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Immobilization of Oxalate-Degrading Enzymes into P(HEMA) for Inhibiting Encrustation on Ureteral Stents

Permanent Link: http://ufdc.ufl.edu/UFE0017575/00001

Material Information

Title: Immobilization of Oxalate-Degrading Enzymes into P(HEMA) for Inhibiting Encrustation on Ureteral Stents
Physical Description: 1 online resource (116 p.)
Language: english
Creator: Mellman, James K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: biomaterial, coating, enzyme, hema, immobilization, kinetics, menten, michaelis, oxalate, stent, ureteral, urology
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

Abstract: Ureteral stents develop calcium-bearing deposits, called encrustations, that diminish their biocompatibility due to complications, such as chronic abrasion to the lumen of the ureter wall and subsequent infection. A reduction of encrustation, namely calcium oxalate, will improve the lifetime, health care costs, and infection resistance of such devices. The purpose of this research project is to study oxalate-degrading enzymes entrapped into a coating material that will control the interface to the urinary environment for ureteral stents. The coating material was a lightly crosslinked poly(2-hydroxyethyl methacrylate) (p(HEMA)) matrix in which the active enzymes were entrapped within the bulk material's free volume. The swelling of p(HEMA) films was comparable in ddH2O and urine. This hydrophilic matrix allows oxalate anions to diffuse into the bulk so that enzyme activity against oxalate can lower its local concentration, and thereby reduce the supersaturation of calcium oxalate. Oxalate oxidase (OxO) and oxalate decarboxylase (OxDc) were the oxalate-degrading enzymes examined herein. Michaelis Menten kinetic models were applied to free and immobilized enzyme activity. A substrate inhibition model was applied to OxO. The free form of OxO had a Vmax of 1.8 ? 0.1 ?M/min-?g, a km of 1.8 ? 0.1 mM, and a ks of 35.4 ? 3.7 mM while the immobilized form had a Vmax of 1.2 ? 0.2 ?M/min-?g, a km of 4.1 ? 0.6 mM, and a ks of 660 ? 140 mM. The free form of OxDc had a Vmax of 23.5 ? 1.4 ?M/min-?g and a km of 0.5 ? 0.1 mM while the immobilized form had a Vmax of 5.0 ? 1.9 ?M/min-?g and km of 23.2 ? 9.1 mM. The enzyme activity was measured to indicate viable application conditions for the coating, such as storing the films in urine over time. The maximum activity was shown at pH 4.2 to 4.5 and activity drops to be negligible by pH 7.0. Storing the enzyme at pH 6.1 exhibited a larger retained activity than storing at pH 4.2, yet storing in urine showed the highest retention. In a six moth trial period in urine, immobilized OxO lost 30% activity to 0.7 ?M/min-?g, whereas the activity for immobilized OxDc fell 50% from about 5.9 to 2.9 ?M/min-?g. Coating p(HEMA) onto polyurethane ureteral stents was applied by dip coating into a monomer-based coating solution. To achieve successful coatings, the viscosity of the coating solution and adhesion to the stent were optimized through a series of experiments with glycerol and superglue to form a primer of p(HEMA). The enzymes were applied to the primer through successive layers without the use of glycerol or superglue. The enzyme activity was used to compare various processing routes, such as dip time, dip cycles, and the use of Triton X-100. An encrustation model was established using artificial and real urine, and an antibiotic/antimycotic solution was added to prevent infection. The solutions were spiked with 0.5 mM oxalate to optimize encrustation conditions. The encrustation study was conducted up to two months in these solutions, and samples were analyzed using polarized light microscopy. Immobilized OxDc inhibited crystal growth up to two-months, although OxO developed encrustation to a similar extent of the control group. This opens the possibility of utilizing the immobilized enzyme as a therapy for degrading oxalate concentrations in urine, which can be employed as a coating on ureteral stents
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by James K Mellman.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Gower, Laurie B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0017575:00001

Permanent Link: http://ufdc.ufl.edu/UFE0017575/00001

Material Information

Title: Immobilization of Oxalate-Degrading Enzymes into P(HEMA) for Inhibiting Encrustation on Ureteral Stents
Physical Description: 1 online resource (116 p.)
Language: english
Creator: Mellman, James K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: biomaterial, coating, enzyme, hema, immobilization, kinetics, menten, michaelis, oxalate, stent, ureteral, urology
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

Abstract: Ureteral stents develop calcium-bearing deposits, called encrustations, that diminish their biocompatibility due to complications, such as chronic abrasion to the lumen of the ureter wall and subsequent infection. A reduction of encrustation, namely calcium oxalate, will improve the lifetime, health care costs, and infection resistance of such devices. The purpose of this research project is to study oxalate-degrading enzymes entrapped into a coating material that will control the interface to the urinary environment for ureteral stents. The coating material was a lightly crosslinked poly(2-hydroxyethyl methacrylate) (p(HEMA)) matrix in which the active enzymes were entrapped within the bulk material's free volume. The swelling of p(HEMA) films was comparable in ddH2O and urine. This hydrophilic matrix allows oxalate anions to diffuse into the bulk so that enzyme activity against oxalate can lower its local concentration, and thereby reduce the supersaturation of calcium oxalate. Oxalate oxidase (OxO) and oxalate decarboxylase (OxDc) were the oxalate-degrading enzymes examined herein. Michaelis Menten kinetic models were applied to free and immobilized enzyme activity. A substrate inhibition model was applied to OxO. The free form of OxO had a Vmax of 1.8 ? 0.1 ?M/min-?g, a km of 1.8 ? 0.1 mM, and a ks of 35.4 ? 3.7 mM while the immobilized form had a Vmax of 1.2 ? 0.2 ?M/min-?g, a km of 4.1 ? 0.6 mM, and a ks of 660 ? 140 mM. The free form of OxDc had a Vmax of 23.5 ? 1.4 ?M/min-?g and a km of 0.5 ? 0.1 mM while the immobilized form had a Vmax of 5.0 ? 1.9 ?M/min-?g and km of 23.2 ? 9.1 mM. The enzyme activity was measured to indicate viable application conditions for the coating, such as storing the films in urine over time. The maximum activity was shown at pH 4.2 to 4.5 and activity drops to be negligible by pH 7.0. Storing the enzyme at pH 6.1 exhibited a larger retained activity than storing at pH 4.2, yet storing in urine showed the highest retention. In a six moth trial period in urine, immobilized OxO lost 30% activity to 0.7 ?M/min-?g, whereas the activity for immobilized OxDc fell 50% from about 5.9 to 2.9 ?M/min-?g. Coating p(HEMA) onto polyurethane ureteral stents was applied by dip coating into a monomer-based coating solution. To achieve successful coatings, the viscosity of the coating solution and adhesion to the stent were optimized through a series of experiments with glycerol and superglue to form a primer of p(HEMA). The enzymes were applied to the primer through successive layers without the use of glycerol or superglue. The enzyme activity was used to compare various processing routes, such as dip time, dip cycles, and the use of Triton X-100. An encrustation model was established using artificial and real urine, and an antibiotic/antimycotic solution was added to prevent infection. The solutions were spiked with 0.5 mM oxalate to optimize encrustation conditions. The encrustation study was conducted up to two months in these solutions, and samples were analyzed using polarized light microscopy. Immobilized OxDc inhibited crystal growth up to two-months, although OxO developed encrustation to a similar extent of the control group. This opens the possibility of utilizing the immobilized enzyme as a therapy for degrading oxalate concentrations in urine, which can be employed as a coating on ureteral stents
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by James K Mellman.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Gower, Laurie B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0017575:00001


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1 IMMOBILIZATION OF OXALATE-DEGRAD ING ENZYMES INTO P(HEMA) FOR INHIBITING ENCRUSTATION ON URETERAL STENTS By JAMES KENNETH MELLMAN 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 2007

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2 2007 by James Kenneth Mellman

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3 To honoring the past, living in the pres ent, and hoping for a better future.

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4 ACKNOWLEDGMENTS First, I must thank my family who prizes ed ucation no matter what the cost, time, or age. My parents always supported me and provided great mentorship throughout my life. I only wish they lived in Gainesville and could have enjoyed some Gator games with me. Yet, my family has truly given me firm roots to grow and I can only hope to keep moving upwards. I am thankful they are with me along the journey. Second, I thank the University of Florida for my education over the last 8 years. The process to achieve my PhD seemed seamless as I earned my bachelors degree and went on to graduate school. The University of Florida offe red me opportunities in a breadth of classes as well as undergraduate and graduate research. I disc overed that University of Florida is diverse enough to do what one wants with an education, and it gave me a solid background for my education in engineering. Also, it has a fantas tic library system that I always cherished. I extend thanks to Dr. Laurie Gower for her supp ort for putting me to work on this project. I enjoyed the hard work in developing this projec t. I appreciate her patienc e in me as I explored this research. Dr. Gower strived for excelle nce, and she set high expectations for me. Finally, I thank the department of materials science & engin eering (MSE) for the classes, faculty and the infrastructure. The office of academic affairs made it easy to come in and feel welcome. The research and classes in MSE gave me a great background to pursue any direction possible. I also thank Dr. John Mecholsky who gave me a start with research in MSE. Dr. Baney was also always a pleasure to discuss research ideas and general science. During graduate school, I had the opportunities to learn from giants so th at I can now stand on their shoulders to attain higher heights. My experience in MSE has aroused my appetite to do more with my education and that is certainly what I will set out to achieve.

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 LIST OF SCHEMES................................................................................................................ ......12 ABSTRACT....................................................................................................................... ............13 CHAPTER 1 MOTIVATION TO DEVELOP AN AC TIVE COATING TO INHIBIT ENCRUSTATION ON URETERAL STENTS.....................................................................15 Project Background............................................................................................................. ...15 Background to Encrustation...................................................................................................16 Encrustation on Urinary Devices.....................................................................................16 Encrustation from Oxalate...............................................................................................17 Societal Need to Inhibit Encrustation.....................................................................................20 A Solution to Encrustation..................................................................................................... .21 2 SYNTHESIS & CHARACTERIZATION OF PHOTOPOLYMERIZED P(HEMA) FILMS WITH VARYING CROSSLINK DENSITY............................................................26 Introduction................................................................................................................... ..........26 Poly(2-hydroxyethyl methacrylate) Hydrogels...............................................................26 Photopolymerization........................................................................................................26 Materials & Methods............................................................................................................ ..27 Materials...................................................................................................................... ....27 Methods........................................................................................................................ ...28 Characterization............................................................................................................... .......28 Equilibrium Weight Content (EWC)...............................................................................28 Scanning Electron Microscopy (SEM)............................................................................29 Conclusion..................................................................................................................... .........30 3 IMMOBILIZATION OF OXALATE-DEGR ADING ENZYMES INTO P(HEMA): EFFECTS ON ACTIVITY KINETICS..................................................................................33 Introduction................................................................................................................... ..........33 Oxalate Oxidase (OxO)...................................................................................................34 Oxalate Decarboxylase (OxDc).......................................................................................34 Enzyme Immobilization..................................................................................................35 Kinetic Model..................................................................................................................35 Experimental Procedures........................................................................................................36

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6 Film Preparation..............................................................................................................37 Enzyme Activity Test......................................................................................................37 Activity Assay for OxO...................................................................................................38 Activity Assay for OxDc.................................................................................................38 Results and Discussion......................................................................................................... ..39 Activity with Respect to Time.........................................................................................39 OxO..........................................................................................................................39 OxDc........................................................................................................................39 Activity with Respect to Oxalate Concentration.............................................................41 OxO..........................................................................................................................41 OxDc........................................................................................................................43 Kinetic Models................................................................................................................44 Conclusions.................................................................................................................... .........44 Supplemental Data.............................................................................................................. ....46 Effects of Ultra-Violet Light Exposure on Activity........................................................46 Product Diffusion from Films over Time........................................................................46 Model for free OxO..................................................................................................52 Model for immobilized OxO....................................................................................52 Model for free OxDc................................................................................................52 Model for immobilized OxDc..................................................................................52 4 DEVELOPMENT OF A P(HEMA) COATING FOR THE PURPOSE OF IMMOBILIZING OXALATE-DEGRADING ENZYMES ONTO A URETERAL STENT.......................................................................................................................... ..........55 Coatings....................................................................................................................... ...........55 Overview....................................................................................................................... ..55 Design Principals.............................................................................................................57 Substrate and coating material.................................................................................57 Application and curing.............................................................................................58 Degradation to enzyme.............................................................................................59 Loading concentration of enzyme............................................................................60 Enzyme activity tests................................................................................................60 Storage and use.........................................................................................................60 Coating for this Project....................................................................................................... ....61 Materials...................................................................................................................... ....61 Primer Studies.................................................................................................................62 Application...............................................................................................................62 Glycerol concentration.............................................................................................63 Secondary Layer Studies.................................................................................................64 FTIR.........................................................................................................................64 Triton X-100.............................................................................................................65 Layering the Coating.......................................................................................................66 Primer.......................................................................................................................66 Immersion time........................................................................................................67 Dip cycles.................................................................................................................68 Conclusion..................................................................................................................... ..69

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7 5 INHIBITION OF ENCRUSTATION BY IMMOBILIZED OXALATE-DEGRADING ENZYMES........................................................................................................................ .....80 Ureteral Stents and Encrustation............................................................................................80 Hydrogel Coatings...........................................................................................................80 Antibiotic Coatings..........................................................................................................82 Encrustation Models........................................................................................................83 Experimental Study of Encrustation.......................................................................................83 Introduction................................................................................................................... ..83 Materials and Methods....................................................................................................84 Films.........................................................................................................................8 4 Artificial urine..........................................................................................................85 Urine.........................................................................................................................8 5 Encrustation crystallizing solutions.........................................................................85 Activity tests.............................................................................................................85 Polarized light optical microscopy...........................................................................86 Results........................................................................................................................ .....86 Oxalate-dependence for encrusta tion in artificial urine...........................................86 Oxalate dependence for encrus tation in real urine...................................................86 Encrustation over time in artificial urine..................................................................87 Encrustation over time in real urine.........................................................................88 Conclusion..................................................................................................................... .........89 Supplemental Data.............................................................................................................. ....96 6 SYNOPSIS..................................................................................................................... ........98 Conclusion..................................................................................................................... .........98 Future Work.................................................................................................................... ......101 APPENDIX A EXPLANTED STENTS.......................................................................................................104 B CALCULATIONS................................................................................................................1 06 LIST OF REFERENCES............................................................................................................. 107 BIOGRAPHICAL SKETCH.......................................................................................................116

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8 LIST OF TABLES Table page 2-1 Composition of p(HEMA) films with varying concentration of DEGDMA.....................31 3-1 Composition for immobilizing OxO and OxDc into p(HEMA) films...............................47 3-2 Constants determined by applying Michae lis Menten kinetics on raw data from Figures 1 and 2. (N.A.-not applicable, n = 2)....................................................................49 4-1 Summary of coating thickness ba sed on composition and dip time..................................72 4-2 Summary of FTIR results...................................................................................................74 4-3 Composition of coating solution and films used for testing effects of Triton X...............75 5-1 Composition of salts for artificial urine.............................................................................91

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9 LIST OF FIGURES Figure page 1-1 Encrustation formed along a ureteral stent........................................................................24 1-2 Molecular structures of oxalic acid and oxalate................................................................24 1-3 Schematic of coating with entrappe d enzymes breaking down the oxalate anion within the bulk of the coating at the devices surface........................................................25 2-1 Molecular reaction and structure of free-radical initiation with HEMA and DEGDMA for p(99m% HEMA-c-1m% DEGDMA)........................................................31 2-2 Molecular structures of the photoinitiat or, DMPA, and its decomposition pathways.......31 2-3 Equilibrium swelling of p(HE MA) with varying DEGMA in ddH2O and urine...............32 2-4 Micrographs of the surface of p(HEMA) f ilms with varying degrees of crosslinker, DEGDMA......................................................................................................................... .32 3-1 Activity of free and immobilized OxO for periods up to 60 minutes................................48 3-2 Activity of free and immobilized OxDc for periods up to 60 minutes..............................48 3-3 Activity of immobilized enzyme in p(HE MA) after being stored in a buffer solution, tested against 50 mM oxalate, a nd restored. (A) OxDc; (B) OxO.....................................49 3-4 Activity tests for reaction rates for Ox O. (A) free OxO; (B) immobilized OxO...............50 3-5 Activity tests for reaction rates for OxDc. (A) free OxDc; (B) immobilized OxDc.........51 3-6 Proposed models for the kinetic mechanisms of oxalate catalysis by free and immobilized OxO and OxDc. The model fo r OxO accounts for substrate inhibition (represented by OxOSS)....................................................................................................52 3-7 Activity profiles of free OxO (2 g) and OxDc (2 g) against 50 mM oxalate after exposed to UV for different periods of time......................................................................53 3-8 Profiles of product release from films over time after the activity test.(A) OxDc; (B) OxO............................................................................................................................ ........54 4-1 General schematic of coating a nd photocuring process for stents.....................................71 4-2 Digital photographs of dip coated primer la yers that had varying amounts of glycerol (0-40%) in the aqueous phase if the reaction solution.......................................................71 4-3 Micrographs of primer layers made from 40 v% glycerol that were coated onto the stent by varying time immersed into coating solution.......................................................72

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10 4-4 Planar view, A (at low and high magnificat ion), and cross-section, B, of the primer coating prepared from 20 % glycerol and 20% ddH2O dipped for one minute..................73 4-5 Planar view, A (at low and high magnificat ion), and cross-section, B, of the primer coating prepared from 40% ddH2O dipped for one minute...............................................73 4-6 Planar view, A (at low and high magnificat ion), and cross-section, B, of the primer coating prepared from 80% monomer and 20% glycerol dipped for one minute..............74 4-7 ransmission spectra from FTIR of primer formed with glycerol and the second layer formed with ddH2O............................................................................................................74 4-8 Activity from stents coated with OxO made from dipping at different amounts of time........................................................................................................................... .........75 4-9 Activity in films made from coating so lutions with varying amounts of Triton X-100....75 4-10 Micrographs of stents coated w ith the primer for layering studies....................................76 4-11 Activity of OxO in coatings that were a pplied by immersing stents with primer into reaction solution for various times.....................................................................................76 4-12 Micrographs of stents coat ed with various dip times.........................................................77 4-13 Activity of OxO in coatings that were appl ied with sequential dip cycles into reaction solution....................................................................................................................... ........77 4-14 Micrographs of coated stents dipped 1 time......................................................................78 4-15 Micrographs of coated stents dipped 2 times.....................................................................78 4-16 Micrographs of coated stents dipped 3 times.....................................................................79 5-1 Polarized images of control p(HEMA) films tested in artificial urine with varying concentrations of oxalate at 1 week...................................................................................91 5-2 Polarized images of control p(HEMA) f ilms tested in real urine against varying concentrations of spiked oxalate at 1 week........................................................................91 5-3 Polarized images of films containing Ox Dc tested in real urine against varying concentrations of oxalate at 1 week...................................................................................92 5-4 Polarized images of films containing Ox O tested in real urine against varying concentrations of oxalate at 1 week...................................................................................92 5-5 Polarized images of control p(HEMA ) films tested in artificial urine...............................93 5-6 Polarized images of OxDc f ilms tested in artificial urine..................................................93

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11 5-7 Polarized images of OxO film s tested in artificial urine....................................................93 5-8 Polarized images of control p(HEMA) films tested in urine.............................................93 5-9 Polarized images of OxDc films tested in urine................................................................94 5-10 Polarized images of OxO films tested in urine..................................................................94 5-11 The effects of pH on free and immobilized OxO..............................................................94 5-12 The effects of pH on free and immobilized OxDc.............................................................95 5-13 The effects of storing films with imm obilized OxO in a buffer of 100 mM Hex-NaCl or real urine, as measure by Vmax.......................................................................................95 5-14 The effects of storing films with i mmobilized OxDc in a buffer of 100 mM HexNaCl or real urine, as measured by Vmax...........................................................................96 5-15 Close-ups of blanks (films without enzyme) in 0.4 oxalate in real urine..........................96 5-16 Close-ups of blanks (films without en zyme) in 0.5 mM oxalate in real urine...................97 A-1 Explanted ureteral sten ts showing encrustation...............................................................104

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12 LIST OF SCHEMES Schemes page 3-1 Decomposition of oxalate by OxO....................................................................................47 3-2 Decomposition of oxalate by OxDc...................................................................................47 3-3 Chemical reaction of OxO assay to measure oxalate degradation.....................................47 3-4 Chemical reaction of OxDc assa y to measure oxalate degradation...................................47

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13 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 IMMOBILIZATION OF OXALATE-DEGRAD ING ENZYMES INTO P(HEMA) FOR INHIBITING ENCRUSTATION ON URETERAL STENTS By James Kenneth Mellman August 2007 Chair: Laurie Gower Major: Materials Science and Engineering Ureteral stents develop calci um-bearing deposits, called encr ustation, that diminish their biocompatibility due to complications, such as ch ronic abrasion to the lu men of the ureter wall and subsequent infection. A reduction of encrus tation, namely calcium oxalate, will improve the lifetime, health care costs, and infection resistance of such devices. The purpose of this research project is to study oxalate-degradi ng enzymes entrapped into a coat ing material that will control the interface to the urinary environment for ureteral stents. The coating material was a lightly cro sslinked poly(2-hydroxyethyl methacrylate) (p(HEMA)) matrix in which the active enzymes were entrapped within the bulk materials free volume. The swelling of p(HEMA ) films was comparable in ddH2O and urine. This hydrophilic matrix allows oxalate anions to diffuse into the bulk so that enzyme activity against oxalate can lower its local concentration, and thereby redu ce the supersaturation of calcium oxalate. Oxalate oxidase (OxO) and oxalate decarboxyla se (OxDc) were the oxalate-degrading enzymes examined herein. Michaelis Menten kinetic models were applied to free and immobilized enzyme activity. A substrate inhibiti on model was applied to OxO. The free form of OxO had a Vmax of 1.8 0.1 M/ming, a km of 1.8 0.1 mM, and a ks of 35.4 3.7 mM while the immobilized form had a Vmax of 1.2 0.2 M/ming, a km of 4.1 0.6 mM, and a ks of 660

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14 140 mM. The free form of OxDc had a Vmax of 23.5 1.4 M/ming and a km of 0.5 0.1 mM while the immobilized form had a Vmax of 5.0 1.9 M/ming and km of 23.2 9.1 mM. The enzyme activity was measured to indica te viable application conditions for the coating, such as storing the films in urine ove r time. The maximum activity was shown at pH 4.2 to 4.5 and activity drops to be negligible by pH 7.0. Storing the enzyme at pH 6.1 exhibited a larger retained activity than st oring at pH 4.2, yet storing in ur ine showed the highest retention. In a six moth trial period in urine, immobilized OxO lost 30% activity to 0.7 M/ming, whereas the activity for immobilized Ox Dc fell 50% from about 5.9 to 2.9 M/ming. Coating p(HEMA) onto polyurethane ureteral stents was applied by dip coating into a monomer-based coating solution. To achieve succe ssful coatings, the viscosity of the coating solution and adhesion to the stent were optimized through a series of experiments with glycerol and superglue to form a primer of p(HEMA). The enzymes were applied to the primer through successive layers without the use of glycerol or superglue. The enzyme activity was used to compare various processing routes, such as di p time, dip cycles, and the use of Triton X-100. An encrustation model was established us ing artificial and r eal urine, and an antibiotic/antimycotic solution was added to preven t infection. The solutions were spiked with 0.5 mM oxalate to optimize encrustation conditio ns. The encrustation study was conducted up to two months in these solutions, and samples we re analyzed using polarized light microscopy. Immobilized OxDc inhibited crystal growth up to two-months, although OxO developed encrustation to a similar extent of the control group. This opens the possibility of utilizing the immobilized enzyme as a therapy for degrading ox alate concentrations in urine, which can be employed as a coating on ureteral stents

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15 CHAPTER 1 MOTIVATION TO DEVELOP AN ACTIVE C OATING TO INHIBIT ENCRUSTATION ON URETERAL STENTS Project Background Ureteral stents are medical prostheses designed to keep the ureter open to drain urine from the kidneys to the bladder. Ureteral stents are hollow cylindrical forms designed to provide relief from obstruction, promote healing and act as a prophylactic against endourological complications [1]. Ureteral stent encrustation of calcium oxalate (CaOx) is shown below (Figure 1-1, A-D). Urinary tract prostheses are typically made of latex, silicone, silicone-copolymers, or polyurethane. A major complication for these devices is encrustati on from high levels of oxalate ions that can normally occur in the urinary tract. The encrusta tion grows on the surface of the stent, and can cause great discomfort a nd risk of infection to the patient. The purpose of this work is to develop an active coating with immobilized oxalatedegrading enzymes that can be used to inhib it growth of CaOx encrustation on such urinary devices. It is hypothesized that si nce the formation of calcium oxala te is due to supersaturated concentrations of calcium and oxalate on the de vice, then using oxalate-degrading enzymes to reduce the local oxalate concentration can provide the device with an active coating that can reduce mineral formation. Activity of the enzyme will be used to assess the development of the coating for various process and application conditions. Urological practitioners seek th e use of long-term indwelling ur inary devices, but it is risky for patients due to the occurrence of encrusta tion and bacterial adhesion, in which the pathologies lead to a risk of blockage of the devices inner lu men and infection throughout the urinary tract [2-4]. Common practic e in urology is to remove devices maybe as often as every 6 to 8 weeks to prevent complications (Persona l conversation with Marv in Andrews, Manager

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16 New Technology Development, Cook Urological Inco rporated, February 2006). Due to frequent device replacement, increased cost and discomfort to patients, more compatible materials for urinary tract prosth eses are required. Complications associated with these devices re sult in increased hospital visits [5]. It has been estimated that 40% of hospital-related inf ections originate from the urinary tract [6]. Additionally, one study of ureteral stents showed that 47% of 141 retrieved showed impassable blockage for urine in the stent lumen [7]. Background to Encrustation Encrustation on Urinary Devices Encrustation is a pathological form of biom ineralization and it occurs on the surface of urinary devices. It is formed by two primary mechanisms [2, 3] The first is caused by the presence of oxalate-a diet-derived ionic prec ursor for the mineral calcium oxalate. Calcium oxalate is predominately found in the upper urinary tract and considered to be formed in sterile conditions. The second mechanism of encrustation is bacterial grow th on the device, usually due to microorganisms in the biofilm [8, 9]. The bacteria can raise urin ary pH, and evoke a condition for the nucleation and growth of the minera ls calcium phosphate (hydroxyapatite) and ammonium magnesium phosphate (str uvite) [10]. Both of these mechanisms that form urinary crystals are independent of one another, yet each mineral can contribute to device blockage and incompatibility for long-term use. Crystals that make up encrusta tion are a result of deposits of ionic and organic components within urine that interact with the biofilm on the device surface [ 11]. An assortment of explanted (removed) ureteral stents that became encrus ted is shown in Appendix A. Urologist Rudy Acosta, M.D. of Tampa, FL, kindly donated th ese explanted stents. In these pictures, encrustation formed as early as 10 days; as ti me elapsed, an accumulation of encrustation was

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17 observed. By one year, the explanted stent s howed bloody encrustation, from chronic irritation by stent encrustation to the ureteral wall. Calcium oxalate is the most predominant mi neral found on ureteral stents. The mineral calcium oxalate precipitates when oxalate ions bi nd to calcium and above a critical concentration of these paired ions, referred to as supersaturation, a mineral can form. Also, encrustation occurs more readily due to the presence of the device from the process of heterogeneous nucleation on a surface, which requires less free energy to form the mineral than a homogenous nucleation, in which the nuclei form in solution. Because of this reduced energy barrier, a smaller supersaturation is required for heterogeneous nucleation, which would occur on the stent. The pH of urine is usually around 6.0, but has a range of pH 4.5-8.0 depending on the health and diet of the person [12]. Calcium oxalate mineral forma tion is considered to be independent of pH but usually forms more below pH 7.0 [3]. Otherwise, phosphate-based minerals form at higher pH levels and are typically induced by bacterial infection [13]. Encrustation from Oxalate Oxalate is introduced to the hum an body through diet, such as l eafy vegetables like spinach and rhubarb [14] and endogenous synthesis in the liver that ac counts for 80-90% of urinary oxalate [15-17]. A high protein (meat) diet ha s even been associated with kidney stone formation because it reduces urinary pH and excret ion of citrate, which are factors for calcium oxalate formation [18]. It has been estimated th at 2-14% of ingested oxalate is absorbed by healthy people whereas 16-20% is absorbed by people who form kidney stones [16, 19]. The human body normally metabolizes oxalate through a bacterium, Oxalobacter formigenes which resides in the intestinal tract [2 0]. An excess of oxalate or a lack of Oxalobacter formigenes results in a variety of disorders, including hyperoxaluria, urolithiasis (kidney stones) and renal failure [20,21]. In one study of 10,000 upper urinary tract kidney

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18 stones, 73% of the urinary cal culi samples were composed calcium oxalate, in which other components included struvite (9%), calcium phosphate (8%), uric acid (7%), cysteine (1%), and some others (2%) [22]. The same process that results in kidney st ones also forms encrustation. A method doctors use to prevent stones and encrusta tion is to prescribe acidic foods, such as cranberry juice with the idea being ascorbate may influence the cr ystal formation [23]. Also, doctors suggest increasing hydration with water in order to lo wer the supersaturation and safely pass oxalate from the body [24]. Perhaps future medicine may include active agents such as Oxalobacter formigenes or oxalate-degrading enzyme s delivered to the gut. Encrustation from calcium oxalate is due to s upersaturated concentrations of oxalate ions in the urinary tract. Oxalate has two pKa values of 2.0 and 4.2 and three molecular (Figure 1-2, A-C). Below pH 2.0, the species oxalic acid is predominant, while between pH 2.0 and 4.2, the monoanionic oxalate species w ould prevail. Above pH 4.2, the dianionic oxalate would increasingly be present. Oxalate-degrading enzymes prefer to have oxalate in the monoanionic form (Personal conversation, Nigel Richards, Biochemistry Prof essor, University of Florida, 23 August 2006). This gives rise to their pH de pendence for activity that will be shown in Chapter 3. Although, the established pKa values for oxalate are used in the literat ure, they are determined in weak buffers, and in solutions like urine, ionic species will have different pH-depende nt behavior in media with a higher ionic strength. It is generally recognized that the chemical composition of urine is a metastable solution that is supersaturated with i ons for certain compounds, including calcium oxalate [25]. Kinetic factors govern the chemical species of the ions and inhibitors or promoters for some specific

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19 compound are within urine and may influence th e crystallization process. When an ionic complex is concentrated above a certain supe rsaturation level, the free energy competition between surface area and volume induces the nuclea tion and formation of a mineral to reduce the surface area of the cluster. The thermodynamic factors prevail over the kinetic ones and a salt precipitates. There is much inte rest to know more about the chemical complexes that these ionic species are forming in urine to predict crystal lization conditions. This phenomenon is referred to as speciation. The thermodynamics of such complexes have been modeled using several software packages, including JESS (Joint Expert Specia tion System) and experimental tests [26-29]. These computer models take into account the th ermodynamic constants for the formation of each species and are considered to be validated models. Cations are calcium, magnesium, sodium, and potassium. Anions are oxalate, phosphate, sulfat e, citrate, and chlori de. Hydrogen is also considered. In these studies, the models showed that calcium oxalate monohydrate is supersaturated independent of pH whereas pH values above 6.5 become supersaturated for hydroxyapatite and struvite [26-29]. Oxalate anion speciation is modeled with hydrogen (H+), sodium (Na+), potassium (K+), magnesium (Mg++), and calcium (Ca++) complexes and the concentra tion for each complex is pH dependent [22-24]. A monoanionic form of oxalate was demonstrated to account for over 20% of the total oxalate from pH 3.07.2 in the species of (NaOx)-, (KOx)-, and (HOx)[22]. This demonstrates that complexes are found far away from their pKa due to the ionic strength, pH, and chemical composition of the solu tion [30]. Another study modeled citrate-based therapy as an additive to control these complexes [28]. It showed that adding citrate would increase the monoanionic form of oxalate and decrease the cal cium oxalate complex as well. Possibly these

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20 complexes can be controlled in the urine for this coating with citrate to reduce calcium oxalate complexes and increase enzyme activity. The size of calcium oxalate stones formed in urine is more sensitive to oxalate ion concentrations than calcium [31]. Oxalate is relevant as a measur e of the clinical diagnosis of various pathologies, e.g. hyperoxaluria, and accura te determination is es sential. In one study between healthy subjects versus subjects with hy peroxaluria, the average plasma oxalate was determined through an oxalate-degrading enzyme activity (oxalate oxidase ). Healthy subjects had an average concentration of plasma oxalate of 1.28 mol/L whereas hyperoxaluric patients had an average of 166 mol/L. [32]. An oxalate diagnostic kit is available commercially (Trinity Biotech) and utilizes oxalate oxi dase in solution to determine oxalate levels. Perhaps a new method that immobilizes oxalate-degrading en zymes onto a device can lead to a new and improved method to measure oxalate amounts b ecause the devices can be reused with a reduction in expensive enzyme consumption. Societal Need to Inhibit Encrustation Reducing encrustation on urinary devices is im portant since once the lumen of the device becomes blocked by encrustation, a painful incont inence arises and removal and replacement of the urinary device is required. Encrustation forms easily in several groups of people that include kidney-stone-formers, elderl y or immobile people, a nd pregnant women [33-35]. Traditionally, clinical endourol ogical management of encrus ted ureteral stents could consist of several repeated surgic al procedures to completely remove the stone and stent from the patient. Extracorporeal shock wave lithotripsy ma y be used to fragment the stones under sonic shock waves concentrated onto the device [36]. The stone fragments should then be able to flow out with urine; otherwise, a urologist may retrieve the st ones using ureteroscopy (Phone

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21 conversation, Rudy Acosta, M.D., 28 March 2005). More recent medical developments have it down to one surgical procedure [37]. Yet, reducing encrustation by an active sten t provides a new method to improve upon the risk management of urinary de vices. A properly modified surface could extend the usefulness of these devices by weeks to months. Great pain and costs could be alleviated, which would improve the market quality for ureteral stents. A Solution to Encrustation Enzymes can act as therapeutic agents for me dical treatment, and as proposed here, such enzymes can be applied to medical devices to inte grate their therapeutic f unction onto the device. This can be achieved by immobilizing the enzyme onto the surface of the de vice or in a coating, thereby localizing the enzyme in an environment, such that the desired interaction with the substrate molecule in the surrounding physiologica l solution is made possi ble (Figure 1-3). The enzyme can activate an otherwise inert device towa rds an enzymes substrate; this functionality can perform some role for the device, perhap s improving the devices biocompatibility or imparting a therapeutic re sponse for the recipient. The chemistry of enzyme catalysis on oxalate occurs at the outer su rface of the ureteral stent, in which urine transports oxalate into the coating through di ffusion (Figure 1-3). The proposed kinetic steps of this system would enta il the diffusion of the oxalate anion into the coatings hydrogel matrix. The enzyme in the bulk material must bind with the oxalate anion to react. Then, the enzyme by-products must diffu se out of the coating and be excreted. The immobilized enzyme imparts its catalyt ic activity to the implants surface. Encrustation primarily takes place at the stents ou tside surface, so only th e outside of the stent will be coated. The application of the coating wi ll involve a method that includes a waterborne, photopolymerization process using a dip coating technique to phys ically entrap the oxalate-

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22 degrading enzymes. This is considered to undertak e as mild as possible conditions to ensure not to denature the enzyme. Whether an enzyme is permanently immobilized or released over time depends on the immobilization method, e.g. adsorption, entrapment, intermolecular cross-linking of enzymes, or covalent linkage [38]. Adsorption releases the enzyme rapidly but usually does not affect enzyme structure or activity profile. Intermolecular cros s-linking of enzymes and covalent linkage to a water-insoluble matrix involves chemically re acting the enzyme with a multifunctional group. It can be a disadvantage to link to an amino acid resi due that may be critical to its active site or substrate binding. Yet, the advantage is that th e enzyme could potentially be attached for the duration of the device. Entrapment is dependent on the free volume within the support matrix and can limit diffusion of enzyme substrate or prematurely re lease the enzyme. The advantage of this method, however, is that the enzyme is not chemically modified (which can re duce activity), yet it can confine the enzyme to the bulk material for long -term activity. It is also conceivable that the open nanostructure of the free volume could help to stabilize the enzymes native structure by constraint. Advantages and disadvantages exist fo r all types of immobilization because of these differences. Each immobilization method could exhibit di fferent enzyme activity profiles for this ureteral stent surface modification. Entrapment was chosen to be the method for immobilizing enzymes for this project because the enzyme coul d be incorporated into a coating solution. The enzyme activity profile was examined for two oxalate degrading enzymes, oxalate oxidase (OxO) and oxalate decarboxylase (OxDc). These oxa late-degrading enzymes were measured as a function of free and immobilized enzyme, time, ultraviolet (UV) exposure, pH, oxalate

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23 concentration, reusability, and urine exposure. Thes e parameters were determined to be probable process and application conditions. The enzymes were entrapped into p(HEMA), which could be applie d as a coating to a ureteral stent. By using a hydrogel matrix to entrap the enzymes, oxalate and the enzyme products were able to diffuse throughout the free vol ume of the material to be accessible to one another. The use of a hydrogel also retained essen tial water and salts to ke ep the protein in its native conformational state. A hydrog el coating provided a soft, lubr icious material to interact with the surrounding tissue upon implantation of the ureteral stent, while the enzyme will impart its functionality to the surface. The coating process reported here was a simple process that entailed mixing enzymes dissolved in buffers with monomers and a phot oinitiator. A photopolymerization method in a waterborne solution was chosen to cure the co ating material. This method allowed for a room temperature, aqueous environment to help ensu re minimal denaturation of the enzyme versus thermal initiation or using an orga nic solvent to cast the coating. The main hypothesis in this pr oject was that enzyme activity would be sufficient enough to reduce encrustation from forming on the sten t surface. The enzyme activity provided a measurable quantity for oxalate catalysis and a qu antifiable way to assess enzyme activity in the coating material. Understanding enzy me behavior from activity stud ies can be used to estimate the ability to inhibit calcium oxalate formation in the in vivo environment. Engineering this biomaterial constitutes its synthesis process a nd testing to correlate enzyme activity against numerous processing parameters to understand and predic t its behavior as a coating material to inhibit encrustation.

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24 A. B. C. D. Figure 1-1 Encrustation formed along a ureteral stent. (A) & (B) Digi tal photographs of a ureteral stent implanted for 28 days. (C) & (D) Micrographs by SEM of a section of the same explanted stent at increasing ma gnifications. Higher ma gnification of stent encrustation reveals calcium oxalate m onohydrate (flat chips) and calcium oxalate dihydrate (jagged points of bi pyramids) as major mineral constituents. Scale Bars: (C) (clockwise from top left) 1mm; 100 m; 100 m;10 m, and (D) 1 m. O O HO O O OH HO O O O O O A C B Figure 1-2 Molecular structures of oxalic acid and oxalate. (A) Oxalic acid; (B) Monoanionic oxalate; (C) Dianionic oxalate.

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25 Figure 1-3 Schematic of coating with entr apped enzymes breaking down the oxalate anion within the bulk of the coa ting at the devices surface.

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26 CHAPTER 2 SYNTHESIS & CHARACTERIZATION OF PHOTOPOLYMERIZED P(HEMA) FILMS WITH VARYING CROSSLINK DENSITY Introduction Poly(2-hydroxyethyl methacrylate) Hydrogels First discovered in the 1960s, 2-hydroxye thyl methacrylate (HEMA) is a hydrophilic monomer that readily forms a pol ymeric hydrogel material at lo w cross-linking densities (Figure 2-1) [39, 40]. Polymers of HEMA (p(HEMA)) have been approved for use by the FDA for cosmetic dyes, contact lenses, and drug release [9 ]. Ureteral stents were coated with p(HEMA) as one of the first polymers that could be us ed to reduce encrustation by adding a hydrophilic surface [41]. Although p(HEMA) has been shown to impart low protein and bacterial adsorption, it is still known to be susceptible to encrustation. Poly(HEMA) shows a weak interaction with proteins, although hydrophobic interactions from the protein to the nonpolar regi ons of the polymer can occur [42]. Proteus mirablilis was found to adsorb significantly less to p(HE MA) than other more hydrophobic polymers [43]. Bacteria in the biofilm eventual ly settle and then cause alkalin e conditions, which predisposes phosphate-bearing stone formation onto a urinary device [44]. Impr oved bacterial resistance is still being sought. This project can determine any enhancement for p(HEMA) to inhibit encrustation of CaOx by usi ng the entrapped oxalate-degrading enzymes within the material. Photopolymerization Photopolymerization involves an excited state process from a molecule that absorbs light and decomposes into an intermedia te species in a triplet state [45] Photoinitiators have different effectiveness throughout the spectrum of light (190-400 nm for UV) [46]. Use of photosensitizers and other initiator concentrations could be studi ed in the future to optimize photopolymerization kinetics [47].

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27 The photoinitiator, 2,2-dimethoxy-2-phenylace tophenone (DMPA) was used in this project. DMPA absorbs in the near UV(A) spectrum ( max = 365 nm, = 94.6), and it breaks down into several reactiv e radical species (Figure 2-2). U pon absorption of the appropriate wavelength of light, there is a cleavage at the C-C bond and a benzoyl radi cal is formed. This radical initiates the polymeriza tion by reacting with the vinyl group in the monomer [45]. The decomposition products of DMPA also break down further to a highly reactive radical methyl group, which has a low molecular weight and high diffusivity even when the polymer chains have formed [48]. Materials & Methods This chapter details the synthetic and physic al characterization me thods for the p(HEMA) material that will be used for the enzyme immobilization. Here, p(HEMA) was lightly crosslinked with diethyelene gl ycol dimethacrylate (DEGDMA) at varying molar ratios (0, 0.5, 1.0, and 2.0%). The films were equilibrated with deionized, distilled water (ddH2O) or urine to evaluate any difference of swelling. The films we re also characterized with scanning electron microscopy (SEM) and FTIR spectroscopy. Materials The main monomer, HEMA (95%, Fluka), was purified by passing the monomer over a column of activated alumina and then stored at 4C. The crosslinke r, DEGDMA (95%, Sigma), was used as received and stored at 22C. The photoinitiator, DMPA (Sigma), was dissolved in N-methyl pyrollidone (NMP) at 600 mg/ml and st ored at 22C. The monomers and photoinitiator solution were stored in amber bottles to reduc e degradation by ambient light. A UV spot lamp was purchased from Fisher Scientif ic (Spectroline, SB-100P, 40 mW/cm2, max ~ 365 nm) and mounted at a fixed distance of ~25cm above the sample during the photopolymerization.

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28 Methods The film was photopolymerized in a solution polymerization, which is composed of a 60/40-volume ratio of monomer to aqueous phase that make up the reaction mixture. The monomer phase consisted of HEMA and DEGD MA with varying compositions of DEGMDA from 0 to 2 mol% (Table 2-1). DMPA was added at 1 wt% of the monomers, in which the density of the monomers was assumed to be 1 mg/mL. The aqueous phase consisted of ddH2O. The mixture was homogenized while being purge d with Argon (g) for at least 15 min. After purging the solutions, they were di spensed into a UV transparent gl ass mold with a spacer of 0.8 mm. Then, the molds were placed under the UV lamp for 15 minutes. Samples were rotated during photopolymerization at 33 rpm. After the film was peeled from the glass mold, it was washed in ddH2O against several exchanges. Then, a bore punch (1 cm diameter) was used to cut discs out of the films to make multiple samples. Approximately 12 discs were produced from each film composition. Samples were stored in fresh ddH2O until used in the study. In some preliminary studies, the length of time for polymerization was determined by polymerizing the reaction mixture on a glass slid e. Scratching the surface of the growing film could monitor the cure process and it revealed that it took over 5 minutes for the waterborne solutions to solidify. This is due to the high surf ace tension of the water resulting in it being a poor solvent for a growing polymer and it has a profound effect on chai n length dependence and polymerization rate [42]. Characterization Equilibrium Weight Content (EWC) Equilibrium water content (EWC), or swelling, was measured to determine its dependence on the crosslinking density (n = 3). Each disk was dried thoroughly under vacuum for 3 days and

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29 then weighed (Wdry). Then, they were placed in a mi crofuge tube with 2 mL of ddH2O or urine (pH 6.1). Samples were incubated at 37C for 2 days, blotted dry and weighed (Wwet). EWC was calculated based on the equation, 100%wetdry wetWW EWC W The p(HEMA) films showed a dependen ce on DEGDMA for its EWC. Results are reported numerically and with a bar graph in Figure 2-3. The films swell until there is equilibrium between the osmotic forces and th e elastic nature of th e network chains. The crosslinker, DEGDMA, provides a retractive force to the chains of p(HEMA), and increasing the crosslinking density thereby re duces swelling and water uptake. It has been stated in the l iterature that p(HEMA) forms hydrogen bonds that can make the swelling independent of low crosslink density but was not the case here [35]. Diffusion of solutes, such as water, through hydrogels is base d on the materials EWC, crosslinking density, and chemical nature due to the physical and chemical interactions with the solutes. A high value for EWC can indicate if the diffu sion of oxalate or the enzyme by-products will diffuse as easily in urine as it does in weak buffers. The norma l range of EWC for p(HEMA) is 3040% water [40], and that was the case here. These p(HEMA) films showed a sim ilar dependence for swelling in ddH2O and urine. From 0 to 2 mol% DEGDMA, the EWC decreased in ddH2O from 41% to 33%, and, in urine, EWC decreased from 40% to 36%. Part of the difference in EWC between urine and water may due to deposits that were visibly formed on the films stored in urine. Scanning Electron Microscopy (SEM) Images of freeze-dried films containing imm obilized enzyme were taken with a field emission SEM (Jeol 6335F). Films were pro cessed for SEM by storing hydrated in ddH2O, flash

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30 freezing with N2 (l), and then placed into a freeze -drier for 24 hours to remove the H2O phase. This technique preserves the microstructure of th e films in the swollen state. Pieces of these freeze-dried samples were coated using a Au/Pd target and stored in a vacuum oven at 25 C and 30 Hg. An accelerating voltage of 5 kV was typically used to prevent the sample from being burned by the electron beam. Films of photopolymerized p(HEMA) showed a single-phase network that had a solid morphology (Figure 2-3, A-D). Wit hout crosslinker, defects were visible on the surface as a beaded residue (Figure 2-3, A). With 0.5% DE GDMA the polymer was much smoother than without (Figure 2-3, B). At 1.0 and 2.0% DEGDMA, the p(HEMA) f ilm was smoothest (Figures 2-3, C and D). Films were generally non-porous although a few pores were noticeable in 2% DEGDMA on the order of 10 m. Conclusion Films of lightly crosslinked p(HEMA) were synthesized via photopolym erization and their characterization were conducted by swelling in ddH2O and urine. Films were evaluated by their EWC and results showed that the films in ddH2O and urine were not sign ificantly different. Yet, at higher crosslink densities, EW C was higher in urine, which wa s possibly due to more deposits accumulating on the film in urine. The surface structure as shown by SEM show ed a residue on the surface of samples without DEGDMA whereas 1.0 and 2.0% DEGDMA were smooth. Pores of 10 m diameter were seen in 2.0% DEGDMA. Since pores can weaken a material, it is ideal that the coating be non-porous and homogenous to be able to withsta nd the shear stresses of being implanted. The p(HEMA) photopolymerized by 1% DEGDMA had th e smooth, continuous surface structure and will be the main composition of the films used in the next chapter of enzyme activity tests.

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31 H2C CH3 O O CH2 CH2 O O H2C n = 2CH3 H2C CH3 O O CH2 CH2 OH + I + 99 1 H2C CH3 O O CH2 CH2 OH H2C CH3 O O CH2 CH2 O H2C y O H H2C xx = 99 y = 1n = 2 Figure 2-1 Molecular reaction a nd structure of free-radical in itiation with HEMA and DEGDMA for p(99m% HEMA-c-1m% DEGDMA). O OCH3 OCH3 O + OCH3 OCH3 +h O OCH3 CH3 h Figure 2-2 Molecular structures of the photoin itiator, DMPA, and its decomposition pathways. Table 2-1 Composition of p(HEMA) films with varying concentration of DEGDMA. P(HEMA-x-DEGDMA (mol%) HEMA (mL) DEGDMA (mL) DMPA (mL) ddH2O (mL) 100-x-0.0 0.600 0.010 0.400 99.5-x-0.5 0.594 0.006 0.010 0.400 99.0-x-1.0 0.589 0.011 0.010 0.400 98.0-x-2.0 0.578 0.022 0.010 0.400

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32 30 32 34 36 38 40 42 00.512 DEGDMA (mol %)EWC (% wt liquid ) ddH2O Urine Figure 2-3 Equilibrium swelling of p(HEMA) with varying DEGMA in ddH2O and urine. A. B. C. D. Figure 2-4 Micrographs of the surface of p(HEMA) films with varying degrees of crosslinker, DEGDMA: (A) 0%; (B) 0.5%; (C ) 1.0%; (D) 2.0%. The scal e bar of all images is 100 m.

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33 CHAPTER 3 IMMOBILIZATION OF OXALATE-DEGRADI NG ENZYMES INTO P(HEMA): EFFECTS ON ACTIVITY KINETICS Introduction Oxalate-degrading enzymes catalyze the breakdo wn of the oxalate anion. There are two main types of oxalate-degrading enzymes, oxala te oxidase (OxO, EC 1.2.3.4), found in plants and some bacteria, and oxalate decarboxylas e (OxDc, EC 4.1.1.2), found in fungi. These enzymes regulate a variety of metabolic pathways in their host organisms, but their ability to degrade oxalate can be exploited for t echnological uses, as discussed here. The oxalate anion can be pathological for diseases such as primary and secondary hyperoxaluria, chronic rena l failure, and calcium oxalate nephroli thiasis. It can also lead to the fouling of urological prostheses by mineral encrus tation [3,4]. The ability to measure oxalate is crucial for diagnosing these conditio ns, and clinicians currently us e the free enzyme of OxO as a common technique. Yet, it would be advantageous to immobilize oxalate-degrading enzymes as a more convenient and reusable type of meas uring technique. Furthermore, it has been envisioned that immobilizing such enzymes can allow them to be used therapeutically, for instance, to be coated on urol ogical prostheses to inhibit calc ium oxalate encrustation. Here, the biocompatible hydrogel p(hydroxyethyl methacrylate) was used as a platform for immobilizing OxO and OxDc. This work studied the effects of the immobilization process on the apparent activity of the enzymes. The rate of enzyme catalysis was init ially determined for the free and immobilized enzyme to determine the linear range of activ ity. Relevant Michaelis Menten models were applied to the free and immobilized enzyme fo r comparison to quantify rate constants for catalysis of each enzyme. This comprehensive analysis demonstrates the difference between

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34 measuring free and immobilized enzyme behavior and highlights the complexities to accurately measure activity of the immobilized enzyme. Oxalate Oxidase (OxO) Oxalate oxidase (EC 1.2.3.4) catalyzes the oxyg en dependent oxidation of oxalate into hydrogen peroxide and carbon diox ide (Scheme 3-1) [49-51]. Its activity depends on dissolved oxygen and hydrogen at the active site [52, 53]. The en zyme is derived from the barley root and is a hexamer with units of 22 kDa (Mw~132kDa), an isoelectric point (pI) of 6.9, and a triad of manganese atoms at its active center, which is wher e oxalate catalysis occurs [51-54]. It has been reported that OxO is pH-dependent for activity w ith an optimum activity of pH 4.0 [52]. Kinetic constants of the free enzyme, such as km, have been reported to be as low as 0.27 mM and high as 1.3 mM [52, 55]. One of these research group s did not detect substrate inhibition whereas another observed it at 4 mM [52, 55]. It has also been repo rted to have a specific activity as high as 34 mol/min/mg [55]. Oxalate Decarboxylase (OxDc) Oxalate decarboxylase (EC 4.1.1.2) converts oxalate into formate and carbon dioxide (Scheme 3-2) [56]. Structural and activity studies of OxDc, similar to OxO in this regard, have shown that Mn2+ plays a cooperative role as a cofactor at the binding site of oxalate [57]. The Mn2+ is bound to a triad of histid ines within OxDc and plays a key role in catalysis [58]. The enzyme is only active in a hexameric stru cture (Mw~264 kDa) and is reported to have an optimal activity range from pH 4.0-5.0 and a specific activity of 21.0 mol/min/mg at pH 5.0 [56, 58, 59]. Crystallography studies have shown th at OxDc has an elliptical shape with focal points being 90 and 85 [60]. Also, it has been reported that OxDc has a pI of 6.1 [61].

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35 Enzyme Immobilization The fundamental parameters to be consider ed for enzyme immobilization include: 1) Relative enzyme activity before and after immo bilization, 2) Favorable coupling conditions, 3) Stability of the protein-substrate complex, and 4) Determination of the active enzyme concentration within its matr ix [38, 62]. Properly designed s upports stabilize enzyme activity over an extended period of time to be used episodically [38, 62, 63]. The immobilization technique applied in this wo rk entrapped the enzymes using a UV-photopolymerizable process for a lightly crosslinked hydroge l composed of poly(2-hydro xyethyl methacrylate-x-1mol% diethylene glycol dimethacryl ate) (p(HEMA-x-DEGDMA)). Kinetic Model The reaction for enzyme catalysis of its substrate is given as, 12 1kk kESESEP (1) where E is the enzyme, S is the substrate, a nd P is the product. The enzyme-substrate complex, ES, forms for the catalytic process to occur, and the constants, ki, denote the rates associated with the formation or breakdown of the ES complex. When Michaelis -Menten kinetics is observed, it can be described as max[] []mS VV Sk (2) The Michaelis Menten constant, km, is defined by, 12 1()mkk k k (3) and represents half the am ount of substrate needed to saturate the enzyme. Vmax is the maximum velocity when the enzyme is saturated by its subs trate. These kinetic cons tants can be used to

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36 compare oxalate activity between the free and i mmobilized enzymes and try to learn what may cause the changes in activity between the two forms. Experimental Procedures Enzymes. Oxalate oxidase from barley seedlings (Sigma) was provided as a lyophilized powder. Stock solutions of OxO were prepared to 1.0 mg/mL in 20 mM Hexamethylenetetramine-HCl, 100 mM NaCl bu ffer (Hex-NaCl). A speci fic activity of 0.71 units/mg solid was indicated for commercial OxO. Specific activity is de fined as the amount of OxO (mg) necessary to produce 1 mol H2O2 from oxalate per minute at 37C at a pH 3.8. Results of protein con centration from a Bradford assay us ing Coomassie Plus indicated only 10% of the dry weight was actual protein, which was accounted for in the reported values herein. The lab of Nigel Richards, Chemistry departme nt, University of Fl orida, provided OxDc, which was prepared through recombinant methods. This enzyme was provided at 1.2 mg/mL in 20mM Hexamethylenetetramine-H Cl, 500 mM NaCl, pH 6.1. As determined by the Richards research group, the OxDc had a specific activit y of 58 units/mg-dissolved solid. One unit is equal to the production of 1 mol formate/min by 1 mg of equivalent solid OxDc in solution measured at 22C in 50 mM acetate, pH 4.2. Polymer Synthesis. HEMA monomer was passed over a column packed with charged dimethacrylate (DEGDMA), was used as received and stored at 20C. The photoinitiator, 2,2dimethoxy-2-phenylacetophenone (DMPA), was di ssolved to 600 mg/mL into N-methyl pyrollidone (NMP) and stored at 20C. All chemicals were stor ed in amber bottles or vials. The UV lamp was mounted at a fixed distance of ~25 cm above the samples. An LP record player was used as a turntable for the samples to get a uniform exposure of UV irradiation on all sides of the sample.

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37 Other reagents and equipment. Chemicals purchased from Sigm a Chemical, USA, include the monomers HEMA and DEGDMA; the enzymes Oxal ate Oxidase, Horseradish Peroxidase (EC 1.11.1.7), and Formate Dehydrogenase (EC 1.2.1.2); and, buffer salts such as hexamethylenetetramine-HCl, sodium chloride sodium acetate, sodium hydroxide, hydrogen peroxide, and potassium formate. All chemicals were processed with deionized, distilled water (ddH20, 18.2 M Barnstead). Activity assays were co lorimetric and performed on Beckman DU-640 spectrophotometer (GMI, Inc) and absorption of all samples was measured in PMMA cuvettes (1.0 cm). Film Preparation Reaction solutions for the films were composed of 40 v% buffer (with dissolved enzyme or without for controls) and 60 v% monomer (Table 1). The same amount of polymer was in each film for this work. Enzyme buffers (1 mg/mL) were diluted with Hex-NaCl by 50 v% (0.5 mg/mL). The photoinitiator, DMPA, was added to the reaction solution at 1 w% to the total weight of HEMA and DEGDMA ( monomer ~ 1 mg/mL). The monome r, photoinitiator, and diluent, such as the en zyme buffer, were mixed and then de gassed with Argon (g) for at least 10 minutes before photopolymerization. Films of ea ch sample were derived from aliquots of 50 L reaction solution pipetted into a mold made of 2 glass slides with 0.8 mm tubing as a spacer. The molds were placed under the UV lamp to cure for 15 minutes while rotated at 33 rpm. The films were then removed, washed, a nd stored in Hex-NaCl (22C, 2 days) before the initial activity test. For free enzyme tests, stock solutions we re diluted with Hex-NaCl buffer immediately preceding the test. Enzyme Activity Test To start the activity tests, the films were rem oved from their storage solution, blotted dry, and immersed in a 2 mL vial with ddH2O (0.5 mL) and 200 mM acetate (0.25 mL). The activity

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38 test was initiated when 200 mM oxalate (0.25 mL) was added (Vt = 1 mL), except for tests that varied oxalate concentrations a nd were diluted accordingly. Activity tests were run at 37C for a predetermined amount of time. Then the sample solutions were treated with their respective assay method to measure the product. Activity Assay for OxO Reaction vessels of the OxO samples were pl aced in boiling water for 5 minutes to stop any further reaction. The samples were then cooled to measure th e total activity. A colorimetric assay was used to measure OxO activity from hydrogen peroxide (H2O2) formed at a 1:1 molar ratio to oxalate (Scheme 3-3). The assay uses a dye, ABTS, (2-2aziisobis-3-ethylbenzthiazoline6-sulphonic acid), which unde rgoes a colorimetric change when it is oxidized by the activity of Horseradish Peroxidase (HRP). The ab sorption of ABTS* was read at 650 nm. The OxO activity assay consisted of an OxO sample or standard (0.5 mL), which was mixed with 5 mM ABTS (0.49 mL) and 2400 U/mL HRP (10 L) in ddH2O (Vt = 1 mL) [64]. A color change from clear to green was immediate and the absorbance was read within 10 minutes of mixing. A stock of 8.8 mM H2O2 was made by diluting 30% H2O2 (1 L) in ddH2O (0.999 mL) (Vt = 1 mL). Standards of 0, 8.8, 44, and 88 nmol H2O2 were prepared by respectively bringing 0, 1, 5, and 10 L H2O2 stock to 0.5 mL with ddH2O and then adding 200 mM acetate, pH 4.2 (0.25 mL) with 200 mM oxalate, pH 4.2 (0.25 mL) (Vt = 1 mL). Activity Assay for OxDc To stop any further reaction of OxDc, 100 L of 1 M NaOH was added to samples and mixed thoroughly (Vt = 1.1 mL). A second colorimetric a ssay was used to measure formate, a 1:1 molar by-product of OxDc, and quantify d ecomposed oxalate (Scheme 3-4). The assay utilized NAD+, which is reduced by FDH activity agains t formate. The absorption of NADH was read at 340 nm.

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39 The OxDc assay was adapted for larger samp le volumes and enzyme concentrations, and consisted of 0.1 mg/mL FDH, 1.0 mg/mL NAD+, 50 mM KiPO4, pH 7.6, (0.945 mL) mixed with a sample or standard (0.055 mL) (Vt = 1 mL) [59, 65]. A change in absorption occurred when NAD+ reduced to NADH. This absorbance was measur ed at 340 nm after incubating overnight at 37C. Formate standards from 0-10 mM were pr epared from a stock of 100 mM formate in ddH2O (0.1 mL), and was mixed with 200 mM acet ate (0.25 mL), 200 mM oxalate (0.25 mL), ddH2O (0.4 mL), and 1M NaOH (0.1 mL) (Vt = 1.1 mL). Results and Discussion Activity with Respect to Time To apply Michaelis-Mente n kinetics to the enzymes, an initi al test needed to be conducted to determine the linear region of activity over time. The purpose was to determine a time point within the linear region to test for km and Vmax in a later experiment. In this activity test, the products of OxO and OxDc were measured over time for a period up to 60 minutes in a solution of 50 mM oxalate in 50 mM acetate, pH 4.2 (1 mL). OxO Free and immobilized OxO tests showed a linear response up to 60 minutes for the production of H2O2 (Figure 3-1). The rate of oxalate c onsumption was higher in the case of immobilized OxO (1.0 nmol/ming) than for free OxO (0.5 nmol/ming) as determined by the slopes of the linear curve. This indicates a positive response by OxO to the immobilization process due to an apparently higher activity. Yet, the immobilization process is unlikely to improve the inherent activity values of OxO. OxDc Free and immobilized OxDc showed measured product that was linear up to 30 minutes, yet they exhibited a decreasing rate from 30 to 60 minutes (Figure 3-2). In the linear region up to

PAGE 40

40 30 minutes, the rate of free OxDc (25 M/ming) was much higher than immobilized OxDc (7.5 M/ming). The decrease in the activity for immobilized OxDc was not due to the photopolymerization process beca use 1) another experiment to study UV exposure on free enzyme activity did not reveal large sensitivity to irradiatio n by either enzyme (supplemental data), and 2) it did not seem to aff ect immobilized OxO in Figure 3-1. The lower rate of immobilized OxDc may be due to the impeded release of formate, which could build up in the film. If the formate were bound, this would obscure the measurable formate in the solution, and explain why the activity is lower overall for the immobilized OxDc. This could also an eventual effect on OxDc activity like product inhibition. The film provides a volume ~10 % than that of the solution, and this concentrates both substr ate and product in the film. If indeed the formate becomes bound, it does not become rel eased from the film (supplemental). The non-linear activity in free OxDc after 30 minutes suggest s that product inhibition could have occurred, the enzyme activity dies as a function of tim e, or the enzyme was no longer saturated by oxalate. The latter point seems un likely by analyzing the da ta. At 30 minutes, free OxDc consumed 800 M/ g oxalate and by 60 minutes 950 M/ g was consumed. Since there was only a loss of 1 mM oxalate from the in itial 50 mM oxalate, more than enough oxalate would be present to saturate th e enzyme. Therefore, the rate lo ss of product formation should not be from a loss of saturation with oxalate. Produc t inhibition has not been reported before for OxDc in the literature. In an experiment to expose the enzyme to oxa late and measure reusability, there was some evidence to suggest OxDc activity dies over continued use (Fi gure 3-3, n = 3). The typical activity test was applied after expos ing the films to a low level of oxalate (1 mM). On day 1, the

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41 film lost 80% of its initial activity. After being ri nsed and put back into solution, the film lost all but slight activity by days 2 and 3. The activity of OxDc is not stable against exposure to oxalate, especially in this buffer. OxO showed a decrease in activity after repeated use but the loss was not as large as OxDc. The other buffers studied for presoaking the films include, the Hex-NaCl storage buffer (pH 6.1), acetic acid at the optimal pH for activ ity (pH 4.2), and urine (pH 6.1). It was shown that films from both enzymes actually preserved their activity longest in urine, which is a solution these enzymes could be used for an appl ication to inhibit encrustation. The increase in activity from being stored in urine is probably du e to the higher ionic strength of the solution and the presence of other proteins in urine like albumin that could stabilize enzyme structure. The trend shows that the enzymes loose activity agai nst oxalate over repeated use in each of the solutions, yet OxDc dies faster than OxO. Activity with Respect to Oxalate Concentration A 30-minute test period was chos en since it was at the upper end of the linear region for OxO and OxDc in the free and immobilized stat e. Oxalate was varied from 0.5 to 100 mM in order to test a wide range of concentrations. The raw data for enzyme activity was compiled with the Michaelis Menten equation to determine the kinetic constants using KaleidaGraph (Table 2). OxO Free OxO clearly exhibits subs trate inhibition after 10 mM oxalate (Figure 3-4a). The activity of free OxO at 50 mM oxalate is about half the activity at 10 mM oxalate. Substrate inhibition for OxO has been reported 4 mM oxalate [55]. The experiments showed that the immobilized OxO had a higher apparent activity at 50 mM oxalate (Figure 3-4b). For example, at 50 mM oxalate the activity appears to be 0.8 M/ming for free and 1.1 M/ming for immobilized OxO.

PAGE 42

42 Substrate inhibition is a phenomenon in whic h the substrate builds up by binding to the non-active sites of the enzyme a nd lowers overall activity. The cons tant for substrate inhibitions formation, ks, is factored into Michaelis Menten models shown below, max[] [] []1m sVS V S kS k (4) The substrate inhibition mode l can explain the higher activity for immobilized OxO in terms of ks. Immobilized OxO exhibits a larger ks value (650 mM) than free OxO (35 mM). This larger ks value for immobilized OxO represents a smalle r effect in substrate inhibition, and this difference in ks highlights the transport resistance of oxalate into the film. The kinetics of free and immobilized OxO are different at concentra tions higher than 10 mM oxalate. Less substrate inhibition can be seen for immobilized OxO. As oxalate increases, immobilized OxO has a constant velocity (Vmax) up to 75 mM oxalate, and substrate inhibition finally appears in the films th rough 100 mM. The activity of immob ilized OxO is twice that of free OxO at these levels of oxalate. Using the substrate inhibition model from equation (4), the Vmax for free OxO (1.8 M/ming) was indeed larger than for immobilized OxO (1.2 M/ming). The km of free OxO (1.8 mM) was about half that of the immobilized OxO (4.1 mM), which indicates a decrease in affinity for oxalate in the immobilized form. If the data is modeled using regular Michaelis Menten kinetics, equation (3), the data is poorly modeled for free OxO. Without accounting for substrate inhibition, the Vmax for immobilized OxO stays the same but the km decreases to 3.1 mM and appears to have a higher affinity for oxalate. It was not expected that the immobilized Ox O would have a higher ac tivity than free OxO. It is assumed that the enzyme would not improve its kinetic pr operties upon immobilization. The

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43 difference in ks between free and immobilized OxO expl ains this behavior. A lower oxalate concentration inside the film could lead to le ss substrate inhibition. A diffusion resistance of oxalate through the film could be due to the negative charge of oxa late at pH 4.2 (pKa,1 = 1.2 and pKa,2 = 4.2). The apparent enzyme activity was m easured higher since it was affected less by substrate inhibition and this is supported by the different ks values. OxDc A 30-minute test period was chosen to test OxDc against oxala te, so that the activity was not in the non-linear rang e of Figure 3-2. The plots for reacti on rate against oxalate concentration (Figures 5a and 5b) show th at the kinetic values for Vmax and km are much slower for the immobilized than the free form of OxDc (Table 2). Also, the activity of immobilized OxDc is not as efficient as the case for immobilized OxO. The Vmax for immobilized OxDc is 20% than that of the free form, and the km of free OxDc is over 40 times less that of immobilized OxDc. It is apparent from that free OxDc is saturated with OxDc by 10 mM oxalate (Figure 3-4), and immobilized OxDc possibly reaches saturation by 50 mM oxalate. The dramatic effect on immo bilized enzyme activity is explained by mass transfer effects of th e substrate and product. Formate, a charged species, would have an ionic interaction with the immobilized OxDc that inhibits its diffusion out of the polymer. OxDc has a pI of 6.1, and has a positive charge at pH 4.2, which could attract the negatively charged formate (pKa of formic acid is 3.8). Although the formate concentration outside of the film did not change over time once the activity was stopped (supplemental), it does not necessarily m ean the ion would desorb from the enzyme. It is hard to determine if oxalate or formate diffusion caused the low activity for immobilized OxDc. It may be a combination of both. But, the volume of the film (100 L) is ten times less than the free enzyme solution. The i mmobilized enzymes have a higher concentration

PAGE 44

44 of ions inside surrounding it in th e film than the solution with free enzyme, and this rise in ionic concentration may have effects that thes e experiments cannot directly measure. Formate concentration inside th e film would influence the en zyme activity since OxDc is susceptible to activity die off or product inhibi tion. If the product accumulates in the film because it does not diffuse sufficiently, the appa rent activity outside the film is lower. When formate diffusion from the film is hindered, it is further possible that less formate is generated over time due to reduced activity or product inhibition. Kinetic Models These following models represent the transport parameters of product and substrate for the free and immobilized enzymes (Figure 3-6). They provide simplified representations of how the enzymes would generally behave according to cert ain rates controlling th e diffusion of substrate or product. Here substrate in hibition is accounted for OxO. Conclusions Oxalate-degrading enzymes (OxO and OxDc ) were entrapped into p(HEMA-x-1mol% DEGDMA) using a photopolymerizati on technique. Enzyme activity was measured for the free and immobilized forms and Michaelis Menten models were applied to study the kinetic constants, including substrate in hibition. These models compared the kinetic constants to understand the effects of the immobilization pr ocess on the entrapped enzyme activity and whether a substrate inhibition model was appropriate. Free OxO was shown to suffer substrate inhi bition with at least 10 mM oxalate. The substrate inhibition model gave a ks of 35 mM for free OxO. The immobilized OxO did not show substrate inhibition during th e 30 minutes test except at 100 mM oxalate and had a ks of 660 mM. Mass transfer effects of oxalate in the film allow less substrate to the enzymes, which enables OxO to have a higher activity (or reduced substrate inhibition). According to the

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45 substrate inhibition model, the Vmax for free and immobilized OxO are 1.8 and 1.2 M/ g, respectively, and the km for the immobilized OxO is half th at of free OxO at 4.1 mM. Without accounting for substrat e inhibition, the Vmax for free and immobilized OxO are 0.9 and 1.1 M/ g, respectively, and the km for the both free and immobilized OxO is lowered to 0.3 and 3.1 mM, respectively. OxDc dramatically looses activity against ox alate and does not display a highly retained enzyme activity. Using the Michaelis Menten model, the free OxDc has a Vmax of 23.5 M/ming and a km of 0.5 mM for free OxDc wher eas immobilized OxDc has a Vmax of 5.0 M/ming and a km is 23.2 mM. The decreased activity meas ured by formate production may be due its diffusion resistance from the film, which could impede ongoing activity by OxDc activity dieing off or through product inhibition. Hydrogen peroxide, measured as the OxO product, has a neutral charge and would not have substantial interactions with the film or enzyme to affect its diffusion from the film. Furthermor e, oxalate diffusion into the film also decreases activity for immobilized OxDc although it helped OxO in these experiments to determine kinetic constants. Enzyme activity could be improved in future tests by changing the characteristics of the film. A change of crosslinker de nsity could elicit a better under standing of oxalate diffusion and ensure substrate inhibition is mi nimized at physiological values of oxalate. Also, the film for OxDc could be changed to include acrylic acid, so that the acid groups can be ionized and repel formate from the bulk. This paper has found that the nature of the film and entrapped enzymes inside its bulk affect the diffu sion of ions. Yet, the overall enzyme activity of the immobilized enzymes reveal this technique is viable for measuring oxalate at physiologically levels of oxalate or to even use it in urine for encrustation resistance.

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46 Supplemental Data Effects of Ultra-Violet Li ght Exposure on Activity Photopolymerization was studied for the e ffects of UV light exposure had on enzyme activity (Figure 3-7). Free enzyme (2 g OxO or OxDc) was mixed with ddH2O and 200 mM acetate similarly to the free enzyme test with time. This solution was placed under the lamp at a distance of 30 cm. After a period of time (0, 1, 5, 10, 20, and 30 min), the samples were taken out of the UV radiation. When samples were finished, they had 200 mM oxalate (0.25 mL) added to initiate the activity test (15 min). Exposure to UV radiation show ed a steadily decreasing enzy me activity. Initially, the enzymes do not lose much initial activity in the first five minutes (~5%). Then, enzyme activity becomes more affected by 10 to 20 minutes. OxO seems to have less appreciable affect until 30 minutes (89 to 77%) where as OxDc shows stead y declining activity fr om 10 through 30 minutes (85 to 73%). Product Diffusion from Films over Time A test was devised to determine if the produc t of enzyme activity was releasing from the films over time to know if the m easured product was a function of time. This would indicate if the product were bound to the film or immobilized enzyme, and how large the measured product deviated over time. The results show that it a ppears there is no significant difference in the measured product for both enzymes (Figure 3-8a and 3-8b for OxDc and OxO, respectively).

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47 O O HO O OxOHOOH OC O Oxalate Hydrogen Peroxide Carbon dioxide O2, H+ (2) Scheme 3-1 Decomposition of oxalate by OxO. O O HO O OxDcC O O OC O H Oxalate Formate Carbon dioxide "cat. O2", H+ Scheme 3-2 Decomposition of oxalate by OxDc. ABTSHOOH HRP ABTS* Scheme 3-3 Chemical reaction of OxO a ssay to measure oxalate degradation. C O O H NAD FDH H NADH Scheme 3-4 Chemical reaction of OxDc assay to measure oxalate degradation. Table 3-1 Composition for immobilizing OxO and OxDc into p(HEMA) films. Composition of Reaction Solution Volume of Reactants (per mL of reaction mixture) Enzyme Buffer (0.5 mg/mL) 0.400 mL 99mol% HEMA 0.589 mL 1mol% DEGDMA 0.011 mL 1w% DMPA 0.010 mL

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48 0 10 20 30 40 50 60 70 80 0102030405060Time (min)Production rate of H2O2 ( M/ g OxO) Free Imm Figure 3-1 Activity of free and immobili zed OxO for periods up to 60 minutes. 0 100 200 300 400 500 600 700 800 900 1000 0102030405060Time (min)Production of formate ( M/ g) Free Imm Figure 3-2 Activity of free and immobili zed OxDc for periods up to 60 minutes.

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49 A. 0 1 2 3 4 5 6 7 20mM Hex 100mM NaCl, pH 6.1 50mM Acetate, pH 4.2 50mM Acetate, 1mM oxalate, pH 4.2 urine, pH 6.1Storage BufferOxDc Activity ( M/ming) Day 1 Day 2 Day 5 Day 7 B. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 20mM Hex 100mM NaCl, pH 6.1 50mM Acetate, pH 4.2 50mM Acetate, 1mM oxalate, pH 4.2 urine, pH 6.1Storge BufferOxO Activity ( M/ming) Day 1 Day 2 Day 5 Day 7 Figure 3-3 Activity of immobilized enzyme in p(HEMA) after being stored in a buffer solution, tested against 50 mM oxalate, a nd restored. (A) OxDc; (B) OxO. Table 3-2 Constants determined by applying Michaelis Menten kine tics on raw data from Figures 1 and 2. (N.A.-not applicable, n = 2) Enzyme (state) Vmax ( M/ming) km (mM) ks (mM) OxO (free) 1.8 0.1 1.8 0.1 35.4 3.7 OxO (imm) 1.2 0.2 4.1 0.6 660 140 OxO (free) 0.9 0.0 0.3 0.0 N.A. OxO (imm) 1.1 0.1 3.1 0.1 N.A. OxDc (free) 23.5 1.4 0.5 0.1 N.A. OxDc (imm) 5.0 1.9 23.2 9.1 N.A.

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50 A. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 020406080100120Oxalate (mM)Production rate of H2O2( M/ming) B. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 020406080100120Oxalate (mM)Production rate of H2O2( M/ming) Figure 3-4 Activity tests for reaction rates for OxO. (A) free OxO; (B) immobilized OxO.

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51 A. 0 5 10 15 20 25 30 020406080100120Oxalate (mM)Production rate of formate ( M/ming) B. 0 1 2 3 4 5 6 020406080100120Oxalate (mM)Production rate of formate ( M/ming) Figure 3-5 Activity tests for reaction rates for OxDc. (A) free OxDc; (B) immobilized OxDc.

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52 Model for free OxO SOxOOxOSOxOP+ + kcatk1k2 +S OxOSS k3k4 Model for immobilized OxO OxOOxO+ + kinkoutkcatk1k2 +S k3k4SoSiPoPiOxOSiOxOSiSi Model for free OxDc OxDcOxDcSOxDcP+ + kcatk1k2S Model for immobilized OxDc OxDcOxDc+ + kinkoutkcatk1k2SoPoSiPiOxDcSi Figure 3-6 Proposed models for the kinetic m echanisms of oxalate catalysis by free and immobilized OxO and OxDc. The model fo r OxO accounts for substrate inhibition (represented by OxOSS).

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53 60 70 80 90 100 05101520253035UV Exposure (min)% Relative Activit y OxDc OxO Figure 3-7 Activity profiles of free OxO (2 g) and OxDc (2 g) against 50 mM oxalate after exposed to UV for different periods of time.

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54 A. 0 2 4 6 8 10 0100200300400500Time After Reaction Stopped (min)Rate of formate production ( M/ming) B. 0.0 0.5 1.0 1.5 0100200300400500Time After Reaction Stopped (min)Rate of H2O2 production ( M/ming) Figure 3-8 Profiles of product release from films over time after the activity test.(A) OxDc; (B) OxO.

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55 CHAPTER 4 DEVELOPMENT OF A P(HEMA) COATING FOR THE PURPOSE OF IMMOBILIZING OXALATE-DEGRADING ENZYMES ONTO A URETERAL STENT Coatings Overview Coatings are products of liquids, pastes, or powders that are a pplied to surfaces, or substrates, and form a film that is protective, decorative, or provides so me other functional role. The main components of a coating are its binders pigments, solvents, and additives [66]. Many types of coating formulations include resins, latex, or mono mers [67]. Use of waterborne coatings over organic solvents has become more popular in recent years since it reduces fire hazards, environmental pollution e.g. ozone laye r degradation, higher insurance premiums and added maintenance on manufacturing equipment [67,68]. Photopolymeriza tion is an emerging field and considered to be a viable way to process materials containing enzymes or other biologics, especially since its fa ster and more benign to enzyme structure than thermal systems [68]. Curing by UV radiation is a simple, relia ble, low energy, and quick method for polymerization [67]. It is widely utilized in the coating industry and traditionally applied for fastdrying protective coatings, printi ng inks, and adhesives [68]. Phot opolymerization is being used in biomedical applications, in particular, to form hydrogels for coatings that can serve various functions such as lubrication, low protein f ouling, or microarrays for patterning cells and proteins [69-75]. Hydrogels s ynthesized by photopolymerization ha ve also been applied as coatings for urological materials to deliver antibiotics or prov ide lubricity [76,77]. Due to a need to minimize stress while processing enzymes and preserve their active structure, a waterborne, photopolym erization method in an aqueous buffer was devised for this project. This method helped to process disso lved enzymes and utilize room temperature

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56 conditions upon polymerization. Waterborne syst ems are becoming more common in biomedical coatings, and many successful in incorporat ing biologics, including p(HEMA) [78-81]. The entrapment of enzymes, growth factors, and cells by photopolymeriz ation is chosen as an alternative to thermal initiation due to the se nsitivity of biologics to high temperatures [38]. Photopolymerization has been previously used to entrap cells, drugs, and proteins [68, 79, 80]. Entrapment occurs when the coating is cure d and the polymer forms around the enzyme, which physically restricts diffusion of the enzyme with in the hydrogel mesh. The immobilized enzyme has been shown to retain signifi cant amount of activity wi th respect to enzyme in the free state. In addition, a higher concentration of enzyme (relative to surface adsorption or chemical bonding to a surface) might be possible due to the three-dimensional st ructure of a hydrogel coating. Therefore, UV photopolymerization for the encapsulation of the oxalate-degrading enzymes into a hydrogel coating seemed like the most viable approach for this project. An advantage of photopolymerization is the ab ility to control when chain propagation is initiated. Photopolymerization requires that a pho toinitiator be mixed into the solution, and upon UV irradiation, it decomposes to generate a free radical, which initiates the propagation of chain formation. The chain length is dependent on initiator and monomer concentration; so small quantities of initiator are used to form long, elastomeric chains between crosslinks [47]. Too much initiator could also cause defects in th e monomer network like lo ops and dangling chains. Typically, initiator is applied at 1w% of the monomer content. Coatings can be applied by dip coating met hods, which provide a low cost, simple, and easy way to control the coating application to a surface [67]. Main parameters that determine coating thickness are viscosity, angl e and rate of withdrawal, and ge ometry of substrate [81, 82].

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57 The geometry of the ureteral stent was a challe nge to apply the reaction mixture by dip coating, so a fundamental approach towards devel oping the coating process was developed. Design Principals A fundamental approach to designing a co ating would entail the following questions: What is the substrate and coating material and how will they react with one another at the interface How will the coating be applied and cured What is the desired coating thickness to optimize substr ate/product diffusion balanced against maximizing the quantity of active enzy me capable of serving the intended function How long will enzyme reside in coating reac tion solution without a significant loss of activity What concentration of enzyme is needed for the intended application What types of activity tests will be applied For how long will the coating be utilized Details of the solutions chosen for these quest ions with respect to stent encrustation are summarized below and indicate the rationale used to develop the composite coating material. Substrate and coating material For this study, Cook Urological Inc. kindly provided ureteral stents to test the coating process. These stents are made of poly(urethane) (PUR). The material for the coating was chosen to be p(HEMA) since it has had prior use as a coating for urological prostheses [41]. These materials could be manufactured together along an assembly line, so the simplest methods were always favored when pursuing the ob jectives of achieving a coating. Initially, wet chemical methods were used to modify the PUR surface and make it reactive. The photopolymerization process ta kes at least 5 minutes and th e reaction solution would drip off the stent before then. This is one reason w hy future studies should try to speed the curing

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58 process. Also, companies use various methods from wet chemistry to coronal discharge in order to modify a surface for reactiv ity. The reaction solution was adhered using superglue, which became the only method that stuck the solution on long enough for it to cure along its length. Stents as long as 10 cm were coated this way successfully in the labor atory. Since chemically modifying the surface didnt make a big di fference at the end, it was abandoned. The immobilization method for the enzymes was chosen to be entrapment, which is in contrast to chemically modifyi ng the protein for binding or adsorb ing the enzyme simply to the coating surface. Entrapment fulfilled the effort to effectively immobilize the enzymes on the stent surface and retain their activity. Application and curing The waterborne coating was to be applied by di p coating the stent into the reaction solution and then curing it by UV photopolymerization. Dip co ating stents with a waterborne solution is not trivial; the coating solution us ually dripped off the stent before polymerizing. Also, water has a high surface tension and is c onsidered to be a poor solvent for flow characteristics during polymerization [67]. A novel tec hnique was employed to affix the reaction solution to the stent. The technique employed to affix the coating onto the stent was based on two concepts: adhesion and viscosity (Figure 4-1). For the first concept, a primer was made to attach the initial layer on the stent (Steps 1-3). Superglue was brushed thoroughly onto clean stents (length = 1to 2 cm) and allowed to partially dry for about 2 mi nutes. Superglue was used as an adhesive to capture the reaction solution onto the stent surface long enough for the photopolymerization to cure the coating. Superglue has been used in various forms before as a medical adhesive, including cyanoacrylate, and there are many ways to characterize its use for a given application and getting FDA approval [83]. Future work can venture into these directions, if necessary.

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59 For the second concept, glycerol was used to increase viscosity of the reaction solution. Added as the aqueous phase in the reaction soluti on, glycerol was used in replacement of buffer while the monomer composition stayed the same. Glycerol changed the reaction solution to a non-Newtonian fluid, which gets less viscous with increasing sh ear rate from dipping. This allowed for more control of thickne ss during application of the coat ing. It also prevented splatter [67]. The stent was dipped into the primer coa ting solution for 30 s econds. Stents were withdrawn slowly by hand and photocured by UV for 30 minutes. More time is allowed for the primer layer to cure than with the enzyme la yers since viscosity is much higher due to the glycerol, which slows the cure rate. Primer coatings were washed sequentially against ddH2O with 25%, 10%, and 0% ethanol. The primer was studied by changing its viscosity and dip time in the reaction solution. The second layers were studied by length of time dipp ing and dip cycles. These studies help deduce how to load this novel coating with enzymes. Enzymes were applied by applying a secondary layer to the primer (Steps 4 and 5). The reaction solution was the same as the f ilms made in Chapter 3 only larger (400 L per 1 cm length). The stents were immersed into the coating solution for a period of time and withdrawn slowly by hand. The secondary layer was finally cured for 15 minutes. Coated stents with enzymes were washed in Hex-NaCl buffer until ready to use. Degradation to enzyme As demonstrated in Chapter 3, the coating solu tion had a short shelf li fe (Figure 3-10). It was also shown to degrade over time from exposure to UV (Figure 3-11). Fresh reaction solutions were polymerized to make the films or coatings in order to limit the exposure of the

PAGE 60

60 enzyme to the denaturing effect of the reaction solution. Exposure to UV radiation was kept to 15 minutes for curing all enzyme samples. Loading concentration of enzyme The concentration of enzyme was based on the composition for the film. In this case, the aqueous phase was limited to 40v% of the reac tion solution. So, the fi nal concentration was determined from its stock solution, which concentration is based on its solubility. For the films used in this study, OxO concentration (0.1 mg /mL) has a maximum concentration at 0.04 mg/mL reaction solution, i.e. 2 g/50 L in the films. A maximum concen tration of OxDc (1.0 mg/mL) was 0.4 mg/mL reaction solution. Concentrations of OxDc can be made higher for this project in the future, but still must be used fr esh to avoid precipitation over time. Enzyme activity tests Colorimetric assays described in Chapter 3 were used to measure enzyme activity. These tests were vigorously studied against varying am ounts of enzyme, oxalate, time, pH and storage buffer. This had provided a guide and baseline for the values determined from enzyme activity in coatings. Coating samples contained much less enzy me than the films characterized in Chapter 3 because less than 50 L reaction solution was applied to the sa mple stent piece. Activity tests for coated stents had to proceed for longer peri ods of time to accumulate enough enzyme activity products to measure (10 hrs for 1 cm stent sample). Storage and use The lifetime of these coatings are designed fo r two periods of time, storage in buffer and application in urine. Storage can be envisioned to be in a steril e package, in which the coated stent is submerged in a storage buffer. The remainde r of the lifetime of the stent will be used in vivo as a ureteral prosthesis in urine.

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61 The goals are to achieve stabile activity of f ilms stored in Hex-NaCl and urine for up to 3 months (Figures 3-26 and 3-28). The President of Cook, Pete Yonkman, acknowledged that it would be useful to show activity for a period up to 3 months to be a competitive product. The data collected for this was better to perform on films rather than stents because films contain a known and larger amount of enzyme to give a strong signal faster. Al so, later trials can utilize a more appropriate manufacturing process for the coated stents. Coating for this Project The p(HEMA) coating developed for this pr oject was designed to provide a proof of principal that immobilization of the active enzyme onto the stent could be achieved. The coating was composed of a primer and secondary layers, in which the primer provided a substrate for the second layer that contains the enzyme to be ap plied. The enzyme was entrapped onto the surface after the secondary solution was ab sorbed into the primer layer a nd was cured in the presence of the enzyme. The main advantage for the primer was that it has an optimal viscosity by using glycerol, and allowed this layer to coat the stent along its length. Both the primer and the secondary layer were mainly composed of p(HEMA). The primer was made with glycerol whereas the secondary layers were made with buffer, in which there were several advantages. First, the primer is a ffixed onto the stent in a quick and simple way and glycerol can help control primer thickness. The buffer dissolved in the secondary coating solution allowed the enzyme to be fully. By dip co ating into the reaction solution with the primer on the stent, the solution was abso rbed into the primer since it was hydrophilic and made of the monomer. This was one way to immobilize the enzyme in an affixed coating on the stent. Materials The coating consisted of 60 v% monomer and 40 v% aqueous phase (e ither glycerol or ddH2O). Monomers included HEMA and DEGDMA. S uperglue was purchased at a local store

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62 and it was composed of cyanoacrylate dissolved in methyl ethyl ket one (MEK). Glycerol (Sigma) was stored at 22 C. It was transferred to a vessel that was placed into boiling water, which lowered its viscosity, so it co uld be pipetted with a micropipettor. Primer Studies The primer was a key for applying the enzyme on to the stent. Preliminary studies were set up to gauge the effect that glyc erol would have on thickness. Gl ycerol could be diluted with ddH2O to reduce its viscosity. This could make the coating thinner, if necessary. Optical photographs were used to show the need for an adhesive like superglu e (see Figure 4-2). Then, SEM was used to assess microstructure and thickness of the coatings after they were photopolymerized (see Figures 4-3 to 4-6). Application The primer was finally developed after a long struggle to get the monomer reaction mixture to adhere to the stent long enough to be cured. After many trials of applying the solution and having a big ring form on the bottom of the st ent after curing, a novel approach had to be taken. Early efforts to hydrolyze the surface with photoinitiators, peroxides, acids, and bases all failed to get a form a full coating along the st ent. Also, Methocel, a cellulose-derivative was added to the reaction solution to increase viscosity. None of th ese methods really provided a robust method for coating. As glycerol was beginn ing to be used as a thickener, the idea came for super gluing the reaction mixture to the stent. Reaction solutions contained either ddH2O (control) and/or glycer ol as the aqueous phase (40v%). Amounts of glycerol va ried by diluting it with ddH2O. Superglue was brushed onto one set of stents before coating with the reaction solution. Stents we re dipped and submerged into the coating solution for 1 minute. St ents were then cured for 20 minutes and washed against ddH2O for 2 days. To stain the coating purple, the stents were dipped into a solution with crystal violet

PAGE 63

63 (0.5w/v%) and rinsed off in water. The stents al one do not absorb stain to an appreciable level. Photos were taken with a di gital camera (Figure 4-2). Glycerol concentration Two parameters were found to affect thickne ss of the primer coating were duration of submersion in coating solution a nd the percentage of glycerol. To observe these effects, SEM was used to characterize various effects from changes in the composition of primer coating solutions. Traditional techniques to analyze coati ngs were taken from planar and cross-sectional views to obtain thickness and porosity. In some ca ses, peeled back films were used, which is common in the analysis of failure in coatings [84]. In Figure 4-3, A-C, stents were immersed for one minute into the reaction solution and then photopolymerized. Figure 4-3, A and B, showed the microstructure, and higher magnification showed the primer to be por ous at the surface and interconnected. Summary of primer thickness was listed in Tabl e 4-1. It showed that the longer the stent was kept in the primer solution, then the more coating adhered. Primer thickness went from 83 to 187 m from a dip time of 30 to 60 seconds. The use of glycerol increased coating thickness, as was also seen from the digital photographs in Fi gure 4-2. From 0 to 40% glycerol, the coating went from 25 to 187 m with one-minute dip times. Variation of primer composition led to severa l effects on thickness and porosity that could be examined. Glycerol could be diluted with ddH2O to change viscosity and be seen be as a thinner primer coating (Figures 4-4 & 4-5). Po rosity could be seen at the surface. With no glycerol and only ddH2O in the aqueous phase, these thinner films exhibited less porosity. Increasing monomer volume fraction in the co ating solution also a ffected thickness and porosity (Figure 4-6). The films were thicke r with 20% glycerol here than with ddH2O. The bulk material seemed non-porous while there was a top layer that exhibi ted an open, cellular structure.

PAGE 64

64 Yet, at planar, low magnifications, it can be seen that it was a denser material throughout the surface than with less monomer content. The kineti cs of the reaction in ad dition to the amount of water in the polymer phase most likely determined final morphology. Secondary Layer Studies The secondary layer was absorbed into the primer, and this way the enzyme could be entrapped onto the surface. The secondary layer wa s initially studied with FTIR and compared to the primer that was applied with glycerol. Triton X-100 was then studied to determine any positive effects it may have for applying the enzyme Extent of enzyme entrapped in coating was assessed with enzyme activity assays. Enzyme wa s applied by submerging stents with primer into the reaction solution for a pe riod of time or by reapplying the coating through layering it. These studies gave an idea of how to best achieve an active enzyme onto a ureteral stent. FTIR Functional groups on samples of p(HE MA) made with glycerol or ddH2O were detected by an FTIR spectrometer. Samples were dried unde r vacuum for 2 days. Films were optically transparent. Peaks from FTIR transmission through the sa mple were shown in Figure 4-7 and summed in Table 4-2. The spectra was similar between the primer and secondary layer of the final p(HEMA) product. Polymerization us ing glycerol did not lead to different FTIR signatures, which could detect a difference in bonding states within p(HE MA). Hydrogen bonding could be expected through hydroxyl groups as a possible difference one c ould see between the spectra. Yet, glycerol seemed to rinse out of the f ilms quickly in wash solutions. Since no hydrogen bonding was detected in the primer spectrum, gly cerol did not affect the final p(HEMA) product.

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65 Triton X-100 Triton X-100 was used as a model surfactant to promote adsorption of the enzyme onto the primer. Triton X-100 is a nonionic su rfactant and can interact with a protein because surfactants have hydrophilic and hydrophobic charac teristics, as are proteins [ 85]. Tests for this experiment were conducted on stents coated with Triton X-10 0 in the formulation as well as tested films made from the coating solution. The reaction solution (Vt = 400 L) was prepared with the OxO stock (1 mg/mL), HexNaCl, and 10v% Triton X-100. Ox O stock concentration was 0.5 mg/mL and final film concentration of OxO was 1 g. Stents had primer already coat ed onto them and hydrated in HexNaCl. These samples were immersed into the reaction solutions for different periods of time, e.g. 1, 10, 30, and 90 minutes (Table 4-3). Results are plotted for absorbance at 650 nm a nd show several trends (Figure 4-8). The profiles between coatings with varying Triton-X 100 are similar at 1 minute and 1 hour and 30 minutes while at 10 and 30 minutes are similar bu t different that the others. Coatings without Triton X-100 are highest from 1 minute and 1 hour 30 minutes dipping while the lowest at 10 and 30 minutes. For all samples, coatings with 0.1% Triton X-100 were higher than 0.5% Triton X-100. A set of samples was made into films from the reaction solutions immediately after the coatings were applied to the stents (n = 3) Samples were photopolymerized and processed as described earlier (Figure 4-9). The ac tivity test was run for 1 hour at 37 C. Results show that film activities from coating solutions without Triton X-100 were highe r by 10-20% than those with Triton X-100. Thus, Triton X100 was not used as an additive to the coating solution.

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66 Layering the Coating The purpose of these experiments was to layer the enzyme onto the primer through a secondary layer and determine how adding the coating layers could maximize enzyme activity. Layering onto the primer was studied two ways. One was to immerse the stent into the reaction solution for a period of time, in which soaking can get more enzyme adsorbed onto the surface. When the reaction cured, the matrix entrapped the enzyme. The second way was to sequentially dip the stent into a reaction solu tion with enzyme. These secondary coating layers were partially cured with UV in between dip cycles. Since the primer and secondary layers are hydrophilic, then they absorb reaction solution for more layers. Primer Primer coatings were prepared by brushi ng superglue onto the st ent (L = 1 cm) and allowing them to dry for about 2 minutes befo re submerging into the coating solution. The coating solution was made with 40% glycerol. Stents were dipped for 30 seconds, and then withdrawn to be cured. This co rrelated to the coatings shown in Figure 4-3, D, that had an average thickness of 83 m. Primers were cured by UV for 30 minutes. The primers were washed against decreasing concentrations of ethanol (25% to 0) and equilibrated with ddH2O prior to coating the secondary layer. One sample of the primer was assessed by SEM (Figure 4-10, A and B). The primer coating seemed to have been applied uniformly with a thickness of about 50 m. Also, the surface was textured in some places as a result of slow curing. Primers were suitable for layering experiments.

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67 Immersion time Coating solutions were prepared with Ox O (1 mg/mL) as the 40v% aqueous phase. Monomers were mixed and the reaction soluti on was purged with Argon (g) for 15 minutes. Stent samples that were already coated with primer were immersed into the solution for various times. Periods of 1, 10, 30, and 60 minutes were tested. After withdrawing from solution, the coatings were cured by UV for 15 minutes. Then, coated stents were washed against several exchanges of Hex-NaCl. Stents were stored in Hex-NaCl until use in activity test. Activity was tested against 50mM oxalate in 50 mM acetate, pH 4.2. Since much less enzyme was applied onto the stents, the activity test against oxalate was allowed to run for 10 hours in order to allow sufficient product to have formed. The results of the activity test showed th ere was a dependence on immersion time (Figure 4-11). At 1-minute immersion, the total activity was to the act ivity at later times. Immersion times of 20 and 60 minutes were similar when accounting for the standard deviations, which were large for all groups. Images of coatings made from various imme rsion times were taken with SEM (Figure 412, A-D). An array of images based on their activity can be compared for their coating morphology. In a qualitative sense, the layers ca n be seen for their to tal coverage, thickness, porosity, and adhesion to the primer. Primer can be seen in Figures 4-12, A-C. The secondary layer is smooth and can be seen in contrast to the primer. In th ese coatings, it was difficult to correlate coating thickness from SEM images to enzyme activity. Although, if the extent of the secondary layer, such as in Figure 4-12, D, was smooth and continuous, then it likely had higher levels of activity.

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68 Dip cycles These stents with primer were immersed into the reaction solution fo r 10 minutes to apply the coating. Then, the coating wa s partially cured for 5 minutes and re-immersed for 10 minutes for each dip cycle. Layering was repeated up to three cycles. Blank coatings without enzyme were also coated onto primers. Results of coating the stent for multiple cycles showed a contradictory result. The activity decreased for increasing dip cycles. This test was repeated and still had similar results. The cause may be to processing issues, such as continue d exposure to UV, or that the enzyme is not accessible within these layers. Coat ings will tend to release particul ates in solution, so further optimization to strengthen the network within th e coating would be requ ired. Dipping one cycle was stronger that dipping extra times. Images were taken with SEM of coatings ma de from up to 3 dip cycles, which showed various levels of activity The highest activity was demonstrated from coatings dipped one time. (Figure 4-14, A-D). In Figure 4-14, A, a low ma gnification image shows the extent of coating along the stent sample that had high activity. Fi gure 4-14, B, showed a higher magnification image of the coating from the same stent. The high activity coating had a full coverage of smooth film. Another stent coated one tim e was shown to not have a full coverage of coating (Figure 414, C). A higher magnification image of this st ent showed that substrate was porous and not dense with a patch of secondary layer. Therefore, an initial second layer that provides a full, smooth coverage of coating was re quired for an active coating. Stents were dipped for 2 or 3 cycles and SEM images were shown in Figures 4-15, A and B, and 4-16, A and B. Although, the average activity of these st ents was lower than one dip, these coatings did retain activity. The full, secondary layer were observed in Figure 4-15, A. In Figure 4-15, B, a high magnification showed the porosity of the sec ondary layer to be less than 1

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69 m. Figure 4-16, A, the image showed the build -up of secondary layers. The wavy primer was seen below the secondary layer in addition to a build up of a ring, which was at the bottom during curing. Figure 4-16, B, showed the secondary layers from the 3 dip cycles at a part of the coating that was broken up a nd exposed the under layers. Conclusion A novel coating was developed using a fundament al approach for designing a coating that was based on understanding the full scope of a pplying and using the coating. The fundamental questions were answered as the research of va rious routes led to the use of superglue and glycerol to form the primer. The primer was required as a result of the poor adhesion and coverage of the stent without these agents. Th e primer provided a means to absorb sequential layers that could include oxalate-degrading enzymes. Studies were established to determine process parameters, such as dip time and glycerol composition. Thickness was measured to show th e dependence on both of these parameters in the coating process. It was conc luded that 40% glycerol was requi red for a full primer coating. Also, a dip time of 30 seconds was chosen fo r activity experiments because it provided a midrange thickness (83 m) compared to the other primer compositions. Activity tests were used to measure the appli cation of the secondary layer, which included OxO and they were required to incubate for 10 hours in order to get sufficient enzyme activity. This was due to a smaller amount of immobilized enzyme in the coating compared to the bulk material studies conducted in Chapter 3. Studies to vary the immersion time showed that the longer the st ent with primer was immersed in the coating solution, then the higher the activity. This seemed appropriate to allow more coating solution to absorb into the prim er. Immersion times of 30 and 60 minutes were similar. Experiments to vary dip cycles of the st ent with primer showed that 1 dip was better than

PAGE 70

70 multiple dips. The standard deviation was large in this study, but the trend was shown in two experiments that activity was not increased upon mu ltiple in cycles in this study. Since activity was shown to increase by increasing the immers ion time, a minimum of 30 minutes should be used for this procedure and the stent will only need to have one secondary layer added to it.

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71 Figure 4-1 General schematic of coati ng and photocuring process for stents. A. B. Figure 4-2 Digital photographs of dip coated primer layers that had varying amounts of glycerol (0-40%) in the aqueous phase if the reaction solution. Stai ned with crystal violet. (A) Top Row: Superglue applied before dip co ating. Bottom Row: No superglue. (B) Magnified picture of stents coated with superglue and reaction solutions containing 30% (left) and 40% (right) glycerol.

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72 A. B. C. D. Figure 4-3 Micrographs of primer layers made from 40 v% glycer ol that were coated onto the stent by varying time immersed into coati ng solution. A, B, & C. Submerged for 60 seconds; D. Submerged for 30 seconds. In A and B, the right image shows a higher magnification view of the coating shown on the left. In C and D, cross-sections of the stent were used to calculate coating thickne ss. Scale bars above are given as follows: (A) 1 mm/10 m; (B) 100 m/10 m; (C) 100 m; (D)10 m. Table 4-1 Summary of coating thickne ss based on composition and dip time. Primer Coating Solution Dip Time (sec) Thickness ( m), n = 5 60 v% monomer; 40% glycerol 30 83 60 v% monomer; 40% glycerol 60 187 60 v% monomer; 20% glycerol, 20% ddH2O 60 36 60 v% monomer; 40% ddH2O 60 25 80 v% monomer; 20% glycerol 60 170 (n = 2)

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73 A. B. Figure 4-4 Planar view, A (at low and high magnification), and cro ss-section, B, of the primer coating prepared from 20 % glycerol and 20% ddH2O dipped for one minute. A) Half the stent is well coated in the left micr ograph. At higher magnification on the right, the surface of this primer is notably porous B) The average thickness (n = 5) at the cross-section is 36 m. Scale bars above are gi ven as follows: A-1 mm/10 m; B-10 m. A. B. Figure 4-5 Planar view, A (at low and high magnification), and cro ss-section, B, of the primer coating prepared from 40% ddH2O dipped for one minute. A) The stent is uniformly coated in the left micrograph. At higher magnification on the right, the surface of this primer has porosity although lower than Figur e 4-4. B) The average thickness (n = 5) at the cross-section is 25 m. Scale bars above are given as follows: A-1 mm/10 m; B-10 m.

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74 A. B. Figure 4-6 Planar view, A (at low and high magnification), and cro ss-section, B, of the primer coating prepared from 80% monomer and 20% glycerol dipped for one minute. A) The stent is shown at low ma gnifications to be uniformly coated with one defect towards the top left. At higher magnification on the right, the surface of this primer does not show porosity but a denser coating. This is due to a higher monomer volume fraction in the composition compared to Figur es 4-4 and 4-5. B) Short arrows show the dense bulk material with an average thickness of 100 m. Long arrows extend to the surface that includes a porous, op en material about an extra 70 m thicker. This is likely due to a difference in polymerization kinetics in these regions of the material. Scale bars above are given as follows: A-1 mm/10 m; B-100 m. -5 0 5 10 15 20 25 30 35 40 010002000300040005000 WavenumberTransmission (%) Primer 2nd layer-Blank Figure 4-7 Transmission spectra from FTIR of prim er formed with glycerol and the second layer formed with ddH2O. Table 4-2 Summary of FTIR results. Wavenumber (cm-1) Functional Group 1718-1735 C=O stretch 2895 CH2, symmetric stretch 2735 CH2, CH3 stretch 3500-4000 OH stretch

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75 Table 4-3 Composition of coating solution and f ilms used for testing effects of Triton X. Sample Variable OxO Stock ( L) Hex-NaCl ( L) Triton X-100 ( L) 0.0v% Triton X-100 80 80 0.1v% Triton X-100 80 78.4 1.6 0.5v% Triton X-100 80 72.0 8.0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1103090Dip time (min)Production of H2O2 (m/min-g) 20mM Hex 100mM NaCl 0.1% Triton X-100 0.5% Triton X-100 Figure 4-8 Activity from stents coated with OxO made from dipping at different amounts of time. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Hex-NaCl0.1% Triton X-1000.5% Triton X-100Production of H2O2 ( M/ming) Figure 4-9 Activity in films made from coating solutions with varying amounts of Triton X-100.

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76 Figure 4-10 Micrographs of stents coated with the primer for laye ring studies. (A) Planar view (at low and high magnification), and (B) High magnification. Notice the thick, full application of the coating. Scale bars are 1 mm/10 m for (A) and 100 m for (B). 0 2 4 6 8 10 12 14 16 18 1103060Immersion time (min)Production of H2O2 ( M/ming) Figure 4-11 Activity of OxO in coa tings that were applied by immersing stents with primer into reaction solution for various times.

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77 A. B. C. D. Figure 4-12 Micrographs of stents coated with various dip times (A) Dipped 1 minute and had low activity; (B) Dipped 10 minutes times and had medium activity; (C) Dipped 30 minutes and had high activity; (D) High magni fication of surface of stent coated for 30 minutes that showed porosity on the nanos cale. Scale bars are as follows: (A) 100 m; (B) 100 m; (C) 100 m; (D) 10 m. 0 5 10 15 20 25 30 1x2x3xDip CyclesProduction of H2O2 ( M/ming) Figure 4-13 Activity of OxO in co atings that were applied with sequential dip cycles into reaction solution.

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78 A. B. C. D. Figure 4-14 Micrographs of coated stents dipped 1 time. (A) High activity; (B) Same stent with high activity at higher magnification; (C) Low activity; (D) Same stent with low activity at higher magnification Scale ba rs are as follows: (A) 1 mm; (B) 100 m; (C) 10 m; (D) 1 m. A. B. Figure 4-15 Micrographs of coated stents dipped 2 times. (A) Hi gh activity; (B) High activity at high magnification. Notice the surface s howing porosity on the nanoscale. Scale bars are 100 m for (A) and 1 m for (B).

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79 A. B. Figure 4-16 Micrographs of coated stents dipped 3 times. (A) High activity; (B) Low activity. Scale bars are 100 m for both images.

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80 CHAPTER 5 INHIBITION OF ENCRUSTATION BY IMMOBILIZED OXALATE-DEGRADING ENZYMES Ureteral Stents and Encrustation The interaction of urinary stents with urine is complex because urine is a highly buffered environment composed of by-products from the ki dney and bladder, like proteins, sugars, and ions [86]. The physical properties of a stent ma terial affect performance and comfort of the patient and these traits include tensile st rength, compliance, flexibility, and surface characteristics such as wettability an d smoothness, i.e. lubricity [87, 88]. Current urinary devices are ma intained by monitoring comfort on a frequent basis, washing out any deposit with acidic soluti on that may include antibiotics, or exchanging the prosthetic device, which requires a hospital or doctors visit [89]. It has ev en been shown that systemic doses of antibiotics in the acid solution washes have had little success inhibiting struvite and hydroxyapatite formation because of diffusion lim itations through the biof ilm [90]. Poly(HEMA) has been used for surface modification to ureteral stents and shown to reduce bacterial adhesion. The nature of the hydrogel may have a positive im pact to the device, and the immobilization of oxalate-degrading enzymes may enhance its resi stance to biofilm by decreasing encrustation. Hydrogel Coatings Hydrogels impart a low coefficient of friction to a surface, and result in easier insertion of the device and less discomfort to the patient [91]. Polymers that have been used as hydrogel coatings for urological prostheses include poly(acrylamide), pol y(ethylene glycol), poly(HEMA), poly(vinyl alcohol), and poly(vinyl pyrollidone) [92]. Fi rst patented by Eckstein in 1975 as a coating to prevent encr ustation, p(HEMA) was chosen as a hydrogel for ureteral stents [41]. Eckstein remarked that this phenomenon wa s due to the shrink-swe ll capacity of p(HEMA) [93]. This work was further established by Block, et al., who observed that p(HEMA) used as a

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81 prosthetic ureter for dogs accumulated no encrus tation in 2 years of se rvice [94]. In 1997, p(HEMA) was re-evaluated as a potential urinary biomaterial and had interpenetrating networks of poly(methyl methacrylate) or poly( -caprolactone) to provide stre ngth to p(HEMA) so it could be used to make the entire sten t [95, 96]. In these studies, only the bulk material was tested. The co-polymer showed the same encrustation resi stance as the control p(HEMA), although the bacterial resistance was superior to p(HEMA). Other initial stent coatings include Hydron, which was a la tex coating applied to latex catheters, and showed le ss encrustation than non-coated control groups [97]. Further studies indicated that latex coated cat heters showed more encrustati on developed here than on noncoated silicone-based catheters [98, 99]. Anot her hydrogel coating, Hydro-plus, was applied to C-flex stents, which reduced encrustation and damage to endothelial cells of the mucosa compared to a control group [100]. Tunney and Gorman applied a coating of pol y(vinyl pyrollidone) (PVP) to poly(urethane) (PUR) ureteral stents. The PVP coating showed complex results when compared to non-coated stents of silicone and urethane [101]. The PVPcoated stents were as good as silicone for resistance to E. faecalis and as good as PUR for E. coli resistance. Also, the PVP coated stents and silicone were superior over PUR for resistance to struvite encrustation. But, the PVP coated stents were less resistant to hydroxyappatite encrustation than both non-coated stents. One study of bulk modifications to urinary devices is Aquavene, a novel poly(ethylene oxide) PUR copolymer. Aquavene was shown to re sist encrustation by en hancing hydrophilicity. Results showed that Aquavene remained unobstructed in its inne r lumen for 24 weeks [76]. Most studies in the literature refer to surface modifica tions of stents by use of a coating, and a variety

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82 of materials and methods have been tested in or der to determine the mech anism that could lead to inhibiting encrustation. The theory that hydrophilic surfaces are essent ial to encrustation resistance has been found to be insufficient since the encrustation process is complex. Silicone, regarded as a superior material for urinary devices, is more hydrophobi c than PUR and resists bacterial deposition [102]. The mixed results from th e studies of PVP-coated cathete rs and other hydrophilic coatings refute the notion that low contac t angles are enough to resist encrustation. Current research focuses more on designing active rather than pa ssive coatings, in which the active coatings interact with the surroundings in a therapeutic way. Antibiotic Coatings In more recent years, urinary biomaterials that have been studie d include novel materials that control the release of antibio tics. The release of antibiotics from a coating could help curb the high rate of urinary tract in fections associated with these devices [103, 104]. These coatings would be especially beneficial for pa tients with impaired immune systems. Ciprofloxacin, an effective antibiotic for urinar y tract infection, was loaded into liposomes that were immobilized in a co ating [104-106]. Results of a seve n-day in vitro study showed a 39 mm inhibition zone to bacterial growth and no su rface growth of bacteria on the material [105]. Yet, in vivo studies found th at the liposome-loaded hydrogel coatings showed no significant difference of encrustation from the control hydrogel [105]. Norfloxacin, another strong antib iotic, was loaded into a PEG/ silicone-based coating [106]. Due to hydrophobic character of norfloxacin, its re lease was sustained for over 1 month. Various bacteria were isolated from patients with urin ary catheters and colonized on an agar plate. Samples were placed on the plates and the inhibition of bacteria growth around them was

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83 measured. This coating demonstrated resistance to Pseudomonas vulgaris a urease-producing bacterium but did not to Staphylococcus epidermis a prevalent bacterium found on skin. Heparin has also been immobilized onto meta llic stents and shown to inhibit encrustation [107]. Heparin is thought to be an inhibitor for crystal formation because it competes with oxalate or phosphate for binding to calcium. Usi ng a rat model, expandable stainless steel stents were encrustation free after 120 da ys whereas control stents with no heparin were extensively covered. There is a potential use of heparin for later studies, in which it could provide a matrix for the enzyme. Encrustation Models Various models exist to study ur inary encrustation in vitro. Mos t models use artificial urine composed of a variety of salts and proteins li ke albumin and urease [108-110]. One researcher who tried to standardize a protocol for encrustation in vitro us ed real urine that was filtered and had antibiotics and antimycotics added. This stud y was run in a 5-day tr ial and actually showed coated stents formed more en crustation than uncoated [111]. Experimental Study of Encrustation Introduction The purpose of this study was to compare th e activity of immobilized enzymes (OxO and OxDc) for their ability to inhibit encrustation comp ared to a control group. Both artificial urine and real urine were tested to determine their effects on calcium oxalate mineralization and enzyme activity against oxalate in these solutions Encrustation is a biolog ically induced process that forms mainly due to supe rsaturation of mineral precurs ors in the urine. This study investigates encrustation in se veral assays against oxalate and time to understand their influence on mineral formation.

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84 The first experiment was for one week and wa s designed to determine oxalate levels that caused encrustation so that the films could be te sted later for inhibition. The normal range of oxalate in urine goes from 0.05 to 0.30 mM, and levels higher than that indicate pot ential stone formation [112]. For artificial urine, the c ontrol groups were tested by varying oxalate concentration over a range of 0 to 1 mM oxalate to evaluate this so lutions dependence on for forming crystals. In parallel to th is test, the control, OxDc, and OxO groups were tested in real urine with oxalate concentration varying from 0.0 to 0.5 mM. Once the oxalate level was decided upon, the second experiment evaluated all the groups for inhibition over 6 weeks. A fresh sample was pulled from its group every week for 1 month, and then 2 weeks later. The degree of encrusta tion was evaluated by polarized light microscopy to determine the extent of mineral crystals that had nucleated on the films. At week 6, the films were tested with SEM/EDS to perform elemental and microstructural analysis. Materials and Methods Films Films made of poly (99m% HEMA-x-1m% DE GDMA) were prepared individually in glass molds as previously described in Chapter 3. A control group with no enzyme was used and tested against OxDc (10 g) and OxO (1 g). The immobilization process consisted of an aqueous phase that provided the enzymes and mo nomers to form a reaction solution for the films. The precursor solutions were miscible and gave rise to a transparent polymer. First, a stock monomer with enzyme buffer wa s purged with Ar (g). A blank film sample was made with the Hex-NaCl (40 v%). These so lutions were mixed and aliquots were pipetted into the glass mold. Samples were cured for 15 mi nutes and washed agains t Hex-NaCl for 2 days before testing in th e encrustation study.

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85 Artificial urine All materials were reagent grade unless otherw ise noted. The composition of artificial urine is listed in Table 5-1. The ddH2O was heated to ~80 C by a hot plate to encourage faster dissolution of the salts, whic h were added one at a time. The pH was adjusted using NH4OH to obtain a final value of pH 6.0. The volume was then brought to 500mL with ddH2O and then allowed to cool to 25 C. Urine Urine was obtained from a single donor (author) and had a pH of 6.0. It was collected and filtered within one day. Encrustation crystallizing solutions The artificial and real urine solutions were filtered in Stericup filtration units (Millipore, Cat. # SCGPU11RE) with a 0.22 m membrane. Note, urine clogs these filters quickly, and several must be used to obtain volumes over 200 mL. A solution of 100X antibiotic/antimycotics (Invitrogen) was mixed into the ur ine-based solutions for a final c oncentration of 1X to prevent microbial growth that could ra ise the pH through their enzymatic activity (no microbial growth or pH change was observed over a period of 6 months). Activity tests Activity tests were run under va ried conditions for films with respect to pH and storage duration to give a reasonable estimate of the amount of activity requi red for inhibition to encrustation. The results at the levels at urinar y pH (6.0) were factored against the activity expected at that particular c ondition and time. These activity te sts were against 50 mM oxalate and conducted as described in Chapter 3.

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86 Polarized light optical microscopy After the test period, samples were removed, rinsed in ddH2O and placed onto a glass slide to observe under an optical microscope. Sample s were first assessed for encrustation using an optical microscope with cross pol ars and the 1st-order red (gypsum) -plate. A polarized optical microscope (Olympus BX-60 with MIT 3CCD camer a) was used to capture images of mineral formation and was ideal to readily assess the de gree of encrustation on films. Transmision mode was used since the films were transparent, and the gypsum wave plate was used because it enables the visualization of both amorphous and crysta lline mineral phases. Results Oxalate-dependence for encrustation in artificial urine The p(HEMA) films used for controls showed a dependence on oxalate in one week as seen in the polarized micrograph images in Figur e 5-1. No minerals formed without the addition of oxalate nor at the lower level of 0.25 mM Ox alate (Figures 5-1a and b). Upon the addition of 0.5 mM oxalate (Figure 5-1c), mineral formation could be detected as small crystallites (~5 m). The amount of mineral formation increased up to 1.0 mM oxalate, in which these crystals could be seen in aggregates. Oxalate dependence for encrustation in real urine In Figures 5-2 to 5-4, the control, OxDc, a nd OxO groups were tested for encrustation by spiking urine with oxalate vary ing from 0.0-0.5mM and testing them for one week. Previous tests (not shown) indicated that this range was suitable for mineralization in these in vitro assays. The controls films shown in Figure 5-2 have the greatest encrustati on change as oxalate increases. All the films without oxalate added to the urine show ed no encrustation. Within one week, the crystal formation did not become eviden t until the addition of 0.3 mM oxalate. At 0.4 mM there was a sheet of single crystals that form ed, and by 0.5 mM, large crystals could be seen

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87 in a few spots. More pictures that show the extent and size of crystal growth from this experiment can bee seen in the supplemental data section. The OxDc films did not show much crystal form ation for any of the oxalate concentrations through 0.5 mM. There was little evidence of crysta l formation from 0.0-0.3 mM oxalate seen in Figures 5-3 a-d. At 0.4 and 0.5 mM encrustation in the form of ti ny crystals did occur, but with much less extent than the controls or OxO. The OxO films did not have much crystal formation up to 0.4 mM oxalate. The big black marks are voids in the film and should not be conf used as crystals. At 0.5 mM a large extent of encrustation developed on the su rface throughout the film (Figure 5-4 f). This big difference of encrustation between 0.4 and 0.5 mM oxalate sugg ests OxO worked well up to a certain point of oxalate. Considering that OxO suffers from substr ate inhibition, and the volume in the film is thought to concentrate oxalate (see chapter 3), th e enzyme activity may have been lessened at this higher level to allow a significant more amount of crystal formation. Encrustation over time in artificial urine Figures 5-5 through 5-7 shows th e growth of crystals on eafh group of films in artificial urine against 0.5 mM oxalate. The control group shown in Figure 5-5, could be seen growing crystals by the first week and continuing to encr ust the film over the two months. The films with immobilized OxDc (Figure 5-6) and OxO (Figure 5-7) exhibited an inhibitory effect for the encrustation through the first week By week 4, crystal growth was apparent for the OxO film, indicating the enzymes did not have a lasting effect for encrustation inhibition under these test conditions. OxDc seemed to maintain an inhibitory effect against crystal formation in artificial urine for the two months. The bette r performance by OxDc over OxO is likely due to its higher activity against oxalate.

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88 Encrustation over time in real urine These sets of images in Figure 5-8 through 5-10 compare the degree of encrustation in real urine with an oxalate concentra tion of 0.5 mM, which induced mi neralization of calcium oxalate. The effect of real urine showed a higher degree of nucleation and growth for encrustation than artificial urine. The number of crystals formed in urine grew faster on the films and became more predominant over time. The control film still exhibi ted a higher density of crystals than films with immobilized OxDc and OxO. Yet, the film s with immobilized enzyme did show crystal growth in both OxDc and OxO. Figure 5-8 shows the encrustation formation of the control group in real urine. In the first week, the films were developing crystals on the surface (1 m), while at the end of the first month, there crystals had grown in size (~5-10 m) and became more predominant on the surface. By the end of the second month, the minerals had developed more coverage but still appeared to be 5 to 10 m. Figure 5-9 shows the development of crystals on the surface of the f ilm with immobilized OxDc. In the first week, there was no discernabl e crystal growth like there was in the control group. At the end of the first month, there app eared to be the formati on of large nuclei for crystals, but there was little birefringence to de note actual crystal growth. At the end of two months, only a few random crysta ls that appeared large (~50 m) were apparent on the surface. Through Oswalds ripening effect, the nuclei seen at one month could have dissolved to continue to provide ions to grow few, large crystals that were apparent in the second month. Figure 5-10 shows the development of crys tals on the surface of the film with immobilized OxO. In one week, a slight number of crystals had formed on the surface. By one month, the surface was covered by crystals, and ju st as covered in two months. The crystals appeared to be 5 to 10 m. There were no large crystals as seen for OxDc, and this may indicate

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89 that Oswalds ripening did occur for OxDc since th ere wasnt a extent of crystal growth as seen in the control and OxO films. Figures 5-11 and 5-12 show the pH profile of the enzymes in the pH range of 4.0 to 7.0. Since the pH of the solutions was pH 6.0, it is cl ear that the enzymes woul d be less active than at pH 4.2, which is optimal. The activity of OxDc and OxO was about 20% at pH 6.0 compared to pH 4.2. This would lead to an expected activity of OxDc of about 10 nmol/min and OxO of 0.2 nmol/min. Yet, the amount of oxalate added to th e artificial and real ur ine systems was 0.5 mM, or 1 mol oxalate in the 2 mL sample. Therefor e, the amount of oxalate degraded by the enzymes should have been enough to eliminat e all of the oxalat e within one day. Several reasons can be considered for the caus e of the development in encrustation on the films. First, it was shown that real urine encrus ted more heavily than artificial. Urine has a high amount of human serum albumin (HSA), which is deposited onto the biomaterials surface [113]. The organic layer from explanted devices has been shown to favor aggregation of crystals from in vitro tests. HSA promotes adhesion w ith the crystals to the device surface [113]. Although one might think that the HSA suppresse d transport of oxalate from urine into the films to prevent its breakdown by the enzymes, this possibility was examined and not the case. An additional set of activity tests were performed on films stored in urine. The results showed that the immobilized enzymes retained high levels of activity after being stored in urine for periods of time as shown in Figur es 5-13 and 5-14. The effects of a storage buffer and urine were compared and a higher level of activity was retained when the films were stored in urine. Although an organic layer such as HSA may form, it apparently di d not affect enzyme activity. Conclusion In vitro studies for calcium oxalate encr ustation were conducted on p(HEMA) and the ability of immobilized OxO and OxDc were comp ared. Artificial and re al urine was used to

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90 develop encrustation to determin e the differences of encrustation from these solutions. Oxalate was varied in both solutions to optim ize the conditions for encrustation. In artificial urine, 0.4 mM oxalate was re quired to form encrustation in one week on control films. In real urine, 0.3 mM oxalate was required. Th e immobilized enzymes showed a dependence on oxalate concentration in urine for their ability to inhibit encrustation for one week. A two-month study monitored the ability of the immobilized enzymes to inhibit encrustation for a longer period of time. The p(HEMA) control group developed encrustation in one week and it continued to develop across the surface over two months. Immobilized OxDc was able to inhibit crysta l growth for the two-month period, although OxO developed encrustation to a similar exte nt of the control group. It was seen in several experiments that OxDc had the ability to inhibit encrustation with 0.5 mM oxalate in urine, even with a reduced acti vity due being in solution at pH 6.0. This opens the possibility of utilizing the immobilized enzyme as a therapy for degrading oxalate concentrations in urine, which can be employed as a coating on ureteral stents. The activity of immobilized OxDc and OxO are retained in urin e over a 6-month period, which also supports the idea that this application is viable.

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91 Table 5-1 Composition of salts for artificial urine. Salt Concentration (mM) Salt Concentration (mM) NaCl 105.6 Na2SO4 17.0 NaH2PO4 29.4 KCl 63.8 Na3Citrate 3.2 CaCl2 5.8 MgSO4 1.9 Na2C2O4 0-1.0 A. B. C. D. E. Figure 5-1 Polarized images of control p(HEMA) films tested in artificial urine with varying concentrations of oxalate at 1 week. (A) O; (B) 0.25; (C) 0.50; (D) 0.75; (E) 1.0 mM oxalate. Magnification was 50x. A. B. C. D. E. F. Figure 5-2 Polarized images of control p(HEMA) films tested in real urine against varying concentrations of spiked oxalate at 1 week (A) 0; (B) 0.1;(C) 0. 2;(D) 0.3;(E) 0.4;(F) 0.5 mM oxalate. Magnification was 50x.

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92 A. B. C. D. E. F. Figure 5-3 Polarized images of films containing OxDc tested in real urine against varying concentrations of oxalate at 1 week. (A) O; (B) 0.1; (C) 0.2;(D) 0.3; (E) 0.4; (F) 0.5 mM oxalate. Magnification was 50x. A. B. C. D. E. F. Figure 5-4 Polarized images of films containi ng OxO tested in real urine against varying concentrations of oxalate at 1 week. (A) O; (B) 0.1; (C) 0.2; (D) 0.3; (E) 0.4; (F) 0.5 mM oxalate. Magnification was 50x.

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93 A. B. C. Figure 5-5 Polarized images of control p(HEMA) films tested in artificial urine: (A) 1 week; (B) 4 weeks; (C) 8 weeks. Magnification was 200x. A. B. C. Figure 5-6 Polarized images of OxDc films tested in artificial urine: (A) 1 week; (B) 4 weeks; (C) 8 weeks. Magnification was 200x. A. B. C. Figure 5-7 Polarized images of OxO films tested in artificial urine: (A) 1 week, (B) 4 weeks; (C) 8 weeks. Magnification was 200x. A. B. C. Figure 5-8 Polarized images of control p(HEMA) f ilms tested in urine: (A) 1 week; (B) 4 weeks, (C) 8 weeks. Magnification was 200x.

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94 A. B. C. Figure 5-9 Polarized images of OxDc films test ed in urine: (A) 1 week; (B) 4 weeks; (C) 8 weeks. Magnification was 200x. A. B. C. Figure 5-10 Polarized images of OxO films tested in urine: (A) 1 week; (B) 4 weeks; (C) 8 weeks. Magnification was 200x. 0 20 40 60 80 100 120 44.555.566.57pHRelative OxO Activity (%) Free Immobilized Figure 5-11 The effects of pH on free and immo bilized OxO. Maximum activity for free OxO was 0.5 M/ming and for immobilized OxO was 1.0 M/ming.

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95 0 20 40 60 80 100 120 4567 pHRelative OxDc Activity (%) Free Immobilized Figure 5-12 The effects of pH on free and immo bilized OxDc. Maximum activity for free OxDc was 10 M/ming and for immobilized OxDc was 4.5 M/ming. 1.1 1.1 1.0 0.7 0.8 0.7 0.5 0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1 Day1 Month2.5 Month6 MonthTime in solutionProduction of H2O2( M/ming) Urine Buffer Figure 5-13 The effects of stori ng films with immobilized OxO in a buffer of 100 mM Hex-NaCl or real urine, as measure by Vmax.

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96 5.9 3.1 2.0 2.9 5.4 3.4 2.6 2.4 0 1 2 3 4 5 6 7 1 Day1 Month3 Month6 MonthTime in solutionProduction of formate ( M/ming) Urine Buffer Figure 5-14 The effects of stor ing films with immobilized Ox Dc in a buffer of 100 mM HexNaCl or real urine, as measured by Vmax. Supplemental Data A. B. C. Figure 5-15 Close-ups of blanks (films without enzyme) in 0.4 oxa late in real urine. (A) 50X; (B) 200X; (C) 200x.

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97 A. B. C. D. E. F. Figure 5-16 Close-ups of blanks (f ilms without enzyme) in 0.5 mM oxalate in real urine: (A) 50X; (B) 100X; (C) 200X; (D ) 50X; (E) 200X; (F) 200X.

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98 CHAPTER 6 SYNOPSIS Conclusion The work contained in this dissertation examines a method to immobilize oxalatedegrading enzymes that retains activity against oxalate in a po lymer film. A hydrogel material, p(HEMA), which had already been used to coat ureteral stents over 30 years ago for inhibiting encrustation, was synthesized by UV photopolymer ization and shown to be benign towards processing the enzymes. Films of p(HEMA) swelled comparably in ddH2O and urine due to the polymers chemical neutral charge. In summary, th is work exhibits the reduction to practice for the immobilization and characteriza tion for oxalate-degrading enzymes. The films were composed of 99% HEMA-x -1% DEGDMA and the two entrapped oxalatedegrading enzymes were oxalate oxidase (OxO ) and oxalate decarboxyla se (OxDc). Oxalate oxidase was bought commercially and found to c ontain 10% protein. Ox alate decarboxylase was provided privately with high purit y. Analytical techniques were developed to compare free and immobilized using Michael is Menten kinetics. Initially, enzyme activity was measured up to one hour to determine the linear response. Both free and immobilized forms of the enzyme s were linear up to one half hour, although OxO seemed linear for at least a period of one hour. Us ing a time period within this linear range (30 minutes), oxalate was varied over a range of con centrations to determine saturated conditions for enzyme activity. This allowed Michaelis Menten kinetics to be examined. The response by OxO to 50 mM oxalate demonstrated that the immobilized form had higher activity than the free form. Michaelis Me nten kinetics was measured by varying oxalate levels through a low to high range, and a subs trate inhibition model was applied to OxO to resolve the difference of free and immobilized enzyme activity behavior. Oxalate oxidase was

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99 exhibited > 10 mM oxalate, which was comparable to a previously reported value of 4 mM. The applied model determined that substrate inhibi tion is more pronounced for the free form of OxO due to the resistance of the film to oxalate diffusion, which th ereby lowers its concentration inside the film duri ng the test period. The kinetic constants, Vmax and km, for OxO were determined using Michaelis Menten model with and without applying th e substrate inhibition. Without applying substr ate inhibition, the free form had a Vmax of 0.9 0.0 M/ming and a km of 0.3 0.0 mM while the immobilized form had a Vmax of 1.1 0.1 M/ming and a km of 3.1 0.1 mM. Applying the substrate inhibition model, the free form had a Vmax of 1.8 0.1 M/ming, a km of 1.8 0.1 mM, and a ks of 35.4 3.7 mM while th e immobilized form had a Vmax of 1.2 0.2 M/ming, a km of 4.1 0.6 mM, and a ks of 660 140 mM. Without applying substrate inhibi tion, the model has a lower Vmax, or rate of activity, for the free form than the immobilized form. By applying the substrate inhibition model, the Vmax for the free form is higher than the immobilized form, which physically makes sense. Also, the km determined by using the substr ate inhibition model is higher for free OxO than without using it, which suggests the affinity for oxalat e is lower, but it also accounts for ks. The ks for free OxO is much lower to indicate its larger inhibition to oxalate than the immobilized OxO. The values for Vmax and km are within the range of previous reports in the literatu re. These are the first known values for ks of a substrate inhibition model applied to OxO. Oxalate decarboxylase showed a linear time response for 30 minutes. The activity of free OxDc was about 5 times larger than immobili zed OxDc. The Michaelis Menten was applied without any other inhibition cons tant accounted since no other i nhibition had been previously reported.

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100 The kinetic constants for OxDc showed the free form had a Vmax of 23.5 1.4 M/ming and a km of 0.5 0.1 mM while th e immobilized form had a Vmax of 5.0 1.9 M/ming and km of 23.2 9.1 mM. The big difference for Vmax and km between free and immobilized OxDc was due to several factors, namely diffusion of format e from the film and the decrease of activity for OxDc over time against oxalate. This was evident in the trial against soaking the films in various buffers, in which activity decreased the most in the buffer that included oxalate. Various buffer conditions were studied and incl uded physiological concentration of oxalate (1mM), pH, an enzyme storage buffer, and real urine. These studies helped to determine the applicability of the enzymes for viability of st orage and application. The maximum activity was shown at pH 4.2 to 4.5 and activity drops to be negligible by pH 7.0. Activity is dependent on pH and requires the form of m onoanionic oxalate. From thermo dynamic models, around 20% of oxalate in urine is in the monoa nionic form; so using the immobilized enzymes in urine should have practicability within its range of pH fr om 4 to 7.0. Also, a reduced activity may be sufficient for inhibiting encrusta tion since urine is normally ar ound pH 6.0. Storing the enzyme at pH 6.1 exhibited a larger retained activity than storing at pH 4.2, yet storing in urine showed highest activity retention. Activity experiments culminated with films stored in urine and Hex-NaCl at 37 C for a period of six months. A sample of film was tested at a specific time after the period of immersion at 37C. Immobilized OxO retained its overall initial activity better than OxDc over the sixmonth period. Both enzymes retained activity more stored in urine than in Hex-NaCl. In urine, the activity for immobilized OxO fell from 1.0 to 0.7 M/ming, a loss of 30%. The activity for immobilized OxDc fell from about 5.9 to 2.9 M/ming, a loss of 50%.

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101 Other work for this project consisted of deve loping a coating that could entrap the enzyme using the above process. A hydrophi lic primer was developed rather than use chemical methods to modify the stent. Superglue was shown to be a superior ad hesive for getting the reaction products to stick. Glycerol help ed to control thickness of the coating application. Experiments using SEM to assess the coatings qualitatively an d quantitatively were performed to determine the best condition for later work. A secondary la yer was applied to affix the enzyme into the coating. Tests devised to study se quential layers in th e coating or immersi on time in the coating solution were used to optimize the coating technique. It was determined that an immersion time of at least 30 minutes was better than th e other conditions for this coating method. The encrustation model was established using artificial and real ur ine. A novel technique was developed to prevent infection in the solu tion by using antibiotic and antimycotics solution. The solutions were spiked with 0.5 mM oxalate to optimize encrustation conditions. The encrustation study was conducted up to two months in these solutions. Samples were analyzed using optical polarized light microscopy. Immobilized OxDc was able to inhibit crys tal growth for the two-month period in both artificial and real urine. Imm obilized OxO developed encrustati on to a similar extent of the control group in real urine but inhibited encrusta tion in artificial urine better than the control group. The results of these encrustation experiment s verify that OxDc has the ability to inhibit encrustation up to 0.5 mM oxalate in urine, ev en though it exhibits a redu ced activity in solution at pH 6.0. This opens the possibility of u tilizing the immobilized enzyme as a therapy for degrading oxalate concentrations in urine, which can be employed as a coating on ureteral stents. Future Work Future work could entail many possible direct ions. From the lessons learned from this project, OxDc exhibited higher ac tivity against oxalate than OxO. In encrustation studies, OxDc

PAGE 102

102 resulted in an encrustation resistant material. Al so, entrapping the enzymes seems to be a viable process method, although other immobilization techniques can be examined. Furthermore, polarized microscopy was a quick way to observe mineralization occurring in these films, and it can be an essential characterization tec hnique for future encrustation studies. First, a collaboration with a urological products manufactur er like Cook, Bard, or Boston Scientific could help apply this coating solutio n onto a manufacturing lin e. The coating can be assessed for its processability in an actual setting in regards to th ickness, cure rate, and stability, in addition to other requirements for FDA for approval. Once an optimal coating has been achieved and retention of enzyme activity verified, then the best encrustation model will be in vivo. In other reported studies, either pigs or rabbits are used as encrustation models. Second, other polymers can be researched for th eir ability to inherent ly resist encrustation or increase diffusion of oxalate and enzyme activ ity products. Varying th e ionic nature of the polymer may influence the release of formate for OxDc immobilized materials, such as with the use of poly(acrylic acid) or poly(vinyl amin e). Also, other hydrophilic monomers, such as poly(vinyl pyrollidone) (PVP), can be added to determine their effects. Well-known antifouling polymers such as poly(ethylene glycol) (PEG) an d mannose could be added to the formulation for studying. Additionally, styrene could be adde d to increase strength in the coating, if necessary. Using a statistical design of experime nt with activity as the main output may help expedite this process. Also, stabilizers in the formulation could be used that lead to interpenetrating networks for the coating. Future work may also entail researching othe r oxalate-degrading enzymes. Oxalate oxidase from the sorghum root had been reported to have optimal activity at pH 6, which would be useful for this application. Furthermore, chemical methods to modify the current enzymes could

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103 possibly shift the optimum pH the enzymes are active. Additionally, covalent linkage to the support may be an advantage although it seemed unn ecessary for the coating in this project since no leakage was detected. Finally, a more thorough biochemical analysis could help understand the mechanisms for the loss of enzyme activity. Specific techniqu es, such as non-denaturing electrophoresis can deduce if the enzyme loses its hexameric form, wh ich is necessary to be active. Also, circular dichroism is a spectral technique to evaluate the secondary struct ure of proteins. Furthermore, oxygen diffusion through the coatin g material may need to be optimized, which could influence enzyme activity. Therefore, developing copolymer s or changing to a different polymer system could be justified. These resear ch directions could lead to determining optimal materials, processing methods, and a deeper understanding of what occurs to th e immobilized enzyme mechanistically.

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104 APPENDIX A EXPLANTED STENTS Figure A-1 Explanted ureteral stents s howing encrustation.

PAGE 105

105 Figure A-1 (continued).

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106 APPENDIX B CALCULATIONS Polymers were calculated in terms of mola r composition by using the monomers physical properties. Since monomers were all liquid, th is calculation made it easy to determine the polymer molar composition. A monomer was provid ed with a given formul a weight (FW, g/mol) and density ( g/mL). When the value of these are brought together as follows, monomerFW A then this value, A, determined a monomer s volume fraction (mL) per mole of a given polymer. Each monomer (A) was desired at a particular molar composition (, mol), e.g. 99%, or 0.99 mol HEMA. The contribution of each monomer was added together for a useful formula weight of the polymer, given as 1 polymerii i F WA in terms of mL/mol and i is any arbitrary monomer. Then, this value was normalized by 60v% due to the monomers composition in the reaction, and calculated by 0.6polymerFW Volume withdrawn, Vi (mL), for a given monomer was de termined from the final product of their respective values from above, given as iiVA

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116 BIOGRAPHICAL SKETCH The journey through school for James Mellman has not been linear, but along the way, he had been warned that life is rarely a linear pa th. So, it can be said, that he knew what he was getting himself into, and even if he didnt, so mehow the alternatives were not much better. Engineering seemed like the right course of st udy to discover. Along th e way, he found his calling to pursue the biomedical sciences with in the framework of engineering, in which biomaterials became the perfect outlet for his interests.