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The synthesis and optimization of functionalized hyperbranched polyglycerols as potential topical hemostatic… Liu, Chu 2018

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THE SYNTHESIS AND OPTIMIZATION OF FUNCTIONALIZED HYPERBRANCHED POLYGLYCEROLS AS POTENTIAL TOPICAL HEMOSTATIC AGENTS by  Chu Liu  B.Sc., Nanjing University, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   March 2018   © Chu Liu, 2018  ii  Abstract  As a classical representative of the hyperbranched polymer family, hyperbranched polyglycerol has been applied in a variety of diagnostics and therapies. After functionalizing the end groups with zwitterionic sulfabetaines (SB) and cationic quaternary amines (QA), the polymer exhibited superior hemagglutination capacity at a concentration ≥ 1 mg/mL and erythrocyte lysis was not observed.  The goal in the present work is to use the monomers above to maximize the polymer adhesion to cells while eliminating the membrane damage caused by multivalent polycation exposure. The desired stickiest structure is a novel attractive bioadhesive material which may serve as a future topical hemostatic agent for bleeding wounds, overcoming the limitations of existing products in the market.  To this end an optimization was performed on the synthesis scheme to achieve a library of HPG based polymer conjugates with a constant amount of one derivative (SB/QA) and a varied number of the other derivative (QA/SB). The efficacy of sulfabetaine (SB) and quaternary amine (QA) was quantified and different hemagglutination behaviors were displayed. Hemolysis, cytotoxicity and inhibition effects as well as the hydration properties of the polymer conjugates were also evaluated. In the blood aggregation analysis, HPG-SB30-QA30-OH40 and HPG-SB30-QA40-OH30 exhibited strong red blood cells aggregation effects even at a concentration as low as 1mg/mL. Thirty percent of SB also reduced the hemolytic activity to an undetectable level and retained high cell viability in the presence of 30% QA. The bound water of each repeat monomer unit was quantified by differential scanning calorimetry and explained the fouling resistance behavior of the cells. The addition of linear polyglycerol sulfate into the HPG-SB-QA-OH iii  system effectively suppressed the red blood cell aggregation, which indicated the possibility of reversibly applying the materials for hemostasis. Overall, the in vitro studies suggest that the stickiest structure of the designed functionalized HPG is HPG-SB30-QA30-OH40, which is biocompatible and harmless to cells.    iv  Lay Summary  In order to enhance the efficacy and safety of hemostatic agents (agents that prevent blood loss), which are essential for treatment of surgical wounds, a novel bioadhesive hyperbrached polyglycerol (HPG) based material was modified with quantified zwitterionic (SB) and cationic (QA) monomers. By controlling the amount of derivatives added, the materials exhibited different aggregation effects to red blood cells, aggregation that could stop blood leaking from a wound. After conducting a series of biocompatibility tests, the structure with 30% SB and 30% QA was determined to be stickiest yet harmless to cells, suggesting its use as the basis of a new hemostasis agent. v  Preface  The work presented in this thesis is based on the experiments and analysis conducted by Chu Liu under the supervision of research supervisor Dr. Donald E. Brooks. Chapter 2 is the chemical synthesis and characterization part, which was performed solely by the author. Dr. Narges Hadjesfandiari assisted me with the interpretation of the spectrum and design of the protocol. Chapter 3 is based on work conducted in Centre for Blood Research UBC and The Centre for Drug Research and Development. The author was responsible for the hemagglutination, hemolysis, cytotoxicity and inhibition tests. The hydration test was performed with the advice and assistance of Dr. Srinivas Abbina. Blood used in the biological tests was drawn from healthy donors by a process approved by the UBC Clinical Ethics Committee (Approval Number: H07-02067 granted to the Centre for Blood Research). vi  Table of Contents  Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Schemes ........................................................................................................................... xiv List of Abbreviations ...................................................................................................................xv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................. xviii Chapter 1: Introduction ................................................................................................................... 1 1.1 Content of blood ......................................................................................................... 1 1.1.1 The formation of red blood cell membranes ....................................................... 2 1.2 Introduction of hemostatic effects .............................................................................. 6 1.2.1 Traditional hemostatic agents ............................................................................. 6 1.2.2 Novel hemostatic agents ................................................................................... 10 1.3 The development of drug delivery agents ................................................................. 12 1.3.1 Stimuli-responsive HPG based materials.......................................................... 14 1.3.2 Click chemistry ................................................................................................. 15 1.3.3 Zwitterioninc materials ..................................................................................... 17 1.4 Thesis goals and structure ......................................................................................... 21 vii  Chapter 2: Synthesis and characterization of zwitterionic functionalized hyperbranched polyglycerols ................................................................................................................................. 24 2.1 Synopsis .................................................................................................................... 24 2.2 Materials and methods .............................................................................................. 24 2.3 Experimental section ................................................................................................. 25 2.3.1 Preparation of HPG (20K) (1a) ......................................................................... 25 2.3.2 Preparation of allyl functionalized HPG (1b) ................................................... 27 2.3.3 Model reaction of HPG-thiol-OH (1c) .............................................................. 28 2.3.4 Preparation of 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium)bromide (2a) ...................................................................................................... 29 2.3.5 Preparation of 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (2b) . 30 2.3.6 Preparation of 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) ......... 31 2.3.7 Preparation of bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) 31 2.3.8 Preparation of 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate (3c) ...... 32 2.3.9 Preparation of HPG-SB (4a) ............................................................................. 33 2.3.10 Preparation of HPG-SB-QA (4b) and HPG-SB-QA-OH (4c) .......................... 33 2.4 1H NMR and 13C NMR characterizations ................................................................. 35 2.4.1 Characterization of HPG (20K) (1a) ................................................................. 35 2.4.2 Characterization of allyl group modified HPG(20K) (1b) ................................ 37 2.4.3 Characterization of HPG-thiol-OH (1c) ............................................................ 38 2.4.4 Characterization of 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium) bromide (2a) ...................................................................................................................... 39 2.4.5 Characterization of 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide . 41 viii  2.4.6 Characterization of 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) . 42 2.4.7 Characterization of bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) (3b) ......................................................................................................... 44 2.4.8 Characterization of 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate ..... 45 2.4.9 Characterization of HPG-SB (4a), HPG-SB-QA (4b) and HPG-SB-QA-OH .. 46 2.4.10 Comparison of HPG-SB30-QA10/20/30, HPG-SB30-QA10/20/30-OH60/50/40 ............ 47 2.5 Summary ................................................................................................................... 49 Chapter 3: In Vitro red blood cell binding and biocompatibility tests .......................................... 50 3.1 Introduction ............................................................................................................... 50 3.2 Experimental section ................................................................................................. 51 3.2.1 Materials preparation ........................................................................................ 51 3.2.2 The aggregation test on red blood cells ............................................................ 52 3.2.3 Hemolytic activity analysis ............................................................................... 53 3.2.4 Quantification of bound water by differential scanning calorimetry ................ 54 3.2.5 Cytotoxicity evaluation of the polymer conjugates .......................................... 55 3.2.6 Reversibility analysis of the red blood cell aggregates ..................................... 55 3.3 Results and discussion .............................................................................................. 56 3.3.1 The aggregation effects on red blood cells ....................................................... 56 3.3.2 Hemolysis activity result................................................................................... 61 3.3.3 Water binding by SB and QA ........................................................................... 63 3.3.4 Cytotoxicity of the polymer conjugates ............................................................ 66 3.3.5 Inhibition effect of the polymer conjugates ...................................................... 68 3.4 Summary ................................................................................................................... 69 ix  Chapter 4: Conclusions and Future Directions ............................................................................. 71 4.1 Conclusions ............................................................................................................... 71 4.2 Future directions ....................................................................................................... 73 Bibliography .................................................................................................................................77  x  List of Tables  Table 3.1 Physicochemical properties of the polymer conjugates for DSC experiments ............. 65 Table 3.2 Hydration properties of the polymer conjugates for each monomer repeat unit .......... 66  xi  List of Figures  Figure 1.1 Composition of blood and components of blood volume2 (© 2007 Vidya V. Sagar, by permission) ...................................................................................................................................... 2 Figure 1.2 Structures of cholesterol and glycosphingolipid ........................................................... 4 Figure 1.3 Structures of phospholipids ........................................................................................... 5 Figure 1.4 Synthetic routes for 2-(methacryloyloxy)ethyl choline phosphate and poly(2-(methacryloyloxy)ethyl choline phosphate)20 (© 2013 Xifei Yu, by permission) ....................... 11 Figure 1.5 Illustration of a phospholipid bilayer containing 1,2-dipalmitoyl-glycero-3-phosphatidyl choline (DPPC)21 (© 2013 Xifei Yu, by permission) .............................................. 12 Figure 1.6 Structures of zwitterionic polycarboxybetaine and polysulfobetaine: a) zwitterionic polysulfobetaine, b) zwiiterionic polycarboxybetaine, c) cationic polycarboxybetaine esters, d) mixed charge polymers ................................................................................................................. 19 Figure 1.7 HPG conjugation structure .......................................................................................... 22 Figure 2.1 GPC chromatogram of HPG (Mn=2.0×104) ................................................................ 26 Figure 2.2 HPG (1a) 1H NMR in D2O .......................................................................................... 35 Figure 2.3 HPG (1a) 13C NMR in D2O ......................................................................................... 35 Figure 2.4 HPG-allyl (1b) 1H NMR in CDCl3 .............................................................................. 37 Figure 2.5 HPG-allyl (1b) 13C NMR in CDCl3 ............................................................................. 37 Figure 2.6  HPG-thiol-OH (1c) 1H NMR in D2O ......................................................................... 39 Figure 2.7 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium) bromide (2a) 1H NMR in D2O ............................................................................................................................................... 39 xii  Figure 2.8 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium) bromide (2a) 13C NMR in D2O ............................................................................................................................................... 40 Figure 2.9 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (2b) 1H NMR in D2O ........ 41 Figure 2.10 3-mercapto-N,N,N-trimethylpropan-1-aminiumbromide (2b) 13C NMR in D2O ...... 41 Figure 2.11 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) 1H NMR in D2O ............. 42 Figure 2.12 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) 13C NMR in D2O ............ 43 Figure 2.13 bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) (3b) 1H NMR in D2O....................................................................................................................................................... 44 Figure 2.14 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3c) 1H NMR in D2O ............. 45 Figure 2.15 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3c) 13C NMR in D2O ............ 45 Figure 2.16 HPG-SB (4a), HPG-SB-QA (4b) and HPG-SB-QA-OH (4c) 1H NMR in D2O ....... 46 Figure 2.17 1H NMR of HPG-SB30-QA10, HPG-SB30-QA20 and HPG-SB30-QA30 in D2O ......... 47 Figure 2.18 1H NMR of HPG-SB30-QA10-OH60, HPG-SB30-QA20-OH50 and HPG-SB30-QA30-OH40 in D2O .................................................................................................................................. 48 Figure 3.1 Erythrocytes aggregation effects of 5mg/mL HPG conjugates after 5 min incubation at 37°C in human whole blood. (I) PBS control (II) HPG-SB30-QA20-OH50, (III) HPG-SB30-QA30-OH40, (IV) HPG-SB30-QA40-OH30 ................................................................................................ 57 Figure 3.2 Erythrocytes aggregation effects of 1mg/mL HPG conjugates after 5 min incubation at 37 °C in human whole blood. (I) HPG-SB30-QA10-OH60 (II) HPG-SB30-QA20-OH50, (III) HPG-SB30-QA30-OH40, (IV) HPG-SB30-QA40-OH30 ............................................................................. 58 Figure 3.3 Erythrocytes aggregation effects of 1mg/mL HPG conjugates after 5 min incubation at 37 °C in human washed blood. (I) PBS control (II) HPG-SB30-QA20-OH50, (III) HPG-SB30-QA30-OH40, (IV) HPG-SB30-QA40-OH30 ...................................................................................... 60 xiii  Figure 3.4 % of RBC lysis after incubation with different types of 5mg/mL polymer conjugates....................................................................................................................................................... 62 Figure 3.5 % of RBC lysis after incubation with different types of 1mg/mL polymer conjugates....................................................................................................................................................... 62 Figure 3.6 Heat profile of water in the presence of HPG-SB20-QA10-OH70 (integration based on the total area under the solid green curves, black line indicated the vertical scope of integration) and heat profile of pure water (integration based on the total area under the dotted blue curves) 64 Figure 3.7 Four types of ending groups on HPG backbone .......................................................... 65 Figure 3.8 % of HUVEC viability after incubation with different types and concentrations of polymer conjugates for 48h .......................................................................................................... 68 Figure 3.9 Erythrocytes incubated under PBS control (I) and 1mg/mL HPG-SB30-QA30-OH40 & LPG-OSO3-(II) at 37°C in human whole blood. ........................................................................... 69 Figure 4.1 List of potential zwitterionic substrates ....................................................................... 75    xiv  List of Schemes  Scheme 2.1 Overall Protocols ....................................................................................................... 24 Scheme 2.2 Preparation of HPG (20K) (1a) ................................................................................. 27 Scheme 2.3 Preparation of Allyl functionalized HPG (1b) .......................................................... 28 Scheme 2.4 Model reaction of HPG-thiolglycerol (1c) ................................................................ 28 Scheme 2.5 Preparation of 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium)bromide (2a)....................................................................................................................................................... 29 Scheme 2.6 Preparation of 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (2b).......... 30 Scheme 2.7 Preparation of 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) ................. 31 Scheme 2.8 Preparation of bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) (3b) 31 Scheme 2.9 Preparation of 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate (3c) .............. 32 Scheme 2.10 Preparation of HPG-SB (4a) ................................................................................... 33 Scheme 2.11 Preparation of HPG-SB-QA (4b) ............................................................................ 33 Scheme 2.12 Preparation of HPG-SB-QA-OH (4c) ..................................................................... 34 Scheme 2.13 Carbon architectures in HPG backbone .................................................................. 36 Scheme 4.1 Synthesis pathway to HPG-SH ................................................................................. 75   xv  List of Abbreviations  AA Acrylamide  ADP Adenosine diphosphate ATRP Atom transfer radical polymerization  CDCl3  Deuterated chloroform CP choline phosphate CuAAC Copper-catalyzed azide/ alkyne cycloaddition  DCM  Dichloromethane DSC Differential scanning calorimetry GPC Gel permeation chromatography h  Hour HPG Hyperbrached polyglycerol HPG-SB Sulfabetaine modified hyprbranched polyglycerol HPG-SB-QA Sulfabetaine and quaternary amine modified hyprbranched polyglycerol MA Methacrylate  MALLS  Multi-angle light scattering MFC Microfibrillar collagen  min  Minute Mn  Number average molecular weight Mw  Weight average molecular weight MWCO  Molecular weight cut off PBS  Phosphate buffered saline PC Phosphatidylcholine  PDI  Polydispersity (Mw/Mn) PEG Poly(ethylene glycol)  PEG  Poly(ethylene glycol) xvi  QA Cationic quaternary amines RBC  Red blood cell  ROMBP Ring-opening multi-branching polymerization SB Zwitterionic sulfabetaines  SDS Sodium dodecyl sulfate  TBAB Tetrabutylammonium bromide TFE 2,2,2-trifluoroethanol TMP  1,1,1-tris(hydroxylmethyl)propane UV  Ultraviolet     xvii  Acknowledgements  First of all, I would like to express my special gratitude and appreciation to my supervisor Dr. Don Brooks, who gave me the opportunity to work in this lab, patiently guided me and encouraged me along my entire studies. I would never have finished this thesis without his invaluable support and effort exerted in reviewing this dissertation. During my past three years in Canada, Dr. Don Brooks kindly provided me with career suggestions and respected my choices, I owe him the sincerest thanks from the bottom of my heart. I am extremely thankful to Dr. Narges Hadjesfandiari. She helped me with the project planning, scheduling, interpretation of the spectrum and editing of the thesis during her pregnancy. I was inspired by her dedication to science and work ethic. I was lucky to be able to finish this work with her tremendous effort to offer every possible help.  My thankfulness is also to my colleagues in Brooks/ Kizhakkedathu lab: Dr. Srinivas Abbina, Dr. Manu Thomas Kalathottukaren, Dr. Kai Yu, Iren Constantinescu, Erika Siren, Usama Abbasi, Lily Takeuchi and Youping Li. I learned from their expertise and thank you for all your support and generous help. Special thanks go to Dr. Elena Polishchuk from UBC Biological Services Laboratory for her kind assistance with the lyophilizer. Finally, I would like to say thank you to all my friends and family on the other side of the earth, especially my parents. Thank you for picking up the late night oversea phone calls and sharing all my sweat and tears. I wouldn’t have been able to survive the past three years without any of you.   xviii  Dedication  To my mother Qiuying, without whose unconditional love and dedication to the family I would never have had the chance to fly over the Pacific Ocean.  To my father Debin, for cheering up everyone in the family and accompanying Qiuying when I was not able to be together with her.1  Chapter 1: Introduction 1.1 Content of blood  Human blood contains a variety of components with different physical characteristics and functions. The constituents can be separated according to the differences of their buoyant density and electric charge to give cell-free plasma, white blood cells, platelets and red blood cells. 1 For a tube of freshly drawn human blood with anti-coagulant agents, after a short time standing still, two layers can be clearly visualized. The supernatant includes plasma, which constitutes 55% v/v of the whole blood. The 1% v/v white blood cells and platelets appear between the yellow top layer and the 45% v/v red cells at the bottom. It’s hard to observe the middle layer due to the low amount. Every 600 red blood cells correspond to 40 platelets and 1 white blood cell in human whole blood.  Plasma is a fluid mixed from more than 90% water and a collection of complex substances including proteins, minerals, salts and hormones. It helps in maintaining the blood pressure and body temperature. White blood cells, also called leukocytes, are derived from the hematopoietic stem cells in the bone marrow. They work as part of the immune system and travel throughout the body to defend against infections and outside intruders. Platelets are somewhat similar to red blood cells in that they have no cell nuclei. They are lens-shaped structures with a diameter of 2-3 m. The function of platelets is to be activated by bleeding and to arrest it by congregating around the wound.  Red blood cells have a well-known unique round shape with an indented center whose diameter is between 6-8 m, like a donut without a hole. Also known as erythrocytes, they play an important role in health by carrying oxygen and carbon dioxide throughout the body bound to intracellular hemoglobin. Hemoglobin is an iron-containing protein which binds oxygen after 2  outside oxygen enters into the blood vessels of lungs and returns carbon dioxide resulting from tissue cell respiration to the lungs for expiration. Unlike regular cells, red blood cells are adapted to contain hundreds of millions of hemoglobin molecules per red cell in order to accommodate the need of oxygen for the body. Thus the nucleus, mitochondria, and ribosomes are lost during the maturation of those cells.  Figure 1.1 Composition of blood and components of blood volume2 (© 2007 Vidya V. Sagar, by permission) 1.1.1 The formation of red blood cell membranes Red blood cell membranes are composed of 40% w/w lipids, 0 to 10 wt. % carbohydrate and the balance is protein. The bimolecular leaflet matrix of the membrane was first proposed by Gorter and Grendel in 1925 and later enhanced by Davson and Danielli.  They stressed the important role played by proteins in the formation of membranes.2 The detailed structure of the lipid bilayer is determined by its amphipathic characteristic. The hydrocarbon chains all aggregate in the middle of the bilayer to form a hydrophobic environment and leave the polar head groups to confront the aqueous surroundings. For the proteins, some are concentrated on the external side of the membrane, while some may also integrate into the lipid bilayer. In order to fit into the hydrophobic internal phase of the bilayer, the transmembrane portion of those integrated proteins is also formed predominantly by hydrophobic amino acids. Also to be 3  compatible with the hydrophilic side of the membrane, charged amino acids are predominantly adopted at the end of the transmembrane portion. This determines how these integrated proteins can be firmly locked in their positions within the bilayer. 3 In order to analyze membrane proteins, and minimize the influence of lipids, sodium dodecyl sulfate (SDS) was introduced as a solubilizing agent to separate the polypeptides from the lipid molecules. The proteins can then be separated according to their molecular weight and visualized as bands on SDS-polyacrylamide gels following electrophoresis since the amount of SDS bound per gram of protein, which provides the charge density that causes movement in the electric field, turns out to be independent of protein structure for most membrane proteins. 4 The drag caused by protein-gel interactions depends on the protein size, providing the molecular weight dependence of the migration distance. The lipid bilayer is mainly formed by three kinds of lipids: cholesterol, glycolipids and phospholipids.  Cholesterol is essential to maintain the integrity and fluidity of membrane structure. On the microscopic level, it enables the cells to change their shape in response to external forces as they encounter barriers to flow in the circulation. On a macro basis, then, it empowers animals to move. In particular, the modulation of biological function of membranes by cholesterol has been extensively studied. When there is a change in the level of cholesterol in the membrane, a proportionate adjustment in membrane protein function can be detected, like Na+- Ca2+ exchange and ATP-ADP exchange although some membrane functions are unresponsive to its level change. Two hypotheses to explain the mechanism are that: 1). the membrane function is inhibited by cholesterol through reducing the available free volume between the interior 4  hydrocarbon chains. The limited free volume constrains the membrane protein conformation change.  2). cholesterol directly binds to membrane proteins to restrain their function.5 Glycolipids are glycosyl derivatives of lipids, which were discovered in 1942 when Ernst Klenk first isolated them from brain tissue. They are comprised of a hydrophobic lipid tail and mono- or oligosaccharide hydrophilic sugar head groups through a glycosidic linker. The hydrophobic nature of the lipid tails enables glycolipids to affiliate with the membrane. N-acylsphingosines (ceramides) are a subtype of glycolipids that have been reported to help with the regulation of cell growth via their interactions with growth factor proteins. Another function exhibited by glycolipids is cell signaling. As receptor components, they can associate with diverse proteins to induce activation of specific kinases. Those kinases will further facilitate the phosphorylation of various substrates. One of the important functions of glycolipids is similar to cholesterol, maintaining the structural integrity of the membranes for the organisms. This function is realized by the inter-lipid hydrogen bonds formed among the glycosyl head groups. Through interacting tightly with cholesterol, they can be successfully segregated from phospholipids which regulate the fluidity of cell membranes. 6    Figure 1.2 Structures of cholesterol and glycosphingolipid Phospholipids comprise the major mass of the lipids and their unique chemical structure determines the bimolecular leaflet matrix of the cell membrane. A typical phospholipid molecule 5  contains a hydrophilic head group and two hydrophilic fatty acid chain tails, each containing 14 ~24 carbons. One tail is usually unsaturated with one or more cis-double bonds, while the other tail is commonly saturated. The head group contains a negatively charged phosphate group and a glycerol, which is soluble in water. Individual lipid molecules are discovered to be able to diffuse freely and exchange places within a monolayer. The test was done by labeling the head group with a nitroxyl group containing an unpaired electron which can be detected by electron spin resonance spectroscopy. Past studies also show that the hydrocarbon chains of the phospholipids are flexible, which enhances their fluid nature. 7  Figure 1.3 Structures of phospholipids The types of phospholipids may vary depending on the nature of the cells and the composition of each layer. For example, in human erythrocytes, the interior side of the plasma membrane facing the cytosol is composed of phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. For the exterior side of the cell, it is constructed mainly of 6  phosphatidylcholine (PC) and sphingomyelin. The head group of phosphatidylcholine contains choline (Fig. 1.3), which makes PC a zwitterionic glycerophospholipid. As the major membrane lipid in eukaryotic cells, PC accounts for nearly 50% of all the membrane lipids. The biosynthesis and significance of PC in signal transduction were studied especially when the role of PC in prokaryotes was gradually revealed in the past decades. 8 1.2 Introduction of hemostatic effects Based on studies of red blood cell surface structure, a deeper understanding has been developed by researchers on hemostatic effects. Hemostasis is defined as the maintenance of blood flow and volume in the circulation through the arrest of bleeding when blood vessels are breached. It is a complicated physiological process which takes place in three successive steps once the circulation is disturbed. Vasoconstriction comes into place first due to the contraction of the muscular wall of the vessels. This step is particularly important in reducing the blood flow and blood loss. At the same time platelets are activated by the blood exiting outside the vessel wall as a result of bleeding. The chemical messengers released by platelets during this process cause more platelets aggregate at the wound site to form a plug. Thus the bleeding is physically blocked by the platelets. The principle of developing either synthetic or biological hemostatic agents to eliminate bleeding is to enhance one or all of the three steps above mentioned. 9,10   1.2.1 Traditional hemostatic agents Hemostatic agents have been developed through extensive and intensive research during the past decades. According to the mechanism of action, the agents are subcategorized into four types, which are physical, absorbable, biological and synthetic agents. For physical agents, bone wax and Ostene are most commonly used since the last century. Bone wax is a kind of sterile mixture of beeswax, paraffin and isopropyl palmitate, which is an 7  emollient, moisturizer and thickening agent. By occlusion of bleeding channels in bone, bone wax achieves hemostasis effectively. But since it is nonabsorbable, it has a negative impact on osteogenesis. Also bone wax was found to impede the clearance of bacteria and may promote infection. 11 Under such circumstance, Ostene was developed and is a form of alkylene oxide polymer which overcomes the problem of impaired bone healing. Animal tests also showed that the infection problem was solved due to its hydrophilicity and resistance to biochemical alteration. 12 Gelatin foams, oxidized cellulose and microfibrillar collagen are three absorbable hemostatic agents commonly used by surgeons. Gelatin foams are animal skin gelatin in a sponge form, which are nonantigenic and work at neutral pH. Therefore gelatin foams can be applied in conjunction with many other pH neutral biologics like thrombin to enhance the effect of hemostasis. Oxidized cellulose is generated from decomposing wood pulp and exhibits great handling characteristics. It can be knitted into any size to tailor the wound. However due to its low pH, red cell lysis can be caused and this also limits the chance to use it in conjunction with other biological topical agents. Microfibrillar collagen (MFC) was derived from bovine corium first by Heit and later continuously modified by various companies in order to give different physical appearances to conform to any need. To date, MFC can be found in the forms of flour, fluff, nonwoven sheet, sponge and pad. MFC depends on platelet activation for its effect, followed by platelet aggregation and then thrombus formation. Once hemostasis is achieved, excessive MFC needs to be removed as it can bind to neutral structures and cause pain. For biologic agents, thrombin is a central enzyme in plasma and has been put into use for more than half a century. Commercial thrombin comes largely from bovine plasma. It works by catalyzing fibrinogen transformation into fibrin to form a kind of clot. Though bovine thrombin 8  worked effectively in stopping bleeding, unlike gelatin foams, some immune responses have been reported.12 A severe coagulopathy was found after human exposure. Later a recombinant human thrombin which was manufactured in vitro through recombinant DNA technology was reported to achieve the same efficacy as bovine thrombin without an immune response. It is expected that after further research and clinical tests, recombinant human thrombin will take the place of bovine thrombin. Based on the study of thrombin, fibrin sealants were formed by adding fibrinogen to thrombin with a dual injector right before application. The ratio of the two components determines the mechanical strength of the clot and the rapidity of clotting. Fibrin sealants are mainly used by urological surgeons to work as complementary adjuncts, which cannot replace the conventional techniques. Thrombin can also cooperate with microfibrillar agents and plasma, which contains fibrinogen and platelets, to form the clot. However the plasma needs to come from the patients themselves, then centrifuged and distributed in a complex delivery system. The intricacy of application limits the acceptance of such kind of agents’ combination. As well as using those naturally occurring polymers and enzymes, scientists have made great strides in developing synthetic hemostatic agents. Cyanoacrylates based materials like Histoacryl® and PeriAcryl® have been approved by the FDA and put into clinical use for over 20 years. Both of these two commercial products take advantage of the highly electron deficient nature of carbon carbon double bonds, which enables the cyanoacrylates to polymerize rapidly once it is applied in the presence of water or water containing substances like human tissue. Feracryl® is another kind of wound care agent which consists of poly(acrylic acid) and an iron content up to 2.5%. The iron content regulates the hemostatic activity and exhibits an 9  antimicrobial activity. The hemostatic mechanism of Feracryl® is through the formation of a Feracryl®/plasma clot. Research focusing on adjusting the content and type of the coordinated metals has also been conducted. 13 Chitosan is a kind of linear polycationic polysaccharide extracted from the chitin shells of shrimp and other crustaceans. In the coagulation studies conducted by Rao et al.14, chitosan formed a coagulum with whole blood by reducing the clotting time to 40% of original without contributing to lysis. The hemostatic mechanism was ascribed to the interaction between the red blood cell membrane and chitosan, which was likely electrostatic in nature and different from the traditional cascade of enzyme activation.  To better use this unique biomaterial, a further study was done in 2011 to enhance its hemostatic capability. A number of catechol groups was conjugated onto the backbone of chitosan using 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) carbodiimide crosslinker. Then Pluronic® F-127 which is a type of antifoaming block polymer based on ethylene oxide and propylene oxide was thiolated by conjugating cysteamine onto the terminal hydroxyl groups. A CHI-C/Plu-SH hydrogel was obtained and showed good antihemorrhagic characteristics. The material showed 20 times higher tissue adhesive ability than regular fibrin glue due to the remarkable hydrogel adhesiveness.  Another type of hydrogel was reported by Kofinas and coworkers15 by inducing Factor VII activation to realize coagulation. Factor VII was formerly known as proconvertin. It’s an enzyme causing blood clotting in the enzyme activation cascade.  A set of six positively charged acrylamide- co- N-(3-aminopropyl)methacrylamide hydrochloride (APM)-co- N,N′-methylenebisacrylamide (BIS) hydrogel compositions were tested. The result indicates that in addition to the positive electrostatic charge, cross-link density and the water content of the 10  hydrogel all play an important role in FVII activation, which further impacts the formation of fibrin.  As well as these polymers, a type of peptide has been developed by Zhang et al16, which can self-assemble into nanofibers as soon as they are exposed to the metal cations in the blood, like Na+. The self-assembling idea here was derived from Lego bricks. Those interlocking toy bricks have both well-designed pegs and holes which can be precisely assembled together into a functional structure. At the molecular level, RADA16-I is designed as a biological scaffold with 5-200 nm pores and high water content. The scaffolding matrix created by the peptides resembles the extra-cellular matrix and shows its efficacy in hemostasis and burnt wound healing.17 1.2.2 Novel hemostatic agents A library of choline phosphate (CP) based novel biomembrane adhesives was also previously published by our group. As it is known that phosphatidycholine is the main phospholipid constituent of erythrocytes, an investigation of the interaction (if any) between PC and its inverse structure CP was designed and carried out. Through synthesizing the identical structure except for the inverse orientation of PC, the electrically neutral zwitterion CP when introduced as a multivalent polymer was found to produce strong binding to the cell surface.18 In order to further enhance the binding strength, a methacrylate containing CP monomer (2-(methacryloyloxy)ethyl choline phosphate) was designed and polymerized through atom transfer radical polymerization (ATRP). The resulting polymer poly(2-(methacryloyloxy)ethyl choline phosphate) (PMCP; Fig. 1.4) exhibited good solubility in water and the target molecular weight could be precisely controlled. PMCP was terminated with an azide group, which could be easily linked to other drugs as a drug delivery agent. In this way, the specific drug would be able to be 11  injected or introduced by catheterizatioin precisely to the cells of interest. In order to confirm the site of binding, a fluorescent molecule Alex Fluor 488 was bonded onto PMCP and tested with erythrocytes. The result from confocal laser scanning microscopy showed that around the periphery of the red blood cells the fluorescence was extremely strong, which clearly supported the membrane binding concept. 19  Figure 1.4 Synthetic routes for 2-(methacryloyloxy)ethyl choline phosphate and poly(2-(methacryloyloxy)ethyl choline phosphate)20 (© 2013 Xifei Yu, by permission) Another CP decorated dendritic polymer was also reported to be able to produce strong binding effects to the cell surface. The aggregation strength was controlled by the CP density. The polymer base used here is hyperbranched polyglycerol (HPG), which is extensively studied and analyzed by our group and will be further detailed in the latter part of this chapter. CP was functionalized onto azide modified HPG through a Copper-Catalyzed Azide-Alkyne Cycloaddition, yielding the new membrane adhesive HPG-CP. Under scanning electron microscope (SEM) a shape distortion of tightly aggregated RBC was observed, which indicated that the inherent elasticity of the cell membrane was defeated by the adhesive forces originating from HPG-CP. The mechanism of CP-PC interaction here was proposed to act as two electrostatic quaternary amine(+)-phosphorus(-) pairs forming a quadrupole to provide the energy. Different experiments were carried out to test the hypothesis. Due to the hindrance from the methyl group on the terminal phosphate, the association of the electrostatic pairs was hard to 12  be observed from the 31P nuclear magnetic resonance. A model reaction using prop-2-ynyle choline phosphate (p-CP) and 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) was performed and analyzed by mass spectrum. A peak representing the p-CP/DPPC association was successfully detected. Also the association was confirmed by the fact that under reduced ionic strength buffer the strength of binding and interaction was spectacularly enhanced. It was deduced that the shielding of the potentials around each charged group was effectively weakened allowing enhanced binding. 20  Figure 1.5 Illustration of a phospholipid bilayer containing 1,2-dipalmitoyl-glycero-3-phosphatidyl choline (DPPC)21 (© 2013 Xifei Yu, by permission)  1.3 The development of drug delivery agents  A dendritic polymer was first reported by P. Smith et al 21 in 1985. The structure possesses three unique characteristics to distinguish it from other traditional oligomers, which include an initiator core, interior layers with repeating units and exterior functionalities. Similar to the dendritic polymer, a hyperbranched polymer is composed of dendritic, linear and terminal 13  units and can be synthesized via only one or two steps from ABx monomers. The architecture of hyperbranched hydrophilic polymers determines their exclusive properties like low intrinsic viscosity, excellent water solubility and multi-functionality. Hyperbranched polyglycerol (HPG) is a classical representative of the hyperbranched polymer family. After a series of developments, it has been applied in a variety of diagnostics and therapies due to its exceptional biocompatibility. 22,23   Initially two strategies were applied for the preparation of HPG. The reaction was carried out either by slow addition of monomers or by adopting a vinyl monomer as an initiating group. The drawback was that both pathways led to extremely high polydispersities and broad distribution of molecular weights. A new approach named ring-opening multi-branching polymerization (ROMBP) was explored by Sunder and coworkers in 1999.24  They first partially deprotonated an alcohol ROH as the initiator, then the commercially available epoxide ring glycidol was ring opened by the limited number of alkoxides on the unsaturated side. Unstable secondary alkoxides were generated and transformed into primary alkoxides via intramolecular or intermolecular electron transfer. The polymerization proceeded until the complete consumption of anionic active species occurred. In this manner, controlled molar mass and low polydispersities were achieved. Compared with poly(ethylene glycol) (PEG), high molecular weight HPG exhibited better thermal and oxidative stability and the same or better biocompatibility, thus there was a growing interest in modifications to HPG for it to better serve as a drug delivery agent.25 The installation of hydrophilic or hydrophobic groups could be either performed at the surface or throughout the inner sphere of the polymer.26 After achieving the ROMBP approach, in the same year, Sunder et al 27 reported the preparation of amphiphilic molecular nanocapsules for 14  hydrophilic agents. Through partial esterification of the exterior hydroxyl groups using fatty acid chlorides with the addition of pyridine as the base, HCl was eliminated and a hydrophobic shell was formed. However the interior core retained its hydrophilic nature and became the host for polar agents.  Another way to transport the active compounds is to focus the drug conjugation on the exterior using cleavable links. The controlled release of low molecular weight molecular drugs depends on certain in vivo stimuli, like pH, temperature, redox potential, enzymes or light. 1.3.1 Stimuli-responsive HPG based materials Unlike the common perception of a constant pH = 7.4 in all the biological systems, actually there is a sharp pH gradient in some pathological states. pH-responsive carriers have been designed by Rainer Haag’s group attaching acetal, ketal or imine functionalities. High stabilities for those linkers were observed at pH=8, but the pH shift to 4~5 in malignant tissues could induce the cleavage of those bonds.28 Thermo-sensitive carriers were built on nanoparticles with a tunable lower critical solution temperature (LCST). Frey and coworkers29 presented an HPG modified with N-isopropylacrylamide (NIPAM) groups which could be coated with gold nanoparticles via a non-covalent bond which displayed a broad LCST range. Adeli et al.30 recently reported a thermo and pH dual-sensitive HPG based supramolecular dendrosome as a dual-phase carrier. The noncovalent interactions between the hyperbranched polyglycerol having a β-cyclodextrin core and bi-chain polycaprolactone were stable at 20-37 °C and pH=7-8. The encapsulated drugs would be quickly released due to the dissociation of the dendrosome block when temperatures higher than 37 °C or pH lower than 7 were tested.  The disulfide bond is the most commonly used linkage in the construction of reduction-sensitive carriers, although its in vivo stability still usually remains to be strengthened in the extracellular environment. Enzyme responsive carriers generally rely on the cleavage of esters by 15  esterases. A polyglycerol based copolymer PG-block-poly(-caprolactone) (PCL) was synthesized by Mao et al.31 Verified by fluorescence intensity, the hydrophobic biodegradable PCL chain was degraded upon the addition of a lipase. Light-receptive carriers demand a dark environment before triggering drug release, which was deemed as the main drawback in clinical uses. The Haag group32 developed a photoresponsive HPG with two different oligoamine shells to control DNA release for gene therapy. HPG was first equipped with a photo-sensitive o-nitrobenzyl functionality, then bis(3-aminopropyl)methylamine and pentaethylenehexamine were separately linked to the HPG core to achieve the carrier. The cationic particles on the peripheral surface had an electrostatic interaction with the nucleic acids and protected them from degradation.  1.3.2 Click chemistry Most of the HPG based drug delivery agents have been modified based on the concept of click chemistry, which is to effectively join small units together by developing heteroatomic links (C-X-C). Ideally, those links are able to work reliably in both small and large scale applications. The concept was first proposed by the well-known chemist Sharpless in 2001.33  Click chemistry has a strict standard that requires all the relevant reactions to be modular, wide in scope, give very high yields and generate only inoffensive byproducts that can be removed by non-chromatographic methods like crystallization and distillation. Also the reaction condition is supposed to be mild, and not sensitive to water or oxygen. What is more favored is to be solvent-free or use a solvent which can be removed easily. The introduction of click chemistry united scientists from different backgrounds and it served as a strong basis and catalyst for interdisciplinary research. 34 16  Copper-catalyzed azide/ alkyne cycloaddition (CuAAC) is recognized as a typical click chemistry reaction. Traditionally the cycloaddition of azide was carried out in toluene under refluxing conditions that demanded a high temperature causing the decomposition of unstable molecules. The problem was solved by Meldal et al. 35 through adding copper as a catalyst, which enabled the reaction to be conducted in various solvents and under mild conditions. Later the reaction was successfully applied in the field of synthetic polymer chemistry in combination with ring-opening metathesis polymerization (ROMP).36 The functionalized monomers bearing an acetylene or -bromoalkyl moiety could be polymerized either before or after the click reaction.  Though CuAAC complemented the existing synthetic methodologies, it was still far from flawless. The presence of Cu became the most cited argument due to its interaction with the resulting triazole ring, making the residual material hard to remove from the reaction products. Also the existence of Cu inhibited some biological enzymatic activity and was not biocompatible in general.  The drawbacks became a strong driver for scientists to look for other potential click reactions. Two important metal- free alternatives to CuAAC are the thiol-ene conjugation and the Diels-Alder cycloaddition reaction. General thiol-ene conjugation includes two steps initiated by the addition of the thiyl radical to the carbon of an ene functionality. A carbon-centered radical will then appear and abstract a hydrogen from a thiol group to give a thiyl radical. Termination occurs by radical-radical coupling.37 Since the revival of thiol-ene chemistry, the potentials of other thiol-based reactions have also been evaluated, especially the UV-triggered thiol-ene reactions, which bear the possibility of remote control. 38 17  In recent years chemists have also witnessed an increasing number of contributions based on the reagent-free Diels-Alder [4+2] cycloaddition between diene and dienophile moieties by intramolecular or intermolecular reactions. The Nobel Prize of 1950 was awarded to Otto Diels and Kurt Alder in recognition of the discovery of this valuable transformation. To date, the reaction has been applied in the architecture of a variety of macromolecules with different topologies.39 In 2008 Barner and his coworkers40 established a reversible hetero-Diels-Alder reaction between a cyclopentadiene and an electron-deficient dithioester. They claimed that the bonding occurred under ambient conditions and the debonding could be realized at 100°C, which enhanced the importance of kinetics in the evaluation of click reactions. Billiet et al. 41 recently reported a triazolinedione (TAD) reaction which combined all those above-mentioned metal free, ultrafast and reversible features together. Due to its unique electrophilic nature, the TAD moiety could act both as a strong dienphile and an enophile in either an Ultrafast Diels-Alder reaction or an Alder-ene reaction. The adduct was thermally stable up to 250°C, when released it could transclick with another diene. This unprecedented clean dynamic behavior has great potential in polymer end-group modification. The elaboration of those “Click” derived methods is still of great interest in the design and preparation of unprecedented high-tech polymeric materials. 1.3.3 Zwitterioninc materials Zwitterionic materials are characterized by bearing both cationic and anionic groups in their structures. The unique charged groups and high dipole moments lead to numerous biological applications and are gaining more attention these days. 42 Zwitterionic polymers are considered to be a substitute for poly(ethylene glycol) (PEG) due to their superior ability to prevent nonspecific protein adsorption, particularly since PEG was 18  found to slowly decompose when exposed to oxygen or transition metal ions. 43 An undesired nonspecific protein adsorption which triggers an organism’s defense cascade system is recognized as fouling behavior. The adhesion and propagation of fouling strongly impact the efficacy of surface based diagnoses. Fouling may also induce other adverse effects on healing processes. The key factors leading to the nonfouling behavior of zwitterionic polymers were reported by Chen et al.44 They found that water plays an important role in surface resistance. A notable amount of water is bound onto zwitterions and forms a hydration layer, which is similar but thermodynamically stronger compared to the one formed by PEG via hydrogen bonds.   Among all the existed zwitterionic materials, carboxybetaine and sulfobetaine polymers show superior nonfouling abilities and have attracted extensive related studies. Methacrylate (MA) and acrylamide (AA) are two universal backbones for polymerization. [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide is a commercialized sulfobetaine monomer. It exhibited different nonfouling abilities when grafted onto a surface via particular methods. 45 Under direct atom transfer radical polymerization the surface demonstrated a nonspecific protein adsorption in single protein solutions at an undetectable level, which is lower than 0.3 ng/cm2. When grafting the monomers to the surface of poly(etherurethane) via free radical polymerization, much weaker nonfouling was detected. The two grafting methods suggested disparate surface packing structures and density of the sulfobetaine monomers. Appropriate packing can minimize the strength of the dipoles in zwitterionic materials, which is another key factor determining their nonfouling behavior.  19    Figure 1.6 Structures of zwitterionic polycarboxybetaine and polysulfobetaine: a) zwitterionic polysulfobetaine, b) zwiiterionic polycarboxybetaine, c) cationic polycarboxybetaine esters, d) mixed charge polymers  Glycine-betaine is a kind of compatible solute in organisms controlling osmotic regulation. Similar to its structure, carboxybetaine polymers are recognized as biomimetic materials. Compared with sulfobetaine polymers, besides the nonfouling feature, the abundant carboxylate functionalities enable conjugation with amine-containing groups like protein using 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry.46 Another asset of carboxybetaine is that the free carboxylate can be esterified to generate hydrolysable zwitterionic ester materials. Due to the positive charge of the ester polymers, they displayed a high fibrinogen adsorption behavior, which showed the completely reverse behaviour compared to the hydrolyzed polymers. Also the cationic ester polymers may serve as a DNA carrier and release DNA at the desired location via hydrolysis of the ester groups.47 Differing somewhat from sulfobetaine polymers, in 2014 Vancso and coworkers introduced a new series of zwitterionic sulfates. The derived polysulfabetaines were first synthesized and exhibited different thermoresponsive behaviors. 48 In the structure of sulfabetaines, carbon and sulfur atoms are bonded together through the link of an oxygen atom. The presence of oxygen increases the distance between the nitrogen 20  cation and sulfur anion, which may lead to unique inter/intramolecular interactions. Especially when the chain length is extended due to the existence of oxygen, intermolecular interactions may become dominant.  The preparation of the sulfabetaine monomers was realized by the ring opening reaction of cyclic sulfate by N-(4-vinylbenzyl)-N,N-dimethylamine. The resulting monomers were polymerized via the radical polymerization of the vinyl groups. Hydrogels could be formed due to the strong ionic charges of the polymers and showed different morphological changes at a high temperature.   Also they found that those sulfabetaine based polymers could act as outstanding capping agents to stabilize metal nanoparticles exposed to salt solutions. The aggregation behavior of those metal nanoparticles caused by environmental factors could be adequately controlled through the coordination with the C=O functionality in ester and amide groups prevailing in derived polysulfabetaines. 49 Later in 2016, the application of sulfabetaines was further explored by the Vancso group 48as an antibacterial, electrospun nanofiber due to its strong hydrolytic stability. An electrospinning technique was used on 10 wt% polymer –trifluoroethanol solutions to obtain the smooth and glossy nanofibers. Then antifouling capacity of polymer embedded bead surfaces were evaluated; it was found that the chemical structures of the hydrating layers played an important role in reducing the bacteria accumulation. The antifouling mechanism of the polysulfabetaines was found to be non-biocidal in nature, which provided the opportunity for them to be used in the future as environmental friendly antifouling materials for marine purposes.  21  1.4 Thesis goals and structure A novel hyperbranched polyglycerol (HPG) functionalized with zwitterionic sulfabetaine (SB) and cationic quaternary amines (QA) was previously synthesized and published by our group. 50 The polymer exhibited superior hemagglutination capacity at a concentration ≥ 1 mg/mL and erythrocyte lysis was not observed. The quaternary ammonium ligands effect as a bioadhesion agent via the interaction with the negative charges on the red blood cell membrane. The biocompatibility of the material was achieved by the zwitterionic sulfabetaine through the assembling a hydration layer as the QA polymers are normally damaging to cell membranes.  In this thesis, the goal was to use the above elements to maximize polymer adhesion to cells while eliminating the membrane damage normally associated with multivalent polycation exposure in order to define the stickiest structure on which to base an improved hemostatic material design. To this end an optimization was performed on the synthesis scheme to achieve a library of HPG based polymers with a constant amount of one derivative (SB/QA) and a varied number of the other derivative (QA/SB). 1-Thioglycerol was introduced as a universal ene-moiety blocking agent to provide free hydroxyl groups. In this way, the efficacy of sulfabetaine (SB) and quaternary amine (QA) could be quantified and different hemagglutination behaviors would be displayed.  22   Figure 1.7 HPG conjugation structure In Chapter 2, a detailed synthesis scheme is presented. In general, HPG was synthesized as the polymer backbone and equipped with an allyl substituent. A quaternary ammonium ligand (QA) and zwitterionic sulfabetaine (SB) were coupled onto the polymer in two successive steps via UV initiated thiol-ene “click” reactions. The polymer was purified by dialysis between each step and ultimately reacted with 1-thioglycerol to maintain the solubility and biocompatibility. The corresponding characterization was done by 1H NMR and 13C NMR and the results are also posted in Chapter 2. In Chapter 3, a series of biological tests regarding the hemagglutination, hemolysis, cytotoxicity, hydration properties and inhibition abilities of the synthesized polymers are presented. According to the results, the stickiest polymer conjugate with a biocompatible QA/SB ratio is defined.  23  In Chapter 4, a summary of this thesis is made. Some potential future work is proposed from the perspective of varying the distance between the quaternary ammonium cation and carboxylic anion. And also altering the functionalities of the click reaction in this synthesis scheme to take the advantage of existing varied betaines.     24  Chapter 2: Synthesis and characterization of zwitterionic functionalized hyperbranched polyglycerols  2.1 Synopsis  This chapter reports the optimized preparation and characterization of the two zwitterionic molecules 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (QA) and 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate (SB) based on the previously published papers from our group50,51. The modification of HPG and the conjugation reactions were documented as well. The final polymer conjugates are intended to serve as a topical hemostatic agent. The overall protocols are shown below in Scheme 2.1.  Scheme 2.1 Overall Protocols 2.2 Materials and methods All the chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON) and were used as received without further purification except for glycidol. It was purified by reduced pressure distillation and stored with molecular sieves to maintain an anhydrous state. The solvents were all HPLC-grade from Fisher Scientific (Ottawa, ON) and directly used except for HPGOHPGOHHPGOOHPGO SNOSO3OSBQA SBQAHPGOSN OSO3O S NOHS OHOHHPGOSNOS NOS OHOSO3OHS N25  acetone, which was redistilled to remove trace amounts of water. Regenerated cellulose dialysis tubing (MWCO 1000 Dalton) was purchased from Spectrum Laboratories, Inc. (Houston, TX). The lamp used for UV irradiation was the BlueWaveTM 50 AS UV light curing spot lamp with a power supply of 50 Watt and an emittance of primarily UVA and blue visible light (300-450 nm) from DYMAX Corporation (Torrington, CT). 1H NMR and 13C NMR were performed on Bruker Advance 300 MHz NMR spectrometers, using deuterated water and chloroform (solvent residual signals: 4.79 ppm, 7.26 ppm). Polymer molecular weights and polydispersities were tested by gel permeation chromatography (GPC) techniques by using the NaNO3 solution as the eluent and a DAWN-EOS multi-angle laser light scattering (MALLS) detector coupled to a refractive index (RI) detector.  2.3 Experimental section 2.3.1 Preparation of HPG (20K) (1a) HPG was synthesized following the published methods as is shown in Scheme 2.2. Being very sensitive to moisture, the whole synthesis process was conducted under the protection of argon. A 250 mL three-neck round bottom flask equipped with a stir bar was flame dried and cooled to room temperature. 1,1,1-tris(hydroxymethyl)propane (TMP) (0.1714 g,1.28 mmol) was added as initiator and heated at 70°C until it melted as a viscous liquid, then it was partially (10%) deprotonated by adding 25% wt potassium methylate methanol solution (0.11 mL). After reacting for 15 min, excess methanol was distilled off under vacuum and the reaction mixture was like a white solid after 3 h distillation. The flask was equipped with a mechanical stirrer and 22 mL (0.34mol) glycidol was injected by syringe pump over 12 h at 90°C. The reaction was quenched by adding 60 mL of methanol and then neutralized by adding 2 M HCl solution. The molecular weight of the crude product was determined by a GPC-MALLS system equipped with 26  Waters ultrahydrogel columns and using 0.1M sodium nitrate aqueous solution as eluent. The polymer was further precipitated by adding acetone to remove the lower molecular weight parts. The precipitate was concentrated under rotavapor, then dissolved and dialyzed against water for 3 days using a 1000 Da MWCO cellulose membrane tubing. The final product was lyophilized and a viscous colorless liquid was obtained with a yield of ~60%. The PDI (1.24) was determined by injecting into the GPC-MALLS and the structure was determined by 1H NMR and 13C NMR. 1H NMR (300 MHz, D2O) δ 4.05, 3.92 (b, -OH), 3.87-3.52 (b, -CH2CHOHCH2-, -CH2-). 13C NMR (100 MHz, D2O) δ 79.64 (L13, -CH-), 78.24 (D, -CH-), 72.35 (L14, -CH2-), 71.10 (D, -CH2-), 70.91 (T, -CH2-), 70.63 (L13, -CH2-), 69.39 (L14, -CHOH-), 69.09 (T, -CH2OH), 62.82 (L13, -CH2OH-).  Figure 2.1 GPC chromatogram of HPG (Mn=2.0×104, PDI=1.24, RI peak shown in blue, LS peak shown in red, which was measured at scattering angle: 90°)   27   Scheme 2.2 Preparation of HPG (20K) (1a) 2.3.2 Preparation of allyl functionalized HPG (1b) Allyl functionalization of HPG was achieved by allyl chloride nucleophilic substitution as it is shown in Scheme 2.3. HPG (1a) (Mn=2.0×104 Da, PDI=1.24, 1.275 g, 17.2 mmol hydroxyl groups), NaOH (2.4 g, 51.6 mmol, 3.5 eq.) and phase transfer reagent tetrabutyl ammonium bromide (TBAB) (0.13 g, 0.4 mmol, 3% mole per mole of –OH group) were measured into a 50 mL two-neck round bottom flask. Seven mL H2O was added to dissolve and stirred at 60°C for one hour to allow the deprotonation of the hydroxyl groups.  The flask was equipped with a refluxing condenser and then 4.6 g allyl chloride (51.6 mmol, 3.5 eq.) was injected via a syringe pump over 1h. The reaction was kept stirring under refluxing at 60°C for 12 h. The viscous reaction mixture was transferred directly into a 1000 Da MWCO cellulose membrane tubing and dialyzed against acetone for one day, then dialyzed against water for another two days. The modified HPG was recovered by lyophilization to give a light yellowish OH OOHOOOOOOOHOOHOHHOOHOOHOOHOOHOOOHOOOOOOHOHOOHOOHOHHOHOOHOHHOHOHOHOHO90°CO1aOHOHOOHOHOOHO28  oil (~1.2 g). The percentage of functionalization was characterized by 1H NMR integration analysis. 1H NMR (300 MHz, CDCl3) δ 5.88 (m, CH2=CHCH2-), 5.28 (s, CH2=CH-), 5.23 (s, CH2=CH-), 5.14 (t, CH2=CH-), 4.14 (s, -OCH2CH=CH2), 3.98 (s, -OCH2CH=CH2), 3.66-3.46 (m, -OCH2-). 13C NMR (100 MHz, CDCl3) δ 135.44 (CH2=CHCH2-), 134.97 (CH2=CHCH2-), 116.93 (CH2=CH-), 72.40-70.42 (-OCH2-).  Scheme 2.3 Preparation of Allyl functionalized HPG (1b) 2.3.3 Model reaction of HPG-thiol-OH (1c) A model reaction was also carried out as presented in Scheme 2.4 in order to help confirm that the peaks were only from 1-thiolglycerol clicked onto HPG-allyl (1b) and to test the reactivity of the UV induced thiol-ene reactions.  Scheme 2.4 Model reaction of HPG-thiolglycerol (1c) The functionalization of HPG-allyl was realized by a UV light photocatalytic thiol–ene reaction. To a solution of HPG-allyl (100 mg, 0.811 mmol) in 2,2,2-trifluoroethanol (5 mL) was added 1-thiolglycerol (175 mg, 1.622 mmol). 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (0.3 mol per mol of allyl groups) was added as photo initiator. The reaction was carried out under UV (365 nm) for 3 h. The excessive catalyst and starting material were removed by dialyzing against in 0.1M NaCl solution and then deionized water with a 1000 29  Da MWCO regenerate cellulose membrane bag. 1c was recovered by lyophilization to give a white foaming solid. All the alkenes were consumed as indicated by 1H NMR analysis. 1H NMR (300 MHz, D2O) δ 4.19 (b, HO-CHCH2-CH2OH), 3.82 (b, HO-CH2-CH(OH)-), 3.75-3.65 (b, -OCH2-), 3.04 (b, HO-CH(CH2OH)-CH2-S-, -CH2-S-CH2-), 2.09 (b, -CH2-S-CH2-). 2.3.4 Preparation of 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium)bromide (2a)  Scheme 2.5 Preparation of 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium)bromide (2a) To a solution of (3-bromopropyl) trimethylammonium bromide (4.90 g, 18.7 mmol) in distilled water (20 mL) was added sodium thiosulfate pentahydrate (5.12 g, 20.6 mmol). The solution was refluxed at 100°C for 24 h and the reaction monitored by 1H NMR. Upon complete consumption of the starting material, the reaction mixture was hydrolyzed and oxidized to the corresponding disulfide salt by adding 2 M H2SO4 solution (20 mL). The solution was refluxed for one more day before neutralizing by adding 2 M NaOH solution. The white powder-like reaction mixture was recovered by lyophilization, which contained both the inorganic salt and the disulfide product. The reaction mixture was washed with methanol three times to remove the insoluble inorganic salt. The solvent was removed in vacuo to give 2a as a white solid. Yield=5.42 g (68%). 1H NMR (300 MHz, D2O) δ 3.57 – 3.47 (m, 4H, -CH2CH2N(CH3)3), 3.20 (s, 18H, -N(CH3)3), 2.86 (t, 4H, S-S-CH2-CH2), 2.37 – 2.22 (m, 4H, -CH2CH2CH2). 30  2.3.5 Preparation of 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (2b)  Scheme 2.6 Preparation of 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (2b) Triphenylphosphine was used here as the reducing reagent to break the disulfide bond in 2a as is shown in Scheme 2.6. 2a (2.02 g, 4.7 mmol) was first dissolved in 14 mL distilled water and then 7 mL of 2,2,2-trifluoroethanol (TFE) was added. To a solution of triphenylphosphine (12.42 g, 47 mmol) in CH2Cl2 (14 mL)  was added the 2a solution. The emulsion was formed by vigorous stirring. After stirring for two days under room temperature, the reaction mixture was extracted with DCM (3 x 50 mL) to remove triphenylphosphine. TFE was removed by rotavapor, and 1H NMR was performed to track reducing progress. When necessary, triphenylphosphine and the solvents were added again for further reducing. The final product was recovered by lyophilization and kept under argon atmosphere in a -20°C fridge to prevent oxidation. Yield=0.98 g (49%). 1H NMR (300 MHz, D2O) δ 3.63 – 3.51 (m, 2H, -CH2CH2N(CH3)3), 3.26 (s, 9H, -N(CH3)3), 2.75 (t, 2H, HS-CH2-CH2), 2.31 – 2.14 (m, 2H, -CH2CH2CH2). 13C NMR (100 MHz, D2O) δ 65.50 (-CH2CH2N(CH3)3), 53.49 (-N(CH3)3), 26.81 (HS-CH2), 20.86 (-CH2CH2CH2). 31  2.3.6 Preparation of 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a)  Scheme 2.7 Preparation of 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) To a solution of 3-dimethylamino-1-propyl chloride hydrochloride (5.34 g, 33.8 mmol) in distilled water (35 mL) was added sodium thiosulfate pentahydrate (9.22 g, 37.2 mmol). The solution was refluxed at 100°C for 24 h and the reaction monitored by 1H NMR. Upon complete consumption of the starting material, the reaction mixture was hydrolyzed by adding 2 M HCl solution to pH=1. The solution was stirred under room temperature for one day, then 2 M NaOH solution was added to adjust the pH to 10~11. Oxygen was bubbled into the solution to oxidize the product to the corresponding disulfide 3a. 3a was recovered by extracting the aqueous phase using DCM (3 x 30 mL) and dried in vacuo to give 3a as a yellowish oil. Yield=2.87 g (72%). 1H NMR (300 MHz, D2O) δ 3.57 – 3.49 (m, 4H, -S-S-CH2), 3.41 (t, J = 7.0 Hz, 4H, -CH2N(CH3)2), 3.15 (s, 12H, -N(CH3)2), 2.47 (t, 4H, CH2CH2CH2). 2.3.7 Preparation of bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) (3b)   Scheme 2.8 Preparation of bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) (3b) 3a (1.07 g, 4.5 mmol) was added into a strictly flame dried flask, dissolved in anhydrous acetone (12 mL) under 0°C. 1,3,2-dioxathiolane 2,2-dioxide (1.45 g, 11.6 mmol) was dissolved in 10 mL anhydrous acetone and added into the solution of 3a by syringe pump in 2 h. The 32  reaction was stirred under 0°C for 2h and then allowed to warm to room temperature. A white precipitate gradually formed during the addition of the 1,3,2-dioxathiolane 2,2-dioxide solution. After 12 h stirring, the reaction mixture was filtered and the precipitate was washed with acetone (15 mL) under centrifuge three times. The solvent was decanted and further removed in vacuo to give 3b as a white solid. Yield=1.83 g (84%). 1H NMR (300 MHz, D2O) δ 4.49 (s, 4H, -CH2OSO3-), 3.83 – 3.72 (m, 4H, CH2CH2OSO3-), 3.60 – 3.49 (m, 4H, -CH2CH2CH2N(CH3)2), 3.21 (s, 12H, -N(CH3)2), 2.82 (t, 4H, S-S-CH2), 2.34 – 2.24 (m, 4H, -CH2CH2CH2N(CH3)2). 2.3.8 Preparation of 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate (3c)  Scheme 2.9 Preparation of 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate (3c) The reducing of 3b was done in the same manner as 2a by using triphenylphosphine as is shown in Scheme 2.9. 3b (1.83 g, 3.8 mmol) was first dissolved in 10 mL distilled water and then 5 mL of 2,2,2-trifluoroethanol (TFE) was added. To a solution of triphenylphosphine (9.9 g, 38 mmol)  in CH2Cl2 (10 mL)  was added the 3b solution. The emulsion was formed by vigorous stirring. After stirring for three days under 35 °C, the reaction mixture was extracted with DCM (3 x 50 mL) to remove triphenylphosphine. TFE was removed by rotavap, and 1H NMR was performed to track reducing progress. Triphenylphosphine and the corresponding solvents had to be added two more times to ensure 100% reduction. The final product was recovered by lyophilization after removal of reducing reagent and kept under argon atmosphere in -20°C to prevent oxidation. Yield=1.02 g (56%). 33  1H NMR (300 MHz, D2O) δ 4.47 (s, 2H, -CH2OSO3-), 3.80 – 3.70 (m, 2H, CH2CH2OSO3-), 3.57 – 3.47 (m, 2H, HS-CH2CH2CH2N(CH3)2), 3.18 (s, 6H, -N(CH3)2), 2.62 (t, 2H, HS-CH2), 2.11 (p, 2H, -CH2 CH2CH2-). 13C NMR (100 MHz, D2O) δ 64.20 (HSCH2CH2CH2), 62.67 (CH2CH2OSO3-), 61.97 (CH2CH2OSO3-), 51.89(-N(CH3)2), 26.35 (HSCH2), 20.64(HSCH2CH2CH2). 2.3.9 Preparation of HPG-SB (4a)  Scheme 2.10 Preparation of HPG-SB (4a) The functionalization of HPG-allyl was realized by a UV light photocatalytic thiol–ene reaction. To a solution of HPG-allyl (100 mg, 0.811 mmol) in 2,2,2-trifluoroethanol (5 mL) was added 3c. 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (0.3 mol per mol of allyl groups) was added as photo initiator. The reaction was carried out under BlueWaveTM 50 AS UV light curing spot lamp with a wavelength of 365 nm for 3 h. Distilled water was gradually added to check the progress of the reaction until the solution didn’t turn turbid. The excessive catalyst was removed by dialyzing against 0.1M NaCl solution and then deionized water with a 1000 Da MWCO regenerated cellulose membrane bag. The percentage of SB installed was calculated by 1H NMR and verified by GPC-MALLS.  2.3.10 Preparation of HPG-SB-QA (4b) and HPG-SB-QA-OH (4c)  Scheme 2.11 Preparation of HPG-SB-QA (4b) 34     Scheme 2.12 Preparation of HPG-SB-QA-OH (4c) Preparation of different percentages of SB/QA modified HPG used 4a as the starting material to keep SB groups always constant, the amount of QA added was varied as shown in Scheme 2.11. The number of each zwitterion group clicked onto HPG was determined by 1H NMR and verified by GPC-MALLS. In order to block the free allyl group, excessive 1-thioglycerol was added in the last step and clicked onto HPG as it is shown in Scheme 2.12.    35  2.4 1H NMR and 13C NMR characterizations  2.4.1 Characterization of HPG (20K) (1a)  Figure 2.2 HPG (1a) 1H NMR in D2O   Figure 2.3 HPG (1a) 13C NMR in D2O 36  The identical –CH and –CH2 groups on HPG backbone with a slightly different chemical environment lead to an imperceptibly different chemical shift for each group, and thus a broad peak is formed on 1H NMR between 3.50-3.85 ppm. According to the analysis of Sunder et al.24 shown below, linear(L13,L14), terminal(T) and dendritic(D) repetitive units are formed during the complicated polymerization process and give a special carbon architecture of HPG as it is reflected on 13C NMR.   Scheme 2.13 Carbon architectures in HPG backbone 37  2.4.2 Characterization of allyl group modified HPG(20K) (1b)  Figure 2.4 HPG-allyl (1b) 1H NMR in CDCl3    Figure 2.5 HPG-allyl (1b) 13C NMR in CDCl3 38                  HPG was allylated with allyl chloride in aq. NaOH under phase-transfer catalyst. The peaks b and c located at 5.9 and 5.2 ppm correspond to the newly installed C=C moiety. The signals at 4.0 and 4.2 ppm are assigned to protons on methylene groups. A trace amount of HDO can be observed at 1.8 ppm. The percentage of allylation on HPG can be calculated by comparing the integration of peak b and HPG backbone. Each repeating unit on HPG back bone contains 5 protons, if the hydroxyl groups on each repeating unit are all modified with allyl groups, then the integration of b should be exactly 1. Here in this batch, the HPG is about 95% modified (5/5.21). The percentage of modification can be adjusted by adding different equivalents of allyl chloride as the starting material or modifying the reaction time.  2.4.3 Characterization of HPG-thiol-OH (1c) As a model reaction, HPG-allyl was clicked with 1-thiolglycerol in order to test the click reaction conditions and exam the reactivity. The chemical shift of each peak for 1c also helps with the peak allocation for the final product 4c. As it is shown in the 1H NMR, after treatment with excessive 1-thiolglycerol, resonances from the C=C moiety have all gone and migrated upfield to 3.1 and 2.1 ppm. The resonance at 3.1 ppm is also partially contributed by the protons of -CH(OH)-CH2 (indicated by italics) due to the weaker electronegativity of S compared to O. Peaks d and c in the downfield are allocated to the protons of carbons bonded to hydroxyl groups.  39   Figure 2.6  HPG-thiol-OH (1c) 1H NMR in D2O 2.4.4 Characterization of 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium) bromide (2a)  Figure 2.7 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium) bromide (2a) 1H NMR in D2O 40   Figure 2.8 3,3'-disulfanediylbis(N,N,N-trimethylpropan-1-aminium) bromide (2a) 13C NMR in D2O A bunte salt was formed by the alkylation on the pendant sulfur of sodium thiosulfate and then further hydrolyzed and oxidized to the corresponding disulfide 2a through using sulfuric acid.  On the 1H NMR all the four peaks can be clearly assigned. The resonance at 2.3 ppm is allocated to peak b due to the lowest deshielding effect. Since nitrogen has a stronger electronegativity than sulfur, peak c besides nitrogen is experiencing a lower electron density and less shielding than peak a. The singlet peak d at 3.2 ppm corresponds to the three identical methyl groups of the quaternary amine.  41  2.4.5 Characterization of 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (2b)  Figure 2.9 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide (2b) 1H NMR in D2O   Figure 2.10 3-mercapto-N,N,N-trimethylpropan-1-aminiumbromide (2b) 13C NMR in D2O 42  The disulfide was reductively cleaved when treated with Ph3P in a TFE/DCM/H2O system. In order to achieve the fastest reducing rate, the reaction condition was altered by conducting it under 35°C or inert gas under room temperature, which both yielded a similar reducing rate. The reaction progress was monitored by 1H NMR using peak a and b as the marks. As the crack of the S-S bonds broke, the peaks at 2.9 and 2.3 ppm gradually migrated upfield to around 2.7 and 2.2 ppm. The completion of reaction was recognized when all the peaks at 2.9 and 2.3 ppm disappeared.  2.4.6 Characterization of 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a)  Figure 2.11 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) 1H NMR in D2O 43   Figure 2.12 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3a) 13C NMR in D2O 3a was synthesized in the same manner as 2a by the alkylation on the pendant sulfur of sodium thiosulfate to form a bunte salt, which was further hydrolyzed and oxidized to the corresponding disulfide using hydrochloric acid.  As is mentioned above, peaks c and a are spotted at 3.55 and 3.45 ppm due to the electronegativity of N and S. The least deshieding methylene group in the middle and the methyl groups of the amine are shown at 2.5 and 3.15 ppm. 44  2.4.7 Characterization of bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) (3b)   Figure 2.13 bis(3-dimethylammoniumdiyl)bis(ethane-2,1-diyl)bis(sulfate) (3b) 1H NMR in D2O  3b was prepared through the ring opening alkylation of 3a by 1,3,2-dioxathiolane 2,2-dioxide. The product was insoluble in the reaction solvent (acetone) and precipitated out. Newly generated peak f and e can be observed at 4.5 and 3.8 ppm. The peak at 2.21 ppm comes from the residual trace amount of acetone.    45  2.4.8 Characterization of 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate (3c)  Figure 2.14 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3c) 1H NMR in D2O  Figure 2.15 3,3'-disulfanediylbis(N,N-dimethylpropan-1-amine) (3c) 13C NMR in D2O 46  3c was obtained by treating with Ph3P in the same way as 2b. The reducing rate was also monitored by the conversion of the two methylene groups. Peaks at 2.8 and 2.3 ppm progressively shift upfield to 2.6 and 2.1 ppm until completion.   2.4.9 Characterization of HPG-SB (4a), HPG-SB-QA (4b) and HPG-SB-QA-OH (4c)  Figure 2.16 HPG-SB (4a), HPG-SB-QA (4b) and HPG-SB-QA-OH (4c) 1H NMR in D2O  Monomers 2b and 3c were conjugated onto 1b successively through UV light induced click reactions by using 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone as the photo initiator. The reaction was conducted for 1h and 3h separately under UV light to compare the conjugation efficiency. The 1H NMR results show that if the reaction is only exposed to UV light for 1h, the unreacted monomers are still trapped in the polymer even after dialysis in regenerated cellulose membrane against water. However, for the 3h long batch, the resonance of the small molecules cannot be observed on 1H NMR after dialysis, which means all of the 2b and 3c 47  monomers were successfully clicked onto 1b.  3c was first conjugated onto 1b to obtain 4a, which is the starting material for conjugation with 2b. According to the previous calculation, around 90% of the –OH groups on HPG were modified with allyl groups to give 1b. The percentage of 3c clicked onto HPG was calculated by comparing the integration of peak i with the HPG backbone after the removal of overlapped peak e. Peak i stands for the allyl groups (-CH2CH=CH2) conjugated on HPG and will be consumed once monomers are clicked onto the polymer. When another kind of monomer 2b is further attached onto HPG-SB to yield 4b, the residual allyl groups will also decease as a result of the click reaction and can be quantified through 1H NMR. Thiol-glycerol was added in the final step to block all the remaining allyl groups. As it is indicated in Figure.2.15-4c, peak i and j disappeared by reason of the complete consumption of allyl groups. 2.4.10 Comparison of HPG-SB30-QA10/20/30, HPG-SB30-QA10/20/30-OH60/50/40  Figure 2.17 1H NMR of HPG-SB30-QA10, HPG-SB30-QA20 and HPG-SB30-QA30 in D2O 48   Figure 2.18 1H NMR of HPG-SB30-QA10-OH60, HPG-SB30-QA20-OH50 and HPG-SB30-QA30-OH40 in D2O The amount of 2b and 3c monomers conjugated onto 1b could be clearly visualized and quantified by peak f and g as they represent the protons of the quaternary amines of SB and QA respectively. As is shown in Figure 2.15, HPG-SB30 was reacted as the starting material with different equivalents of QA, which contributed to 10%, 20% and 30% QA installation. The resonances of remaining allyl groups were still at 6.0 and 5.4 ppm. The materials in Figure 2.16 were further clicked with 1-thiolglycerol to consume all the remaining C=C bonds. In Figure 2.17 it shows the spectrums of HPG-SB30-QA10-OH60, HPG-SB30-QA20-OH50 and HPG-SB30-QA30-OH40, indicated by the appearances of peaks k and m. Those two new peaks represent the signals of -O-CH2-CH2-S-(indicated by italics), accordingly the downfield signals of the allyl groups completely disappeared. 49  2.5 Summary In this chapter, two zwitterionic molecules 3-mercapto-N,N,N-trimethylpropan-1-aminium bromide and 2-((3-mercaptopropyl)dimethylammonio)ethylsulfate were synthesized and installed onto HPG-allyl through UV induced click reactions. For the monomer synthesis, different reaction time and air sensitive conditions were evaluated to ensure the complete cleavage of the disulfide bonds. Also by adjusting the pH and performing multiple extractions, the zwitterions were obtained in an economical, green and column-free method. The allylation of HPG was up to 90% through controlling the addition of allyl chloride and sodium hydroxide.   The click reactions were conducted in a sequential manner to make sure the amount of QA or SB could be precisely controlled. A series of SB constant polymers: HPG-SB30-QA10-OH60, HPG-SB30-QA20-OH50, HPG-SB30-QA30-OH40, HPG-SB30-QA40-OH30 and etc., as well as QA constant polymers HPG-QA30-SB10-OH60, HPG-QA30-SB20-OH50, HPG-QA30-SB30-OH40, HPG-QA30-SB40-OH30 and etc., were successfully obtained for further biological tests.  50  Chapter 3: In Vitro red blood cell binding and biocompatibility tests 3.1 Introduction In Chapter 2 the design of polymer conjugates with different zwitterionic and cationic moiety ratios was introduced. This chapter describes their interactions with biological systems. The polymer conjugates produced were applied to test the in vitro hemostatic effects on red blood cells which takes advantage of the negative charge on the red blood cell surface due to sialylated glycoproteins embedded in the lipid bilayers.52 Those negative charges work effectively in preventing the interactions between red cells and other kinds of cells. In this chapter results are described for experiments in which a series of polymer conjugates containing the same percentage of SB and a variable amount of QA were incubated with red blood cells to determine the least amount of QA required to show its aggregation efficacy.   In case of red blood cell membrane lysis, the hemolytic potential of the polymer conjugates was also evaluated through controlling the contact time and comparison with different controls. The cyanmethemoglobin method was applied for hemoglobin determination and the absorbance at 540 nm was examined. The best amount of SB was determined with almost no detectable hemolytic activity induced.  The hydration properties of the polymer conjugates were also measured by using differential scanning calorimetry. The ice to water fusion enthalpy of the sample solution is reduced if a significant number of water molecules is bound to each repeat monomer unit. The hydration layer contributes to the fouling resistance characteristic of the zwitterions and helps provide a better understanding of the excellent biocompatibility exhibited by the polymer conjugates.  A cytotoxicity evaluation of the polymer conjugates was also performed on a human 51  umbilical vein endothelial cell line through using propidium iodide and Hoechst 33258 as the chromosome staining dyes. When the integrity of the cell membrane was compromised, the emitted fluorescence was able to be detected by the imaging system. The cytotoxicity test was performed to investigate the safety of the polymer conjugates on the vascular system and the feasibility of their in vivo exposure. To exam the mechanism proposed for the hemostatic activity of the polymer conjugates, a kind of linear sulfated polyglycerol was synthesized and applied as a possible reversal agent. The negative charges on the sulfate were supposed to be able to neutralize the positively charged moieties on the polymer conjugates and compete with its electrostatic interaction with the red blood cell membrane. The resulting inhibition of the strong aggregation contributed to our understanding of the electrostatic interaction on the cell surface and showed the potential to apply the polymer conjugate with best SB/QA ratio as a topical hemostatic dressing on the basis of further in vivo tests. 3.2 Experimental section 3.2.1 Materials preparation  The polymer conjugates were synthesized as described in Chapter 2. Blood was drawn from healthy donors by a process approved by the UBC Clinical Ethics Committee (Approval Number: H07-02067 granted to the Centre for Blood Research). In the case of coagulation measurements, freshly drawn blood was stored in a 3.8% sodium citrated tube (BD VacutainerTM). Through inverting the tube, nine parts blood and one part sodium citrate mixed together and the binding of calcium by the salt induced anticoagulation immediately. Phosphate-buffered saline (PBS) was used as the buffer to mimic the osmolality and ion concentration of human body to maintain the erythrocytes in good condition. PBS solution was obtained by 52  dissolving the PBS tablet from Sigma-Aldrich Canada Ltd. (Oakville, ON) in 200 mL deionized water, which was double purified by a Milli-Q Plus water purification system (Millipore Corp., Bedford, MA) before using. The final solution yielded 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C. Drabkin’s Reagent was obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON) in a powder form and each vial was reconstituted with 1000 mL of deionized water to prepare the desired solution. Propidium iodide and Hoechst 33258 HUVEC were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON). 3.2.2 The aggregation test on red blood cells Red blood cell aggregation observation was conducted by using freshly drawn citrated anticoagulated blood according to the previously published procedures from our group25. Five milligrams and 25 mg of polymer conjugates were measured and dissolved in 0.5 mL PBS solution to achieve the desired polymer stock solutions (10mg/mL and 50mg/mL). Ten microlitres of the stock solutions were transferred into 90 L of citrated whole blood and completely mixed by aspiration. In this way the final concentrations of the polymer conjugates were 10 times diluted to 1mg/mL and 5mg/mL respectively. The mixtures were incubated at 37 °C in a thermostatic water bath for 5 min. After incubation, the polymer-whole blood mixtures were separated by centrifuge at a speed of 8000 rpm for 3 min, then 6 L of the supernatant was dropped onto the glass slide and 2 L of the red blood cells resuspended in that fluid. Microscopic images were captured by Axioskop2 plus microscope (Carl Zeiss Microimaging Inc.) at 40x magnification.  Washed red blood cells were prepared from the citrated whole blood. One half millilitre of PBS solution was added into 0.5 mL of the citrated whole blood and gently mixed. The mixture was centrifuged at 1000 g for 5 min. The supernatant was then removed and replaced by 53  a new PBS solution. The washing process was repeated for three times to achieve the washed red blood cells. The aggregation of washed red blood cells was performed in the same way as whole blood cells. PBS incubated red blood cells without polymer addition was used as the negative control for the aggregation tests.   3.2.3 Hemolytic activity analysis The whole blood sample was drawn into a Micro-Hematocrit Capillary Tube and centrifuged at 13000 rpm for 7min. The hematocrit was measured by using a micro-capillary reader and the whole blood diluted to 10% v/v accordingly. Cell lysis was assessed as follows. Thirty microliters of 50 mg/mL polymer conjugate stock solution was added into 270 L of 10% hematocrit RBC suspension to achieve a 5 mg/mL final concentration. The mixture was incubated at 37 °C for one hour. A RBC suspension at 10% hematocrit incubated with PBS solution formed the blank while a fully lysed sampled was made by water addition; these were employed as negative controls and positive controls respectively. To 1mL Drabkin’s Solution, 20 L of the polymer/whole blood solution was added and mixed well, inducing lysis. The mixture was allowed to stand for 20 min at room temperature for complete reaction. Another 20 L of the polymer/whole blood solution was centrifuged and the supernatant was separated and treated with another 1 mL Drabkin’s Solution. The absorbance of the whole blood mixture and the supernatant were read under 540 nm by using the SpectraMax® M3 Multi-Mode Microplate Reader in a costar 96 well plate. The extent of lysis was calculated by using the absorbance of supernatant sample divided the absorbance of the whole blood sample. A 1 mg/mL polymer conjugate stock solution was also tested by using the same method. 54  3.2.4 Quantification of bound water by differential scanning calorimetry  The hydration properties of the polymer conjugates were measured by using a Q2100 differential scanning calorimetry (DSC) (TA instruments, New Castle, DE, USA). The polymer conjugates were all prepared in a concentration of 10 mg/mL with deionized water. An empty Tzero pan with a Tzero hermetic lid was weighed by using the balance of a thermogravimetric analysis (TGA) instrument (TA instruments, New Castle, DE, USA) as the empty reference. Twenty microliters of each 10%w/w sample was loaded into the pre-weighed Tzero pans equipped with Tzero hermetic lids. All the pans were properly sealed by using a die set. The exact weight of the samples loaded was obtained by difference.  The method used was previously developed by our group53. Basically the temperature was cooled down at 20°C/min to -30°C and held isothermally for 10 minutes, then the temperature was ramped up at 1°C/min to -10°C. The sample was then continuously heated at 0.2°C/min to 6°C, thereafter at 1°C/min to 25°C. The ice to water fusion heat enthalpy (∆𝐻𝑝) was calculated from the integration of the peak at the negative temperatures around 0°C from TA Universal Analysis 200054. The number of water molecules bound to each polymer (N) was calculated by plugging ∆𝐻𝑝 into the following equation53: 𝑁 =∆𝐻0(1−𝐶𝑝 )−∆𝐻𝑝𝑀𝐻2 𝑂∆𝐻0𝐶𝑝 ∙ 𝑀𝑛           (Eq. 3.1) Where ∆𝐻0 is the fusion enthalpy of pure water obtained under the identical experiment conditions, 𝐶𝑝  is the weight percentage of each polymer sample, 𝑀𝐻2 𝑂 is the molecular weight of pure water and  𝑀𝑛 represents the molecular weight of the polymer conjugates.  55  3.2.5 Cytotoxicity evaluation of the polymer conjugates For all experiments, human umbilical vein endothelial cell viability was determined by staining cells with propidium iodide and Hoechst 33258.55 Concentrations of 5, 10 and 20 mg/mL polymer conjugates in PBS solutions were prepared. The cell-containing T-75 flask was placed in a 37°C, 5% CO2 humidified incubator for 4 hours and then the medium was changed to 20 mL fresh Endothelial Cell Growth Medium. Twenty-four hours later the medium was removed by aspiration and 2 mL Trypsin/EDTA solution was added. After incubation for 2 min, 40 mL of the medium was added and the solution was split into two T-75 flasks. Two hundred microliters of the solution was transferred to the wells of a 96-well plate. After 24h incubation at 37°C, the old medium was removed and 180 L fresh medium was added. Twenty microliters of each stock solution was added to the appropriate wells to give final concentrations of 0.5, 1 and 2 mg/mL. Twelve wells of the cells were treated with 50% DMSO to induce cell death as positive controls. Another 12 wells were addition-free and kept as negative controls. The plate was incubated for another 48h before staining. After chemical exposure, the staining propidium iodide and Hoechst 33258 were added directly to the medium to achieve a final concentration of 2.5 mol/L and 12 g/mL respectively. After 15 min incubation at 37°C, the live cell nuclei were labeled blue and dead cells were labeled red due to the dyes different cell membrane permeability. The cells were imaged using a Cellomics Arrayscan VTI high content imaging system. The viability was calculated as the percentage of cells which were not stained by propidium iodide.  3.2.6 Reversibility analysis of the red blood cell aggregates LPG-OSO3- (Mn:4700, PDI:1.6) was synthesized following the method in a previously published paper50. Five milligrams of the positively charged polymer conjugate (HPG-SB30-56  QA30-OH40) and LPG-sulfate were measured and dissolved in 0.5 mL PBS solution to achieve a concentration of 10mg/mL. Ten microlitres of the stock solution was transferred into 90 L of citrated whole blood and completely mixed by aspiration. After a 5-min long incubation at 37 °C, the mixture was centrifuged. The red blood cells were resuspended into the supernatant and examined under the microscope.  3.3 Results and discussion 3.3.1 The aggregation effects on red blood cells Based on what was found in previous studies50 it was decided to test polymer conjugates containing 30% of the zwitterion SB prepared to maintain the biocompatibility and different percentages of positive charged QA added onto HPG-SB30 to obtain HPG-SB30-QA10-OH60, HPG-SB30-QA20-OH50, HPG-SB30-QA30-OH40 and HPG-SB30-QA40-OH30 . With the percentage of SB being constant, the content of cationic quaternary amine was gradually increased to determine a reasonable amount able to cause aggregates while minimizing damage to the cell membranes. The representative optical microscopic images of the red blood cells incubated with the HPG conjugates are shown in Figure 3.1 and 3.2. In Figure 3.1(I), without the addition of HPG conjugates, PBS incubated whole red blood cells exhibited the original rouleaux status. As 5 mg/ mL HPG-SB itself was not observed to be able to induce aggregates, positively charged QA was responsible for the aggregation effects observed. The positive charge density of the lowest amount of QA (10%) was not strong enough to formulate the aggregates (data not shown). In Figure 3.1 (II), very mild aggregation was observed when the content of QA was elevated to 20%. As the percentage of QA was increased to 30%, red blood cells started to clump together without forming strings of cells and the positive charge was high enough to induce moderate 57  hemagglutination. When the amount of QA reached 40% as it was shown in Figure 3.1 (IV), a large sized clot was formed due to the strong affinity. A similar trend is also observed in Figure 3.2, when the concentration of the polymer conjugates was decreased to 1mg/mL. When the content of QA was as low as 10~20%, hardly any aggregates could be observed and the red blood cells stayed in their typical rouleaux form. Small aggregate size and many single cells were characteristic of these conditions.  c      Figure 3.1 Erythrocytes aggregation effects of 5mg/mL HPG conjugates after 5 min incubation at 37°C in human whole blood. (I) PBS control (II) HPG-SB30-QA20-OH50, (III) HPG-SB30-QA30-OH40, (IV) HPG-SB30-QA40-OH30 spc  (I) (II) (III) (IV) 58  The aggregation effects were also tested on washed blood cells as is shown in Figure 3.3. After washing in PBS, the rouleaux construction of the red bloods was not observed and they were dispersed evenly in the solution. After incubation at 1mg/mL polymer conjugates, weak aggregation could be detected when the content of QA was 20%. When it came to 30%, obvious large area aggregation is exhibited. The effect became stronger as the percentage of cationic QA was elevated to 40, which agreed with the phenomenon noticed for whole blood cell tests.  However, compared with whole blood cell suspensions, the same polymer conjugates s         Figure 3.2 Erythrocytes aggregation effects of 1mg/mL HPG conjugates after 5 min incubation at 37 °C in human whole blood. (I) HPG-SB30-QA10-OH60 (II) HPG-SB30-QA20-OH50, (III) HPG-SB30-QA30-OH40, (IV) HPG-SB30-QA40-OH30    (I)  (II) (III) (IV) 59  under all concentrations displayed better aggregation efficacy. A proposed interpretation50 is that the existence of plasma in whole blood neutralizes part of the positively charge supplied by QA, which is essential in forming the electrostatic affinity with the cell surface. From the blood tests, strong and positive correlations could be expected between the content of QA and the extent of aggregation as the percentage of SB was always holding constant. Twenty percent of QA in both 1mg/mL and 5mg/mL exhibited very mild efficacy, whereas 30% of QA was good enough to cause robust, large areas of clumping. The proposed mechanism of RBC aggregation induced by HPG-SB-QA-OH was similar to that given for the aggregation of RBC by polylysine56, which was also positively charged. While the negative charges originating from carboxyl groups of the sialic acid on cell membrane cause repulsive forces between each other, they are also able to form an electrostatic adsorption force with the cationic macromolecules.56 In the linear aggregates, cells maximize the cell-cell contact area without much change in the shape of the individual cells in the aggregates. The strong electrostatic energy provided by the membrane negative charges and the polymer positive charges deforms the cells from the biconcave discoid shape that they naturally attain when not stressed. Under the forces of strong binding the cells are deformed and form three dimensional aggregates that are not all in focus as it is shown in Figure 3.3 (III)&(IV).   Experiments were done by Jan and Chien 57 using neuraminidase to remove the membrane surface charge due to sialic acid. Results indicated that after the treatment the RBC aggregation induced by neutral macromolecules could be enhanced but the aggregation formed by positively charged molecules was reduced. The adsorption force for the neutral macromolecule was concluded to be weak van der Waals force or hydrogen bonding, which was not so strong as the electrostatic adsorption force generated by the cationic macromolecules. This 60  may also explain the relatively low HPG-SB-QA-OH concentration (1mg/mL, 30% QA) needed for the RBC aggregation and cell deformation allowing the three-dimensional morphology of the clot instead of linear aggregates of minimally deformed cells maximizing the area of cell surface contact.                                             (I)                                                                                                   (II)                                           (III)                                                                                                (IV) Figure 3.3 Erythrocytes aggregation effects of 1mg/mL HPG conjugates after 5 min incubation at 37 °C in human washed blood. (I) PBS control (II) HPG-SB30-QA20-OH50, (III) HPG-SB30-QA30-OH40, (IV) HPG-SB30-QA40-OH30  61  3.3.2 Hemolysis activity result The biocompatibility of the polymer conjugates is an important consideration in developing a safe topical hemostatic agent. Hemolysis is defined as the disruption of the red blood cell membrane and the release of hemoglobin into the plasma58. Several factors leading to red blood cell destruction have been recognized: mechanical fragmentation, spherocytosis, fixation of specific globulins to RBC surfaces, stasis, splenic activity, injury of cell by exogenous chemicals and metabolic hemolysins.59 Some undesired medical reactions like anemia, kernicterus and jaundice may be caused by the increased plasma hemoglobin level. So it’s necessary to evaluate the hemolytic potential of the polymer conjugates before they are administered. The method of choice to determine the hemoglobin content was the cyanmethemoglobin method, which is a type of colorimetric method using Drabkin’s Reagent. At alkaline pH in the presence of potassium ferricyanide (K3[Fe(CN)6]), the iron in blood hemoglobin is oxidized to form methemoglobin (Fe3+), which is able to combine with the potassium cyanide (KCN) in Drabkin’s Reagent to form cyanmethemoglobin (HiCN).60 The color of HiCN has maximum absorption at 540 nm and the intensity is proportional to the content of hemoglobin. Equation 3.2 was used to determine the percentage of hemolysis as shown below.  % 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 =𝐴𝑃𝐴𝑊× 100%                       (Eq. 3.2) AP stands for the absorbance of the supernatant after the incubation with polymer conjugates. Aw is the absorbance of the polymer/whole blood sample. When mixing with Drabkin’s Solution, the cell membranes lose their integrity and the hemoglobin is all released so Aw indicates the total amount of hemoglobin in the 20 L polymer/whole blood solution. 62  In this hemolysis test, HPG conjugates with 5 different SB/QA ratios were evaluated. According to the aggregation assay, 30% of QA on HPG is able to show its extraordinary efficacy in RBC aggregation so the QA content was controlled to be constant at 30% and the amount of SB was varied from zero to 40%.  Figure 3.4 % of RBC lysis after incubation with different types of 5mg/mL polymer conjugates  Figure 3.5 % of RBC lysis after incubation with different types of 1mg/mL polymer conjugates 18.9811.034.982.24 2.18 2.0579.750102030405060708090SB 0 SB 10 SB 20 SB 30 SB 40 PBScontrolWater%of RBC lysisTypes of samples and controls11.387.433.70 2.18 2.03 2.0580.750102030405060708090SB 0 SB 10 SB 20 SB 30 SB 40 PBScontrolWater%of RBC lysisTypes of samples and controls63  At the 5mg/mL level, the results in Figure 3.4 show that when there was no protection of the zwitterionic ligands, severe hemolysis was caused (19%). As the amount of SB was gradually elevated, the hemolytic activity was dramatically suppressed. Though 10% SB still induced mild lysis (11%), 20% SB effectively decreased the lysis level to 5%. 30% and 40% SB both exhibited excellent biocompatibility with negligible 2% lysis detected. Negative control PBS also gave a lysis level at around 2%, which indicated that no detectable undesired reaction happened on the RBC membrane surface when a moderate content of zwitterionic ligand was used. The results in Figure 3.5 for 1mg/mL also follow the same trend and are consistent with the previous findings for 5mg/mL. The hemolytic experiment further justified the model that the aggregates are caused by the electrostatic adsorption force generated by the cationic macromolecules only on the surface and no penetration seems to occur. It is believed that the hydration property of zwitterionic ligands plays an important role in fouling resistance. A notable amount of water molecules binds onto SB and forms a hydration layer which contributes to rendering surface proteins inert. As shown below a concentration providing 30% of SB proved to be sufficient to grant the polymer conjugate a promising biocompatibility.  3.3.3 Water binding by SB and QA  In order to confirm the hydration properties of the polymer conjugates, DSC was used to measure the number of water molecules bound onto each polymer molecule. The idea is to quickly freeze the polymer-water solution and then slowly ramp up the temperature to thaw the ice. During this process, the water molecules interacting strongly with HPG do not participate in the freeze-thaw heat exchange or contribute to the fusion enthalpy. Thus compared with the pure water control under identical experiment conditions, the fusion enthalpy of polymer-water 64  solution (∆𝐻𝑝) is reduced. The bound water molecules are responsible for the difference between pure water fusion enthalpy (∆𝐻0) and ∆𝐻𝑝 as shown in Figure 3.6.   Figure 3.6 Heat profile of water in the presence of HPG-SB20-QA10-OH70 (integration based on the total area under the solid green curves, black line indicated the vertical scope of integration) and heat profile of pure water (integration based on the total area under the dotted blue curves)    The equation 3.3 is a modified form of equation 3.1, which is derived to the following format by the author: 𝑁 =[∆𝐻0(1−𝐶𝑝 )−∆𝐻𝑝]∙𝑚𝑠𝑜𝑙𝑀𝐻2 𝑂∆𝐻0÷𝐶𝑝 𝑚𝑠𝑜𝑙𝑀𝑛     (Eq. 3.3) where 𝑚𝑠𝑜𝑙 stands for the weight of the solution and cancels out of the final equation. The first term represents the moles of water which don’t contribute to the ice to water fusion enthalpy. The divisor is the moles of polymer conjugates in the solution. The physicochemical properties of the polymer conjugates are listed in Table 3.1.  65   Table 3.1 Physicochemical properties of the polymer conjugates for DSC experiments Sample # of SBa,c # of QAa,c # of thiol-OHa,c # of glycerol-OHa,c Mna×104 Mnb×104 HPG - - - 270 - 2.0 HPG-thiol-OH - - 240 27 5.6 6.4 HPG-SB20-QA10-OH70 54 27 160 27 6.5 7.8 HPG-SB30-QA20-OH50 81 54 110 27 6.9 8.0 HPG-SB40-QA30-OH30 108 81 54 27 7.4 8.4 aFrom 1H NMR integration bFrom GPC at 25 °C in 1 N NaNO3 cMolecular parameters except MW are groups per macromolecule. For each typical polymer conjugate, there are four types of ending groups as shown in Figure 3.7. indicated by four different colors. Glycerol-OH group is the original type which is of a constant number for all the modified HPG due to the reason that HPG was initially 90% modified with allyl groups. SB and QA monomers are variable depending on the extent of functionalization and the leftover is thiol-OH group.  Figure 3.7 Four types of ending groups on HPG backbone The number of water molecules bound to each glycerol-OH repeat unit is calculated by using unmodified HPG as a reference, which is an average value rounded off to an integer 6.24 66  Similarly HPG-thiol-OH (See Scheme 2.4 for structure) is used for determining the number of water molecules associated per thiol-OH group. DSC measurements were carried out and showed that a thiol-OH group has also an average value rounded off to an integer 6 bound water molecules, which indicated that thiol-glycerol is an effective agent with which to block the excessive allyl groups during the synthesis process. Having the hydration properties of those two types of ending groups, the amount of water bounded to SB and QA is solved by multiple linear regression using Origin 10 and summarized in Table 3.2. Table 3.2 Hydration properties of the polymer conjugates for each monomer repeat unit Sample Cp/(g/g) ΔHp/(J/g) NSBa NQAa Nthiol-OH Nglycerol-OH H2O  406 12 5 6 6 HPG 0.097 307 HPG-thiol-OH 0.089 352 HPG-SB20-QA10-OH70 0.091 350 HPG-SB30-QA20-OH50 0.096 346 HPG-SB40-QA30-OH30 0.101 343 aThe molecular of water molecules bond per SB/QA unit calculated using multiple linear regression with p=0.05 for both of the two independent variables.  For each positively charged QA monomer, the ionic solvation effect50 causes an average value rounded off to an integer 5 water molecules to be bound. For the zwitterionic molecule SB, due to the dual ion effects 12 water molecules are dramatically bound. The positively charged quaternary amine and negatively charged sulfate on SB provide more interaction sites and form a strong hydration layer, which contributes to the non-fouling characteristic.  3.3.4 Cytotoxicity of the polymer conjugates The potential of the synthesized polymer conjugates to cause cytotoxicity of the endothelial cells in the vascular system was evaluated by using an in vitro screening method. The 67  viability of cells was measured by assessing the cell membrane integrity by using propidium iodide and Hoechst 33258 as the staining dyes.61 As a red-fluorescent nuclear and chromosome counterstain, propidium iodide is not able to permeate live cells, so it’s normally excluded from the inside of healthy cells. The dye can cross the membrane and bind to intracellular DNA to detect dead cells in a population only when the cell membrane is compromised. Hoechst 33258 is permeable to cell membranes and can bind to DNA in living cells. The blue fluorescence emitted by the counterstain labels all cell nuclei.  A human umbilical vein endothelial cell line (HUVEC) was used for the cytotoxicity investigation due to its major function in forming the endothelium. The endothelium plays an important role in controlling the gas exchange on the pulmonary level and controls the circulating of red blood cells.62  The influence of the synthetic polymer conjugates on HUVEC is summarized in Figure 3.8. The result shows that without the protection of zwitterionic SB, the cytotoxicity of the HPG-QA30-OH70 was not negligible and the cells lost their viability increasingly with the higher chemical addition to the media. On the contrary, with the protection of zwitterionic SB, 30% SB exhibited good cell retention compared with the 20% SB addition, even when the concentration of the polymer conjugate was relatively high at 2mg/mL. The cell viability maintained at an 80% level, the value observed when no polymer was present and indicated the great potential of applying HPG-SB-QA-OH using in vivo studies. Presumably the compactly bound water molecules are organized as a protective hydration layer around the zwitterionic moieties as it was shown in the blue curve by applying HPG-SB30-OH70 were maintained in a high viability state. Derivatizing with QA groups instead of SB (orange line) reduced the viability but including SB30 with QA30 (grey line) produced a conjugate known to aggregate red cells while not damaging HUVECs excessively as shown in Fig 3.8. 68    Figure 3.8 % of HUVEC viability after incubation with different types and concentrations of polymer conjugates for 48h  3.3.5 Inhibition effect of the polymer conjugates It is concluded from the previous aggregation and hemolysis analyses that HPG-SB30-QA30-OH40 has the best suitability in causing the red blood celling binding while maintaining the integrity of the cell membrane. HPG-SB30-QA30-OH40 was treated with a kind of sulfated linear polyglycerol (LPG-OSO3-)63 to examine the proposed binding mechanism. The LPG-sulfate bears a high density of negative charges which are presumed to be able to neutralize the cationic quaternary amines on the HPG-QA and eliminate its electrostatic interaction with the red blood cell surface, inhibiting the aggregation. Figure 3.9 shows the PBS control and the HPG-SB30-QA30-OH40  & LPG-OSO3- microscopic images for red cells incubated with these preparations. In the previous hemagglutination test, HPG-SB30-QA30-OH40 exhibited strong aggregation efficacy when there was no anionic LPG-OSO3- addition. However in this case Figure 3.9 (I) and Figure 3.9 (II) show similar rouleaux morphology for the red blood cells, which indicates that the anionic 69  sulfate could effectively neutralize the positive charge and inhibit the binding of the quaternary amine to cell surface.  3.4 Summary  In this chapter, low concentrations of polymer conjugates were tested for their ability to cause red blood cell aggregation. It was found that when QA was maintained at over a 30% level, obvious aggregation effects were observed at a low concentration of 1mg/mL. Similar behavior was also observed on washed red blood cells. Based on this finding, the amount of SB was also varied to evaluate the hemolytic behavior of the material. Thirty percent of SB could effectively decrease the percentage of lysis to a negligible level implying that the polymer conjugate didn’t penetrate into the cells. Cytotoxicity tests were performed on human umbilical vein endothelial cells and 30% of SB was found to able to strongly reduce the cytotoxicity of the cationic quaternary ammonium ligands. The HUVEC viability was preserved at more than 80% even when the addition of HPG-SB30-QA30-OH40 was 2 mg/mL. The excellent biocompatibility       Figure 3.9 Erythrocytes incubated under PBS control (I) and 1mg/mL HPG-SB30-QA30-OH40 & LPG-OSO3-(II) at 37°C in human whole blood.   (I) (II) 70  of the polymer conjugates was explained by the hydration tests which showed that around 12 water molecules were bounded to each SB ending group and they effectively protect the cell membranes from fouling. Finally, the inhibition of the aggregations was tested by applying a kind of anionic linear polyglycerol- sulfate to neutralize the positive charge of the cationic quaternary ammonium ligand. No aggregations were observed after the addition of the counter ions, which further confirmed that it was the electrostatic affinity between the cell membrane and the cationic ligand that induced the hemagglutination.  The quaternary amine here worked as a glue that when multiplied on a polymer background binds all the red blood cells together while the zwitterion SB was able to eliminate the damaging effects to cells. Through a series of tests, the best stickiest and biocompatible ratio was identified: 30% of QA and ≥ 30% SB would be ideal for serving as a potential candidate for a topical hemostatic agent. 71  Chapter 4: Conclusions and Future Directions 4.1 Conclusions In this thesis, a library of HPG based polymers with a constant ratio of one derivative (SB or QA) and a varied number of the other derivative (QA/SB) was successfully synthesized. The polymer conjugate can be potentially applied as a topical hemostatic agent for the bleeding wound. The cell membrane damage caused by cationic quaternary ammonium ligand exposure could be effectively eliminated by the installation of the zwitterionic sulfabetaine through the formation of a hydration layer. The stickiest structure was defined based on a series of biological examinations. As it was descried in Chapter 2, the synthesis of QA and SB moiety was optimized through controlling the amount and reaction time using triphenylphosphine as the reducing agent for the cleavage of disulfide bond. HPG was functionalized with alkene moieties, which provided substantial double bonds for further functionalization. UV induced thiol-ene click reactions were concluded to be able to efficiently complete in three hours. The highlight of performing the reactions with SB and QA on a sequential basis was that the amount of one conjugate could be held constant, which made it more reliable to investigate the efficacy of the other conjugate. On the final functionalized HPG, any mole fractions of zwitterionic sulfabetaine and cationic trimethyl ammonium ligands could be realized.1-Thiolglycerol was applied as a universal ene-moiety blocking agent and effectively consumed the free double bonds.  During the whole synthesis process, various column-free and environmental friendly purification methods were used like precipitation, extraction, filtration and dialysis. A combination of different characterization techniques was used to determine the structure and composition of desired products.  72  In Chapter 3, the hemagglutination, hemolysis, cytotoxicity and inhibition effects as well as the hydration properties of the polymer conjugates were evaluated. In the blood aggregation analysis, HPG-SB30-QA30-OH40 and HPG-SB30-QA40-OH30 exhibited strong red blood cells aggregation effects even at a concentration as low as 1mg/mL. They even displayed stronger aggregates for the washed blood cells due to the absence of plasma, which neutralized a portion of polycations supplied by QA and inhibited the electrostatic affinity between polymer conjugate and cell membrane.  The hemolysis test proved that with the percentage of zwitterionic sulfabetaine ligand increasing, the hemolytic activity gradually decreased, and ultimately to an undetectable level when SB reached 30% level. The force between the aggregates was due to the electrostatic adsorption of the quaternary amine ligands by cell surface acid groups and the hemolytic experiment indicated that there was no penetration of the cell surface. The membrane was protected by the hydration layer formed from the zwitterions.  Similarly, in the cytotoxicity test performed on human umbilical vein endothelial cells, the protection from the bound water layer induced by 30% SB successfully retained a high cell viability in the presence of 30% QA. The hydration test further quantified the bounded water molecules of each repeat monomer unit and especially provided firm evidence of the existence of the hydration layer for SB. In order to further authenticate the binding mechanism proposed, an inhibition test was carried out to neutralize the positive charge of the quaternary amine ligand. The addition of linear polyglycerol sulfate into the HPG-SB-QA-OH system effectively suppressed the red blood cell aggregation. Instead of clumped together, the cells remain in the original rouleaux 73  morphology. The negatively charged LPG-sulfate competed with the negative charge on cell surface and stabilize the system through electrostatically interacting with cationic QA.  As an overall result, the stickiest structure of the designed functionalized HPG was proposed to be HPG-SB30-QA30-OH40, which fulfills the requirements of eliminating the membrane damage to a minimum level and exhibited potential in working as a topical hemostatic agent. The structure of the polymer conjugate is flexible and the amount of SB can be further elevated in terms of future needs. The 1-thiolglycerol block enhanced the hydrophilicity of the polymer functionalities, which are ready to be applied in all kinds of biological environments. 4.2 Future directions During the past years, the pioneer’s dedication provided an overview to this kind of HPG based zwitterionic hemostatic agent and I worked on giving a deeper understanding of defining the structures. We intended to develop a series of user-friendly, highly effective, easy to produce and economical topical hemostatic agents. The long-term plan for realizing that goal was to either further improve the hydration capability of the zwitterionic functionality or alter the potential HPG molecular weight to yield better efficacy.  The future work on investigating the zwitterionic ligand can go in two directions, one is to vary the distance between the quaternary ammonium cation and coupled anion. The other is to develop different other types of betaine.  The distance between the positive and negative charges in the SB designed by our group was two –CH2- units. From the computation and molecular dynamics simulation work done by Qian et al 64, it was reported that in the carboxybetaine zwitterionic trimers, the strongest hydration occurred when that distance reached 6-8 –CH2- trimers. The trimer was utilized to mimic the interacting zwitterion chains grafted on a substrate, like the polymers. In their 74  simulation experiments, the carbon spacer length between the anions and cations was increased from 1-8, incrementally by 1 methylene group, and then increased by 2 units from 8 to 20. The number of water molecules was counted when they were within 3.5 angstroms from the zwitterion surface. Within this distance a voluntary hydrogen bond would be formed at an angle greater than 150°. They calculated the net charge when the distance varied and found that the net charge on the carboxylic anion was always higher than that of the quaternary ammonium cation. Due to the trimer structure, the anions at the chain ends repelled each other. The balance between the repel force and the attractive interaction of the counter ions was the bending of the carbon chains. The hydrophobic nature of the hydrocarbon chain collapsed onto each, especially when there was a long carbon spacer length. The competition between the electrostatic interaction and the hydrophobic interaction dictated the hydration-dehydration transitions of the trimer. Inspired by this simulation, the carbon chain length in our sulfabetaine can also be varied to identify the best hydration capacity. Also on real polymer surface, the degree of repulsion would be more complicated compared with the trimer model, which remains to be further explored.  Inspired by the direct polymerization of commercially available acrylamide, methacrylate and vinyl pyridine functionalized zwitterionic monomers, the modification of HPG can be carried out by another route. In this thesis, allyl groups were installed onto HPG and then reacted with the thiol groups of the substrates. In the scheme below, HPG is functionalized with thiol groups in a two-step reaction. By tosylation of the –OH groups and nucleophilic substitution of the intermediary tosylate with NaSH, the target thiol can be obtained ready to “click” with a variety of existed functionalities under UV irradiation.65  75   Scheme 4.1 Synthesis pathway to HPG-SH A variety of readily used zwitterionic substrates are listed in Figure 4.1.  Figure 4.1 List of potential zwitterionic substrates In such an approach, a library of previously reported or purchasable zwitterionic substrates can be conveniently added onto the HPG backbone and screened to get the best fouling resistance.  Due to the readily oxidizable characteristic of thiol groups, another type of azide-alkyne cycloaddition reaction may be applied as a substitutable method to link the HPG-N3 and alkyne functionalized substrates together through a triazole linker.  The molecular weight of the HPG core applied in this thesis was around 20K Da and synthesized by using trimethylolpropane as the initiator and partially deprotonated by potassium methylate. The preparation of higher molecular weight HPG (up to 700K) was realized and reported by our group51 through adding the diethylene glycol dimethyl ether or dioxane as an emulsifying solvent to reduce the viscosity of the reaction mixture and improve mixing. This scale of high molecular weight HPG has a low intrinsic viscosity and an extremely large number 76  of free hydroxyl groups which can be derivatized. 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