DEVELOPMENT OF NOVEL NITRIC OXIDE RELEASING MATERIALS AND POLYMERIC COATINGS
FOR BLOOD CONTACTING BIOMEDICAL APPLICATIONS
by
Zhengrong Zhou
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in The University of Michigan 2006 Doctoral Committee:
Trang 2UMI Number: 3224797 Copyright 2006 by Zhou, Zhengrong All rights reserved INFORMATION TO USERS
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Trang 3Zhengrong Zhou
—— 2006
Trang 4To Mom, Dad and Yue—
Your continuous love and support have made this endeavor possible
Trang 5ACKNOWLEDGEMENTS
First and foremost, I must express enormous gratitude to my mentor, Dr Mark Meyerhoff, for his gracious support and expert guidance throughout this endeavor As a role model, his energy and enthusiasm for truly understanding the fundamental chemistry behind every research project have always encouraged me to achieve my best in the course of this work I am extremely grateful for the freedom, respect and the trust he has given me on the research I have pursued I truly believe that the guidance he has given me during the past five years will benefit me as a scientist for the rest of my life
I would also like to thank Drs Zhan Chen, Adam Matzger and Shuichi Takayama for being members of my dissertation committee I especially want to thank Dr Chen, who recruited me into this wonderful program five years ago, and allowed me to work with his students and use the instruments in his laboratory for part of my thesis project
Sincere thanks also go to my collaborators and co-workers, without whom this dissertation research would not have been possible I want to first thank our collaborators at the Medical Center of the University of Michigan for their support with animal-related experiments: Dr Gail Annich for providing hemodialyzers and carrying out animal studies for the NO releasing dialysis project; Dr Robert Bartlett and his group members, especially Amy Skrzypchak and Nathan Lafayette, for preparing blood samples and performing animal experiments for testing the NO/Heparin polymers I am also very
Trang 6grateful to Zhihong Shen from Professor Xiangqun Zeng’s lab at Oakland University for carrying out the QCM experiments for the surface-bound heparin, and my fellow group members Yiduo Wu and Dr Megan Frost for planning/conducting biocompatible evaluations for the NO/heparin materials I would also like to thank Dr Scott Merz and Dr Melissa Reynolds (a former group member) at MC3 Inc., who have always been extremely supportive to our basic research and our goal toward employing NO releasing polymers to improve biocompatibility of medical devices A special thanks to Melissa, with whom I greatly appreciated the opportunity that we had worked together on various projects Many thanks also to Wei Tang, Xiaoyun Chen and Dr Kai Sun at the University of Michigan for assisting me with characterizing a number of materials using SFG, fluorescence and X-ray photoelectron spectroscopies
On a personal note, I am indebted to many past and all the present members of the Meyerhoff research group for their support and friendship I feel so lucky and grateful to have such a supportive and excellent group of co-workers Special thanks go to Megan
Frost, Jeremy Mitchell-Koch, Yiduo Wu, Biyun Wu and Meredith Anderson (also thanks
for working with me on the dialysis project) for their proofreading/revision of this dissertation and previous manuscripts I also want to acknowledge the previous/current NO-group members for their valuable suggestions throughout this dissertation work, especially to Pawel Parzuchowski, Huiping Zhang, Megan Frost, Melissa Reynolds, Sangyeul Hwang, Wansik Cha, Yiduo Wu and Biyun Wu I really enjoyed the time working together with all of you at Michigan!
Finally, I want to express my deep appreciation to my parents, Chaosheng Zhou and Yali Sun, for their tremendous care and love I am sure you are proud of your son! My
Trang 8TABLE OF CONTENTS DEDICATION ACKNOWLEDGEMENTS LIST OF FIGURES LIST OF SCHEMES LIST OF TABLES ABSTRACT CHAPTER 1 INTRODUCTION 1.1 Current Challenges with Synthetic Polymeric Materials 1.2 Blood-Polymer Interactions
1.3 Unique Functions of the Non-Thrombogenic Endothelium 1.4 Mimicking the Endothelium to Improve Blood Compatibility 1.5 Statement of Dissertation Research
1.6 References
2 POLYMETHACRYLATE-BASED NITRIC OXIDE DONORS WITH
Trang 93 DEVELOPMENT OF WATER-SOLUBLE POLYMERIC NITRIC OXIDE DONORS FOR POTENTIAL APPLICATIONS IN HEMODIALYSIS 3.1 3.2 3.3 3.4 3.5 Introduction Experimental Section Results and Discussion Conclusions References 63 63 66 73 91 93
4 NOVEL POLYMERIC COATINGS WITH COMBINED NITRIC OXIDE
RELEASE AND IMMOBILIZED ACTIVE HEPARIN 4.1 4.2 4.3 4.4 4.5 Introduction Experimental Section Results and Discussion Conclusions References 5 CONCLUSIONS AND FUTURE DIRECTIONS 5.1 5.2 5.3, APPENDIX SYNTHESIS AND CHARACTERIZATION OF DIALKYLDIAMINE Conclusions Future Directions References 96 96 99 109 127 129 133 133 136 141 BIS-DIAZENIUMDIOLATES AND THEIR 0?-METHYL-PROTECTED DERIVATIVES A.I Introduction
Trang 10FIGURE 1.1 1.2 1.3 1.4 1.5 1.6 1.7 2.1 2.2 LIST OF FIGURES
Biological responses to an implanted material in blood [Figure adapted from Zhang”']
Schematic of healthy EC surface (left) and structures of associated agents that impart exceptional hemocompatibility (right)
The formation and the inhibition (by heparin, TM, PGI; and NO) of thrombus
Schematic of NO production by nitric oxide synthase (NOS) within the ECs that line blood vessels NO diffuses into the flowing blood and also the surrounding smooth muscle cells where it inhibits platelet adhesion and activation on the endothelium surface as well as smooth muscle cell proliferation
Summary of a variety of NO releasing/generating strategies examined to devise potentially more blood compatible polymeric coatings Incorporation of diazeniumdiolate functional group into polymers (X=amine or amino group, yyy = polymer chain) [Figure adapted from Smith et al.?]
Schematic of two types of biomedical applications using different types of N-diazeniumdiolate-based NO releasing polymers
Schematic representation of NO release chemistry that occurs with newly developed polymethacrylate-based NO donors in the presence of water
Trang 112.3 2.4 2.5 2.6 2.7 2.8 2.9
Schematic of NO-reactor system used for all the NO addition reactions described in this thesis work All reactor parts, except the argon-line, are made from stainless steel, Teflon, or glass, all of which are inert to NO@) and yet can sustain at least 80 psi pressure (@ stands for a valve in the diagram.) [Figure adapted from Zhang’? | Typical IR (D and 'H NMR (ID spectra of monomer (6d) (spectra A), N-Boc-protected copolymer (7d) (spectra B), and deprotected copolymer (8d) (spectra C) (‘H NMR solvent= CDCl)
An example of the 'H NMR spectra showing the methylene groups adjacent to nitrogens in the side chains of diamine copolymer (8c) and its nitrosamine product that forms when the copolymer is reacted with NO in the absence of NaOMe *One cluster of the nitrosamine signals b at 3.5-3.6 ppm is overla apped by the signal of the methoxy] groups on the polymer backbone (H NMR solvent= CDC13)
An example of decomposition of diazeniumdiolates within copolymer (9b) with time as determined by UV spectroscopy at room temperature (in deoxygenated methanol)
An example of decomposition of diazeniumdiolates in terms of nitrosamine formation within copolymer (9d) with time under ambient conditions as determined by IR spectroscopy (A) diamine copolymer (8d) (starting material); (B) diazeniumdiolated copolymer (9d), immediately after NO loading; (C) (9d), after work-up; (D) exposed to ambient conditions for few hours; (E) exposed to the ambient conditions for few days
NO release from different diazeniumdiolated copolymers at 37 °C (I), and the same copolymer at different temperatures (II) Five mg of each material as measured by the CL NOA) in 4 mL of deoxygenated 0.1 M PBS buffer (A) (9d) made with 2 eq NaOMe; (B) (9d) with 1 eq NaOMe; (C) (9d’) with 2 eq NaOMe; (D) (9d) with no NaOMe (** showing no significant NO release)
Trang 122.10 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8
NO surface fluxes (I) and total NO release curves (II) for (9d) (8 % wt) embedded in SR matrix (with a thickness of 150-200 pm) as measured by CL in PBS buffer (pH 7.4) at 37 °C (A) PLGA as additive (8 % wt); (B) KTpCIPB as additive (8 % wt)
A schematic diagram illustrating the concept of improving the blood compatibility of hemodialyzer fibers via NO-release from a polymeric NO releasing agent added to the flowing dialysate solution
A schematic diagram of the NO-release dialysis set-up
Typical !H NMR (solvent=D,0) and FT-IR spectra of HMwPEI (2) and its NO addition product HMwPEI/NO (5)
NO release from 5 mg of HMwPEI/NO (5) in PBS buffers with various buffer capacities The measurements were performed in 4 mL deoxygenated PBS buffer at 37 °C via the CL NOA
Decomposition kinetic study of diazeniumdiolates within polymer HMwPEI/NO (5) as measured via UV in 100 mM deoxygenated PBS buffer (0.1 mg/mL, pH=7.4) at 26 °C; each spectrum was collected at a 10 min interval Inset: Plot used to determine a near-first-order rate constant of k=3.9x10° s"' (each absorbance data was collect at A=250 nm with an interval of 15 s)
NO release from 5 mg of HMwPEI/NO (5), PEI-COONa/NO (8) and PEI-PRO/NO (11) as measured in 4 mL deoxygenated PBS buffer (pH=7.4, 100 mM) at 37 °C via the CL NOA; Inset: Total NO release of the three compounds
Proton-stimulated spontaneous NO releases as measured at 26 and 37 °C via the CL NOA by mixing a 100 uL solution of PEI/NO (I), PEI- COONa/NO (II) or PEI-PRO/NO (IID) (NOD soln.: 1 mg/mL in phosphate buffer, pH=8.0) with a 3300 wL of PBS (10 mM or 100 mM, pH=7.4) in the presence and absence of PAANa additive (30 pg/mL)
Trang 133.9 3.10 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8
Interfacial NO fluxes generated across the dialysis membranous fibers within a Minntech™ mini-filter at room temperature as measured via the CL NOA HMwPEI/NO (5) and PEI-PRO/NO (11): 1 mg/mL in phosphate buffer (pH=8) at a rate of 0.5-1 mL/min; dialysate solution: PBS buffer (pH=7.4) at a controlled rate of 15 mL/min
Accumulated nitrite release from 5 mg of HMwPEI/NO (5), PEI- COONa/NO (8) and PEI-PRO/NO (11) under the basic and oxygen- free conditions at room temperature as measured via the Griess assay The measurements were conducted at an interval of 1 h for the first four hours and then at an interval of 1 d for three days
Illustration of dual acting biomimetic coating with combined NO release and surface-bound heparin (a) top layer: aminated polymer with surface-bound heparin (10 wm); (b) middle layer: a polymer matrix with NO donor DBHD/N,Q) (structure as shown above) (approx 80 um); (c) bottom layer: dense polymer (10 wm)
Scheme used to covalently bind heparin to aminated polymer surfaces using EDC and NHS as coupling agents
(I) Typical IR spectra of PU (CarboSil) and its derivatives; (II) Close- up comparison of characteristic IR bands centered at 1578 and 1112 cm’, respectively CarboSil (spectrum A), CarboSil-NCO (spectrum B), CarboSil-PEO-NH)2 (spectrum C) and NH2-PEO-NH) (spectrum D)
Typical 'H NMR spectra of CarboSil (spectrum A) and aminated CarboSil (spectrum B) as measured in DMSO-ds
(I) Representative 0-1100 eV survey XPS spectra of blank PVC (spectrum A), aminated PVC (spectrum B), and heparinized PVC (spectrum C); and (II) smoothed high-resolution S23 spectra of PVC and the surface-modified PVC materials
Overview of energy dispersive X-ray microanalysis (EDX) spectra of aminated CarboSil (spectrum A) and heparinized CarboSil (spectrum B)
Normalized UV-vis spectra of Toluidine Blue solution (0.0005 wt %) (spectrum A); Toluidine Blue solution fully titrated with free heparin (spectrum B); and heparinized CarboSil film after exposed to Toluidine Blue solution (smoothed spectrum C)
Trang 144.9, 4.10 4.11 4.12 5.1 A.l A42
The binding process of immobilized heparin with AT III as measured by QCM (1) Frequency change vs time when 10 yl, 20 pl, 30 pl, 30 ul of 20 g/ml AT III were added to Carbosil-PEO-Hep modified Au QCM surface in 1 ml PBS buffer at 25 °C, respectively; (II) [AT ITJ/AM vs [AT TT] of AT HĨI binding with Carbosil-Hep
(I) NO surface flux profile generated from SR catheter sleeves coated with plasticized PVC containing 6.9 wt % KTpCIPB and 4.4 wt % DBHDN;O;, with or without top-coated aminated-PVC; (II) NO release before and after (*) heparin immobilization for formulas C and D The catheter sleeves were soaked in PBS at 37 °C and NO release was measured by chemiluminescence NO analyzer
(I) NO surface flux profile generated from SR catheter sleeves coated with various PU formulations (matrix/top-coating) containing 6.9 wt
% KTpCIPB and 4.4 wt % DBHD/N20O2, with or without top-coated
aminated-PU; (II) NO release before and after (*) heparin immobilization for formulas A and C The catheter sleeves were soaked in PBS at 37 °C and NO release was measured by chemiluminescence NO analyzer
(I) NO surface fluxes generated from SR catheter sleeves coated with
PurSil formulations containing various amounts of DBHD/N2O2 and
equal molar amount of KTpCIPB with plain PurSil top-coatings as measured via the CL NOA in PBS at 37 °C (mean + SD, n2 3); (ID In vitro platelet adhesion onto various NO releasing PurSil coatings as indicated by activities of the LDH from the lysed platelet that adhered on such coatings (mean + SD, n = 8 for all experiments, using at least 2 different blood lots as the source for the PRP)
A schematic illustration of a multi-functioning thromboresistant polymeric coating containing anti-platelet agents: nitric oxide (NO) and prostacyclin (PGI;) as well as anticoagulant agents: heparin (Hep) and thrombomodulin (TM)
Structures of amines and diazeniumdiolates where R, R' and R" are
side groups, x is an integer, and M” is a positively charged species (i.e., Na”, K”, NH¿”, etc.)
Trang 15LIST OF SCHEMES
SCHEME
2.1 Synthesis of N-Boc-protected methacrylate monomers (6a-d) 2.2 Three-step synthesis of NO releasing copolymers’ including:
polymerization, deprotection and NO addition
2.3 The hydrolysis of poly(lactide-co-glycolide) in the aqueous environment
3.1 Synthesis of diazeniumdiolated branched PEIs 3.2 Synthesis of diazeniumdiolated carboxylated-PEI
3.3 Synthesis of diazeniumdiolated L-proline-incorporated PEI 3.4 Proposed mechanism by which use of carboxylate site speeds NO
release
Trang 16TABLE 2.1 2.2 2.3 3.1 3.2 4.1 4.2 4.3 A.1 A.2 A43 LIST OF TABLES
Compositions and yields of various methacrylate copolymers with pendant diamine side chains
Molecular weights of the N-Boc-protected copolymers
UV and chemiluminescence (CL) characterization of the copolymers after NO addition reaction
Yields and UV/chemiluminescence (CL) characterization of the NO addition products of PEIs and their derivatives
Decomposition parameters of the water-soluble diazeniumdiolated polymers
Elemental composition of the blank PVC and the surface-modified PVCs following amination and heparin immobilization *
Elemental composition of the blank CarboSil and the surface-modified CarboSils following a series of particular reaction steps “
Air-water contact angles at the polymer surfaces °
Potential clinical applications of the diazeniumdiolates: in vivo proof- of-concept studies [Table modified from Keefer’ ]
Trang 17ABSTRACT
To devise more biocompatible materials for use in blood contacting devices, novel polymers with either nitric oxide (NO) release alone or NO release in concert with surface-bound active heparin have been developed, with the goal of mimicking the non- thrombogenic properties of the endothelial cell (EC) layer that lines all blood vessels
New NO releasing polymethacrylates with pendant N-diazeniumdiolated alkyldiamine moieties were synthesized and characterized The NO releasing polymeric coatings were formulated by doping such polymer-based NO donors within PVC and silicone rubber matrices, and employing a biodegradable poly(lactide-co-glycolide) as a harmless proton-generating additive to greatly prolong the NO release of such coatings Polymer coatings with a desired NO surface flux at/above intact EC NO levels (0.5-4 x 10'° mol-cm?-min”) for at least 24 h could be prepared using this approach Such coatings may be employed to improve blood compatibility of some short-term biomedical devices via the sustained NO release with minimal potential toxicity
A new approach to potentially resolve serious thrombosis issues associated with hemodialysis therapies was also developed New water-soluble polymeric NO donors, based on the diazeniumdiolated branched poly(ethylenimine)s and their derivatives, were prepared and characterized These macromolecular NO donors (with up to 4.15 umol/mg of total NO release) were utilized as additives to the dialysate solution of model dialysis
Trang 18filters It was demonstrated that steady, controllable and physiologically relevant NO fluxes could be generated at the high surface area dialysis fiber/blood interface within hemodialysis filters Such localized increase in NO levels may greatly decrease the risk of thrombosis
Finally, a new polymeric coating that combines NO release with surface-bound active heparin was developed, with the aim of more closely mimicking the normal functions of the EC layer The dual acting polymeric coatings, using PVC and polyurethanes as model matrices, were formulated to be capable of releasing tunable and physiological levels of NO for up to one week The outermost layer of the coating also possesses surface-bound heparin with synergistic anticoagulant activity Such EC biomimetic coatings are capable of functioning by two complementary anti-thrombotic mechanisms and, therefore, are expected to exhibit greatly enhanced thromboresistivity compared to polymers that utilize either immobilized heparin or NO release alone
Trang 19CHAPTER 1
INTRODUCTION
Synthetic polymers are the most important and largest family of materials used in the medical field to construct or coat a wide variety of biomedical devices According to a 2002 market research review conducted by Business Communications Company, Inc., US demand for medical polymers will increase nearly 3.0 percent per year to 3.6 billion pounds in 2008, valued at $5.0 billion Indeed, medical devices that employ such polymers have an even larger and more rapidly increasing market, reported to be $43 billion in 2001 in the US and projected to grow at a compound annual rate of between 6 % and 8 % through 2007.! Yet, with all of these tremendous opportunities available for the use of polymeric materials in biomedical applications, great challenges exist with respect to overcoming biocompatibility issues associated with use of these materials
1.1 Current Challenges with Synthetic Polymeric Materials
Trang 20evolving new polymers with even better intrinsic biocompatibility.” A persistent problem with current synthetic polymers is that a wide range of biological responses arise at the body/polymer interface as the polymer-fabricated or coated medical devices are in contact with physiological fluids or tissues.°° For devices that are in direct contact with blood, such biological responses can cause serious complications (e.g., platelet activation, thrombosis, embolism, etc.) in patients and ultimately functional device failure.®? Asa result, systemic or localized anticoagulation treatments (e.g., heparin) or use of anti- platelet agents (e.g., Plavix™) are always required to reduce the risk of clot formation during most extracorporeal therapies (where blood is removed from the body and passed through a device) and/or for more long-term intravascular implants.!® !Ì The long-term use of exogenous anticoagulants, however, can often lead to hemorrhagic complications For example, in dialysis treatments, when the patient is systemically anticoagulated with heparin to reduce risk of clot formation during such clinical procedures, dialysis-induced 12,13 Tn addition, even when bleeding may occur, including intracranial hemorrhage
anticoagulant levels can be managed effectively, heparin-induced thrombocytopenia (HIT), a complex process which results in loss of platelets and excess bleeding can still occur Some of the significant challenges facing current medical devices include the following: [List adapted from Ratnerf]
« Small diameter vascular grafts fail early due to thrombolic occlusion, « Embolic complications are noticed with artificial hearts,
Trang 21= Blood contacting biosensors fail due to thrombus accumulation, « Long-term implants are seen to be continuously platelet reactive,
= Significant blood damage is observed during hemodialysis and extracorporeal blood oxygenation,
« Venous prostheses cannot be made at all,
« Blood interaction problems are associated with endoluminal stents
Therefore, the development of polymeric materials that can be used to fabricate medical devices without the concern of either clotting or excess bleeding is of vital importance Indeed, this is the main focus of this dissertation
1.2 Blood-Polymer Interactions
As a special case of biocompatibility, hemocompatibility (blood compatibility) describes how materials foreign to an organism interact with the natural defense mechanisms of the organism when the materials are in contact with blood.'* > Thrombus formation at the polymer/blood interface is a common and major problem for implanted polymers The blood coagulation cascade is initiated by protein adsorption on the surface of the material, followed by platelet adhesion and activation Then, a series of coagulation factors ultimately convert soluble fibrinogen to insoluble fibrin that entraps activated platelets resulting in thrombus formation (see Figure 1.1)
Trang 22materials that are truly compatible with blood.” ‘618 Tdeally, polymeric materials used to construct or coat a wide variety of blood-contacting devices (e.g., vascular grafts and
stents, hemodialyzers, catheters, extracorporeal circuits, membrane oxygenators,
intravascular chemical sensors, etc.) should be made of materials that exhibit excellent blood compatibility
red blood
- fibrin and
protein platelet thrombus
adsorption adhesion formation
—> ——> —>
4 (seconds) (hours)
polymer platelet
Figure 1.1 Biological responses to an implanted material in blood [Figure adapted from Zhang'”]
1.3 Unique Functions of the Non-Thrombogenic Endothelium
Trang 23potent anti-platelet agents nitric oxide (NO) and prostacyclin (PGI2), as well as the anticoagulants thrombomodulin (TM) and heparan sulfates (analogs of heparin) (see Figures 1.2) Relevant background related to the biological functionality of each of these EC derived agents is reviewed below to understand how the normal endothelium serves as an ideal non-thrombogenic surface Nitri oO id CH,S0; G00" GH, SS" CHSOy -N=O: tric Oxide " + =n Prostacyclin on MB SOF Nitric Oxide (NO) Heparan/Heparin (Hep) NO oon và Prostacyclin (PGI,) HO” Endothelial Cell HO” Thrombomodulin (TM)
Figure 1.2 Schematic of healthy EC surface (left) and structures of associated agents that impart exceptional hemocompatibility (right)
Coagulation cascade Platelet activities
Platelet adhesion CNo) : <—Cacnan) Phospholipase C Xa — activation TM | AT III Platelet activation | Cytoplasmic Ca? Increasing "\@Gi) Platelet shape
Prothrombin Thrombin (IIa)
Fibrinogen Fibrin monomer change/secretion
Xia |
Fibrinclot ; Platelet aggregates Red cell
THROMBUS
Trang 24Heparan sulfates/heparin Heparan sulfate, a structural and functional analog of the major clinical anticoagulant heparin, is rich on the EC surface.”””? Heparan sulfate binds antithrombin III (AT III) to form a complex which in turn accelerates the inactivation of thrombin (Factor IIa) and consequently inhibits formation of fibrin Heparan sulfate/AT III complex also binds coagulation Factor Xa, thereby inhibiting its catalytic generation of thrombin from prothrombin (see Figure 1.3).°%”> Since thrombin is a pivotal enzyme in coagulation cascade, and the most potent of all platelet activators, reducing thrombin activity and inhibiting its formation is known to be one of the most effective ways of decreasing thrombus formation on the vessel luminal surface
Thrombomodulin (TM) Thrombomodulin is an integral membrane protein, which is present on the vascular EC layer and serves to disrupt the clotting cascade (see Figure 13)? Each EC surface has approximately 30,000-100,000 molecules of thrombomodulin.”” TM binds to thrombin to form a 1:1 complex Thrombin bound to TM consequently loses its procoagulant and proinflammatory functions: it cannot cleave fibrinogen or activate platelets and Factor x.” 7! Simultaneously, the TM—thrombin complex proteolytically triggers protein C activation The catalytic efficiency of thrombin to activate protein C is accelerated about 10,000 times more in the presence of TM than alone.** Activated protein C (APC), in the presence of protein S, will in tum selectively inactive coagulation Factor Va and VIIla, and affects immune reactions, 33-37 as well as inflammatory processes.*® Additionally, thereby controlling hemostasis
Trang 25Prostacyclin (PGI2) Endothelial cells also produce prostacyclin, a member of the prostaglandin family.“ More specifically, PGI, is synthesized by an EC and a smooth muscle cell bound enzyme, known as prostacyclin synthase The greatest production of PGI, occurs at the intimal surface and decreases towards the underlying smooth muscle cells Prostacyclin’s physiological functions are very similar to NO (see Figure 1.3), as it’s not only an inhibitor of platelet activation and aggregation” but also a vasodilator It inhibits platelet activation by stimulating receptors on the surface of the platelet, which in turn increases the production of platelet intracellular cAMP (cyclic adenosine monophosphate), and consequently decreases the intracellular Ca?” level.” Further, PGh, like NO, has also been shown to be a potent inhibitor of smooth muscle cell
*2, 41 also by creating an increase in intracellular cAMP levels proliferation
Nitric oxide (NO) Nitric oxide is an extremely reactive free radical that is endogenously produced in many cells in the body Nitric oxide serves as a “4 4 cytotoxic agent (i.e., anti-bacterial and anti-cancer agents),"° neurotransmitter,
vasodilator*’ and inhibitor of platelet activation and aggregation." Under basal conditions, the normal EC layer produces NO via reaction of L-arginine and molecular oxygen by NO synthase (NOS), a calcium dependent enzyme.*Š * The generated NO điffises into the blood vessel lumen as well as into surrounding vascular smooth muscles (see Figure 1.4) Based on measurements using cultured ECs, it has been estimated that the basal flux of NO from the surface of such cells is 1 x 10” mol-em ”-min'."°
Trang 26activity of phospholipase C, the two key substances required for platelet activation (see Figure 1.3).7: >!) °? Nitric oxide can also down-regulate the functions of some platelet receptors, which in turn prevents platelet aggregation and adhesion onto blood vessel walls Beyond binding to hemoproteins, NO also reacts with S-thiols (RSH) in the blood, which results in the formation of S-nitrosothiols (RSNO) This formation prolongs the half-life and biologic activity of NO and allows for further platelet inhibition
Blood Vessel Vascular Endothelial Cell Vascular Smooth
lumen Cat 4 HP sre Muscle Cell 5
Red Blood Cell #> <>~calmodulin
©) L-Arginine
Platelet
Figure 1.4 Schematic of NO production by nitric oxide synthase (NOS) within the ECs that line blood vessels NO diffuses into the flowing blood and also the surrounding smooth muscle cells where it inhibits platelet adhesion and activation on the endothelium surface as well as smooth muscle cell proliferation
When NO reaches the underlying vascular smooth muscle cells, it binds to the heme group of the enzyme guanylate cyclase This results in a production of cGMP The increased level of cGMP leads to vascular relaxation that allows the vessel to dilate and thereby lowers blood pressure.”
In addition to its anti-platelet and vasodilation activities, NO is also known to
54-56
Trang 27medical devices (e.g stents, vascular grafts).°° Although its lifetime in blood is very short (< 1 sec) owing to its rapid reaction with hemoglobin,’ among all the endogenous EC agents involved in controlling platelet function and cell proliferation, NO is most attractive since it possesses both functions
1.4 Mimicking the Endothelium to Improve Blood Compatibility
Numerous approaches have already been examined to improve the biocompatibility of polymeric materials for blood contacting applications These approaches can be generally discussed in two categories: (1) development of inert surfaces that should not interfere with mechanisms in blood and that delay/avoid adverse defense reactions (e.g., approaches to physically or/and chemically modify surfaces for suppressing either protein adsorption or cell adhesion); and (2) so-called “bioactive” surfaces that are intended to actively support natural control mechanisms in order to prevent unwanted and uncontrolled responses of the host to the foreign materials.'’ In fact, approach 1 has had virtually no success in true in vivo situations Hence, the bioactive approach is, therefore, much more promising Indeed, materials that mimic the functions of the endothelium, i.e possess immobilized anticoagulants (such as heparin and TM) and/or release anti-platelet agents (such as NO and PGI,), can be considered as a proactive way that can result in surfaces that will not induce thrombus formation during clinical procedures
Trang 28years have suggested the use of immobilized heparin species on polymer surfaces to decrease thrombus formation, and several commercial coating products (i.e., Duraflo I, Carmeda BioActive Surface™, etc.) were developed based on this concept However, the clinical effectiveness of immobilized heparin alone is questionable at best.”’ Indeed, this approach has not been successful in eliminating thrombus formation mainly because the amount of heparin on the polymer surfaces may not be adequate to effectively prevent coagulation, or/and the immobilized species is not able to bind fully with AT III and thrombin, simultaneously, a requirement for inhibiting fibrin formation In addition, use of heparin as a surface-active agent does not significantly decrease platelet activation and adhesion In fact, it has been reported that immobilized heparin as individual agent can actually enhance platelet activation
Thrombomodulin Immobilized on Polymers Another approach exploited to enhance a material’s blood compatibility is to immobilize TM onto polymer surfaces.°'~”
Some research reported using this approach have shown positive results For example, Kishida et al.°!® clearly showed enhanced thromboresistance for polyurethane materials possessing immobilized TM, and Lo et al demonstrated that PTFE grafts with crosslinked TM were more patent after 7 d compared to controls using a porcine model However, data on use of human TM immobilized on various surfaces is still rather limited Compared to the heparin approach, immobilized TM seems potentially more effective, as TM is much more potent than heparin with respect to its anticoagulation activity (see the above biochemistries of heparin and TM) Further, recent studies in this laboratory found that polyurethane-immobilized TM possesses stable protein C activation
Trang 29cofactor activity This activity was retained even after several months of long-term storage under appropriate conditions.®
Prostacyclin-Incorporated Polymers Prostacyclin-releasing/immobilized polymers have also been investigated, aimed at utilizing prostacyclin’s anti-platelet activity in preventing thrombosis.” Biomedical polymers loaded with prostacyclin and related prostaglandins release these species at controlled rates (for periods of up to 40 d), and such polymers have been shown to exhibit greatly reduced platelet adhesion and aggregation on their surfaces in vitro.’’ Similarly, polymers with immobilized forms of prostacyclin have been prepared that also have demonstrated improved thromboresistivity via in vitro experiments 7 However, such polymers did not exhibit improved blood compatibility in vivo, likely due to either an insufficient amount of PGI) released or loss of biological function after immobilization.”
Recent Advances in NO Releasing/Generating Polymers Recent research carried out in this laboratory”"” and elsewhere®!** has demonstrated the efficacy of NO producing polymers in preventing platelet adhesion and activation The materials developed are capable of releasing/generating low but controllable levels of NO at the polymer/blood interface (as summarized in Figure 1.5), which are comparable to that generated by the healthy EC layer under the physiological conditions (ca 1x10"? mol-cm “-min' `)
N-Diazeniumdiolates (complexes of NO with primary/secondary amines) are one of the most useful NO donors that have been employed within a variety of polymer matrices
85, 86
to prepare NO releasing polymeric coatings (see Figure 1.5-I) Such species were first synthesized by Drago et al in 1961," and later investigated by Keefer et al., who
Trang 30performed some of the earliest studies using such NO donors as agents for the controlled biological release of NO to demonstrate its anti-platelet effect." N-Diazeniumdiolates have been shown to decompose and release NO by two mechanisms, proton-driven®® and thermal” dissociation One mole of diazeniumdiolate species readily dissociates into two moles of NOg) and one mole of the donor amine when exposed to water’’ or at relatively high temperature.”! Chemistry Polymer coatings Blood phase | Proton-mediated NO release SN: rar / “ NH xé ho H wf ÌÌ © diazeniumdiolate-based NO donors Platelet Il Photoinitiated NO release ®-: Đ NO fumad silica ` (“` HN lửv) a S-nitrosothiol derivatized ` ny [ fumed silica Il Catalytic NO generation ) ^= LM NO cat or L CE catalytic species (Cu/Se) NH ° immobillzed polymere RSNO Red Blood Cell NO
Figure 1.5 Summary of a variety of NO releasing/generating strategies examined to devise potentially more blood compatible polymeric coatings
Previously, N-diazeniumdiolated secondary amine segments were reported to be either physically dispersed within polymer matrices (a) or covalently linked to various polymeric structures—on backbones (b) or on pendant side chains (c}—o create a range
Trang 31of new NO releasing polymers/polymeric compositions (see Figure 1.6).2' It was shown that the blood compatibility of these materials is dramatically improved, with a significant decrease in platelet adhesion and activation on surface of such materials when
85, 86
tested in vivo More detailed information and considerations related to the
development of such NO donors will be discussed in the Introduction to Chapter 2
PAL ISL PPL SS APIS - X—([N;O¿} X-N2Oa] noi PAP PL LDS SPS SSSI PAIN KI X-[N203] ° - AAA X—[N202] a b c
Figure 1.6 Incorporation of diazeniumdiolate functional group into polymers (X=amine or amino group, yw = polymer chain) [Figure adapted from Smith et al.®"],
An alternate new NO releasing material that utilizes S-nitrosothiols as the NO-donor group was recently reported” 3 (see Figure 1.5-II S-Nitrosothiol derivatives of cysteine, N-acetylcysteine and N-acetylpenicillamine were covalently tethered to the surface of fumed silica, a polymer filler particle,” as well as the backbone of methacrylate-based polymers.” The derivatized particles were blended into hydrophobic polymers (i.e., polyurethane, silicone rubber) to create polymeric coating that can release NO in the presence of copper ions, ascorbate or light Among them, photoinitiated NO release from the polymer coatings may be a potentially powerful tool in biomedical research and applications, as the light can be used as an external on/off trigger to control NO fluxes generated from these materials.”
Although the previous two types of NO releasing materials can be employed in a wide range of medical applications, the finite NO donor reservoirs may limit their
Trang 32potential applications in some medical devices that require an ultra-thin coating but long- term implantation (i.e., vascular stents) The limited amount of NO donor loading may influence the level and duration of the NO release possible to effectively prevent platelet adhesion and activation on such device surfaces To overcome this shortcoming, our group recently discovered a completely new approach to produce NO from polymers The new polymers contain a lipophilic ligand complex with Cu(II) ions that can catalytically generate NO from a variety of nitrosothiol species that are known to exist in blood.” This concept was further extended to polymethacrylate-type hydrogels with covalently linked Cu(II)-cyclen complex for in situ generation of NO from nitrosothiols in blood” (see Figure 1.5-IID While, in principle, the generation of NO at the blood/polymer interface could be infinite in duration, it is likely that some leaching and/or fouling of the catalytic sites may limit the length of time that physiologically relevant NO levels can be generated when such polymers are in contact with blood To address this issue, further studies are required
In addition, very recently, a selenium-based NO generation chemistry has been integrated with polymer surfaces/matrices”’ (see Figure 1.5-III) Polymers (i.e., cellulose filter paper, poly(ethylenimine)s, and heparin) that possess immobilized organoselenium species were found to catalytically generate NO from nitrosothiols in the presence of a reducing agent Further, certain organotellurium compounds have also been discovered as catalysts to decompose nitrosothiols to produce NO.” Both efforts are potentially valuable additions to the toolbox for creating polymeric coatings that are ideally capable of generating NO from blood containing species for considerably long period (i.e., years)
Trang 33Multifunctional EC Biomimetic Coatings for Biomedical Applications As summarized in the previous sections, molecules contributing non-thrombogenic and anti- cell proliferation properties of the EC can be incorporated into polymer matrices or immobilized on polymer surfaces in such a way that they are either released from 71, 85, 86, 99-101 Gy are present in biologically active forms on the polymer polymers
surfaces.>® °!-63 6: 69, 72, 102, 103 Tndeed, recent research has demonstrated that superior biocompatibility (in terms of inhibition of platelet activities and cell proliferation) can be achieved when medical devices are coated with NO releasing polymers.*’ Others have shown that polymers possessing immobilized heparin, thrombomodulin, and prostacyclin (as individual agents) offer some promise of reducing thrombosis™ °° © 7 '°2 However, such a single agent approach is not likely to solve the complexity of the biocompatibility problem of intravascular devices Rather, the use of a multivalent strategy, in which two or more of these naturally occurring EC derived anti-platelet, anticoagulant and/or anti- cell proliferation agents are used synergistically, is likely to provide the ultimate route to safely prevent thrombosis and smooth muscle cell proliferation that occur when vascular devices (i.e., grafts and stents) are implanted This multivalent strategy could greatly extend the lifetime and reduce the risks associated with such implants Indeed, this is one new direction that is examined in this dissertation work.’ Ultimately, the use of NO releasing/generating chemistries can be employed in concert with coatings that possess immobilized active heparin and/or thrombomodulin and/or slow release prostacyclin as means to achieve optimal reduction in thrombosis and restenosis associated with blood contacting extracorporeal devices and implants
Trang 341.5 Statement of Dissertation Research
The primary goal of the research described in this dissertation is to develop and investigate novel polymer-based NO donors that are capable of releasing low but controllable levels of NO when exposed to physiological conditions Two types of N- diazeniumdiolate-based NO releasing polymers and their potential biomedical applications were studied in this research work (see Figure 1.7), including (1) water- insoluble (more lipophilic) NO donors that can be incorporated into various polymeric coatings that are in contact with blood;’® and (2) water-soluble polymeric NO donors that can be added to dialysate solutions that equilibrate with blood flowing through membranous fibers within dialysis filters Additionally, new polymeric coatings with combined NO release and surface-bound active heparin were developed, aimed at more closely mimicking the nonthrombogenic properties of the endothelium by two complementary anti-thrombotic mechanisms, one based on NO’s potent anti-platelet activity and the other the result of the anticoagulant ability of immobilized heparin.‘
Chapter 2 describes the preparation and characterization of completely new NO releasing polymethacrylates, based on newly synthesized methacrylate monomers with alkyldiamine structures that can be diazeniumdiolated.'°° Novel NO releasing polymeric coatings were formulated by doping one of the new polymethacrylate-based NO donors into inert polymeric matrices (i.e, PVC, polyurethanes, silicone rubber, etc.) Biodegradable poly(lactide-co-glycolide) was employed as a film additive to greatly prolong the NO release of such coatings by continuously generating protons within the organic phase of the polymeric films, thereby driving decomposition of the
diazeniumdiolates
Trang 35Dialysate with water-soluble NO releasing polymer Polymer matrix Red blood cell Red Blood Cell \ ® | ® i H,0 ®@ Toxic species I 2 Platelet Platelet | z | Ẵ I 8 — NO I Blood
Add Modified surface ;
itive Blood flow
NO donor Dialysis fiber wall
(I) Water-insoluble NO donors (II) Water-soluble NO donors
used as coating dopants used as dialysate additives
Figure 1.7 Schematic of two types of biomedical applications using different types of N-diazeniumdiolate-based NO releasing polymers
A novel approach to potentially resolve serious thrombosis issues associated with kidney dialysis (hemodialysis) therapies is described in Chapter 3 Three new types of water-soluble polymeric NO donors, based on the diazeniumdiolated branched poly(ethylenimine)s and their derivatives, were prepared and characterized These macromolecular NO donors with high NO loadings were utilized as additives to the dialysate solution of dialysis filters The presence of these species creates a localized increase in NO levels at high surface area dialysis fiber/blood interface within the hemodialyzers, which is expected to greatly decrease the risk of thrombosis within the dialysis filter when patient blood is pumped through the device
Chapter 4 presents the first data on a new direction to devise thromboresistant polymer coatings via use of NO release materials The concept described in this chapter is to create polymer films that have tunable NO release properties, but also possess immobilized active heparin on their outermost layer The specific goal of this
Trang 36initial work is to demonstrate that the chemistries required to create NO release coatings with diazeniumdiolate-type NO donors are compatible with the surface chemistry necessary to immobilize active heparin It is believe that the combination of having NO release in concert with immobilized heparin may ultimately lead to coatings that are more thromboresistant than those that have only NO release or immobilized heparin alone Further, the presence of the active heparin may decrease the levels of NO required to achieve the desired biological effect (to prevent platelet activation and adhesion)
Conclusions of the entire research work are presented in Chapter 5, with further discussions on future directions toward the development of a new generation of polymeric coatings that closely mimic the EC with respect to combining NO release/generation with immobilized and/or released forms of other naturally occurring EC agents Such a multi-functioning biomimetic approach may ultimately lead to a truly nonthrombogenic synthetic endothelium Additionally, combining NO release/generation with localized delivery of pharmaceutical agent(s) to devise more biocompatible polymeric coatings is also discussed in this chapter
The appendix included in this dissertation discusses an indirect method to prove the authenticity of a series of newly synthesized sodium-based bis-diazeniumdiolates, whose identities have been difficult to verify by elemental analysis and other methods.’ As described in the appendix, the bis-diazeniumdiolates were first converted to their stable O’-methyl-protected forms and, subsequently, fully characterized For the first time, a single crystal structure of such bis-O’-methylated diazeniumdiolates is reported, confirming the authenticity of the bis-diazeniumdiolate structures
Trang 37Finally, it should be noted that this dissertation was prepared in a multi-manuscript format All of the chapters (as well as the appendix), except Chapters 1 and 5, were converted from either the published journal articles or manuscripts to be submitted to the related journals The work described in Chapter 2 has already been published as a full 105 and the work in Chapter 3 has already been paper in Biomacromolecules (2005),
prepared as a manuscript, intended for publication as a full paper within the same journal The results in Chapter 4 are from a full paper that has already been published in Biomaterials (2005).'“ Further, the Appendix is a summary of one side project All the synthetic data (including characterization) are a part of a co-authored paper published in Organic Letters (2005),' and the new single crystal structure data are intended for submission as a communication to another journal
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