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The chemical modification of hyperbranched polyglycerols for improved bioadhesive and hemostatic properties Wen, Jiying 2015

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THE CHEMICAL MODIFICATION OF HYPERBRANCHED POLYGLYCEROLS FOR IMPROVED BIOADHESIVE AND HEMOSTATIC PROPERTIES by  Jiying Wen  B.S., Beijing Normal University, 2012  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)   August 2015  © Jiying Wen, 2015 ii  Abstract  Enhancement of hemostasis at the site of wound is a very attractive method to limit bleeding and to reduce the need for blood transfusion support. However, many commercially available bioadhesives and hemostatic agents fail to fulfill the design requirements of efficacy, safety and cost.  There is a need to develop novel bioadhesive and hemostatic agents that would overcome these limitations. A library of hyperbranched polyglycerol (HPG) based macromolecular structures functionalized with different mole fractions of zwitterionic sulfabetaine and cationic quaternary ammonium ligands were synthesized and characterized. A post-polymerization method was employed that utilized double bond moieties on the HPG backbone for the coupling of thiol-capped functional groups via UV initiated thiol-ene “click” chemistry. The proportions of different ligands were precisely controlled by varying the monomer concentration during the irradiation process. The effect of the polymer on hemostasis has been investigated using whole blood. It was found that polymer with 40% or more positive charged groups caused hemagglutination without causing red blood cell lysis. The quaternary ammonium groups can interact with the negative charged sites on the membranes of erythrocytes, which improves the bioadhesiveness. The zwitterionic sulfabetaine can provide a hydration layer to partially mask the adverse effects that are likely to be caused by cationic moieties on the integrity of cell membrane. The conjugate was also found to be able to enhance platelet aggregation and activation in a concentration and positive charge density dependent manner, which would contribute to the initiation of hemostasis. The polymer-induced hemostasis is obtained by a process independent of the normal iii  blood coagulation cascade but dependent on red blood cell agglutination, where the polymers promote hemostasis by linking erythrocytes together to form a lattice to entrap the cells. iv  Preface The work presented in this thesis is based on the unpublished work conducted by Jiying Wen under the supervision of Dr. Donald E. Brooks at the Centre for Blood Research UBC. Chapter 2 is about the chemical synthesis and characterization of the designed macromolecules. I performed the all the synthesis and characterization experiment, and analyzed the spectra data. Dr. Marie Weinhart assisted me in the interpretation of the results.  Chapter 3 describes the in vitro analyses of bioactivity, blood compatibility and toxicity of the polymers. Benjamin Lai and I performed the biological assays, and I analyzed the data. Manu Thomas helped with the ITC experiment. Dr. Donald Brooks helped with the design of research project, interpretation and discussion of the results, and editing of the writing. A manuscript based on Chapter 2 and Chapter 3 is in preparation.  Ethics approval was received from UBC for studies conducted at the Centre for Blood Research (UBC Ethics approval no: H07-02198). v  Table of Contents  Abstract .......................................................................................................................................... ii	  Preface ........................................................................................................................................... iv	  Table of Contents .......................................................................................................................... v	  List of Tables ................................................................................................................................ ix	  List of Figures ................................................................................................................................ x	  List of Schemes ............................................................................................................................ xv	  List of Symbols and Abbreviations .......................................................................................... xvi	  Acknowledgements .................................................................................................................... xix	  Dedication .................................................................................................................................... xx	  Chapter 1: Introduction ............................................................................................................... 1	  1.1	   Properties of Mammalian Blood .................................................................................... 2	  1.1.1	   Surface Characteristics of Red Blood Cells ............................................................ 2	  1.1.2	   Platelets in Hemostasis ........................................................................................... 5	  1.2	   Cationic Polymers for Biomedical Application ............................................................. 7	  1.2.1	   Natural Cationic Polymers: Chitosan ...................................................................... 8	  1.2.2	   Synthetic Cationic Polymers ................................................................................... 9	  1.3	   Biocompatible Materials .............................................................................................. 12	  1.3.1	   Biocompatibility and Hemocompatibility ............................................................. 13	  1.3.2	   Polyethylene Glycol .............................................................................................. 15	  vi  1.3.3	   Hyperbranched Polyglycerol ................................................................................ 16	  1.3.4	   Zwitterionic Materials ........................................................................................... 19	  1.4	   Thesis Aim and Structure ............................................................................................. 23	  Chapter 2: Synthesis and Characterization of Multifunctional Hyperbranched Polyglycerols ............................................................................................................................ 25	  2.1	   Introduction .................................................................................................................. 25	  2.2	   Experimental Section ................................................................................................... 28	  2.2.1	   Materials and Methods .......................................................................................... 28	  2.2.2	   Synthesis of HPG .................................................................................................. 28	  2.2.3	   Synthesis of Allyl Functionalized HPG ................................................................ 30	  2.2.4	   Synthesis of Cationic Ligand: 3-Mercapto-N, N, N-Trimethyl Bromide ............. 31	  2.2.5	   Synthesis of Zwitterionic Ligand: Sulfate Betaine ............................................... 32	  2.2.5.1	   Synthesis of Bis(3-Dimethylaminopropyl)Disulfide Precursor ..................... 32	  2.2.5.2	   Synthesis of Sulfabetaine 2-Propanethiol ...................................................... 33	  2.2.6	   Polymer Functionalization: Thiol-ene “Click” Reaction ...................................... 34	  2.2.7	   Quantification of Bound Water ............................................................................. 36	  2.3	   Results and Discussions ............................................................................................... 36	  2.3.1	   Synthesis and Characterization of HPG ................................................................ 36	  2.3.2	   Synthesis and Characterization of HPG-allyl Conjugate ...................................... 38	  2.3.3	   Characterization of Cationic Ligands: 3-Mercapto-N, N, N-Trimethyl Bromide 39	  2.3.4	   Characterization of Zwitterionic Ligands: Sulfabetaine 2-Propanethiol .............. 41	  2.3.5	   Chemistry and Structure of HPG-Zwitterionic/Cationic Molecules ..................... 43	  2.3.6	   Quantification of Bound Water of Polymers ........................................................ 47	  vii  2.4	   Summary ...................................................................................................................... 50	  Chapter 3: In Vitro Characterization of Functional Hyperbranched Polyglycerol .............. 51	  3.1	   Introduction .................................................................................................................. 51	  3.2	   Experimental Section ................................................................................................... 53	  3.2.1	   Materials ............................................................................................................... 53	  3.2.2	   Blood Analysis ...................................................................................................... 54	  3.2.2.1	   Blood Preparation .......................................................................................... 54	  3.2.2.2	   Red Blood Cell Aggregation .......................................................................... 55	  3.2.2.3	   Hemolysis Assay ............................................................................................ 55	  3.2.2.4	   Complement Activation ................................................................................. 56	  3.2.2.5	   Platelet Activation .......................................................................................... 57	  3.2.2.6	   Platelet Aggregation ....................................................................................... 57	  3.2.2.7	   Coagulation .................................................................................................... 58	  3.2.3	   Cytotoxicity Measurement .................................................................................... 59	  3.2.4	   Isothermal Titration Calorimetry Analysis ........................................................... 60	  3.2.5	   Statistical Analysis ................................................................................................ 61	  3.3	   Results and Discussions ............................................................................................... 61	  3.3.1	   Effects of Polymers on the Aggregation Morphology of Red Blood Cells .......... 61	  3.3.2	   Evaluation of the Hemolytic Activity of Polymers ............................................... 67	  3.3.3	   Effect of Polymers on the Aggregation and Activation of Platelets ..................... 69	  3.3.3.1	   Effect of Polymers on the Aggregation of Platelets ....................................... 69	  3.3.3.2	   Effect of Polymers on the Activation of Platelets .......................................... 71	  3.3.3.3	   Relationship Between Platelet Aggregation and Activation .......................... 72	  viii  3.3.4	   Effect of the Polymers on Coagulation ................................................................. 73	  3.3.4.1	   PT and aPTT .................................................................................................. 73	  3.3.4.2	   Thromboelastography (TEG) ......................................................................... 74	  3.3.5	   Effect of the Polymers on Complement Activation .............................................. 77	  3.3.6	   Investigation of the Cytotoxicity of the Polymers ................................................ 78	  3.3.7	   Reversal of Red Blood Cell Binding .................................................................... 79	  3.3.8	   Effect of Polyelectrolyte Complex on Platelet Activation .................................... 81	  3.3.9	   Investigation of the Binding of Polymers and LPG-sulfate by Isothermal Titration Calorimetry ....................................................................................................................... 83	  3.4	   Conclusion ................................................................................................................... 85	  Chapter 4: Concluding Remarks and Future Directions ........................................................ 88	  4.1	   Conclusions .................................................................................................................. 88	  4.2	   Future Directions ......................................................................................................... 89	  Bibliography ................................................................................................................................ 92	   ix  List of Tables Table 2.1 Physicochemical properties of macromolecules used in this study. ............................. 47 Table 2.2 The enthalpy changes (ΔH) of water as measured by DSC for aqueous solutions of different polymers. Pure water was used as a normal control. ................................................. 48 Table 3.1 Clotting kinetics parameter values of human whole blood mixed with polymer solutions. ................................................................................................................................... 75 Table 3.2 Molecular characteristics of sulfated linear polyglycerol used in the study. ................ 80 Table 3.3 Thermodynamic parameters for the interaction of HPG-SB-QA45 with LPG-sulfate measured by ITC in sodium phosphate buffer at 37 °C. ........................................................... 84   x  List of Figures Figure 1.1 Sialic acid residues on the surface of red blood cells. The spheres in orange and yellow represent sugars attached to proteins (protein + sugar = glycoprotein). On the right is the chemical structure of sialic acid, the last sugar in a chain attached to a protein and galactose, the next sugar beside it. .............................................................................................. 4	  Figure 1.2 Cartoon illustration of the formation of blood clots. ..................................................... 6	  Figure 1.3 Chemical structure of chitosan. Chitosan is the copolymer with n > 50. ...................... 8	  Figure 1.4 Chemical structures of linear PEI, typical branched PEI fragment, PLL, and PDMAEMA. All of these polymers will be protonated at physiological pH. .......................... 10	  Figure 1.5 Simplified illustration of the mechanism of polymer-induced hemolysis. Movement of ions (K+, Na+, Cl- etc) is omitted in the model. ........................................................................ 12	  Figure 1.6 Diagram illustrating the interactions between host, material, and clinical application of the material that are important in biocompatibility, modified from a previous publication56.................................................................................................................................................... 14	  Figure 1.7 Illustration of the entropy model: protein adsorption is not favorable due to an entropy loss caused by a compression of the restricted volume of polymer chains by protein. ............ 16	  Figure 1.8 Approach for designing dendrimer-based therapeutics. Modified from a review by Duncan et al65. .......................................................................................................................... 17	  Figure 1.9 Examples of dendrimers and hyperbranched polymers. .............................................. 18	  Figure 1.10 Illustration of non-fouling property given by chain hydration of zwitterionic polymers. ................................................................................................................................... 19	  Figure 1.11 Chemical structures of phosphorylcholine betaine, sulfobetaine, carboxybetaine, and sulfabetaine. .............................................................................................................................. 20	  xi  Figure 1.12 Illustration of the interactions between choline phosphate and phosphatidyl choline.................................................................................................................................................... 21	  Figure 1.13 Multifunctional HPG for specific applications. ......................................................... 24	  Figure 2.1 Schematic representation of the structure of polymer conjugate designed in this thesis.................................................................................................................................................... 27	  Figure 2.2 1H NMR spectrum of HPG-allyl conjugate. Ratio of the peak at 5.88 ppm, representing the protons at CH2=CHCH2- (indicated by Italic H), to protons on the HPG backbone is used to determine the percentage of functionalization. ........................................ 38	  Figure 2.3 13C NMR spectrum of HPG-allyl conjugate. ............................................................... 39	  Figure 2.4 1H NMR spectrum of 3-mercapto-N, N, N-trimethyl bromide. .................................. 40	  Figure 2.5 13C NMR spectrum of 3-mercapto-N, N, N-trimethyl bromide. ................................. 41	  Figure 2.6 1H NMR spectrum of sulfabetaine 2-propanethiol. ..................................................... 42	  Figure 2.7 13C NMR spectrum of sulfabetaine 2-propanethiol. .................................................... 42	  Figure 2.8 1H NMR spectra of HPG-SB and HPG-SB-QAx where x indicates the percentage of QA. ............................................................................................................................................ 44	  Figure 2.9 13C NMR spectra of HPG-SB and HPG-SB-QA40. ..................................................... 45	  Figure 2.10 1H NMR spectrum of the control polymer. ............................................................... 46	  Figure 2.11 A proposed model of the possible interactions between water and the polymers. Two-dimensional illustration here but the real world is three-dimentional. ............................. 49	  Figure 3.1 Optical microscopic images of human whole blood incubated with polymers. All images are at 40×magnification. (A) PBS control, (B) HPG-SB, (C) HPG-SB-QA30, (D) HPG-SB-QA35, (E) HPG-SB-QA40, (F) HPG-SB-QA45; polymer concentration: 5 mg/mL. ............ 62	  xii  Figure 3.2 Optical microscopic images of human whole blood incubated with polymers. All images are at 40×magnification. (A) HPG-SB-QA30, (B) HPG-SB-QA35, (C) HPG-SB-QA40, (D) HPG-SB-QA45; polymer concentration: 1 mg/mL. ............................................................ 63	  Figure 3.3 Optical microscopic images of washed RBCs incubated with or without polymers. All images are at 40×magnification. (A) PBS, (B) HPG-SB, (C) HPG-SB-QA40, (D) HPG-SB-QA45; polymer concentration: 1 mg/mL. .................................................................................. 65	  Figure 3.4 Optical microscopic images of washed RBCs incubated with or without polymers in saline with different ionic strength. All images are at 40×magnification. (A) physiological saline (0.9% NaCl), (B) HPG-SB-QA15 in physiological saline, (C) low ionic strength saline (0.18% saline + 4% glucose), (D) HPG-SB-QA15 in low ionic strength saline; polymer concentration: 1 mg/mL. ........................................................................................................... 66	  Figure 3.5 Illustration of the hemagglutination reaction induced by positively charged polymer.................................................................................................................................................... 67	  Figure 3.6 (A) Hemolysis assay for SB/QA-modified HPG. Polymer concentration is 5 mg/mL. Relative hemolysis is expressed considering the hemolysis of PBS as 0% and hemolysis of water as 100%. Asterisk indicates significant difference, **p < 0.01. (B) Images of RBCs incubated with or without polymers (5 mg/mL) at 37 ˚C for 1 h. ............................................ 68	  Figure 3.7 Aggregation traces of platelets in plasma. Platelet aggregation was measured in the microplate reader at 37 ˚C (wavelength 595 nm, n = 6). Polymer concentration: (A) 1 mg/mL, (B) 0.1 mg/mL. ......................................................................................................................... 70	  Figure 3.8 (A) Maximal percentage aggregation of PRP measured by light transmittance with a microplate reader; (B) Platelet activation in PRP measured by the expression of platelet xiii  activation marker CD62P using anti-CD62P antibody by flow cytometry; ns means p > 0.05.................................................................................................................................................... 71	  Figure 3.9 Relationships between CD62P expression and PRP aggregation. Each point represents the mean values of 5 independent experiments. See the text for the corresponding equations and statistical significance. ....................................................................................................... 72	  Figure 3.10 Effect of polymers on coagulation (A) prothrombin time (PT); (B) activated partial thromboplastin time (aPTT). Polymer concentration is 1 mg/mL. ** means p < 0.01, ns means p > 0.05. .................................................................................................................................... 74	  Figure 3.11 (A) Normal TEG trace; (B) Representative TEG traces for polymers with concentration of 1 mg/mL. Black line shows the PBS buffer. Green shows HPG-SB-QA30. Pink shows HPG-SB-QA40. Brown shows HPG-SB-QA45; (C) Representative TEG traces for polymers with concentration of 0.1 mg/mL. Black line shows the PBS buffer. Green shows HPG-SB-QA30. Pink shows HPG-SB-QA45. ............................................................................ 76	  Figure 3.12 Complement activation determined in human serum using sheep erythrocyte based complement consumption assay. Polymer concentration: 1 mg/mL. IgG and PBS were used as positive and normal control respectively. ** means p < 0.01, ns means p > 0.05. ................... 77	  Figure 3.13 Dose-response curves for cell viability of HUVECs (A) and CHO cells (B) after being treated 48 h with polymers by the propidium iodide live cell staining assay. Results are means ± SD (n = 6). .................................................................................................................. 78	  Figure 3.14 Optical microscopic images of human whole blood incubated with or without polymers. All images are at 40 × magnification. (A) PBS control, (B) HPG-SB-QA45 1 mg/mL, (C) mixture of HPG-SB-QA45 and LPG-sulfate, (D) whole blood first incubated with 1mg/mL HPG-SB-QA45 for 5 min, then LPG-sulfate added and incubated for another 5 min.81	  xiv  Figure 3.15 (A)The formation of PEC between HPG-SB-QA45 and LPG-sulfate; (B) The neutralization of platelet activation upon incubating with 0.1 mg/mL HPG-SB-QA45 and increasing concentrations of LPG-sulfate. Each data point is the platelet activation in PRP measured by the expression of platelet activation marker CD62P using anti-CD62P antibody by flow cytometry. Curves were fit to binding isotherms with GraphPad Prism. .................... 82	  Figure 3.16 ITC analysis of LPG-sulfate binding to HPG-SB-QA45. Upper panel: raw data showing heat change due to the heat of dilution during each injection as LPG-sulfate binds to HPG-SB-QA45.  Lower pannel: plot showing the integrated area corresponding to the heat change (ΔH) during each injection normalized as a function of molar ratio (ligand/macromolecule); the best least-squares fit of the data to a one-site binding model is given by the solid red line. ........................................................................................................ 85	   xv  List of Schemes Scheme 1.1 Simplified scheme of blood clotting reactions. ........................................................... 7	  Scheme 2.1 Mechanism of UV initiated thiol-ene coupling. Modified from a review by Hawker et al106. ....................................................................................................................................... 26	  Scheme 2.2 The synthetic scheme for HPG. ................................................................................. 29	  Scheme 2.3 The synthesis for allyl functionalized HPG. ............................................................. 30	  Scheme 2.4 The synthetic scheme for 3-mercapto-N, N, N-trimethyl bromide. .......................... 31	  Scheme 2.5 The synthetic scheme of bis(3-dimethylaminopropyl)disulfide. ............................... 32	  Scheme 2.6 The synthetic scheme for sulfate betaine. ................................................................. 33	  Scheme 2.7 The scheme of UV initiated thiol-ene “click” reaction. ............................................ 35	  Scheme 2.8 Mechanism of anionic polymerization of glycidol to form well-defined HPGs by slow monomer addition. Modified from a review article by R. Hagg 107. ................................ 37	   xvi  List of Symbols and Abbreviations ADP adenosine diphosphate aPTT activated partial thromboplastin time  CHO Chinese hamster ovary  CP choline phosphate DCM dicholoro methane DMSO dimethyl sulfoxide DSC differential scanning calorimetry  EOS enhanced optical system fL femtolitre, 10-15 liters GPC gel permeation chromatography  GVB gelatin veronal buffer HUVEC human umbilical vein endothelial cell HPG hyperbranched polyglycerol IgG immunoglobulin G ITC isothermal titration calorimetry  kDa kilo Dalton LPG linear polyglycerol LT light transmittance MA maximum amplitude MALLS multi-angle laser light scattering Mn number average molecular weight xvii  MPC 2-methacryloyloxyethyl phosphorycholine  MW molecular weight MWCO molecular weight cut-off  NMR nuclear magnetic resonance PAMAM polyamidoamine PBS phosphate buffered saline  PC phosphatidyl choline  PDI polydispersity index PDMAEMA poly[2-(N,N-dimethylamino)ethyl methacrylate] PEC polyelectrolyte complex PEG poly(ethylene glycol)  PEI poly(ethyleneimine)  PI propidium iodide  PLL poly-L-(lysine)  PPP platelet poor plasma  PRP platelet rich plasma  PSBMA poly(sulfobetaine methacrylate) PT prothrombin time  QA quaternary amine QELS quasi-elastic light scattering RBC red blood cell Rh hydrodynamic radius xviii  ROMP ring-opening multi-branching polymerization SB sulfate betaine SD standard deviation  TBAB tetrabutylammonium bromide TEG thromboelastography TFE 2,2,2-trifluoroethanol TGA thermogravimetric analysis TLC thin-layer chromatography TMP 1,1,1-tris(hydroxylmethyl)propane UV ultraviolet VWF von Willebrand factor  WBC white blood cell  xix  Acknowledgements First and foremost, I want to express my sincerest gratitude for my supervisor, Dr. Donald Brooks, for giving me the opportunity to work in his group and guiding me through many challenges, not only in this project, but also in my entire graduate studies. It has been a great honor to be mentored by him, and his unfailing confidence in me in the form of simple kindness, consideration and encouragement has made this a thoughtful and rewarding journey.  Every member of the Brooks/Kizhakkedathu Lab has been involved in my graduate school life in some form, sharing the struggles or satisfaction of lab life with me. I am forever in indebted to Dr. Marie Weinhart and Benjamin Lai. Marie showed me nearly all the techniques needed to complete the synthetic part of the project, and Ben taught me nearly every biological assay and protocol needed to complete the biological part of the thesis. Moreover, I appreciate the supportive and positive working environment fostered by all my group members, past and present, including Narges, Mahsa, Manu, Prashant, Erika, Iren, Irina, Sonja, Rajesh, Srinivas, Anil, Nima, Bo, Kai, David and many others. I would also take the opportunity to thank Dr. Suzana Straus, Dr. Russ Algar and Dr. Keith Mitchell for being my thesis committee, generously giving their time and expertise to be my thesis readers and participate in my defense. Above all else, I am forever grateful to my parents, who were always willing to lend an ear to my troubles, encourage me when everything seems hopeless, and cheer me on when I succeed. I could not have made it this far without their unconditional love and support.   xx  Dedication       To my parents, for their endless support and encouragement.1  Chapter 1: Introduction  A dangerous complication during a surgery is uncontrolled bleeding as it poses significant fatality risks and usually requires numerous units of transfused blood1. Minimizing the utilization of allogeneic blood in surgical patients has become an important topic in transfusion medicine, not only due to the lack of absolute safety but also to the increasing problem of donor blood availability. Hemostasis is the body’s multifaceted response to hemorrhage, and the enhancement of hemostasis at the site of the surgical wound is a very attractive method to limit bleeding and to reduce the need for blood transfusion support. A number of natural or synthetic topical hemostatic agents based on different mechanisms and theories have been developed, but most of the currently available products have problems such as high cost or the adhesion is too weak to arrest massive bleeding, which can stress the blood supply2-4. The quality of cellular blood products is directly related to their time in storage, particularly for platelets where it is known that behavior in vivo is better the shorter their storage period. The ideal hemostatic agent would be easy to use, highly efficacious, nonantigentic, fully absorbable and inexpensive4-5. Despite profuse research in the field of hemostasis and a plethora of hemostatic agents, the search for the ideal hemostatic agent that can be easily and rapidly applied to severed vessels to reduce perioperative blood loss at all scales still exists. Development of a facile method for sealing off surgically related bleeds from organs or vessels would provide some relief from the effect of the resulting demands on stores of blood products with a concomitant improvement in the quality of transfused blood products.  2  1.1 Properties of Mammalian Blood Blood is a specialized circulating fluid that provides the body with oxygen, nutrients, and waste removal, helps to fight infection, and facilitates wound healing. Blood can be separated into two parts: plasma and blood cells. About 55% of the total volume of whole blood is plasma that consists mostly of water, protein macromolecules, i.e. fibrinogen, globulins, and albumin, and salts. The cellular elements that are suspended in plasma consist of three main classes, red blood cells (RBCs), white blood cells (WBCs) and platelets6.  RBCs, or erythrocytes, are hemoglobin filled blood cells that occupy approximately 40% of the total blood volume, primarily known to perform the important task of delivering oxygen to body tissues and transporting carbon dioxide to the lungs. Healthy human RBCs are anucleate deformable biconcave disks with a flattened center, about 8 µm in diameter, 1 µm thickness in the center, 2 µm at their thickest location, and 92.8 fL for the mean cell volume. WBCs, also called leucocytes, are nucleated, larger than RBCs, roughly 12 µm in diameter, whose main function is to protect body from infection7. Platelets are not actual cells but rather small fragments of cells with no nucleus, and they are shaped like small plates in the non-active form. The mean diameter of platelets is 1-2 µm, and the mean cell volume is 5.8 fL8. Platelets play an important role in hemostasis, as will be discussed. For our purpose, we focus on the characteristics of RBCs and platelets.  1.1.1 Surface Characteristics of Red Blood Cells The abundance of RBCs in whole blood makes them a crucial component to study when developing blood-contact materials. RBCs in blood samples from health donors are known to form reversible linear aggregates that look similar to a stack of coins, which are referred as 3  rouleaux. The deformability of the discoid RBCs gives them a large surface area to make contact with and stick to each other with characteristic face-to-face morphology. This usually happens with increased serum proteins, especially fibrinogen and globulins. As for the mechanism of rouleaux formation, two mutually exclusive mechanistic models exist, cross-bridging and formation of a depletion layer9. The phenomenon that rouleaux formation happens in whole blood but not in washed RBC suspension favors the bridging model10, which assumes the aggregation arises when protein macromolecules in plasma especially fibrinogen and globulins become adsorbed into RBC surfaces thus bridging the surfaces of adjacent RBCs. When the bridging force overcomes the disaggregation forces such as electrostatic repulsion and mechanical shearing force, aggregation occurs11.  To better understand the behavior of RBC, it is crucial to understand the characteristics of the RBC membrane. Like other cell membranes, the membrane of RBC is a fluid structure composed of a phospholipid bilayer with a two-dimensional asymmetrically organized mosaic of proteins12. The negative electrical charge originates from extracellular antigens composed of glycosylated proteins, mostly due to the ionization of the carboxyl group of sialic acid residues13-15, as illustrated in Figure 1.1. The terminal sialic acid has a pKa of 2.6, resulting in its complete ionization under physiological conditions.  This negative layer creates a zeta potential, which assures the stability of RBCs suspended in the blood and governs the spacing between erythrocytes. The negative electrical charge of RBCs is also suitable to induce the adsorption of cationic macromolecules. 4   Figure 1.1 Sialic acid residues on the surface of red blood cells. The spheres in orange and yellow represent sugars attached to proteins (protein + sugar = glycoprotein). On the right is the chemical structure of sialic acid, the last sugar in a chain attached to a protein and galactose, the next sugar beside it.  The shape of red cells is encoded in the mechanical properties of the membrane16. The membrane of RBCs is highly deformable, which allows them to repeatedly undergo extensive, reversible shape changes while passing through blood vessels with a diameter of 2-3 µm during circulation12. The ability to undergo shape change without increasing surface area is due to the extra surface provided by their biconcave-discoid shape16. Although RBCs are a major component of blood clots, as shown in Figure 1.2, their participation in the clotting process is unclear. Recently, Cine and colleagues17 discovered that RBCs lost their typical biconcave shape and turned into an unusual polyhedral shape with several flat surfaces and straight edges when they were incorporated into the clot, which could be packed much more tightly in small spaces and make an almost perfect seal at the center of the blood clot to reduce blood loss.     OCCCHOH H OHH HHOHNCCH3O HO C O OOH OOHOHOOSialic Acid Galactose  5  1.1.2 Platelets in Hemostasis Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair, allowing an organism to close off damaged blood vessels, keep the blood in the fluid state and remove blood clots after restoration of vascular integrity18. Hemostasis is a complex mechanism that involves a synchronized action of various plasma proteins, cells, signaling molecules and platelets. Platelets are key blood components involved in hemostasis. Platelets contribute to the hemostatic process in two different ways. Firstly, the adhesive and cohesive functions of platelets can lead to the formation of a hemostatic plug, which is the primary event in hemostasis19-20. Secondly, they can activate coagulation systems through the exposure of a phospholipidic surface, acting as a catalytic site for the development of coagulation and the consolidation of the hemostatic plug, known as secondary hemostasis21.  When circulating under physiological conditions, platelets are smooth discs with a number of membrane bound glycoprotein receptors on their surface. As soon as the wall of a blood vessel is damaged, platelets adhere to the subendothelial components of blood vessel wall22. The “glue” that holds platelets to the vascular subendothelial is von Willebrand factor (VWF), a protein produced by the cells of the vessel wall. Collagen exposure and thrombin act at the site of the injury to induce platelet activation. As platelets accumulate at the site, they form a initial hemostatic plug that plugs the injury20. The platelets change shape from round to spiny, the membrane becomes ruffled with cytoplasmic projections, and the granules are centralized and discharged, releasing proteins and other substances such as adenosine diphosphate (ADP), that attract more platelets and blood cells, producing a clot that plugs the break.  6   Figure 1.2 Cartoon illustration of the formation of blood clots.  Formation of a clot also involves the initiation of the coagulation cascade, which is divided into two enzymatic pathways, the intrinsic (contact activation) and extrinsic (tissue factor). Both intrinsic and extrinsic pathways can converge into the common pathway and activate factor X to factor Xa, which in turn cleaves prothrombin to form thrombin. Thrombin converts fibrinogen, a blood clotting factor that is normally dissolved in blood, into insoluble cross-linked fibrin that forms a net to entrap more platelets and blood cells. The fibrin strands add bulk to the developing clot and help hold it in place to keep the vessel wall plugged23. Platelet activation and blood coagulation are complementary, mutually dependent, interactive processes in hemostasis24. Scheme 1.1 illustrates how the coagulation cascade (initiated by either the intrinsic or extrinsic pathway) and the activation of platelets interact with each other and form a blood plug as a result.    Platelet Red blood cell Broken blood vessel wall A B Activated platelet Clot Fibrin 7            Scheme 1.1 Simplified scheme of blood clotting reactions.  1.2 Cationic Polymers for Biomedical Application Cationic polymers have been appealing for biological applications due to their strong cellular interactions. All cells naturally have a negatively charged cell surface. Cationic substances therefore can interact with cell membranes more easily than neutral and anionic substances due to the interaction of opposite charges25. Moreover, cationic polymers are able to form polyelectrolyte complexes (PEC) with negatively charged macromolecules such as nucleic acids, proteins or drugs, showing great potential in drug delivery and the gene delivery field26. Their inherent bioactive properties such as antimicrobial or anti-inflammatory characteristics further enhanced the therapeutic potential of cationic polymers25. There is a wide range of cationic polymers that have been investigated for various therapeutic applications; they are generally divided into two categories: natural or synthetic.  Tissue damageTissue factor:VIIaXXaProthrombin ThrombinFibrinogen FibrinXIIIaFibrin clot Platelet adhesionPlatelet shape change/granule secretionPlatelet aggregationRed blood cellsStable hemostatic plug/Blood clotSurface contactCascade ofclotting factors Primary hemostatic plug8  1.2.1 Natural Cationic Polymers: Chitosan        Figure 1.3 Chemical structure of chitosan. Chitosan is the copolymer with n > 50.  Chitosan is an abundant natural cationic polysaccharide, obtained by deacetylation of the naturally occurring substance chitin. Being non-toxic with good biocompatibility and biodegradability, chitosan is one of the most widely studied natural cationic polymers for use in drug delivery27, tissue engineering28, and gene therapy29. When the degree of deacetylation of chitin is higher than 50%, it is generally regarded as chitosan, as shown in Figure 1.3. Chitosan is a weak polybase with a pKa around 6.5, implying that the primary amine groups on chitosan would be protonated at acidic pH, and the resulting positive charge could form strong electrostatic associations with the negatively charged mucins, which counts for its excellent mucoadhesive properties30-31.  The hemostatic potential of chitosan was first reported by Malette et al32, following studies have shown that the cationic chitosan can act as a hemostatic agent and can be used in hemostatic bandages33. Previous studies indicated that cationic chitosan agglutinated RBCs by electrostatic attraction in solution, resulting in the formation of a cellular hemostatic plug that O OOHOO OH NHH3C OH NH2HOO 100-n nn - degree of deacetylationchitin          n < 50%chitosan     n > 50%9  stopped bleeding. As for the mechanism of the function of chitosan to stop bleeding, a lot of studies have been done both in vitro and in vivo, but there is still no clear answer. Several studies report that the hemostatic activity of chitosan is independent of the classic blood coagulation cascade34, but it could promote the adhesion and aggregation of platelets35.  Currently one of the FDA approved commercially available hemostatic agents HemCon® is chitosan based and has been widely used by the military and some successful cases in treating human wounds have been reported36. However, chitosan-based wound dressings also have several issues: (a) Chitosan is soluble at acidic pH but insoluble at physiological pH, making its use in solution with living cells and tissues problematic; (b) the primary amines in chitosan are not as effective as quaternary amines against bacterial infection37; (c) the batch to batch variation of the natural polymer makes it difficult for mass production. Quaternization of the primary amine on chitosan could solve the first two issues, and this can be achieved by the reaction of chitosan with methyl iodide under basic conditions. This strategy makes the solubility and positive charge of chitosan independent of pH. The main difficulty of the batch-to-batch variability of natural polymers can be overcome by synthetic polymers.  1.2.2 Synthetic Cationic Polymers Synthetic polymers make it possible to have good control over the structure and function relationship, since the functional groups and bioactive moieties can be readily incorporated into the synthetic polymer system. The use of synthetic polycations in biomedical engineering can be dated back to approximately sixty years ago38, and the investigation of the hemostatic potential of polycations also started in the 1950s39. Widely studied synthetic cationic polymers include poly(ethyleneimine) (PEI), poly-L-(lysine) (PLL) and poly[2-(N,N-dimethylamino)ethyl 10  methacrylate] (PDMAEMA), all of them bearing amine moieties that can be protonated under physiological conditions thus exhibiting good mucoadhesive/bioadhesive properties. Their chemical structures are shown in Figure 1.4. Nearly all of the synthetic polycations mentioned above have been used for gene delivery applications40, but there are also others that have been used in different areas of biomedical engineering such as tissue engineering41 and drug delivery25, 42.                         Figure 1.4 Chemical structures of linear PEI, typical branched PEI fragment, PLL, and PDMAEMA. All of these polymers will be protonated at physiological pH.  All of the aforementioned synthetic polycations have achieved levels of success in biomedical applications, but they have also been faced with the challenge of biocompatibility. Their relatively high cytotoxicity would often result in the disruption of the cell membrane43, which has limited their therapeutic applications. Although PECs are usually less cytotoxic than the non-complexed polycations, it is always better to consider the worst-case scenario for HN NH2H2N nHN NH NH2R ONH2 O NH2nNH N N N NHNH2NHH2N N NH2H2Nlinear poly(ethyleneimine) (PEI) typical branched PEI fragmentpoly-L-lysine (PLL) ON npoly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA)pKa: ~ 10 pKa: ~ 7.5pKa: ~ 10-11 pKa: 4.5 for primary,         6.7 for secondary        11.6 for tertiary amine groups11  toxicity. Moreover, the administration of PECs to blood could result in their dissociation yielding native polycations. Fischer et al44 conducted a comparative in vitro cytotoxicity study with a series of water-soluble polycations and found that the molecular weight and the cationic charge density were the key factors for the interactions with cell membrane, thus inducing cell damage, and an increase in the molecular weight and/or charge density of polycation results in an increase in toxicity as well. Also the magnitude of the cytotoxicity effect was time and concentration dependent.  Low hemocompatibility is another issue with synthetic cationic polymers. Polycations may interact with negatively charged blood components such as RBCs, platelets, and distinct plasma proteins, provoking responses such as hemagglutination, hemolysis, complement activation, platelet activation and aggregation. The negatively charged RBC surface favors the adsorption of cationic polymers, thus hemagglutination occurs. It is well known that most polycations induce RBC aggregation, and the aggregation strength depends strongly on polymer concentration and molecular weight45. A variety of polycations have shown hemolytic properties, including PEI, PLL, and PDMAEDA. Hemolysis occurs as the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to irreversible rupture of the membrane46, as illustrated in Figure 1.5. Studies also showed that cationic polymers could induce platelet aggregation and complement activation47, and inhibit extrinsic blood coagulation by interaction with some clotting factors that have an acidic pI at physiological pH48.    12         Figure 1.5 Simplified illustration of the mechanism of polymer-induced hemolysis. Movement of ions (K+, Na+, Cl- etc) is omitted in the model.  In order to address the issue of toxicity, great effort has been taken to look into the structure-toxicity relationship and redesign of the polycation component to reduce the cytotoxicity while maintaining the effect of charge to achieve the desired applications. Simple modifications include varying the molecular weight, composition and architecture of the cationic polymer. Specifically, imparting cationic moieties to a biocompatible polymer backbone49, grafting the biocompatible polymers like chitosan to synthetic polycations50 or modifying cationic backbones with biocompatible moieties such as PEG modified PEI51 have been utilized as common practice. Potentially biocompatible synthetic materials will be discussed in the following section.   1.3 Biocompatible Materials For applications in biomedical sciences, there is always a strong demand for the development of biocompatible materials. As most of the naturally derived polymers are present in the structural tissues of living organism, they usually offer several advantages compared with synthetic polymers, including biodegradability, bioactivity, and biocompatibility52. However HemoglobinRed cells H2O H2O H2OH 2OH 2O+ Polymer13  naturally occurring polymers also bear the problem of low reproducibility due to batch-to-batch variation and normally their preparation is difficult to scale up52. Therefore synthetic polymers present an attractive avenue for biocompatible biomaterials due to their well-studied synthesis and modifiable properties. Currently biomaterial science has moved in the direction of using characteristics of the biological environment to help design novel biomaterials for specific applications53, which requires a significant understanding of cellular signaling and cell interactions. For example, one could introduce cell adhesion motifs, and growth factors into biomaterials for tissue engineering applications54. With the progressive advancement of understanding of the biological environment, biomaterials will continue to evolve and lead to further breakthroughs in the healthcare field.  1.3.1 Biocompatibility and Hemocompatibility Biocompatibility is a key concept in the development of materials contacting the human body. A material contacting living tissues must be well accepted by the tissues. However there are no truly inert biomaterials, as when a material is placed into living tissue, interactions with the complex biological systems around it will occur, resulting in some sort of biological response.  Because of the multifaceted applications of biomaterials, a universal strict definition of biocompatibility is almost impossible, and it has to be tailored with regard to specific applications. Williams gave a very good definition of biocompatibility of biomaterials:  “ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy, 14  but generating the most appropriate beneficial cellular or tissue response in that specific situation, and optimizing the clinically relevant performances of that therapy”55. This definition reveals that biocompatibility is a dynamic and multifactorial ongoing process involving not only the biomaterial and its function, but also the response of the host. All the factors including the host, materials and the applications need to be taken into account to determine the biocompatibility of the material.                  Figure 1.6 Diagram illustrating the interactions between host, material, and clinical application of the material that are important in biocompatibility, modified from a previous publication56.   For a biomaterial that has potential application in blood, hemocompatibility is a major requirement, which means that the material must be non-toxic to any of the cellular components in blood. Hemocompatibility focuses on blood-biomaterial interactions and their consequences, describing the property of the material to not provoke changes in blood function. Thus, as with biocompatibility, the definition of hemocompatibility should also be adjusted according to the material’s specific function. For example, the material possesses chemical and physical properties that promote or avoid specific cell substrate interactions, i.e. either cell adhesion or non-adhesion depending on the final application. For cell adhesion applications like blood Application Biocompatibility 15  sealants or adhesives, red blood cell aggregation is a desired phenomenon, but it should be avoided for the application of non-fouling surfaces.  1.3.2 Polyethylene Glycol Biomaterials with anti-fouling properties are always highly desirable in biomedical applications, and poly(ethylene glycol) (PEG) is considered as the gold standard for stealth polymer in the biological field. PEG decorated surfaces have been studied extensively for their non-fouling properties to increase biocompatibility by masking the surface charge57-58. The protein resistance of PEG was explained by the entropy model proposed by De Gennes and co-workers59, which is illustrated in Figure 1.7. As the protein approaches the surface it compresses the PEG chains, which leads to an entropy loss of the polymer chain and accordingly, in the absence of enthalpy effects, to an increase in the Gibbs free energy of the system, which makes the diffusion of the protein into the layer energetically unfavorable. More recently, it is generally agreed that the hydration ability of PEG plays a key role in its non-fouling property60, and PEGylation on nanoparticles can increase their water solubility and stability and decrease the interactions with proteins, etc61. However, there are certain drawbacks that limit the application of PEG. For example, a PEGylated surface faces the problem of chemical stability, as the PEG groups are susceptible to decomposition in the presence of oxygen and transition metal ions and can lose their function in biological media62. Thus significant effort has been invested in the search for alternative non-fouling materials with improved stability compared to PEG.    16         Figure 1.7 Illustration of the entropy model: protein adsorption is not favorable due to an entropy loss caused by a compression of the restricted volume of polymer chains by protein.   1.3.3 Hyperbranched Polyglycerol Dendrimers are highly branched macromolecules with well-defined interior and exterior regions. The high level of control over the three dimensional architecture and the multifunctional surface-tailoring capability make dendrimers attractive carriers in biomedical applications such as drug delivery63 and gene delivery64. The bioactive agents may either be encapsulated into the relatively empty intramolecular cavity of the dendrimers or be chemically attached onto the dendrimer surface to tailor the properties of the carrier to the specific needs of the active materials and its therapeutic applications65, as illustrated in Figure 1.8.       Protein RepulsionPEG Chain17            Figure 1.8 Approach for designing dendrimer-based therapeutics. Modified from a review by Duncan et al65.  However the synthesis of perfect dendrimers is tedious and complicated, requiring multistep procedures of protection, deprotection and purification66-67. Since structural perfection is not a strict prerequisite for most applications, the less structurally perfect hyperbranched polymers synthesized via one-pot reactions have been considered as a possible alternative. Figure 1.9 presents a typical example of the architecture of dendrimer and hyperbranched polymer. Our group has focused its attention on the study of hyperbranched polyglycerols (HPGs)49, 68-70, which are glycerol-based dendritic macromolecular architectures that are structurally similar to PEG, but with a high degree of branching and hydroxyl end group functionality. Due to its globular nature, the hydration ability of HPG is higher than that of PEG71. HPG can be obtained with various molecular weights with polydispersity index (PDI) typically below 1.5. The terminal hydroxyl groups can be directly used as linker functionalities for many applications in organic synthesis, or further be functionalized with other reactive Drug encapsulationin the core Surface modification with targeting ligandSurface modificationwith polymer, oligomer or fatty acid Surface modificationwith additional chemistry(maybe linker) and drug attachment18  groups, allowing the coupling of multiple copies of bioactive molecules and chemical entities towards the design of multifunctional nanostructures.          Figure 1.9 Examples of dendrimers and hyperbranched polymers.  The pioneering work exploring the biocompatibility of HPG was presented by our group in 200672. We also investigated the blood compatibility of HPG/PEG-based polymers containing multivalent cationic sites73, finding that compared to standard cationic polymers such as PEI, HPG-based polymers showed much lower cytotoxicity, which is an essential feature for biomedical applications. Due to the low degree of molecular weight dispersity, good chemical stability, high hydrophilicity, inertness under biological conditions, and flexible design74, HPG has emerged as a scaffold with a broad range of possible structural designs to meet current shortcomings or deficits in modern applications, including drug conjugates75-76, erythrocyte surface camouflage70-71, 77, antidote for anticoagulant49, and antibacterial peptide78.   hyperbranched polymersdendrimers19  1.3.4 Zwitterionic Materials Zwitterionic materials have been emerging as a new class of anti-fouling material and have attracted significant attention in this field. The idea of zwitterionic materials was inspired by the mammalian cell membrane, whose lipid components are mainly phospholipids bearing zwitterionic phosphatidyl choline (PC) headgroups. A general characteristic of the zwitterionic structure is that it has a balance of positively and negatively charged moieties within the same segment side chain, rendering an overall neutral charge. Zwitterionic surfaces can be highly resistant to protein adsorption and bacterial adhesion, exhibiting high biocompatibility79. The high degree of hydration of zwitterionic materials, which exceeds the hydration ability of PEG80, is responsible for the non-fouling properties of zwitterionic surfaces. The tightly bound water layer dictated by electrostatic attractions between the charges on the pendant groups and water molecules81 forms a physical and energetic barrier to prevent protein adsorption on the surface, as shown in Figure 1.10.         Figure 1.10 Illustration of non-fouling property given by chain hydration of zwitterionic polymers.   20  The work on 2-methacryloyloxyethyl phosphorycholine (MPC) based surfaces82 was the first step towards the development of zwitterionic materials and introduced the importance of dual ions. The MPC structure composed of a methacrylated polymer and PC headgroup, the side chain consisting of a phosphate anion and a quaternary ammonium cation, and has been widely used as a coating material to reduce unwanted nonspecific adsorption83-84. In addition to MPC, other types of biomimetic polymers containing a quaternary ammonium cation similar to that of PC but bearing a different anionic group, such as sulfobetaine, and carboxybetaine (structures shown in Figure 1.11), have received growing attention for use in a new generation of blood-contacting materials.            Figure 1.11 Chemical structures of phosphorylcholine betaine, sulfobetaine, carboxybetaine, and sulfabetaine.   Almost all of the zwitterionic materials that have been reported are known to have excellent non-fouling property, except the methyl decorated choline phosphate (CP)85-87, whose amino and phosphate groups are in the reverse orientation of PC, displaying a strong affinity for O P OOO CH2 Nn N CH2 SO OOnN CH2 CO On N CH2 SO OOn OSulfobetaineCarboxybetaine SulfabetainePhosphorylcholine betaine21  biological membranes, leading to extreme RBC aggregation. Figure 1.12 presents the illustration of the electrostatic PC-CP interaction. Interestingly, this feature is only effective for a structure with a methyl group connected to one of the oxoanions. The exact same structure but with butyl groups is much easier to synthesize, but it behaves the same as other polybetaines88.               Figure 1.12 Illustration of the interactions between choline phosphate and phosphatidyl choline.    Among the various kinds of betaine structures, sulfobetaine is the most widely studied. It was reported that poly(sulfobetaine methacrylate) (PSBMA) surfaces were ultralow fouling for adsorption from both single protein solutions and complex media such as human blood plasma89. Lalani and Liu90 reported that the electrospun PSBMA membrane is ideal for a novel type of nonadherent, superabsorbent and antimicrobial wound dressings. A number of zwitterionic sulfobetaine coated nanoparticles have been synthesized and characterized for demonstration of their biocompatibility and hemocompatibility by reducing nonspecific protein adsorption in various biological applications91-92. Another extensively studied zwitterionic material with nonfouling properties is R1O OO OP OO ONOPO OO NRCholine phosphate Phosphatidyl cholineR2O22  carboxybetaine with side chains composed of cationic quaternary ammonium and anionic carboxylate functional groups. The difference between carboxybetaine and sulfobetaine is that its negatively charged groups are based on the carboxylic acid (-COOH) moiety, which may give poly(carboxylbetaine) a wider application as the carboxylic acid groups can be easily conjugated to amine-containing functional molecules. Wang and colleagues93 modified PAMAM dendrimers with carboxylbetaine, and found a thin compact layer of the zwitterionic moieties could significantly reduce the cytotoxicity of PAMAM through minimizing the interaction with protein and cell membrane. Compared to sulfobetaine and carboxybetaine, sulfabetaine has not been widely reported. Unlike the sulfobetaines where the sulfur atom of the sulfonate group is directly linked to carbon, in polysulfabetaines, the carbon atom is linked to sulfur through an oxygen atom. Theoretically, this additional oxygen atom can be expected to introduce more hydrophilicity to the polymer. The presence of an additional heteroatom in the form of oxygen also increases the size of the anionic moiety resulting in a lower charge density. Vasantha et al94 reported the synthesis of polysulfabetaine and studied its thermoresponsive behavior. To the best of our knowledge, the interaction of sulfabetaines with biomolecules like proteins has not been published yet. Although Jiang and coworker95 employed molecular modeling techniques to claim that sulfate betaine would specifically interact with some protein residues which might influence their ability to resist nonspecific protein adsorption, we believe that with a similar zwitterionic structure, sulfabetaine should be able to exhibit the stealth effect to some extent. This thesis is mainly focused on the ability of zwitterionic sulfabetaines to reduce the cytotoxicity of polycations however.  23  1.4 Thesis Aim and Structure The primary goal of the research described in this thesis is to develop a novel class of multivalent water soluble HPG-based polymeric materials that combine several functions by incorporating multiple anti-fouling and/or adhesive bioactive agents, as shown in Figure 1.13, and investigate its potential biological applications. These structural components, whether the HPG backbone, the zwitterionic moieties, or the quaternary ammonium groups, are intended to function synergistically to be a potential biocompatible hemostatic wound dressing agent. Our hypothesis is that the conjugate possess hemostatic functions with high efficacy since cationic moieties can attract the negatively charged red blood cells and enhance the formation of blood clots while both the zwitterionic ligands and HPG backbone can impart hemocompatibility to the conjugate to attenuate the cytotoxicity of polycations and also absorb wound fluids due to their strong hydration property. To achieve this, HPG was used as the polymer backbone to which was coupled different numbers of cationic quaternary ammonium and zwitterionic sulfate betaine functional groups using UV initiated thiol-ene “click” post-polymerization modification technique. The physical and chemical properties of the polymer conjugates were characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC). The results of this study are described in Chapter 2 of the thesis.     24     Figure 1.13 Multifunctional HPG for specific applications.  After developing and characterizing the series of novel HPG-based mixed zwitterionic/cationic conjugates, studies aimed at defining the bioactivity, hemocompatibility and toxic effect of the polymer conjugates were conducted in vitro. The results are used to demonstrate that we achieved a structure with an optimized zwitterionic/cationic composition that can aggregate erythrocytes and platelets, and activate platelets but will not activate the complement system and is non-toxic. The details of the in vitro studies are presented in Chapter 3. Since the polymer we develop is proposed to have the potential to work as a topical hemostatic dressing, emphasis was placed on defining the ability of the polymer conjugate to help form a blood clot to seal wounds without causing any undesired side effects.  Chapter 4 provides concluding remarks of the work presented in the thesis, emphasizing its significance and directions for future work.   Anti-fouling & HemocompatibilityCell adhesionHPG25  Chapter 2: Synthesis and Characterization of Multifunctional Hyperbranched Polyglycerols  2.1  Introduction The synthesis of macromolecules with precise control over molecular weight, architecture, composition, and functional groups is always a challenge in polymer chemistry96. Post-polymerization modification provides a more versatile platform for the preparation of functional materials97. Such chemical modification can be easily performed on HPG, a hyperbranched macromolecule with multiple derivatizable hydroxyl groups, using classical hydroxyl group chemistry to change the HPG hydroxyl groups to alkynes98, alkenes99, or amines100.  In 2001, Sharpless et al. introduced the concept of “click” chemistry101, which broadly describes the highly efficient coupling reactions that proceed to quantitative conversion without any side products or reactions, are highly orthogonal, and are preferably conducted in benign solvent media. Huisgen 1,3-dipolar cycloaddition of azide and akyne is the model of “click” chemistry and has been widely used in the surface modification of polymers102. This reaction is usually catalyzed by copper ion but concerns about the cytotoxicity of copper have led to investigation of other possibilities for “click” reactions. The thiol-ene reaction appears a useful alternative of the methods available for the post-polymerization reaction of synthetic polymers, and has been demonstrated to functionalize surfaces in a facile and efficient manner103-104. It usually gives high, often quantitative yields, and produce limited byproducts, leading to an accelerated procedure without the need for chromatographic purification66. Scheme 2.1 shows 26  the mechanism of a UV initiated thiol-ene coupling reaction. One of the advantages of thiol-ene “click” reactions is that it tolerates a variety of solvents, including water, and can proceed in the presence of oxygen. Thiol-ene “click” chemistry also provides an additional advantage over other “click” methods in that various thiol-containing moieties can be easily introduced to the polymer backbone without extra chemical modification or toxic catalysts, which is very attractive in the synthesis of bioconjugates for biomedical applications105.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Scheme 2.1 Mechanism of UV initiated thiol-ene coupling. Modified from a review by Hawker et al106.  The goal of this work was to develop a series of novel water-soluble hyperbranched polyglycerol-based multivalent polymer conjugates, as shown in Figure 2.1, using tandem post-polymerization modification. First, HPG, a polymer bearing multiple hydroxyl groups, was prepared via anionic ring-opening multibranching polymerization. This precursor polymer was then used as a reactive scaffold to introduce pendant alkene groups in the first post-R1 SH + photo-intiator R1 S R1 S R2hv R1 S R2 R1 SHR2thiol-ene product27  polymerization modification step using allyl chloride. Subsequently, this latent alkene-functional polymer was used as a linker for further functionalization, which was demonstrated via the thio-ene “click” reaction using thiol-functionalized zwitterionic/cationic ligands. This method allows for efficient, facile tuning of the surface of HPG, making it possible to tailor the ratio of zwitterionic/cationic moieties and achieve optimal binding efficiency and blood compatibility.  With this methodology we could possibly create a library of compounds by changing the concentration of cationic groups within the polymer.    Figure 2.1 Schematic representation of the structure of polymer conjugate designed in this thesis.  OOOO OO O O OHO OO O OHO O OOOHO O OO OOOOHOO O OOO OHOO O O OHO S NSNSNSN S N O S OOOS N O S OOOS N OS OOOSNOSOO OSNOSO O O SNOSO OO S N O SO OOSNOSOO SNOSO OO SNOS OO OO O S NO O O S N O SHO O OOO S NHPG core Linker Zwitterionic/cationic Shell28  2.2  Experimental Section 2.2.1 Materials and Methods All solvents and reagents were of analytical grade and purchased from commercial suppliers (Sigma-Aldrich, Fisher Scientific, and Alfa Aesar) and used as received without further purification unless otherwise stated. Glycidol (96%) was purified by distillation under reduced pressure and stored in the refrigerator (4 °C). Molecular weight cut-off (MWCO) membrane (Spectra/Por Biotech cellulose ester dialysis membranes, MWCO 1000 Da or 3500 Da) was obtained from Spectrum Laboratories, Inc. (Rancho Dominguez, CA).  The analytical thin-layer chromatography (TLC) plates used were aluminum-backed ultrapure silica gel 60 Å and the thickness is 250 µm.  The lamp used for irradiation of samples was a BlueWave™ 50AS UV light curing spot lamp from DYMAX Corporation (Torrington, CT), which emits 365 nm light at 50 W. 1H and 13C NMR spectra were acquired at ambient temperature (25 °C) using Bruker AV300 or AV400 instruments. Chemical shifts are expressed as parts per million (ppm), relative to D2O (4.79 ppm for 1H) or CDCl3 (7.24 ppm for 1H and 77.2 ppm for 13C). Molecular weight and PDI of polymers were determined by GPC with a DAWN EOS multi-angle laser light scattering (MALLS) detector from Wyatt Technology Corporation (Santa Barbara, CA) in aqueous NaNO3 solution. Hydrodynamic radius of the polymers was measured using quasi-elastic light scattering (QELS) detector associated with the DAWN-EOS system.   2.2.2 Synthesis of HPG HPGs were synthesized following published methods72, the procedure is shown in Scheme 2.2. Generally, the initiator molecule 1,1,1-tris(hydroxymethyl)propane (0.1542 g, 1.15 mmol) was first transferred into a 100 mL flame-dried three-necked round bottom flask equipped 29  with a mechanical stirrer under argon, and then partially (10%) deprotonated using potassium methylate methanol solution (0.10 mL, 25 wt % in methanol). Excess methanol was distilled off under vacuum for 4 h. Then, the flask was heated to 95 °C and an aliquot of glycidol (20 mL, 0.3086 mol) was added dropwise for 12 h via syringe pump and stirred for another 8 h. The mixture was then dissolved in methanol, and neutralized by adjusting pH using 1 M aqueous HCl solution. The polymer was precipitated three times from methanol solution into acetone to lower the polydispersity. Then the precipitate was dissolved in water and further purified by dialysis against water for 2 days using a regenerated cellulose membrane with MWCO of 1000 Da. The final solution was lyophilized to recover the polymer. 1H NMR and 13C NMR were used to determine the chemical structure and branching. The molecular weight (MW) was determined by a GPC-MALLS system equipped with Waters ultrahydrogel columns and 0.1 N NaNO3 as an eluent. A dn/dc of 0.12 mL/g was used for the MW determination. A viscous colorless liquid was obtained as the HPG (20 K) with the yield of ~ 70% (~ 14 g).           Scheme 2.2 The synthetic scheme for HPG. OOOO OHHO O O OHHO OHO O OHHOHO O OHOOHO OHOHO O OHHOOOOHOHOHO OOHO OHOOHOH O OHHOOHOHHO CH3O K O OHargon atmosphere, 95 oC HPG OH30  2.2.3 Synthesis of Allyl Functionalized HPG Allyl functionalized HPGs were synthesized by the allylation of HPG with allyl chloride, as shown in Scheme 2.3. HPG (Mn = 20 K, PDI = 1.4, 6.5 g, 8.78 × 10-2 mol hydroxyl groups), powdered NaOH (17.56 g, 0.439 mol, 5 mol per mole of hydroxyl group) and distilled water (17.56 g, the same mass as NaOH), tetrabutylammonium bromide (TBAB) (0.8491 g, 2.63 × 10-3 mol, 3% molar on each mole hydroxyl group), were added into a two-neck round-bottom flask equipped with a condenser and a dropping funnel. The mixture was heated to 60 °C and stirred at this temperature for 1 h to get a viscous slurry. TBAB was used as a phase-transfer catalyst. Next, allyl chloride (33.59 g, 0.439 mol, 5 mol per mole of hydroxyl group) was transferred into the dropping funnel and added in drops to the mixture over a period of 2 h. The mixture was kept under reflux with vigorous stirring. After 12 h of reaction, DCM (200 mL) was added to the mixture. The organic phase was then separated, washed with water, dried over Na2SO4, filtered, and concentrated by rotary evaporator to yield the crude product. The crude product was further purified by dialysis against acetone for two days, and acetone was removed via rotary evaporation. The final product was obtained as a colorless oil after drying in vacuo (~ 9 g). For long-term storage, it was necessary to keep the allyl ether product under an inert atmosphere, i.e. argon, at -20°C to prevent the slow cross-linking polymerization. The allyl functionalized HPG was characterized by NMR analysis. The number of allyl groups on the polymer was calculated from 1H-NMR integration values.    Scheme 2.3 The synthesis for allyl functionalized HPG. HPG Cl+ NaOH, dH2OTBAB, 60 oCOH HPG O31   1H NMR (δ: ppm, 300 MHz, CDCl3) 5.84 (m, CH2=CHCH2-), 5.24(s, CH2=C), 5.19(s, CH2=C-), 5.10 (t, CH2=C-), 4.10(s, -OCH2CH=C-), 3.95 (s, -OCH2CH=C-), 3.30 – 3.80 (m, -OCH2-)  13C NMR (δ: ppm, 75 MHz, CDCl3) 135.5 (CH2=C-), 135.1(CH2=C-), 116.9 (CH2=C-), 77.7 (-OCH2-), 77.2 (-OCH2-), 76.8 (-OCH2-), 72.4 (-OCH2-), 72.1 (-OCH2-), 72.0 (-OCH2-), 71.8 (-OCH2-), 71.4 (-OCH2-), 70.4 (-OCH2-)  2.2.4 Synthesis of Cationic Ligand: 3-Mercapto-N, N, N-Trimethyl Bromide     Scheme 2.4 The synthetic scheme for 3-mercapto-N, N, N-trimethyl bromide.  The cationic ligand, 3-mercapto-N, N, N-trimethyl bromide, was synthesized following Scheme 2.4. The starting material, (3-bromopropyl) trimethylammonium bromide (5.20 g, 0.02 mol, 1 equiv.), was added into a 100 mL flask. Sodium thiosulfate pentahydrate (5.46 g, 0.022 mol, 1.1 equiv.) was dissolved 20 mL deionized water and added into the flask containing (3-bromopropyl)trimethyl ammonium bromide. The mixture was heated to 100 °C and kept under reflux and stirring for total of 12 h. The resulting Bunte salt was then hydrolyzed and oxidized to the corresponding disulfide by adding H2SO4 solution to give an acid concentration of 1M in the mixture. Thiol was obtained by reducing the disulfide with triphenolphosphine in a H2O/DCM/TFE mixture (20 mL, water: DCM: TFE = 2: 2: 1, v/v) at room temperature for 12 h. Br N Br Na2S2O3 S NSOOONa Br+ 100 oC, H2Oreflux, overnightN S S NBr BrH2SO4 HS N BrH2O/DCM/TFEtriphenolphosphine[O]32  The water phase was collected and TFE was removed by rotatory evaporation. The product was recovered by freeze-drying and stored under an inert atmosphere. 1H NMR (δ: ppm, 300 MHz, D2O) 3.47 (t, 2H, -CH2CH2N-), 3.16 (s, 9H, -N(CH3)3), 2.65 (t, 2H, HS-CH2-), 2.22 (m, 2H, -CH2CH2CH2-) 13C NMR (δ: ppm, 75 MHz, D2O) 65.34 (-CH2CH2N-), 53.13 (-N(CH3)3), 26.62 (HS-CH2-), 20.61 (-CH2CH2CH2-)  2.2.5 Synthesis of Zwitterionic Ligand: Sulfate Betaine 2.2.5.1 Synthesis of Bis(3-Dimethylaminopropyl)Disulfide Precursor       Scheme 2.5 The synthetic scheme of bis(3-dimethylaminopropyl)disulfide.  A scheme for the synthesis of the precursor is given in Scheme 2.5. Sodium thiosulfate pentahydrate (5.46 g, 0.022 mol, 1.1 equiv.) was first dissolved in 20 mL of deionized water and then added into a 100 mL round-bottom flask containing 3-dimethylamino-1-propyl chloride hydrochloride (3.16 g, 0.02 mol, 1 equiv.). The mixture was heated to 100 °C and kept under reflux with stirring overnight. Cold aqueous hydrochloric acid solution (20 mL, 1 M) was added dropwise to the mixture and the Bunte salt was hydrolyzed to the corresponding thiol. Then the Cl N HCl Na2S2O3 S NSOOONa HClHS N HCl HS NHCl NaOHpH = 1 pH = 10S NSN+ 100 oC, H2Oreflux, overnight[O]33  mixture was kept stirring for 2 h and again neutralized by adding NaOH solution (1 M) until the pH of the mixture reached 10. Dimethylamino-1-propyl chloride hydrochloride was achieved by bubbling air into the flask under room temperature for 12 h. The product was extracted with DCM and the organic phase was combined, dried over Na2SO4, filtered, and concentrated under vacuum. 1H NMR (δ: ppm, 300 MHz, CDCl3) 2.64 (t, 4H, (-SCH2-)), 2.26 (t, 4H, -CH2N(CH3)2), 2.13 (s, 12H, -N(CH3)2), 1.76 (m, 4H, -CH2CH2CH2-) 13C NMR (δ: ppm, 75 MHz, CDCl3) 58.30 (-CH2N(CH3)2), 45.57 (-N(CH3)2), 36.78 (-SCH2-), 27.34 (-CH2CH2CH2-)  2.2.5.2 Synthesis of Sulfabetaine 2-Propanethiol      Scheme 2.6 The synthetic scheme for sulfate betaine.  Scheme 2.6 shows the synthetic route of sulfate betaine. All glassware was flame-dried and protected by argon. Bis(3-dimethylaminopropyl)disulfide (2.36 g, 0.01 mol, 1 equiv.) was added to a 100 mL Schlenk flask, cooled to 0 °C in the ice bath. The five-membered cyclic sulfate ester 1,3,2-dioxathiolane (2.73 g, 0.22 mol, 2.2 equiv.) was added to another Schlenk flask, and 20 mL anhydrous acetone was added to dissolve it. The resulting acetone solution was transferred into the flask containing bis(3-dimethylaminopropyl)disulfide through a long needle O OSO O S N OSO3S N OSO3dry acetoner.t. overnight+S NS Ntriphenolphosphine35 oC, 48 h HS N OSO334  in a dropwise manner. The reaction was continued for 1 h at 0 °C then allowed to warm to room temperature overnight. A white precipitate was obtained during the reaction, which was filtered, washed thoroughly with acetone and dried under vacuum (4.4 g, 91%).   The product from the previous step (2.20 g, 0.0045 mol, 1 equiv.), triphenolphosphine (12 g, 0.045mol, 10 equiv.), water/DCM/TFE (20 mL, water: DCM: TFE = 2: 2: 1, v/v) were transferred into a 50 mL round-bottom flask and reacted at 35 °C overnight. The water phase was collected and TFE was removed by rotatory evaporation. Sulfabetaine 2-propanethiol was recovered by freeze-drying (3.5 g, 80 %) and stored under an inert atmosphere.  1H NMR (δ: ppm, 300 MHz, D2O) 4.50 (t, 2H, -CH2SO4), 3.76 (t, 2H, SO4-CH2N-), 3.55 (t, 2H, - CH2CH2CH2N-), 3.20 (s, 6H, -N(CH3)2), 2.65 (t, 2H, HSCH2-), 2.14 (m, 2H, -CH2CH2CH2-) 13C NMR (δ: ppm, 75 MHz, D2O) 64.10 (-CH2SO4-), 62.54 (SO4-CH2CH2N-), 61.88 (-CH2CH2CH2N-), 51.76 (-N(CH3)2), 26.22 (HSCH2-), 20.51(-CH2CH2CH2-)  2.2.6 Polymer Functionalization: Thiol-ene “Click” Reaction The functionalization of allyl-HPG was explored using a UV-initiated radical-mediated thiol-ene “click” reaction. A typical reaction was carried out in a mixture solvent of methanol/TFE using 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a room temperature photo-initiator. The synthetic route of the polymer functionalized with sulfabetaine only is elaborated in Scheme 2.7. Briefly, allyl functionalized HPG (100 mg, 3.43 × 10-6 mol, 7.41 × 10-4 mol allyl groups), sulfabetaine 2-propanethiol (0.3735 g, 1.53 × 10-3 mol, 2 mole per mole of allyl groups), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (34 mg, 1.53 × 10-4 mol, 0.2 mole per mole of allyl groups), methanol/TFE (5 mL, methanol: TFE = 1:4, 35  v/v) were transferred to a 20 mL reaction vial and stirred until the mixture was clear. The reaction was carried out under the irradiation of UV light with a wavelength of 365 nm for 45 min. The reaction vessel was checked every 5 min by gradually adding drops of deionized water to ensure the homogeneity of the mixture. When the mixture no longer became turbid after the addition water, the reaction could be considered as completed, as the product was water-soluble but the reactant allyl-HPG only dissolved in organic solvents. Finally, excessive zwitterionic ligands and the photoinitiator were removed by dialysis. The mixture was first dialyzed against 0.15 M aqueous sodium chloride solution and then deionized water with a cellulose dialysis membrane (MWCO = 3500). The molecular weight was determined by both calculation from 1H NMR and a GPC-MALLS system equipped with Waters ultrahydrogel columns and 1 N NaNO3 as an eluent.      Scheme 2.7 The scheme of UV initiated thiol-ene “click” reaction.  Hyperbranched polymers with different compositions of zwitterionic/cationic groups were prepared by varying the ratio of one component to the other. The number of each group on the polymer was calculated from the 1H-NMR integration values. The control polymer was synthesized in a similar procedure, except that the zwitterionic ligands were replaced by equal moles of 1-thiolglycerol. HPG O + HS N OSO3 HPG O S N OSO3photo-initiator, UV, 45 minMeOH/TFE/H2O photo-initiator: O OHOHO36  2.2.7 Quantification of Bound Water The amount of water bound to polymers was quantified using a Q2000 differential scanning calorimeter (DSC) from TA Instrument (New Castle, DE). Polymer was first dissolved in deionized water with a 5 (w/w)% polymer concentration, the exact mass fraction of polymer in aqueous solution was obtained from thermogravimetric analysis (TGA) using a TGA Q500 from TA Instrument (New Castle, DE). Around 15 µL of polymer solution was pipetted into a Tzero pan to evaluate the ice-water fusion enthalpy. An empty pan was used as the reference. Scanning was conducted from -30 °C to 30 °C at a rate of 2 °C/min, the power (µW) was recorded as a function of temperature and the fusion enthalpy was evaluated from the peak integral at negative temperatures around 0 °C using the TA Universal Analysis Software. The number of water molecules bound to each polymer molecule was calculated from the following equation: 𝑁   =   ∆𝐻? 1− 𝐶?   −   ∆𝐻?18.02∆𝐻?𝐶? ∙   𝑀?                                                                                      (Eq. 2.1) Where ∆𝐻? is the fusion enthalpy of pure water, ∆𝐻? is the fusion enthalpy of free water in the polymer solution, 𝐶? is the mass fraction of polymer in solution, and 𝑀? is the molecular weight of the polymer.  2.3 Results and Discussions 2.3.1 Synthesis and Characterization of HPG HPG with a molecular weight of 20 kDa was synthesized via anionic ring-opening multi-branching polymerization (ROMP) of glycidol. 1,1,1-Tris(hydroxylmethyl)propane (TMP), a starter molecule with three -OH groups, was used as a polyfunctional initiator. Generally, TMP 37  was first partially deprotonated with potassium methoxide to control the concentration of the active sites in the polymerization, thus achieving simultaneous chain growth leading to well-defined HPG with fairly narrow polydispersity. Then glycidol was added in a dropwise manner over a period of 12 – 15 h using a syringe pump. The slow monomer addition approach makes the control of the average molecular weight of the resulting polymer possible by adjusting the ratio of monomer and initiator, as in the ideal case every newly formed glycerol unit is attached to the branched glycerol already formed in the reaction68. Due to the controlled polymerization conditions, the monomer exclusively reacts with the growing multifunctional hyperbranched polymer, leading to a “living” type growth of the macromolecule. The mechanism is shown in Scheme 2.8. Polydispersity could be further lowered by removing the low molecular weight fraction via dialysis against water. The HPG was obtained in good yield (~70%), with Mn = 20000, PDI = 1.3. By calculation there are ~ 270 hydroxyl groups per HPG molecule.             Scheme 2.8 Mechanism of anionic polymerization of glycidol to form well-defined HPGs by slow monomer addition. Modified from a review article by R. Hagg 107. OH RO O RO OHOH OH O KKPG RO KO OHROH CH3O KCH3OHInitiation PropagationIntermolecular transfer Intramolecular transfer38  2.3.2 Synthesis and Characterization of HPG-allyl Conjugate The allylated HPG was prepared via etherification of the hydroxyl endgroups by allyl chloride using aqueous conditions and sodium hydroxide as a base.  In Figure 2.2, all the chemical shifts corresponding to protons on the macromolecule are clearly assigned. The resonance at 2.14 ppm is due to protons from the methyl group of the leftover acetone. The signals between 3.3 to 3.8 ppm are assigned to the protons from the backbone of HPG. The two peaks located at 5.2 ppm and 5.8 ppm in 1H NMR correspond to the protons on the ene-moiety. Meanwhile, the two protons from the methylene group next to the double bond are visible at 3.96 and 4.10 ppm. The percentage of allylation was calculated according to the integration of the protons at CH2=CHCH2- (indicated by Italic H), compared to the integration of protons on the HPG backbone. Specifically, if all the hydroxyl groups are functionalized with double bond, the ratio of the integration of protons at 5.8 ppm and HPG backbone should be 1:5. Thus here ~80 % of the hydroxyl groups have been converted into allyl groups, resulting in ~216 terminal double bonds.                 Figure 2.2 1H NMR spectrum of HPG-allyl conjugate. Ratio of the peak at 5.88 ppm, representing the protons at CH2=CHCH2- (indicated by Italic H), to protons on the HPG backbone is used to determine the percentage of functionalization. 39  The structure of allyl-HPG was also confirmed by 13C NMR spectrum, as shown in Figure 2.3. The triplet at 77.0 ppm represents the chemical shift of CDCl3 solvent. The two carbons in the ene-moiety are easily spotted at 116 ppm and 135 ppm respectively. The methylene group next to the double bond and the HPG backbone are shown around 70 – 73 ppm.  Figure 2.3 13C NMR spectrum of HPG-allyl conjugate.  2.3.3 Characterization of Cationic Ligands: 3-Mercapto-N, N, N-Trimethyl Bromide The cationic ligand, 3-mercapto-N, N, N-trimethyl bromide, was generated from the alkyl halide via a Bunte salt. Alkyl halide was treated with sodium thiosulfate in aqueous solution until complete conversion into the corresponding thiosulfate S-ester (Bunte salt) occurred as detected by TLC analysis. The SN2 displacement of the alkyl bromide with thiosulfate anion proceeded in reasonable yield to afford the corresponding Bunte salt. Thiolsulfates are known to be cleanly and quantitatively cleaved to the corresponding thiol under acidic conditions108. Therefore, the crude Bunte salt was subjected to hydrolysis using 1M H2SO4 at room temperature. Vacuum filtration was utilized to filter off the salt, mostly NaBr and Na2SO4, generated in the reaction. The product was dissolved in the water phase and recovered by freeze-drying. Since the 40  hydrolysis reaction was not carried out under an inert atmosphere, most of the thiols were simultaneously oxidized into the corresponding disulfide. We applied an additional step to cleave the disulfide S-S bond into the corresponding thiol using the phosphorus nucleophile, triphenolphosphine. Figure 2.4 and 2.5 show the 1H NMR spectrum and 13C NMR spectrum respectively.    Figure 2.4 1H NMR spectrum of 3-mercapto-N, N, N-trimethyl bromide. 41   Figure 2.5 13C NMR spectrum of 3-mercapto-N, N, N-trimethyl bromide.   2.3.4 Characterization of Zwitterionic Ligands: Sulfabetaine 2-Propanethiol The sulfabetaine disulfide was prepared in a single step via the ring opening alkylation of bis(3-dimethylaminopropyl)disulfide by 1,3,2-dioxathiolane 2,2-dioxide. Here 1,3,2-dioxathiolane 2,2-dioxide was acting as an alkylsulfating agent. Sulfabetaine 2-propanethiol was obtained by reduction with triphenylphosphine at room temperature, giving a highly hygroscopic powder. The reducing agent triphenylphosphine was used due to the easy phase separation. The 1H NMR spectra and 13C NMR spectra are shown in Figure 2.6 and Figure 2.7 respectively.      42               Figure 2.6 1H NMR spectrum of sulfabetaine 2-propanethiol.            Figure 2.7 13C NMR spectrum of sulfabetaine 2-propanethiol. 43  2.3.5 Chemistry and Structure of HPG-Zwitterionic/Cationic Molecules In this study, double bond-carrying HPG was further modified with different amounts of cationic and zwitterionic molecules bearing terminal thiols via a UV initiated radical thiol-ene “click” reaction under mild conditions. The feeding ratios of cation/zwitterion ranging from 0 to 1 were used to control the degree of modification. An excess of thiol-containing components were used in the reaction mixture to suppress the undesired cross-linking reactions between the double bonds on the backbone. A photo-initiator was used to increase the radical concentration and thus increase the rate of reaction. The residual thiol-containing molecules and photo-initiator were removed by subsequent dialysis. During the irradiation, it was necessary to adjust the ratio of water and organic solvent to ensure the reaction mixture was in a homogenous state. This was because the conversion efficiency of thiol-ene “click” reaction depends on the miscibility of the polymer phase with the reagents employed and the accessibility of surface reactive groups. The synthesized library consisted of 6 structures containing a HPG core, an alkyl spacer, and thiol-ether-linked zwitterionic/cationic ligands. The molecules were colloquially named as follows: HPG-SB is the molecule in which all the double bonds in HPG-allyl are conjugated with  SulfaBetaine head groups; HPG-SB-QA20 is the one that 20% of the double bonds in HPG-allyl are conjugated with Quaternary Ammonium and rest of the double bonds are functionalized with SulfaBetaine.  The functionalized ratio of SB/QA on the HPG backbone can be investigated from 1H NMR spectrum, shown in Figure 2.5. Compared to the 1H NMR of HPG-allyl (Figure 2.3), the new peak at 3.23 ppm represents the protons on the two methyl groups connected to the nitrogen of sulfabetaine, and the peak at 3.17 ppm represents the protons on the three methyl groups connected to the nitrogen of quaternary amine. It is very clear that as the proportion of QA 44  increases, the peak at 3.17 ppm goes up while the peak at 3.23 ppm goes down. These two signals of the resulting end groups do not interfere with other signals in the spectrum and therefore permit reliable integration of the 1H NMR spectrum for the determination of the ratio between zwitterions and cations on the polymer backbone as well as the molecular weight. There are other new peaks at 2.70 ppm corresponding to the ester linkage after full reaction of the alkene groups to form a saturated bond, indicating the successful conjugation of thiol head groups with the peripheral double bonds. Simultaneously, the original two peaks located at 5.1 ppm and 5.8 ppm in Figure 2.3 representing the alkene protons at the periphery disappeared, indicating the alkene side groups were quantitatively consumed during the thiol-ene addition.   Figure 2.8 1H NMR spectra of HPG-SB and HPG-SB-QAx where x indicates the percentage of QA.  45  Figure 2.9 shows the 13C NMR spectra of HPG-SB and HPG-SB-QA40. It is clear that the conjugation of the QA introduces two additional peaks in the 13C NMR spectrum compared to the one functionalized with SB only: 53.2 ppm represents the three methyl groups on the nitrogen, and 65.6 ppm shows the methylene group next to the quaternary amine.   Figure 2.9 13C NMR spectra of HPG-SB and HPG-SB-QA40.  The charge bias property of the polymer could be controlled by regulating the SB and QA ligand ratios. The quantification of the ratio of SB and QA on the HPG backbone was represented by the relative mole fraction of QA segment, as summarized in Table 2.1. The relative proportion of the ligands in the reaction (SB/QA ratio of 100/0, 90/10, 75/25, 70/30, 60/40, and 50/50) was close to those of the modified HPG with relative SB/QA ratios of 100/0, 46  85/15, 80/20, 70/30, 60/40, and 55/45, respectively. This indicated that the relative proportion of zwitterions and cations was effectively and precisely controlled. The number of SB/QA groups was calculated based on the percentage of the two ligands, with the assumption that all of the 216 terminal alkene groups have been converted. A control polymer was synthesized with cationic ligands only and the remaining double bonds were conjugated with 1-thioglycerol; the chemical structure and 1H NMR spectrum of control polymer is shown in Figure 2.7.                        Figure 2.10 1H NMR spectrum of the control polymer.  As shown in Table 2.1, the hydrodynamic radius of the series of polymer conjugates ranged from 4.5 to 6.2 nm, and the hydrodynamic radius of the core HPG is around 3 nm. For HPG-SB, the contribution of the ligands to the overall hydrodynamic radius is ~ 1.5 nm, which is reasonably close to that estimated from the fully extended geometric length of the sulfabetaine ligand (~1.7 nm). As the proportion of the QA moieties increases, the hydrodynamic size as well 47  as the polydispersities of the polymer get larger, suggesting the formation of a tiny fraction of aggregates in the solution, which may also explain why the Mn determined by GPC is generally higher than that calculated from 1H NMR spectra for the HPG-SB-QAX series.  Table 2.1 Physicochemical properties of macromolecules used in this study. Polymer # SB a # QA a Mn a Mn b PDI b Rh (nm) c HPG NA NA NA 2.0 × 104 1.3 3.0 HPG-SB 216 0 8.2 × 104 8.0 × 104 1.4 4.5 HPG-SB-QA15 184 32 7.8 × 104 8.4 × 104 1.5 4.7 HPG-SB-QA20 173 43 7.7 × 104 7.8 × 104 1.6 4.7 HPG-SB-QA30 150 66 7.4 × 104 8.9 × 104 1.5 4.9 HPG-SB-QA40 130 86 7.2 × 104 1.0 × 105 1.7 5.8 HPG-SB-QA45 120 96 7.1 × 104 1.1 × 105 1.8 6.2 Control polymer NA 130 5.4 × 104 NAd NAd NAd a Calculated from 1H NMR integration b Determined by GPC at 25 °C in 1 N NaNO3 c Determined by dynamic light scattering (QELS) at 25 °C in 1 N NaNO3 d The GPC measurement of the control polymer failed due to the positive charge  2.3.6 Quantification of Bound Water of Polymers The amount of bound water on native and modified HPGs was analyzed by DSC, and the results are summarized in Table 2.2. There are three forms of water existing during the interaction between water and polymer molecules: non-freezable water, freezable bound water and free water. Among them only free water would show similar melting/crystallization temperatures as bulk water. In other words, only free water would contribute to the enthalpy 48  change during the melting process at around 0 °C. Thermal transitions were observed for all of the native and modified HPGs, indicating free water exists when all the binding sites of polymers are close to being saturated by water molecules, which gives the transition over a temperature range similar to that of the ice-to-water transition for bulk water. By comparing the enthalpy change of polymer/water system and pure water, the number of bound water per polymer can be calculated.  Table 2.2 The enthalpy changes (ΔH) of water as measured by DSC for aqueous solutions of different polymers. Pure water was used as a normal control.  Polymer C (g/g) ΔH (J/g) Nb Nc Nd Ne Pure H2O NA 362 NA ~ 5 ~ 14 ~ 4 HPG 0.096 289 1200 HPG-SB-QA20 0.089 302 3700 HPG-SB-QA30 0.090 301 3600 HPG-SB-QA40 0.088 303 3300 HPG-SB-QA45 0.086 304 3200 Control polymer 0.098 298 2400         a: the polymer concentration in aqueous solution determined by TGA         b: the number of moles of water bound per mole of polymer, calculated using Eq. 2.1         c: the number water molecules bound per glycerol unit         d: the number of water molecules bound per SB unit, calculated using multiple linear regression, p = 0.002         e: the number of water molecules bound per QA unit, calculated using multiple linear regression, p = 0.08   The hydration of a material plays a key role in its biocompatibility. The functionalization of HPG with zwitterionic/cationic ligands greatly increased its ability to hold water molecules. In 49  addition to the water molecules bound to the HPG backbone via hydrogen bonding, the charged ligands mainly interact with water molecules via ionic solvation, which can generate a strong hydration layer.  For the cationic quaternary ammonium ligand, water molecules should bind to the positively charged group (-N+(CH3)3). For the zwitterionic sulfate betaine ligand, water molecules would simultaneously bind to the positively charged group (-N+(CH3)2-) and the negatively charged group (-SO4-). As illustrated in Figure 2.7, a zwitterionic ligand is able to hold more water molecules compared to a cationic ligand because of the dual ions, which might provide more interaction sites. This is also confirmed by the data shown in Table 2.2, as the percentage of cationic ligand increases, the moles of water bound per mole of polymer decreases. The number of water molecules bound per SB/QA ligand is obtained using multiple linear regression, and the result shows that there are on average 5 water molecules bound to per glycerol unit in the HPG backbone, which is comparable with pervious publication71, 14 water molecules bound to one SB ligand, and 4 water molecules bound to one QA ligand.         Figure 2.11 A proposed model of the possible interactions between water and the polymers. Two-dimensional illustration here but the real world is three-dimentional.  O S NHPG O SOO OO OH HO S NO HH OHH OH HOHHO HHOHH OH HOHH O HH OH HO HHOH H OHH OH H50  2.4 Summary Here the concept of tandem post-polymerization modification was utilized to synthesize a series of novel water-soluble HPG-based multifunctional polymer conjugates. Narrowly dispersed HPG was synthesized using a one-pot reaction. Post-polymerization modification of HPG with allyl chloride resulted in a well-defined hyperbranched polymer with pendant allyl functionality, which could not be obtained by direct polymerization of double-bond containing monomers due to the cross-linking side reactions. This polymer was further functionalized via thiol-ene “click” reaction with a mixture of thiol-terminated zwitterionic sulfabetaine and cationic quaternary ammonium ligands. NMR and GPC analysis confirmed the structure and molecular weight of the library of conjugates. DSC result showed that the SB/QA functionalized HPG had a higher ability to bind water molecules compared to native HPG, which was mainly contributed by the dual ion on SB ligand. The enhanced hydration observed would account for the improved biocompatibility of the polymers.  The synthesis template presented here represents a strategy to improve the biocompatibility of polycations through rationale design. This rationale design attempted to attenuate the cytotoxicity of cationic moieties while still keeping their essential traits, fine-tuning the compositions of zwitterionic and cationic ligands on the surface by varying the monomer concentration in the reaction. This methodology supports the future investigation of synthesizing multifunctional polymers for predetermined composition, functionality, molecular weight and architecture. The biocompatibility and potential application of the conjugates was investigated and is reported in the next chapter. 51  Chapter 3: In Vitro Characterization of Functional Hyperbranched Polyglycerol  3.1 Introduction Hemostatic agents have proven to be quite attractive as topical wound dressings2, 4, 109. Hemostatic agents perform their task by enhancing one or several stages in the hemostasis process, including concentrating the platelets and coagulation factor at the site of bleeding by absorbing water, activating and aggregating platelets to form clots at the bleeding site, creating a mechanical barrier to bleeding by adhering strongly to the cell surfaces, and accelerating the production of one or more coagulation factors5. Although an explosive of hemostatic agents have been developed due to the advances in biotechnology, hemorrhage is still a major cause of morbidity and mortality. This is because none of the present generation of hemostatic agents is free of drawbacks. Synthetic hemostatic agents such as cyanoacrylates and glutaraldehyde cross-linked albumin are reported to exhibit toxicity110 and potential mutagenicity4 or carcinogenicity. The application of widely used zeolite-based QuickClot generates heat that can cause burn injuries111. Fibrin-derived hemostatic agents overcome these problems, yet they still have some limitations such as high cost2. Thus there is a need for developing novel synthetic biocompatible adhesives that could overcome these limitations.  Positively charged polymers of various architectures interact with blood components including erythrocytes and platelets due to their overall negative surface charge. Such ionic, multivalent interaction of the polymer with the cell membrane can cause aggregation of cells in a polymer concentration and surface charge density dependent manner. Defined and adjustable 52  polymer/biomembrane interactions yield control over the strength of cell aggregation and adhesiveness and thus contribute a valuable tool to the biomedical and tissue engineering field. However, the strong interaction of positively charged polymers with biomembranes can be toxic to cells.  Disruption of the cell membrane and lysis are often encountered (occasionally desired) effects of positively charged polymers on eukaryotic and prokaryotic cells.  Polybetaine modified, charge neutral polymers of linear or branched architecture are considered stealth polymers within the biomedical community due to their very weak interaction with bioentities, such as proteins and cells112-113. In part the high degree of hydration of such betaines which exceeds the hydration of PEG80, in general, is held responsible for the observed “stealth” effect of polymers or surfaces decorated with dipolar groups81.    In Chapter 2, we showed that engineering the surface of HPG through conjugating different numbers of cationic quaternary ammonium and zwitterionic sulfabetaine resulted in multivalent hyperbranched polymers with strong hydration ability. In this chapter, the blood compatibility and hemostatic potential of the series of polymers were investigated. We hypothesize that randomly distributed positive charges on the polymer backbone can be screened by multiple “stealth” betaine groups. These betaine groups render the polymer biocompatible and attenuate adverse charge-related toxic effects. At the same time ionic charge interactions of these polymers with negatively charged biomembranes are not completely hindered by the betaine groups. The model system is tested via regulating the ratio of a positively charged quaternary ammonium group and a charge neutral sulfabetaine group quantitatively to control the charge bias property of the polymer.   Since the polymer we develop is proposed to have the potential to work as a topical hemostatic dressing, emphasis was placed on defining the ability of the polymer to help form a 53  blood clot to seal the wounds without causing any undesired side effects. The ability of the polymer to aggregate red cells and platelets, and to activate platelets was evaluated. The influence of the polymer on red cell membrane integrity, on the complement system, and on cytotoxicity to endothelial cells were studied. Coagulation assays including prothrombin time (PT), activated partial thromboplastin time (aPTT) and thromboelastography (TEG) were also performed to investigate whether the polymer-induced hemostasis is related to the classic blood coagulation cascade. We also investigated the possibility of reversing this strong aggregation effect using a polymer bearing multiple anionic sites. These results contribute to our understanding of the interactions between the polymer and blood components, and are an important first step in determining the suitability of these polymers for further testing in an in vivo bleeding model.  3.2 Experimental Section 3.2.1 Materials The polymer conjugates used were synthesized as described in Chapter 2. The aPTT and PT reagents were purchased from Dade Behring. Anti-CD62PE and goat anti-mouse PE antibodies were purchased from Immunotech. GVB2+ (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN3 with 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.3) and antibody-sensitized sheep erythrocytes were purchased from CompTech (Tyler, TX). Human umbilical vein endothelial cells (HUVECs) were obtained from Lorus Pharmaceuticals (Allendale, NJ).  Phosphate buffered saline (PBS) was prepared by dissolving PBS tablets in distilled water. Physiological saline was prepared by making 0.9% NaCl solution. Low ionic strength saline was prepared by making 4% glucose in 0.18% NaCl solution.   Sodium phosphate buffer 54  (pH = 7.4) was prepared by dissolving 0.31 g NaH2PO4•H2O, 1.09 g Na2HPO4 and 1.75 g NaCl in 200 mL of deionized water.  Water used in all experiments was purified using a Milli-Q Plus water purification system (Millipore Corp., Bedford, MA).  3.2.2 Blood Analysis 3.2.2.1 Blood Preparation Blood was drawn from healthy consented volunteers into a 3.8% sodium citrated tube (BD VacutainerTM) with a blood/coagulant ratio of 9:1 at the Centre for Blood Research, by a protocol approved by the University of British Columbia clinical ethics committee. For serum preparation, blood was collected in serum tubes without anticoagulant. After 20 min, the clotted whole blood sample was centrifuged at 1200×g for 30 min in an Allegra X-22R centrifuge (Beckman Coulter, Canada). The supernatant serum was transferred into a plastic tube and stored at room temperature until use. Platelet poor plasma (PPP) was isolated by centrifugation of citrated whole blood samples at 3000×g for 15 min at room temperature. Platelet Rich Plasma (PRP) was isolated by centrifuging the citrated blood at 150×g for 15 min. Packed RBCs were separated by centrifugation of whole blood at 1000×g for 5 min. The plasma and buffy coat were removed, and RBCs were further washed three times with sterile PBS solution by centrifugation. RBC suspensions were prepared by resuspending one part of packed RBCs in seven parts of the corresponding buffer (PBS or saline) to yield a hematocrit (volume percentage of RBCs in the blood) of 10%.  55  3.2.2.2 Red Blood Cell Aggregation Red blood cell aggregation was performed using both whole blood and washed RBC suspensions. RBC aggregation in whole blood was performed according to a previous publication of our lab69. Ten µL stock solution (50 mg/mL and 10 mg/mL) of polymer in PBS was first transferred into an Eppendorf tube, and then 90 µL of whole blood was added into the polymer solution with a micropipette to achieve a final concentration of 5 mg/mL and 1 mg/mL of polymer in the blood, resulting in a 36% hematocrit. To prevent high local concentration of the polymer, the mixture was homogenized by several up-and-down aspirations and then incubated for 5 min at 37 °C. Whole blood incubated with PBS was used as the negative control. After incubation, the RBCs and plasma/polymer were separated by centrifugation at 8000 rpm for 3 min. Aliquots of 2 µL of the red blood cells were resuspended in 10 µL of plasma/polymer and examined by bright field light microscopy (Zeiss Axioskop 2plus) using wet mounted slides. Images were captured with a digital microscope camera (AxioCam ICc 1, Carl Zeiss Microimaging Inc.) at 40× magnification of ten different observation fields on the same chip for statistically sound results.  RBC aggregation with washed RBCs was performed by mixing one part of polymer stock solution with nine parts of 10% hematocrit washed RBC suspension. Then aliquots of 5 µL of the mixture were used for microscope examination.  3.2.2.3 Hemolysis Assay The membrane disruption of RBCs was estimated to determine the hemocompatible nature of polymers. The hemolysis assay was performed according to a procedure modified from previous references114. A 40 µL aliquot of the stock polymer solution prepared in PBS (50 56  mg/mL) was added to 360 µL of the 10% w/v RBCs suspension to make the final dendrimer concentration 5 mg/mL and then incubated at 37 °C for 1 h. After incubation, the mixture was centrifuged and the supernatants were transferred to a 96-well plate (150 µL per well). Release of hemoglobin was measured by spectrophotometric analysis of the supernatant at 540 nm. Deionized water and PBS were used as positive and negative controls, respectively. The percent of hemolysis was calculated as follows: 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠  %   =   𝐴𝑏𝑠™? , ™ ⌡    ™????   –   𝐴𝑏𝑠™? , ™?𝐴𝑏𝑠™? , ™???   –   𝐴𝑏𝑠™? , ™?   ×  100%                          (Eq. 3.1)    3.2.2.4 Complement Activation The level of complement activation was measured by CH50 sheep erythrocyte complement lysis assay in human serum, according to a previous publication by our lab115. Stock solutions of polymer at concentrations of 10 mg/mL and 1 mg/mL were prepared. Aliquots of 10 µL stock solutions were mixed with 90 µL of GVB2+ diluted human serum to obtain a final polymer concentration of 1 mg/mL and 0.1 mg/mL respectively. Heat-aggregated human IgG and PBS mixed with diluted human serum were used as positive and negative controls respectively. After 1 h incubation at 37 °C, 75 µL of the polymer/serum mixture was further mixed with 75 µL antibody-sensitized (Ab-sensitized) sheep erythrocyte and then incubated for another hour. Then 300 µL of cold GVB-EDTA buffer was added to each sample. Ab-sensitized sheep erythrocytes incubated with distilled H2O were regarded as the 100% lysis control. Intact Ab-sensitized sheep erythrocytes were then centrifuged at 8000 rpm for 3 min and the supernatants were sampled in a 96-well plate (100 µL per well). Percentage sheep erythrocyte lysis was calculated using average absorbance values at 540 nm as follows: 57  %𝑙𝑦𝑠𝑖𝑠 =    ™? ™? , ™??    ™???? ? ™? ™? , ™?™? ™? , ™??? ? ™? ™? , ™?   ×  100%                                                  (Eq. 3.2)  Percentage of complement consumed by polymer was expressed as 100 - %lysis.  3.2.2.5 Platelet Activation P-selectin (CD62P) surface expression is commonly used as a marker to quantify the level of platelet activation. For a typical experiment, 90 µL of PRP was mixed with 10 µL of stock polymer samples (10 mg/mL and 1 mg/mL) to achieve a final polymer concentration of 1 mg/mL and 0.1 mg/mL respectively. After 1 h incubation at 37 ˚C, aliquots of the incubation mixtures were removed for assessment of the platelet activation state. Aliquots of 5 µL post-incubation platelet/polymer mixture were added to 45 µL PBS buffer, and further incubated for 20 min in the dark at room temperature with 5 µL of fluorescently labeled monoclonal anti-CD62-PE. The incubation was then stopped by adding 300 µL of PBS buffer. The level of platelet activation was analyzed in a BD FACSCanto II flow cytometer (Becton Dickinson) by gating platelet specific events based on their light scattering profile. Activation of platelets was expressed as the percentage of platelet activation marker CD62-PE fluorescence detected in the 104 total events counted. Duplicate measurements were performed for each donor, and three different donors were used. The results were reported as mean with standard deviation.  3.2.2.6 Platelet Aggregation Aggregation of platelets was performed in a flat-bottomed 96-well plate, the method is modified from an experimental procedure that has been previously published116. Aliquots of 90 µL of PRP were pipetted per well into the microplate and 10 µL of polymer stock solution was 58  added into the appropriate wells. Adenosine diphosphate (ADP) with a concentration of 5µM and PBS were used as a positive and negative control respectively. For calibration, 100 µL of PRP and 100 µL of PPP were placed in some wells. After the addition of agonists, kinetic reading of the plate was started immediately. The light transmittance (LT) was then measured at 595 nm at an interval of 15 seconds for 20 minutes at 37 ˚C using a Spectramax PLUS plate reader (Molecular Devices). During the run time the plate was mixed every 3 seconds using the automix function of the reader. The percentage of aggregation was calculated using the formula: %  𝑜𝑓  𝑎𝑔𝑔𝑟𝑒𝑔𝑎𝑡𝑖𝑜𝑛 =    ™ ™? , ™??    ™???? ? ™ ™? ,   ℡?™ ™? ,       ℡? ? ™ ™? ,       ™?   ×  100%                                    (Eq. 3.3)   3.2.2.7 Coagulation 3.2.2.7.1 PT and aPTT Activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured using a coagulation analyzer using mechanical end point determination (ST4, Diagnostica Stago) according to the procedure published previously115. The effect of each polymer on the intrinsic pathway of coagulation was studied after mixing PPP with the polymer stock solution (9:1 v/v polymer: plasma) in the cuvette-strips at 37 °C for 3 min before adding the partial thromboplastin reagent (Actin® reagent) and calcium chloride (CaCl2) solution. Actin® reagent contains phospholipids, ellagic acid, stabilizers and preservative. For PT determination, stock solution of the polymer was first mixed with PPP (9:1 v/v PPP: polymer), and the extrinsic coagulation pathway was activated by incubating plasma with innovin® reagent and the clotting time then measured. Innovin® is a lyophilized reagent consisting of recombinant human tissue factor and synthetic phospholipids (thrombosplastin), calcium ions, a heparin-neutralizing 59  compound, buffers and stabilizers (bovine serum albumin). A control experiment was done by adding identical volume of PBS buffer to plasma. Each measurement was performed in triplicate, using plasma from at least three different donors.  3.2.2.7.2 Thromboelastography The effect of polymers on blood clotting was examined using thromboelastography (TEG) analysis. TEG is used to assess the viscoelastic changes in whole blood clotting under low shear conditions after adding a specific coagulation activator117. In a typical experiment, 40 µL of stock polymer solution was mixed with 360 µL of freshly drawn citrated whole blood (within 5-10 min of venipuncture). Aliquots of 340 µL of the polymer/blood mixture were loaded into TEG cups in the analyzer. The coagulation analysis began when the citrated whole blood was re-calcified with 20 µL of CaCl2 solution (0.20 M). PBS (40 µL) mixed with citrated whole blood (360 µL) served as the normal control for experimental study.  Each experiment was performed at least three times with different donors. All TEG experiments were performed at 37°C and ended after 2 h. The detailed procedure was published previously118.  3.2.3 Cytotoxicity Measurement The cytotoxicity of the polymers was studied by using the propidium iodide live cell staining assay119. The cells were seeded on 96-well plate at a density of approximately 5×103 cells per well. After 24 h of cell-seeding incubation at 37 °C, polymer stock solution (dissolved in cell culture media and filtered) was added to the appropriate wells to achieve a final concentration of 0.25, 0.5, 1 and 2 mg/mL and incubated for 48 h. A positive control was included using 50% DMSO in six wells per plate to induce cell death. Twelve wells in each plate 60  were also reserved as a negative control with no chemical addition. Cell toxicity was determined by staining cells with propidium iodide (PI) and a Hoechst dye solution. After chemical exposure, 100 µL of staining solution (1 µL of 20 mg/mL Hoechst, 100 µL of 50 µg/mL PI, 10 mL pre-warned medium) was added directly into the wells without aspirating the treatment medium and incubated for 30 min at 37 ˚C. Hoechst binds to DNA of all living/dead cells, and thus is used as a blue fluorescent marker for the nuclei of all cells. PI is a red fluorescent nuclear and chromosomal counterstain that is not permeable to live cells and therefore only stains cells with compromised cell membranes. Cells were then imaged using the CellomicsTM Arrayscan VTI (Thermo Scientific, Pittsburgh, PA) high content imaging system. Cell viability was calculated as the proportion of all cells not stained with PI. The untreated wells (media) were regarded as reference wells during the scan and the data output %responder indicated as a percentage of cells with average intensity in cytoplasmic greater than the average intensity in the untreated cells. Absolute values are reported along with the media control.  3.2.4 Isothermal Titration Calorimetry Analysis Isothermal titration calorimetry (ITC) was performed using a MicroCal iTC200 calorimeter from GE Healthcare (Northampton, MA). Sodium phosphate buffer (pH = 7.4) was used to dissolve the compounds for ITC study. Titrations were performed by injecting consecutive 10 µL of ligand solution into the ITC cell containing HPG-SB-QA45. The ITC data were corrected for the heat of dilution of the titrant by subtracting mixing enthalpies for 10 µL injections of ligand solution into macromolecule-free buffer. At least two independent titration experiments were performed at 37 °C to determine the binding constant of ligand to HPG-SB-QA45. Isotherm data were analyzed with Origin7 software (MicroCal) supplied by the 61  manufacturer. Binding stoichiometry (N), enthalpy (ΔH), and equilibrium association constant (Ka), were determined by fitting the corrected data to an independent binding model. The stoichiometry and binding constants were calculated with respect to the LPG-sulfate binding units per HPG-SB-QA45 molecule. The Ka value then permitted calculation of the change in free energy (ΔG), which together with the ΔH allowed the calculation of the entropic term TΔS.  3.2.5 Statistical Analysis Data were expressed as means ± standard deviation (SD). Measurements were conducted in at least triplicate if not stated. All statistical analysis was performed using the Prism software program (GraphPad, San Diego, CA). Data were statistically analyzed using repeated measures analysis of variance with the Tukey test at a 95% confidence interval. A p value < 0.05 was considered as statistically significant.  3.3 Results and Discussions 3.3.1 Effects of Polymers on the Aggregation Morphology of Red Blood Cells The role of red cells in hemostasis has recently attracted renewed attention. It was previous recognized that increased aggregation of RBCs upon interaction with nanomaterials was a highly undesired phenomenon that could induce serious circulatory disorders45, thus absence of hemagglutination was considered as a main criterion for the evaluation of hemocompatibility of blood-contacting materials. However, RBCs do not appear to be a mere bystander in hemostasis; there are studies showing the aggregation of RBCs can not only influence fibrin network structure and pore size120 but also change to a polyhedron shape to form a perfect seal17.   62                                                  Figure 3.1 Optical microscopic images of human whole blood incubated with polymers. All images are at 40×magnification. (A) PBS control, (B) HPG-SB, (C) HPG-SB-QA30, (D) HPG-SB-QA35, (E) HPG-SB-QA40, (F) HPG-SB-QA45; polymer concentration: 5 mg/mL. (A) (C) (D) (E) (F) (B) 63   Figure 3.2 Optical microscopic images of human whole blood incubated with polymers. All images are at 40×magnification. (A) HPG-SB-QA30, (B) HPG-SB-QA35, (C) HPG-SB-QA40, (D) HPG-SB-QA45; polymer concentration: 1 mg/mL.  Figures 3.1 and 3.2 include representative images of RBC morphology in whole blood observed in the presence or absence of conjugates. In the absence of polymer, as shown in Figure 3.1(A), native RBCs stacked together in groups of 4 – 10 cells and formed typical rouleaux. Similar features were observed upon the addition of 5 mg/mL charge neutral HPG-SB solution (Figure 3.1(B)), indicating that it would not induce hemagglutination.  As the percentage of positively charged QA on the polymer was increased from 20 to 45, native RBCs clumped together and led to aggregates. The higher percentage of cationic groups, the greater the size of (A) (B) (C) (D) 64  the aggregates. The strongest aggregation activity was observed with HPG-SB-QA45, shown in Figure 3.1(E), where RBCs lost their original biconcave morphology and showed an unusual affinity towards each other, with almost no monodispersed cells. When the concentration of HPG-SB-QA45 is reduced to 1 mg/mL, there are still visible aggregates, as shown in Figure 3.2(D), but the size of the aggregate is much smaller compared to those in the 5 mg/mL one. More monodispersed cells are observed for the 1 mg/mL polymer solution compared to the 5 mg/mL one. The higher the polymer concentration, the greater the size of the agglutinates and the smaller the number of monodispersed cells. The results showed that the degree of hemagglutination increases in relation to the increment of polymer concentration and positive charge density. As whole blood was induced to aggregate by the addition of HPG-SB-QAX, we also investigated the aggregation of washed erythrocytes, shown in Figure 3.3. As expected there was no rouleaux formation in washed erythrocytes, and RBCs retained their original biconcave shape when dispersed in PBS buffer. It is of interest to point out the aggregation happens at lower concentration in washed RBCs. The aggregates in Figure 3.3(C) and (D) were much easier to identify compared to those in Figure 3.2(C) and (D). This can be explained by a partial neutralization of the cationic ligands through their interactions with plasma proteins, which are mostly negatively charged as well. Thus theses plasma proteins might compete with RBCs for the cationic ligands.     65               Figure 3.3 Optical microscopic images of washed RBCs incubated with or without polymers. All images are at 40×magnification. (A) PBS, (B) HPG-SB, (C) HPG-SB-QA40, (D) HPG-SB-QA45; polymer concentration: 1 mg/mL.  Polymer with a lower proportion of cationic ligands, for example HPG-SB-QA15, does not cause RBC aggregation under physiological ionic strength (0.9% NaCl), shown in Figure 3.4(B); but induced severe aggregation under low ionic strength (0.18% NaCl + 4% glucose, glucose was added to retain isosmotic conditions), as shown in Figure 3.4(D). This is because in physiological saline where more ions are presented, the negatively charged chloride ions will seek the positively charged moieties on the polymer, surrounding and shielding them, thus weakening the interactions between the macromolecule and RBC surface. On the contrary, in low ionic strength saline where fewer chloride anions are available to interact with the positively (A) (C) (B) (D) 66  charged moieties on the polymer, stronger binding of the cationic ligands to the negatively charged RBC surfaces can occur.               Figure 3.4 Optical microscopic images of washed RBCs incubated with or without polymers in saline with different ionic strength. All images are at 40×magnification. (A) physiological saline (0.9% NaCl), (B) HPG-SB-QA15 in physiological saline, (C) low ionic strength saline (0.18% saline + 4% glucose), (D) HPG-SB-QA15 in low ionic strength saline; polymer concentration: 1 mg/mL.  The RBC surface has a net negative charge, which is mainly because of the ionization of carboxyl group of N-acetylneuraminic acid residues of the membrane glycoprotein14-15. These charges help prevent the interaction between RBCs and other blood components, majority of which are also negatively charged. The binding between positively charged macromolecules and (A) (B) (C) (D) 67  negatively charged RBC membrane was assigned to electrostatic attractions. It could be pictured as the polymer is acting as a physical bridging among RBCs through the cationic ligands that can be attached to adjacent cells, as shown in Figure 3.5.  For agglutination to occur, the attractive interactions provided by oppositely charged polyionic structures have to overcome the repulsive forces generated between the negative surfaces of RBCs.              Figure 3.5 Illustration of the hemagglutination reaction induced by positively charged polymer.  3.3.2 Evaluation of the Hemolytic Activity of Polymers Having demonstrated the aggregation of erythrocytes induced by the HPG-SB-QAX polymers, we were interested in evaluating the extent of RBC membrane damage. For this purpose, a hemolysis assay was performed. Hemolysis is the disruption of the RBC membrane, hence causing the release of hemoglobin, which can be detected by absorbance at 540 nm. Hemolysis was measured and compared with the PBS control. A control polymer with no zwitterionic groups was included. The results were shown in Figure 3.6.    Red blood cells Positively charged polymer Aggutination68   Figure 3.6 (A) Hemolysis assay for SB/QA-modified HPG. Polymer concentration is 5 mg/mL. Relative hemolysis is expressed considering the hemolysis of PBS as 0% and hemolysis of water as 100%. Asterisk indicates significant difference, **p < 0.01. (B) Images of RBCs incubated with or without polymers (5 mg/mL) at 37 ˚C for 1 h.  Although the polymers, namely HPG-SB-QA40 and HPG-SB-QA45, showed strong RBC aggregation, the hemolytic activity of polymers is at the undetectable level (less than 1%), with almost no measurable undesired reaction following the RBC membrane contact. The release of hemoglobin was not significantly different from the control. On the contrary, the control polymer with no zwitterionic ligands caused severe hemolysis (~ 23%), with a supernatant that was clearly red as shown in Figure 3.6(B).  This could be explained by the fact that cell aggregation involved only the outer layer of RBC membrane, but the release of hemoglobin would require disturbance of the inner part of the membrane structures sufficiently to affect cell permeability. This suggests the SB/QA modified HPG does not penetrate into the RBC membrane which would result in cell lysis; instead it adsorbs on the RBC surface, keeping a more open conformation with flexible cationic ligands able to bridge adjacent RBCs. Due to the strong hydration property, the water molecules bound to the zwitterionic ligands may create an energy barrier that contributes to reducing the cross-linking action mediated by the cationic ligands. 69  There is no such camouflage provided by dipolar groups in the control polymer, thus severe disruption of RBC membrane occurs. The excellent compatibility with RBCs indicates that the polymer could be a promising candidate for applications in vivo.  3.3.3 Effect of Polymers on the Aggregation and Activation of Platelets Compared with other blood cells, platelets are one of the most sensitive kinds to react with foreign surfaces, giving rise to their activation and aggregation. Platelets are key blood components that are involved in hemostasis. We hypothesize that the positive charge on the surface of HPG-SB-QAX could bind to platelets and enhance their adhesion and activation, which results in the formation of a cell embolus and promotes blood clotting.   3.3.3.1 Effect of Polymers on the Aggregation of Platelets To test the extent of aggregation induced by polymer, PRP was incubate with HPG-SB-QAX polymers and the transmittance at 595 nm was continously measured for 20 minutes. Following exposure to an agonist, formation of platelet aggregates results in an increase in light transmission through the sample. ADP, which has long been recognized as a stimulus for platelet adhesion and aggregation, was used as a positive control. The aggregation traces of PRP stimulated by different concentration of HPG-SB-QAX polymers at 37 ˚C monitored in the microplate reader (averages from the two simultaneously run measurements of three different donors) are shown in Figure 3.7. The maximal extent of aggregation is shown in Figure 3.8 (A). 70   Figure 3.7 Aggregation traces of platelets in plasma. Platelet aggregation was measured in the microplate reader at 37 ˚C (wavelength 595 nm, n = 6). Polymer concentration: (A) 1 mg/mL, (B) 0.1 mg/mL.   Maximum percentage of platelet aggregation induced by HPG-SB-QA20-45 in human PRP, as shown in Figure 3.8(A),  was significantly different compared to the PBS control, even at 0.1 mg/mL. The ability to induce platelet aggregation by HPG-SB-QA45 was not significantly different from the ADP control. On the contrary,  HPG-SB bearing no positively charged moieties could not cause platelet aggregation, and neither did HPG-SB-QA15, whose ability to induce platelet aggregation was not significantly different from PBS buffer, indicating only high concentration of cationic groups per polymer, not neutral or low concentration of cationic groups per molecule, would induce the aggregation of human platelets in plasma. The percentage of aggregation was clearly related to the concentration of surface ammounium groups.   0 500 1000020406080100Time (s)Light Transmittance (%)ADPHPG-SB-QA45HPG-SB-QA40HPG-SB-QA30 HPG-SB-QA20HPG-SBPBS control0 500 1000020406080100TimeLight Transmittance (%)(B) (A) 71   Figure 3.8 (A) Maximal percentage aggregation of PRP measured by light transmittance with a microplate reader; (B) Platelet activation in PRP measured by the expression of platelet activation marker CD62P using anti-CD62P antibody by flow cytometry; ns means p > 0.05.  3.3.3.2 Effect of Polymers on the Activation of Platelets Platelet activation has been another line of study for understanding the hemostatic potential of HPG-SB-QAX  polymers. Activation level of platelets in human PRP in the presence of polymers with proportions of cationic ligands higher than 20%, as determined by measuring the expression of the platelet activation marker P-selectin (CD62P) using fluorescently labeled anti-CD62P antibody, was significantly different compared to that of control platelets incubated with buffer solution, while the neutral polymer HPG-SB and the polymer with 15% cationic ligands showed no difference compared to PBS control. This result agrees with that from platelet aggregation. Platelets have a net negative charge and as a result, they could attract the positively charged polymers. Decreasing the QA moieties on the HPG backbone reduced the number of activated platelets. It was shown that almost no activated platelets were caused by HPG-SB with overall charge neutrality. The results indicate that the SB/QA modified HPG has a proaggregant PBS BufferHPG-SBHPG-SB-QA15HPG-SB-QA20HPG-SB-QA30HPG-SB-QA40HPG-SB-QA45Control PolymerADP0204060801001200.1 mg/mL1 mg/mLControlnsns% AggregationPBS BufferHPG-SBHPG-SB-QA15HPG-SB-QA20HPG-SB-QA30HPG-SB-QA40HPG-SB-QA45Control Polymer020406080100120% of CD62 +ve platelets0.1 mg/mL1 mg/mLPBSns(A) (B) 72  and proactivating effect on platelets, and the initial contact between the polymer and platelets could be an agglutination leading to platelet activaton and granule secretion.  3.3.3.3 Relationship Between Platelet Aggregation and Activation To study the relationship between HPG-SB-QAX  induced platelet activation and aggregation, flow cytometric protocols and optical aggregometry were applied to establish a mathematical relationship between platelet activation and PRP aggregation, after stimulation with different polymers.                 Figure 3.9 Relationships between CD62P expression and PRP aggregation. Each point represents the mean values of 5 independent experiments. See the text for the corresponding equations and statistical significance.  A linear regression characterized the relationship between PRP maximal aggregation and CD62P surface expression was obtained as follows: 𝑦   =   1.064𝑥  –   9.310 with R2 = 0.9477 and P < 0.0001. 0 20 40 60 80 100020406080100% of CD62P platelets% of PRP aggregation73  Our results showed the existence of a positive linear correlation between platelet aggregation and the release reaction of α-granules, indirectly quantified as surface expression of the P-selectin molecule. The result was consistent with previous publication121.  3.3.4 Effect of the Polymers on Coagulation 3.3.4.1 PT and aPTT A change in plasma coagulation properties upon incubation with the polymer is an indication of its interactions with blood components. Accepted in vitro blood coagulation tests include prothrombin time (PT), activated partial thromboplastin time (aPTT). PT is used to evaluate the extrinsic and common coagulation pathway whereas aPTT is used to evaluate the intrinsic pathway.  PT measures the amount of time it takes factor VIIa to form a complex with tissue factor and proceed to clot formation122, with 11-14 s as the normal range. As shown in Figure 3.10(A), the presence of HPG-SB-QAX polymers in PPP did not produce any statistically significant difference in the time taken for clots to form compared to the PBS buffer control, suggesting that the extrinsic pathway of coagulation is not affected by these polymer conjugates. However the control polymer almost doubled the time and fell outside the normal range. The results of aPTT are expressed in seconds and measure the speed of the contact pathway122. As shown in Figure 3.10(B), the neutrally charged HPG-SB did not affect aPTT but all positively charged HPG-SB-QAX conjugates increased aPTT significantly compared to the PBS control, indicating the presence of HPG-SB-QAX would somehow affect the intrinsic coagulation pathway. This suggested the positively charged HPG-SB-QAX might form polyelectrolyte complexes with key proteins governing the intrinsic coagulation pathway, but the detailed mechanism is still 74  unknown to us. The aPTT of the control polymer fell outside the measurement range of the instrument (The measurement stopped automatically after 500 s).  Figure 3.10 Effect of polymers on coagulation (A) prothrombin time (PT); (B) activated partial thromboplastin time (aPTT). Polymer concentration is 1 mg/mL. ** means p < 0.01, ns means p > 0.05.   3.3.4.2 Thromboelastography (TEG) PT and aPTT alone cannot always reflect overall biomaterial-induced blood clotting activity. Further studies are required to better understand these effects. TEG characterizes the formation and strength of blood clot in fresh human whole blood as a function of time, providing more detailed information on blood coagulation compared to aPTT and PT. TEG is used to investigate the following parameters in the blood clotting process123, shown in the standard TEG curve in Figure 3.11(A). R represents the time until the first evidence of a clot is detected after calcium is added back into the citrated whole blood and is measured at the point that the two lines have separated 2 mm. K represents the clot propagation and is measured as the time from R until the curves are 20 mm apart. The angle α represents the tangential angle of the curve with the horizontal and also represents clot propagation. MA is the maximal amplitude represented as PBS BufferHPG-SBHPG-SB-QA30HPG-SB-QA40HPG-SB-QA45Control Polymer050100150400600aPTT (sec)ns**> 500PBS BufferHPG-SBHPG-SB-QA30HPG-SB-QA40HPG-SB-QA45Control Polymer0510152025PT (sec)ns**(A) (B) 75  the distance in millimeters between the two curves once they are parallel and represents peak clot rigidity. LY30 is the rate of amplitude reduction 30 min after maximum amplitude, and assesses the clot breakdown.  Here the influence of SB/QA modified HPG on the whole blood clotting process were measured, using PBS buffer as a normal control. The TEG data is listed in Table 3.1. Additionally, representative TEG traces are given in Figure 3.11(B) and (C). TEG traces for PBS control, and HPG-SB-QA30 (0.1 mg/mL) did not differ. For HPG-SB-QA30 (1 mg/mL) and HPG-SB-QA45 (0.1 mg/mL), the initial clotting time (R) was prolonged by 2-3 min, but still considered within normal range. TEG traces did not lose their characteristic shape but the blood clotting time was prolonged significantly when positive charge density and concentration of the conjugate both increased, which agreed with the aPTT result. Upon the treatment of HPG-SB-QA40/45, the initial “artificial” clot formed by the aggregation of RBCs and platelets would help plug the wound; the coagulation system will be initiated although it takes longer time.   Table 3.1 Clotting kinetics parameter values of human whole blood mixed with polymer solutions. Polymer (mg/mL) R (min) K (min) α (deg) MA (mm) Reference Range124 3 - 22 2 - 7 47 - 78 48 - 70 PBS control 12.1 ± 1.8 3.9 ± 1.4 50.7 ± 9.4 55.4 ± 4.1 HPG-SB-QA30, 0.1 HPG-SB-QA30, 1 11.9 ± 1.0 3.2 ± 0.8 50.4 ± 7.2 53.1 ± 8.6 14.9 ± 3.6 3.4 ± 1.2 47.7 ± 10.4 54.4 ± 6.9 HPG-SB-QA45, 0.1  13.8 ± 3.1 3.6 ± 1.0 47.3 ± 6.9 49.6 ± 1.2 HPG-SB-QA40, 1 23.9 ± 4.1 8.2 ± 1.9 19.1 ± 5.5 47.7 ± 0.4 HPG-SB-QA45, 1 62.0 ± 5.9 NA 5.4 ± 0.8 20.9 ± 2.2  76                                                 Figure 3.11 (A) Normal TEG trace; (B) Representative TEG traces for polymers with concentration of 1 mg/mL. Black line shows the PBS buffer. Green shows HPG-SB-QA30. Pink shows HPG-SB-QA40. Brown shows HPG-SB-QA45; (C) Representative TEG traces for polymers with concentration of 0.1 mg/mL. Black line shows the PBS buffer. Green shows HPG-SB-QA30. Pink shows HPG-SB-QA45. (A) (B) (C) Ca2+ add to the citrated  whole blood in the TEG 77  3.3.5 Effect of the Polymers on Complement Activation Activation of the complement system is a major mechanism in the immune response to facilitate the elimination of bacteria, viruses and other foreign bodies125. About 30 proteins in plasma or on the surface of the immune cells may be involved in this complex immunological machinery. Complement activation is one of the major barriers limiting the use of synthetic cationic polymers in the biomedical field. Activation of the complement system could produce anaphylatoxin that would activate the immune system126. Complement activation is a major parameter to evaluate since it has been shown that polycations could activate complement47. Biomaterial-induced complement activation is an undesirable effect that should be avoided as it may provoke rapid clearance of the biomaterials and inflammatory reactions127.                   Figure 3.12 Complement activation determined in human serum using sheep erythrocyte based complement consumption assay. Polymer concentration: 1 mg/mL. IgG and PBS were used as positive and normal control respectively. ** means p < 0.01, ns means p > 0.05.    The 50% hemolytic complement assay (CH50 assay) is a screening assay for the activation of the classical complement pathway128. CH50 screens for the activation of the PBS BufferHPG-SBHPG-SB-QA30HPG-SB-QA40HPG-SB-QA45Control PolymerIgG020406080100% complement consumedns**78  complement pathway by detecting the hemolysis of antibody sensitized sheep erythrocytes. The effect of the HPG-SB-QAX polymers and control polymer HPG-QA on complement activation in human serum at 37 °C were investigated. Immunoglobulin G (IgG) and PBS buffer are used as positive and normal controls respectively. The results are presented in Figure 3.12. The results showed that none of the HPG-SB-QA polymers activate complement pathways compared to the positive control (IgG), and the level of complement activation between HPG-SB-QA polymer conjugates and buffer control were comparable. In contrast, the control polymer showed significant complement consumption.  3.3.6 Investigation of the Cytotoxicity of the Polymers   Figure 3.13 Dose-response curves for cell viability of HUVECs (A) and CHO cells (B) after being treated 48 h with polymers by the propidium iodide live cell staining assay. Results are means ± SD (n = 6).  Cell membrane damage is an important aspect of nucleated cell toxicity upon polymer treatment. The in vitro cytotoxicity of polymers was measured as a function of polymer concentration by using the propidium iodide (PI) live cell staining assay. When cells have membrane damage, PI in the assay solution passively diffuses into the cytoplasm and binds with intracellular RNA or DNA. By quantitating the percentage of PI positive cells, we could deduct 0.0 0.5 1.0 1.5 2.00255075100Concentration (mg/mL)Cell Viability (%) HPG-SBHPG-SB-QA30HPG-SB-QA40HPG-SB-QA45Control PolymerCHO cells(B)0.0 0.5 1.0 1.5 2.00255075100Concentration (mg/mL)Cell Viability (%)HPG-SBHPG-SB-QA30HPG-SB-QA40HPG-SB-QA45Control PolymerHUVECs(A)79  the percentage of cells experiencing membrane damage in the total cell population. As endothelial cells line the entire vascular system, the influence of HPG-SB-QAX on the membrane of human umbilical vein endothelial cell (HUVEC) was investigated. The results are summarized in Figure 3.13(A), showing that for the control polymer, cytotoxicity increases with increasing polymer concentration. Greater than 30% of the cell population lost viability at the highest polymer concentration (2 mg/mL). In contrast, cells incubated with HPG-SB-QAX polymers slightly decreased with increasing concentration, retaining greater than 80% of their viability even when the concentration used was up to 2 mg/mL. The results were consistent with the Chinese hamster ovary (CHO) cell line, shown in Figure 3.13(B). Cell cytotoxicity assays demonstrated the safety of the HPG-SB-QAX polymers even at the concentration of 2 mg/mL, suggesting that shielding the positively charged surface quaternary ammonium groups with zwitterionic moieties is effective in reducing the interaction force between the polymer and cell membranes, thus reducing the cytotoxicity.   3.3.7 Reversal of Red Blood Cell Binding As discussed above, the hemostatic potential of the polymers is driven by the aggregation of erythrocytes via electrostatic interaction between the positively charged HPG-SB-QA and the negatively charged cell membrane. This also suggests a mechanism to reverse the aggregation, which is via species bearing a higher density of negative charges that can preferentially bind to the positive moieties of the polymer and thereby disengage the positive ligands from the cells. For this purpose, we designed two microscope-based experiments to investigate the potential of the reversal of erythrocytes aggregation. Sulfated linear polyglycerol129 (LPG-sulfate, molecular characteristics shown in Table 3.2) was used as the reversal agent. As the highest aggregation 80  activity was observed with the polymer HPG-SB-QA45, it was used as a model structure for this study.   Table 3.2 Molecular characteristics of sulfated linear polyglycerol used in the study. Sample Repeating Unit Mn PPPDI # Sulfate Groups per Polymer Degree of Sulfation LPG-sulfate  4700 1.6 27 100%   We first mixed the positively charged HPG-SB-QA45 with LPG-sulfate in equal mass, then added 10 µL of the mixture into 90 µL whole blood, and examined the morphology of RBC with microscope. As shown in Figure 3.14(C), RBC formed typical rouleaux, similar to the one incubated with PBS buffer (Figure 3.14(A)). This was because the positively charged HPG-SB-QA45 and negatively charged LPG-sulfate formed a neutral or negatively charged PEC, thus would not induce hemagglutination.  Later we found that the aggregates could also be reversed by subsequent exposure to this low molecular weight negatively charged LPG-sulfate. Figure 3.14(D) shows the result of adding LPG-sulfate (final concentration 1mg/mL) to whole blood aggregates formed by adding 1 mg/mL HPG-SB-QA45. From the visual observations, it was clear that the aggregates could be instantly reversed by this low molecular weight negatively charged LPG-sulfate. As indicated by ITC study (Figure 3.16, Table 3.3), the small molecule LPG-sulfate bound to HPG-SB-QA45 with a binding affinity in the order of 105 M-1, which is strong enough to cause the positively charged ligand on the polymer to “unhook” from RBC membrane and bind to the negatively charged LPG-sulfate instead. As a result, the adjacent cells are no longer connected by polymer chain thus able to flow freely.  OO3SO81                                    Figure 3.14 Optical microscopic images of human whole blood incubated with or without polymers. All images are at 40 × magnification. (A) PBS control, (B) HPG-SB-QA45 1 mg/mL, (C) mixture of HPG-SB-QA45 and LPG-sulfate, (D) whole blood first incubated with 1mg/mL HPG-SB-QA45 for 5 min, then LPG-sulfate added and incubated for another 5 min.  3.3.8 Effect of Polyelectrolyte Complex on Platelet Activation To confirm the explanation given in Section 3.3.7, we also investigated the effect of the polyelectrolyte complex formed between HPG-SB-QA45 and LPG-sulfate on platelet activation. Both HPG-SB-QA45 and LPG-sulfate are soluble in water, and when the two are mixed in solution, they precipitated out from aqueous solution to form a complex coacervate (picture shown in Figure 3.15(A)), the concept first described by the Dutch chemist H.G. Bungenberg de (A) (B) (C) (D) 82  Jong130. We mixed 0.1 mg/mL HPG-SB-QA45 with different concentrations of LPG-sulfate ranging from 0 to 0.5 mg/mL, checked the level of CD62P expression by flow cytometry, and plotted the percentage of CD62P against the concentration of LPG-sulfate, as shown in Figure 3.15(B). The expression of P-selectin remained at a high level (above 70%) until the concentration of LPG-sulfate reached 0.03 mg/mL, where the moles of the cationic ligands and negatively charged sulfate groups were in almost a 1:1 ratio, then continued to drop to the same level as the PBS control when the anions were in excess. As the concentration of LPG-sulfate increased, more positive sites on HPG-SB-QA45 would be expected to be complexed with the negative moieties, resulting in a lower level of platelet activation compared with the uncomplexed state. The PEC of HPG-SB-QA45/LPG-sulfate featured low hemostatic activity.   Figure 3.15 (A)The formation of PEC between HPG-SB-QA45 and LPG-sulfate; (B) The neutralization of platelet activation upon incubating with 0.1 mg/mL HPG-SB-QA45 and increasing concentrations of LPG-sulfate. Each data point is the platelet activation in PRP measured by the expression of platelet activation marker CD62P using anti-CD62P antibody by flow cytometry. Curves were fit to binding isotherms with GraphPad Prism. 83  3.3.9 Investigation of the Binding of Polymers and LPG-sulfate by Isothermal Titration Calorimetry To further understand the interaction of LPG-sulfate and HPG-SB-QA45, we adapted the isothermal titration calorimetry (ITC) technique to determine the binding affinity of the two molecules. Figure 3.16 presents the representative raw ITC titration traces  and integrated heats fit to an independent one-site binding model, with the assumption that n ligands bind per macromolecule with identical thermodynamics. Each negative peak shown in the heat signal curves in the upper pannel represents an exothermic process, which denotes the heat released in one injection of the aqeous LPG-sulfate into HPG-SB-QA45 solution as a function of time. As the sites available on the surface of HPG-SB-QA45 become progressively occupied during titration, the exothermicity of the peaks decreses and eventually saturates. The sigmoidal enthalpy curve in the lower pannel corresponds to the site-to-site, uncooperative and instantaneous binding of LPG-sulfate molecules to the quaternary ammonium groups on HPG-SB-QA45 driven by eleactrostatic attraction.  Table 3.4 provides the corresponding regressed values for the binding stoichiometry (N), binding constant (Ka), free energy (ΔG), binding enthalpy (ΔH), and the entropy component (TΔS). The binding of LPG-sulfate to HPG-SB-QA45 could be enthalpy driven, indicated by the larger value of ΔH compared to TΔS. The binding stoichiometry reported was calculated on the basis of the number of LPG-sulfate molecules bound to HPG-SB-QA45 polymer at saturation. ITC data revealed that HPG-SB-QA45 exhibited strong binding to LPG-sulfate with a binding affinity in the order of 105 M-1. HPG-SB-QA45 binds LPG-sulfate with 1: 3.63 stoichiometry, indicating some of the HPG-SB-QA45 macromolecues can bind with 3 molecules of LPG-sulfate, and some of them can interact with 4 molecules of LPG-sulfate thus on average 1 molecule of 84  HPG-SB-QA45 can bind approximately 3.63 molecules of LPG-sulfate. This value corresponds to the fact that there are about 96 positively charged ligands on the HPG-SB-QA45 and 27 negatively charged groups on LPG-sulfate, as 96/27 ≈ 3.56. It is worth pointing out that the ITC binding curve has been interpreted with a very simple and unrealistic binding model: a one to one stoichiometry between anionic and cationic sites, all of the binding sites are identical and independent, and every ligand molecule binds to the macromolecule in identical thermodynamics with the same binding constant. However this is unrealistic in electrostatic binding as while the cationic sites on the surface of macromolecules are progressively occupied by the anionic ligands, the surface potential of the macromolecule would change, resulting in the change of the binding affinity of the ligand to the next available cationic site on the macromolecule.  Table 3.3 Thermodynamic parameters for the interaction of HPG-SB-QA45 with LPG-sulfate measured by ITC in sodium phosphate buffer at 37 °C.  Polymer Ligand Na Ka (M-1)  ΔGb (kcal/mol) ΔH (kcal/mol) TΔS (kcal/mol) HPG-SB-QA45 LPG-sulfate 3.63 ± 0.16 6.32 ± 0.56×105 -8.23 ±0.05 -8.21 ±0.42 0.017 ±0.36 a N is the number of ligand-binding sites per macromolecule.   b ΔG and ΔS are calculated from the equation ΔG = -RTlnK = ΔH - TΔS.        85                             Figure 3.16 ITC analysis of LPG-sulfate binding to HPG-SB-QA45. Upper panel: raw data showing heat change due to the heat of dilution during each injection as LPG-sulfate binds to HPG-SB-QA45.  Lower pannel: plot showing the integrated area corresponding to the heat change (ΔH) during each injection normalized as a function of molar ratio (ligand/macromolecule); the best least-squares fit of the data to a one-site binding model is given by the solid red line.     3.4 Conclusion The hemostatic potential and hemocompatibility of a series of HPG-based polyvalent cationic polymers was evaluated, and the polymers have been found to have hemostatic potential as well as biocompatibility, depending on the type of assay. Employing well-defined and extremely purified polymeric materials, our data have allowed us to draw clear relationships between the macromolecular properties of the zwitterionic/cationic mixed charge polymers and their hemoreactivity. Based on these systematic and extensive observations, we are in a position -1 0 1 2 3 4 5 6 7 8-8.00-6.00-4.00-2.000.00-0.50-0.40-0.30-0.20-0.100.000 10 20 30 40 50 60 70Time (min)µcal/secMolar Ratiokcal mol-1 of injectant86  to propose, or confirm, the mechanisms underlying different kinds of blood reactions. When the percentage of cationic sites is higher than 40%, the conjugates showed high whole blood aggregation activity. When the components of blood, i.e. erythrocytes and platelets were examined individually, the highest aggregation activity was observed with HPG-SB-QA45, the polymer that bearing 45% of the cationic ligands. Though bearing ~ 100 positively charged quaternary ammonium groups, HPG-SB-QA45 showed vanishingly weak hemolytic activity, and the complement system was not activated. This was attributed to the presence of the tightly bound layer of water brought by the zwitterionic sulfabetaine ligands, which acted as an energy barrier that attenuate the interactions between cationic ligands and cell membranes. Moreover, the strong erythrocyte aggregation induced by HPG-SB-QA45 could be reversed by the sequential application of LPG-sulfate, a polymer with a high negative charge density. This characteristic is very attractive for use in local drug delivery or as a tissue sealant. Binding studies using isothermal titration calorimetry confirmed that LPG-sulfate binds to HPG-SB-QA45 with high affinity, displaying an apparent association constant in the order of 105 M-1. From our perspective, the hemostatic property was attributed to electrostatic interactions with the negative charges on the erythrocyte surface charges induced by the cationic nature of the conjugate, which enabled the conjugate to promote hemostasis by linking erythrocytes together to form a lattice mesh to entrap more blood cells and enhance platelet and erythrocyte aggregation, a feature necessary to initiate blood hemostasis. Microscopic observation also demonstrated that the red blood cells treated with the polymer solution lost their typical biconcave morphology and coalesced into a tight aggregate. In addition, we have shown that modifying the cationic surfaces with zwitterionic molecules is an efficient strategy that masks the cytotoxicity of the cationic sequence. The high hygroscopic capability brought by the 87  zwitterionic ligands contributes to the absorption of water from blood with the subsequent concentration of platelet and coagulation factors at the site of bleeding. The high adhesiveness towards cell membrane allows the conjugate to adhere firmly to the surrounding tissues, providing a physical barrier to prevent the blood from escaping the vessels. Both of the attributes would enhance the antihemorrhagic effect. It is of interest to point out, however, that the positive charge on HPG-SB-QAX acted as a double-edged sword, in that it promoted erythrocyte aggregation, platelet activation and aggregation, but inhibited the activation of the contact system and thus affected the intrinsic coagulation pathway. As a result, upon the application of the conjugate, the blood takes longer time to clot, but the initial plug formed by the aggregation of RBCs and platelets would be able to seal the wound and possibly stop bleeding. 88  Chapter 4: Concluding Remarks and Future Directions 4.1 Conclusions In this thesis, we developed a family of novel multifunctional hyperbranched polymers that were functionalized with multiple zwitterionic sulfabetaines and cationic quaternary ammonium ligands. These polymer conjugates could potentially be used as a topical hemostatic agent for wound sealing. The camouflaging strategy relies on the fact that the quaternary ammonium moieties should bind to the negative charged sites on the surface of RBC while the zwitterionic sulfabetaine moieties should contribute to provide a hydration layer to protect the integrity of the membrane. In Chapter 2, we aimed to optimize surface modification strategies to adjust the ratio of cationic/zwitterionic ligands on the hyperbranched polyglycerols backbone. UV-initiated thiol-ene “click” chemistry has emerged as a powerful and facile method to conjugate a layer of water-soluble ligands on a broad range of substrates. We tested the application of this strategy to incorporate different mole fractions of zwitterionic sulfabetaine and cationic trimethyl ammonium ligands on the allylated hyperbranched polyglycerols backbone. The structures and composition of the series of conjugates were characterized by various techniques. Importantly, DSC was used to evaluate the water binding property of the polymers and the results indicated the conjugation of charged ligands, especially the zwitterionic groups, greatly enhanced the ability of the HPG backbone to hold water molecules, which is a desired property for a hemostatic reagent. The strong hydration layer brought by zwitterionic ligands was also expected to contribute to the enhancement of biocompatibility of the conjugate.  The in vitro hemocompatibility and bioactivity of the polymers were evaluated in Chapter 3. The polymers exhibit abilities to aggregate erythrocytes, aggregate and activate platelets in a 89  concentration and positive charge density dependent manner. The polymers play a role in inducing the aggregation of erythrocytes by interaction with erythrocyte’s surface charges and promoting hemostasis by activation and aggregation of platelets. The presence of the tight hydration layer brought by zwitterionic sulfabetaine moieties on the polymer backbone partially prevents the adverse effects that are likely to be caused by cationic moieties on cell membrane integrity. As a result, none of the polymers induces significant hemolysis or endothelial cell lysis. Furthermore, the polymer does not affect the complement system. In addition, the high hydrophilicity allows the absorption of a high amount of water from the blood, which concentrates both the platelets and coagulation factors at the bleeding site and thus accelerates hemostasis. It is also noticeable that the aggregation effect induced by the polymer is reversible, and can be reversed by using another molecule bearing a higher negative charge density. This is a desired feature for the application as blood vessel sealant.  4.2 Future Directions Our long-term goal is to develop a topical hemostatic agent that is easy to use, highly efficacious, nonantigenic, fully absorbable and inexpensive. The synthesis of HPG-SB-QA is a stepping stone to realize this long-term goal. I call it “stepping stone” because it is far from perfect and more effort should be made to improve it. We anticipate this will involve the combination of advances in (1) surface engineering approaches to incorporate therapeutic biomolecules that promote hemostasis and the regeneration of cellular components critical to restore the surface characteristics and native blood vessel; and (2) material science and engineering to generate scaffolds which, as well, will exhibit industrial manufacturing 90  specifications such as scalability and off-the-shelf availability. Towards this end, we propose three major areas for future exploration. In Chapter 3, we showed the polymer played a role in catching erythrocytes and platelets to form aggregates or a clot, but the in vitro coagulation tests such as aPTT and TEG showed it would somehow retard the intrinsic coagulation pathway and delay the coagulation time. The mechanism that is responsible for this phenomenon is still unclear to us. This might suggest that the polymer promoted hemostasis is independent from the classic coagulation pathway, but it would be nicer if the material were able to enhance the coagulation process, or at least do not interfere with the coagulation process. To resolve this, we could further investigate the effect of the macromolecule on the activity of various clotting factors involved in the intrinsic or contact coagulation pathway in a more rigorous manner. A more comprehensive library of polymers, for example keeping the quantity of one ligand constant and varying the number of the other ligand, needs to be synthesized. This would help us to further engineer the surface of the macromolecule according to the structure-property relationship to overcome this defect.  The model system we employed in this study was evaluated via regulating a positively charged quaternary ammonium group and a charge neutral sulfabetaine 2-propanethiol group quantitatively to control the charge bias property of the polymer. Similar experiments could also be carried out using other betaine groups, with more methylene groups between the charged centers, or different negative moieties like the sulfonate ion. Molecular simulation studies showed that the length of the carbon spacer would affect the hydration ability of the carboxylic groups in the carboxyl betaine131. Both sulfate and sulfonate anion bearing one negative charge, but the size of the sulfonate anion is smaller than that of the sulfate anion, resulting in a higher anionic charge density.  The first structure in Figure 4.1 is the one we have studied in this thesis, 91  and the remaining three are the possible structures of thiol-based zwitterionic ligands that are worth to investigating.          Figure 4.1 Structures of thiol-based sulfabetaine and sulfobetaine.  A final area for future research will be the optimization of industrial manufacturing specifications to generate scaffolds for wound dressing application. Electrospinning is a simple and effective method to produce a fibrous membrane, which provides multiple desirable features for wound dressings, including high absorptivity due to high surface-area-to-volume ratio, high gas permeation due to a porous structure, and conformability to contour of the wound bed132-134. One approach is to introduce alkene end groups on the surface of a nanofibre mat that is approved to be suitable for tissue engineering applications, for example poly(ε-caprolactone) or poly(lactic-acid), and click on our cationic/zwitterionic ligands. Another approach is to redesign the structures of the ligands and copolymerize them and then electrospin the copolymer into a fibre mat. 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