also developed by adding ethanolamine groups to an HPG using similar chemistry outlined for the HPG-DFO conjugates. Additionally, the results of previous studies of low MW chelators in zebrafish were compared to HPG-DFO. Unlike the low MW chelators LI and ICL-670 previously described, exposure to polymeric chelators did not result in any visible defects in embryos throughout the duration of the study. Embryos exposed to up to 1 mM of HPG-DFO and 40SDO2 remained active and viable throughout the course of the 96 h study, regardless of chelator concentration (Figure 35-36). In contrast, the ethanolamine modified HPG caused a significant increase in mortality of zebrafish embryos and a reduction in hatch successs (Figure 37). Moreover, there was no significant difference in embryo morphology, the number of hatched fish or the time taken to hatch. Swim behavior between chelator treated embryos and controls did not differ throughout the course of the study (p=ns for all) (Figure 35-38).   117    Figure 35: Toxicity of HPG-DFO conjugates in zebrafish embryos.  HPG conjugation to DFO does not enhance DFO toxicity in zebrafish embryos. The mortality of DFO, HPG-DFO and 40SDO2 treated embryos did not vary significantly after 96 h of exposure (A-D), indicating that polymer conjugation to HPG and 40SDO2 did not enhance toxicity (p=ns for all).  118     Figure 36: HPG conjugation to DFO does not influence the hatching success of zebrafish embryos.  DFO, HPG-DFO and 40SDO2-treated embryos hatched successfully; there were no significant defects observed in DFO treated embryos. There were no behavioral and morphological defects observed, up to 1 mM of chelator.    119  4.4.6 Positive Control HPG Increases Mortality and Reduces Hatching Success of Zebrafish Embryos   Figure 37: The effect HPG-NH2 on zebrafish embryo mortality and hatching.  Zebrafish embryos (n=45) were incubated with HPG-NH2 for 96 h and the mortality and hatching success was determined as previously described. Embryos treated with HPG modified with ethanolamine showed increased mortality and hatching compared.    120    Figure 38: Zebrafish larvae morphology after 72 h of exposure to polymeric iron chelators.  Representative optical images of zebrafish morphology after 72 h exposure to DFO, HPG-DFO and 40SDO2. Zebrafish kept in fish water was used as control. Embryos treated with up to 1 mM polymeric chelators did not demonstrate any morphological defects upon hatching. Images were captured at 2X magnification.   121  4.5: Discussion  Iron is an essential micronutrient for living organisms and functions as an integral component of many biological reactions. Iron chelators are used to reduce iron levels in patients that are susceptible to iron overload. Although a wide range of toxicities have been reported to occur, information on the effect of iron chelators in embryo development is lacking. Thus we investigated the effects of iron chelator exposure on mortality, hatching success and morphology of zebrafish embryos. We also investigated possible mechanisms of toxicity by measuring the changes in cardiac function and the expression of hepcidin, FPN and DMT-1, genes anticipated to be sensitive to iron status. Different chelators (DFO, L1, ICL-670) exerted distinct effects in zebrafish embryos and differed in the extent of toxicity exerted (Figures 29-34). While DFO treated embryos did not differ from control in any of the parameters measured; ICL-670 treated embryos exerted a range of toxicity which increased with increasing dose and longer duration of exposure. Exposure to L1 was associated with small changes to morphology.  The permeability and chemical properties of pharmacological agents and the characteristics of the zebrafish chorion may have separate, additive or synergistic effects on the chelator-induced toxicities (144-147). The size, polarity and specificity of each chelator for cations may vary and may influence the ability of chelators to permeate the zebrafish embryo, thus influencing the observed toxicities. The notable differences in size of chelators (DFO-560 Da, L1-139 Da and ICL-670-373 Da) may also be an important factor influencing the intensity of chelator exposure in embryos (21, 144-146). The zebrafish embryo is multi-compartmental by nature with each compartment possessing distinct permeability characteristics. Harvey et al demonstrated that the chorion of 122  the embryo may act as a permeability barrier (147). The chorion has three layers and is nanoporous. The middle and inner layers are pierced by pore canals; thus, some particles can cross the chorion through the 0.5-0.7 μM chorionic pores (148). However, it has been shown that the rate of permeation by DMSO is several fold greater after dechorionation; indicating that the chorion can retard the passage of solutes. Another study showed that two chemically dissimilar fluorescein molecules dissolved in DMSO crossed the chorions of zebrafish at different rates. While DMSO appeared to support the passage of fluorescein across the chorion, it did not influence uptake of 2,7-dichlorofluorescein across the chorion (149). However, this it is not likely that DMSO influenced any observed toxicity as our control experiments with DMSO and DFO showed that there was no influence of the carrier solvent in the observed behavior (Figures 25-28).  Another important aspect of toxicity in relation to iron chelation therapy may be the removal or displacement of iron or essential other metals. In the absence of iron overload, iron chelators can interfere with zinc, copper and other micronutrient binding although the binding constants for these chelators to other metal ions are relatively small (the log cumulative stability constant of DFO-Fe is 30.6 versus 11.1 for DFO-Zn 2+) (21). Additionally, reducing essential iron in the cell can result in reduced cell proliferation by inhibiting intracellular ribonucleotide reductase (150). Thus hydrophilic iron chelators such as DFO may not readily enter cells to elicit this pattern of toxicity, probably because of their inability to penetrate cell membranes. This would explain the lack of toxicity observed for DFO at the concentrations studied. Further, it has been shown that a direct correlation exists between the rate of cellular uptake of a drug and its potential toxicity (145).  A recent study on the evaluation of cardiotoxic 123  drugs in zebrafish embryos showed higher toxicity for injected drugs than the soaked solutions, supporting this argument (96).   Although the precise daily iron requirements of teleost fish are unknown, the daily loss of iron is comparable to that in humans (151). For example, zebrafish loose 14 μg of iron per kg/day, a value that is similar to that in man which typically loose 14-28 ug/kg/day (151). It has also been shown that developing fish embryos receive sufficient iron from maternal stores in the yolk and that iron acquisition by fish embryos is generally limited, while studies show that mature teleost fish can become iron limited (151-152). Roeder and Roeder showed that retarded growth results when swordtail and platyfish were fed iron poor foods, with growth rates returning to normal when the diet was supplemented with iron salts (153). Thus, although the effects of chelators were less pronounced in adults the significant change in heart function observed upon treatment with ICL-670 (Figure 33) may have resulted due to the depletion of essential cardiac iron or perturbation of metal balance (other essential metal ions) in heart tissue. Furthermore, the higher lipophilicity of ICL-670 has been shown to cause accumulation in some tissues and related organ damage in humans (154).  The seemingly mild effects of chelators on mRNA expression (Figure 34) are likely due to the short duration of exposure. Hepcidin is a multifunctional peptide with a key role in iron metabolism. FPN exports iron to the blood stream during absorption while DMT-1 functions as a carrier for most divalent metals across the apical surface of the cell. DFO produced a slight but insignificant increase in FPN expression after 24 h of exposure while gene expression in L1 and ICL-670 treated fish did not differ from control. Because DFO is known to be efficient at hepatocellular iron removal this finding is not surprising (10, 12). This may mean that DFO caused a reduction in the zebrafish liver iron causing an iron poor state.  124  DFO and L1 also induced the greatest change in hepcidin expression in the gut; causing a significant down-regulation of the gene compared to control, while ICL-670 did not appear to significantly affect hepcidin expression. When iron levels fall in the body, hepcidin levels are also decreased and more FPN is available to bring iron into the body and to release it from storage. Down regulation of hepcidin implies that cells are iron poor and causes enhanced iron absorption through increased FPN expression. This would be expected if these iron chelators chelate significant amounts of iron. Thus, it is likely that increasing the dose of chelator would result in a corresponding down regulation in hepcidin with the effect being increased iron absorption. Interestingly, ICL-670 did not have the same effect.  DMT-1 functions as an iron transporter in vertebrates. It can also transport other divalent metals. For example, rat DMT-1 has been shown to transport a range of divalent metal cations, including Fe, Pb, Zn, Cu and Cd (136, 140). DMT-1 has a critical function in iron metabolism as it allows the entrance of iron through the duodenal enterocyte, and enables the utilization of iron by cells via the transferrin receptor mediated iron uptake. DMT-1 is also required for transporting iron out of the endosome and into the cytosol where it is incorporated into proteins or stored in ferritin (136, 155).  Not surprisingly, juvenile zebrafish were more susceptible to toxic effects than adults. This may have resulted from the longer duration of chelator exposure in the embryos compared to the single dose given to adult zebrafish. The short duration of exposure in adults may also account for the slight alterations in gene expression observed in adults in the organs tested. Further studies of gene expression which take advantage of increased exposure time in adults (chronic exposure) would help to improve our understanding.  125  Zebrafish studies showed that FDA-approved chelators L1 and ICL-670 may exert toxicity in developing zebrafish embryos (157). Embryos, larvae and adult zebrafish were all more sensitive to exposure to smaller MW chelators. However, similar studies conducted with 40SDO2 and HPG-DFO showed that the early life stages of zebrafish were not negatively affected by 40SDO2 and HPG-DFO exposure. Thus, the HPG component used to generate HPG-DFO appears to be non-interactive with the zebrafish embryo to an extent that causes significant changes in mortality or morphology over our 96 h study period.  Analysis of these findings, when interpreted in light of the findings of small molecular weight chelators suggest that molecular weight and lipophilicity of drug particles may be key determinants of chelator toxicity. These findings are also consistent with earlier studies carried with other DFO polymer conjugates and other similar drugs (158). Lipophilicity has had a significant effect on chelator toxicity and was reported to be a factor within the DADFT family of ligands, in which case the more lipophilic ligands were more toxic (158). Our studies indicate that polymeric chelators do not interact with the embryo to the extent that small molecular weight chelators like L1 and ICL-670 do. Our previous studies of low MW chelators in zebrafish showed that higher MW, hydrophilic DFO did not cause any toxicity to exposed embryos but that lower MW chelators with more hydrophobic properties  did influence the development and mortality of embryos.  The response of zebrafish to chemicals such as small molecules, drugs and environmental toxicants can be similar to that of mammals; however, it is important to conduct further studies that are aimed at determining how the effect concentrations in zebrafish are related to those of mammalian models. This is especially relevant as the exposure to drugs in fish embryos is static, and internal concentrations are established by partition equilibrium. 126  While in mammals, drugs are administered by single or repeat doses, and exposure is not static. Thus, it is important to appropriately translate all findings obtained in experimental models. Koren et al provide an excellent review regarding this (156).  4.6: Conclusions  This study demonstrates that clinically approved iron chelators vary in their ability to induce toxicity in zebrafish embryos and adults, and that the time and dose of exposure are major factors influencing toxicity. DFO and HPG-DFO exposure did not induce any toxicity; there was no change in mortality, morphology, or percentage of hatched embryos. L1 did not significantly affect mortality but caused morphological alterations at higher concentrations. While ICL-670 caused significant morphological deformities and hatching problems in zebrafish embryos above certain concentrations such that these iron chelators are influencing the development into larvae. Unlike other chelators, ICL-670 caused significant change in the heart rate and cardiac output in adult zebrafish when injected at concentration of 100 µM (10 µL). Changes in expression of hepcidin, FPN and DMT-1 genes were observed in adults, however, the effect are not pronounced as in embryos.  However, these studies do not define the mechanisms underlying the reduced toxicity of higher MW chelators in zebrafish. It is most likely that a combination of factors including the differential permeability of the components of the embryo; the size, charge, lipophilicity and chemical structure of HPG-DFO, DFO, L1 and ICL-670 influenced their ability to induce toxic effects in zebrafish. Studies focused on characterizing the permeability barriers in the zebrafish chorion will further elucidate the factors that influence the observed chelator toxicity. Future studies must focus on deconstructing the role of the zebrafish embryo chorion in modulating solute uptake into the embryo and understanding the interactions between HPG 127  and cellular components. Additionally, future studies which investigate the method of chelator exposure, as well as the absorption, metabolism and excretion profile of each chelator in zebrafish are recommended. Such studies can elucidate scaling factors which may allow more suitable and accurate comparisons and correlation to rodents, and other relevant experimental models.  128  Chapter 5: Conclusions and Future Directions 5.1: Conclusions Iron chelation therapy is clinically indicated for the treatment of transfusion associated iron overload; a common condition in patients with severe hemoglobin disorders like β-TM, SCD, and the MDS (9-10, 14-17, 20, 26-34). DFO was the first iron chelator to become available and remained the only option for patients for almost 40 years. Iron chelation therapy with DFO has transformed the management of transfusion associated iron overload. Studies show that chelation therapy reduced glucose intolerance, liver disease and heart failure, and extended the lifespan for transfusion dependent patients (9-16). Prior to this time, the life expectancy of transfusion dependent patients was up to the second decade, with heart failure due to iron-induced myocardial damage accounting for the majority of deaths (9-12, 14-17).  However, DFO use was associated with some challenges. The short circulation half-life of ~20 minutes necessitated continuous DFO exposure for patients to reap the benefits. As such, patients are required to daily, subcutaneously infuse DFO for 8-12 hours. This demanding regimen has remained a major challenge for many patients and the greatest hindrance to DFO use. Additionally, DFO use at high doses is associated with visual and auditory toxicities, and subcutaneous infusions lead to allergic reactions at the infusion site. Due to these challenges, iron overload continued to be a major source of morbidity and mortality for patients that could not comply with DFO therapy (22-25, 159).  There has been significant advancement in chelation therapy over the past ten years. Most noteworthy is the advent of two novel, orally active iron chelators; L1 and ICL-670 (26-38). The availability of oral iron chelation has radically improved compliance in many patients and has also significantly improved quality of life. Additionally, availability of orally active 129  chelators with differing properties and modes of action allow improved, more effective iron removal than DFO alone. For example, the lipophilic properties of L1 and ICL-670, which enable their easy passage inside of cells of the heart; coupled with the hydrophilic, high chelation efficiency of DFO have led to new possibilities like chelator combination therapy (10, 119-121, 160-161). Chelation combination therapy has proven highly beneficial in allowing patients inadequately chelated by a single chelator to achieve sufficient iron excretion. In patients that become unresponsive, or in those that experience severe side effects of one chelator, combination therapy or switching to a different chelator has proven to be helpful alternatives. Due to these increased options for chelation, there have been significant improvements in life expectancy and quality of life in chronically transfused patients that comply with therapy. Today, the life expectancy of adequately chelated patients can be up to the 5th decade (162).  Although there has been substantial improvement in patient compliance, quality of life, and lifespan in transfusion dependent patients, there are many areas in iron chelation therapy that remain under-explored. One such case is in the area of chelator toxicity during early development.  This area is likely to become of great importance as the interest in and likelihood of reproduction among thalassemic women continues to increase. Secondly, there is still the need to develop more efficient iron chelators with improved safety than LI and ICL-670 and better pharmacokinetics than DFO.  Current iron chelation therapy requires the daily administration of almost maximum tolerated doses of DFO, L1 and ICL-670 in order to ensure that the rates of transfusion associated iron loading and iron excretion in transfusion dependent patients are well matched. Not surprisingly, this causes patients to experience a wide range of toxicity. Additionally, in 130  many cases the administered doses are still insufficient to mobilize the required amount of iron and ensuring negative iron balance. Most importantly, it is estimated that 80% of births of the patients with transfusion dependent anemias occur in low-or middle-income countries (LMICs), where access to oral chelation therapy, primarily ICL-670, is virtually impossible due to high costs (163-164). Patients in these countries are dependent on subcutaneous DFO or L1, the shortcomings of which have been well described by studies showing that adherence to DFO therapy is problematic and that L1 is not effective in all groups of patients and with prolonged use (16, 22-32, 102).  Given these circumstances, iron chelators that have improved properties would advance therapy for a great number of patients. Chelators that have reduced toxic side effects, better administration regimens than DFO and are more efficient and consistent at iron mobilization among all patients would prohibit the accumulation of labile body iron. More importantly, such chelators would prevent iron overload from progressing to severe organ damage, the major cause of death in inadequately chelated patients. 5.2 Significance of Thesis  The work described in thesis begins to address these challenges. To address the need for innovative approaches to iron chelation, the results described in Chapter 2 describe the development and properties of a new, polymeric, DFO-containing iron chelator. Furthermore, this thesis highlights the role of polymers like HPG in modifying the properties of small MW drugs like DFO. These findings build on current knowledge of the influence of polymers on DFO chelating properties and advances knowledge by characterizing the thermodynamics that dictate iron binding when DFO is conjugated to polymers. In addition, this was the first study which probed the efficiency of iron removal from ferritin by DFO-polymer conjugates. The 131  discovery that polymer conjugation does not affect the ability of DFO to chelate iron under physiologically relevant conditions (temperature and pH) and that the properties of several HPG-DFO conjugates are superior to that of DFO and 40SDO2, highlights the potentially high clinical utility of HPG-DFO (105).  The findings reported in Chapters 3 provide a biological evaluation of these novel polymeric, HPG-DFO chelators. Previous studies demonstrate that the MW of the polymer components may influence biocompatibility. Moreover, very high MWs may cause several complications and thrombotic events (165). In addition to large polymer size, the large number of functional hydroxyl groups that are present on the periphery of the HPG could potentially affect interactions with biological macromolecules or cells. Thus, understanding and defining the biocompatibility of these novel nanomedicines is very important. We selected high MW HPGs of various sizes and with various degrees of modification and studied their biocompatibility properties in detail. These studies build upon previously characterized methods of drug modification and specifically explore the role of HPG as a useful option for modifying the toxicity of iron chelators. The findings that HPG-DFO were highly biocompatible were important for defining the utility of HPG as a modifier of iron chelator toxicity. Moreover, this assessment served to guide the development of future experiments in more advanced models. For example, subsequent studies conducted in iron overloaded mice have further demonstrated the clinical utility of HPG-DFO.  The results of experiments conducted in zebrafish embryos described in Chapter 4, investigated iron chelator toxicity at early stages of development and defines possible mechanisms of chelator-induced toxicity. Although this is a preliminary study, these finding will direct future studies aimed at improving our understanding of the mechanisms, processes, 132  and factors underlying iron chelator toxicity in early development. Moreover it will allow a correlation to be made between chelator structure and toxicity in this life stage. The observed changes in mortality and morphology in chelator treated embryos described in this thesis suggest the need for a deeper understanding of the influence of permeability barriers like the zebrafish chorion on chelator exposure. Therefore, future research focused on measuring the rate of chelator entry into the embryo, or the integral changes in the permeability barriers of embryos with prolonged exposure would help to advance our understanding of chelator induced toxicity. Furthermore, comparing the effects in different experimental models would also help to more appropriately translate the findings and advance understanding. Similarly, the significant change in heart function observed with acute exposure to chelators in adult zebrafish warrants further exploration and may expand knowledge about the organ systems that are first affected by iron depletion early in development as well as methods of intercepting toxicity.  The discovery that HPG-DFO are non-toxic in zebrafish embryos lends supports the conclusion that HPG-DFO are biocompatible and confirms that the HPG component is not associated with toxicity.  The important discovery that DFO chelation properties remain unaffected while toxicity and half-life are greatly improved suggests that the use of nanomedicines in iron chelation may produce a paradigm shift in chelation therapy. The retention of high chelation ability and extended half-life observed with HPG-DFO could allow once-weekly dosing of chelators and more efficient iron removal than is possible with small MW chelators or any previously derived DFO-polymer conjugate. This would likely reduce drug costs and undoubtedly cause improved compliance among patients. Table 9 below compares the 133  properties of HPG-DFO to DFO and more clearly depicts the potential value and clinical utility that such a chelator is anticipated to have.     134  Table 9: The anticipated reduction in drug needed with increased half-life of DFO through polymer conjugation.       Chelator Property Ideal Chelator DFO HPG-DFO Cost Affordable for patients in low income countries Moderate  Likely to be lower than DFO based on reduction of dose Route of Administration Oral i.v. injection or s.c. infusion i.v. injection or s.c. infusion Circulation t1/2 Long enough to allow once-daily dosing and effective iron removal Short (~20 min) requires all-day (8-12 h) delivery  Long (up to 44 h in mice), enough to allow once-weekly dosing Therapeutic index High High at high doses in patients with high burden High in all patients  Toxicity  None Neurotoxic, swelling at infusion sites, bone deformities Should be alleviated due to reduced dosing Unsaturated Iron Binding Capacity  (UIBC) High: Long enough to prevent drastic fluctuations in LIP None Similar or higher than that reported for 40SDO2 Ability to remove iron from heart, liver etc High Low High although indirect due to sustained chelator concentrations over time 135  5.3: Implications of Increasing Chelator Half-life In many aspects, polymeric chelators are shown to be significantly more effective than low MW chelators at in vivo iron removal due to extended plasma circulation times.  Although none of the previously designed polymeric chelators have advanced to the clinic, the successful phase Ib clinical trials conducted by Harmatz et al suggest the potential that exists for high MW of chelators generated by DFO modification. This study demonstrated that chelators which circulate for extended periods provide comprehensive coverage against the development of labile plasma iron pools several days after receiving the initial dose (71). This highlighted the advantage of polymeric chelators in depleting iron and providing coverage against rebound iron overload.  One of the most important properties of an iron chelator is its half-life which influences the unsaturated iron binding capacity of the chelator in the plasma and ultimately, the rate of NTBI generation and removal (166). As iron is constantly being turned over due to RBC catabolism in macrophages or the breakdown of ferritin within cells, these pools of iron are redox active and are mainly responsible for the iron loading of plasma and tissues. Thus, in order to achieve effective iron chelation and the removal of labile iron, 24 h chelation coverage is the ideal (Figure 39). The importance of unsaturated iron binding capacity in the circulation has also been demonstrated in studies that show that before major changes in cardiac iron occur, cardiac failure is reversed via continuous administration of  DFO and that NTBI appears within minutes of a chelator being cleared from the body (168). These observations demonstrate the need for continuous chelator exposure in patients susceptible to iron overload which is adequately provided by higher MW chelators.   136  Increased half-life of DFO through the conjugation to HPG is anticipated to have important positive effects on the unsaturated iron binding capacity and the compliance to therapy for patients treated with DFO. Due to previous studies conducted with 40SDO2, and the results obtained in mice, it is anticipated that HPG-DFO will have an unsaturated iron binding capacity that can reduce the need for daily subcutaneous infusion. Although further long-term studies that have increased duration are needed to confirm this, it is likely that the reduction in plasma iron will also reduce cellular iron uptake. This means that prolonged half-life will also result in reductions in cellular iron levels due to a reduced LIP in the plasma.  In addition to significant iron removal from the plasma, DFO is known to enter liver cells and promote iron excretion via the bile. Previous animal studies of HPG-DFO in mice have also indicated iron excretion from vital organs (105). Therefore, in addition to removing considerable amounts of iron from the plasma HPG-DFO are also expected to promote some degree of iron excretion from liver cells.  Our findings indicate that polymers and in particular HPG, are useful for obtaining significant improvements to toxicity, in vivo half-life and iron mobilization with DFO. These studies have furthered our understanding of the potential benefits of conjugating polymers of various sizes to DFO. It builds upon existing knowledge of DFO modification and has demonstrated that the properties of such conjugates can be tunable, allowing the development of optimized chelators. Furthermore, given that the current arduous regimen of s.c. DFO has proven challenging for many patients, HPG-DFO holds promise as an optimized version of DFO for advancing therapy.  137   Figure 39: Polymeric iron chelators result in a higher unsaturated iron binding capacity.  High MW chelators have longer half-lives and are not readily taken up by cells. In contrast, small MW chelators are highly permeable to cells and may be rapidly taken up, metabolized and removed from the circulation. 138  5.4: Future Directions One major aspect of this work was investigating embryo toxicity of low and high MW chelators using the zebrafish model (Chapter 4). Our studies indicate that the MW and surface groups of HPG-DFO chelator conjugates may be factors that mediate embryo toxicity upon chelator exposure. Due to the pores present on the zebrafish chorion, and the previously reported differences in permeability of the chorion and embryo, future studies that investigate the role of each barrier are necessary. These speculations can be addressed in several ways. To determine whether size is a single mediating factor, radiolabelled polymeric chelator conjugates with a greater variation in MW (5kDa to 500kDa) than previously tested can be designed and tested using similar protocols as those described in Chapter 4. Due to the μM size of the chorionic pores, it is most likely that smaller particles will be taken into the chorion at a faster rate and radiolabelling will allow an efficient way of tracking the location and accumulation of chelators over time.  Previous studies have shown that the chorion can act as a permeability barrier in developing zebrafish embryos. Therefore, to determine whether exclusion from the chorion mediates toxicity of higher MW chelator conjugates, the studies reported in Chapter 4 can be repeated using zebrafish that have had their chorions removed mechanically. This will allow higher MW chelators to directly interact with the embryo without the hindrance of the chorion. This can also be done for lower MW chelators to determine the impact of the chorion on the embryo exposure and toxicity induced by chemicals. In addition to chorion removal, microinjection of chelators into the chorion may also provide a means of measuring the influence of the chorion on embryo exposure and chelator toxicity. This will ensure that embryos have direct exposure to the respective chelators. However, since microinjection does 139  not allow effective testing of chronic exposure, such a study would likely have to be coupled with studies that expose embryos to chelators via direct soaking, in order to enable effective interpretation of the data.  The role of polymers in biomedicine has grown over the past three decades. Polymers are used in drug delivery, in tissue engineering as components of artificial organs, in medical devices and cosmetics (169-170). Through appropriate design and modification of biocompatible polymers, several high MW chelators have been developed and characterized. These conjugates take advantage of the biophysical properties of the polymers that can extend plasma circulation and reduce dose dependent toxicity, while retaining excellent chelating properties.  Our experience with modifying the properties of low MW iron chelators like DFO through nanoengineering with polymers has grown only moderately from the first attempt in 1989 (68). It is well established that the undesirable properties of DFO can be significantly transformed by modification with starch and dextran, with the improvements in chelating properties varying with the properties of the biomaterials used (68-75). The possibility of engineering other small MW iron chelators like L1 and ICL-670 to improve their toxicity profile remains uncertain but such exploration may advance chelation therapy also.   In addition to high chelation efficacy and reduced toxicity of the chelator component, it is essential that polymer components are rigorously tested for safety, can be reproduced consistently, easily and on a large scale. Additionally, polymers should be suitable for transformation into effective pharmaceutical formulations that are practical to use clinically in areas with high numbers of transfusion dependent patients. Therefore, future studies must answer several important questions. First, it is important to determine whether polymers such 140  as HPG can be further modified to generate more targeted high MW. This is important because inadequate chelation leads to organ toxicity and damage in many patients. Moreover, iron induced cardiac and liver failure causes a majority of deaths among iron overload patients with such damage occurring, in some instances, even if patients are intensively chelated (159). As a result, the development of iron chelators which are targeted to select organs susceptible to iron overload would greatly optimize iron removal from patients.  The HPG-DFO conjugates developed in this thesis have demonstrated biocompatibility, low toxicity and the structural flexibility to allow further optimization via the numerous OH groups present. These OH groups can be modified to allow the development of high MW, liver targeting HPG-DFO chelators. This may be achieved through conjugation of the targeting ligand on the HPG-DFO. Hepatocytes express an endocytic receptor (ASGPR) which has been shown to effectively enhance drug delivery to the liver (171-172). Thus, future experiments focused on modifying the surface of HPG-DFO to mimic the endogenous ligand of the ASGPR by adding galactose or N-acetylgalactosamine moieties may allow liver targeting. These novel HPG-DFO-Gal conjugates would be expected to interact with the hepatic lectin which would internalize the chelators and enhance iron excretion from the LIP, as depicted by Figure 40. In addition to producing more targeted chelators, it is also important to determine whether the high MW chelators have specific degradation routes or are prone to accumulation with chronic use. Since the MW of drug molecules can play a critical role on their cellular and tissue accumulation, determining the degradation/accumulation profile of HPG-DFO, and their likelihood of accumulating with chronic use will be of great importance for future studies.  Research has shown that nanoparticles may be trafficked to the lysosome for degradation (173). In the case of chelators, the lysosome may also provide a target for iron 141  chelation as the lysosome was shown to accumulate iron due to the degradation of ferritin. Therefore, it is important to conduct studies which track the cellular uptake and degradation of HPG-DFO as this will enable better understanding of its mechanism of iron chelation and the iron pools that are accessible to polymeric chelators.  Cellular iron levels can be monitored in response to iron chelation therapy using molecular markers such as iron regulatory proteins (IRPs). IRPs regulate iron uptake and storage by binding to iron responsive elements on the mRNA of proteins involved in iron uptake and storage; for example transferrin receptor 1 (TfR1) and ferritin (174). Therefore, the effect of high MW chelators on the molecular changes of cells like macrophage and hepatocytes may help to further elucidate the mechanisms and molecular changes that occur when exposed to novel polymeric chelators. Additionally, effects of HPG-DFO on preventing the uptake of or directly chelating radiolabelled iron from transferrin would also allow direct assessment of the ability of these chelators to influence transferrin iron and would also indicate which iron pool is targeted by HPG-DFO to promote the enhanced iron excretion observed in mice observed in our study (105). However, previous reports have shown that DFO does not chelate significant amounts of transferrin iron and that the physiological release of iron from transferrin requires acidification. Thus, while chelators may influence transferrin iron uptake, they are unlikely to directly chelate transferrin iron (100).   Our previous work has demonstrated that it is possible to create biodegradable HPG polymer conjugates (175). Using ketal linkages, previous studies in our lab have developed a class of HPG-based polyether polyketals containing different ketal linkages in their backbone that degraded over time, predictably in solution within CHO cells under physiologically relevant conditions. Similar approaches may be used to generate high MW, biodegradable iron 142  chelators. Figure 40 shows the proposed features of an optimized, liver-specific, biodegradable HPG-DFO-Gal.    Figure 40: The proposed intracellular degradation of liver-specific HPG-DFO.  Galactose containing HPG-DFO (HPG-DFO-Gal) will be recognized by the hepatic lectin (ASGPr) and subsequently taken up into liver cells via receptor mediated endocytosis. Intracellular Fe pools of ferritin, hemosiderin and Fe-citrate will be chelated by Gal-HPG-DFO prior to its entrance into the lysosome. The reduced pH of the lysozome will cause the S-S bonds to be broken, thus fragmenting the HPG-DFO-Gal conjugate into smaller fragments. It is anticipated that upon degradation, the low MW fragments will eliminate through kidney rapidly.  5.5: Polymeric Iron Chelation: Beyond Transfusional Iron Overload Iron is an essential element for several metabolic processes and the disruption of iron metabolism has been shown to be a major factor in many diseases. Iron removal has been shown to be a useful approach for the treatment of microbial infectious diseases, reducing the growth rate of some cancer cells and neurodegenerative diseases (176-179). Additionally, iron 143  chelation may be useful in malaria treatment and treatment of the HIV virus (180-181). Therefore, the ability to modify polymers to enhance iron chelation, minimize toxicity and maximize blood circulation may prove beneficial in depleting iron stores in these disease states also.  Polymeric iron chelators may also be modified to enhance targeting to specific areas of the body requiring iron depletion.  For example, polymeric chelators may be uniquely suited for iron depletion at the site of tumors owing to their large size that can be used to passively target tumors through their compromised endothelial junctions via the enhanced permeation and retention (EPR) effect (182). Likewise, the increased residence time associated with polymeric iron chelators may have utility in treating chemotherapy patients (105, 177). Patients receiving chemotherapy for the treatment of cancer may experience an elevation of NTBI, which may occur as toxicity is exerted on bone marrow cells. This can reduce the demand of marrow cells for iron and cause transferrin to become saturated, increasing NTBI and rendering the host more susceptible to oxidative damage. While low MW chelators are prone to enter cells, high MW drugs are almost exclusively restricted to the vascular and extracellular spaces due to the poor cellular uptake. Thus, polymeric chelators are advantageous for a wide range of diseases beyond iron overload diseases.    144  References  1.  Crichton RR, Boelaert JR (2001). Inorganic biochemistry of iron metabolism: from         molecular mechanisms to clinical consequences. John Wiley & Sons.  2. Wang J and Pantopoulos K. Regulation of cellular iron metabolism, Biochem J 2011; 434:365-381. 3. Aisen P, Enns C, Wessling-Resnick M. Chemistry and biology of eukaryotic iron metabolism. Int. J. Biochem Cell B 2001; 33: 940 – 959. 4. Aisen P, Leibman A, and Zweier J. Stoichiometric and site characteristics of the binding of iron to human transferrin. J. Biol. Chem 1978; 253: 1930-1937. 5. Frazer DM, Anderson GJ. The regulation of iron transport. Biofactors 2014; 40 (2): 206-214. 6. Chasteen ND, Harrison PM. Mineralization in ferritin: An efficient means of iron storage. J Struct Biol 1999; 126 (3):182-194. 7. Winterbourn CC. Toxicity of iron and hydrogen peroxide: The fenton reaction. Toxicol Lett 1995; 82(0):969-974. 8. Kehrer JP. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 2000; 149(1):43-50. 9. Olivieri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. Blood 1997; 89 (3):739-761. 145  10. Hoffbrand AV, Taher A, Capellini MD. How I treat transfusional iron overload. Blood 2012; 120: 3657-3669. 11. Adams et al. The Optimizing Primary Stroke Prevention in Sickle Cell Anemia (STOP 2) Trial Investigators. N Engl J Med 2005; 353: 2769-2778. 12. Hershko C, Link G, Konijn AM, and Cabantchik ZI. Objectives and Mechanisms of Iron Chelation Therapy. Ann. N.Y.Acad. Sci 2005; 1054 :124-135.  13. Hershko C, Graham G, Bates GW, Rachmilewitz EA. Non-Specific Serum Iron in Thalassaemia: an Abnormal Serum Iron Fraction of Potential Toxicity. Brit J Haematol 1978; 40: 255-263.  14. Porter JB, Garbowski M. The Pathophysiology of Transfusional Iron Overload.  Hematol Oncol Clin North Am 2014; 28(4): 683-701. 15. Modell B, Khan M, Darlison M. Survival in beta-thalassaemia major in the UK: data from the UK Thalassaemia Register. The Lancet 2000; 355: 2051–2052. 16. Brittenham, GM. Iron-Chelating Therapy for Transfusional Iron Overload. N Engl J Med 2011; 364:146-56.  17. Kontoghiorghes GJ, Pattichi K, Hadjigavriel M., Kolnagou A. Transfusional iron overload and chelation therapy with deferoxamine and deferiprone (L1). Transfus Sci 2000; 23(3): 211-223.  18. Morehouse LA, Thomas CE, Aust SD. Superoxide generation of NADPH-Cytochrome P-450 reductase: The effect of iron chelators and the role of superoxide in microsomal lipid peroxidation. Arch Biochem Biophys 1984; 232(1):366-377.  146  19. Link G, Athias P, Grynberg A, Pinson A, Hershko C. Effect of iron loading on transmembrane potential, contraction and automaticity of rat ventricular muscle cells in culture. J Lab Clin Med 1989; 113:103-111.  20. Borgna-Pignatti C, Rugolotto S, De Stefano P, Zhao H, Cappellini MD, Del Vecchio GC, Romeo MA, Forni GL, Gamberini GR et al. Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine. Haematologica 2004; 89(10): 1187-1193. 21. Zhou T, Ma Y, Kong X, Hider RC. Design of Iron Chelators with Therapeutic Applications. Dalton Trans 2012; 41: 6371–6389.   22. Lee P, Mohammed N, Marshall L, Abeysinghe A D.,  Hider RC, Porter JB, Singh S. Intravenous Infusion Pharmacokinetics of Desferrioxamine in Thalassemic Patients. Drug Metab Dispos. 1993; 21(4): 640-644. 23. Porter JB, Faherty A, Stallibrass L, Brookman L, Hassan I and Howes C.  A Trial to Investigate the Relationship between DFO Pharmacokinetics and Metabolism and DFO-Related Toxicity. Ann. N.Y.Acad. Sci 1998; 850(1): 483-487.  24. Kaplinsky C, Stark B, Goshen Y, Yaniv I, Bashara S and Zaizov, R. Deferoxamine (Desferal)-induced ocular toxicity. Pediatr Hematol Oncol 1988; 5(4): 293-297. 25. Levine JE, Cohen A, MacQueen M, Martin M, Giardina PJ. Sensorimotor neurotoxicity associated with high-dose deferoxamine treatment. J Pediatr Hematol Oncol 1997; 19: 139-41. 147  26. Kontoghiorghes GJ, Aldouri MA, Hoffbrand AV, Barr J, Wonke B, Kourouclaris T, Sheppard L. Effective chelation of iron in beta thalassaemia with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one. Br Med J (Clin Res Ed) 1987; 295(6612):1509. 27. Motekitis RJ, Martell AE. Stabilization of the iron (III) chelates of 1,2-dimethyl-3-hydroxypyrid-4-ones and related ligands. Inorg Chim Acta 1991; 183: 71-80. 28. Rombos Y1, Tzanetea R, Konstantopoulos K, Simitzis S, Zervas C, Kyriaki P, Kavouklis M, Aessopos A, Sakellaropoulos N, Karagiorga M, Kalotychou V, Loukopoulos D. Chelation therapy in patients with thalassemia using the orally active iron chelator deferiprone (ICL-670). Haematologica 2000; 85(2):115-117. 29. Hoffbrand AV, Cohen A, Hershko C. Role of deferiprone in chelation therapy for transfusional iron overload. Blood 2003; 102(1):17-24. 30. Cohen AR, Galanello R, Piga A, De Sanctis V and Tricta F. Safety and effectiveness of long-term therapy with the oral iron chelator deferiprone. Blood 2003; 102(5):1583-1587. 31. Kontoghiorghes GJ, Bartlett AN, Hoffbrand AV, Goddard JG, Sheppard L, Barr J, Nortey P. Long-term trial with the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1). I. Iron chelation and metabolic studies. Br J Haematol. 1990; 76(2):295-300. 32. Hoffbrand AV, AL-Refaie F, Davis B, et al. Long-term trial of deferiprone in 51 transfusion-dependent iron overloaded patients. Blood 1998; 91(1):295-300. 148  33. Nisbet-Brown E, Olivieri NF, Giardina PJ, Grady RW, Neufeld EJ, Séchaud R, Krebs-Brown AJ, Anderson JR, Alberti D, Sizer KC, Nathan DG. Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised, double-blind, placebo-controlled, dose-escalation trial. The Lancet 2003; 61: 1597–602. 34. Capellini MD. Iron-chelating therapy with the new oral agent ICL670 (Exjade®). Best Pract Res Cl Ha 2005; 18(2): 289–298. 35. Galanello R, Piga A, Alberti D, Rouan M, Bigler et al. Safety, Tolerability, and Pharmacokinetics of ICL-670, a New Orally Active Iron-Chelating Agent in Patients with Transfusion-Dependent Iron Overload Due to B Thalassemia. J Clin Pharmacol 2003; 43: 565-572.  36. Bruin GJM, Faller T, Wiegand H, Schweitzer A, Nick H et al. Pharmacokinetics, Distribution, Metabolism and Excretion of Desferasirox and Its Iron Complex in Rats. Drug Metab Dispos 2008; 36:2523-2538.   37. Gaboriau F, Leray A, Ropert M, Gouffier L, Cannie I et al. Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG, Biometals 2010; 23:231-245.  38. Gattermann N, Finelli C, Della Porta M, Fenaux P, Ganser A et al. Deferasirox in iron-loaded patients with transfusion-dependent myselodysplastic syndromes: Results from the large 1-year EPIC study. Leukemia Research 2010; 34: 1143-1150.  149  39. Wei HY, Yang CP, Cheng CH, Lo FS. Fanconi syndrome in a patient with B-thalassemia major after using desferasirox for 27 months. Transfusion 2011; 51: 949-954.  40. Sánchez-González PD, López-Hernandez FJ, Morales AI, Macías-Nu˜nez JF and López-Novoa JM. Effects of deferasirox on renal function and renal epithelial cell death. Toxicol. Lett 2011; 203: 154-161.  41. Galanello R, Campus S, and Origa R. Desferasirox: pharmacokinetics and clinical experience.  Expert Opin. Drug Metab. Toxicol 2012; 8: 123-134. 42. Yoshikawa T, Hara T, Araki H., Tsurumi H, Oyama M., Moriwaki H.  First report of drug-induced esophagitis by deferasirox.   Int. J. Hematol 2012; 95 (6): 689-691. 43. Kontoghiorghes GJ. A record number of fatalities in many categories of patients treated with deferasirox: loopholes in regulatory and marketing procedures undermine patient safety and misguide public funds? Expert Opin. Drug Saf 2013; 12(5):605-609. 44. Riva A, Comment on: A record number of fatalities in many categories of patients treated with deferasirox: loopholes in regulatory and marketing procedures undermine patient safety and misguide public funds? Expert Opin. Drug Saf 2013; 12(5):793-795. 45. Maxton DG, Bjarnason I, Reynolds AP, Catt SD, Peters TJ, Menzies IS. Lactulose, 51Cr-labelled ethylenediaminetetra-acetate, L-rhamnose and polyethyleneglycol 400 150  [corrected] as probe markers for assessment in vivo of human intestinal permeability. Clinical Science 1986; 71(1): 71-80. 46.  Bergeron RJ, Weigand J, Brittenham GM. A Potential Alternative to Deferoxamine for Iron-Chelation Therapy. Blood 1998; 91: 1446-1452. 47. Samuni AM, Afeworki M, Stein W, Yordanov AT, DeGraff W, Krishna MC, Mitchell JB, Brechbiel MW. Multifunctional Antioxidant Activity of HBED Iron Chelator. Free Radical Biol Med 2001; 30(2): 170-177. 48. Bergeron RJ, Wiegand J, Brittenham GM. HBED: The continuing development of a potential alternative to deferoxamine for iron-chelating therapy. Blood 1999; 93(1): 370-375. 49. Richardson DR, Ponka P. Pyridoxal isonicotinoyl hydrazone and its analogs: potential orally effective iron-chelating agents for the treatment of iron overload disease. J Lab Clin Med  1998; 131(4): 306-315. 50. Brittenham GM. Pyridoxal isonicotinoyl hydrazone: an effective iron-chelator after oral administration. Sem hematol 1990; 27(2):112. 51. Bláha K, Cikrt M, Nerudová J, Fornusková H, Ponka P. Biliary Iron Excretion in Rats Following Treatment With Analogs of Pyridoxal Isonicotinoyl Hydrazone. Blood 1989; 91(11): 4368-4372. 52. Hider RC. Recent developments centered on orally active iron chelators. Thalassemia reports 2014; 4:2.  151  53. Baker E, Vitolo ML, Webb J. Iron chelation by pyridoxal isonicotinoyl hydrazone and analogues in hepatocytes in culture. Biochem pharmacol 1985; 34(17): 3011-3017. 54. Buss JL, Hermes-Lima M, Ponka P. Pyridoxal isonicotinoyl hydrazone and its analogues. Iron Chelation Therapy. Springer US 2002; 205-229.  55. Rienhoff Jr HY, Viprakasit V, Tay L, Harmatz P, Vichinsky E, Chirnomas D, Neufeld EJ. A phase 1 dose-escalation study: safety, tolerability, and pharmacokinetics of FBS0701, a novel oral iron chelator for the treatment of transfusional iron overload. Haematologica 2010: Haematol-2010.  56.  Neufeld EJ, Galanello R, Viprakasit V, Aydinok Y, Piga A, Harmatz P, Rienhoff HY. A phase 2 study of the safety, tolerability, and pharmacodynamics of FBS0701, a novel oral iron chelator, in transfusional iron overload. Blood 2012; 119(14): 3263-3268. 57. Ferrer P, Tripathi AK, Clark MA, Hand CC, Rienhoff Jr HY, Sullivan Jr DJ. Antimalarial iron chelator, FBS0701, shows asexual and gametocyte Plasmodium falciparum activity and single oral dose cure in a murine malaria model. PloS One 7.5 (2012): e37171. 58. Taher AT, Musallam KM. A new chelator in the house. Blood 2012; 119(14): 3191-3192. 152  59. Hider R, Kong X, Luker T, Conlon K. SPD602 Is a Selective Iron Chelator Which Is Able To Mobilise The Non-Transferrin-Bound Iron Pool. Blood 2013; 122(21): 1673-1673. 60. Chansiw N, Pangjit K, Phisalaphong C, Fucharoen S, Evans P, Porter J B. Toxicity study of a novel oral iron chelator: 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in transgenic b-thalassemia mice. Vitam Miner 2013; 2: 1-6. 61.  Srichairatanakool S, Pangjit K, Phisalaphong C, Fucharoen S. Evaluation of a novel oral iron chelator 1-(N-acetyl-6-aminohexyl)-3-hydroxypyridin-4-one (CM1) for treatment of iron overload in mice. Adv Biosci Biotechnol 2013; 4: 153. 62.  Pangjit K, Banjerdpongchai R, Phisalaphong C, Fucharoen S, Srichairatanakool  S. Efficacy of 1-(N-acetyl-6-aminohexyl)-3-hydroxypyridin-4-one (CM1) in treatment of iron-loaded hepatocyte cultures. Adv Biosci Biotechnol 2012; 3: 1060. 63.  Kulprachakarn K, Chansiw N, Pangjit K, Phisalaphong C, Fucharoen S, Hider RC, Srichairatanakool S. Iron-chelating and anti-lipid peroxidation properties of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in long-term iron loading β-thalassemic mice. Asian Pac J Trop Biomed 2014; 4.8: 663. 64. Padhye S, Kauffman G B  Transition metal complexes of semicarbazones and thiosemicarbazones. Coord. Chem. Rev. 1985, 63, 127-160. 153  65. Kalinowski D S, Richardson DR. Future of toxicology iron chelators and differing modes of action and toxicity: the changing face of iron chelation therapy. Chem. Res. Toxicol. 2007; 20: 715-720. 66. Yuan J,  Lovejoy DB, Richardson DR. Novel di-2-pyridyl–derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment. Blood 2004; 104(5):1450-1458.  67. Beraldo H, Gambinob D. The wide pharmacological versatility of semicarbazones, thiosemicarbazones and their metal complexes. Mini Rev Med Chem 2004;  4(1): 31-39.  68. Hallaway PE, Eaton JW, Panter SS, Hedlund BE. Modulation of deferoxamine toxicity and clearance by covalent attachment to biocompatible polymers. Proc Natl Acad Sci USA 1989; 86: 10108–10112. 69. Polomoscanik SC, Cannon CP, Neenan TX, Holmes-Farley SR, Mandeville W H, Dhal PK. Hydroxamic acid-containing hydrogels for nonabsorbed iron chelation therapy: synthesis, characterization, and biological evaluation. Biomacromolecules 2005; 6: 2946–2953. 70. Rossi NAA, Mustafa I, Jackson JK, Burt HM, Horte SA, Scott MD, Kizhakkedathu JN. In vitro chelating, cytotoxicity, and blood compatibility of degradable poly 154  (Ethylene glycol)-based macromolecular iron chelators. Biomaterials 2009; 30: 638-648.  71. Harmatz P, Grady RW, Dragsten P, Vichinsky E, Giardina P Madden J, Jeng M, Miller B, Hanson G and Hedlund B.  Phase Ib clinical trial of starch-conjugated deferoxamine (40SD02): a novel long-acting iron chelator. Br J Haematol 2007; 138(3):374-81.  72. Winston A, Varaprasad, DV, Metterville JJ, Rosenkrantz H. Evaluation of polymeric hydroxamic acid iron chelators for treatment of iron overload. J Pharmacol Exp Ther 1985; 232: 644–649.   73. Zhou T, Kong XL, Liu ZD, Liu DY, Hider RC. Synthesis and iron (III)-chelating properties of novel 3-hydroxypyridin-4-one hexadentate ligand-containing copolymers. Biomacromolecules 2008; 9: 1372–1380.  74. Zhou T, Neubert H, Liu DY, Liu ZD, Ma YM, Kong XL, Hider RC. Iron binding dendrimers: a novel approach for the treatment of haemochromatosis. J Med Chem 2006; 49: 4171–4182.  75. Young SP, Baker E and Huehns ER. Liposome entrapped desferrioxamine and iron transporting ionophores: a new approach to iron chelation therapy. Brit J Haematol 1979; 41, 357-363.  76. Green-top Guideline No. 66: Management of Beta Thalassaemia in Pregnancy. The Obstetrician & Gynaecologist 2014; 16: 148. doi: 10.1111/tog.12100. 155  77. Aessopos A, Karabatsos F, Farmakis D, Katsantoni A, Hatziliami A, Youssef J, Karagiorga M. Pregnancy in patients with well-treated β-thalassemia: outcome for mothers and newborn infants. Am J Obstet Gynecol  1999; 180(2): 360-365. 78.  Bosque MA, Domingo JL and Corbella J. Assessment of the developmental toxicity of deferoxamine in mice. Arch Toxicol 1995; 69: 467-471. 79. Singer ST, Vichinsky EP. DFO Treatment during Pregnancy: Is it Harmful? Am J Hematol 1999; 60:24–26. 80. Berdoukas V, Bentley P, Frost H, Schnebli HP. Toxicity of Oral Iron Chelator ICL-670. The Lancet 1993; 341, 1088. 81. Domingo JL. Developmental toxicity of metal chelating agents. Reprod Toxicol 1998; 12: 499-510.  82. Jensen CE, Tuck SM, Wonke B. Fertility in beta thalassaemia major: a report of 16 pregnancies, preconceptual evaluation and a review of the literature. Br J Obstet Gynaecol 1995; 102:625-9.  83. Ho PJ, Tay L, Lindeman R, Catley L, Bowden DK. Australian guidelines for the assessment of iron overload and iron chelation in transfusion-dependent thalassaemia major, sickle cell disease and other congenital anaemias. Internal Medicine Journal 2011; 41: 516-524. 84. Vini D, Servos P, Drosou M. Normal pregnancy in a patient with β-thalassaemia major receiving iron chelation therapy with deferasirox (Exjade). Eur J Haematol  2011; 86(3): 274–275. 156  85. Farmaki K, Tzoumari I, Berdoukas V. Rapid iron loading in a pregnant woman with transfusion-dependent thalassemia after brief cessation of iron chelation therapy. Eur J Haematol 2008; 81: 157–9. 86. Kainthan RK, Hester SR, Levin E, Devine DV, Brooks DE. In Vitro Biological Evaluation of High Molecular Weight Hyperbranched Polyglycerols. Biomaterials 2007; 28: 4581–4590.  87. Kainthan RK, Muliawan EB, Hatzikiriakos SG, Brooks DE. Synthesis, Characterization and Viscoelastic Properties of High Molecular Weight Hyperbranched Polyglycerols. Macromolecules 2006; 39: 7708–7717.  88. Calderon M, Quadir MA, Sharma SK, Haag R.  Dendritic polyglycerols for biomedical applications.  Adv. Mater 2010; 22 (2): 190-218.    89. Brooks DE, Kizhakkedathu JN, Kainthan RK.  Polymer-based serum albumin substitute. PCT Int Appl, WO 2006130978, A1 20061214; 2006.   90. Chapanian, R.; Constantinescu, I.; Brooks, D. E.; Scott, M. D.; Kizhakkedathu, J. N.  In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.  Biomaterials 2012; 33 (10): 3047-3057.   91. Rossi NAA, Constantinescu I, Kainthan RK, Brooks DE, Scott MD, Kizhakkedathu JN.  Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols.  Biomaterials 2010; 31(14): 4167-4178.  92. Chapanian R, Constantinescu I, Rossi NAA, Medvedev N, Brooks DE, Scott MD, Kizhakkedathu JN.  Influence of polymer architecture on antigens camouflage, CD47 157  protection and complement mediated lysis of surface grafted red blood cells. Biomaterials 2012; 33 (31): 7871-7883.  93. Shenoi RA, Kalathottukaren MT, Travers RJ, Lai BFL, Creagh AL, Lange D, Yu K, Weinhart M, Chew B, Du C, Brooks DE, Carter CJ, Morrissey, JH, Haynes CA, and Kizhakkedathu JN. Affinity-based Design of Synthetic Universal Reversal Agent for Heparin Coagulants. Sci. Transl. Med. 6.260 (2014): 260ra150-260ra150. 94. Hill AJ, Teraoka H, Heidman W, Peterson RE. Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol. Sci 2005; 86: 6-19.  95. Zon LI, Peterson RT. In vivo drug discovery in the zebrafish. Nat Rev Drug Discov 2005; 4: 35-44.  96. Zhu JJ, Xu YQ, He JH, Yu HP, Huan CJ et al. Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish. J Appl Toxicol 2014; 34: 139-148.  97. Tan JL and Zon LI. Chemical screening in zebrafish for novel biological and therapeutic discovery. Methods Cell Biol 2010; 105: 493-516.  98. Haldi M, Harden M, D’Amico L, DeLise A, Seng W L. Developmental toxicity in zebrafish. In Zebrafish: Methods for assessing drug safety and toxicology 2012. (McGrath, P. Ed.)  John Wiley & Sons, pp.15-25.   99. Peterson RT and Macrae CA. Systematic approaches to toxicology in the zebrafish. Annu. Rev. Pharmacol. Toxicol. 2012; 52:433–453.  158  100. Keberle H. The biochemistry of desferrioxamine and its relation to iron metabolism. Ann NY Acad Sci 1964; 119:758-768.  101. Robotham JL, Lietman PS. Acute Iron Poisoning. Am J Dis Child 1980; 134 (9):875-879.  102. Aydinok Y, Nisli G, Kavakli K, Coker C, Kantar M, Cetingül N. Sequential use of deferiprone and desferrioxamine in primary school children with thalassaemia major in turkey. Acta Haematol 1999; 102(1):17-21.  103. Gehlbach PL, Purple RL, Hallaway PE, Hedlund BE. Polymer conjugation reduces desferrioxamine induced retinopathy in an albino rat model. Invest Opthamol Vis Sci 1993; 34: 2871-2877.  104. Balla G. Ferritin: A cytoprotective antioxidant stratagem of endothelium. J. Biol. Chem. 1992; 267(25): 18148-18153.  105. Imran ul-haq M, Hamilton JL, Lai BF, Shenoi RA, Horte S, Constantinescu I, Leitch HA, Kizhakkedathu JN. Design of long circulating nontoxic dendritic polymers for the removal of iron in vivo. ACS Nano 2013; 7(12):10704-10716.  106. Hoy TG, Harrison PM and Shabbir M. Uptake and Release of Ferritin Iron Surface effects and Exchange within the Crystalline Core. Biochem. J 1974; 139: 603-607. 107. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. J Biol Chem 1951; 193: 265.  108. Macara IG, Hoy TG, Harrison PM. The formation of ferritin from apoferritin. Kinetics and Mechanism of iron uptake. Biochem J 1972; 126: 151-162.  159  109. Ghosh MM, O'Connor JT, Engelbrecht RS. Bathophenanthroline method for the determination of ferrous iron. Journal (American Water Works Association) 1967; 897-905.  110. Drabkin DL, Austin JH. Spectrophotometric Studies: II. Preparations from Washed Blood Cells; Nitric Oxide Hemoglobin and Sulfhemoglobin. J. Biol. Chem 1935; 112: 51-65.  111. Winterbourn, C. C. Free-Radical Production and Oxidative Reactions of Hemoglobin. Environ. Health, Perspect. 1985; 64: 321–330.  112. Scott MD and Eaton JW. Thalassemic erythrocytes: cellular suicide arising from iron and glutathione-dependent oxidation reactions? Br J Hematol 1995; 91(4): 811-819. 113. Gouin S, Winnik FM. Quantitative Assays of the Amount of Diethylenetriaminepentaacetic Acid Conjugated to Water-Soluble Polymers Using Isothermal Titration Calorimetry and Colorimetry. Bioconjugate Chemistry 2001; 12 (3): 372-377.  114. Majonis D, Herrera I, Ornatsky O, Schulze M, Lou X, Soleimani M, Nitz M, Winnik MA Synthesis of a Functional Metal-Chelating Polymer and Steps toward Quantitative Mass Cytometry Bioassays. Anal. Chem. 2010; 82 (21): 8961-8969.  115. Ladbury JE and Chowdhry BZ. Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chemistry & Biology 1996; 3(10):791–801. 160  116. Dobrawa R, Lysetska M, Ballester P, Grüne M, Würthner F. Fluorescent Supramolecular Polymers:  Metal Directed Self-Assembly of Perylene Bisimide Building Blocks. Macromolecules 2005; 38 (4):1315-1325 117. Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini A.; Vacca, A. Coord. Chem. Rev.  1999; 184: 311-318. 118. Bou-Abdullah F, McNally J, Liu XX and Melman A. Oxygen catalyzed obilization of iron from ferritin by iron (III) chelate ligands. Chem Commun 2011; 47: 731-733. 119. Link G, Konijn AM, Breuer W, Cabantchik ZI, Hershko C. Exploring the “iron shuttle” hypothesis in chelation therapy: Effects of combined desferioxamine and deferiprone treatment in hypertransfused rats with labeled iron stores and iron-loaded rat heart cells in culture. J Lab Clin Med 2001; 38: 130-138.  120. Wonke B, Wright C and Hoffband AV. Combined therapy with deferiprone and desferrioxamine. Br J Haematol 1998; 103:361-364. 121. Cario H, Janka-Schaub G, Janssen G, Jarisch A, Strauss G, Kohne E. Recent developments in iron chelation therapy. Klin P ä diatr 2007; 19(1):258-165.  122. Brady MC, Lilley KS, Treffry A, Harrison PM, Hider RC, Taylor PD. Release of Ferritin Molecules and their iron-cores by 3-hydroxypyridinone chelators in vitro. J. Inorg. Biochem 1989; 35: 9-22.  123. Tufano TP, Pecoraro VL Raymond KN. Ferric Ion Sequestering Agents. Biochimica et Biophysica Acta 1981; 668:420-428.  161  124. R. C. Hider, A. D. Hall, in Perspectives on Bioinorganic Chemistry, ed. R. W. Hay, J. R. Dilworth and K. B. Nolan, 1991, pp. 209–254.  125. Haag R, Kratz F. Polymer Therapeutics: Concepts and Applications Angew. Chem. Int. Ed. 2006; 45:1198 – 1215.  126. Knop K, Hoogenboom R, Fischer D, and Schubert US. Poly (ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives. Angew. Chem. Int. Ed., 2001; 49: 6288 – 6308.  127. Kamaly N, Xiao Z, Valencia PM, Radovic-Moreno AF, Farokhzad OC. Targeted polymeric therapeutic nanoparticles: design, development and clinical translation. Chemical Society Reviews 2012; 41(7): 2971-3010.  128. Scarpelini S, Rhind S.G, Nascimento B, Tien H, Shek PN, Peng HT, Huang H, Pinto R., Speers V, Reis M and Rizoli SB. Normal range values for thromboelastography in healthy adult volunteers. Braz J Med Biol Res 2009; 42: 1210-1217. 129. Salooja N, Perry DJ. Thromboelastography. Blood Coagul. Fibrinolysis 2001; 12:3217–337. 130. Imran ul-haq M, Lai BFL, Chapanian R, Kizhakkedathu JN. Influence of Architecture of High Molecular Weight Linear and Branched Polyglycerols on Their Biocompatibility and Biodistribution. Biomaterials 2012; 33: 9135–9147.  131. Kainthan RK, Gnanamani M, Ganguli M, Ghosh T, Brooks DE, Maiti S, Kizhakkedathu JN. Blood Compatibility of Novel Water Soluble Hyperbranched 162  Polyglycerol-Based Multivalent Cationic Polymers and Their Interaction with DNA. Biomaterials 2006; 27: 5377–5390. 132. Poot M, Rabinovitch PS, Hoehn H. Free radical mediated cytotoxicity of desferrioxamine, Free Radic Res Commun 1989; 6:323–328.  133. Reichert S, Welker P, Calderón M, Khandare J, Mangoldt D, Licha K, Haag R. Size‐Dependant Cellular Uptake of Dendritic Polyglycerol. Small 2011; 7(6): 820-829. 134. Strähle U, Bally-Cuif L, Kelsh R, Beis D, Mione M, Panula P, Figueras A, Gothilf Y, Brösamle C, Geisler R, Knedlitschek G. EuFishBiomed (COST Action BM0804): a European network to promote the use of small fishes in biomedical research. Zebrafish  2012; 9(2):90-3. doi: 10.1089/zeb.2012.0742.  135. Chapin R, Augustine-Rauch K, Beyer B, Daston G, Finnell R, Flynn Tet al. State of the art in developmental toxicity screening methods and a way forward: a meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish. Birth Defects Research Part B: Developmental and Reproductive Toxicology 2008; 83(4): 446-456. 136. Donovan A, Brownlie A, Dorschner MO, Zhou Y, Pratt SJ et al. The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1 (DMT1). Blood 2002; 100: 4655-4659. 163  137. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004; 306: 2090-2093. 138. Shike H, Shimizu C, Lauth X, Burns JC. Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates. Dev Comp Immunol 2004; 28: 747–754. 139. Fraenkel PG, Traver D, Donovan A, Zahrieh D, Zon LI. Ferroportin1 is required for normal iron cycling in zebrafish. J  Clin Invest   2005; 115:1532–1541 140. Ganz T. Frontiers in Nephrology: Molecular Control of Iron Transport. J Am Soc Nephrol  2007; 18: 394-400.  141. Steinhauser S, Heinz U, Bartholomä M, Weyhermüller T, Nick H et al. Complex formation of ICL670 and related ligands with Fe III and Fe II.  Eur J Inorgan Chem 2004; 21: 4177-4192.  142. Livak KJ, Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2[-Delta Delta C (T)] method. Methods 2001; 25: 402-408. 143. Craig PM, Galus M, Wood CM, McClelland GB Dietary iron alters waterborne copper-induced gene expression in soft water acclimated zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol 2009; 296: R362–R373. 144. Walter A, Gutknecht J. Permeability of Small Nonelectrolytes through Lipid Bilayer Membranes. J Membr Biol 1986; 90:207-217. 164  145. Kansy M, Senner F, Gubernator K. Physicochemical High Throughput Screening: Parallel Artificial Membrane Permeation Assay in the Description of Passive Absorption Processes. J Med Chem 1998; 41:1007-1010.  146. Camenisch G, Alsenz J, van de Waterbeemd H, Folkers G. Estimation of permeability by passive diffusion through Caco-2 cell monolayers using the drugs’ lipophilicity and molecular weight. Eur J Pharm Sci 1998; 6: 313–319.  147. Harvey B, Norman Kelly R, Ashwood-Smith MJ. Permeability of Intact and Dechorionated Zebrafish Embryos to Glycerol and Dimethyl Sulphoxide. Cryobiology 1983; 20: 432-439.  148.  Bonsignorio D, Perego L, Giacco LD, Cotelli F. Structure and Macromolecular Composition of the Zebrafish Egg Chorion. Zygote 1996; 4(02): 101-108. 149. Kais B, Schneider KE, Keiter S, Henn K, Ackermann C, Braunbeck T. DMSO modifies the permeability of the zebrafish (Danio rerio) chorion-Implications for the fish embryo test (FET). Aquat Toxicol 2013; 140: 229-238.  150. Cooper CE, Lynagh GR, Hoyes KP, Hider RC, Cammack R et al. The Relationship of Intracellular Iron Chelation to the Inhibition and Regeneration of Human Ribonucleotide Reductase. J Biol Chem 1996; 271: 20291–20299.   151. Bury N, Grosell M. Iron acquisition by teleost fish. Comp Biochem Physiol Phamacol 2003; 135: 97–105.  165  152. Andersen O. Accumulation of waterborne iron and expression of ferritin and transferrin in early developmental stages of brown trout (Salmo trutta). Fish Physiol Biochem 1997; 16: 223-231. 153. Roeder M, Roeder R H. Effect of iron on the growth rate of fishes. J Nutr 1966; 90: 86-90.  154. Kontoghiorghes GJ, Kolnagou A, Peng C-T, Shah SV, Aessopos A. Safety issues of iron chelation therapy in patients with normal range iron stores including thalassemia, neurodegenerative, renal and infectious diseases. Expert Opin. Drug Saf. 2012; 9:201-206.  155. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388: 482–488.  156. Koren G, Pastuszak A, Ito S Drugs in pregnancy. N Engl J Med 1998; 338:1128–1137. 157. Hamilton JL, Hatef A, Imran ul-haq M, Nair N, Unniappan S,  Kizhakkedathu JN. Clinically Approved Iron Chelators Influence Zebrafish Mortality, Hatching Morphology and Cardiac Function. PLoS One 2014; 9(10):e109880. 158. Bergeron RJ, Wiegand J, McManis JS and Bharti N. Lipophilicity can have a profound effect on toxicity, within the DADFT family of ligands the more lipophilic ligands were more toxic. J Med Chem 2006; 49: 7032-7043. 166  159. Anderson LJ, Westwood MA, Prescott E, et al. Development of thalassaemic iron overload cardiomyopathy despite low liver iron levels and meticulous compliance to desferrioxamine. Acta Haematol 2006; 115(1-2):106-108. 160. Voskaridou E, Komninaka V, Karavas A, Terpos E, Akianidis V, and Christoulas D. Combination therapy of deferasirox and deferoxamine shows significant improvements in markers of iron overload in a patient with b-thalassemia major and severe iron burden. Transfusion 2014; 54(3): 646-9. 161. Berdoukas V, Carson S, Nord A, Dongelyan A, Gavin S, Hofstra TC, Wood JC, Coates T. Combining two orally active iron chelators for thalassemia. Ann Hematol 2010; 89 (11):1177-1178.  162. Ladis V, Chouliaras G, Berdoukas V, Chatziliami A, Fragodimitri C, Karabatsos F, Youssef J, Kattamis A, Karagiorga-Lagana M. Survival in a large cohort of Greek patients with transfusion-dependent beta thalassaemia and mortality ratios compared to the general population. Eur J Haematol 2011; 86(4):332-8. 163. Cohen AR. Iron Chelation: you gotta have heart. Blood 2010; 115 (12): 2333-2334. 164. Weatherall DJ. The challenge of haemoglobinopathies in resource-poor Countries, Br J Haematol 2011; 154(6): 736–744. 165. Gorbet MB and Sefton MV. Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes. Biomaterials 2004; 25: 5681–5703. 167  166. Murtagh LJ, Whiley M, Wilson S, Tran H, and Bassett ML. Unsaturated Iron Binding Capacity and Transferrin Saturation Are Equally Reliable in Detection of HFE Hemochromatosis. Am. J. Gastroenterol.  2002; 97(8): 2093-2099.  167. Baran GR, Kiani MF, Samuel SP. Biomaterials Applications in Medicine and Case Studies. Healthcare and Biomedical Technology in the 21st Century 2014; 249-285. 168. Breuer W, Ronson A, Slotki IN, Abramov A, Hershko C, Cabantchik ZI. The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation. Blood 2000; 95(9): 2975-2982. 169. Meddahi‐Pellé A, Legrand A, Marcellan A, Louedec L, Letourneur D, Leibler L. Organ Repair, Hemostasis, and In Vivo Bonding of Medical Devices by Aqueous Solutions of Nanoparticles. Angewandte Chemie 2014; 126(25): 6487-6491. 170. Ramakrishna, S., et al. Biomedical applications of polymer-composite materials: a review. Compos Sci Technol 2001; 61(9): 1189-1224. 171. Han J, Oh Y, Kim D, Kim C. Enhanced hepatocyte uptake and liver targeting of methotrexate using galactosylated albumin as a carrier. Int. J. Pharm 1999; 188: 39–47.  172. Wu D, Lua B, Chang C, Chen C, Wang T, Zhang Y, Cheng S, Jiang X, Zhang X, Zhuo R. Galactosylated fluorescent labeled micelles as a liver targeting drug carrier, Biomaterials 2009; 30: 1363–1371.  168  173. Panariti A, Miserocchi G, Rivolta, I. The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions? Nanotechnol Sci Appl. 2012; 5, 87. 174. Kühn, Lukas C. Iron regulatory proteins and their role in controlling iron metabolism. Metallomics (2015). 175. Shenoi RA, Jayaprakash KN, Hamilton JL, Lai BFL, Horte S, Kainthan RK, Varghese JP, Kallanthottathil GR, Manoharan M, Kizhakkedathu JN. Molecular Control of Degradation of Multifunctional Polymers within Live Cells by the Bonding Structure of Ketal Linkages. J. Am. Chem. Soc 2012; 134(36):14945-14957. 176. Whitnall M, Howard J, Ponka P, Richardson D R.  A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics.  Proc. Natl. Acad. Sci 2006; 103(40): 14901-14906.  177. Bergan T et al. Chelating agents. Chemotherapy 2001; 47: 10–14.  178. Whitnall M, Richardson DR.  Iron: a new target for pharmacological intervention in neurodegenerative diseases. Semin. Pediatr. Neurol 2006; 13 (3): 186-197.   179. Neupane GP, Kim DM.  In vitro time-kill activities of ciprofloxacin alone and in combination with the iron chelator deferasirox against Vibrio vulnificus.  Eur. J. Clin. Microbiol. Infect. Dis 2010; 29(4): 407-410.  169  180. Debebe Z, Ammosova T,  Jerebtsova M, Kurantsin-Mills J, Niu X, Charles S, Richardson  D R, Ray PE, Gordeuk VR, Nekhai S.  Iron chelators ICL670 and 311 inhibit HIV-1 transcription. Virology 2007; 367 (2): 324-333.  181. Gordeuk VR et al. Iron chelation with desferrioxamine-B in adults with asymptomatic Plasmodium-falciparum parasitemia. Blood 1992; 79: 308–312.  182. Fox ME, Szoka FC and Fréchet JMJ. Soluble Polymer Carriers for the Treatment of Cancer: The Importance of Molecular Architecture. Acc. Chem.  Res.  2009; 42(8): 1141-1151.   170  Appendix A Table 10: The properties of HPG-DFO molecules tested for Biocompatibility in Chapters 2-4.    Nomenclature Size of HPG Number of DFO Rh 25K-65 25 65 2.7 44K-1 44 10 4.2 44K-3 44 45 4.5 44K-4 44 85 -- 50K- 25 50 10 -- 50K- 200  50 44 -- 100K-100 100 17 -- 200K-100 200 20 -- 300K-100 300 21 -- 500K-100 500 26 -- 500K- 150 500 55 7.3 500K-3/500K-300 500 129 7.7 500K-400  500 120 -- 500K- 500  500 180 -- 500K- 700 500 330 -- 500K- 900 500 228 -- 700K-100 700 28 -- 171  Appendix B  Table 11: Species present in ITC titration of Fe(NTS) and DFO, HPG-DFO and 40SDO2.   Species Formula Log β Fe NTA H Hepes  7.55 0 0 1 1  9.29 0 1 1 0  11.94 0 1 2 0  13.68 0 1 3 0 Fe1NTA1 15.9 1 1 0 0 Fe1NTA1OH 11.54 1 1 -1 0 Fe1NTA1(OH)2 3.96 1 1 -2 0 Fe1NTA1(OH)3 -6.76 1 1 -3 0 Fe1NTA2 23.97 1 2 0 0  -13.77 0 0 -1 0  172    Figure 41: Speciation plot of Fe-NTA complexes existing in the syringe.  The conditions of the titration were specified and simulated using HySS (116). The above figure shows that at pH 7.00 there are two dominant species in the syringe. The values of binding constants for Fe-NTA used to generate the plot were obtained from: Motekaitis, R. et al (27).    173  Appendix C  The in vivo toxicity of HPG-DFO   LDH level of mice serum after exposure to 44k-85DFOA BFigure 42:  Tolerance studies of 44K-85 chelator in Balb/C mice.   (A) The body weights of mice administered (i.v., bolus injection, 200 µL) with escalating doses of 44K-85 remained within the normal range 14 days post-injection. (B) LDH levels of mice serum (N=3 per group) injected (i.v., bolus injection) with 44k-85DFO with different dose range (100 mg/kg to 1000 mg/kg DFO equivalent).  174                            Figure 43: Tolerance studies of 44K-85 chelator in Balb/C mice.   Histology of fixed liver tissues of control mice and mice injected with 1000mg/kg after hematoxylin and eosin (H&E) staining showing normal morphology (C). (D) Histology of fixed kidney tissues of control mice and mice injected with 1000 mg/kg after hematoxylin and eosin (H&E) staining showing normal morphology. Data showed that 44 kDa conjugated with 85 DFO molecules is well tolerated even at 1000 mg/kg where as 250 mg/kg DFO leathal to mice as reported in literature (105).   1000mg/kg 44k-85DFOSalineCH & E stained of liver sectionsDH & E stained of kidney sectionsSaline1000 mg/kg     44k-85DFO

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