Heparin-mimicking reverse thermal gel as a potential cationic growth factor delivery vehicle

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Heparin-mimicking reverse thermal gel as a potential cationic growth factor delivery vehicle
Hockensmith, Lindsay Hunt ( author )
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Denver, CO
University of Colorado Denver
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Heparin ( lcsh )
Growth factors ( lcsh )
Addition polymerization ( lcsh )
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )


Growth factor delivery is an important venture in the field of tissue engineering because of its widespread applications and promising therapeutic outcomes. Bolus injection, natural polymers, and synthetic polymers have all been utilized to deliver these specialized proteins to target cells/tissues, but each vehicle category has its own limitations. Bolus injection can deliver growth factors through minimally invasive ,means, but this method requires multiple injections for therapeutic relevance, has the potential for growth factor influence in non-target tissue, and poses a risk of delivering too high a protein concentration and exerting undesirable effects. Natural polymers are known for their biocompatibility and biodegradability but their batch-to batch variation can lead to unpredictable growth factor release profiles. Synthetic polymers allow for specific material modification for protein release optimization but can lack biodegradability and biocompatibility possibly require an invasive means of implantation, and introduce harmful or irritating chemicals through in-situ polymerization. General concerns that apply to every delivery vehicle, no matter what type, include the necessity for retaining the bioactivity of the growth factor and providing a means through which the protein can be released over an extended time period. To address these limitations, a series of sulfonated reverse thermal gels were investigated for their potential to deliver growth factors. Specifically, the sulfonate groups on the polymer provided a negative charge with which a positively charged growth factor could electrostatically interact - mimicking the natural interaction seen in the body between heparin and cationic growth factors. 1H-NMR showed that the polymer was available for conjugation with the reverse thermal gelling polymer, PNIPAAm, and with the sulfonate groups through a sulfonation reaction. FTIR indicated the presence of sulfonate groups through peak comparison with the original reverse thermal gel before sulfonation. The sulfonated polymers retained their gelation properties after the sulfonation reaction, which confirms that this polymeric solution can be administered in a minimally invasive manner. Furthermore, the release profiles of sulfonated reverse thermal gels were compared to a non-sulfonated reverse thermal gel using BSA. White the non-sulfonated reverse thermal gel performed similarly to a sulfonated reverse thermal gel, each of the reverse thermal gel contains sulfonate groups that have the potential to mimic heparin and protect positively charged growth factors from proteolytic degradation in the body.
Thesis (M.S.)--University of Colorado Denver. Bioengineering
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Department of Bioengineering
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by Lindsay Hunt Hockensmith.

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HEPARIN MIMICKING REVERSE THERMAL GEL AS A POTENTIAL CATIONIC GROWTH FACTOR DELIVERY VEHICLE by LINDSAY HUNT HOCKENSMITH B.S. Materials Science & Engineering Virginia Tech 2010 A thesis submitted to the Faculty of the Graduate School of the Univ ersity of Colorado in partial fulfillment of the requirements for the degree of Master of Science Bioengineering 2014


ii This thesis for the Master of Science degree by Lindsay Hunt Hockensmith has been approved for the Bioengineering Program by Micha el Yeager Chair Daewon Park Advisor Danielle Soranno July 2 4 2014


iii Lindsay Hunt Hockensmith (M.S ., Bioengineering ) Heparin mimicking Reverse Thermal Gel as a Potential Cationic Growth Factor Delivery Vehicle Thesis directed b y Assistant Professor Da ewon Park ABSTRACT Growth factor delivery is a n imp ortant venture in the field of tissue engineering because of its widespread applications and promising therapeutic outcomes. Bolus injection, natural polymers, and synthetic polymers have all been utili zed to deliver these specialized proteins to target cells/tissues but each vehicle category has its own limitations. Bolus injection can deliver growth factors through minimally invasive means but this method requires multiple injections for therapeutic relevance, has the potential for growth factor influence in non target tissue, and poses a risk of delivering too high a protein concentration and exerting undesireable effects. Natural polymers are known for their biocompatibility and biodegradability b ut their batch to batch variation can lead to unpredictable growth factor release profiles. Synthetic polymers allow for specific material modification for protein release optimization but can lack biodeg radability and biocompatibility possibly require an inv asive means of implantation, and introduce harmful or irritating chemicals through in situ polymerization. General concerns that apply to every delivery vehicle, no matter what type, inclue the necessity for retaining the bioactivity of the growth fac tor and providing a means through which the protein can be released over an extended time period To address these limitations, a series of sulfonated reverse thermal gels were investigated for their potential to deliver growth factors. Specifically, the sulfonate groups on the polymer provided a negative


iv charge with which a positively charged growth factor could electrostatically interact mimicking the natural interaction seen in the body between heparin and cationic growth factors. 1H NMR showed that t he polymer was available for conjugation with the reverse thermal gelling polymer, PNIPAAm, and with the sulfonate groups through a sulfonation reaction. FTIR indicated the presence of sulfonate groups through peak comparison with the original reverse the rmal gel before sulfonation. The sulfonated polymers retained their gelation properties after the sulfonation reaction, which confirms that this polymeric solution can be admistered in a minimally invasive manner. Furthermore, the release profiles of sul fonated reverse thermal gels were compared to a non sulfonated reverse thermal gel using BSA. While the non sulfonated reverse thermal gel performed similarly to a sulfonated reverse thermal gel, each of the reverse thermal gel contains sulfonate groups t hat have the potential to mimick heparin and protect positively charged growth factors from proteolytic degradation in the body. The form and content of this abstract are approved. I recommend its publication. Approved: Daewon Park


v ACKNOWLEDGMENTS I would like to thank Dr. Daewon Park for allowing me to work in his lab and pushing me to always do my best. My journey here has been an incredible learning experience, both professionally and personally. I would also like to thank Dr. Michael Yeager a nd Dr. Danielle Soranno They have been instrumental in my success in this program and have helped me prepare for future endeavors My lab mates of the Translational Biomaterials Research Lab also deserve gratitude their optimistic attitudes helped me mo re than they may know. Enough cannot be said about my family and friends. Their unwavering support and encouragement has been a constant source of motivation and joy Without them, this work would not have been pos sible. E verything that I accomplish is in dedication to them


vi TABLE OF CONTENTS CHAPTER I INTRODUCTION ................................ ................................ ................................ ............ 1 Overview ................................ ................................ ................................ ................. 1 Clinical Motivation ................................ ................................ ................................ 3 Anatomical and Physiological Considerations ................................ ................. 6 Growth Factor Considerations ................................ ................................ .......... 7 Relevant Literature ................................ ................................ ................................ 12 Current Growth Factor Delivery Approaches ................................ ................. 12 bFGF Delivery for Angiogenesis ................................ ................................ .... 15 Polymeric Protein Delivery Systems ................................ .............................. 18 Thermally Induced G elling Systems ................................ ............................... 22 PNIPAAm Considerations ................................ ................................ .............. 24 FDA Approved Synthetic Polymers ................................ ................................ ..... 24 Polymeric Backbone Considerations ................................ ................................ .... 26 Specific Aims For The Thesis ................................ ................................ ............... 27 II MATERIALS AND METHODS ................................ ................................ .................. 28 Materials ................................ ................................ ................................ ............... 28 Polymer Synthesis ................................ ................................ ................................ 28 Polymer Characterization ................................ ................................ ...................... 32 BSA Release Study ................................ ................................ ............................... 33 III RESULTS AND DISCUSSION, CONCLUSION, AND FUTURE WORK .............. 34 Results and Discussion ................................ ................................ ......................... 34 PSHU and dPSHU NMR ................................ ................................ ................ 34 Polymer Synthesis SRTG 1 9 ................................ ................................ ......... 37


vii Gelation Test for SRTG 1 9 ................................ ................................ ............ 40 Zeta Potential Results SRTG 5 9 ................................ ................................ .... 43 Fourier Transfrom Infrared Spectroscopy (FTIR) RTG, SRTG 5, 8, and 9 ... 45 LCST Measurement RTG, SRTG 5, 8, and 9 ................................ ................. 46 BSA Release Study RTG, SRTG 5, 8, and 9 ................................ .................. 48 Conclusion ................................ ................................ ................................ ............ 50 Future Work ................................ ................................ ................................ .......... 52 Polymer Characterization ................................ ................................ ................ 52 Tube Formation Assay ................................ ................................ .................... 53 Release Test Using bFGF ................................ ................................ ............... 53 REFERENCES ................................ ................................ ................................ ................. 55 APPENDIX ACETATE BUFFER ................................ ................................ ................................ ........ 61 PNIPAAM MOLECULAR WEIGHT TITRATION ................................ ........................ 62 BSA CALIBRATION CURVE ................................ ................................ ........................ 63 PSHU SYNTHESIS, DEPROTECTION, AND CONJUGATION OF PNIPAAM ........ 64 FTIR FOR RTG, SRTG 5, SRTG 8, AND SRTG 9 ................................ ......................... 65


viii LIST OF TABLES T able III.1 SRTG 1 9 ...39 III. 2 SRTG 1 9 Synthesis Conditions and G


ix LIST OF FIGURES Figure I. 4 Plaque build I. 5 Electrostatic potential maps (in units of 8 I. 6 9 I. 7 Polymer solubility behavior at the Lower Critical III.1 1 H NMR of PS ....35 III. 2 1 H NMR of dPSHU (green) and PS III. 3 Theoretical synthesis of sulfonated RTG (SRTG 1 8 III. 4 Example of gel sh III. 5 Illustration of zeta potential charge loc III. 6 Zeta potential measurements for SRTG 5 III. 8 7 III. 9 BSA release profiles for RTG, SRTG 5, SRTG 8 and SRTG 9 4 9 Appendix D PSHU Synthesis, Deprotection, a 4 Appendix E FTIR for RTG, SRTG 5 5


x LIST OF ABBREVIATIONS ACA Azobis(4 cyanovaleric acid) bFGF Basic fibr oblast growth f actor FGF 2 BM Basal media BME Basement membrane extract BSA Bovine serum a lbumin CABG Coronary artery bypass g raft CHD Coronary heart disease CM Complete media DMF Dimethylformamide DMSO Dimethyl sulfoxide d PSHU Deprotected PSHU EC M Extracellular matrix EGF Epidermal growth factor FBS Fetal bovine serum FDA Food and Drug Administration HDI Hexamethylene diisocyanate HSPGs Heparan sulfate proteoglycans HUVEC Human umbilical vein endothelial cell MI Myocardial infarction NaOH Sodium hydroxide NIPAAm N Isopropylacrylamide PCL Poly caprolactone PDGF Platelet derived growth factor PEAD Poly (ethylene argininylaspartate) PEG Poly ethylene glycol PLGA Poly (lactic co glycolic acid) PNIPAAm Poly (N isopropylacrylamide) PS 1, 3 Propanesultone PSHU Poly (serinol hexamethylene urea) RTG Reverse thermal gel (dPSHU PNIPAAm) SRTG Sulfonated reverse thermal gel (sulfonated dPSHU PNIPAAm) STP Sulfur Trioxide Pyridine THF Tetrahydrofuran


1 CHAPTER I INTRODUCTION Overview Growt h factor delivery, as it pe rtains to tissue engineering, has garnered much enthusiasm in scientific research 1 Growth factors are important signaling molecules that can exert their influence on cells through their specific cell surface binding Because t issue regeneration is ultimately a result of said biological environment c ontrol over this would result in forcing the cell to respond in a desireable manner D esigning a material that can successfully transport specific signaling molecules in the exact location and concentration necess ary for a cellular response could lead to advantageous whole tissue and organ re action s depending on the protein intention s Due to advances in other scientific fields, the signaling mechanisms, relevant tissues, and overall biological results linked t o a multitude of growth factors have been elucidated. For example, basic fibroblast growth factor (bFGF, also named FGF 2) is known for its pro angiogenic signaling. Accordingly, many studies and clinical trials have been dedicated to the delivery of gro wth factors as a means of medical treatment 1 A suitable delivery vehicle for the growth factor must be fashioned to ensure adequate spacio temporal release for the targeted cells A reverse thermal gelling polymeric system is fit for the task To this end, a sulfonated deprotected poly (serinol hexamethylene urea) conjugated to poly ( N isop ropylacrylamide) (SRTG) was designed as a growth factor delivery material. A reverse thermal gel ling polymer provides a minimally invasive way to deliver growth factors because it acts as a sol below body


2 temperature and forms a gel above body temperature Not only that, but polymers can be synthesized with functional groups available for subsequent modification depending on the application (i.e. conjugated with a charge, polypeptide, and other polymers) In particular, t he objective of this study was t o fashion an anionic polymeric system that electrostatically interact s with and subsequently deliver s a steady amount of positively charged protein Specifically concerning the charge a negatively charged reverse thermal gel derived from sulfonate group s would mimic heparin, a na tural protein found in the body that can protect the growth factors from proteolytic degradation. The com prehensive theory is that if the polymer can take advantage of the well known binding between growth factors and the sulfon ate groups on heparin, all the while remaining in a gelled state, then a sustained and localized concentration of growth factor can be delivered to desired tissues. This introduction discusses the clinical motivation for this study and the relevant growth factor details Subsequently, a literature review cover s the status of research regarding growth factor delivery as a whole, the types and general mechanisms for in situ polymeric gelling, and the use of reverse thermal gels applicable in the medical fiel d Additiona l ly, a few Food and Drug Administration (FDA) approved polymers are introduced A brief section detailing the proposed polymeric system investigated by this work is explored b ackbone Lastly, the specific aims for this research are listed to provide the goals for this work.


3 Clinical Motivation The overall clinical motivation for this research concerns the treatment of coronary heart disease (CHD) by route of angiogenesis, or new blood vessel growth, through sustained delivery of a known pro angiogenic growth factor, fibroblast growth factor 2 (FGF 2) While other approaches to angiogenesis exist, like cell therapy and gene therapy, delivery of growth factors is considered to be the most simple and direct method because it require s no additional processing means associated with cells or viral vectors 2 In 2010, an estimated 15.4 million Americans were currently living with a CHD diagnosis, and almost 400,000 people died that same year as a result 3 CHD is defined as a plaque build up in the coronary arteries. The ability for the heart to sustain blood flow to the entiry of the body depends on the blood flow to the heart itself through the right and left coronary arteries vessel s extend ing from aortic dilatations, known as the sinuses of Valsalva, located above the aortic valve cusps 4 Lifestyle chang es, p harmaceutical means, and surgical interventions are all available for patients suffering from CHD. The surgical options are the most relevant treatments to this study, as they are physical ways of either clearing the blockage through angioplasty or r evascularizing the ischemic myocardial tissue that results from CHD through coronary artery bypass graft (CABG) surgery Angioplasty, shown in Figure I.1, employs a balloon at the end of a catheter and is routed through the cardiovascular system The b alloon is then distended at the artery narrowing to open up the vessel. Complications including bleeding, development of infection, and reoccurrence are all risks co ncerning this procedure the one year patient cost for someone undergoing this


4 procedure is $52, 673 3 Likewise, there are many inherent risks like bleeding and infection, involved with vascular surgery through CABG shown in Figure I.2 This procedure has a one year patient cost of $86,914 dollars 3 CABG surgery is a revascularization technique that improves the function of the heart by circumventing the ischemic heart tissue with an existing section of vein or artery taken from elsewhere in the body, typically a vein from the leg or an artery within the chest wall and re routing oxygenated blood directly from the aorta to tissue downstream of the plaque build up Figure I.1 Diagram of a ngioplasty 5


5 Figure I.2 Diagram of CABG surgery 6 T he proposed polymeric system in this work has the intention of promoting angiogenesis and could theoretically be used alone as a replacement for CABG surgery or in conjunction with an gioplasty or CABG surgery The growth factor delivery vehicle SRTG, has the advantageous property of being a reverse thermal gel so its deployment would be in a minimally invasive manner through intracardiac injection thereby removing the risks associa ted with CABG surgery Also, as it sustains the release of protein, it has the potential to only be injected once and it reduce s the amount of hospital care post treatment To provide a better understanding of the biological factors associated with t he clinical motivation, anatomical and physiological concerns and growth factor considerations are addressed in the following subsections.


6 Anatomical and Physiological Considerations Lack of blood flow to the heart tissue is characterized as myocardial i schemia. This occurs when coronary arteries become partially or fully stenosed by atheroma, which is a cholesterol rich, subintimal plaque 4 As shown in Figure I.3, from within the lumen of the artery the layers moving outward are called tunica intima, tunica media, and tunica adventitia. Importantly, endothelial cells, the type of cells on which FGF 2 can exert their pro angioge nic influence, are cells located in the tunica intima and in direct Figure I.3 Wall structure of a healthy artery 7 Atheromas primarily affect the behavior of the intima la yer and can be attributed to diabetes, hypertension, obesity, cigarette smoking, hypercholesterolaemia, and genetic predisoposition for cardiovascular disease 4 If the atheroma is only partially blocking the lumen of the coronary artery then the downstream effects on myocardium can range from asymptomatic ischemia to angina and a decrease in exercise tolerance. An example of t he plaque build up is presented in Figure I.4


7 Figure I.4 Plaque build up in the subintimal wall 8 In more dangerous situations, atheromas can lead to myocardial infarctions by is (the formation of an organized blood clot) by prompting acute cardiac failure 4 As a result, myocardial cells that ar e not receiving adequate blood flow can become necrotic within hours of this event. In order to circumvent the damage from the myocardial infarction or as a way to prevent such damage from occurring by a stenosed vessel, we investigated the potential for an anionic polymer system to deliver a pro angiogenic positively charged growth factor that can endothelial cells. Growth Factor Considerations Growth factors are signalin g molecules that instruct a cell or group of cells on how to behave. The presence and/or cooperative interaction of different growth factors with cells has precise control over the fate of the cell itself, its desired actions, or what it


8 will produce and subsequently release to be used elsewhere in the body. A simplified explanation of growth factor signaling involves protein protein interaction between the growth factor and a specific transmembrane receptor on the cell surface. Through a series of phosp horylations, ion fluxes, changes in metabolism, gene expression, and subsequent protein synthesis, the cell has integrated the biological instructions initiated 1 The particular protein that prompted this work was basic fibroblast growth factor (bFGF, also known as FGF 2). bFGF is a cl inically relevant protein due to its role in angiogenesis, cell adhesion, wound healing, bone regeneration, and tissue regeneration 9 As mentioned previously, the polymeric system was designed to contain negatively charged functional moieties attributed to sulfonate groups, which could electrostatically interact with a po si tively charged growth factor, in this case, FGF 2. Sangaj et al showed the positively charged basic residues (K27, K120, R121, K1 26, K130, and K136) that intera ct with sulfonate groups 10 T he relevant electrostat ic map can be seen in Figure I.5 where the color blue indicates positive residues on bFGF and the color red denotes negative residues on heparin Figure I. 5 Electrostatic potential maps (in units of kT /e) for bFGF (left) and heparin (right) 10


9 Basic fibroblast growth factor has been shown to promote angi ogenesis, defined as the formation of new bloo d vessels from existing vessels. FGF 2 exerts its influence on endothelial cells through binding to a fibroblast growth factor receptor and transmitting a signal (through the MAPK/ERK signaling pathway) for the cell to secrete proteases which facilitate the dissolution of ba sement membrane 11; 12; 13 This activates chemoattractants and mitogens, thereby promoting endothelial cell migration, proliferation and ultimately, new tube formation with a reconstructed basement membrane 11; 12 A key biological interaction that this study exploits is the binding action of heparin and/or heparan sulfate proteoglycans (HSPGs) to FGF 2. Sulfonate groups, shown in Figure I. 6 are present on both of these b inding proteins. Th rough electrostatic attraction they sequester the FGF 2 from its free form and prote ct it from proteolysis. FGF 2 can be presented to the cell surface FGFRs to begin the signaling pathway through the cooperative binding between FGFRs and cell bound HSPGs, ECM bound HSPGs releasing FGF 2 after proteolytic event, or heparin protected FGF 2 release 10 Figure I. 6 carbon bound to sulfur as well as the remaining chemical makeup of the polymer. FGF 2 is present in n ormal cardiac environments but it is sequestered from endothelial cells in an inactive form by heparan sulfate proteoglycans in the extracellular


10 matrix. In diseased cardiac environments, the concentration of FGF 2, a long with VEGF, another pro angiogenic growth factor, has been recorded to be higher considered to be a result of a physiological response to myocardial ischemia 14; 15 Andres et al investigated inflammatory responses and how they are tightly linked to angiogenesis 15 Their findings pointed to further confirm ation that inflammatory chemokines play a key role in the promotion of angiogenesis through endothelial stimulation by route of FGF 2 production and release. Furthermore, they indicate that inflammatory cell may produce FGF 2 proteins. Additionally, hypoxic conditions promote cell interaction with FGF 2. Two mechanisms are generally considered to be the rea son for increased bFGF availablility for cell attachment: heparanase or other glycosaminoglycan degrading enzymes and bFGF binding protein chaperone interaction 16 Both of these actions have the potential to release bFGF from its sequestered inactive form to an active form that can bind to FGF recept ors and exert its signaling potential. In the case of coronary heart disease, plaque build up is a pro inflammatory action. Additionally, the myocardial ischemia due to the narrowing of the vessel lumen is also pro inflammatory. As a result, the body is responding to an inflammatory response by eliciting bFGF release from the heparan sulfates so that it is free to interact with the FGF receptors on endothelial cells to promote angiogenesis. The natural biological response can not always overcome the new demand for revascularization due to CHD ; therefore, delivery of additional FGF 2 could encourage an advantageous environment for therapeutically relevant angiogenesis since FGF 2 auto up regulates its own production through autocrine signaling 15


11 As stated before, bFGF is a pro angiogenic growth factor that has been shown to promote collateral vessel formation in animal models under experimental conditions 14; 17; 18 Unger et al investigated human tolerability of intracardiac injection of bFGF by route of the left coronary artery 17 Their findings indicate that bFGF has a serum half life of 85 minutes increased coronary arterial diameter and led to a decrease in mean arterial pressure. This hypotension ph enomenon is attributed to bFGF stimulating endothelial nitric oxide production, a known vasodilator. Similarly, another clinical trial organized by Laham et al showed cases of patient hypotension, but their study also included quality of life assessment in accordance with the Seattle Angina Questionnaire (SAQ) 18 According to their results, patients that received bFGF intracardiac injections experienced increased exercise intolerance consistent with results after CABG surgery or angioplasty. Furthermore, patients from th is study showed a decreased myocardial ischemic area as shown through magnetic resonance imaging. The st udies mentioned above prove in vivo tolerance for bFGF as well as some of its advantages concerning an increase of exercise tolerance I n a differen t study Laham et al. went even further because they proved that an intrapericardial injection of bFGF does in fact promote collateral vessel generation 14 Through use of a porcine model, bFGF treatment resulted in significant increase of coronary blood flow and collateral formation. Limitations of bFGF delivery include thrombocytopenia (abnormally low platelet count), proteinuria (excessive protein in urine) 17 and non target angiogenesis 19 Moreover bFGF delivery alone may not result in a maximal therapeutic effect that could rival patient outcomes when compared to other forms of CHD treatment because


12 a ngiogenesis is a complex manifestation of multiple growth factors all working in concert to produce new blood vessels. While co nsiderable research has shown that bFGF delivery can produce collateral vessels in animal models and result s in higher exercise tolerance in humans, delivery of bFGF in conjuction with other pro angiogenic growth factors like VEGF, PDGF etc. may prove to be the most clinically useful treatment. Considering the design of our polymeric system, co release with another heparin binding growth factor that is relevant to angiogenesis, like VEGF, for enhanced therapeutic treatment could be a future application f or this vehicle. Basic fibroblast growth factor is the most commonly used isoform in the FGF family investigated for angiogenic potential 20 and is the best studied 16 The FGF family consists of 23 isoforms and only three are necessarily associated with cardiac revascularization: FGF 1, FGF 2, and FGF 4. Other pro agiogenic growth factors exist, like vascular endothelial g rowth factor (VEGF) and platelet derived growth factor (PDGF) for example, but bFGF 2 is a more potent angiogenic factor than those two which are also commonly studied 20 For these reasons, bFGF was our target growth factor in designing our anionic polymeric system. Rele vant Literature Current Growth Factor Delivery Approaches Different methods of growth factor delivery include bolus injection as well as natural and synthetic polymer vehicles. Examples and details relevant t o eac h type are discussed herein


13 Bolus inject ion of protein has been extensively studied as a therapeutic practice in the medical field 21 Pept ide treatments require that multiple doses/injections be administered frequently because growth factors are susceptible to proteolytic degradation. E ven when they are not conformationally altered by the microenvironment specifically therapeutic growth fa ctors have a short half life in free form within in the body due to proteolytic degradation 9 A biological compensation for this occurance can be seen through heparin binding Heparin sequesters growth factors away from trypsin and other proteases to avoid said protelytic degradation. An example of this includes, but is not limited to, fibroblast growth factor 2 (FGF 2) being sequestered from the extracellular matrix by heparin and heparin related agents 22 Due to their short half lives, slow diffusion within the bo dy and susceptibility to proteolytic degradation, b olus injection of growth factors does not typically yield the complete desired effect of the protein 1; 23 In an effort t o compensate for this, the use of hepari n and heparin mimicking polymers to distribute growth factors amongst target tissue has ga rnered much attention due to their potential for biomedical applications 24 Chu et al successfully designed a polycation, poly ( ethylene argininylaspartate diglyceride) (PEAD) that binds to heparin, which subsequently binds to FGF 2 and forms a coacervate upon injection Their results indicate d that the relationship between heparin and FGF 2 maintains the bioactivity of FGF 2 after polymer incorporation and controls an extended release profile versus the non heparin PEAD 25 Researchers have utilized but heparin can vary greatly from batch to batch and can have unexpected, even unwant ed biological interactions with non target tissues/cells 9 Notab ly, heparin has been shown to prevent


14 the growth of human umbilical vein endothelial cells and human dermal fibroblasts this action of heparin would be in direct opposition to the intended effects of bFGF 9 Other natural polymers have been used for growth factor delivery, including gelatin and alginates. Hori et al u tilized a biodegradable hydrogel comp rising cationized gelatin to release epidermal growth factor (EGF) in rabbit corneas with epidermal defects 26 In their fi ndi ngs, the hydrogel release of EGF was successful in accelerating cornea healing. Likewise, Heo et al successfully delivered EGF through alginate, a natural polymer, which was photocrosslinked using visib le light, to the growth factor to promote wound h ealing 27 While natural polymers like gelatin and alginate have the advantage of being biocompatible and biodegradable, they are often associated with the same batch to batch variability as heparin. Due to the mentioned issues with delivering growth factors with a naturally occurring vehicle, in this case heparin, the potential use for synthetic polymers in this application has been investigated. Polymer scaffolds and microspheres made of poly(lactic co glycolic acid) (PL GA) have been used frequently in research studies because it has proved to be a highly biocompatible material 2 Richardson et al took advantage of a PLGA derivative to create a scaffold microsphere composite that successfully delivered growth factors vascular endot helial growth factor (VEGF) and platelet derived growth factor (PDGF) simultaneously 28 While the results were promising as the study showed the advantage of delivering two growth f actors working towards intitiating and sustaining angiogenesis, the fact remains that using a scaffold requires surgical implantation and has the potential to limit its applications due to accessibility within the body.


15 Nguyen et al. designed a heparin m imicking polymer that successfully protected bFGF from various internal and external stressors 9 The protection vehicle was a copolymer synthesized by styrene sulfonate units and methyl methacrylate units containing poly (ethylene glycol) side chains. In their study, the sulfonate gr oups were the deciding factor dictat ing the overall bFGF stability. By their analysis, bFGF was able to bind to the polymer on the negatively charged sulfonate groups, thereby mimicking the negatively charged heparin found in the body that attracts positively charged growth factors like bF GF. Ultimately, the bFGF interacti ng with polymer containing sulfonate groups retained their bioactivity, whereas, the bFGF interacting with polymer that was void of these negatively charged groups lost their bioactivity after stressors like heat, low pH, and proteolytic degradation were introduced 9 To reiterate, t hi s work focused on a sulfonated reverse thermal gel that could potentially accomplish the same protein protection that Nguyen et al. provided but with the added bonus of being a reverse thermal gel. To this end, SRTG has a negative charge that at tracts pos itively charged bFGF and the thermogelling component PNIPAAm allows the polymer to be administered through a minimally invasive injection. bFGF Delivery for Angiogenesis Ito et al. developed an FGF 2 apatite coated implant and successfully delivered the growth factor in an effective and controlled manner in vivo to decrease the area and impact of a brain infarction 29 Related growth factors, like vascular endothelial growth factor (VEGF) and other chemokines were quantified after implantation results suggest that an increase in FGF 2 signals for the added release of chemokines and growth factors are needed for angiogenesis. The mechanism with which this group proposes endothelial


16 cell i nteraction with implant released FGF 2 is by route of FGF absorption into the vessel through arachnoid granulations, which are specific to the brain. The accepted explanation for angiogenesis in this case is that there is a period of rapid proliferation o f collateral arteries which represents a scenario in which a tissue is perfused by means of a network of arteries. Collateral arteries are known for being originally microvascular, thin, and only composed of endothelial cells, i nternal elastic lamina, and several layers of smooth muscle cells. This form of FGF 2 delivery, while successful, does not fit the surgical techniques for our intended deployment. The polymer in this study was designed with the intention of it being deployed through an intercardia c injection which would neither require surgery n or involve surgical associated risks. Shute et al. performed studies to elucidate the differences in lung tissue in normal and asthmatic patients 30 Their findings indicate a physiological remodeling that could be due to the presen ce of FGF 2. Asthmatic patients exhibit an increased subepi thelial collagen deposition that promotes an inflammatory response. This phenomenon shown in lung tissue also has the potential to appear in the diseased vessel area of coronary heart disease. W hen the plaque forms, the body is going to naturally mount an inflammatory response to clear the build up. However, considering that endothelium which allow s excessive plaque accumulation may be structurally or physiologically damaged and that inflammato ry conditions lead to a potential increase of the internal elastic lamina, a significant and naturally mounted inflammatory response might only worsen the problem. Furthermore, FGF 2 is a growth factor known for its proliferative effects on different cell types namely, fibroblasts, which are the cells responsible for creating extracellular matrix components (tpes of components found in basement membranes). If FGF 2 is not


17 deployed and kept to a specific localized area, then an additional amount of FGF 2 c ould be detrimental and further the conditions of the coronary heart disease. Accordingly, a balance between FGF 2 angiogenic action and FGF 2 induced inflammation must be found to produce a clinically useful treatment. Wang et al used an injectable temperature sensitive chitosan hydrogel in conjunction with bFGF to enhance angiogenesis and improve cardiac function in an ischemic heart rat model 31 After ligation of the left coronary artery, the chitosan hydrogel solution with bFGF was injected in to the left ventricular wall As a result, the group found that the b FGF activity was sustained for four weeks after injection, neo angiogenesis was initiated in the infarcted tissue area, there was a reduction of the infarcted region compared to other conditions tested (PBS, PBS an d bFGF, and chitosan only), and overall cardiac function like left ventricular ejection fraction, was improved. Chitosan was chosed as the desired material for this application because it is biodegradable, biocompatible, and can easily be delivered alon gside bioactive agents, like growth factors, but the actual mechanism explaining why and how this system worked still remains unclear. The left ventricle may have received structural and mechanical support due to the chitosan hydrogel injection and the bF GF promoted perfusion of the infarct affected tissue but the bioactivity of the bFGF was not recorded in vivo, the mechanical properties of the hydrogel were not investigated, and most importantly for our application, this material has not been investigat ed for angiogenic potential on the coronary arteries, which are subject to a different mechanical and chemical environment than within the left ventricle wall.


18 Katayama et al published research on the co treatment of CABG surgery with bFGF injection an d CABG surgery without bFGF injection 32 Importantly, this research applies to our study because this polymeric system could be utilized in conjunction with this surgical route of revascularization. Their findings indicate that patients with the bFGF injections showed a significantly more developed collateral vessel system and increased myocardial perfusion. The examples reviewed in this section provide evidenc e for the potential of bFGF to create collateral vessels after deployment in the body of animals and humans alike Moreover, the introduction of this extra vessel network allows for more perfusion of the heart tissue and has the capability to increase qua lity of life since the original problems experienced with CHD are due to ill perfused myocardium resulting from stenosed vessels. Polymeric Protein Delivery Systems General Co nsiderations Polymers are defined as large molecules, or macromolecules, whi ch co mprise many repeating subunits or monomers and the process of combining said monomers is called polymerization 33 The specific types of monomers, as well as the polymerization reaction mechanisms dictate the behavior of the final polymeric material. For example, biocompatibility and degradability are two of the most important factors when designing biomaterials as these are the means by which the polymer interacts with cells and the cellular environment. Biocompatibility ensures that the polymer is not e liciting an


19 undesirable immune response and degradability directs the release behavi or of the therapeutic cells, protei ns, or drugs being delivered. Polymers are desireable amongst drug delivery systems because they lend themselves to chemical alterations through manipulation of their function al groups. The functional groups of a poly mer refer to the specific part of the molecule that determines how the polymer as a whole reacts in certain environments or in the presence of other polymers or materials 33 In terms of this work, fashioning a negative surface charge to the RTG through a sulfonate functional group allows for ionic interactions with a positively charged growth fac tor Subsequently, when the ionic bonds weaken the growth factor can be released and exert its potential on the immediately surrounding tissue. In situ Polymeric Gelling Systems In situ gelling polymer systems have garnered extensive attention because they can be deployed in a minimally invasive manner and have the potential to deliver drugs, encapsulated cells, or therapeutic proteins steadily over a long period of time with one dose 34; 35; 36; 37 As a whole t hese types of polymeric systems share the polarizing endpoints of the sol and gel state s but the mechanisms with which they transition from these states can vary greatly. The major reaction classifications representing these systems are thermoplastic pastes, in situ cross linked polymer systems, in situ polymer precipitation, and thermally induced gelling systems 35 T he advantages and disadvantages of each category are discussed herein. Thermoplastic pastes take advantage of polymers that have low melting points (25 60 C) ; they are administered through a needle when the material is in its melted state


20 and as the mate rial cools to body temperature it then turns into a semi solid. Notable but the major disadvantage of this delivery is the high temperature the pastes must reach to be mel ted 34 Temperatures above or nearing 60 C have the potential to be painful and pos e a risk for tissue necrosis and scar tissue formation. More recently though, AP Ph arma has developed a new generation of poly (ortho) esters that exist as semi solids at room temperature and do not require extensive heating before injection, th us removing the negative associations with high melting temperatures pre deployment 35 In situ cross linked polymer systems c an be further subcategorized into thermosets, photo cross linked gels, and ion mediated gels. Covalent crosslinks form into a polymeric network when thermosets are heated (by the body) shape. Dunn et al experimented with copolymerizing D,L lactide or L lactide with caprolactone using an initiator and a catalyst 34 The polymerization time ranged from 5 30 min utes and as a result, a large burst release was realized because the polymer could not form quickly enough to withhold the drug flurbiprofin Also, the introduction of an initiator can be dangerous within the body because it inherently breaks down into a free radical species and has been thought to cause tumor formations. Hubbel et al. produced a photopolymerizable hydrog el using free radi cal chemistry a mechanism that is prompted by a light source stimulating the photoinitiator 34 T he polymerization was very quick and reduced the risk posed by the free radical species shown in thermosets but this pr ocess also made the polymer brittle and it exhibited more shrinkage conditions of which would favor faster drug expulsion Concerning a different type of in situ polymerization, ion mediated gels, or a lginate s are


21 polysaccharides that t ransition to a gel state in the presence of calcium ions The complication that this type of polymerization pose s include s the availability of such ions for gelation and the large burst release that can occur when sufficient calcium ions are indeed present. In situ polymer precipitations involve injections of non water miscible polymers that are in organic solvent solutions in the body When the solu tion is accosted by the naturally aqueous environment i n the body, the water invades the solution and the polymer forms a precipitate as the organic solvent is expelled from the solution. Many groups like ARTRIX Laboratories and ALZA Corporation have deve loped functioning in situ polymer precipitations but the general concerns with this type of polymer system include painful injections due to the organic solvents and possible myotoxic (muscle necrosis) effects 35 Thermally induced gelling systems have garnered extensive attention because their gelation occurs solely due to body temperature. Chen et al designed a tribl ock copolymer, consisting of PLGA PEG PLGA that releases porcine growth hormone at a generally constant rate (serum levels of 3 6 ng/ml) for 4 weeks with no initial burst release 38 The gel physically entraps the hormone and allows release primarily through erosion and diffusion. The protein release me chanism in this work is aiming to releas e prote in due in particular to the break down of the electrostatic interactions in addition to erosion and diffusion. Not only can temperature dictate gelling, but it can be used in conjunction with pH sensitive polymers to further increase the specifi c conditions promoting gelation. Li et al. created a temperature/pH sensitive hydrogel that releases insulin in a sustained


22 fashion for 28 days 37 The polymer only gels when both the pH is 7.4 and the temperature is 37 degrees Celcius so that the hydrogel does not gel prematurely and clog the needle when it is being deployed deep into the body. The insulin kept its bioactivity and the hydrogel was non toxic. This dual sensitivity approach could lead to further advances with our thermal gel for future applications. Thermally induced gelling systems are of particular importance in this study because this is the exact mechanism by which our polymer system transitions from a sol to a gel state. The details are discussed in the subsequent section but the underpinning idea is that below the lower critical solution temperature (LCST), the hydrophilic behavior and keep it in solution with water but above the LCST the hydrophobic interactions dominate and the polymer comes out of solu tion to form a gel. Thermally Induced Gelling Systems General Considerations Thermally induced gelling systems also termed thermogels, avoid some of the major disadvangtages posed by other types of in situ polymeric gelling systems 21 Ultimately, thermogels are defined by their ability to transition from a sol to a gel state solely from the influenc e of temperature 39 Because of this, the physical gelation is void of harmful free radicals, high polymerization temperatures, and potentially irrit ating organic solvents. The lower critical solution temperature (LCST) is the fulcr um upon which the physical state of a thermogel is decided LCST refers to the temperature that determines


23 the miscibility of two constituents in a mixture Thermogel solutions comprise polymers or both (ampiphilic). The naturally antagonistic features of hydrophilicity versus hydrophobicity would normally prevent one polymer or monomer from dissolving into a solvent (i.e., a hydrophobic po lymer is non miscible in water) but the components of the mixture are miscible In contrast, above the LCST the components become non miscible and can precipitate out of the solution. These occurances can be attributed to t he domina nt interactions above and below the LCST. Below the LCST, the hydrogen bonds on the hydrophilic moieties and force the hydrophobic moieties into solution. Conversely, abov e the LCST, the hydrogen bonds are weaker and the hydrophobic moieties interact with one another to repel the water and shrink together, thereby precipitating out of the solution and forming a gel 40 A physical representation of this phenomenae can be seen below in Figure I 7


24 Figure I 7 Polymer solubili ty behavior at the Lower Critical Solution Temperature (LCST). Left hand side shows hydrated polymer below LCST with entropic loss of 41 Synthesizing a polymer with both hydrophobic and hydrophilic end groups can be accomplished by block copolymerization, where hydrophilic monomer A and h ydrophobic mo nomer B could be conjugated in ABA, BAB or a similar fas h ion, or if the polymer itself possesses both types of end groups (ampiphilic). This study focuses on poly (N isopropylacrylamide) (PNIPAAm ) an ampiphilic polymer PNIPAAm Consideration s In the field of thermoresponsive polymers, PNIPAAm is one of the most commonly studied materials i ts ability to conform to any shape when exposed to a temperature above its LCST (~32 C) makes i t an ideal injectable polymer 42 Specifically, at body temperature the polymer dissolved in water transitions from a solution to a gel form. This shift in state is the result of the polymer chains undergoing a coil globule transition in which the hydrophobic interactions amongst the hydrophobic groups of the polymer dominate the originally domina n t hydrophilic interactions that kept the polymer in solution with water 42 FD A Approved Synthetic Polymers Synthetic polymers are advantageous materials for use in the medical field because they typically possess functional groups that can be specifically fashioned for a particular application. For example, this work looks to co njugate a sulfonate group as the


25 functional group on the polymeric backbone. A functional group is the part of the polymer that will directly interact with its environment. In our case, the sulfonate group is present to electrostatically interact with a positively charged protein in hopes of mimicking the heparin growth factor r elationship found in the body. This paper will discuss herin a few FDA approved synthetic polymers and the decision s for not using them in this work Poly lactic co glycolic acid (PLGA) is an FDA approved polymer known for its biodegradability and biocompatibility 43 The PLGA vehicles used for drug delivery include nanoparticles, microparticles, an d scaffolds. While it does possess the ability to be fashioned for different applications, the vehicle itself is what makes PLGA unsuitable for our proposed method of treating CHD. Scaffolds need to be surgically implanted. This fact would negate the mi nimally invasive route of delivery that this work looks to take. Additionally, microparticles and nanoparticles have the potential to act in highly cell specific means, but because we are using a growth factor that can act on such a common cell type, endo thelial cells, particle drug delivery may lead to unexpected or unwanted tissues being affected. Poly ethylene glycol (PEG) is also a biocompatible FDA approved polymer This material can undergo a reaction, called PEGylation, which describes coval ent bonding of bioactive materials, like proteins 44 PEG has been shown to successfully deliver proteins in a controlled manner but there have been instance s where the bioactivity of the protein has been reduced or changed due to the effects of polymer binding. Our polymeric system looks to avoid this occurance by encouraging polymer protein interaction through


26 electrostatic interactions, which theoreticall y would not interfere with the conformation of the protein. The last FDA approved polymer to discuss is polycaprolactone (PCL). PCL is a biodegradable polymer known for its ease of manufacturing and manipulation 45 The form this vehicle takes includes scaffolds and micro and nanoparticles. While these vehicles have been shown to exhibit advantageous protein release profiles and cellular integration, the same co ncerns that apply for PLGA would be present when using PCL. Scaffolds do not allow for the minimally invasive deployment that is desired and the particles conjugated with bFGF might affect non target tissues. All of the discussed concerns led us to ch oose a different backbone for this application which is addressed in the next section. Polymeric Backbone Considerations The decision for utilizing poly (serinol hexamethylene urethane) (PSHU) as the backbone polymer was based on the following informa tion: it has been shown to exhibit good biocompatibility, its primary amine groups lend themselves to modification in regard to further polymer, drug, or peptide conjugations, and the components used to create PSHU are nontoxic and commonly used in other b iomedical applications 37; 46 The chemistries involved with primary amine conjugation are well established mechanisms throughout the organic chemistry field. Specif ically relevant to this study is the conjugatio n of sulfonate groups by the ring opening reaction of 1,3 Propanesultone (PS) initiated by a nucleophilic attack by the dPSHU primary amine group. As a result, the polymer contains a negative charge from the sulfonate group. A key factor in


27 sustaining gr owth factor delivery depends on the increased interaction between the polymer and the protein through electrostatic interactions 47 Specific Aims For The Thesis The list of specific aims below is meant to be used as a guide through this work The purpose, results, and discussion of each tested aim are discussed in subsequent sections. The three specific aims: 1) Synthesize a reverse thermal gel that is capable of further conjugation through sulfonation of primary amine group chemistries 2) Characterize sulfona ted polymers 3) Obtain a release profile using a positively charged protein for the test.


28 CHAPTER II MATERIALS AND METHODS Materials Serinol, urea, hexamethylene diisocyante (HDI), Acetic Acid, Sodium Acetate, 1,3 Propane sultone, trifluoroacetic acid (TFA), triethylamine, azobis(4 cyanovaleric acid) (ACA), methanol, phenolphthalein and dimethyl sulfoxide (DMSO) were purchased fr om Sigma Aldrich Di tert butyl dicarbonate, ethyl acetate, N hydroxysuccinimide (NHS), N (3 dimethylaminopropyl) ethylc arbodiimide hydrochl oride (EDC), sodium carbonate and dimethyl sulfoxide d6 (DMSO d6) were purchased from Alfa Aesar Ethanol, hexane, diethyl ether and sodium hydroxide (NaOH) were purchased from Fisher Scientific Dimethylformamide (DMF) was obtained from BDH Chemicals N i sopropylacrylamide (NIPAAm) and sulfur trioxide pyridine were obtained from Acros Organics Chloroform d (CDCl 3 ) was obtained from Millipore Endothelial cell growth media kit (EGM 2 BulletKit) was obtained from Lonza Walkersville Inc. Recombinant human FGF 2 (146 aa) was obtained from R&D systems and cultrex basal membrane extract (without phenol red) was obtained from VWR International LLC Polymer Synthesis N Boc Serinol Synthesis N Boc serinol is an important component used in the synthesis of poly ( serinol hexamethylene urea) (PSHU). S erinol (21.5mmol) was dissolved in 20 mL of 200 proof ethanol in a round bottom flask A solution of 15 mL of 200 proof ethanol and Di tert butyl dicarbonate (26.0 mmol) was made in a 25 mL scintillation vial Whil e maintaining


29 the solutions at 4 C, the contents of the scintillation vial were added in a dropwise fashion into the round bottom flask whose cont e nts were constantly stirring a process that lasted for almost an hour. The combined solution was heated to 37 C for an hour while vigourously stirring. Ethanol was subsequently removed by rotary evaporation at 50 C and 40mbar vacuum. The remaining white solid was redissolved in a 1:1 mixture of ethyl acetate and hexane and brief heating at 50 C. Hexane was add ed dropwise until crystalline structures were formed. Additional hexane was added to ensure complete precipitation and left overnight in a 4 C environment. Vacuum filtration was employed to separate and dry N Boc serinol product from hexane. PSHU Synt hesis PSHU is the backbone of the polymer system used in this study. Urea ( 6 mmol) and N Boc Serinol ( 6 mmol) were lyophilized for 24 hours and then dissolved in 6 mL of anhydrous DMF under nitrogenous atmosphere in a round bottom flask Once fully disso lved, HDI (12 mmol) was added dropwise to the flask. The contents were left gently stirring for seven days at 90 C under nitrogenous atmosphere. At the end of the reaction time, the polymer was precipitated twice in cool diethyl ether and once in water. T he product was subsequently lyophilized and recovered for further conjugation. Deprotecting PSHU In order to conjugate other polymers/chemicals to PSHU it was deprotected by removing the tert Butyloxycarbonyl ( BOC ) groups and exposing the primary ami ne groups through the following process. PSHU (1.00 g) was dissolved in equal parts


30 methylene chloride (15 mL) and trifluoroacetic acid (15 mL) in a round bottom flask. The co mponents were left gently stirring with no heat and no lid at room temperature for 30 minutes. Rotary evaporation was used to remove solvents at 45 C and 10 mmbar. The remaining contents were dissolved in DMF (1 mL) and diethyl ether was added to the flask to precipitate the polymer out of solution. Excess ether was poured off wh ile taking care to keep polymer within the flask. After rotary evaporation was used to remove residual ether, the precipitation and evaporation was carried out once more. The deprotected PSHU (dPSHU) was fully dissolved in TFE, precipitated in diethyl et her, and s haken until the PSHU looked white and broken up. Excess ether was carefully poured out of the flask and then evaporated. At the end of this process, the polymer should look dry and white. PNIPAAm Synthesis PNIPAAm was synthesized by combini ng ACA (0.062g), NIPAAm (5.0 g) and 25 mL of methanol in a round bottom flask. N itrogen gas was bubbled through the solution for 30 minutes at room temperature The reaction proceeded at 68 C for three hours while stirring and under nitrogen gas (not bub bled). For purification, the solution was precipitated dropwise in 60 water and then dialyzed for 48 hours in 12,000 14,000 Da dialysis tubing. The polymer, while in the tubing, was in a 1L beaker filled with stirring water. T he solution was then lyoph ilized for two days.


31 PSHU PNIPAAm Conjugation ( RTG Synthesis) PNIPAAm was conjugated to 25% of the free amine groups on dPSHU. EDC/NHS chemistry was used to accomplish this conjugation. PNIPAAm was dissolved in anhydrous DMF (approximately 10ml DMF / 1 g PNIPAAm ) in a round bottom flask. EDC (3 times PNIPAAm moles) and NHS (3 times PNIPAAm moles) were added to the flask and left stirring under nitrogenous atmosphere for 24 hours. The appropriate amount of dPSHU was lyophilized for 30 minutes and dissolve d in anyhydrous DMF in a scintillation vial ( 10 mL DMF/1g dPSHU ). The dPSHU DMF solution was then added to the round bottom flask containing PNIPAAm while stirring. The resulting solution was capped (no vent) and left stirring for 24 hours to complete th e reaction. The polymer was precipitated three times in diethyl ether, and solvent was removed each time by rotary evaporation. The dried polymer was dissolved in milliQ water at 4 C and then purified by putting that solution into 12 14K dialysis tubing and left stirring for 48 hours in a beaker filled with 1L of milliQ water The solution in the tubing was then dried by the lyophilizer for 48 hours. SRTG 1 9 Synthesis The following protocol is based on the Wu et al sulfonating protocol 48 PS and t BuOK were dissolved in anhydrous D MF (3 mL) at 50 C under nitrogenous atmosphere. In a scintillation vial, RTG was di ssolved in anhydrous DMF ( 10ml DMF/1 g RTG ) When the contents of the round bottom flask were sufficiently dissolved the RTG/DMF solution was added slowly to the round bott om flask while stirring. The reaction proceeded for 48 hours at 60 C under nitrogenous atmosphere. After the contents cooled


32 down to ambient temperature, the polymer was precipitated into cold diethyl ether ; the solvents were then removed by rotary evapo ration. Subesequently, the dried polymer was dissolved in milliQ water at 4 C and then dialyzed (12 14K tubing) against 1L of milliQ water for 48 hours. The process was officially finished after the solution in the dialysis tubing was removed and lyophil ized for 48 hours. Polymer Characterization Experimental titration was used to define the molar mass of PNIPAAm per prepared batch PNIPAAm (0.005 g) was dissolved in a 10 mL dH 2 O in a 4 C environment overnight. Phenolphthalein was added to the PNIPA Am solution to provide color change. The amount of NaOH needed to reach a neutral pH of 7 when the solution would turn pink, was used to calculate the molar mass. Appendix B presents t he detailed procedure and equations The removal of the BOC groups on PSHU wa s confirmed by the proton nuclear magnetic resonance ( 1 H NMR) spectra produced on an INOVA 500 MHz instrument (Varian) with a 5 mm triple resonance proton detector. Samples were prepared either by dissolving the appropriate polymer in 100% DMSO d6 or in 90% chloroform and 10% DMSO d6 The resulting spectra was analyzed using ACD 1D NMR Processor software (Advanced Chemistry Development, Inc.). C arbon nuclear magnetic resonance (C NMR) was used on this same machine to locate sulfur carbon bonds on SRTG polymers. SRTG and RTG samples were evaluated using Fourier Transfo rm Infrared (FT IR). Samples were dissolved in tetrahydrofuran ( THF ) and placed on polyethylene windowed cards were then analyzed by a Nicolet 6700 (Thermo Fisher Scientific ).


33 Gelation tests were performed by dissolving an appropriate weight percent of polymer (RTG or SRTG) in milliQ water and then exposing the vial to 37 C and noting any color change or solution movement after inverting the vial. LCST measurements were made on a Cary UV Vis Spectrophotometer usi ng quartz cuvettes with 1 wt% solutions of polymer. Zeta potential measurements for SRTG and RTG samples were taken using a Malvern Zetasizer Nano Series in conjunction with Zetasizer Software (Malvern Instruments Ltd). BSA Release Study 20 wt% p olymer ic solutions were created using acetate buffer with and without bovine serum albumin (BSA) protein. Solutions were mixed and left to dissolve overnight at 4 C. 100 l gels were formed in 1ml syringes in a 37 C i ncub ator for 20 minutes and then added to acetate buffer (release solution) already warmed to 37 C. Pre forming the gels was based off of the Chen et al protocol 38 At predetermined timepoints, 1ml of release solution was removed from each sample and 1ml of fresh acetate buffer was added. Each 1ml release solution sample was analyzed under UV spectroscopy at 280nm and recorded. The absorbance values acquired could then be compared to the values created by the calibration curve, Appendix C shows this


34 CHAPTER III R ESULTS AND DISCUSSION, CONCLUSION, AND FUTURE WORK Results and Discussion In this work a series of sulfonated reverse thermal gel ( SRTG ) polymers w as synthesized and then characterized for the application of delivering positively charged growth factors. The backbone of the SRTG, dPSHU was copolymerized to PNIPAAm and then next sulfonated using different ratios of moles of amine groups to moles of PS The following subsections detail the results and decision criteria used for each subsequent test. PSHU and dPSHU NMR PSHU and dPSHU were analyzed by proton nuclear magnet ic resonance ( 1 H NMR). 1 H NMR utilizes the fact that when under the influence of an external magnetic field, proton species will either align themselves in the same or opposite direction of the applied magnetic field. The particular alignment, paired wi th the inherent electron density near each hydrogen atom determines where the chemical shift for hydrogen peaks will present themselves 49 The PSHU spectrum is shown in Figure III.1 and the presence and subsequent removal of the BOC group peak is shown in Figure III.2. dPSHU is the product of PSHU after it has fully undergone a deprotection reaction which consists of the rem oval of the BOC protecting groups The confirmation of peak labels


35 A was determined through the use of an NMR simulation package (Advanced Chemistry Development, Inc ). Figure III.1 1 H NMR of PSHU. Inset: The chemical structure of PSHU. The proton peak associated with B OC chemical structure shown in the to p left hand corner of the structure


36 Figure III 2 1 H NMR of PSHU (green ) and dPSHU ( red ). Inset: The chemical structure of dPSHU. The magnified spectra help to see the presence of the BOC group (denoted In Figure III. 2 abov e, the dPSHU spectrum, shown in red, clearly lacks a peak associated with hydrogens in a BOC group The absence of the BOC group is evidence that our polymer now contains primary amine groups that are ready for subsequent conjugation with other polymers. The particular monomer that originally gave this polymer the BOC group, N BOC serinol, was chosen so that in synthesizing PSHU, those nitrogen atoms would not unexpectedly interact with other monomers. However, the deprotection step is performed in a tim ely manner so that now the PSHU nitrogen atoms are reactive.


37 Polymer Synthesis SRTG 1 9 As shown through 1 H NMR, the primary amine groups a re available for further conjug ation. The polymer that gives the entire system its thermal properties, PNIPAAm was conjugated to 25% of the available primary amine groups the remaining primary amine groups were left for the attachment of sulfonate groups. The overall PSHU synthesis, deprotection, and PNIPAAm conjugation are presented in Appendix D. Figure III. 3 below details the sulfonation of R TG The potassium on the tert butoxide hydroxyl group dissociates from the oxygen and that same oxygen compels the hydrogen to leave the primary amine group on dPSHU. From here, the now negatively charged nitrogen is able to carry out a nucleophilic attack on the carbon ring, specifically on the carbon nearest the oxygen. The electrons rearrange themselves so that the ring opens up and the polymer is left with a negative charge on the oxygen in the sulfonate group. The positively charged potassium in solution will be electrostatically attracted to the negatively charged oxygen in the sulfonate group but theor etically this interaction should be transient and not interfere with the overall negative charge.


38 Figure III. 3 Theoretical synthesis of sulfonated RTG (SRTG 1 9 ). As shown in F igure III.3 one sulfonate group is covalently attached to a nitrogen atom on dPSHU. Because there is still a nother hydrogen associated with the nit rogen this study investigated the possibility of using different molar ratios of mole amines to mole 1,3 Propanesultone (PS), thought of as NH:PS to conjugate more sulfonate groups Table III.1 describes the varying conditions with which nine SRTGs were created.


39 Table III.1 SRTG 1 9 Synthesis Conditions Polymer Name NH 2 : PS ratio NH 2 : t BuOK ratio Time/Temp SRTG 1 1:1 1:1 24 hr/60C SRTG 2 1:2 1:2 24 hr/60C SRTG 3 1:3 1:3 24 hr/60C SRTG 4 1:5 1:5 24 hr/60C SRTG 5 1:1 1:0. 1 24 hr/60C SRTG 6 1:0. 5 1:0. 1 48 hr/60C SRTG 7 1:10 1:0. 1 48 hr/60C SRTG 8 1:1 1:0. 1 48 hr/60C SRTG 9 1:2 1:0. 1 48 hr/60C The actual progression of this SRTG series began by synthesizing SRTG 1 4 a nd then realizing that a change was needed While the SRTG 1 4 polymers were reacting, the solutions turned from transparent to cloudy, a n indication of crosslinking. The design of our polymer is predicated on it being linear so even though the reactions were completed, the polymers subsequently purified, and gelation tests performed on all polymers SRTG 1 4 specifically, were not characterized by other techniques or used in the release study. The appropriate change that was made for future SRTG reactions was the amount of potassium tert butoxide being used. The ratio of 1:0.1 denotes 10% of the molar amines available for conjugation 10% represents a catalytic amount of initiator. For the synthesis of SRTGs using catalytic amounts of potassium ter butoxide, the reacting


4 0 solution stayed transparent. The crosslinking wi th high amount s of t BuOK could be due to the over activation of amines, leading to more nucleophilic attacks on unexpected carb ons of the po lymer chain itself. Likewise, crosslinking could occur because the existing negatively charged oxygens after pota ssium dissociation, were capable of performing nucleophilic attacks on carbons along the polymer backbone because the available hydrogens attached to amines had potentially been displaced. Another change to note was the amount of reaction time for SRTG 6 9. The amount of reaction time was doubled to more closely match the synthesis conditions 48 SRTG 5 was allowed to proceed for 24 hours so that a comparison could be made between SRTG 5 and SRTG 8 since their conditions are the same except for the reaction time. The following subsection will cover the results of different techniques used to elucidate the structure and behavior of these polymers Gelation Test for SRTG 1 9 This gelation test used only 10 wt% and 20 wt% polymeric solutions. All 10 wt% so lutions failed to form a stable gel (results not shown) This was confirmed by exposing the vial containing the polymeric solution to 37 C for 1 minute and then inverting the vial to see if the sol ution was in sol or gel state remaining in the sol state t ranslates to not forming a stable gel. At 20 wt% solution, however, all polymeric solutions resulted in stable gels. The information from Table III.1 and the results from this gelation test were combined to form Table III.2 below.


41 Table III. 2 SRTG 1 9 Synthesis Conditions and Gelation Test Results Polymer Name NH 2 : PS ratio NH 2 : t BuOK ratio Time/Temp Form Stable Gel? Gel Shrinkage? SRTG 1 1:1 1:1 24 hr/60C Yes Yes SRTG 2 1:2 1:2 24 hr/60C Yes Yes SRTG 3 1:3 1:3 24 hr/60C Yes Yes SRTG 4 1:5 1:5 24 hr/60C Yes Yes SRTG 5 1:1 1:0.1 24 hr/60C Yes Yes SRTG 6 1:0.05 1:0.1 48 hr/60C Yes No SRTG 7 1:10 1:0.1 48 hr/60C Yes No SRTG 8 1:1 1:0.1 48 hr/60C Yes No SRTG 9 1:2 1:0.1 48 hr/60C Yes No Comp ared to Table III.1, Table III. 2 has an addi tional column for confirmation of a stable gel formed, as well as a column noting if the gel shr a nk after stabilization. PNIPAAm has the ability to shrink in size if it expels a portion of water after formin g a stable gel. This phenomenon can be seen bel ow in Figure III. 4.


42 Fi Figure III. 4 Example of gel shrinkage. The gel on the left has not shrunk (SRTG 9) whereas the gel on the right represents a gel that did shrink (SRTG 3). Upon first analysis of Table III.2, every SRTG that was considered to be crosslinked (SR TG 1 4) had shrunk after gelation. Crosslinking due to high amounts of potassium tert butoxide however, cannot be the only explanation for gel shrinkage because SRTG 5 used a catalytic amount of t BuOK and it also shra nk. The factor that separate s the shrunken gel s from the non shrunken gels is the reaction time condition. The polymers did not shrink w hen sulfona tion was carried out for 48 hrs instead of 24 hrs It could be speculated that a longer reaction time produces a more uni form polymer matrix that can withhold water much easier than a polymer produced under a shorter reaction time with a potential varied structure. Furthermore, sulfonate groups are hydrophilic. In theory, the addition of more hydrophilic features to this p olymeric system could encourage the gel to retain more water. This is in accordance with a polymer having more domina n t hydrophibic features wanting to expel more water (no addition of sulfonate groups), whereas a polymer ha s additional hydrophilic featur es wanting to expel comparatively less water (addition of sulfonate groups).


43 Zeta Potential Results SRTG 5 9 Zeta potential was employed to detect a surface charge o nly on the polymeric solutions SRTG 5 9 because SRTG1 4 were removed from further test ing due to suspected crosslinking Zeta potential measures the electric charge, typically recorded in millivolts, devolping as a result of a charged surface contacting a liquid phase 50 An electric potential develops between the fixed layer and the mobile layer, shown in Figure III. 5 A limitation of zeta potential includes the fact that it cannot be used to quantify available functional groups so this experiment was only used to de tect charge differences in the polymers. Figure III. 5 Illustration of zeta potential charge location 50 Zeta potential measurements were performed on SRTG 5 9 at 2.5 wt% solution s. The reason behind such a low concentration of polymer is so that the polymeric solution is not overly viscous. As mentioned previously, the surface charge is measured by the difference in charge of the polymer versus the liquid (water), so the lower t he weight percent, theoretically, the more clear the reading w ill be due to sufficient liquid to polymer contact. Figure III.6 is a graph presenting the zeta potential results


44 Figure III.6 Zeta potential measurements for SRTG 5 9. Error bars denote st andard error for each polymer type. The values for the graph produced in Figure III.6 were taken from the zeta potential repots that Zetasizer software provided after each sample run. Outliers were removed using the Thompson Tau Technique 51 No decisions for further testing were made based on the zeta potential results. Speculation dictates that the chains of PNIPAAm conjugated to dPSHU are so large that they are able to block an accurate charge reading from the zeta potential equipment. Furthermore, PNIPAAm allows the polymer to be soluble in water. This feature would decrease the accuracy of a charge derived from a material layer in contact with a solvent layer becaus e the polymer is now solub l e in water. Despite no decisions being made based on zeta potential measurements, SRTG 6 and SRTG 7 were not considered for further characterization or experiments at this point in the study SRTG 6 had a molar ratio of 1:0.5 amine groups to PS. This condition is


45 not conducive to our overall goal of creating multiple SRTG polymers because our theory is that the more sulfonate groups we have, the more interaction there is with a cationic growth factor and the better the releas e profile of said growth factor. Conversely, SRTG 7 has the potential to render too many sulfonate groups attached to the primary amine groups. Nitrogen has the capability to make four overall bonds, three with sulfonate groups and one to the polymer bac kbone. In this case, nitrogen would possess a positive charge and the addition of this charge could negate the effects of a negative charge on the oxygen l ocated in the sulfonate group. As a result, SRTG 6 has the potential to give us too few sulfonate g roups, and SRTG 7 has the potential to gives us too many sulfonate groups so they were removed from the study. Fourier Transfrom Infrared Spectroscopy (FTIR) RTG, SRTG 5, 8, and 9 FTIR is used as a chemical structure characterization method. An infrare d beam i s directed towards a sample t he subsequent bond stretching, bending, or contracting result s in specific infrared radiation absorbance and is presented as a line spectrum based on absorbance or transmittance versus wave number 50 In this study, FTIR was used to determine if the sulfonate group is present on the polymer. Specifically, a bond for this confirmation would be a sulfur (single) bond to oxygen FTIR was performed on RTG to provide a baseline spectrum with which to compare the sulfonated polymers, SRTG 5, 8, and 9 Peaks appea ring in the sulfonated polymer spectr a would represent changes in bonds due to the sulfonation process.


46 Figure III.7 FTIR results for RTG, SRTG 5, SRTG 8, and SRTG 9. The orange rectangle denotes the location of an S O bond peak. Figure III. 7 highlights a peak area between 1050 1025 cm 1 that includes a peak present in all of the SRTG polymers and absent in RTG. At this wavenumber, t he small peak corresponds to a sulfur (single) bond to oxygen. The existence of this peak for each sulfonated polymer is sufficient evidence to move forward with these three SRTGs for the protein release test. Appendix E shows t he full spectra with all f our polymers LCST Measurement RTG, SRTG 5, 8, and 9 LCST measurements were investigated through material absorption on UV Spectrometry as the material temperatu re was raised to determine if the polymers, post


47 sulfonation, sustained their gelation prope rties The presence of gelation activity is indicated by a visible difference in a mostly clear solution transforming to an increasingly opaque solution (as temperature increases). For this reason, the transmittance should go down as the polymer begins t o gel The rapid decrease in transmittance at approximately 32C indicates that the polymeric solution is making the transition from sol to gel state. As seen in Figure III.8 all of the polymers SRTG 5, SRTG 8, SRTG 9, PNIPAAm, and RTG exhibit the same LCST. Figure III. 8 LCST with 1 wt% solutions. The important information to be derived from Figure III.8 is that all of the materials are making the same transition from sol to gel at the same temperature as PNIPAAm and the shape of the line associate d with that transition is similar. Note:


48 differences in starting or ending transmittances of the materials are inconsequential. Different quartz cuvettes were used for each material and even though the measurements were all zeroed to water, specific mea surements can vary from cuvette to cuvette. Solutions made up of 1 wt% polymer were used so that the polymer system would not gel during the test. It is known that reverse thermal gels synthesized with PNIPAAm can expel the water trapped within the po lymeric system, therefore leading to phase separation. This phenomenon would have pointed to conflicting measurements because the spectrophotometer could have been measuring uniform polymer solutions or only the solvent, water in this case, which would g ive a higher transmittance than expected. BSA Release Study RTG, SRTG 5, 8, and 9 BSA is a model protein that is commonly used in polymer release studies 52 A sodium acetate buffer causes the BSA to be positively charged because the isoelectric point of BSA dictates that at pH of 4.5, the BSA will exhi bit cationic behavior Because of the nature of the polymeric system under investigation for this study, it is imperative to have a positively charged protein to see the interaction with the negatively charged sulfonate groups on the polymer. In theory, with more sulfonate groups, the polymer should have more/stronger electrostatic interactions and release the BSA slower than the standard RTG system.


49 Figure III. 9 BSA release profiles for RTG, SRTG 5, SRTG 8, and SRTG 9. As stated previously, the theory concerning the release profile is that with more sulfonate groups there would be more polymer protein interaction, which would result in a lower cumulative release. This trend does exist in Figure III.9 between the three SRTG polymers. Although t he exact percentages of sulfonate groups were not quantified in this work, the reaction conditions would ideally produce increasing amounts of sulfonate groups in the order of SRTG 5, SRTG 8 and SRTG 9, respectively. The unexpected result derived from F igure III. 9 is that RTG performed similarly to SRTG 9, whereas, according to our theory, all of the sulfonated polymers should out perform RTG. Since RTG has no sulfonate groups, we think the viscos ity is changing after sulfonation and that fact could ex plain why the release profiles are as such.


50 Comparitively, a sulfonated reverse thermal gel would be adding hydrophilicity to the polymer due to the sulfonate groups. Because of this, the polymer is more likely to interact with water, decrease the viscos ity, and lead to a higher protein release amount due to the increased water interactions. Along the same lines, RTG would have a higher viscosity, less interaction with water, and higher amount of protein kept within its gel. Appendix C presents t he cal ibration curve with which the results from the release test were c alculated Conclusion The purpose of this work was to synthesize a reverse thermal gel that has the capability to be further conjugated with sulfonate groups, investigate the characteris tics of the sulfonated polymer, and determine its release profile using a model protein. 1 H NMR provided evidence that protecting BOC groups on our polymer backbone, PSHU, were successfully removed through a deprotection reaction intended to produce p rimary amine groups on dPSHU. These primary amine groups are available for further manipulation. PNIPAAm, the material giving the polymer its gelation properties, was successfully conjugated to dPSHU through means of amine group chemistry as established by successfully gelling polymers through a gelation test Furthermore, sulfonate groups were also conjugated to dPSHU through amine group chemistry A series of sulfonated polymers, SRTG 1 9 were synthesized based on varying molar ratio s between amine g roups and PS. All SRTG 1 9 20 wt% solutions made stable gels but only SRTG 6 9 did not shrink during the test possibly due to additional hydrophilic features from the sulfonate groups that decreas e the tendency to expel water. SRTG 1 4 were removed from further testing because of suspected crosslinking.


51 Zeta potential measurements for SRTG 5 9 were shown but no decisions on how to continue with research were made based on these results. PNIPAAm is suspected of interfering with the measurements, both because of its size and its hydrophilic features that allow the polymer to be soluble in water. At this point, SRTG 6 and SRTG 7 were removed from this study because their theoretical synthesis conditions were not desireable. Moving forward, only SRTG 5, SRTG 8, and SRTG 9 were used for FTIR, LCST measurements, and the release test. FTIR spectr a revealed that in comparison to RTG, the sulfonated polymers, SRTG 5, 8, and 9 all exhibited a peak correlating to a sulfur to oxygen single bond in the region of 1050 1025 cm 1 range. As a result, each of these polymers was considered for further testing. The LCST measurements confirmed that RTG, SRTG 5, SRTG 8, and SRTG 9 all retained the same sol to gel transition that PNIPAAm exhibits (~32 C). The shape o f the transition and the transition temperature, were the used to reach this conclusion. The results of the BSA release test show that a trend exists between the theoretical RTG performed as well as SRTG 9 due to suspected changes in viscosity after sulfonation. With this in mind, SRTG 9 still possesses the desired sulfonate groups that will provide protein protection in a biologically activ e environment within the body, and it still presents itself as a desireable candidate for cationic growth factor release. The results presented herein are promising benchmarks for a successful cationic growth factor delivery vehicle, but further investigation should be performed to truly g auge their potential in this medical application.


52 Future Work Further investigation of the sulfonated polymers, SRTG 5, 8, and 9 is imperative to truly elucidate the behavior of the synthesized materials. The three main categories of future work inclu de more polymer characterization a tube formation assay, and a bFGF release study. Polymer Characterization Carbon nuclear magnetic resonance ( 13 C NMR) should be used to s how the existence of the carbon to sulfur single bond that would be present after s ulfonation. This idea stems from the results that Sangaj et al acquired after confirming their own sulfonation process using 13 C NMR spectrums 10 Sulfonated dPSHU alone should be investigated due to the fact that PNIPAAm will eclipse the spectrum and not allow this carbon sulfur bond to be seen, even if it is present. The FTIR results in this work gave enough evidence to believe that sulfonate groups were conjugated to dPSHU PNIPAAm, but presence of a C S bond would further confirm these beliefs. Elemental a nalysis should be employed to determine t he percentage of sulfur content within the polymers. The molar ratio conditions for the series of sulfonated reverse thermal gels were developed with the expectation that a 1:2 ratio of NH 2 : PS would give a higher amount of sulfonate groups versus a 1:1 r atio of NH 2 :PS. Naturally, more sulfonate groups would provide more sulfur content to the polymer. By this relationship, the amount of sulfur content could be used to confirm or deny the motivation for varying the molar ratio of amine groups to PS. V iscosity differences from RTG to SRTG were used to explain the results from the BSA release study. For this reason, it would be informative to use a viscometer and


53 actually measure the viscosity of the polymeric solutions to determine if this is in fact a plausible explanation as to why RTG and SRTG 9 perform similarly in the release test. Tube Formation Assay Tube formation assay is a powerful in vitro method to gauge what conditions promote or inhibit angiogenesis 53 Human umbilical vein endothelial cells (HUVECs) would be used for this assay because they not only have the ability to form tubes that represent potential vessel growth in vivo, but the y also are the most commonly used endothelial cells for in vitro assays concerning endothelial cell lines 53 According to the typical assay setup, HUVECs would be see ded on basement membrane extract (BME) which forms a gel at 37C and mimicks the extracellular matrix conditions found naturally in the body. Three types of media conditions would need to be used for this test: basal media, minimal media, and basal media plus gel Basal media wells would consist of BME, HUVECs, and media without any growth factors. Minimal media would consist of BME, HUVECs, and media with only bFGF (the same amount loaded in the gel). Basal media plus g el would consist of BME, HUVECs and media covering a gel loaded with bFGF that is located on the side of the well. This assay setup would help show what influences the gel has on bFGF bioactivity, as well as the degree of tube formation that exists when a polymer is introduced into the experimental system. Release Test Using bFGF A release test using FGF 2 should be performed. FGF 2 is positively charged in water; therefore, an acetate buffer would not be used. BSA is a convenient and useful


54 model protein for release tests, but it would be worthwhile to see how or if the release profile would change if water was used as the release sol ution instead of acetate buffer and if the specific interactions of sulfonate groups and FGF 2 proteins are that much stronger than sulfonate groups a nd BSA proteins. FGF 2 is also considerably smaller than BSA (~20 kDa versus ~60 kDa) ; it would be in formative to see if the size of the prote in changes the release profile from the polymer. The general release test setup would be s imilar to the release test performed in this work, except for the factors to which I alluded FGF 2 would be used in place of BSA and water would be employed as the release solution and solvent for dissolving polymer instead of acetate buffer. Furthermore, an enzyme linked im munosorbent assay (ELISA) would be used in place of UV Spectroscopy to calculate the presence of protein. At the wavelength that was used for the release test in this work, 280 nm, the machine is able to detect small amounts of polymer in the release solu tion. For this reason, a set of samples with just polymer gels dissolved in acetate buffer were used to create a baseline number, which was then subtracted from the value acquired by the polymer plus protein gel sample. An ELISA employs an anti bFGF anti body that is attached to a well plate. When the release solution is added to the well, the bFGF can bind to the plate through antibody interaction Subsequently, a detection antibody is added, which can form a complex with the bFGF attached to the origin al antibody. An enzymatic substrate can then be added that a cts upon the protein complex in the well and produces a visible signal that can be read through a plate reader. In this format, the bFGF is specifically detected and polymer interaction would b e cancelled out.


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61 APPENDI X A ACETATE BUFFER MATERIALS: Sodium acetate (Sigma) Acetic Acid Glacial (reagent grade from Sigma) milliQ water pH meter PROCEDURE: 1. Use the equation C 1 V 1 =C 2 V 2 to determine the amount of acetic acid needed to d ilute the stock solution to 0. 0 1M with milliQ wat er (keep in mind the total amount of acetate buffer you desire will be V 2 ) 2. Prepare 0.01M sodium acetate solution with milliQ water 3. U se the Henderson Hasselbalch equation (below) to determine the amount of 0.01M sodium acetate solution that should be added to the solution created in step number 1 4. Add appropriate amount of sodium acetate to solution and mix well 5. Clean pH meter with ethanol and then thoroughly clean pH meter by submerging it into milliQ water multiple times 6. Place pH meter in the acetate buffer solution to check that the pH is 4.5 7. Adjust the solution accordingly Henderson Hasselbalch equation where [A 1 ] denotes the molarity of the acid and [HA] denotes the molarity of the base:


62 APPENDI X B PNIPAA M MOLECULAR WEIGHT TITRATION MATERIALS: Sodium hydroxide (NaOH) (0.01N) ( Fisher Scientific ) milliQ water P henolphthalein (Sigma Aldrich) Stir plate Stir bar PROCEDURE: 1. Dissolve small amount of PNIPAAm (~6 mg) with milliQ water (10 ml) in the cold room (4 C) in a scinti llation vial 2. Add phenolphthalein (10 l) and stir bar to solution 3. Place vial on stir plate and mix gently 4. Add 10 l of NaOH and note any color change 5. Keep track of how much NaOH has been added and repeat Step 4 until the solution turns to a light pink shade 6. Calculate the molecular weight as s hown below


63 APPENDI X C BSA CALIBRATION CURVE MATERIALS: BSA protein (Sigma Aldrich) Polymer Acetate Buffer Quartz Cuvette UV Spectrophotometer PROCEDURE: 1. Make a series of standard concentrations with BSA and acetate buf fer (0.5mg/ml 0.1mg/ml) 2. Use 0mg/ml in quartz cuvette to as a standard at 280nm with the spectrophotometer 3. Analyze each standard concentration at this same wavelength and plot the values 4. Use a linear trendline to formulate an equation describing the valu es and display the R 2 value y = 0.6266x 0.0054 R = 0.997 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0 0.1 0.2 0.3 0.4 0.5 0.6 Absorbance (Au) Concentration (mg/ml)




65 APPENDI X E FTIR FOR RTG, SRTG 5, SRTG 8, AND SRTG 9 Note: The clump of peaks on RTG in between 2500 2000 cm 1 is representative o f carbon dioxide. The machine uses liquid nitrogen to purge the container that holds the sample but occasionally, the first sample run can have this recognizable and familiar result. Also, in order to see the difference between these four peaks, the grap h has to be magnified from wavenumber 1200 750 cm 1 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Wavenumber (cm^ 1) RTG SRTG 9 SRTG 8 SRTG 5