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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-12-31.

Permanent Link: http://ufdc.ufl.edu/UFE0021756/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-12-31.
Physical Description: Book
Language: english
Creator: Odukale, Anika
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Anika Odukale.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Batich, Christopher D.
Electronic Access: INACCESSIBLE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021756:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021756/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-12-31.
Physical Description: Book
Language: english
Creator: Odukale, Anika
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Anika Odukale.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Batich, Christopher D.
Electronic Access: INACCESSIBLE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021756:00001


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SURFACE FUNCTIONALIZED AND MOLECU LARLY IMPRINTED POLYMERS FOR PHOSPHATE REMOVAL IN PATIENTS WITH END STAGE RENAL DISEASE By ANIKA ASSATA ODUKALE EDWARDS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Anika Assata Odukale Edwards 2

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To those who kept me along the way. 3

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ACKNOWLEDGMENTS Realizing that no one makes this journey alone is one of the gr eatest gifts that I have been given. Recognizing those who will never know how much of an impact that they have had on me academically, and more-so personally, is th e best way in which I can express my lifelong gratitude. I thank my advisor, Dr. Christopher Batich, for the opportunity to work on a project that has allowed me to learn how to creatively an alyze science based problems from a unique and nontraditional point of view. My greatest gratitu de goes to my committee: Dr. E. Goldberg, Dr. H. El-Shall, Dr. L. S. Hollid ay, and Dr. W. Sigmund, for their patience and understanding. I am extremely thankful to Dr. Hassan El-S hall, Dr. Eugene Gol dberg, and Dr. Anne Donnelly for their support and open doors. They will probably never know how much their advice and encouragement has meant to me. I am appreciative to the Batich group, especi ally Tara Washington, Jompo, Taili, and to members of the Goldberg group. Very special th anks to Jennifer Wrighton for always making sure that all of the is were dotted and ts crossed. I would li ke to acknowledge the facility directors and trainers at MA IC and the ERC for use of th eir equipment and expertise. I am grateful for the support from Martha, Do ris, and Jennifer, in our Graduate Office. You have always shared in my successwe did it! And to Joni Nattiel, for always making sure that I received a paycheck. I would also like to express gratit ude to the Department Chairman, Dr. Kevin Jones, who allowed me the opportunity to TA many different cla sses in an effort to provide various experiences that would support my goal of teaching. I would like to thank my family, who have b een my always cheerleaders. My mother, Jabari, always told me that I could do anything growing up, and even ventured on the internet to learn about polymers, so that sh e could tell everyone what her da ughter studied. To my brothers 4

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Kao, whose humor could shed light on the bright side of anything, Go Vikes!, Baba, J-Rock, and Femi, for being proud of their Sis. My greatest appreciation goes to my husband, best friend and confidant, laughing partner, motivator, reality checker and constant compani on, Jesse. I simply could not have done this without him. He will never know how much I adore him, or how grateful I am for everything that he has done for me. In the immortal words of Piglet; Well be friends forever, wont we, Pooh asked Piglet. Even longe r, Pooh answered. I give thanks to you for my precious son Akil, and for beloved my step-son and Little Buddy Tyler, and for the blessed family that we are. I would like to acknowledge a nd dedicate this to Eddie Ll oyd, Jr., Constance Fabunmi, Mark Maykoski, and Brenda Sm ith, who are all greatly missed. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES .........................................................................................................................10 LIST OF FIGURES .......................................................................................................................11 LIST OF ABBREVIATIONS ........................................................................................................15 ABSTRACT ...................................................................................................................................16 CHAPTER 1 INTRODUCTION................................................................................................................. .18 Rationale .................................................................................................................................18 Specific Aims..........................................................................................................................19 Specific Aim 1: Develop and Characteri ze Surface Functionalized Polymers for Phosphate Removal in Aqueous Systems. ...................................................................19 Specific Aim 2: Develop and Characteri ze Molecularly Imprinted Polymers for Phosphate Removal in Aqueous Systems. ...................................................................20 Specific Aim 3: Determine the Efficacy of Phosphate Uptake with these Functionalized Polymers In-Vitro ................................................................................21 2 BACKGROUND................................................................................................................... .22 Introduction .............................................................................................................................22 Emulsions ...............................................................................................................................22 Phosphate Poisoning and Chronic Renal Failure ....................................................................23 Surface Functionalization of Polymeric Microparticles .........................................................25 Molecular Imprinting ..............................................................................................................25 Covalent vs. Noncovalent Synthesis ...............................................................................27 Radiation Polymerization ................................................................................................29 Previous Related Work ....................................................................................................30 3 RENAGEL CHARACTERISTICS........................................................................................33 Understanding Renagel ........................................................................................................33 Structural Properties of Renagel ..........................................................................................33 Renagel Swelling Behavioral Studies ..................................................................................34 Experimental Methods ............................................................................................................34 Effect of pH on Phosphate Uptake .........................................................................................35 Phosphate Binding Mechanism ..............................................................................................37 Molecular Dynamics .......................................................................................................39 Vacuum system ...............................................................................................................39 6

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Solvated System ..............................................................................................................39 Monte Carlo .....................................................................................................................40 Determining the Shell Volume .................................................................................40 Generating Random Numbers ..................................................................................41 Determining the Ratio ..............................................................................................41 Determining the Volume of the Molecule ................................................................41 Results .............................................................................................................................42 Conclusions .....................................................................................................................47 4 POLYMERIZATION PARAMETERS..................................................................................50 Experimental Design and Material Selection .........................................................................50 Synthesis Parameters ..............................................................................................................51 Template Molecule ..........................................................................................................51 Functional Monomer .......................................................................................................53 Initiator ............................................................................................................................56 Crosslinking Agent ..........................................................................................................57 Genipin crosslinked microspheres ...........................................................................58 Glutaraldehyde crosslinked microspheres ................................................................59 Dextran .....................................................................................................................60 D,L-glyceraldehyde ..................................................................................................61 Porogen ............................................................................................................................63 Determine the Efficacy of Phosphate Uptake with the Polymers in-vitro ..............................64 Assessment of the Reaction Parameters ..........................................................................64 Recovery ..........................................................................................................................64 In-Vitro Analysis of Phosphate Uptake in the Functionalized Polymer.........................64 Instrumentation .......................................................................................................................65 ICP...................................................................................................................................65 UV/VIS ............................................................................................................................65 Phosphate meter ...............................................................................................................65 Characterization of bulk polymer ....................................................................................66 Scanning Electron Microscopy (SEM)....................................................................66 Infrared (IR).............................................................................................................66 5 GELATIN MICROSPHERES VIA EMULSIFICATION.....................................................67 Water in Oil Emulsions ..........................................................................................................67 Crosslinked Gelatin Microspheres ..........................................................................................69 Synthesis of Gelatin Microspheres ..................................................................................69 Crosslinking Gelatin Mi crospheres with Genipin...........................................................70 Crosslinking Gelatin Micr ospheres with Glutaraldehyde...............................................70 Crosslinking Gelatin Micros pheres with additional agents .............................................71 Dextran .....................................................................................................................71 Glyceraldehyde .........................................................................................................72 7

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Gelatin Microsphere Processing .............................................................................................72 Removal of the Template ................................................................................................72 Base Wash Extractions ....................................................................................................72 Acid Wash Extractions ....................................................................................................73 Results .....................................................................................................................................74 Gelatin Inverse Emulsions ...............................................................................................74 Genipin Crosslinked Gelatin Polymerization ..................................................................79 Glutaraldehyde Crosslinked Gelatin ................................................................................81 Dextran Crosslinked Gelatin ...........................................................................................82 Glyceraldehyde Crosslinked Gelatin ...............................................................................83 Conclusions .....................................................................................................................83 6 MOLECULARLY IMPRINTED POLYMERS.....................................................................86 Microwave Fabricated Gelatin Micr ospheres as Phosphate Binding Agents .........................86 Crosslinked Gelatin Microsphe res via Microwave Radiation ................................................86 Microwave Treatment .............................................................................................................87 Results .....................................................................................................................................87 Gelatin Emulsion Microspheres ......................................................................................87 Genipin and Glutaraldehyde Crosslinked Microspheres .................................................88 Conclusions .....................................................................................................................92 7 GUANYLATED POLYMERS..............................................................................................94 Functionalized Gelatin Micr ospheres via Guanylation ..........................................................95 Imprinted Gelatin Microspheres ......................................................................................96 Room Temperature Guanylation ..............................................................................96 Heat Assisted Guanylation.......................................................................................97 Surface Guanylated Gelatin Microspheres ......................................................................97 Results .....................................................................................................................................97 Imprinted Polymers .........................................................................................................97 Glutaraldehyde................................................................................................................99 Surface Modified Crosslinked Polymers .......................................................................101 Genipin ..........................................................................................................................101 Glutaraldehyde..............................................................................................................101 Glyceraldehyde ..............................................................................................................103 Conclusions ...................................................................................................................104 8 DISCUSSION................................................................................................................... ....107 Synopsis ................................................................................................................................107 Conclusions ...........................................................................................................................112 Future Work ..........................................................................................................................115 Crosslinking Agent ........................................................................................................115 Substrate Imprinting ......................................................................................................116 Instrumentation ..............................................................................................................116 8

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Polymer Binding ............................................................................................................116 Enzyme Studies .............................................................................................................117 APPENDIX CALIBRATION OF A P HOSPHATE MEASURING APPARATUS..................118 Experimental Methods ..........................................................................................................118 Conclusions ...........................................................................................................................121 LIST OF REFERENCES .............................................................................................................122 BIOGRAPHICAL SKETCH .......................................................................................................129 9

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LIST OF TABLES Table page 3-1 Interatomic distance values (mean, sta ndard deviation, high, and low) for the NVT ensemble simulation in vacuum for four phosphate system with dielectric 1 debye. ........47 3-2 Interatomic distance values (mean, sta ndard deviation, high, and low) for the NVT ensemble with dielectric 78 debye to mimic a solvated system (H2O) .............................48 5-1 Phosphate uptake in gelatin crosslinked microspheres that have been either acid or base washed prior to trials. .................................................................................................84 6-1 Phosphate uptake in microwave irradiated gelatin crosslinked microspheres that have been either acid or base washed prior to trials. ..................................................................92 7-1 Phosphate uptake in surface functiona lized and molecularly imprinted gelatin crosslinked microspheres prepared by e ither emulsion or microwave irradiated. ...........105 A-1 Phosphate calibration values using the Hannah HI 93717 meter. ..................................119 A-2 Phosphate readings from the Hannah HI 93717 meter compared to those obtained from the Shimadzu UV/Vis. ............................................................................................120 10

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LIST OF FIGURES Figure page 1-1 Surface functionalized crosslinked gelati n microsphere interacting with phosphate molecules ...........................................................................................................................20 1-2 Formation of imprinted cavities for phospha te molecules in gelatin microsphere used to bind phosphate molecules ..............................................................................................20 2-1 Hormonal control loop for calcium metabolism ................................................................25 2-2 Concept of molecular imprinting of polymers ...................................................................27 2-3 Simplified schematics of covalent and noncovalent imprinting procedures.....................28 2-4 Depiction of an HPLC column packed w ith a MIP stationary phase used for chiral separations ..........................................................................................................................31 2-5 Nucleation of calcite at the polymer surface ......................................................................31 3-1 Chemical structure of sevelamer hydrochloride ................................................................34 3-2 Phosphate (P) uptake in Renagel.. ..................................................................................37 3-4 Observed swelling of dry particles ....................................................................................38 3-5 Van der Waals representation of the space filled model system with four phosphate ions bound to the polymer..................................................................................................43 3-6 Interatomic distance between Nitrogen (126) and Phosphorous (237) in the model system Renagel and four triphosphate ions, with an N-P separation adjustment of the system at about 480 ps. ................................................................................................44 3-7 Interatomic distance between Nitrogen (204) and Phosphorous (237) in the model system Renagel and four triphosphate ions ....................................................................45 3-8 The change of interatomic di stance between N(89) and P(237). .......................................45 3-9 The P(237)-P(216) interatomic se parations versus simulation time.. ................................46 4-1 Common polyprotic molecular st ructures of the phosphate ion. .......................................52 4-2 Titration curve for phosphoric acid. ...................................................................................52 43 Relative fractions of the various fo rms of phosphate at different pH levels. ....................53 4-4 Molecular structures of common amino acids found in-vivo .............................................55 11

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4-5 Structure of Gelatin ............................................................................................................56 4-6 The molecular structure of Genipin and Geniposide. ........................................................59 4-7 Crosslinked structures of geni pin crosslinked gelatin microspheres .................................60 4-8 The molecular structure of glutaraldehyde. .......................................................................60 4-9 Crosslinked structures of glutaral dehyde crosslinked gelatin microspheres .....................61 4-10 Molecular structure of dextran. ..........................................................................................62 4-11 Preparation of gelatine h ydrogels crosslinked with partially oxidized dextrans...............62 4-12 Molecular structure of D,L-glyceraldehyde. ......................................................................63 5-1 An inverse emulsion system. .............................................................................................67 5-2 Interfacial tension and em ulsion variations versus formulation (as HLD) at near unit water-to-oil-ratio. ...............................................................................................................69 53 Template removal from the active binding site. ................................................................73 5-4 SEM and corresponding EDS analysis of raw Type B (bovine) gelatin. ...........................74 5-5 SEM and corresponding EDS analysis of ge latin microspheres prepared via water in oil emulsion. .......................................................................................................................75 5-8 FTIR spectrum of unproce ssed Type B (bovine) gelatin. ..................................................77 5-9 FTIR spectrum of gelatin microspheres via water-in-oil emulsion. ..................................78 5-10 FTIR spectrum of gelatin microsphe res containing phosphate via water-in-oil emulsion. ............................................................................................................................79 5-11 Gelatin microspheres crosslinked with 15% genipin without phosphate: before and after phosphate testing, and with phosphate: before a nd after phosphate testing. ...........80 5-12 FTIR spectrum of genipin cr osslinked gelatin microspheres. ...........................................81 5-13 FTIR spectrum of genipin crosslinked gelatin microspheres containing phosphate. ........81 5-14 FTIR spectrum of glutaraldehyde crosslinked gelatin without phosphate, and with phosphate. ..........................................................................................................................82 6-1 SEM of gelatin crosslinked micros pheres via microwave processing without phosphate : before and after phosphate testing, and with phos phate: before and after phosphate testing. ...............................................................................................................89 12

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6-2 Protonated amine cage surrounding a phosphate ion. ........................................................90 6-3 Modeled system of the phosphate binding polymer. .........................................................91 6-4 FTIR spectrum of glutaraldehyde crosslinked gelatin, via microwave irradiation: without phosphate, and with phosphate. ............................................................................91 7-1 Synthetic route used to obtain a guanylated polymer. .......................................................95 7-2 Zwitterionic interaction of phosphate with a guanyl group. ..............................................95 7-3 SEM images of uncrosslinked gelatin, na tive, guanylated without phosphate, and guanylated with phosphate .................................................................................................98 7-4 Predicted proximity of 4 phosphate ions w ithin the polymer structure, and a guanine derivative self assembly into G-ribbo n, G-quartet, and G-quadruplex formed by stacking of G-quartets around a column of cations ...........................................................99 75 Glutaraldehyde crosslinke d gelatin microspheres prepared via microwave irradiation, room temperature surface gua nylation: after guanylation, after template extraction, and after phosphate upta ke testing. Without phos phate, and with phosphate ................100 7-6 Glutaraldehyde crosslinked gelatin microspheres prepared via microwave irradiation, heat initiated surface guanylation: after guanylation, after template extraction, and after phospha te uptake testing. Wit hout phosphate, and with phosphate. ........................................................................................................................100 7-7 Glutaraldehyde crosslinked gelatin microspheres prepared via emulsion polymerization, room temperature surf ace guanylation: after guanylation, after template extraction, and after phosphate upt ake testing. Without phosphate, and with phosphate .........................................................................................................................102 7-8 Glutaraldehyde crosslinked gelatin microspheres prepared via emulsion polymerization, heat initiated surface guanylation: after gua nylation and after template extraction. Without phosphate, and with phosphate. ........................................102 7-9 FTIR spectrum of surface guanylated ge latin crosslinked with glyceraldehyde. ............103 8-1 The synthetic pathway followed for the development of the gelatin microspheres. .......108 8-2 The synthetic pathway followed for the development of the molecularly imprinted gelatin microspheres. .......................................................................................................108 8-3 Phosphate uptake results for acid washed polymeric samples synthesized without and with the phosphate template ion. .....................................................................................110 8-4 Phosphate uptake results for base wash ed polymeric samples synthesized without and with the phosphate template ion. ...............................................................................110 13

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8-5 Phosphate uptake results for room te mperature guanylated polymeric samples synthesized without and with the phosphate template ion. ..............................................111 8-6 Phosphate uptake results for acid washed polymeric samples synthesized without and with the phosphate template ion. .....................................................................................111 8-7 Phosphate uptake in surface functionalized polymers using genipin as a crosslinking agent. ................................................................................................................................112 8-8 Phosphate uptake in surface functiona lized polymers using glutaraldehyde as a crosslinking agent. ...........................................................................................................113 A-1 Hannah HI 93717 handheld photometer. .........................................................................118 A-2 Actual vs. Theoretical concentratio ns for phosphate ion using the Hannah HI 93717 phosphate meter. ..............................................................................................................119 A-3 Calibration curve for the Hannah HI 93717 phosphate meter. .......................................120 A-4 Phosphate uptake in Renagel samples as measured by the Hannah HI 93717 phosphate meter. ..............................................................................................................121 14

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LIST OF ABBREVIATIONS m Micron Angstrom ATR Attenuated Total Reflectance Ca 2+ Calcium CSSR Cambridge Structure Search and Retrieval EDS Energy Dispersive Spectroscopy ESRD End Stage Renal fs Femto second FTIR Fourier Transform Infrared Spectroscopy HCl Hydrochloric Acid ICP Inductively Coupled Plasma meq Milli equivalent MIP Molecularly Imprinted Polymer MIT Molecular Imprinting Technology mmol Milli mole NaOH Sodium Hydroxide PO 4 3Phosphate ps Pico second SEM Scanning Electron Microscope T Half life temperature 15

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SURFACE FUNCTIONALIZED AND MOLE CULARLY IMPRINTED POLYMERS FOR PHOSPHATE REMOVAL IN PATIENTS WITH END STAGE RENAL DISEASE By Anika Odukale Edwards December 2008 Chair: Christopher D. Batich Major: Materials Science and Engineering Patients with end stage renal disease (E SRD) commonly use nonabsorbable synthetic polymers to sequester excess phosphat e on a large scale basis. Howeve r, the cost of these agents at 10-15 pills/day/patient, and approximately $1 USD/pill, makes them an alternative not affordable to the greater general public. Thus the need to develop a low cost therapeutic alternative exists. One way to achieve such a system is to design surface functionalized molecules with specific molecular targeting ab ilities that are capable of mimicking natural binding phenomena found in livi ng organisms. Thus, a technique for the synthesis of novel surface functionalized gelatin microparticles as oral phosphate binding agents was developed that employs the use of a modified general i nverse emulsion schema, or a microwave-assisted synthetic technique, biocompatible crosslinking ag ents, and guanylating agents. Furthermore, this unique template capturing phenomenon was maximized by creating highly stable molecularly imprinted polymers (MIPs) that possess selective bindi ng properties due to recognition sites within the polymer matrix. Highly crosslinked, su rface tailored gelatin microspheres, on the order of 10 200 m, were produced with high-affinity phosphate attracting functional groups at the polymer surface. Serum phosphate uptake was evaluated using the modified microspheres in an in-vitro milieu which simulated an in-vivo physiological 16

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environment. Characterization of functionalized polymers was performed using FTIR, SEM, and ICP analytical techniques. Pote ntially therapeutic polymers micros pheres for oral ingestion were thus realized with an effectiv e phosphate binding ability of 12 meq/g polymer. This research suggests the feasibility of this novel low cost technology for the therapy of reducing phosphate levels in patients with acute renal disorders. 17

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CHAPTER 1 INTRODUCTION Rationale This body of work explores selective phosphate binding polymers and their potential use in treating dialysis-related hyper phosphatemia. The ability to distinguish among molecules in solution is central to many areas of research and plays a vital role in t echnologies ranging from biotechnology to materials scien ce. Though there are many syntheti c approaches to address this issue, surface functionalized polymeric molecules were investigated as a vehicle for these studies due to their well establis hed synthetic routes and chemical understanding. Polymeric microspheres which are chemically ta ilored at the surface as a means to interact with specific molecules provide an elegant and e fficient approach to achieve targeted molecular recognition. Surface functi onalization of polymeric particles with partic ular molecular groups has quickly become a requirement in the treatment of polymer particles for biotechnological applications. The immobilization of a hydrophilic layer as well as the po ssibility of coating particles with specific ligands (e .g., saccharide moieties, lipids, pr oteins) provide very attractive possibilities. 1 Particles can be created by a variety of methods which allo w a vast array of microspheres to be developed with customized size, shape, and composition. In addition, a broad selection of functionalitie s can be integrated onto the particle interface due to the availability of a large amount of functional molecular species a nd flexibility of manufacturing tools. Molecular imprinting technique (MIT) is a rapidly growing a pplication that involves the design of molecularly imprinted polymers (MIPs) to produce recognition sites whose selectivity rivals that of natural antibodies. 2 Target molecules for recognition include hormones, 3 smallmolecule drugs, metal ions, 4 proteins, 5 nucleic and amino acids. 6 Therefore, MIPs potential for 18

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enhanced stability, lower cost, and selectivity at lower concentrations in comparison to their natural derivatives make them attractive materials. 7 The creation of such recognition sites is based on the fundamental characteristics of a functional imprinted po lymer such as: stiff structure, good accessibility of binding sites, reversible chemical binding, mechanical and thermal stability, and the ability to distinguish between molecules, even enantiomers. These properties can be controlled th rough the way in which the MIP is synthesized, thus production parameters are significant. A vast array of research has focused on the optimization of imprinted polymer synthesis conditions. Th e reaction variables studied incl ude the type and amount of print molecule, functional monomers, cross-linking agent, chemical initiator, porogen, and all other reaction conditions i nvolved in polymerization. 8 A good deal of interest has been directed towards the field of MIT because of the significant role that they play in surface modification and stability. 9 Extensive studies show that one way to successfully achieve stable forma tion of imprinted polymers is by means of noncovalent imprinting. 10-12 Specific Aims Specific Aim 1: Develop and Characterize Su rface Functionalized Polymers for Phosphate Removal in Aqueous Systems. The explicit goals of this research require a logical progression of microsphere synthesis with surface immobilized species that provide potential phosphate binding in relevant aqueous milieus. Gelatin microspheres were produced an d tested under various conditions for phosphate capturing ability. Assays were repeated with part icles that have been crosslinked with different biocompatible agents for any positive change in phosphate sequestration. Particles were then surface functionalized with a phosphate interac ting ligand (Figure 1-1), and uptake was again examined. 19

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Figure 1-1. Surface functionalized (w hite lines with pink ends) crosslinked gelatin microsphere (gold) interacting with p hosphate molecules (green) (adapted from Adobe Stock Photos). Specific Aim 2: Develop and Characterize Mo lecularly Imprinted Polymers for Phosphate Removal in Aqueous Systems. Results from Specific Aim 1 were used to de fine parameters necessary to synthesize insoluble phosphate-imprinted polymers. The ro le of each process parameter was determined, and a valid synthesis scheme was defined (similar to Figure 2). Figure 1-2. Formation of imprinted cavities for phos phate molecules in gelatin microsphere used to bind phosphate molecules (g reen). Magenta regions de note the rigid cavities, both within and on the surface of the gelatin sphere (gold), created by the imprinting process (adapted from Adobe Stock Photos). Surface at tached functional groups are represented by the white lines with pink ends. 20

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Specific Aim 3: Determine the Efficacy of Phosphate Uptake with these Functionalized Polymers In-Vitro Hyperparathyroidism, secondary to chronic ki dney disease, has etiol ogic factors such as hyperphosphatemia, calcium balance, and 1,25 dihydr oxyvitamin D deficiency as typical targets for intervention. 13 By examining the effect that these surface tailored insoluble polymer particles have on the binding specificity of phosphate, th eir potential influence on hyperphosphatemia can be determined. To prevent hyperphosphatemia, the phosphate serum level should not exceed 400-800 mg/dL, however the average pa tient intake is >1 g/day. 14 Thus, the microsphere must be capable of binding and removing these excess amounts of phosphate in-vivo without significant deleterious side effects, and subsequently excreted through the urinary tract and feces. In-vitro studies were conducted in solutions that mi mic a physiological environment, and phosphatebinding efficiency was determined on a meq/ g polym er base. Data from these experiments were used to investigate the therapeutic feasibility of this surface functionalized polymer system for patients with ESRD. 21

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CHAPTER 2 BACKGROUND Introduction Over the past 20 years there have been many advances in the development of MIPs, as their practical applications ha ve far-reaching appeal. These include non-covalent and covalent imprinting and catalysis using imprinted polymer s. Progression in the field has continued through the work of many researchers such as Wulff, Mosbach, and Shea. 15 Collectively, these groups have come up with a basic reaction sche me for developing highly selective, imprinted polymers. Extensive research has focused on the optimization of imprinted polymer synthesis conditions. 2 This scheme concentrated on describing how surface modified polymers were prepared for targeting phosphate ions, and examined their potential applications in chronic renal failure patients with elevated serum phosphate levels. Emulsions Traditionally, some of the most common me thods used to prepare surface modified polymeric materials have been emulsion technology, 16 bulk, 17 and precipitation polymerizations, 18 each with its own advantages and disadvantages. Bulk polymerizations are rather straight forward, however they require an aggressive grinding and sieving step prior to use, which can destroy the binding sites and redu ces reproducibility from batch-to-batch. With precipitation polymerization, there is more c ontrol over the final si ze and morphology of the resulting particle, which reduces the batch-to-batch variability, but they are restricted by slow kinetics and low yields. 19 The use of surface modified polymeric resins prepared using an emulsion method has been a long standing industry standard for producing microspheres due to th e simplicity of the process, the multiple guest binding sites created as a result of this surface functionalization 22

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technique, and elimination of the grinding and sieving process involved in other traditional techniques as a means to expose available bindi ng sites. The partic le size and morphology are reproducible from batch-to-batch, and the monomer : template ratio can be manipulated to obtain the maximum number of template binding sited per microsphere. 20 Microspheres in the range of 1 200 m will be targeted for development, Renagel reports a 23 m 21 average product particle size to bind phosphate most effectively. An inverse (wat er-in-oil) emulsion polymerization method was used to fabricate micr on sized particles for investigation. Since spherical microparticles can be produced with guest binding sites on the surface, and each particle has a significant surface area, phosphate-binding phenomena should be observed. 22 Phosphate Poisoning and Chronic Renal Failure The irregularities of uremic bone disease ar e usually referred to as renal osteodystrophy, which occur in patients with chronic renal fail ure. The two major types of bone disorders in renal osteodystrophy include: a low-turnover osteodystrophy charac terized by osteomalacia of adults and rickets of children (or renal ricket s), and a high-turnover os teodystrophy characterized by osteitis fibrosa and other bone changes of secondary hyperparathyroidism; osteopenia, and osteosclerosis. Bone is a dynamic organ, constantly remodeled under the actions of the basic multicellular unit, a complex of osteoclasts and osteoblasts. 23 Osteoclasts, the cells responsible for bone resorption, originate from haematopoietic stem cells. They are recruited at a leading edge, dissolving the mineral and matrix alike, forming the Howships lacunae. These cavities are then filled in by recruited osteoblasts, which are derived from local mesanchymal cells. However, patients with ESRD retain phosphorous, which disturbs the kidneys normal regulation cycle. This condition is highl y likely to occur in ESRD pa tients and effects about 90% (230,000 US patients a year) of all patients on dialysis. 23 23

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Serum Ca 2+ represents less than 1% of the total body calcium, however its concentration is extremely important to maintain regular cellular functions. The average person filters approximately 250 mmol of ionized calcium thro ugh the kidneys daily. Three major hormones help regulate serum Ca 2+ metabolism: parathyroid hormone (PTH), which is excreted from parathyroid glands found in the neck, 1,25-di hydroxyvitamin D, and calcitonin. Improper maintenance of calcium and phosphorous in the bl ood can cause the kidneys to fail. When PTH levels decrease, calcium absorption in the gastroin testinal tract decreases, phosphate in the renal tubule increases, and stimulates 1,25-dihydroxyv itamin D production is adversely affected (Figure 2-1). 14 This results in a decrease in serum Ca 2+ which triggers a Ca 2+ sensor that initiates the extraction of this mineral from the bones in an effort to raise the blood Ca 2+ levels back to a normal concentration. If this cycle continues for long periods of time, the decrease in the Ca 2+ in the bones causes them to weaken, 14 and also indirectly c ontrols the excretion of urinary phosphate. 24 Chronic renal failure also prevents the kidneys from making calcitrol, which prevents the body from absorbing dietary calcium into the blood and bones. 23 Regulating the amount of blood serum phosphate levels in patients through the oral administration of phosphate binding polymers, can reduced or avoi d the onset of hyperphosphatemia, which can lead to the regulation of a normal serum Ca 2+ PO 4 3equilibrium cycle. Current alternatives to treat hyperphosphatemia include aluminum, lanthanum, and calcium based binders, selected ion exchange resins, nonabsorbable synthetic polymers, and reduced dietary phosphate intake. 23 Significant drawbacks of the pr oducts include aluminum toxicity, hypercalcemia, and poor binding effi ciency. Thus, a need exists for affordable, improved insoluble phosphate binders that can be orally administered in appropriate dosage amounts, with reduced side effects. 24

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Figure 2-1. Hormonal control loop for calcium metabolism. 25 Surface Functionalization of Polymeric Microparticles Surface modified microspheres have been prove n to have great impact applications in regards to the synthesis of new and exciting biom aterials. Characteristically, microspheres are superior materials due to thei r various tunable properties. Their inherent high surface area provides an ideal platform for attaching specific s ite attachment of ligands which can be used in various systems in which targeted molecular recognition is paramount. Through this process, relatively inert microspheres can be altered to have precise interactions with other molecules. Molecular Imprinting The concept of using molecular imprinted polymers (MIPs) for chemical recognition actually originated from Linus Pa ulings template-and-cast theory. 26 In the early 1940s, Pauling speculated that when an antigen is introduced into a blood system it is recognized as a foreign object. The system creates antibodies that surrou nd the antigen and form a rigid structure, thus 25

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holding the antigen in place and rendering it inef fective. He described possible bonding that allows the antibodies to encase th e antigen in a 3-dimensional c onfiguration, and proposed that the antigen can eventually dissociate from th e antibody structure, thus leaving behind a welldefined antigen-binding cavity. When the antige n is re-introduced to the system, it can be selectively recognized and re-t rapped by the same antibody struct ure. Though Paulings theory was not correct for antibodies, his idea eventually found fulfillment in the synthesis of molecular imprints. 27 The challenge in molecular imprinting has b een to determine which materials are best suited to be a molecular cast for desired templa te molecules and subse quent release of the template for future recognition. In addition, the determinations of the optimal processing parameters for these reactions are not yet know n. Many researchers began to address this challenge in the 1970s. Gunter Wulff and his group were among th e first to report the imprinting of cross-linked organic polymers as a method to synthesize molecular recognition sites in 1972 4 The overall concept is very si mple, as shown in Figure 2-2. 28 A template molecule is introduced into a solution containing functionalized monomer, and functional groups of the monomer bind to reactive sites on the template mol ecule. A cross-linking agent is added to the solution and polymerization occurs around the te mplate molecule. When the template is removed, a well-defined cavity remains in the rigi d cross-linked polymer. Once re-introduced to the polymer, the template molecule is capable of selectively binding to the site and thus is recognized Both non-covalent and covalent bonds can be formed between a MIP and a print molecule, which are usually the same as those that happen during the initial steps of polymer synthesis. 26

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The major requirement in these interactions is that they be reversible. Figure 2-2. Concept of mol ecular imprinting of polymers. 28 Covalent vs. Noncovalent Synthesis For most applications, two methods are generally considered for the synthesis of imprinted polymers: the covalent and non-covalent technique (Figure 2-3). 29 Each approach has its own set of advantages and disadvantages, and the overall choice ul timately depends on the type of template molecule targeted for imprinting. In covalent imprinting, the functional m onomer and template molecule are linked together through covalent binding, and subsequently polymerized. Upon cleaving, the covalent bond is broken, and the template molecule is released from the polymer. When the guest molecule (template molecule) is re-introduced to the polymer matrix, the imprint site will recognize the molecule and bind it th rough the same type of covalent bond. In terms of template uptake, binding sites are only s ituated inside the cavity. Upon removal of the template, the cavities usually swell, which ensure a high pr oportion (90-95%) of reupt ake after the initial removal. This concurrently faci litates a rapid mass transfer duri ng equilibration of the template with the polymer. Upon reuptake of the target molecule, the ca vity shrinks to its original volume. Advantages of using this scheme are that the monomer-template conjugates are stable and stoichiometric, which makes imprinting re latively straightforward. In addition, many 27

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different polymerization environments can be sup ported due to the use of such stable covalent bonds. Disadvantages include diffi cult synthesis of the monomer-t emplate conjugate, the limited number of available imprinted sites produced during synthesis, cleaving the guest molecule from the imprinted polymer can be de structive to the site, and the overall slow kinetics of the removal/binding process. 30, 31 Figure 2-3. Simplified schematics of (a) covale nt and (b) noncovalent imprinting procedures. 29 With non-covalent imprinting (hydrogen bonding, electrostatic interaction, and coordination-bond formation), the functional mono mer is first allowed to self-assemble around the template molecule, and then the mixture is polymerized. The guest molecule is then extracted using either an acid or a base. Once re-introduced, th e guest molecule can be bound to the MIP through non-covalent inte ractions. Noncovalent imprinting requires a four-fold excess of binding sites in order to guarantee good select ivity. The binding sites are distributed among the entire polymer. However, only 15% of the ca vities are available for reuptake of the template molecule, with the rest being irreversibly lost for use. 32 This is possibly due to the shrinking of the majority of the host sites. Advantages in clude eliminating the monomer-template conjugate bonding process, as required by the covalent imprinting proce ss, easy removal of the guest 28

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molecule from the imprinted polymer, and the ki netics of the removal/binding process is fas Disadvantages include a loosely defined process since the stoichiometry is not strict, reaction conditions have to be clearly defined in order to maximize the number of imprinting sites developed, and selectivity of these sites is reduced due to the excess functional monomer present. t. ovalent imprinting was chosen for th ese studies, since the easy removal of phosp the photopolymerization, or redox r s in s n Polymerization ing radiation initia tion are becoming a popular alternative to tradit itiate ined 30, 31 Nonc hate to produce the MIP, and fast binding of the molecule in-situ are paramount to management of hyperphosphatemia in chronic renal failure patients. MIPs are often prepared via methods such as thermal initiation, polymerization techniques. 33 Several advantages that ther mal initiation has over the othe methods are that it has faster enantiomeric separation times, thus th e detection times are shorter. However, the other techniques pr ovide slightly better adsorption capacities, which provide a medium that is more selective towards the template molecule. MIPs are su perior to antibodie terms of their enhanced affinity towards a number of important analytes, higher loading capacities, reusability, reproducib ility and stability. For this reason, thermal initiation wa chosen. Radiatio Polymerizations involv ional chemical initiators due to their decreas ed dependency on external additives to in polymerization. This provides a process in whic h green polymerizations can occur, which can reduce the amount of byproducts produced during synthesis. For imprinted polymers, using radiation initiation provid es spatially controlled polymerization in situ, with rigid, clearly def imprint sites. 34 Incorporation of this strategy into the imprinti ng regime may improve the 29

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stability, thus the overall success, of the phospha te reuptake process with the functionalized microspheres. Polymerizations reactions were conducted us ing a simple countertop kitchen microwave, which was purchased from a local department st ore, with remarkably great efficiency. The irradiation energy from the microwave is suffici ent enough to promote radical or step growth initiation in many different polymer systems. In this study, chain crosslinking is induced to an extent that is MIT compliant. Previous Related Work Many research groups 31, 35, 36 have successfully developed im printed polymers for a variety of applications in different areas of technol ogy, which show promise for the probable success of fabricating a phosphate imprinted polymer. One such use involves MIPs as stationary phases in chromatography for chemical detection. 37, 38 Figure 2-4 shows the achievement of MIPs in enantiomeric chromatographic applications. 29 Another practical use of imprinted polymers is in the directed nucleation of calcite. Functionalized monomers that cont ain a crosslinking agent are intr oduced to a calcite structure, and subsequently polymerized. The calcite is washed away leaving behind a well-defined imprinted polymer. The resulting imprinted cav ities serve as preformed nucleation sites for calcite, and upon exposure to a solution of calcium chloride and sodium carbonate, rhombohedral calcite crystals are formed (Figure 2.5). 39 MIPs have also found an interesting niche in many different bio-inspired applications, such as biomimetic glucose sensors. 40 In this technological development, a molecularly imprinted poly (allylamine hydrochloride) hydroge l was synthesized with d-glucose 6-phosphate monobarium salt as the template molecule. The resulting MIP displayed enhanced binding affinity towards glucose over fructose, indicating enantiomeric selectivity. 30

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Figure 2-4. Depiction of an HPLC column packed with a MIP stationary phase used for chiral separations. 29 Figure 2-5. Nucleation of calcite at the polymer surface. Scanni ng electron micrographs (Hitachi S570) showing: a, template calcite crysta ls; b, Calcite imprinted polymer (PI-1) as obtained; c, d, PI-1 after HC l/CH3OH wash; e, f, nucleati on of calcite crystals on PI1; g, h, control polymers CP-1 and CP -2 respectively, after immersion in supersaturated CaCO3(aq.). 39 31

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The hydrogels ability to demonstr ate preferential template uptak e in a replicated physiological environment prove its potential use in the de velopment of a new hand held glucose sensor device. 32

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CHAPTER 3 RENAGEL CHARACTERISTICS Understanding Renagel Phosphate binding remedies have been extens ively used as a means to reduce the excess blood serum phosphate that is commonly found in individuals suffering with chronic renal failure. Sevelamer HCl is a highly effective bi nder due to the cationic charge of protonated amine groups, removing phosphate in-vitro at approximately 3 meq (288 mg/g drug). 41 Rigorous pharmacokinetics has not been published so as to de scribe the need for the rather high doses of this expensive medication in clinical practic e. This study was designed to examine the pharmaceutical success of Renagel, which is one of the most successful phosphate sequestering agents in current circulation. In order to provi de an accurate and well-formed evaluation of the phosphate binding polymeric microspheres that have been developed for this project, a study of the effect of pH on swelling and phosphate uptake in Renagel is presented. Structural Properties of Renagel Renagel is the trade name for the chemical Sevelamer Hydrochloride, an oral medication for patients on dialysis. Its major binding mechanism reduces the amount of excess serum phosphate that is absorbed by the intestine in patients with hyp erphosphatemia, thus lowering the overall serum phosphate concentration. Se velamer HCl is manufactured by GelTex Pharmaceuticals in Waltham, MA, and distributed by the Genzyme Corporation in Cambridge, MA. 42 It is an extremely popular phosphate binding polymer due to its exclusion of calcium or aluminum as binding agents. Structurally, Renagel is poly(allylamine hydrochloride) crosslinked with epichlorohydrin, containing about 40% forty percent of proton ated amines separated by one carbon from the polymer main chain (Figure 3-1) The amines are partially protonated and 33

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interact with phosphate molecu les through ionic and hydrogen bonding. 43 Chemically, it is properly referred to as poly(allylamine-co-N,N'-diallyl1,3-diamino-2-hydroxypropane) hydrochloride. Sevelamer hydrochloride is hydrophilic, and insoluble in water 44 because of the crosslinking. Figure 3-1. Chemical structur e of sevelamer hydrochloride. 45 Renagel Swelling Behavioral Studies Polymer swelling studies were also performe d to examine possible influences associated with phosphate uptake. Experimental Methods Particles for swelling analysis were obtain ed by placing a Renagel tablet in 100 mL of deionized water at pH=7 for 1 hour at room temperat ure. This led to disruption of the tablet, and release of the smaller particles of polymer. Th e solution was filtered, and the particles were air dried overnight. Once dried, particles were spr ead over double-sided stick tape, and a flow cell was constructed (attach the tape to the middle of a microscope slide, pl ace a bead of silicon 34

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around the circumference of the slide, place anot her slide on top, and le t dry for at least 24 hours). Solution flow was set to 100 mL/hr, which was regulated via a syringe pump, and attached to the flow cell thr ough syringe needles. Swelling was monitored with an optical microscope using Zeiss imaging software. Partic le swelling was analyzed after flowing solutions of 0.1M HCl at a pH level of 1, 2.5, or 7 for one hour over them. Additional trials involved a combination of solution environments to determ ine the effect that changing pH conditions had on swelling. This was apparent through flowing a solution of pH=7 through the cell for one hour, followed by a solution at pH=1 for an additional hour, and vice versa. Careful attention was paid to the cell to insure that it was not allo wed to dry during any part of the procedure, as to maintain true swelling for the analysis Effect of pH on Phosphate Uptake Polymer-based drugs present unique challenges in that they may swell or dissolve in aqueous solutions. Their efficacy can thus de pend on the kinetics of ligands in solution interacting with moieties having varying degrees of hydration. Conditions or medications that impair gastric acid might then par tially explain the patient-to-patient variability in the number of tablets needed to control plasma phosphate levels with ostensibly similar dietary intakes. To investigate this effect, we studied phosphate up take by Renagel at pH 7, after an initial incubation phase in solutions at different levels of acidity. Incubated solutions at pH of 1, 2.3 and 7 were created to simulate potential human gastric conditions: Renagel 800mg tablets were each placed unalt ered in 400 ml of the incubation solution, with an n=7 for each of the 3 pH levels for 1 hour, with agitation, at 37 C. The pH was adjusted to 7, and all fluid was removed by vacuum filtration. Phosphate uptake was then tested using a pH 7 solution simula ting a gastric environment (20 mM KH 2 PO 4 80mM NaCl and 30mM Na 2 CO 3 ) for 3 hours, with agitation, at 37 C, and filtered. Phosphate content was assayed using atomic emission by ICP, to obtain a mean of 5 repetitions reported in this work. 35

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Incubated solution at pH 0.3: Experiments at acidic levels outside of th e normal human gastric range were conducted to examine maximal protonation. Identical sampling and ICP measurements were performed at this pH. Statistics: Descriptive and nonparametric statisti cs (2 tailed Kruskal-Willis test) were performed, with a p < 0.05 considered st atistically significant. Uptake values were calculated as milliequiv alents, meq, which describes moles of phosphate multiplied by its valency (1.8) in solution. Mean values were expressed to one SD. Figure 3-2 illustrates tablets in a pH 7 incubation demons trated an average phosphate uptake level of 1.85 0.46 meq/g drug. This increased to 2.72 0.35 meq/g with pH 2.3 incubation, and 3.13 0.21 meq/g with pH 1 inc ubation. These changes were statistically significant: pH 7 versus either pH 2.3 (p=0.01) or pH 1 (p=0.0003); and pH 2.3 versus pH 1 (p=0.01). The difference between the pH 7 and pH 1 uptake was approximately 1.28 meq/g drug. This represents 123 mg phosphate/g of the medication, or an increase of 69% in binding. The dry polymer demonstrated significant sw elling of 50-70%, upon 1-hour exposure to a aqueous pH solution ranging from 1 to 7. Howe ver, not as much swelling (2-10%) occurred when a hydrated sample in a low pH acidic soluti on, was further exposed to a dilute HCl solution of pH 7. The majority of phosphate uptake was concluded to occur with in the first hour of exposure to an aqueous environment. Altering pH had a significant effect on th e amount of phosphate uptake in our studies. Polymer swelling was examined at different solu tion pH levels, as to imitate small intestine conditions in-vivo Swelling was consistent with the litera ture, which reports an overall increase in swelling of various polymers as the pH falls below the pKb of the molecule. Although Sevelamers phosphate binding has been characterized at different pH levels, it is speculated that transient pre-exposure to an acidic environment enhances hydration, which may lead to an 36

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increase in phosphate uptake. The hydrogels ch ains expand to their physical limit, but remain intact. Figure 3-2. Phosphate (P) uptake in Renagel. Solutions were at a pH of 1, 2.3 or 7, and incubated at 37 o C, to simulate human gastric conditions. At this point there is minimal interaction betw een the chains, which in turn is offset by the hydrogel matrix crosslinkers. As a result, phosph ate uptake is likely the result of binding to available protonated sites via diffusion of phosphate molecules into the network, from the aqueous environment, which creates an overall hydrating type effect of the polymer. These results also indicated that it may be possible for the polymer particles to crosslink to an extent such that large-scale swelling is not physically attainable. Phosphate Binding Mechanism Computational techniques were used to determine mol ecular binding in the Renagel polymer system. The established model defined the mechanism of phosphate ion capture, upon which a direct comparison was made to the expect ed binding phenomena in this surface modified microsphere system. A relatively simple m odel was designed with a dimer of Sevelamer Hydrochloride and four phosphate ions for binding simulation, which was based on electrostatic 37

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interactions. Molecular dynamics and Monte Carl o simulations were used to determine average structure, intermolecular interaction regions, and the volume change associated with the phosphate capturing mechanism. This computa tional study was performed and interpreted in conjunction with Dr. Reginald Parker, formerly of Florida State University, and Dr. Jesse Edwards III, Assistant professor of Ch emistry at Florida A&M University. a b c Figure 3-4. Observed swelling of dry particles (a) exposed to an acidic solution at pH = 1 for 1 hour (b), followed by additional exposure to a pH = 7 solution (c), using an optical microscope. The amine groups in Sevelamer Hydrochloride are protonated by HCl, resulting in a set of positively charged cages in which phosphate anion capture occurs. The hydroxyl groups serve as additional binding sites through hydrogen bond forming interactions. Using these parameters 38

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and a predictive model of 1.5 repeat units (Figure 3.5), molecular dynamics, 46, 47 and Monte Carlo methods, 46, 48 insight into the interactions and bonding structure of this system were defined. Monte Carlo simulations were specific ally used to conduct volume calculations which can be compared to experimental swelling measurements by of this model. 49 Molecular Dynamics Vacuum system Sybyl 6.9 Software 50 was used to perform minimization and molecular dynamics simulations on SGI Origin 2000 using a Tripos Fo rcefield. The energy of a molecule derived from a Tripos force field is defined as the su m of energy contributions that extend over all bonds, bond angles, torsion angles, and non-bonded inter actions between atoms not bound to each other or to a common atom. 51 Energy minimization was calculate d with Sybyl 6.9 force-field and Gasteiger-Huckel 52 charges with a non-bond cutoff distance of 8 using the conjugate gradient method until the root mean square deviation (RMS) < 0.05 kcal/mole/. An NVT ensemble 46 molecular dynamic simulation was conducte d in vacuum with Gasteiger-Huckel 53 charges assigned at 300 K for 400 ps, with a time step of 1 fs. Data was collected at intervals of 200 fs. Solvated System The dielectric constant of the system was set to a value of 78 debye to simulate an aqueous solvated system with a homogenous field. Gast eiger-Huckel charges were assigned to the atoms of the system. Using this treatment, the large dielectric constant diminishes the coulombic interactions of the charged atoms, which allows the van der Waals inte ractions to be probed, absent strong coulombic effects. Thus, the e ffect of the van der Waal s attraction on phosphate capture is determined, and the overall binding event is elucidated. 39

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Monte Carlo Monte Carlo simulations were used to determine the volume of each molecule. The Monte Carlo method was broken into four parts: First, a spherical shell was formed around the molecule. Second, several random values were generated as points in space, bound by the dimensions of the sphere, which allowed all de rived points to fall within the sphere without necessarily having to fall within the molecule. Third, an algorithm was used to assess if the values actually fell within the molecule. Last ly, the volume of the sphere was multiplied by the ratio of points falling within the molecule to the total number of available points. The resultant product was assessed to be the ag gregate volume of the molecule. Determining the Shell Volume The molecular systems 3-D spatial coordinate s are extracted from the molecular modeling program in the CSSR format (Cambridge Struct ure Search and Retrieval). This layout was comprised of a numbering column, an atom identification column, spatial coordinates, connectivity matrix, the potential charge of each atom, and several other miscellaneous chemical information attributes that help define the system. The spatial location was given in terms of Cartesian coordinates. The center of the molecule (x,y,z) was determined by averaging the values in the coordinate space. The molecule was then translated by spatial subtractio n of each value by the origin. This resulted in an origin of the molecules center being set to (0,0,0). The coordinate space was subsequently transformed into spherical coordinates. The la rgest value of r was determined, and 2 were added in order to determine r shell From this, the volume of the shell was calculated. 33 4shell shellrV (3-1) 40

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Thus, the Monte Carlo method was used to extract the molecular volume based on the computationally derived shell volume. Generating Random Numbers The stock random number generator of Matlab was used to generate three random numbers for each point in the simulation. The first two numbers were bound by (-, ) with angles given in radians, and the last nu mber was bound by the radius of the shell. Determining the Ratio The solution technique was evaluated in it erative segments in order to maximize the efficiency of the treatment. The first step assessed the generated radius of the molecule. If the derived radius was larger than that on the exis ting molecule, the value was determined to be on the outside of the molecules spherical area and ad ded only to the total count. If the value was less than those found within the molecule, it was c onsidered to contribute to the molecule itself. In addition, radii could also contri bute to the molecule if one or more points have a radius greater than the determined angle, and are within 0.07 ra dians (~2 degrees) of any of those points. This effective method was run several times until the ratio values (Equation 3-2) become relatively constant and reproducible. total insidecount count ratio (3-2) Determining the Volume of the Molecule The ratio of molecules determined using E quation 3-2 was then used to calculate the volume of each molecule, as set up in Equation 3-3. ratioVVshell molecule (3-3) The volume of the molecule was found by using a ne sted for-loop towards the average of several runs. The first for-loop generated a ratio, while the second for-loop determined the stability of 41

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the Monte Carlo method prediction for the volume of the molecule. The method was deemed valid of it had a covariance less than 3%. Results Results compared the actual experimental swelli ng measurements to calculated values obtained from running the simulations. Mole cular dynamic simulations were us ed to create a model of the average structure of the proposed system interacting with four triphosphate ions. The derived average structure was used in a Monte Carlo si mulation to calculate th e volume of the model system. The percent change of volume (% sw elling) was calculated by comparing the volume difference of the model system with and without the presence of phosphate ions. The space filled representation of the mode l system (Figure 3-4) suggested that approximately 75% of the phosphate ions were tr apped in the amine cages found in the center of the system, and the remaining phosphate ions or iented at a surface amine site on the model molecule. This was a strong indication that Van der Waals interactions were the primary binding forces for the phosphate ion capturing mechanism in this model system. Mo re specifically, these results supported the notion that this was a zw itterionic interaction. Zwitterions carry both formal positive and negative charges that, in e ssence, compensate for each other making the overall molecule electrically neutral. Their association was polar in nature. Further examination was performed by plotting the interatomic distances between various nitrogen and phosphate atoms, and evaluati ng the dynamic motion of the amine groups and sequestered phosphate ions. Interatomic distances were isolated c hoosing specific nitrogen (Nitrogen 126) and phosphorous (Phosphorous 237) atoms, wh ich are listed in Figure 3-6. The atom labels represent the atom type and number for the total selected system. The data demonstrated that system stabilized and maintained a bond distance be tween 2.7 4 upon the resolution of the 42

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reorganization energy. In addition, the plot illust rates a system adjustment around 480 ps, which allows the Nitrogen Phosphorous separation to fall below 3.5 Eventually the system adjusted back to an average separation of appr oximately 3.5 This provided a mechanism for hydrogen bonding between the phosphate ions an d the hydrogen atoms on the amine groups through a zwitterion association. Figure 3-5. Van der Waals representation of the space filled model system with four phosphate ions bound to the polymer. The green spheres re present chloride ions (counter ions to amines), light blue spheres are hydrogen atoms, dark blue spheres are nitrogen atoms, red spheres are oxygen atoms, and the or ange spheres are phosphate ions. Figure 3-7 represents a similar type of pl ot, except the distances of Nitrogen 204 and Phosphorous 237 was calculated. In this exampl e the nitrogen group was bound to the end of the polymer, which allowed more freedom in the wagging motion in comparison to the previous calculation. This allowed the phosphate motion to be held constant, thus the resulting motion was attributed to the amines ab ility to wag in and out of the proximity of the phosphate. The periodic interatomic separation between the phosphate ion and the amine nitrogen were due to 43

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the flapping motion of the amine on the polymeric ta il and varied between a separation of 5 for 200 ps to below 4.3 for 400 ps. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 100 200 300 400 500 600 700 800 900 1000 Time (ps)Distance (Angstrom) Figure 3-6. Interatomic distance between Nitr ogen (126) and Phosphorous (237) in the model system Renagel and four triphosphate ions with an N-P separation adjustment of the system at about 480 ps. The reorganization and stabilization that occu rred during the simulation was represented in Figure 3-8, which expressed the interatomic distance between Nitrogen (89) and Phosphorous (237), which display typical behavi or for amine groups and phosphate ions in the central cages of the polymer. After the reorganization of the polymer, the molecular separation was maintained at a distance which was roughly 3.5-4 Tables 3-1 and 3-2 show values of various interatomic separations obtained from the results of a NVT ensemble simulation in vacuum for the four phosphate ion system. Phosphate ions bound in the central region of the polymer exhibit nitrogen-phosphorous separations on the order of 3-4 which indicated minimal m ovement of these sequestered phosphates. 44

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0 1 2 3 4 5 6 7 0 100 200 300 400 500 600 700 800 900 1000 Time (ps)Distance (Angstrom) Figure 3-7. Interatomic distance between Nitrog en (204) and Phosphorous (237) in the model system Renagel and four triphosphate ions This figure shows the periodic motion of end amine group flapping in and out of the proximity of the Phosphorus 237 atom of a triphosphate ion. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 1002003004005006007008009001000 Time (ps)Distance (Angstrom ) Figure 3-8. The change of in teratomic distance between N(89) and P(237). Shown is the reorganization and stabilizati on that occurred during the simulation. An interatomic separation of approximately 3.5-4 was maintained after the reorganization of the polymer. 45

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Figure 3-9 shows the stable separation be tween Phosphorous 237 and Phosphorous 216 of about 6-7 when measuring the distances between a set of inte rior (captured) phosphate ions, which remained constant after the stabilization of the simulation. 0 1 2 3 4 5 6 7 8 0 100 200 300 400 500 600 700 800 900 1000 Time (ps)Distance (Angstrom) Figure 3-9. The P(237)-P(216) inte ratomic separations versus simulation time. The plot shows a stable separation after about 100 ps between 6-7 This separation was constant between all of the phosphorus atoms within the phosphate ions confined to the center of the polymer. Due to the large dielectric constant in th e simulated solvated system, considerable reorganization takes place before being stabilized. Equation 3-4 is used to compute the coulombic interactions, which significantly decrease. 2 21D r qq U (where D is the dielectric constant) (3-4) In Table 3-1 the standard deviation of the nitrogen phosphorous separations for the centrally captured phosphate amines was relatively low, where as the deviations in Table 3-2 were much higher due to the mimicked solvatio n using the change in dielectric from 1 to 78 debye. This was due to the fact that the bindi ng of phosphate is attri buted to van der Waals 46

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interactions, which required structural rearrangem ent within the polymer to properly incorporate the phosphates in an amine cage. Table 3-2 summarizes such results. Table 3-1. Interatomic distance values (mean, st andard deviation, high, and low) for the NVT ensemble simulation in vacuum for four phosphate system with dielectric 1 debye. NVT Ensemble 4 PO4 dielectric=1 Interatomic Distances [Nitrogen Phosphorous, Phosphorous Phosphorous, Hydrogen Chlorine] () Atom Types and ID Number Mean Standard Deviation High Low N126-P237 3.35 0.28 4.21 2.82 N39-P237 3.16 0.18 4.02 2.76 N89-P237 3.66 0.19 4.27 2.96 N30-P237 3.35 0.14 3.81 2.92 N81-P237 3.13 0.12 3.73 2.69 N204-P237 4.33 0.68 6.00 3.34 N102-P2421 3.15 0.11 3.63 2.79 N204-P242 3.36 0.16 4.00 2.78 N165-P242 4.10 0.17 5.32 3.49 N89-P226 3.44 0.42 5.12 2.86 N165-P226 3.08 0.09 3.51 2.79 N188-P226 3.19 0.13 4.03 2.81 N139-P226 5.84 1.03 7.01 2.97 N201-P216 3.03 0.14 3.82 2.71 N16-P216 3.29 0.23 4.56 2.82 N65-P216 3.18 0.17 4.30 2.74 P237-P216 6.21 0.53 7.04 4.12 P226-P242 5.90 4.09 0.19 4.88 Cl106-H143 3.19 0.13 4.03 2.81 The calculated average volume from the molecular dynamic simu lation of the solvated four phosphate systems produced a percent swelling of 140% which was good agreement with some of the extreme values of the experimental results. Conclusions Molecular dynamic and Monte Carlo simulations were used to develop a simple model of phosphate binding events in the Renagel polymer. Our results showed an average swelling value of 25% in vacuum and over 140% in water during the simulations, which were relatively 47

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consistent with experimental results of betw een 10-200%, with the average being approximately 50-60%. Data was also obtained using these simulations that suppor t the view that the predominant molecular interactions in this syst em are via van der Waals forces, specifically zwitterionic in nature. Table 3-2. Interatomic distance values (mean, st andard deviation, high, and low) for the NVT ensemble with dielectric 78 debye to mimic a solvated system (H2O). This system shows the capture of four phosphates internally and exte rnally for several hundred picoseconds. NVT Ensemble 4 PO4 dielectric=1 Interatomic Distances [Nitrogen Phosphorous, Phosphorous Phosphorous, Hydrogen Chlorine] () Atom Types and ID Number Mean Standard Deviation High Low N16-P223 10.22 2.23 21.41 6.72 N131-P228 4.52 0.97 9.28 3.19 N84-P228 4.59 1.06 9.40 3.37 N96-P228 5.55 0.78 8.45 3.70 N144-P228 9.19 1.04 13.67 4.06 N155-P228 10.48 1.42 14.61 4.54 N77-P228 7.82 0.89 12.33 6.01 N77-P202 4.69 1.59 10.59 3.03 N177-P202 5.66 2.28 12.81 3.19 N62-P202 8.52 4.84 20.28 3.24 N155-P202 5.92 2.70 14.42 3.39 N119-P202 12.44 1.99 15.98 4.73 N77-P223 6.40 3.49 20.10 3.15 N119-P223 9.88 3.78 21.27 3.36 N29-P212 4.99 1.12 11.35 3.42 N37-P212 4.76 1.17 12.46 3.32 P223-P212 9.80 2.77 18.99 5.73 N77-P212 4.91 0.74 8.18 3.26 N29-P223 8.00 3.72 17.18 3.40 P228-P202 9.41 1.66 12.27 4.16 P223-P228 8.47 1.96 17.60 4.91 These results exemplify the capture mechanism of the phosphate ions with minimal electrostatic interactions, which will be used to envisage the various forms of the protonated phosphate ion available for uptake in the synthe sized surface altered polymers. The simulated 48

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polymer model indicates that in ternally, amine cages form aro und the phosphate in the center of the polymer for binding events, while the extern al capture of the phosphates is accomplished through interactions about one of the P-O bonds with the amines on the exterior of the polymer. These results are used in subse quent chapters to structure the method of phosphate capture that occurs in the different MIPs that are produced, and to a nd support the use of molecular imprinting technology in this research. 49

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CHAPTER 4 POLYMERIZATION PARAMETERS Experimental Design and Material Selection The main goal of this research was to de velop a robust system in which a functionalized polymer could be synthesized for the selective binding of phosphate ions. Upon modification of this polymer, and other process parameters, th e polymer could be produced and applied to invitro and in-vivo testing. This novel technique may be es pecially valuable in the development of resorbable polymer technology in which highly selective chemical recognition is possible and, due to the highly crosslinked nature of the host polymer, the system degradation will be minimal. This is especially important for in-vivo applications, where the polymer must remain in tact while removing the molecule of interest in a gastrointestinal environment. In-vivo processing of these polymers would include or al ingestion, processing through the digestive tract, binding of phosphate in the gastrointestinal tract to prevent phosphate from being absorbed, and eventual urinary and bowel excretion. Gelatin was chosen as a potentially useful candidate. Granular gelatin was weighed and placed in a reaction vial with a predetermined amount of boiling water, whose pH had been adjusted to approximately 7.4, until it dissolved. Potassium phosphate monobasic (KH 2 PO 4 ) was added to the gelatin solution for phosphate containing samples only, otherwise the control sample was the viscous gelatin blend alone. The gelatin mixture was slowly added to a round bottom flas k containing common grade oil (vegetable, corn, etc.) at 60 o C to create a water-in-oil emulsion, where no additional emulsifier was required. The reaction flask was stoppered with a rubber septa, purged with a nitrogen stream, and allowed to stir for approximately 60 minutes to allow a st able emulsion to form. 10 50% w/v of the crosslinking agent was added, and the blend wa s mixed for an additional amount of time. Upon visible formation of microspheres, the crosslinked mini emulsion was aggressively washed in 50

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order to remove any residual amount of crosslinking agent. The potassium phosphate monobasic, Type B (225 bloom) bovine gelatin, 50% w/v glutaraldehyde, D, L-glyceraldehyde ( 90%), and cottonseed oil all came from Sigma-Aldrich chemical company. The crosslinking agent, genipin, was ordered from Wako Chemicals. All chemicals were used as received. The common grade oil, either vegetable or olive, was purchased from the local grocery store. Synthesis Parameters Template Molecule The choice of guest molecule depends on the use of the intended polymer. 54, 55 However, studies show that imprinting of small molecules is more successful than that of larger biological molecules, such as proteins, because their size and fragility make it almost impossible to apply either of the two classical approaches using covalent or noncovalent binding approaches. 56 With this in mind, careful consideration must be taken to ensure that the system chosen is compatible with a successful molecular te mplating strategy. For targe ting phosphate, potassium phosphate monobasic was chosen. According to well established polyprotic acid data based on phosphoric acid, the phosphate ion can exist in four different molecular fo rms (Figure 4-1), depending on the number of hydrogen atoms that are protonated. This can be affected by the pH of the surrounding solution, as predicted by the Henders on-Hasselbalch Equation: (4-1) which is the equivalent of: (4-2) 51

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Figure 4-1. Common polyprotic mol ecular structures of the phos phate ion. There are three hydrogen atoms that dissociate at different pH values; thus, phos phoric acid has three pK a values. Figure 4-2. Titration curve for phosphoric acid. Phosphoric acid (H 3 PO 4 ) has three hydrogens and therefore three midpoints at pH 2.1 (pK 1 ), pH 7.2 (pK 2 ), and pH 12.4 (pK 3 ). 57 To generate in-vitro assays that mimic the human in-vivo gastric environment, phosphate uptake trials were run at pH = 7.4, which mean t that the expected phosphate ion available for binding was primarily in the form of H 2 PO 4 (80%) with some HPO 4 2(20%). These values were obtained from extrapolat ions using Figure 4-3. 58 52

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Figure 43. Relative fractions of the various forms of phosphate at different pH levels. Functional Monomer The monomer system was chosen based on its ability to remain intact with the other components (template, crosslinker, etc.) upon polymer ization, and remain bi ologically inert. The system varies with the intended use of the nona bsorbable polymer, as well as the type of imprinting or surface functiona lization technique used. One of the major concerns with de veloping this type of polymer for in-vivo use was biocompatibility, which has been a major problem with traditional synthetic routes that use potentially toxic crosslinking agen ts. The original plan was to develop the system which would be refined so that it was biocompatible, which le d to the thought of simp ly starting with a known material that would suit this purpose. Common monomers exte nsively used as recognition networks include methacrylic acid, itaconic acid, acrylamides, 4-vinyl pyridine, and acrylamide. 59 After some consideration, gelatin was identified as a logical, well studied substance with over 100 years of biocompatible use in humans, which was used as the main functional monomer backbone that would provide enhanced rebinding selectivity. 53

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The industrial preparation of gelatin is through thermal denaturation of collagen from animal skin, bones, and the less common fish scales. 60 Its structure is mostly comprised of glycine (arranged every third residue) and proline. Gelatin contai ns extended left-handed proline helix conformations incorporated with 300 to 4000 amino acids. Gelatinous solutions undergo coil-helix transitions and then aggregate, cau sing the formation of higher level pyrrolidines, which results in the formation of stronger gels. The approximate amino acid (Figure 4-4) composition of gelatin is: glycine 21%, proline 12%, hydroxyproline 12%, glutamic acid 10%, alanine 9%, arginine 8%, aspartic acid 6%, lysine 4%, serine 4%, leucine 3%, valine 2%, phenylalanine 2%, threonine 2%, isoleucine 1%, hydroxylysine 1%, methionine and histidine <1% and tyrosine <0.5%. These values vary, depending on the source of the raw material and pro cessing technique. 61 There are two main types of gelatin; Type A, which is derived from porcine skin, and Type B, which is a bovine derivative. Type A usually undergoes an acid pretreat ment, while Type B is exposed to an alkaline pretreatment in which asparagines and glutamine residues are converted to their respective acids and results in higher viscosity. Gela tin is amorphous and amphoteric, thus has a broad range of materials applications. Gelatin based materials have recently f ound biomaterial applications for use in bioadhesives, wound dressing material, and bone substitute. Gelatin is a natural polymer which has a low antigenicity and is biodegradable. Howe ver, gelatin is limited by the fact that it rapidly swells and dissolves in aqueous e nvironments, which ultimately leads to rapid in-vivo degradation. 62 54

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Figure 4-4. Molecular structures of common amino acids found in-vivo 63 One way to address this issue is to treat the gelatin with crosslinking materials. Chemical crosslinking can be achieved by creating bonds be tween functional groups of amino acids, such 55

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as transglutaminase to link lysine to glutamine residues, and glutaraldehy de to link lysine to lysine. 64 The experimental design used for this work is based on the notion th at phosphate groups will bind to the arginine groups on the gelatin chain. Upon the addition of the crosslinking agent, there should be spontaneous reactions with the amino acids, which will hold the template in place. NH2CH3O NH O N O NH NH2 +NH NH2NH O NH O O N O O O-OH NH O N O Figure 4-5. Structure of Gelatin Crosslinking density is obviously affected by the amount of crosslinking agent available, but it is believed that every possibl e crosslink site will not react. Initiator In developing templated microspheres for vari ous applications, the viscosity of the product must be a tunable property. Though a solid, rigid polymer ma y be desired, extraction and subsequent use may be more difficult than that of a more porous and flexible hydrogel-like product. Temperature, duration of polymeriza tion, stirring speed, and altering the type and amount of initiator all affect pol ymer rigidity. The amount of in itiator has an influence on the number of attachment sites and hence the amount of target molecule that is capable of binding to the polymer. 65 This will also effect the rate in whic h polymerization is initiated and terminated, which assists in determining the final composition of the templated polymer. 56

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For these reasons, thermal initiation was chosen to induce polymerization. In this instance, the temperature at which polymers begin to crosslink depends on the physical properties of the crosslinking agent, and not the half-life, T of the potential initiator used. 66 It is also important to note that the binding of mono mers to templates can cause a reduction in chain termination, thus an overall increase in reaction rate. Ul timately, the amount of crosslinked, imprinted polymer that produced is strongly affected by the polymerization temperature. Crosslinking Agent Gelatin microsphere swelling was dictated by the extent of crosslinking between the polymer chains. The greater the degree of cro sslinking, the shorter th e bond distance of the chains, which causes a more dense spherical structure. 67 Thus the type and amount of crosslinker used has a significant a ffect on the overall microstructure. The major requirement for specific ion imprin ting is that the host polymers possess rigid guest-binding sites that are stab le under a host of chemical environments. The crosslinking agent is primarily responsible for this feature in that it influences th e solubility of the polymer in solvents, 68 which allows the them to be used in many different technical applications. In molecular imprinting, selec tivity is also mainly influen ced by the type and amount of crosslinking agent used. Below a certain amount of crosslinking in the polymer ( 10%), selectivity usually is not observed due to the un stable nature of the cavities. Typically, selectivity steadily increases when crosslinking is greater than 10%, and between 50-60%, an unexpectedly high increase in selectivity occurs. 69 Varying the type and amount of crosslinker used directly controls the structure and environment around the guest-binding site. The crosslinking agent used should be similar in structure to the type of the host monomer in order to provide adequate random copolymerization, and ev enly distribute functional sites throughout the polymer network. 57

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The molar stoichiometry of the crosslinking r eagent to the functional monomer is also an important controlling property of the final templated structure. 70 When the crosslinker is in great excess, the imprint sites become too close together and actually hinder rece ptor recognition. In the other extreme, not enough imprint sites are fo rmed, and specific template recognition is not possible. The amount of cross-linker used is usually between 25 80% by weight, depending on the combined weight of crosslinking and mono mer. Glutaraldehyde, genipin, glyceraldehyde and dextran were thoroughl y investigated as crosslinkers becau se they have been reported in literature as being effective crosslinking agents fo r proteins, especially gelatin. The crosslinker used during synthesis should provi de mechanical and thermal stability, good wettabity in most rebinding media, rapid mass transfer with good recognition properties, and be biologically compatible. Genipin crosslinked microspheres Genipin is a naturally occurri ng crosslinking agent used comm only for proteins, collagen, gelatin, and chitosan. It is derived from geniposide present in fruit of Gardenia jasmindides Ellis. It has a low acute toxici ty, and is significantly less toxic than many other commonly used synthetic cross-linking regents. 71 The structure of genipin (Figur e 4-6) was discovered in the 1960s by Djerassi. It has a molecular formula of C 11 H 14 O 5 a molecular weight of 226.227 g/mol, and contains a dihydropyran ring. 72 Genipin itself is colorless but it r eacts spontaneously with amino acids to form blue pigments. It is so luble in ethanol, methanol, acetone and slightly soluble in water. 71 The components of the Gardenia fruit have been used in traditional Chinese medicine and as a blue colorant by food industries in East Asia. It is also used for pharmaceutical purposes because it is a naturally occurring, biodegradab le molecule with low cytotoxicity. Recent explorations into the us e of genipin crosslinked gelatin (Figur e 4-7) for the use as a bioadhesive, 58

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wound dressing, and as bone substitutes, have shown it to have potential as a new and safe crosslinking agent. Figure 4-6. The molecular structure of Genipin (top) and Geniposide (bottom). Glutaraldehyde crosslinked microspheres Glutaraldehyde has a wide variety of industr ial uses as a disinfectant, preservative, fixative, crosslinking agent, and as a chemical intermediate in the synthesis of pharmaceuticals and pesticides. It is also used for industrial water treatment and as a chemical preservative. However, it can be toxic upon de gradation, causing severe eye nose throat and lung irritation, as well as headaches drowsiness and dizziness. 73 Glutaraldehyde is an oily liquid at room temperature ( density 1.06 g/ mL ). It has a molecular formula of C 5 H 8 O 2 and a molecular weight of 100.11 g/mol. 74 It is a colorless liquid with an overpowering smell. Glutaraldehyde is frequently used in bioche mistry applications as an amine-reactive 59

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homobifunctional crosslinker and has been the gold standard used for crosslinking gelatin (Figure 4-9) for decades. Crosslinking happe ns through the reaction of the aldehyde group of glutaraldehyde and the free amino groups of lysine or hydroxylysine amino acid residues 75 on the gelatin chain. The oligomeric state of proteins can be examined through this application. It is miscible in water alcohol and benzene Figure 4-7. Crosslinked st ructures of genipin cross linked gelatin microspheres 76 Figure 4-8. The molecular structure of glutaraldehyde. Dextran Dextran is a branched polysaccharide composed of glucose, with the main chains being formed by 1 6 glycosidic linkages and the side branches are attached by 1 3 or 1 4 linkages (Figure 4-10). Dextran is a bacterial product whose st ructure is dependent on its inherent source. It is also used commercially as food additives, an anti-pla telet, and increases the volume of blood in plasma. 77 60

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Figure 4-9. Crosslinked stru ctures of glutaraldehyde cr osslinked gelatin microspheres 76 Dextran has a molecular formula of n(C 6 H 10 O 5 ) is water-soluble, and been found to have an average molecular weight of 75,000 in its pu rified form. Lower weight dextrans display slightly less branching 78, 79 and have a more narrow molecular weight distribution. Dextrans with molecular weights greater than 10,000 have highly branched char acteristics and as the weight increases, the dextran molecules attain greater symmetry. 80 At molecular weights below 2,000 dextran has a rod-like behavior, 81 while those with 2,000 molecular weight 10,000 exhibit the properties of an expandable coil. The primary crosslinking of gelatin by dext ran occurs through a Schiff base formation between the -amino lysine groups and the hydroxylysin e residues of gelatin and the free aldehyde. 82 D,L-glyceraldehyde Another nontoxic altern ative to chemically crosslinking gelatin along the macromolecular chains is through the use of D,L-glyceraldehyde (Figure 4-12). Recent studies show the successful crosslinking of chitosan microspheres using d,l-glyceraldehyde, 83 and its proposed 61

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broad application as a biocompatible crosslinking agent for protein microspheres. 84 Glyceraldehyde is also a metabo lic product of fructose in the human organism, which indicates complete biocompatibility. It has a crystalline appearance, chemical formula of C 3 H 6 O 3 and a molecular weight of 90.08 g/mol. As an aldehyde, crossli nking mainly occurs through lysine residues. Figure 4-10. Molecular structure of dextran. gelatine + dextranaldehyde gelatin crosslinked with dextran Figure 4-11. Preparation of ge latine hydrogels crosslinked with partially oxidized dextrans. 85 62

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OH H OH O Figure 4-12. Molecular stru cture of D,L-glyceraldehyde. It has been demonstrated that, on average, na tural polymers crosslinked with glyceraldehydes do not swell as much as th ose using glutaraldehyde. 83 The extent of crosslinking can vary, depending on the amount of crosslinking agent used and the crosslinking time period. Porogen The use of solvents in polymerization is central to the success of the imprinting process, and vary depending on the imprintin g technique employed. Though an important application of solvents is that they dissolve components fo r polymerization, greater significance is placed on the role that they play in creating porous structures. 86, 87 During polymerization, solvent molecules are incorporated into the polymer structure, and removed during the post-treatment process, l eaving behind a porous space. Without this, the polymer would be too rigid a nd dense, making template bind ing impossible due to too few accessible cavity sites. The solvent used during polymerization affects the selectivity of template recognition in that it may either enhance or destabilize the specific interactions th at lie at the center of the imprinting effect. Another role of porogens is that they disp erse the heat of reaction associated with polymerization. 88 Without this, the temperature of reaction for the polymerization would increase, which may create detrimental side effects that could impede or terminate the reaction. The porogen should be the least polar solvent capable of dissolving the components of the 63

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mixture as to maximize the non-covalent intera ctions between the te mplate and functional monomer. Acetone and water will be used as porogens for microsphere synthesis. Determine the Efficacy of Phospha te Uptake with the Polymers in-vitro Once the polymer system was defined in Specific Aim 1, examination of the effect that the polymers had on the binding specificity of phosphate, and their possible influence on hyperphosphatemia, was determined. This was accomplished by determining the amount of phosphate the surface modified microsphere could bind, and ascertaining if this was sufficient enough to reduce the excess amount of phosphate in patients with chro nic renal failure. Assessment of the Reaction Parameters The gelatin monomer system from specifi c aim 1 was modified until a functionalized polymer sphere was fabricated in which extractio n and reuptake of the template was optimized. This included adjusting each of the parameters described for synthesis. Success was established by identifying polymers that could bind solution phosphate at a minimal level of 1.00 mEq/ g polymer. Recovery The success of polymer impr inting was determined through in-vitro analysis in an aqueous phosphate milieu. Once synthesized, the polymer was washed with an acidic or basic solution in order to extract most of the phosphate present during synthesis. The concentration of the phosphate ion in each successive wash was probed using ICP, UV/VIS, or a handheld phosphate meter. Once extracted, the polymer was washed with deionized water to remove any excess solvents from the system, and dried in-vacuo overnight to eliminate excess moisture. In-Vitro Analysis of Phosphate Upta ke in the Functionalized Polymer For in-vitro studies, 800 mg of the polymer were placed in 400 mL of a pH = 7 solution of 20 mM phosphate, 80 mM sodium chloride, and 30 mM sodium carbonate, which mimicked the 64

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conditions of the small intestine, 89 for a period of 3, 24, and 48 hours. An aliquot of the test solution was collected and the concentration anal yzed using either ICP or a Hanna high range hand held phosphate meter. The amount of phosphate bound to the polymer was calculated by subtracting the phosphate found in the filtrate so lution from the original phosphate present. [PO 4 3Bound ] = [PO 4 3Initial ] [PO 4 3Final ] Instrumentation The instrumentation used for analysis were: ICP and IR at the Particle Engineering Research Center (PERC), SEM at the Major Anal ytical Instrumentation Center (MAIC), UV in the Goldberg laboratory, and the phosphate meter in the Batich lab. All facilities were located on the campus of the University of Florida. ICP Inductively coupled plasma was used to dete rmine the concentrati on of phosphate in the collected aqueous washes of the test samples. A calibration curve wa s constructed using a phosphorous standard, and the phosphate test samples were analyzed against this. UV/VIS Ultraviolet visible sp ectroscopy was also used with an EnzChek phosphate assay kit E6646 (Molecular Probes) to quantify the inorgani c phosphate in solution. The absorbance peak of the substrate appeared ar ound 330 nm against a calibration curve prepared at a 360 nm absorbance. Phosphate meter A HI 93717 phosphate high range phosphate meter (Hanna Instruments), was used for the fast and direct analysis of phosphate concentratio ns in mediums inclusive of, but not limited to water. The reagent sets for analysis were included with the instrument. 65

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Characterization of bulk polymer Scanning Electron Microscopy (SEM) Due to the expected small size of the imprint sites, the polymer was imaged using scanning electron microscopy (SEM) with EDS capability in order to examine the morphology and possible molecular content of the sample. Th e particles were mounted on aluminum stubs, attached to the surface using double-stick tape and coated with gold prior to imaging. Infrared (IR) Fourier transform infrared sp ectroscopy (FTIR) analysis, using a Perkin Elmer Spectrum 100 Series FTIR spectrometer, was used to in vestigate the sample composition by determining the presence and type of bonds in the molecule using attenuated total re flectance (ATR). ATRIR is used to probe the surface of materials. Sa mples were measured at room temperature in the range of 4000 6000 cm -1 at a resolution of 4 cm -1 66

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CHAPTER 5 GELATIN MICROSPHERES VIA EMULSIFICATION Water in Oil Emulsions A water-in-oil emulsion system, commonly referre d to as an inverse emulsion, was chosen due to its known ability to produce fast and relatively simple polym er particles of high molecular weights, and to allow a rapid transfer water laden spheres to the aqueous phase. 90 In water-in-oil emulsions, an aqueous solution of a water soluble monomer is emulsified in a hydrophobic medium (Figure 5-1). Emulsificati on is dependent on the reaction temperature, concentration of the ingredients, and can be initiated through the introduction of either a water or oil soluble chemical. The attraction of the synthetic scheme used in these experiments is that the reaction is thermodynamically driven, thus a chemical initiator is not required. Instead, a water soluble crosslinking agent was used to control the extent of cr osslinking, producing a dispersion of spheres. In addition, an emulsifier free emulsion was carried out, which does not require the use of a surfactant. Figure 5-1. An inverse emulsion system. 67

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Reverse phase emulsions mainly transpire within the monomer swollen particles, contain a discontinuous aqueous phase wh ich usually has two or more droplet size distributions, 91 provide high polymer solids with reduced oil content, low bulk viscos ity, and high physicochemical stability. However, these particles are on aver age less stable than those prepared by the more common oil-in-water emulsion technique due to the electrostatic forces involved. In this formulation, the aqueous solution cont ains the phosphate and/or gelatin, and oil is the nonpolar phase, which is a simp le hydrocarbon medium. Smaller size drops can be affected by many different variables, such as the rate of stirring, duration of r eaction, decreasing the interfacial tension, or by the c oncentration of the components in the water phase. Some of these variables have a greater influe nce on the emulsion polymerization process more than others, however they can all be modified to achie ve the best product characteristics. To better understand the physicochemical formulation concerns of a water-in-oil emulsion system, a set of parameters are examin ed which are indicative of the nature of the components. Although emulsions are not in equilibr ium, the length of the reaction often leads to a physicochemical type equilibrium in which th e formulation determines the properties and phase behavior 92 of the mixture. In the past 50 years, researchers have made intensive efforts to ch aracterize the physical and chemical behavior of emulsion polymerizatio ns, resulting in the identification of the HLD (Hydrophilic Lipophilic Devia tion), which is a measure of the relative affinity of the surfactant for the aqueous and oil phase. At HLD = 0 the su rfactant affinities are exactly matched, and a minimum interfacial tension is attained, as sought in enhanced oil recovery processes. 93 Since surfactants are not used in this specific a pplication, it was assumed that the system was functioning at the HLD = 0 level, thus the a pparent performance was based on the features 68

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provided in Figure 5-2, 91 which summarizes a survey of data available in literature. 94, 95 Figure 5-2. Interfacial tension and emulsion variations versus fo rmulation (as HLD) at near unit water-to-oil-ratio. Thus the interfacial tension of the particle, the emulsion viscosity, and the emulsion drop size should all be at their minimum values, whic h provide a state for maximum efficiency for the reaction. It is important to recognize that as the emulsion stability concomitantly decreases, the drops coalesce instantly upon contact, which favors a larger particle size. Ultimately, this mixed phase phenomena results in a bimodal distribution of drops. Th is effect can be addressed by increasing the stirring energy, which widens the area where th e decreasing tension produces smaller drops, thus favoring the production of a decrease in microsphere size. Crosslinked Gelatin Microspheres Synthesis of Gelatin Microspheres A modified synthetic scheme 96 was developed for gelatin microsphere fabrication. Dissolve 2.5g of 225 Bloom bovine gelati n in 10 mL of deionized water at 100 o C, whose pH has been adjusted to 7.4 using dilute NaOH or HCl so lutions, and place in an ultrasonic water bath at 60 o C. The gelatin completely dissolved, resulti ng in a viscous solution. 10 mM of potassium phosphate monobasic is added to the sample for impr inting, while the sample that did not contain phosphate served as the control. The solution wa s added dropwise to 20 mL of vegetable oil at 60 o C, and the mixture was placed in an oil bath at 60 o C. Stir for approximately 1 hour, upon 69

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which a clearly defined gelatin emulsion was appa rent. The emulsion was placed in a water bath at 4-13 o C, while stirring, for 30 minutes. Particles were dehydrated by adding 30 mL of cold (4 o C) acetone to the solution, and allowed to stir for 30-60 minutes. They were filtered, rinsed with precooled acetone, and vacuum dried overnight in order to remove any residual water or oil left on the particles. Some particles were crosslinked using differe nt amounts of crosslinking agent in order to determine if the appropriate amount to use for microsphere formation concurs with the established parameters detailed earlier in Chapter 4. Once the optimum amount of crosslinker to use was ascertained, it will be the st andard for future experiments. Crosslinking Gelatin Microspheres with Genipin Genipin is a naturally occurri ng crosslinking reagent for gelatin with very low cytotoxic effects. 97 For this reason, there has been a recen t significant shift towards using it as an alternative to other cros slinking agents that are known to be cytotoxic. 2.0 g of the gelatin microspheres were placed in 20 mL acetone/water (2:1, v/v) mixture at pH = 7.4., to which was added 300 mg of genipin to the mixture and stirred for 15 hours at 4-10 o C, or until crosslinking was evid ent by a pigment change of the pr otein to a dark blue color. The solution was then allowed to stir at room temperature for 10 hours. The resulting microspheres are filtered, rinsed with cold acetone, and dried under vacuum overnight. The resulting product will be a ri gid, dark blue microsphere. Genipin crosslinked gelatin microspheres were prepared using 2, 15, and 75% (w/w) genipin. The samples were prepared using pure gelatin (control), and gelatin with phosphate microspheres. Crosslinking Gelatin Microspheres with Glutaraldehyde The most commonly used crosslinking reagen t for gelatin is the synthetic reagent 70

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glutaraldehyde due to its effici ency in crosslinking collagen based materials, and its relatively cheap cost. 98 However, glutaraldehyde is often consider ed to be cytotoxic due to the release of formaldehyde upon degradation. Nevertheless, many articles cite the use of glutaraldehyde crosslinked gelatin microsphere s as being relatively safe in-vivo, providing that their synthesis includes a rather vigorous washing procedure to remove unreacted reactant. Even so, there has been a widespread shift from its use to crosslinking agents that are known to have little to no cytotoxic side effect. Gelatin spheres can be crosslinked with gl utaraldehyde during the emulsion process. Uncrosslinked particles were prepared, as desc ribed above, and stirred for an hour in the prechilled acetone. 30 mL of 25% gl utaraldehyde (w/v) in DI water wa s added, and allowed to stir overnight. The appearance of bur nt orange colored spheres were present after several hours of mixing. The particles were then be filtered washed with 100 mL portions of ethanol to terminate crosslinking reaction, and washed w ith DI water. The microspheres were then dehydrated by adding 30 mL of cold (4 o C) acetone to the solution, and allowed to stir for 30 minutes. The supernatant was filtered off, and the particles washed with precooled acetone. The spheres were dried overnight in vacuo. Crosslinking Gelatin Microspheres with additional agents In an effort to provide a thorough analysis of crosslinked gelatin polymers that have potential in-vitro and in-vivo applications, dextran and glyceral dehyde were used as crosslinking agents with low toxicity. Dextran Oxidized dextran can form a cross-linked gelatin network through s ugar mediated crosslinking, which can reduce the dissolution of gelatin. 99 Uncrosslinked gelatin microspheres were prepared using an inversion emulsion scheme, a nd crosslinked using 15% w/w dextran dissolved 71

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in 3 ml of DI water. Crosslinking was carried out 5 o C for 24 hours. The resultant white powdery-like microspheres were rinsed several tim es with precooled acetone, and allowed to dry in vacuo for 24 hours. Glyceraldehyde Uncrosslinked gelatin microspheres were prepared in the usual fashion, and crosslinked using 2% w/v glyceraldehyde sin ce a highly crosslinked polymer wa s desired. Crosslinking was carried out in an acetone-wate r mixture (40:20, v/v) at 5 o C for 24 hours. The resultant bright pumpkin colored microspheres (darker burnt orange if phosphate is used) were rinsed several times with precooled acetone, and al lowed to dry in vacuo for 24 hours. Gelatin Microsphere Processing Removal of the Template After the spheres were crosslinked, extrac tion of the phosphate template molecule was performed with a simple washing procedure. Assays were performed on acid washed and base washed microspheres to determine the most effect template extraction procedure to provide the most suitable polymeric microenvironment for the phosphate binding mechanism. Since 100% crosslinking was not attained, there should be amines remaining from arginine groups on the gelatin chain on the surface and within the polymeri c particle (Figure 53), both of which should be available for phosphate uptake. Base Wash Extractions Approximately 200 mg of the gelatin polymer sa mple was measured, placed in 20 mL of a 1:1 solution of 0.1 M NaOH: MeOH, and allowed to stir for 45 minutes. The solution was decanted off, and the procedure was repeated two more times. The solution was decanted, 20 mL of DI water was added, adjusted to pH = 7 and allowed stir for 30 minutes. The particles were filtered, 72

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rinsed with neutral DI water, and dried overnight in a vacuum oven at room temperature. Particles were then used for phosphate uptake trials. NH NH NH2CH3 NH NH NH2CH3 NH NH NH2CH3 NHNH CH3NH2 NHNH CH3NH2 NHNH CH3NH2 NH2NH NH+ -O-O-OH O P NH2NH NH+ O-O-OH O P NH2NH NH+ NH2NH NH+ OH O-OH O P NHNH NH2CH3 NH NH NH2CH3 NH NH NH2CH3 NH NH NH2CH3 O-O-OH O P Figure 53. Template removal from the active binding site. Acid Wash Extractions Measure approximately 200 mg of polymer sa mple. Place sample in 20 mL of a 1:1 solution of 1 M HCl: H 2 O. Let stir for 30 minutes (agitate occasionally if a rotating apparatus is not available). Decant off solution and take read ing on the phosphate meter. Repeat process two more times. Add 20 mL of DI water. Adjust to pH = 7. Let stir for 30 minutes. Filter particles, rinse with neutral DI water, a nd dry particles overnight in a vacuum oven. Particles are then used for phosphate uptake trials. 73

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Results Water-in-oil emulsions have proven to be a relatively simple and straightforward method used to prepare crosslinke d gelatin microspheres. Gelatin Inverse Emulsions Initially, gelatin microspheres prepared via w/o emulsions were synthesized and evaluated for compositional informati on using SEM and ATR-IR. These non-crosslinked samples dissolved during the in-vitro testing at 37 o C, as expected. Figure 5-4. SEM and corresponding EDS analys is of raw Type B (bovine) gelatin. 74

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SEM analysis provided a sound survey of th e change in the size and morphology of the gelatin as it was exposed to different proces sing conditions. One noti ceable feature was the decrease in particle size from the raw gelatin to the gelatin spheres. This could be attributed to the fact that the gelatin was dissolved in a hot water medium and placed in hot oil under turbid stirring conditions. 20 m 250 m Figure 5-5. SEM and correspondi ng EDS analysis of gelatin micr ospheres prepared via water in oil emulsion. 75

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250 m 20 m Figure 5-6. SEM and corresponding EDS analysis of gelatin microspheres containing phosphate, prepared via water-in-oil emulsion polymerization. This procedure allowed the formation of a n ew gelatinous material whose size could be altered. Also, the microspheres that contai ned phosphate had seve ral puckered looking spheres, which may have been the result of bound phosphate ions in the gelatin chain. EDS, while indicating the presence of phosphate in the gelatin containing polymer 76

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particles, did little more than confirm the presence of a larg e amount of carbon and oxygen. Therefore, EDS analysis was discontinued. IR spectral analysis was used to monitor the change in the gelatin structure as it is processed. In general, the amide bonds in protein -helices absorb around 1660 cm -1 sheets at 1640 cm -1 and near 1675 cm -1 -turns in the range 1695 cm -1 and unordered structures and -helices at 1650 cm -1 100 Therefore, the amide I band region ( 1650 cm -1 ) was closely monitored for possible change s in the gelatin sec ondary structure as different processing methods and crosslinki ng agents were used. The amide II band ( 1550 cm 1 ) positions were analyzed to get informa tion about the environment and hydrogen-bonding characteristics of the peptide CNH bonds. The amide III band ( 1240 cm -1 ) intensity was monitored as an indication of intermolecula r associations between the gelatin and the crosslinking agent. 90 91 92 93 94 95 96 97 98 99 100 650 1150 1650 2150 2650 3150 3650 Wavenumbers (cm-1)% Transmittanc e Figure 5-8. FTIR spectrum of unpro cessed Type B (bovine) gelatin. 77

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The raw Type B (bovine) gelatin show ed characteristic peaks at 3514 cm -1 3313 cm and 3071cm -1 due to -NH stretching of secondary amide. The amide I band region contains amide carbonyl C=O stretching at 1680 cm and 1630 cm with some contribution of C-N stretching and C-C-N deformation. The amide II region has -NH bending at 1526 cm and C-N stretching at 1542 cm There is also -NH out -of-plane wagging at 670 cm C-H stretching at 2924 cm and 2853 cm C-C stretching and C=N ring stretching at 1452 cm -1 (Figure 5-8). It can be observed from the spectrum of th e processed gelatin spheres in Figure 5-9, that the peaks remain similar to those of the unproces sed particles, indicating no apparent change in structure. The gelatin microspheres containing phosphate in Figure 510 illustrate the first noticeable difference in the increase of the p eak intensity, and the peak at 1030 cm confirms the presence of the phosphate ion. 99 99.5 100 100.5 101 101.5 102 102.5 650 1150 1650 2150 2650 3150 3650 Wavenumbers (cm-1)% Transm ittanc e Figure 5-9. FTIR spectrum of gelatin microspheres via water-in-oil emulsion. 78

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65 70 75 80 85 90 95 100 105 650 1300 1950 2600 3250 3900 Wavenumbers (cm-1)% Transmittance Figure 5-10. FTIR spectrum of gelatin microspheres containing phosphate via water-in-oil emulsion. Figure 5-10 showed that there was a noticeabl e increase in the intensity of the spectral peaks when the phosphate was added to the gela tin. This was rather unexpected since the presence of template molecules should have a minimal effect on a non-cr osslinked structure. The gelatin content was anticipated to be simila r. This difference could simply be due to a difference in contact of the sample with the crystal on the IR. Genipin Crosslinked Gelatin Polymerization Gelatin was crosslinked with 2%, 15%, and 75% genipin in order to pr obe the stability of the particle. The physical appear ance of the resulting crosslinke d spheres varied. When using 2% genipin, the particles we re cloudy blue, semi-opaque, gel-like particles. The 15% crosslinked particles were dark bl ue and spherical, and the 75% pa rticles were dark blue, small, 79

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and very rigid. Upon washing with a 1:1 mixture of HCl : DI water, the 2% particles dissolved, while the 15% and 75% particles remained intact, though hydrated. Figure 5-11. Gelatin microspheres crosslinked wi th 15% genipin without phosphate (top): before (right) and after (left) phosphate testing, and with phos phate (bottom): before (right) and after (left) phosphate testing. 100 m 100 m 100 m 20 m Genipin crosslinked microspheres FTIR spectra indicated an incr ease of the ratio between the amide I (1627 cm-1) and amide II (1532 cm-1), and a C-O vibration around 1080 cm-1, which are all indicative of crosslinking in the sample. Typical gelatin peaks remained, however the intensity of the spectra incr ease. The peak at 1030 cm confirmed the presence of the phosphate ion. 80

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88 90 92 94 96 98 100 102 650 1150 1650 2150 2650 3150 3650 Wavenumber (cm-1)% Transmittanc e Figure 5-12. FTIR spectrum of genipin crosslinked gelatin microspheres. 99 99.5 100 100.5 101 101.5 650 1150 1650 2150 2650 3150 3650 Wavenumber (cm-1)% Transmittance Figure 5-13. FTIR spectrum of genipin cross linked gelatin microspheres containing phosphate. Glutaraldehyde Crosslinked Gelatin The glutaraldehyde crosslinked gelatin micr ospheres were confirme d by the presence of characteristic peaks at 2925 cm -1 and 1744 cm -1 which represent C-H stretching of methyl and 81

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methylene groups, and C=O stretching 101 respectively. The CO vibration around 1080 cm -1 is indicative of crosslinking in the sample. Typical gelatin peaks remained, however the intensity of the spectra increase. The peak at 1030 cm confirms the presence of the phosphate ion. 88 90 92 94 96 98 100 102 650 1150 1650 2150 2650 3150 3650 Wavenumber (cm-1)% Transmittanc e Figure 5-14. FTIR spectrum of glutaraldehyde crosslinked gela tin without phosphate (top), and with phosphate (bottom). Many of the subsequent spectra taken were ve ry similar in appearance, due to the strong presence of the gelatin bands, with slight differen ces in characteristic peak appearance. For this reason, the band appearance will be reported for each sample, but the FTIR spectra will not be presented. Dextran Crosslinked Gelatin Typical dextran spectra exhibit wide absorp tion bands in the region of vibrations of hydroxyl groups (3700-3000 cm -l ), and a band of stretching vibrations of CH-groups with the main maximum at 2920 cm -1 The dextran crosslinked gelati n microspheres were confirmed by the presence of hydroxyl groups around 3080 cm -1 and 3007 cm -1 and characteristic peaks of 82

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gelatin, however the intensity of the spectra increase. The presence of phosphate was indicated by a peak at 1015 cm -1 Glyceraldehyde Crosslinked Gelatin The typical spectra of glyceraldehyde displa y absorption bands in the region of 2956, 2924 and 2855 cm -1 corresponding to C-H stretch vibration bands, and a C=O band at 1743 cm -l The glyceraldehyde crosslinked gela tin microspheres were confirmed by the presence of peaks at 2925 cm -1 and 1744 cm -1 which represent C-H stretching of methyl and methylene groups, and C=O stretching, respectively. The C-O vibration around 1080 cm -1 is indicative of crosslinking in the sample. Characteristic peaks of gelatin were displayed, however the intensity of the spectra increase. The presence of phospha te was indicated by a peak at 1015 cm -1 Phosphate uptake trials were performed in the usual manner. A stock solution of 20 mM phosphate, 80 mM NaCl, and 30 mM Na 2 CO 3 was made to mimic conditions in the small intestine environment, and adjusted to pH = 7.4 to replicate phys iological conditions. Microspheres were exposed to this solution for 3-48 hours at 37 o C. Table 5-1 displays results for phosphate uptake in the gelatin polymer s. Each reported value represents the average of 3 samples. Overall, the results de monstrate no significant phosphate uptake by the polymers crosslinked using various reagents. Conclusions The 2% genipin crosslinked samples did not have enough crosslinker in them to remain intact for these studies. Thus, upon heating, they simply became extremely hydrated and microsphere swelling occurred so much so, that the matrix formed a hydrogel like material, which hindered mixing during the ph osphate uptake trials. The 75% crosslinked samples were crosslinked at such a high degree, that the matr ix is too closed in, which actually restricts the phosphate ion from entering. Also, using such an excess of genipin makes the production of 83

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these polymers cost prohibitive for the intended applications. The results obtained using the 15% genipin crosslinked st ructure appears to be a feasible line to continue. Though appreciable phosphate uptake does not occur, the physical charac teristics indicate that it may be possible to manipulate the surface of this polymer in future experiments. Table 5-1. Phosphate uptake in gelatin crosslinked microspheres that have been either acid or base washed prior to trials. Sample Acid treatment Base treatment 48 hour uptake mEq / g polymer 2% Genipin swelled to a gel 2% Genipin swelled to a gel 2% Genipin w/ Phosphate swelled to a gel 2% Genipin w/ Phosphate swelled to a gel 15% Genipin 0.15 0.04 15% Genipin 0.14 0.06 15% Genipin w/ Phosphate 0.41 0.35 15% Genipin w/ Phosphate 0.08 0.07 75% Genipin 0.23 0.12 75% Genipin 0.00 75% Genipin w/ Phosphate 0.00 75% Genipin w/ Phosphate 0.00 25% Glutaraldehyde 0.45 0.24 25% Glutaraldehyde 0.43 0.45 25% Glutaraldehyde w/ Phosphate 0.73 0.35 25% Glutaraldehyde w/ Phosphate 0.83 0.14 Dextran dissolved in wash Dextran dissolved upon heating Dextran w/ Phosphate dissolved in wash Dextran w/ Phosphate dissolved upon heating Glyceraldehyde 0.00 Glyceraldehyde 0.00 Glyceraldehyde w/ Phosphate 0.90 .060 Glyceraldehyde w/ Phosphate 0.68 0.34 Glutaraldehyde and glyceral dehydes crosslinked spheres expressed a trend towards increased levels of phosphate binding in the samp les prepared with the phosphate imprints, thus, these materials will be further surface modified as well. 84

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The dextran crosslinked samples most likely undergo hydrolysis during the base wash and heating of the acid treated samples, which is why they dissolve. It is likely that this polymer is unsuitable for any type of heat processing, even if the surface could be favorably modified for binding phosphate ions. 85

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CHAPTER 6 MOLECULARLY IMPR INTED POLYMERS Microwave Fabricated Gelatin Micros pheres as Phosphate Binding Agents Biomimetic sensors can be tailored for use in-vivo and in-vitro. These mechanisms can be utilized in areas such as dete cting the presence of cells that cause tumors cancer chemotherapy, or monitoring the amount of glucose in the blood stream in diabetic patients. 40 The extensive list of in-vivo applications of MIPs, w ithout significant patient side effect s, show that it is feasible to design phosphate MIPs for oral administration fo r patients with chronic renal failure. A novel microwave-assisted technique has been developed for the synthesis of molecularly imprinted gelatin microparticles as oral phosphate binding agents on the order of 10 200 m, is described. After extraction of the template mo lecule, an imprint cavity of specific shape and memory remains in the polymer. Crosslinked Gelatin Microspheres via Microwave Radiation Thermal denaturation has been examined as a way in which proteins can be physically crosslinked. 102 Extended dehydration of ge latin at elevated temperat ures and reduced pressures have been known to induce the spontaneous formation of interchain amide links 103 within the matrix. Crosslinking occurs through a condensation reaction between a free carboxyl group on the gelatin backbone and an amino acid on an adjacent chain. However, crosslinking can take more than 24 hours under ambient conditions, wh ich can be accelerated by the use of microwave radiation. Previous studies have shown that gelatin can be thermally crosslinked after 10 minutes using a CEM microwave oven (model MAS 7000) with an inlet temperature of 250 o C 104 Such an oven was not available for experiments, so tr ials were simulated using a regular, department store purchased, small kitchen microwave (1 .2 kW Samsung Simply microwave oven, model 86

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#MW5470W). This procedure was developed to investigate the synthesis of molecularly imprinted polymers via microwave treatment, in order to decrease the synthetic reaction time in comparison to standard step-growth or radical polymerization procedures. 105 With success, an alternative synthetic route was provided that is cheaper, more time efficient, and can be performed under a gentler chemical environment as compared to traditional schemes. A major benefit of this particular regime was that particles prepared this way may allow increased imprinted sites for binding. However, in order to comply with the ideal of highly crosslinked MIPs, crosslinking agents will be used as well. Microwave Treatment 500 mg of gelatin microspheres, produced as pr eviously described, were placed in a glass vial containing 2 ml of a 1:1 (v/v) mixture of acetone : crosslinking agen t (a polar medium was used to obtain uniformity of microwave energy during the trial). The vial was placed in a microwave for the determined amount of time ( 2 minutes), with a glass microscope slide loosely placed on top, as to prevent total solven t evaporation. The microspheres were collected and washed five times in 10 ml acetone, or until the supernatant was clear (cloudy indicates excess glutaraldehyde still being released). Th e particles were then washed once with 1:1 solution of 0.1 M NaOH: MeOH, three times with DI water, filtered, and rinsed with acetone. The spheres were dried at room temperature, in-v acuo, in order to remove any residual moisture. Results Gelatin Emulsion Microspheres Using plain gelatin spheres for the microwave tr eatment resulted in particles that appeared to have low crosslinking density character at about 25 minutes, and started to cook around 30 minutes. Spheres expanded, took on a rice cris py like appearance, a nd had a pungent odor. A 87

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slight decrease in swelling wa s apparent after 40 minutes of microwave time, and a definite crosslinked form occurred around 50 minutes. Th is was indicated by the slight darkening in color of the sphere which is associated with the slight reduction in partic le diameter. However, particles eventually swell upon long term exposure to water ( 24 hours). Thus, it was apparent that, in order to accomplish the synthesis of th is type of crosslinked gelatin without using a crosslinking agent, the microw ave technique reported in litera ture should probably be used. Therefore, since crosslinking c ould not be established using the Samsung microwave, the overall feasibility of crosslinking gelatin particles without the use of a chemical crosslinking agent could not be determined at this time Genipin and Glutaraldehyde Crosslinked Microspheres The experiment was repeated using genipin and glutaraldehyde as crosslinking agents. Noticeable crosslinking appeared to happen in le ss than 2 minutes using glutaraldehyde, which was associated by the change in color of the s pheres to burnt orange, and the emergence of a pungent smell. Visible crosslinking is notably minimal when micr owaving genipin for as long as 10 minutes, thus the production of these polymeric microspheres was discontinued. The glutaraldehyde crosslinked particles were processed using different amounts of crosslinking agent in order to determine if the appropriate amount to use for microsphere formation concurs with the established parameters detailed earlier in Chapter 4. Once the optimum amount of crosslinker to use was ascertained, it was es tablished as the standard for future experiments. SEM highlighted several intere sting morphological features that suggest phosphate binding in the microspheres (Figure 6-1). The microsphe res synthesized without phosphate appeared to be relatively smooth on the surface, even after they had been tested for phosphate uptake. This indicated minimal phosphate binding within the sa mple. Conversely, microspheres created with 88

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phosphate showed a textured surface prior to uptake trials, and an agglomeration of microstructures on the surface of pa rticles after uptake testing. Figure 6-1. SEM of gelatin crosslinked mi crospheres via microwave processing without phosphate (top): before (left) and after (right) phosphate testing, and with phosphate (bottom): before (left) and af ter (right) phosphate testing. The mechanism for phosphate binding in the gelatin microspheres for this study was proposed in Chapter 4. This could be further explained by comparing th e SEM in Figure 6-2 to the modeled system. Figure 62 illustrates th e way in which phosphate is bound by a protonated amine cage, and Figure 6-3 demonstrates multiple phosphates bound by the microsphere structure, which resemble the imprint sites for the template. It was reasonable to conclude that there were recognition sites within the polymer as well as on the surface. If phosphate was 89

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bound at the surface, it could function as a nucleati on site for additional phosphate ions to grow from. Individual phosphate ions are too small to be visible through SEM, however phosphate clusters at the surface of the microsphere will appear mineralized. This phenomena was seen in the images. Figure 6-2. Protonated amine cage (blue) surrounding a phosphate ion (red/orange). The glutaraldehyde crosslinked gelatin micr ospheres were confirme d by the presence of characteristic peaks at 2925 cm -1 and 1744 cm -1 which represent C-H stretching of methyl and methylene groups, and C=O stretching 101 respectively. The CO vibration around 1080 cm -1 is indicative of crosslinking in the sample. Typical gelatin peaks at 1630 cm -1 1535 cm -1 and 1237 cm -1 (amide I, amide II, and amide III, respectively) were represented in the spectra, and the presence of phosphate is shown around 1030 cm -1 The microspheres that were acid washed prior to phosphate uptake testing had largely similar IR spectra, however there were small band shifts, which may be attributed to the presence of phosphate. 90

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Figure 6-3. Modeled system of the phosphate binding polymer. 95.5 96 96.5 97 97.5 98 98.5 99 99.5 100 100.5 650 1150 1650 2150 2650 3150 3650 Wavenumber (cm-1)% Transmittanc e Figure 6-4. FTIR spectrum of gl utaraldehyde crosslinked gelatin, via microwave irradiation: without phosphate (top), and with phosphate (bottom). 91

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The microspheres that were base washed pr ior to phosphate uptake testing had largely similar IR spectra. The peaks for the particles that did not contain phosphate appear less intense in comparison to those that did contain phosphate however peak ratio an alysis indicated that they there was no significant difference in the spectra. Phosphate uptake trials were performed in the usual manner. A stock solution of 20 mM phosphate, 80 mM NaCl, and 30 mM Na 2 CO 3 was made to mimic conditions in the small intestine environment, and adjusted to pH = 7.4 to replicate phys iological conditions. Microspheres were exposed to this solution for 3-48 hours at 37 o C. Table 6-1. Phosphate uptake in microwave irradi ated gelatin crosslinked microspheres that have been either acid or base washed prior to trials. Sample Acid treatment Base treatment 48 hour uptake mEq / g polymer 10% Glutaraldehyde 0.20 0.04 10% Glutaraldehyde 0.61 0.54 10% Glutaraldehyde w/ Phosphate 1.05 0.85 10% Glutaraldehyde w/ Phosphate 0.62 0.40 25% Glutaraldehyde 0.23 0.32 25% Glutaraldehyde 0.39 0.24 25% Glutaraldehyde w/ Phosphate 1.98 1.38 25% Glutaraldehyde w/ Phosphate 0.95 .38 40% Glutaraldehyde 0.45 0.24 40% Glutaraldehyde 0.60 0.13 40% Glutaraldehyde w/ Phosphate 0.82 0.68 40% Glutaraldehyde w/ Phosphate 1.07 0.24 Table 6-1 displays results for phosphate uptake in the gelatin polymer s. Each reported value represents the average of 3 samples. Conclusions There were several noticeable trends that emerged for the microspheres after phosphate testing. First, the phosphate binding capacity increased as the amount of crosslinking agent increases. This was accurate in terms of informa tion represented in the literature. In order to produce an efficient MIP, a high degree of cro sslinking must be present such that a non92

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destructible template cavity is formed that allows multiple binding and extraction events. However, polymers that are too highly crosslinked restrict template recogn ition, and crosslinking that is too low to not provide a mechanic ally stable enough environment for template sequestration. The 40% w/w glutaraldehyde com position seemed to provide a feasible formula for creating MIPs via microwav e irradiation. Though there was a great degree of error associated with the 25% w/w acid treated sample, the uptake was so great, that it could not be ruled out as an insignif icant result. Second, the based wa shed microspheres displayed overall enhanced phosphate binding in comparison to their acid washed counterparts. This indicated that the base wash provided an environment that wa s more conducive to removing the phosphate ions from the imprinted polymer, thus providing additi onal available binding s ites for ion extraction from solution. 93

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CHAPTER 7 GUANYLATED POLYMERS This chapter describes a technique for th e synthesis of novel guanylated gelatin microparticles as oral phosphate binding agents Using modified processing schemes, various biocompatible crosslinking agents, and high affi nity phosphate binding functional groups, highly crosslinked, tailored, gelatin microspheres on the order of 10 200 m were produced. Previous experiments performed involving the use of gelatin, crosslinked with either genipin, glutaraldehyde, glycer aldehyde, or dextran have shown limited phosphate binding capacity during uptake trials. Thus, the need for adding a f unctional group to the gelatin backbone to increase phosphate uptake became a forefront consideration. To address this issue, a guanidine derivative was chosen to structurally modify the gelatin microspheres. Guanylating reagents are compounds that chemically transform a specific reactive group, in this case an amine, into guanidine. Th is reaction can take place in organic solvents as well as water, and usually under relatively mild conditions. One major set back with the use of guanidines is that they are highly basic, with a pKa of 12.5, and highly nucleophilic. 106 However, this same feature allows them to be fully protonated under physiological conditions 107 which provides a foundation for its ligand-receptor interaction. Recent applications of guanidine derivates include biocidal polymers, 108 bile acid sequestrants, 109 and potential gene delivery carriers. 107 There has also been interest and some success in using amidinium based m onomers in molecular imprinting 110-113 for phosphonates. 1-H-pyrazole-1-carboxamidine hydrochloride was chosen as the guanidinating reagent for the direct preparation a gelati n-guanidinium compound, which reacts with an amine group on the polymer according to the following general scheme in Figure 7-1. The primary amines are readily converted, while sec ondary amines usually require heat to initiate guanylation. 114 The 94

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pyrazole byproduct is soluble in water, which allows the insoluble guanylated polymer to be easily removed. Figure 7-1. Synthetic route used to obtain a guanylated polymer. In turn, the guanidine group can form a str ong association with th e external oxygen groups on the phosphate ion, which make binding possi ble. The use of guanidinium groups for oxyanion recognition and binding 115 has attracted a great deal of interest over the past years. Their ability to sequester these ions thr ough cationic and hydrogen bonding through Zwitterionic interactions make them unique biocompatible binding agents. Figure 7-2. Zwitterionic interacti on of phosphate with a guanyl group. The synthesis of low molecular weight gua nidinium compounds has been extensively reported in literature. However, there is onl y a small body of work that has inspected the synthesis of polymeric guanidinium salts. Functionalized Gelatin Microspheres via Guanylation The first step in this synthetic route was to prepare previously descri bed crosslinked gelatin microparticles for surface functionalization. Du e to the basic nature if the 1-H-pyrazole-195

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carboxamidine, the spheres were first base treat ed which resulted in a freebase surface environment that was more amenable to the high pKa of the guanylating agent. There were two approaches to particle synthe sis: noncrosslinked gelatin mi crospheres were prepared for guanylation modification and subseq uently crosslinked for molecular imprinting, and the already crosslinked gelatin particles were surface gua nylated. SEM and IR were used for surface morphology and bond characterization. In the typical IR spectrum of guanineHCl, bands at 2725 cm -1 2520 cm -1 and 2485 cm -1 represent N-H stretching of weak NH + -Cl intermolecular hydrogen bonds. 116 The absence of these characteristic peaks i ndicated molecular conversion. Imprinted Gelatin Microspheres Room Temperature Guanylation A freebase sample of uncrosslinked gela tin was prepared by placing 1.5 g of the microspheres in 30 mL of acetone, and NaOH was used to adjust the solution to a pH of 12. The solution was stirred for 1 hour, rinsed 3 times with DI water, and dried overnight in vacuo. A cloudy white solution of 20 mM 1-Hpyrazole-1-carboxamidine: 10 mM KH 2 PO 4 in a water : acetone (1:2) mixture was adjusted to pH = 7.4, and stirred for 3 hours under nitrogen in order to form a complex between the guanidine and the ph osphate. The freebase gelatin particles were added to the guanidine mixture, which was purged with nitrogen and turned clear after 1 hour. The reaction was allowed to stir for 24 hours at room temperature, under a nitrogen stream, cooled to room temperature, filtered, and wash ed 3 times for 20 minutes in 25 mL acetone. The sample was then washed with a 1:1 solution of 0.1 M NaOH: MeOH, neutra lized with DI water, filtered, and dried in vacuo. The final product was off white and spherical. In addition, this sample was further processed by crosslinking. 96

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Heat Assisted Guanylation A freebase sample of uncrosslinked gela tin was prepared by placing 1.5 g of the microspheres in 30 mL of acetone, and NaOH was used to adjust the solution to a pH of 12. The solution was stirred for 1 hour, rinsed 3 times with DI water, and dried overnight in vacuo. A cloudy white solution of 20 mM 1-Hpyrazole-1-carboxamidine: 10 mM KH 2 PO 4 in a water: acetone (1:2) mixture was adjusted to pH = 7.4, and stirred for 3 hours under nitrogen in order to form a complex between the guanidine and the ph osphate. The freebase gelatin particles were added to the guanidine mixture, which was purged with nitrogen and turned clear after 1 hour. The reaction was allowed to stir for 24 hours at 60 o C, under a nitrogen stream, cooled to room temperature, filtered, and washed 3 times for 20 minutes in 25 mL acetone. The sample was then washed with a 1:1 solution of 0.1 M NaOH: MeOH, neutralized with DI wate r, filtered, and dried in vacuo. The final product was off white and sp herical. In addition, this sample was further processed by crosslinking. Surface Guanylated Gelatin Microspheres The surface guanylated gelatin microspheres we re prepared both with and without the introduction of heat, as described in the prev ious section. Howeve r, crosslinked spheres synthesized according to the descriptions in Chapters 5 and 6 were used instead of the uncrosslinked gelatin microspheres. These guanyla ted moieties are further treated with an acid or base to remove the template as described in Chapter 5. Results Imprinted Polymers Uncrosslinked gelatin microspheres were prepared and guanylated so that they could be further crosslinked and processed as MIPs. Batches of control gelatin microspheres, which did not contain phosphate, and sphere s that contained the phosphate template molecule, were 97

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synthesized. Because the uncrosslinked microspheres are susceptible to hydrolysis and degradation under heat, these batches were guanyla ted at room temperature. For this same reason, in-vitro phosphate uptake testing was not possible. As a result, these materials were probed using SEM and FTIR instruments. 20 m 250 m Figure 7-3. SEM images of uncrosslinked gelati n, native (left), guanyla ted without phosphate (middle), and guanylated with phosphate (right). Bottom images are magnified versions of top images. SEM analysis provided visual evidence concerning the modificat ion of gelatin side chains using guanylating agents. Raw, uncrosslinked gelatin spheres have relatively smooth surfaces in comparison to the gelatin microspheres that ha ve been guanylated, which have rough and bumpy surfaces. The guanylated particles that are s upposed to have bound phosphate in them had a rough and bumpy surface as well, however the magnification of these samples clearly show the presence of crystal shaped rods on the surface, which were believed to be phosphate. The SEM images are taken of microspheres that have been used in phosphate uptake trials, and subsequently air dried. Thus, the bound phosphate in solution becomes more concentrated at the 98

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surfac el ate crystals. It is proposed o Figure 7-4 and a guanine derivative self a ssembly into (a) G-ribbon, (b) G-quartet, and (c) Grartets the hich prom ote formation and increa se stability, and the e G-rich moieties. GlutaThe uncrosslinked, guanylated gelatin microsphe res were further processed by crosslinking e, which contributes to the amount of crystals that are imaged. This visual phenomenon can be explained by the suggested phospha te ion binding mod proposed in Chapter 3. With the proposed cl ose proximities of all of the phosphate binding events (Figure 7-4) it is possible that there ar e template ion cages th at stack on top of each other, thus promoting the nucle ation and upward growth of phosph that these cages can also orientat e themselves to f rm G quartets. Predicted proximity of 4 phosphate ions within the pol ymer structure (right), quadruplex formed by stacking of G-quartets around a column of cations. 117 DNA and RNA are guanine rich, which allows them to form interand intramolecular fou stranded structures, referred to as G-quartets. 118, 119 G-quartets evolve from the association of four guanine bases into a cyclic Hoogsteen H-bonding arrangement (Figure 7-4). The G-qu stack on top of each other to give rise to tetrad-helical structures, which would explain the presence of the phosphate crystal structures. Th e stability of G-quartet structures depends on presence of the monovalent cations, w concentration of th raldehyde 99

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Figure 75. Glutaraldehyde crosslinked gelatin microsphe res prepared via microwave irradiation, room temperature surface gua nylation: after guanylation (left), after template extraction (middle), and after phosphate uptake testing (right). Without phosphate (top), and with phosphate (bottom). Figure 7-6. Glutaraldehyde crosslinked gelatin microsphe res prepared via microwave irradiation, heat initiated surface guanylation: after guany lation (left), after template extraction (middle), and after phosphate upt ake testing (right). Without phosphate (top), and with phosphate (bottom). 100

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with glutaraldehyde according to the microwave irradiation proce res. The spheres hav the change in microsphere m dure (Chapter 6). Resultant polymeric particles were dark burnt orange sphe e a translucent off-white surface hue. We used SEM to visually confirm orphology as it is processed late extraction, and ul timately tested in a phosphate ion solution. ere the template was extracted, which suggested was successful. The vi sible crystals on the surface of the particles phosphate, which was most likely both bound by the gelatin polymer bed on the surface. Surface Modified Crosslinked Polymers Samp without the section. ng ing to the reverse emulsion procedure that was thoroughly described in Chap wed after template extraction had relatively was not a considerable change in the surface struct phosphate. from guanylation to temp Pitting is present in several of the samples wh that phosphate ion binding represented the presence of sphere as well as physisor les of gelatin crosslinked microspheres were taken and guanylated either with or use of heat, as de tailed in the above Genipin The uncrosslinked, guanylated gelatin microsphe res were further processed by crosslinki with genipin accord ter 5. The resultant polymeric particles were dark teal, w ith either spherical or chunk like shapes. Glutaraldehyde SEM was used to visually confirm the ch ange in microsphere morphology as it is processed from guanylation to template extractio n, and ultimately tested in a phosphate ion solution. Samples that were guanylated or vie smooth surfaces, which indicated that there ure of the sample between processing step s. The visible crystals on the surface of the particles after uptake testing repr esented the presence of 101

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polymerization, room temperature surface gua nylation: after guanyl ation (left), after esting (right). Without phosphate (top), and with phosphate (bottom). Figure 7-7. Glutaraldehyde crosslinked gelatin microsphe res prepared via emulsion template extraction (middle), and after phosphate uptake t Figure 7-8. Glutaraldehyde crosslinked gelatin microsphe res prepared via emulsion polymerization, heat initiate d surface guanylation: after guanylation (left) and after template extraction (right). Without phos phate (top), and with phosphate (bottom). 102

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In samples tals most likely is the result of physis orption. In samples that did contain phosphate in the original st likely due to phosphate both bound by the gelatin polymer sphere as well as phGlyceraldehyde that did not contain phosphate in the or iginal composition, the presence of the crys composition, the presence of the crystals was mo ysisorbed on the surface. 70 75 80 85 90 95 100 105%Transmittanc e Room Temp Heat Room Temp + P 650 11501650 2150 26503150 3650 Wavenumber (cm-1) Heat + Pe d by the increase in intensity in the 2925 cm-1 peak, which represented C-H stretching of methyl and methylene groups, and C=O stretching, respectively. Th e C-O vibration around 1080 cm-1 is indicative of crosslinking in the sample. Characteristic peak s of gelatin are present. Phosphate uptake trials were performed in the usual manner. A stock solution of 20 mM phosphate, 80 mM NaCl, and 30 mM Na2CO3 was made to mimic conditions in the small intestine environment, and adjusted to pH = 7.4 to replicate phys iological conditions. Microspheres were exposed to this solution for 3-48 hours at 37 oC. Figure 7-9. FTIR spectrum of su rface guanylated gelatin cross linked with glyceraldehyde. The typical spectra of glyceraldehyde displa y absorption bands in the region of 2924 and 2855 cm -1 corresponding to C-H stretch vibr ation bands, and a C=O band at 1743 cm -l The glyceraldehyde crosslinke d gelatin microspheres were confirm 103

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Conclusions Microspheres that were glut araldehyde crosslinked using microwave irradiation were processed to produce a molecularly imprinted product. Though phosphate uptake occurred in these samples, the variability in the heat tr eated trials was great, and the significant error associated with the standard deviations demonstrat ed that this procedure should be discontinued. However, room temperature guanylated micros pheres indicate reasonable phosphate binding in the 25% crosslinked batch, even though uptake is below the previously established minimum success value. This indicated that polymer imprinting is achieved through this synthetic pathway. Template recognition may have been hindered by probable small size, buried position, lems with M re The r d ples support the data. A markable physical feature of th e samples after phosphate testing is that the impri re, and or spatially restricted pos ition of the imprinted site, whic h are all reported common prob IP rebinding studies. Glutaraldehyde crosslinked microspheres, prep ared by an inverse emulsion process, we surface guanylated and the phosphate binding capacity was measured. Interestingly enough, the heat treated microspheres were able to attract pho sphate to a greater degree in comparison to the room temperature processed particles. In a ddition, the heat processed samples containing phosphate had less phosphate uptake in them than di d the samples that did not contain phosphate. everse trend was seen in the ro om temperature processed samples. Table 7-1 displays results for phosphate uptake in the gelatin polymer s. Each reporte value represents the average of 3 samples. SEM images of the guanylated glutaraldehyde crosslinked sam particularly re nted microspheres appeared to have phosphat e emanating from within the microstructu and the surface modified spheres appeared to ha ve phosphate attached onto the surface. This would verify the viability of imprin ted and surface templated techniques. 104

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Table 7-1. Phosphate uptake in surface func tionalized and molecularly imprinted gelatin Sample Room Heat 48 hour uptake crosslinked microspheres prepared by e ither emulsion or microwave irradiated. Temperature mEq / g polymer Emulsion Processed 15% Genipin 0.62 0.09 15% Genipin 0.14 0.14 15% Genipin w/ Phosphate 1.19 0.18 15% Genipin w/ Phosphate 0.32 0.05 25% Glutaraldehyde 0.68 0.26 25% Glutaraldehyde 1.08 0.02 25% Glutaraldehyde w/ Phosphate 0.90 0.23 25% Glutaraldehyde w/ Phosp hate 0.63 0.17 0.73 0.21 Glyceraldehyde Glyce raldehyde 0.34 0.17 Glyceraldehyde w/ Phosphate 0.79 0.48 Glyceraldehyde w/ Phosphate 0.11 0.07 Microwave Irradiated 10% Glutaraldehyde 0.87 0.42 10% Glutaraldehyde 1.07 0.1 6 10% Glutaraldehyde w/ Phosphate 0.85 0.29 10% Glutaraldehyde w/ Phosphate 1.78 1.62 25% Glutaraldehyde 0.21 0.15 25% Glutaraldehyde 0.85 0.55 25% Glutaraldehyde w/ Phosphate 0.62 0.11 25% G lutaraldehyde w/ Phosphate 0.74 0.09 40% Glutaraldehyde 0.54 0.42 0.29 0.17 40% Glutaraldehyde 40% Glutaraldehyde w/ Phosphate 0.69 0.31 40% G lutaraldehyde w/ Phosphate 1.21 1.21 d gua nylated using heat. ot a difference between microspheres ntain the tee ion in co Though there was no appreciable phosphate up take in the genipin samples guanylate using heat, results indicate that gelatin spheres guanylated at room temperature show successful phosphate binding. There was minimal phosphate uptake in the gl yceraldehyde samples The gelatin spheres guanylated at room temperat ure show phosphate binding, but there is n much of that c o mplat mparison to those 105

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that do not. Ph osphate uptake is below the mi mum established value if signifi y his hypothesis wa not tested. ni cance, but ma be more selective, although t s 106

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CHAPER 8 DISCUSSION Synopsis Functionalized gelatin microsphere s were designed to address the need for a cost effective alternative for phosphate removal in aqueous sy stems, specifically for patients with ESRD, where hyperphosphatemia is a common physiological encumbrance. To the best of this authors knowledge, previ ous work has not been reported in literature concerning the development of these novel polymers. Various synthetic mechanisms were proposed and evaluated in order to derive a method to develop a robust material that could effectiv ely bind phosphate. At the conclusion of each synthetic scheme, the attributes of the polymer s that had the highest binding capacity were incorporated into the next design, as to deve lop a rigorous procedure for the development of tailored gelatin microspheres that u tilized the best processing parameters. There were two hypothesis that were tested : Could a surface functionalized gelatin polymer for phosphate removal in aqueous system s be developed and characterized, and could a molecularly imprinted polymer be developed and characterized for phosphate removal in aqueous systems. Each synthetic route investig ated is illustrated in Figures 8-1 and 8-2. The efficacy of phosphate uptake with these functionalized polymers was determined invitro, and three samples were identified as a synthe tic success due to their statistical significance verified at p 0.05. These polymeric microspheres did no t degrade in an artificial environment created to simulate the physiologi cal conditions of the ga strointestinal tract, which indicated their possible success for in-vivo applications. Initial trials employed the use of genipin as a crosslinking ag ent. While crosslinking was easily accomplished, phosphate uptake was minimal. In an effort to establish the feasibility of T 107

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Can gelatin microspheres for phosphate removal be synthesized? Microwave Water in oil Treatment Glutaraldehyde Crosslinking agent Genipin Crosslinking agent emulsion Glutaraldehyde crosslinking agent Genipin crosslinking agent Acid wash vs. Base wash Guanylate at RT vs. Guanylate using heat Acid wash vs. Base wash Guanylate at RT vs. Guanylate using heat Acid w vs Base ash wash Guanylate at RT vs. Guanylate using heat Acid wash vs. Base wash Guanylate at RT vs. Guanylate using heat Figur es. Figure 8-2. The synthetic pathway followed for the development of the molecularly imprinted Glyceraldehyde crosslinking agent Dextran crosslinking agent Acid wash vs. Base wash Guany Gu late at RT vs. anylate using Acid wash vs. Base wash Guanylate at RT vs. Guanylate using e 8-1. The synthetic pathway followed for the development of the gelatin microspher Can a MIP for phosphate removal synthesized? using gelatin be Microwave Treatment Water in oil emulsion gelatin microspheres. Glutaraldehyde Glutaraldehyde Crosslinking agent Genipin Crosslinking Crosslinking agent agent Acid wash Guanylate at RT Acid wash ash Guanylate at RT vs. Guanylate using heat Acid wash vs. Base wash Guanylate at RT vs. Guanylate using heat vs. Base wash vs. Guanylate using heat vs Base w 108

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using gelatin polymers as phosphate binders, add itional crosslinker were investigated. These crosslinking agents chosen were glutaraldehyde, glyceraldehyde, and dextran, due to their biocompatibility and various appli cations in crosslinking gelatin. From these results, samples were selected for complete evaluation that represented the best of the acid washed, base washed, room temper ature guanylated, and heat assisted guanylated trials. The samples assessed were as follows: 1. Acid Washed: 25% glutaraldehyde crosslinked gela tin, microwave treated, with and without phosphate 15% genipin crosslinked gelatin, with and without phosphate 2. Base Washed: 25% glutaraldehyde crosslinked gela tin, microwave treated, with and without phosphate 15% genipin crosslinked gelatin, with and without phosphate Guanylation: and without phosphate 3. Room Temperature 25% glutaraldehyde crosslinked gela tin, microwave treated, with 15% genipin crosslinked gelatin, with and without phosphate 4. Heat Assisted 25% glutaraldehyde crosslinked gela tin, microwave treated, with Guanylation: and without phosphate 15% genipin crosslinked gelatin, with and without phosphate Each polymer set included a c ontrol, which was the crosslinked polymer, and a sample of the crosslinked polymer with phosphat e as an initial reactant. Samp les were tested in triplicate, and the average and standard deviation values were calculated using a p = 0.05 statistical significance limit obtained from a statistical two tailed T-test. 109

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The following figures summarize the body of da ta collected from all of the conducted experiments. 0 0.5 1 1.5 2 2.5 3.5m/g p 3P microwavemicrowave microwavemicrowave microwavemicrowaveUptake (Eqol) Figure 8-3. Phosphate uptake 4 15% Genipin 15% Genipin + 10% Glut. + 10% Glut. + 25% Glut. + 25% Glut. + 40% Glut. + 40% Glut. + + P + P + P Sample results for acid washed polymeric samples synthesized without and with the phosphate template ion. 0 2 0.2 15% Genipin 25% Glut. w/o emuls. 10% Glut. + microwave 25% Glut. + microwave 40% Glut. + microwave 0. 0. 4 6 1 1.4 1.8 SampleUptake (mEq/g pol) Figure 8-4. Phosphate uptake re sults for base washed polymeric samples synthesized without (left) and with (right) the phosphate template ion. 1.6 1.2 0.8 110

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0 0.2 0.4 0.6 0.8 1 1.2 1.4 15% Genipin 25% Glut. w/o emuls. 10% Glut. + microwave 25% Glut. + microwave 40% Glut. + microwave SampleUptake (Eq/g pol) Figure 8-5. Phosphate uptake results for room temperature guanylated polymeric samples synthesized without (left) and with (right) the phosphate template ion. m Figure 8-6. Phosphate uptake re sults for acid washed polymeric samples synthesized without (left) and with (right) the phosphate template ion. 111

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Conclusions The 15% genipin crosslinked polymers show ed minimal phosphate uptake in most samples, except for those guanylated at room te mperature. The room temperature guanylated polymeric microspheres reported values that were statistically significant, which indicate the successful guanylation of the arginine groups in the crosslinked polymer. Figure 8-7. Phosphate uptake in surface functio nalized polymers using genipin as a crosslinking agent. These microspheres prepared by a simple wa ter-in-oil type emulsion process and surface functionalized with guanine groups if desired. However, this did not account for the fact that, on average, the particles synthesized with phosphate as an initial component had a slightly higher phosphate ion concentration in comparison to thei r counterpart control particles. Thus, this feature must be attributed to th e fact that the phosphate in the s phere is extracted in a manner in which cavities are created in which phosphate can be subsequently bound. The room temperature guanylated microspheres report significantly significant data. 112

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The samples crosslinked with glutar icrowave irradiation are believed to be molec in aldehyde vi a m ularly imprinted polymer microspheres, wh ich would explain the average difference phosphate uptake between the samples cont aining phosphate, and those without. Figure 8-8. Phosphate uptake in surface functionalized polymers using glutaraldehyde as a crosslinking agent. Crosslinking may have created rigid pores that are amenable to binding phosphate in microwave treatment initiates crosslinking in a wa y in which many of the arginine groups on the gelatin structure are polymer bound, thus are not as available for subsequent guanylation at room temperature in comparison to the heat assisted process. Statistically significant results were calcu lated for both room temperature and heat processed guanylated polymers. The majority of the reported phosphate upt ake success results evaluated were centered about the genipin and glutaralde hyde processed gelatin microsphere s. The different chemical crosslinking mechanisms with gelatin could e xplain the increased stab ility and capacity to structurally manipulate the samples within this subset. solution after an acid or base extraction of the in itial phosphate molecule. It is possible that the 113

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The entire body of data colle cted indicated that, in most cases, an overall phosphate binding increase in samples that contained phosphate in comparison to their control counterpa One possible explanation may be that the phosphate present duri ng synthesis create rts. s additional cavities that, once extracted, are available for phos phate binding. Another reason may be that the phosphate acts as a spacer per se which promotes polymer crosslin king to a greater extent than the control samples. Genipin typically sells for $20 USD/ g. Usi ng the microspheres prepared in these studies, it would require 250 mg of genipin to produce a product that would bind phosphate in-vivo as efficiently as Renagel. Including the price of gelatin and oil, the microspheres prepared during these trials were approximately $5.50 USD/ g. Though the genipin particles were the most y preclude th alternatives for use by those with acute renal failure. uld be surpassed through aggressive washi ffective acute renal failure. biocompatible polymer synthesized during these studies, the potential cost of production ma em as cost effective Glutaraldehyde typically sells for $0.035 USD/ g. Using the micr ospheres prepared in these studies, it would require 3.0 g of glutaraldehyde to produce a product that would bind phosphate in-vivo as efficiently as Renagel. Includi ng the price of gela tin and oil, the microspheres prepared during these trials we re approximately $0.25 USD/ g. Although the glutaraldehyde particles had cy totoxic concerns, they co ng procedures. Thus, the co st of production makes them viable candidates as cost e alternatives for use by those with Comparative analysis of the two systems re sults in an overall recommendation of the genipin crosslinked microspheres as the more favorable system. T hough the glutaraldehyde 114

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produ the tion facilities, etc. Howeve r, the cost summary does provide preliminary inform er s ens, ent, and the effects that changing the amou nt of e identified. ced can be prepared at a cost that is significantly lower than their genipin alternative, inherent biocompatibility of genipin make th em far more appealing as a pharmaceutical alternative, and is in accor d with engineering aspect. The potential cost analysis of the polymeric ma terials prepared during this research can not be directly compared to that of Renagel becau se they do not include factors such as patent development, produc ation about which MIT can possibly lead to the more low-cost technology. One very important aspect to consider is th at, even though the cost of producing a polym that binds phosphate in solution may be greater than that of Renagel, the selective template binding may not disqualify their usefulness as potential therapeutic agents. Future Work Crosslinking Agent There are several suggestions for the future in corporation of different crosslinking agent for these studies. Glyceraldehyde and dext ran should be further examined in greater compositional concentrations to establish thei r overall suitability for this application. EDC, which is a zero length crosslinker that is extensively used with gelatin and collag may be investigated as a chem ically efficient alternative. Free amino/carboxylic acid determination studi es should be performed to identify the extent of crosslinking achieved with each different ag nt of crosslinker used aff ects the overall gelatinous micros phere. The maximum amou each crosslinker required to provide the best gela tin microstructure for phosphate uptake can b 115

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Substrate Imprinting This scheme is used to imprint phosphate mol ecules via a surface grafted thin polyme strategy. r layer er film would be grafted from d particle size. rmed using NMR. The selectivity of the gelatin micro nvestigated using HPLC, which will on, height, width, and tai ling, whether or not phosph ate has been favorably bound to the gelati ined by incorporating ions that are comp of t of the gastrointestinal system. This information would be instrumental in the development of an ideal set of polymer proce ssing parameters, and should lend itself to the results obtained from the kinetic assays. 36 The phosphate molecules are on a substrat e, and a polym this surface. Once the substrate is removed, the guest molecule could be extracted from the host site, leaving behind a surface imprinte d polymer, capable of recognition upon guest reintroduction. This may ultimately increase the number of imprint cavities available for phosphate uptake. The polymer could be ground and sintered to produce the desire Instrumentation Structural elucidation studies for these nove l polymers should be perfo spheres can be i show by peak separati to the gelatin microsphere in comparison to other ions th at may be in the solution. Polymer Binding Kinetic studies should be perf ormed that establish the rate of phosphate binding n microspheres. Binding selectivity of phosphate should also be exam etitive in nature to phosphate into the solution to be tested for phosphate uptake. Swelling studies, as reported in Chapter 3 on Renagel, should be unde rtaken. The pH the uptake solutions should be varied, and pol ymer swelling and phosphate uptake evaluated at each level. In particular, the pH of the testing so lution should be lowered, as to mimic the acidic environmen 116

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Enzyme Studies est Prior to actual in-vivo trials, which can be expensive, various enzymes which are known to exist in the gast rointestinal surroundings should be incorp orated into the phosphate uptake t solution. Features such as polymer degradation and biocompatibility can be further investigated. 117

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APPENDIX A CALIBRATION OF A PHOSPHATE MEASURING APPARATUS The process of determining phosphate (PO4) ion uptake in polymer samples has been well established and can be can be performed by methods such as ICP, UV/Vis, and other spectrophotometric techniques. We have chosen to use the Hannah HI 93717 High Range ISM phosphate meter for measuring phosphate ion concentration largely becau se it is cost effective in relation to the previously mentioned instruments and the mobile na ture of this handheld me ter allows it to take real time, in field measurements with minima l sample preparation. The Hannah HI 93717 is a small, portable photometer that measures phos phate uptake in aqueous solutions upon exposure to an amino acid based developing reagent kit. 3Figure A-1. Hannah HI 93717 handheld photometer. However, limited information was availabl e concerning the robustness of using data obtained using the HI 93717 in comparison to that from traditional, well established analytical devices. In an effort to addr ess this, several calibration proc esses were tested in order to determine the efficiency, accuracy, and precision of the Hannah phosphate meter readings. Experimental Methods Initially, the Hannah HI 93717 meter was calib rated against a phosphate solution sample set of known concentrations (Table A-1). 118

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Table A-1. Phosphate calibration va lues using the Hannah HI 93717 meter. Sample C onc. at 0 ppm Conc. at 5 ppm Conc. at 10 ppm Conc. at 20 ppm (mg/ L) (mg/ L) (mg/ L) (mg/ L) Trial 1 0.00 4.70 9.90 19.90 Trial 2 0.00 5.20 10.10 19.80 Trial 4 0.00 5.00 9.70 19.80 0 9.80 19.80 9.92 19.63 Trial 3 0.00 4.90 10.10 19.20 Trial 5 0.00 4.8 Average 0.00 4.92 Stand Devia ard tion 0.00 0.19 0.18 0.28 Great care was taken in determining a sound se t of testing parameters that would provide elapsed to reading were standardized until reprod ucible numbers were consistently obtained. repeatable data values. Factors such as glassware treatment, reagent amounts used, and time The data was graphically examined (Figure A-2), and the R2 analysis obtained from the calibration curve (Figure A-3) showed ve ry good agreement within the numbers. Figure A-2. Actual vs. Theore tical concentrations for phospha te ion using the Hannah HI 93 717 phosphate meter. solutions were developed using a tradit ional ascorbic acid method. The data obtained is presented in Table A-2. Next, standard solution sets were tested on Shimadzu UV-2401PC UV-Vis spectrophotometer. The tested phosphate 119

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Figure A-3. Calibration curve for the Hannah HI 93717 phosphate meter. Table A-2. Phosphate readings from the Ha nnah HI 93717 meter compared to those obtained from the Shimadzu UV/Vis. Phosphate meter vs. UV/Vis Sample meter (ppm) UV/VIS (ppm) 2.5 ppm 2.6 5.0 ppm 5.5 5.8 10.0 ppm 10.4 10.4 20 ppm 20.6 20.2 20 ppm (different phosphate source) 19.9 19.7 The data indicated reasonable, acceptable devi ation between the techniques. The use of the Hannah meter appeared to be an acceptable apparatus for phosphate determination. Finally, industry standard Renagel samples were measured for phosphate uptake. A set of 10 different 800 mg pills were dissolved in 300 mL of deionized water at a pH of 6.98. The 10 different 100 mg samples of the powder were measured and placed in 50 mL centrifuge tubes, to each of which was added 30 mL of a phosphate st andard solution at a pH of 7.37. The tubes were placed in a ro tating incubator at 37 oC for 3 hours, removed, and phosphate uptake was slurry obtained was filtered, dried, and stored under vacuum to obtain a white, powder product. 120

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measured using the HI 93717 meter and determined using equation A-1. The results are found in Figure A-4. PO4 3uptake = [PO4 3-]standard solution [PO4 3-]sample after 3 hours (A-1) 0.00 1.50 2.00 (mE 0.50 1.00 uptak2.54 2.50 3.00 3.50 4.00 123PO4eq/ g polymer) Phosphate uptake iagel samples as measured by the Hannah HI 93717 phosphate meter. Conclusions lative deviation in the results obt ained using the HI 93717 meter and U h meter will c Uptake (mEq/ g polymer) 3.54 3.48 2.88 3.32 3.03 2.92 3.00 3.11 3.03 Average STD 3.08 0.30 456 Sample 78 910 Figure A-4 n Ren Overall, the minimal re V/Vis allowed us to establish the use of the meter for analysis as a sound technique capable of providing accurate resu lts for phosphate analysis for our studies. The Hanna ontinue to be used as the initial, pr imary instrument for measuring phosphate in our experiments. 121

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BIOGRAPHICAL SKETCH Anika Odukale is a native of Minnesota, where she graduated from the Summatech program te umber of African-Americans, chemistry, and to accelerated ath and science areas. She also was the pr esident of the first NOBCChE chapter at the university. While enrolled as an undergraduate, Anika cons istently held Summer research Internship positions at the 3M Company, Burroughs Wellc ome (currently Glaxo-Smith-Kline), and Interpoll Laboratory, as a means to further he r research experiences and supplement herself financially for College. Through her combined instructional and resear ch experiences, Anika first realized her passion for teaching and scientific exploration. She became dedicated to learning how to inspire Critical Thought in students th rough non-traditional educational and scientific methods Anika obtained a Masters' degree in Analytical Chemistry from Michigan State University. During her tenure there she successfully completed required courses, taught chemistry recitation courses and labs, and started a fr ee tutorial program for first semester chemistry classes. She also served as an Instructor for the Charles Drew Tutorial Assistance in Chemistry program, which provided intensive supplemental instruct ion for gifted and ta lented students from underrepresented groups in science and engineer ing. This program was highly successful, attracting the attention and funding from the National Science Foundation. at Minneapolis North Community High School. She then attended North Carolina Sta University, where she received a B.S. degree in chemistry, with a minor in African-American studies. To address the noticea ble trend of an alarmingly low n specifically women, involved in th e sciences, Anika tutored students in math and taught for a pilot program that introduced Afri can-American children aged 8-15 m 129

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Upon degree completion at ika attended the University of Florid e and aterials ilities Network (www.mrfn.org), an d work on polymeric encapsulants for organic light ity Dr. Jesse Edwards III, an Associate Professor in Chemistry at Florida A&M Michigan State Univ ersity, An a and enrolled in the Doct oral degree program in the depart ment of Materials Scienc Engineering. She received a cer tificate in Forensic Science in the Drug Chemistry concentration, and a Masters Degree in Materials Science and Engineering while ma triculating through the Doctoral degree program. Anika has obtained a postdoctoral research posi tion at the University of California at Santa Barbara. She is working under the direction of Professor Craig T. Hawker at the Materials Research Laboratory (MRL), wher e she is the program coordinato r for the NSF funded M Research Fac emitting devices (OLEDs) for the Mitsubishi Chemical Corporation. Anika will continue to serv e the community through mentor ing and tutoring univers students, specifically women and mi norities, in the STEM areas. Anika is married to University, who share a 1 year old son, Ak il, and 12 year old stepson, Tyler Edwards. 130