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Synthesis and Engineering of Polymeric Latex Particles for Medical Applications

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PAGE 1

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS By SANGYUP KIM 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 2005

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Copyright 2005 by Sangyup Kim

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To all who made this work possible.

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ACKNOWLEDGMENTS I owe what I am today and this moment to many people, without whom I could not have been here. First of all, I would like to express my sincere and deepest appreciation to my parents, wife Mikyung, daughters Heehyung and Erin, whose love, patience, and support helped me pursue my dream. I gratefully acknowledge my advisor, Dr. H. El-Shall, who gave me an opportunity to become part of an exciting and beneficial research project and exert myself in the research work. His guidance, knowledge and stimulating scientific and critical thinking have contributed to my success to a great extent, and inspired me to dedicate myself to research work. I especially appreciate Dr. R. Partch as a co-advisor, who has helped me conduct experiments with valuable discussion for my research from my first year. I am greatly thankful to Dr. C. McDonald, whose idea and valuable experimental advice and discussion in the area of polymer chemistry and membrane technology helped cultivate my interest in this area and were vital to performing this research. Without his contribution this work could not have been achieved. I also thank my doctoral committee members, Dr. R. Singh, Dr. A. Zaman, and Dr. B. Koopman for appreciated comments. Financial support and equipment use for research from the Particle Engineering Research Center at the University of Florida were vital in continuing my study for more than 4 years. My sincere thanks are due to my former advisor Dr. Kiryoung Ha, in Keimyung University, who helped me open my eyes, and broaden my thought and introduced me into a limitless polymer field. I would like to also thank to past and current iv

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members in Dr. El-Shalls research group for their assistance, advice, and support. I sincerely appreciate Stephen for the assistance of thesis writing. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ...........................................................................................................viii LIST OF FIGURES .............................................................................................................x 1 INTRODUCTION........................................................................................................1 2 BACKGROUND AND LITERATURE SURVEY......................................................5 2.1 The Significance of End Stage Renal Disease (ESRD) ..........................................5 2.2 Hemodialysis (HD) Treatment ...............................................................................6 2.3 Advances in Membrane Technology ......................................................................8 2.4 Sorbent Technology ..............................................................................................16 2.5 Limitation of Current Hemodialysis Treatment ...................................................19 2.6 Latex Particles ......................................................................................................20 2.6.1 The Components for Emulsion Polymerization .........................................21 2.6.2 Particle Nucleation .....................................................................................21 2.6.3 Types of Processes for Emulsion Polymerization ......................................23 2.6.4 Chemistry of Emulsion Polymerization .....................................................24 2.6.5 Seed Emulsion Polymerization ...................................................................25 2.6.6 Polystyrene Latex Particles ........................................................................26 2.6.7 Various Applications of Latex Particles .....................................................28 2.7 Proteins .................................................................................................................30 2.7.1 Interaction Forces between Proteins ...........................................................33 2.7.2 -Microglobulin (M) 2 2 ..............................................................................35 2.7.3 Serum Albumin ..........................................................................................36 2.8 Protein Adsorption ................................................................................................38 2.8.1 Interaction between Protein Molecule and Latex Particle ..........................38 2.9 Hypothesis for Toxin Removal .............................................................................39 2.9.1 Toxin Removal by Size Sieving Based on Monodispersed Pore Size .......40 2.9.2 Toxin Removal by Selective Adsorption on Engineered Latex Particles ..41 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY..................44 3.1 Materials ...............................................................................................................44 3.2 Latex Particle Preparation .....................................................................................46 3.2.1 Preparation of Seed Latex Particles ............................................................47 vi

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3.2.2 Preparation of Seeded Latex Particles ........................................................48 3.2.3 Preparation of Core Shell Latex Particles ..................................................48 3.2.4 Purification of Synthesized Latex Particles ................................................48 3.3 Characterization ....................................................................................................49 3.3.1 Degree of Conversion .................................................................................49 3.3.2 Fourier Transform Infrared (FTIR) Spectroscopy ......................................49 3.3.3 Quasielastic Light Scattering (QELS) ........................................................50 3.3.4 Field Emission-Scanning Electron Microscopy (FE-SEM) .......................50 3.3.5 Zeta Potential Measurement .......................................................................50 3.3.6 Protein Adsorption ......................................................................................51 3.3.7 Blood Biocompatibility by Hemolysis Test ...............................................53 4 RESULTS AND DISCUSSION.................................................................................55 4.1 Polymerization of Latex Particles .........................................................................55 4.1.1 Polystyrene Seed Latex Particles ................................................................55 4.1.2 The Growth of Polystyrene (PS) Seed Latex Particles ...............................61 4.1.3 Core Shell Latex Particles ..........................................................................71 4.2 Characterization of Latex Particles .......................................................................75 4.2.1 Fourier Transform Infrared Spectroscopy (FTIR) ......................................75 4.2.2 Zeta Potential Measurements .....................................................................75 4.3 Protein Adsorption Study .....................................................................................85 4.3.1 Adsorption Isotherm ...................................................................................87 4.3.2 Adsorbed Layer Thickness .......................................................................103 4.3.3 Gibbs Free Energy of Protein Adsorption ................................................104 4.3.4 Kinetics of Adsorption .............................................................................107 4.4 Blood Compatibility ...........................................................................................112 5 SUMMARY OF RESULTS, CONCLUSION, AND RECOMMENDATION FOR FUTURE WORK......................................................................................................115 5.1 Summary of Results ............................................................................................115 5.2 Conclusions .........................................................................................................121 5.3 Recommendation for Future Work .....................................................................121 LIST OF REFERENCES .................................................................................................122 BIOGRAPHICAL SKETCH ...........................................................................................141 vii

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LIST OF TABLES Table page 4-1. The polymerization recipe of polystyrene (PS) seed particles. ..................................57 4-2. Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size. ............................................................63 4-3. Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size. .........................................................64 4-4. The preparation recipe of PS core with various shell latex particles. .........................72 4-5. The isoelectric point (IEP) of common proteins. .......................................................83 4-6. The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model. ......................93 4-7. The equilibrium concentration values (q) of BSA adsorption on PS/PMMA particles calculated the Langmuir-Freundlich isotherm model. m 100 ...............................96 4-8. The equilibrium concentration values of BSA adsorption on PS/PMMAPAA particles calculated the Langmuir-Freundlich isotherm model. 90 10 ...............................98 4-9. The equilibrium concentration values of BSA adsorption on PS/PMMAPAA particles calculated the Langmuir-Freundlich isotherm model. 75 25 .............................100 4-10. The equilibrium concentration values of M adsorption calculated the Langmuir-Freundlich isotherm model. 2 ..................................................................102 4-11. Absorbed BSA layer thickness () of BSA in phosphate buffer (PB) at 37C. o .....104 4-12. Absorbed BSA layer thickness () of BSA in phosphate buffered saline (PBS) at 37C. o .......................................................................................................................104 4-13. The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37C. o ..........................................................................................................105 4-14. The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37C. o ...............................................................................105 viii

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4-15. The values of Gibbs free energy change of M adsorption in phosphate buffere (PB) at 37C and pH 7.4. 2 o ........................................................................................106 4-16. Fitting parameters of second-order kinetic model. .................................................111 ix

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LIST OF FIGURES Figure page 1-2. Stereo drawing of the carbon backbone of M 2 .......................................................2 2-1. An image of the location and cross section of a human kidney. ..................................5 2-2. A schematic draw of the hemodialysis route ................................................................7 2-3. Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes ........................11 2-4. The chemical structure of polysulfone (PSf). .............................................................12 2-5. The chemical structure of polyamide (PA) .................................................................13 2-6. Chemical structure of polyacrylonitrile (PAN). .........................................................14 2-7. Emulsion polymerization system ................................................................................24 2-8. L--amino acid ...........................................................................................................31 2-9. Ribbon diagram of human M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ) 2 .....................36 2-10. Secondary structure of human serum albumin (HSA) with sub-domains ................37 2-11. The relationship between pore size and particle size in body-centered cubic and closed packed arrays .................................................................................................41 2-12. Schematic representation of the protein adsorption on the core shell latex particles at pH 7.4 .....................................................................................................42 3-1. Chemical structure of main chemicals. .......................................................................45 3-2. Experimental setup for semi-continuous emulsion polymerization. ..........................46 3-3. The particle preparation scheme in various types and size ranges of latex particles. 47 3-4. Schematic of the procedure for a protein adsorption test. ..........................................52 3-5. Separation of RBC from whole blood by centrifuge process. ....................................53 x

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3-6. Schematic of the procedure for hemolysis test. ..........................................................54 4-1. Schematic representation of semi-continuous seed latex particles preparation and growth. ......................................................................................................................56 4-2. Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PS S2.59 S2.33 S2.07 S1.81 .................................................................59 4-3. SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm ....................................................................................................65 4-4. SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm ....................................................................................................68 4-5. Dependence of the particle size on the surfactant to monomer ratio. .........................70 4-6. Schematic of core shell latex particle structures. .......................................................71 4-7. Scanning Electron Micrograph of latex particles (A) PS (B) PS/PMMA (C) PS/PMMAPAA (D) PS/PMMAPAA 100 90 10 75 25 ............................................................73 4-8. FTIR spectra of polymerized latex particles. (A) bare polystyrene (PS) (B) PS/PMMA (C) PS/PMMAPAA (D) PS/PMMAPAA 100 90 10 75 25 ...............................76 4-9. Schematic representation of ion distribution near a positively charged surface. .......78 4-10. Schematic representation of zeta potential measurement .........................................79 4-11. Zeta potential of PS latex particles at 25C. o .............................................................80 4-12. Zeta potential of PS/PMMA latex particles at 25C. 100 o ............................................81 4-13. Zeta potential of PS/PMMAPAA latex particles at 25C. 90 10 o ..................................82 4-14. Zeta potential of PS/PMMAPAA latex particles at 25C. 75 25 o ..................................82 4-15. The decay of surface potential with distance from surface in various electrolyte concentrations: (1) low (2) intermediate (3) high ....................................................84 4-16. High-affinity adsorption isotherm of typical flexible polymer on solid surface. .....86 4-17. Overall schematic representation of the protein adsorption on the synthesized latex particles. ...........................................................................................................86 4-18. The dependence of UV absorbance on BSA concentration. (A) 1.0mg/ml (B) 0.6mg/ml (C) 0.2mg/ml ............................................................................................87 4-19. Standard curve of net absorbance vs BSA sample concentration. ...........................88 xi

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4-20. Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37C in phosphate buffer media. o ...............................89 4-21. Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37C. o ..................................................................................................90 4-22. Adsorption isotherm of BSA adsorption 37C in phosphate buffered saline (PBS) on PS latex particles. o .....................................................................................91 4-23. Conformation of bovine serum albumin (BSA) on latex particles ...........................92 4-24. Adsorption isotherm of BSA 37C in PB on PS/PMMA core shell latex particles. o 100 ...................................................................................................................94 4-25. Adsorption isotherm of BSA at 37C in PBS on PS/PMMA core shell latex particles. o 100 ...................................................................................................................95 4-26. Adsorption isotherm of BSA at 37C in PB on PS/PMMAPAA core shell latex particles. o 90 10 ...........................................................................................................97 4-27. Adsorption isotherm of BSA at 37C in PBS on PS/PMMAPAA core shell latex particles. o 90 10 ...........................................................................................................98 4-28. Adsorption isotherm of BSA 37C in PB on PS/PMMAPAA latex particles. o 75 25 ....99 4-29. Adsorption isotherm of BSA 37C in PBS on PS/PMMAPAA core shell latex particles. o 75 25 ...................................................................................................................99 4-30. Adsorption isotherm of M onto PS and PS/PMMA latex particles in PB at 37C and pH 7.4. 2 100 o ....................................................................................................101 4-31. Adsorption isotherm of M onto PS and PS/PMMAPAA latex particles in PB at 37C and pH 7.4. 2 90 10 o ..........................................................................................101 4-32. Adsorption isotherm of M onto PS and PS/PMMAPAA latex particles in PB at 37C and pH 7.4. 2 75 25 o ..........................................................................................102 4-33. Gibbs free energy of adsorption of proteins on latex particles in PB at 37C and pH 7.4. o ....................................................................................................................107 4-34. The kinetics of protein adsorption in PB on PS latex particles at 37C and pH 7.4. o ..........................................................................................................................109 4-35. The kinetics of protein adsorption in PB on PS/PMMA latex particles at 37C and pH 7.4. 100 o .............................................................................................................109 4-36. The kinetics of protein adsorption in PB on PS/PMMAPAA latex particles at 37C and pH 7.4. 90 10 o ....................................................................................................110 xii

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4-37. The kinetics of protein adsorption in PB on PS/PMMAPAA latex particles at 37C and pH 7.4. 75 25 o ....................................................................................................110 4-38. Image of red blood cells .........................................................................................113 4-39. Hemolysis caused by latex particles, n=5. ..............................................................114 xiii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS By Sangyup Kim December 2005 Chair: Hassan El-Shall Major Department: Materials Science and Engineering Latex particles with well-defined colloidal and surface characteristics have received increasing attention due to their useful applications in many areas, especially as solid phase supports in numerous biological applications such as immunoassay, DNA diagnostic, cell separation, and drug delivery carrier. Hemodialysis membrane using these particles would be another potential application for the advanced separation treatment for patients with end stage renal disease (ESRD). It is desirable to remove middle molecular weight proteins with minimal removal of other proteins such as albumin. Thus, it is necessary to understand the fundamental interactions between the particles and blood proteins to maximize the performance of these membranes. This improvement will have significant economic and health impact. The objective of this study is to synthesize polymeric latex particles of specific functionality to achieve the desired selective separation of target proteins from the human blood. Semi-continuous seed emulsion polymerization was used to prepare monodisperse xiv

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polystyrene seed particles ranging from 1267.5 to 216.3 nm in size, which are then enlarged by about 800nm. Surfactant amount played a key role in controlling the latex particle size. Negatively charged latex particles with a different hydrophobicity were prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid monomer. The prepared polymeric particles include bare polystyrene (PS) particles, less hydrophobic PS core and PMMA shell particles, and more hydrophilic PS core and PMMA-co-PAA shell latex particles with a 370nm mean diameter. SEM, light scattering, and zeta potential measurements were used to characterize particle size and surface properties. Adsorption isotherms of two proteins, bovine serum albumin (BSA) and 2-microglobulin ( 2 M), on latex particles were obtained as a function of pH and ionic strength using the bicinchoninic acid (BCA) assay method. The Langmuir-Freundlich adsorption model was used to determine the adsorption amount of protein at equilibrium. The thickness of adsorbed BSA layer on latex particles was obtained in order to investigate the adsorption orientation such as end-on or side-on mode. Adsorption kinetics experiments for both proteins and all latex particles were also performed. The adsorption kinetic constant determined from the Langmuir-Freundlich adsorption isotherm model was used to calculate Gibbs free energy of adsorption to compare the competitive adsorption of BSA and 2 M. Hemolysis tests were performed to investigate the blood compatibility of synthesized latex particles. PS/PMMA 90 PAA 10 and PS/PMMA 75 PAA 25 core shell latex particles had desirable material properties with, not only a large amount and high rate of selective 2 M adsorption over BSA but also high blood compatibility showing less than 3% hemolysis. xv

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CHAPTER 1 INTRODUCTION End stage renal disease (ESRD) is a chronic condition in which kidney function is impaired to the extent that the patients survival requires removal of toxins from the blood by dialysis therapy or kidney transplantation. The National Kidney Foundation estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF]. The number of people with ESRD is rapidly increasing in the United States with approximately 96, 295 incidents and 406, 081 prevalent patients, including 292, 215 on dialysis and 113, 866 with a functioning graft in 2001. It is projected that there will be more than 2.2 million ESRD patients by 2030 [USRDS 2003]. The expenditure for the ESRD treatment program had reached $22.8 billion, 6.4% of the Medicare budget in 2001. Due in part to the limited availability of kidneys for transplantation, hemodialysis is the primary clinical treatment for the patients with ESRD. The central element of a hemodialysis instrument is the semipermeable membrane that allows for selective transport of low molecular weight biological metabolites less than 5,000 Da such as urea and creatinine as well as excess water and electrolytes [Baker 2004] from the blood. One limitation of current dialysis technologies is the inability to efficiently remove middle molecular weight toxins such as 2 -Microglobulin ( 2 M) and interleukin 6 (IL-6). 2 M is a causative protein of dialysis-related amyloidosis (DRA), a disease arising in patients with chronic kidney failure as a serious complication of long-term hemodialysis treatment [Gejyo et al. 1985]. 2 M deposition in tissue is the primary cause 1

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2 of destructive arthritis and carpal tunnel syndrome [Vincent et al. 1992; Drueke 2000]. The 2 M structure is shown in Figure 1-2. Figure 1-2. Stereo drawing of the carbon backbone of 2 M [Becker et al. 1985]. Although attempts have been made to increase the efficiency of middle molecular weight toxin removal by changes in the membrane pore size and the use of innovative materials to adsorb these toxins [Samtleben et al. 1996; Ronco et al. 2001], removal efficiency is not as high as those achieved by a normal healthy kidney. Traditional membranes have a number of processing and performance limitations [Westhuyzen et al. 1992; Leypoldt et al. 1998], such as a restricted choice of surface chemistries and limited control of porosity. The development of novel engineering membrane technology is needed to remove middle molecule toxins. Polymeric latex particles have received increasing attention in medical application areas, especially as solid phase supports in biological applications [Piskin et al. 1994]. Examples of these applications include immunoassay [Chen et al. 2003; Radomske-Galant et al. 2003], DNA diagnostic [Elassari et al. 1998], cell separation, drug delivery carrier [Luck et al. 1998; Kurisawa et al. 1995; Yang et al. 2000], etc. This is because of

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3 the well-defined colloidal and surface characteristics of the particles. By using a seed emulsion polymerization method, it is possible to synthesize monodisperse latex particles with various particle size ranges and surface chemistry. Functionalized core-shell latex particles can be introduced by multi-step emulsion polymerization. Core particles are synthesized in the first stage of the polymerization and the functional monomer is added in the second stage. This is done without any emulsifier addition to prevent the production of new homopolymer particles [Keusch et al. 1973]. The core-shell particles are useful in a broad range of applications because of their improved physical and chemical properties over their single-component counterparts [Lu et al. 1996; Nelliappan et al. 1997]. Through the development of a hemodialysis membrane using monodisperse latex particles, improvements in advanced separation treatment for patients with end stage renal disease (ESRD) can be realized. This requires the maximum removal of middle molecular weight proteins with minimal removal of other beneficial proteins such as albumin. Thus, an understanding of the fundamental interactions between the particles and biopolymers is vital to maximize the performance of this membrane technology. The field of material science and biotechnology is based on fundamental chemistry has developed over the past three decades into todays powerful discipline that enables the development of advanced technical devices for pharmaceutical and biomedical applications. This novel and highly interdisciplinary field is closely associated with both the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]. Hemodialysis membrane using these particles would lead to improvements in the advanced separation treatment for patients with end stage renal disease (ESRD). The interdisciplinary nature of this approach enables a more complete understanding of the

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4 phenomena of protein adsorption and the material properties necessary for selective separation. This innovative approach to membrane fabrication has the potential of making inexpensive, highly efficient membranes for both industrial and specialized separation processes. The goal of this study is to prepare polymeric latex particles with tailored properties to maximize separation of toxin molecules and to investigate the fundamental interactions between the particles and molecules in the biological system in order to optimize the performance of a membrane material for these applications. Polymeric latex particles were synthesized with specific functionality in an attempt to achieve selective separation. Seeded emulsion polymerization was used to synthesize functionalized monodisperse latex particles in various sizes. Negatively charged hydrophobic polystyrene latex particles were synthesized by the same method. Core shell latex particles, PS/PMMA 100 PS/PMMA 90 PAA 10 PS/PMMA 75 PAA 25 were also synthesized to differentiate the degree of hydrophobicity of particles. Scanning Electron Micrograph (SEM) and light scattering measurements were used to characterize particle size and shape, and zeta potential measurements were conducted to measure the electrical surface property of synthesized particles. Adsorption isotherms of target proteins, bovine serum albumin (BSA), and 2 M on latex particles were obtained as a function of pH, ionic strength, and protein concentrations using the BCA assay method. Adsorption kinetics for both proteins on the latex particles were also measured. Finally, hemolysis tests were run to determine the biocompatibility of polymer latex particles with human blood. This research will be described in more detail in chapters 3 and 4.

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CHAPTER 2 BACKGROUND AND LITERATURE SURVEY 2.1 The Significance of End Stage Renal Disease (ESRD) The kidneys are responsible for removing excess fluid, minerals, and wastes from the blood regulating electrolyte balance and blood pressure and the stimulation of red blood cell production. They also produce hormones such as erythropoietin (EPO) and calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005; Kurbel et al. 2003]. EPO acts on the bone marrow to increase the production of red blood cell in case of bleeding or moving to high altitudes. Calcitriol mainly acts on both the cells of the intestine to promote the absorption of calcium from food, and also bone to mobilize calcium from the bone to the blood. When kidneys fail, harmful waste builds up, blood pressure rises, and body retains excess fluid. The body also does not make enough red blood cells. When this happens, treatment is needed to replace the function of failed kidneys. Figure 2-1. An image of the location and cross section of a human kidney. 5

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6 The National Kidney Foundation estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF]. Chronic renal disease is a gradual and progressive loss of the function, unlike acute renal disease where sudden reversible failure of kidney function occurs. Chronic renal failure usually takes place over a number of years as the internal structures of the kidney are slowly damaged. In the early stages, there can be no symptoms. In fact, progression may be so gradual that symptoms do not occur until kidney function is less than one-tenth that of a normal health kidney. If left untreated, chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal Disease (ESRD). ESRD is a rapidly growing heath-care problem in the United States. In 2001, approximately 96,295 incidents and 406,081 prevalent patients were diagnosed with ESRD, including 292,215 patients on dialysis and 113,866 patients with a functioning graft [USRDS 2003]. The projected number of patients with ESRD is expected to exceed 2.2 million patients by 2030 with much of this growth being driven by the increasing prevalence of major contributing factors such as diabetes and high blood pressure [USRDS 2003]. A great extent of ESRD program cost and Medicare budget have been spent in 2001. Due in part to a limited availability of kidneys for transplantation, hemodialysis (HD) is the primary method of treatment for ESRD and is currently used for approximately 61% of U.S. ESRD patients. 2.2 Hemodialysis (HD) Treatment HD removes toxins from the body by extracorporeal circulation of the blood through a semipermeable membrane, referred to as a dialyzer. The toxins are removed primarily by diffusion across the membrane to a dialysate solution which is circulated on the opposite side of the membrane. The cleaned blood is then returned to the blood stream. A surgically constructed vascular access connects the extracorporeal circuit to the

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7 patients system. Treatments are almost always performed three times per week in specially equipped dialysis facilities for 3-4 hours per each treatment. Figure 2-2 shows the scheme of the hemodialysis route. The critical element of a HD instrument is the semipermeable membrane, which allows for selective transport of low molecular weight biological metabolites from blood. igure 2-2. A schematic draw of the hemodialysis route ESRD patients who undergo dialysis therapy often experience several other problems associated with the treatment such as anemia, fatigue, bone problems, joint problems, itching, sleep disorders, and restless legs. Anemia is common in patient with kidney disease because the kidneys produce the hormone erythropoietin (EPO), which stimulates the bone marrow to produce red blood cells. Diseased kidneys often do not produce enough EPO to stimulate the bone marrow to make a sufficient amount of red blood cells. This leads to bone disease, referred to as renal osteodystrophy and causes bones to become thin and weak or malformed and can affect both children and adults. Older patients and women who have gone through menopause are at greater risk for this disease. Uremic toxins, which cannot be remove from the blood by the current dialyzer membranes can lead to itching. This problem can also be related to high levels of F

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8 parathyroid hormone (PTH). Dialysis patients can also experience day-night reversis, they have insomnia at night and sleep during the day. This can be related to possible nerve damage in the body and a chemical imbalance in the blood due to the excess toxinOxidative stress is a problem for patients on maintenance dialysis. This problem is due to an imbalance between proand antioxidant factors [Roselaar et al. 1995; Cristol et al. 1994]. Oxidative stress affects oxidation of low-density lipoproteins which are the main factor for atherogenesis [Huysmans et al. 1998] and is also involved in the development of malignancies and diabetes mellitus [Rice-Evans et al. 1993]. In order to reduce antioxidant defense, dialysis is needed to contribute to help stimulate free radical production or eliminate antioxidants. Dialysis-related amyloidosis (DRA) is also acommon and serious problem for people who have been on dialysis for more than 5years. DRA develops when proteins in the blood deposit on joints and tendons, causipain, stiffness, and fluid in the joint, as is the case with arthritis. Normally the healthy kidneys can filter out these proteins, but dialysis filters are not as effective. 2.3 Advances in Membrane Technology al, that s. ng Dialysis for blood of ESRD. Hemofiltration s ysis using a cellulose based membrane occurred 1913. John Abel [1990] from the Johns Hopkins Medical School, described a method purification is widely used in the treatment dialysis (HD) techniques use a semi-permeable membrane to replace the role of the kidney. The membranes used in HD can be broadly classified into those basedon cellulose and those manufactured from synthetic copolymers. These membranes come in various shapes such as sheets, tubular structures or hollow fiber arrangements. The hollow fiber type is the most popular and is incorporated into over 800 different devicein world wide [Ronco et al. 2001]. The first attempt at blood dial

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9 wheresing e om ysis was performed, until the mid 1960. The basic molecrial in een benzyl) was used for the membrane with hepar by the blood of a living animal may be submitted to dialysis outside the body ua membrane based on cellulose and returned to the natural circulation without exposurto air, infection by microorganisms or any alteration that would necessarily be prejudicial to life. This same technique is still used to today, however the device used has been modified over the years as better membranes were developed and the anti-coagulant, heparin, has become available. Cellulose membranes have been widely used for the treatment of renal failure fr1928, when the first human dial ular structure of cellulose is made of a long chain containing hydroxyl (OH) groups. The realization that such groups imparted undesirable qualities on the materespect to blood contact behavior was discovered in the early 1970s and since has bthe focus of development. These modified cellulose membranes used the partial substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt to reduce their negative effect. The result is a molecular mosaic of hydrophobic (and hydrophilic (hydroxyl and cellulose) regions. Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients with acute renal failure in 1943. Cellophane tubing in as the anticoagulant. For the next 17 years, hemodialysis therapy was performed by this method but only for the patients with acute reversible renal failure. Vascular access required repeated surgical insertions of cannulas (slender tubes) into an artery and vein, and limited the number of treatments for a patient could receive in order to minimize the amount of vascular damage.

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10 Initially, the need for dialysis in patients with acute renal failure was determmainly by the development of signs and sym ined ptoms of uremia. After dialysis, some time might et al. ent improvement. In 1960, the development of a shunt [Quinton et al. 1960] fone (PSf), polyamide (PA), and polyacrylonitrile (PAN) by phase inversion or preciplidone e elapse before uremic manifestations returned to warrant a sequential treatment ofdialysis. Many patients with acute renal failure, secondary to accidental or surgical trauma were hypercatabolic, but the interdialytic interval might be prolonged because of anorexia or use of a low-protein diet. However, Teschan and his coworkers [Obrien 1959] showed that patient well-being and survival were improved by what they termed prophylactic daily hemodiaysis, or administration of the treatment before the patient again became sick with uremia. Their report in 1959 was the first description of daily hemodialysis. Development of membrane accessories such as a shunt has also been an area of focus for treatm a flexible polytetrafluoroethylene (PTFE or Teflon ) tubing, made many more hemodialysis treatments possible for chronic kidney failure patients. PTFE has a non-stick surface and is relative biocompatibility leading to minimized blood clotting in theshunt. Synthetic membranes are prepared from engineered thermoplastics such as polysul itation of a blended mixture resulting in the formation of asymmetric and anisotropic structures. Figure 2-3 shows a fiber type of the Polyflux S membrane consisting of poluamide (PA), polyacrylethersulfone (PAES), and polyvinylpyrro(PVP) with the integral three-layer structure. The skin layer on the inside fiber typmembrane contacts blood and has a very high surface porosity and a narrow pore size

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11 distribution. This layer constitutes the discriminating barrier deciding the permeabiliand solute retention properties of the membrane. The skin layer is supported by thick sponge-type structure larger pores, providing mechanical strength and very low hydrodynamic resistance. ty Figure 2-3. Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al. 1998] n [Malchesky 2004], because of its thermal stability, mechanical strength, and chemical inertnciated ls. m, PSf is a widely used membrane material for the hemodialysis applicatio ess. According to a report from the National Surveillance of Dialysis-AssoDisease (NSDAD) in the US, over 70% of hemodialysis membranes were PSf based [Bowry 2002]. This is most likely because PSf has many advantages over other materiaThis synthetic polymer is one of few materials that can withstand sterilization by steaethylene oxide, and -radiation. PSf membrane can be prepared by conventional immersion precipitation methods into many different shapes including porous hollow

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12 fiber or flat sheet hemodialysis membranes. The material also has a high permeablow molecular weight proteins and solute, and high endotoxin retention. The chemical structure of PSf is shown in Figure 2-4. ility to Figure 2-4. The chemical structure of polysulfone (PSf). There is one major disadvantage to PSf. The hydrophobic nature of the PSf causes ment alternative pathway leadins not 3; et al. in O acrylate serious complications through the activation of the comple g to the adsorption of serum proteins onto the membranes [Singh et al. 2003]. Anticoagulants are added during dialysis therapy to avoid blood clotting, but this doecompletely eliminate the problem. In order to overcome this disadvantage of the PSf membrane, various studies have been performed to change the materials surface properties. These investigations include hydrophilic polymer coating [Brink et al. 199Kim et al. 1988; Higuchi et al. 2003], layer grafting onto PSf membrane [Wavhal 2002; Song et al. 2000; Pieracci et al. 2002; Mok et al.1994], and chemical reaction of hydrophilic components onto the membrane surface [Higuchi et al.; 1990; 1991; 1993; Blanco et al. 2001; Nabe et al. 1997; Guiver et al. 1993]. Hydrophilic monomers, 2-hydroxy-ethylmethacrylate (HEMA), acrylic acid (AA), and methacrylic acid (MMA), have also been grafted onto PSf membrane to increase flux and Bovine Serum Album(BSA) retention [Ulbricht et al. 1996]. Hancock et al [2000] synthesized polysulfone/poly(ethylene oxide) (PEO) block copolymers to improve the resistance to platelet adhesion. Kim et al. [2003] also studied blending a sulfonated PEdiblock copolymer into PSf in order to reduce platelet adhesion and enhance

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13 biocompatibility. PEO is a commonly used biomaterial due to its excellent resistanceprotein adsorption and inherent biocompatibility [Harris 1992]. Kim et al. [20a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf membrane with an ultra-thin skin layer. The polymer had an entrapped diblock copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO to 05] studied ate lic graft l). e of thier aromatic or/and 3 acryland a hydrophobic block of octadecylacrylate (OA). Molecular dynamic (MD) simulations were performed as a function of copolymer density to optimize interfacial structure information. McMurry [2004] developed a strategy using an amphiphicopolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glycoWhen compared to unmodified PSf, these graft copolymer and resulting blend membranes are found to hold promise for biomedical device applications. Polyamide (PA) membranes have also been used for hemodialysis becausmechanical strength in both wet and dry conditions. Polyamide consists of aliphatic monomers with amide bonding (-CONH-), also known as a peptide bond.The basic amide bond in polyamide is shown in Figure 2-5. R 1 and R 2 can be either aromatic or aliphatic linkage group. CN H O~ RR 12~ CN H O~ RR 12~ Figure 2-5. The chemical structure of polyamide (PA). Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane ompatible technique due to the use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of and concluded that PA hemofiltration was a highly bioc

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14 the ace e is n is a semi-crystalline polymer and the mechanical properties strong 2CN tivated anaphylatoxins and 2 -Microglobulin ( 2 M). The PA based membrane, Polyflux (manufactured by Gambro GmbH, Germany) blended with polyamide, polyarylethersulfone and polyvinylpyrrolidone (PVP), was able to clean small molecules such as urea, dreatinine, and phosphate, as well as decrease 2 M amount by 50.2% [Hoenich et al. 2002]. Due to the non-selectivity of the membrane removal of thesunwanted materials also led to the undesirable loss of beneficial proteins during therapy. Meier et al. [2000] evaluated different immune parameters using a modified celluloslow-flux hemophan and synthetic high-flux PA membrane during a 1 year period in chronic hemodialysis patients. They found that the 1-year immunological evaluation of hemodiaysis membrane biocompatibility was associated with changes in the pattern ofchronic T-cell actiovation. Polyacrylonitrile (PAN) is another commonly used membrane material because it inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio[Scharnagl et al. 2001]. PAN ly depend on the crystalline structures. The chemical structure of PAN is shown in figure 2-6. CHCH n2CN CHCH n Chemical structure of polyacrylonitrile (PAN). The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming agent, gives PAN membranes more flexible processing parameters and increased e has been optimized through copolymerization with many other vinyl monomers including glycidyl methacrylate Figure 2-6. performance [Jung et al. 2005]. PAN membrane performanc

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15 [Godj acid for for a non-solub this in oid underneath the inner surface of membrane decreased by increasing the amount of solvent DMSA in the evargova et al. 1999; Hicke et al. 2002], N-vinylimidazole [Godjevargova et al. 2000], hydroxyl ethyl methacrylate [Ray et al. 1999; Bhat et al. 2000], methacrylic[Ray et al. 1999], vinyl pyrrolidone [Ray et al. 1999], acrylic acid [Trotta et al. 2002], acrylamide [Musale et al. 1997], and vinylchloride [Broadhead et al. 1998]. These monomers provide a reactive group for enzyme immobilization, improved mechanical strength, solvent-resistance, pervaporation, permeation flux, anti-fouling and bio-compatibility. Because of this, PAN-based copolymer membranes have great potential the treatment of hemodialysis in an artificial kidney. This material can also be usedother applications like the treatment of wastewater, the production of ultra-pure water, biocatalysis together with separation, and methanol separation by pervaporation. Lin and his coworker [2004] studied the modification of PAN based dialyzer membranes to increase the hemocompatibility by the immobilization of chitosan and heparin conjugates on the surface of the PAN membrane. When a foreign material is exposed to blood, plasma proteins are adsorbed, clotting factors are activated, and le fibrin network, or thrombus, is formatted [Goosen et al. 1980]. The result ofresearch was that the biocompatible chitosan polymer and a blood anticoagulant heparprevented blood clotting. They showed prolonged coagulation time, reduced platelet adsorption, thrombus formation, and protein adsorption. Nie et al. [2004] studied PAN-based ultrafiltration hollow-fiber membranes (UHFMs). In order to improve the membrane performance, acrylonitrile (AN) was copolymerized with other functional monomers such as maleic anhydride and -allyl glucoside. They found that the number and size of macrov

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16 internerum and d a rface. The second step is the transf after yjak et embrane 1978; Malchesky et al. 1978] has been further developed to increase the efficiency of al coagulant. The water flux of the UHFMs also decreased while the bovine salbumin rejection increased minutely. Godjevargova et al. [1992] modified the PANbased membrane with hydroxylamine and diethylaminoethylmethacrylate to improve membrane dialysis properties. Formed functional groups like primary amine, oximetertiary amine groups, provided the membrane with more hydrophilic properties ansubstantial increase in the permeability of the membranes. The wide use of filtration in practice is limited by membrane fouling. Solute molecules deposit on and in the membrane in the process of filtration causing dramaticreduction in flux through the membrane. Fouling occurs mostly in the filtration of proteins. Three kinetic steps are involved in the fouling of UF membranes according toNisson [1990]. The first step is the transfer of solute to the su er of solute into the membrane until it either finally adsorbs or passes througha set of adsorption-desorption events. The third step includes surface binding accompanied by structural rearrangement in the adsorbed state [Ko et al. 1993]. Bral. [1998] studied the surface modification of a commercially available PAN membrane to develop superior filtration properties with less fouling by proteins. The PAN membrane was immersed in excess NaOH solution to convert some of the surface nitrile groups into carboxylic groups by the hydrolysis process. This modified PAN mwas not so severely fouled in the Bovine Serum Albumin (BSA) filtration test. The pore size, however, decreased during the hydrolysis process leading to a significant reduction in flux and made the membrane less productive in the ultrafiltration (UF) mode. 2.4 Sorbent Technology Over the last three decades, sorbent technology [Castino et al, 1976; Korshak et al.

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17 dialysis, or replace it, for the treatment of ESRD. Sorbents remove solutes from solution through specific or nonspecific adsorption depending on both the nature of the solute and the sorbent. Specific adsorption contains tailored ligands, or antibodies, with high selectivity for target molecules. een used in autoimmune disordand e al. 1996], various resins [Ronco et al. 2001], albume as Specific adsorbents have b ers such as idiopathic thrombocytopenic purpura [Snyder et al. 1992] and for the removal of lipids in familial hyper cholesterolemia [Bosch et al. 1999]. Nonspecific adsorbents, such as charcoal and resins, attract target molecules through various forces including hydrophobic interactions, ionic (or electrostatic) attraction, hydrogen bonding, and van der Waals interactions. New dialysate with sorbents has become an accepted modification of dialysis, sorbent hemoperfusion is gaining ground as a valuable addition to dialysis, especially asnew sorbents are developed [Winchester et al. 2001]. Hemoperfusion is defined as thremoval of toxins or metabolites from circulation by the passing of blood, within a suitable extracorpoteal circuit, over semipermeable microcapsules containing adsorbents such as activated charcoal [Samtleben et in-conjugated agarose etc. Novel adsorptive carbons with larger pore diameters have been synthesized for potential clinical use [Mikhalovsky 1989]. Newly recognized uremic toxins [Dhondt et al. 2000; Haag-Weber et al. 2000] have resulted in several investigations on alternatives to standard, or high-flux, hemodialysis to remove thesmolecules. These methods include hemodiafilteration with [de Francisco et al. 2000] or without [Ward et al. 2000; Takenaka et al. 2001] dialysate regeneration using sorbents,well as hemoperfusion using such adsorbents as charcoal and resins.

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18 Kolarz et al. [1989; 1995] studied the hyper-crosslinked sorbent prepared from styrene and divinylbenzene (DVB) for a hemoperfusion application. They found that thepore structure of a swelling sorbent was changed by additional crosslinking with -dichloro-p-xylene in the presence of a tin chloride catalyst and in a di chloroethane solutiand en decyl groups that attract 2M through a hydrophobic interaction. The adsor of 5]. d penet on. They also realized that the hemocompatibility was useful for nemoperfusion could be imparted to the sorbents by introducing sulfonyl groups at a concentration of about 0.2mmol/g. A special polymeric adsorbing material (BM-010 from Kaneka, Japan) has beinvestigated by another group [Furuyoshi et al. 1991] for the selective removal of 2M from the blood of dialysis partients. The adsorbent consists of porous cellulose beads modified with hexa ption capacity of this material is 1mg of 2M per 1ml of adsorbent. Using a hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a high-flux hemodialyzer, several small clinical trials were performed. During 4-5 hourstreatment, about 210mg of 2M were removed, thus reducing the concentration in the blood by 60-70% of the initial level [Nakazawa et al. 1993; Gejyo et al. 1993; 199RenalTech developed a hemoperfusion device, BetaSorb containing the hydratecross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure designed to remove molecules between 4 and 30 kDa [Winchester et al. 2002]. In this case, solute molecules are separated according to their size based on their ability to rate the porous network of the beaded sorbents. The resin beads were prepared with a blood compatible coating, and confirmed to be biocompatible in vivo in animals [Cowgill et al. 2001].

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19 2.5 Limitation of Current Hemodialysis Treatment Hemodialysis is a widely used life-sustaining treatment for patients with ESRD. However, it does not replace all of the complex functions of a normal healthy kidnea result, patients on dia y. As lysis still suffer from a range of problems including infection, accelerated cardioion, anemia, chrone se middle alysis sessios f vascular disease, high blood pressure, chronic malnutrit ic joint and back pain, and a considerably shortened life span. One significant limitation of the current dialysis technology is the inability to efficiently remove larger toxic molecules. This is mainly because of the broad pore size distribution reducing thselective removal of toxins, and unsatisfied biocompatibility causing lots of complications such as inflammation, blood clotting, calcification, infection, etc. Dialysis purifies the patient's blood by efficiently removing small molecules, like salts, urea, and excess water. However, as toxic molecules increase in size, their removalrate by hemodialysis substantially declines. Typically, only 10% 40% of themolecular weight toxins (300-15,000 Da) are removed from the blood during a di n [Vanholder et al. 1995]. These toxins then reach an abnormally high level and begin to damage the body. One such toxin, 2 M, causes destructive arthritis and carpal tunnel syndrome, by joining together like the links of chain to form a few very large molecules and deposit damaging the surrounding tissues [Lonnemann et al. 2002]. This ialso a main cause of mortality for long-term dialysis patients. Other middle molecule toxins appear to inhibit the immune system and may play a significant role in the high susceptibility to infections in dialysis patients. Still others are believed to impair the functioning of several other body systems, such as the hematopoietic and other endocrine systems. This may contribute to accelerated cardiovascular disease, the leading cause o

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20 death among dialysis patients, as well as clinical malnutrition, which affects up to 50% of this patient population. Over the last decade, polymeric dialysis membranes have been developed to increase the capacity for removing middle molecular weight toxins by changing the pore size of dialyzer membra nes and using new materials that adsorb these toxins for improse styrene made during World War II in the United States. The Dow chemical compananufacturer of polystyrene, includd of on rug ntrol of ved removal characteristics. However, removal efficiency is not as high as thoachieved by a normal healthy kidney. 2.6 Latex Particles The first synthetic polymer synthesized using emulsion polymerization was a rubber composed of 1,3-butadiene and y has been a major m ing latex, which they used in paint formulations. The theory of emulsion polymerization, in which a surfactant is used, was established by Harkins [1948] and by Smith and Ewart [1948]. By 1956 the technology was complete, including the methobuilding larger diameter particles from smaller ones. The product by this emulsipolymerization is referred to as latex, a colloidal dispersion of polymer particles in water medium [Odian 1991]. Latexes are currently undergoing extensive research and development for a broad range of areas including adhesives, inks, paints, coatings, ddelivery systems, medical assay kits, gloves, paper coatings, floor polish, films, carpet backing and foam mattresses to cosmetics. The relatively well known and easy cothe emulsion process is one of main advantages for these applications. Therefore, polymeric latex particle prepared by emulsion polymerization can be a candidate for themedical applications because of the easy control of the particle size and morphology as well as flexible surface chemistry to be required.

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21 2.6.1 The Components for Emulsion Polymerization The main components for emulsion polymerization process are the monomer, a dispersing medium, a surfactant, and an initiator. A vailable monomers are styrene, sing medium is usually water, whichticles n ation. To monomer droplets but in micelles because the initiators are insolule and olution. butadiene, methylmethacylate, acryl acid, etc. The disper will maintain a low solution viscosity, provide good heat transfer, and allow transfer of the monomers from the monomer droplets into micelles and growing parsurrounded by surfactants, respectively. The surfactant (or emulsifier) has both hydrophilic and hydrophobic segments. Its main functions is to provide the nucleatiosites for particles and aid in the colloidal stability of the growing particles. Initiators are water-soluble inorganic salts, which dissociate into radicals to initiate polymerizcontrol the molecular weight, a chain transfer agent such as mercaptan, may be present.2.6.2 Particle Nucleation Free radicals are produced by dissociation of initiators at the rate on the order of 10 13 radicals per milliliter per second in the water phase. The location of the polymerization is not in the ble in the organic monomer droplets. Such initiators are referred to as oil-insolubinitiators. This is one of the big differences between emulsion polymerizationsuspension polymerization where initiators are oil-soluble and the reaction occurs in the monomer droplets. Because the monomer droplets have a much smaller total surface area,they do not compete effectively with micelles to capture the radicals produced in sIt is in the micelles that the oil soluble monomer and water soluble initiator meet, and is favored as the reaction site because of the high monomer concentration compared to that in the monomer droplets. As polymerization proceeds, the micelles grow by the addition of monomer from the aqueous solution whose concentration is refilled by dissolution of

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22 monomer from the monomer droplets. There are three types of particles in the emulsion system: monomer droplets; inactive micelles in which polymerization is not occurring; and active micelles in which polymerization is occurring, referred to as growing polymerparticles. The mechanism of particle nucleation occurs by two simultaneous processes: micellar nucleation and homogeneous nucleation. Micellar nucleation is the entry of radicals, ei ther primary or oligomeric radicals formed by solution polymerization, from the aqed the r s n is coagulation with other particles rather than polymerization of monomer. The driving force for coagulation of precursor particles, ueous phase into the micelles. In homogeneous nucleation, solution-polymerizoligomeric radicals are becoming insoluble and precipitating onto themselves or ontooligomers whose propagation has ended [Fitch et al. 1969]. The relative levels of micellaand homogeneous nucleation are variable with the water solubility of the monomer and the surfactant concentration. Homogeneous nucleation is favored for monomers with higher water solubility and low surfactant concentration and micellar nucleation is favored for monomers with low water solubility and high surfactant concentration. It hasalso been shown that homogeneous nucleation occurs in systems where the surfactant concentration is below CMC [Roe 1968]. A highly water insoluble monomer such astyrene [Hansen et al. 1979; Ugelstad et al. 1979] has probably created by micellar nucleation, while a water soluble monomer such as vinyl acetate [Zollars 1979] has beeformed by homogeneous nucleation. A third latex formation reaction mechanism has been proposed, referred to as coagulative nucleation. In this reaction, the major growth process for the first-formed polymer particles (precursor particles)

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23 severles. yer es r a l the beginning of the polymerization. As soon as the initiator is gins with the formation and growt and ved added at one time, during the later stages of the polymerization, prior to complete al nanometers in size, is their relative instability compared to larger sized particThe small size of a precursor particle with its high curvature of the electrical double lapermits the low surface charge density and high colloidal instability. Once the particlbecome large enough in size, maintaining the high colloidal stability, there is no longedriving force for coagulation and further growth of particles takes place only by the polymerization process. 2.6.3 Types of Processes for Emulsion Polymerization There are three types of production processes used in emulsion polymerization: batch, semi-continuous (or semi-batch), and continuous. In the batch type process, alcomponents are added at added and the temperature is increased, polymerization be h of latex particles at the same time. There is no further process control possible once the polymerization is started. In the semi-continuous emulsion polymerization process, one or more components can be added continuously. Various profiles of particlenucleation and growth can be generated from different orders of component addition during polymerization. There are advantages to this process such as control of the polymerization rate, the particle number, colloidal stability, copolymer composition,particle morphology. In the continuous process, the emulsion polymerization components are fed continuously into the reaction vessel while the product is simultaneously remoat the same rate. High production rate, steady heat removal, and uniform quality oflatexes are advantages of the continuous polymerization processes. Other available methods include the intermittent addition and shot addition of one or more of the components. In the shot addition process, the additional components are

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24 conversion of the main monomer. This method has been used successfully to develowater-soluble functional monomers such as sodium styrene sulfonat p e [Kim et al. 1989]. 2.6.4 lsion Chemistry of Emulsion Polymerization Emulsion polymerization is one type of free radical polymerization and can be divided into three distinct stages: initiation, propagation, and termination. The emupolymerization system is shown in Figure 2-7. Aqueous phase Aqueous phase Micelle withmonomerI R EmulsifierPolymer particle swollenWith monomer Monomer droplet IR Micelle withmonomer IR EmulsifierPolymer particle swollenWith monomer Monomer droplet Monomer droplet IR Figure 2-7. Emulsion polymerization system. [Radicals (R) are created from initiators (I). Monomer is transferred from larger monomer droplet into micelles by emulsifier. Initiated polymer particle by radicals is keep growing until monomers are all consumed. The reaction is performed in aqueous media] In the initiation stage, free radicals are created from an initiator by heat or an ultraviolet radiation source. The initiator with either peroxides groups (-O-O-), such as odium persulfate, or azo groups (-N=N-) such as azobisisobutyronitrile, is commonly used for emt with the mer chainere s ulsion polymerization. The primary free radicals created from initiator reaconomer for initiation of polymerization. In the propagation stage, the polym grows by monomer addition to the active center, a free radical reactive site. Thare two possible modes of propagations, head-to-head addition and head-to-tail addition

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25 The head-to-tail mode is the predominant configuration of the polymer chain, a result osteric hindrance of the substitute bulky group. In the termination stage, polymer chain growth is terminated by either coupling of two growing chains forming one polymer molecule or transferring a hydrogen atom (dispropotionation) from one growing chain toanother forming two polymer molecules, one having a saturated end group and the other with an unsaturated end-group. 2.6.5 Seed Emulsion Polymerization Polymer latex particles have received increasing interest because of the versatiliof the many applications heterophase polymerization processes like emulsion, dispersion, micro-emulsion, seeded emulsion, precipitation, etc. Especially, to prepare well-defined microspheres having monodispe f ty rse and various particle sizes as well as surface group ed particles prepared by emulsion polymerization and es hological design. In seede to control n functionalilties, it is necessary to use se nlarge them to a desired size in the further stages of reactions. Polymeric particles are required to have a uniform particle size in many applications, such as chromatography, where they are used as a packing material. Morphological control of latex particle is also important for many practical application[Schmidt 1972]. Seed emulsion polymerization (or two-stage emulsion polymerization) is a useful method to achieve both monodisperse particle size and morp d emulsion polymerization [Gilbert 1995], preformed seed latex is usedthe number of particles present in the final latex. The advantage of seeded emulsiopolymerization is that the poorly reproducible process of particle nucleation can be bypassed, so that the number concentration of particles is constant and known. Various mechanisms have been proposed for the growth of latex particles [Ugelstad et al. 1980; Okubo et al. 1992] using this polymerization technique. The initial seed particle

PAGE 41

26 preparation step is well known, and relatively easy to perform because, at the relativesmall particle sizes (0.2 to 0.5 micron), the particle growth process can be readily controlled by the use of an emulsifier. Enough emulsifier is used to prevent coagulation but not enough to cause the formation of new particles. As the particles are grown to larger sizes in successive seeding steps it becomes increasingly difficult to maintastable, uncoagulated emulsion without forming new particles and thereby destroying monodisperisty of the latex. Recently, there have been several reports concerning treaction kinetics of seed emulsion polymerization and the development of latex morphologies over the course of the reaction [Chern et al. 1990; Delacal, et al. 1990; Let al. 1995; Lee 2000; 2002]. 2.6.6 Polystyrene Latex Particles A number of papers have described the synthesis of the polystyrene latex particlbearing various functional surface groups such as carboxyl [Lee et al. 2000; Tun2002; Reb et al. 2000], hydroxyl [Tamai et al. 1989], marcapto [Nilson 1989], epoxy [Shimizu et al. 2000; Luo et al ly in a he ee es cel et al. 2004], acetal [Izquierdo et al. 2004; Santos et al. 1997], ometyl [Izquierdo et al. 2004; Park et al. 2001; Sarobh the d thymine [Dahman et al. 2003], chlor e et al. 1998], amine [Counsin et al. 1994; Ganachaud et al. 1997; Ganachaud et al. 1995; Miraballes-Martinez et al. 2000; 2001; Anna et al. 2005], ester [Nagai et al. 1999], etc. In order to produce these functionalized particles, different methods for particle preparation must be used. The polymer emulsion with core-shell morphology of latex particles is one of them. This is a multistep emulsion polymerization process in whicpolystyrene core particle is synthesized in the first stage and the functional monomer is added in the second stage of the polymerization without any emulsifier postfeeding to prevent the production of new homopolymer particles, thus forming the functionalize

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27 polymer shell on the core particle [Keusch et al. 1973]. There are requirements to limit secondary nucleation and encourage core-shell formation in seeded emulsion polymerization including the addition of smaller seed particles at high solid content to increase particle surface area; low surfactant concentration to prevent formation of micelles; and the semi-continuous addition of monomer to create a starved-feed conditioand keep the monomer concentration low. There are some advantages [Hergeth et al. 1989] of dispersions with polymeric core-shell particles: First, it is possible to modiinterfacial properties of polymer particles in the aqueous phase by the addition of only very small amounts of a modifying agent during the last period of the reaction. Thusthese core-shell particles are useful in a broad range of applications since they always exhibit improved physical and chemical properties over their single-component counterparts [Lu et al. 1996; Nelliappan et al. 1997]. In this way, the improvement of surface properties of such dispersions is straightforward and inexpensive. The other is that polymers with a core-shell structure are perfect model systems for investigating thmaterial properties of polymer blends and composites because of their regular distribution of one polymer inside a matrix polymer and because of the simple spgeometry of the system. Their properties usually depend on the structures of latex particles. Chen and his coworkers [Chen et al. 1991; 1992; 1993] reported the morphological development of core shell latex particles of polystyrene/poly(methyl methacrylate) during polymerization. Before the research by Chen and his coworker, Min et al. [Min et al. 1983] reported the morph n fy the e herical ological development of core shell latex of polystyrene (PS)/polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

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28 additiof ity of graft ing reagents. Thus protein adsorption on polymeric solid surfaces has st are microspheres, defined as fine polym can be n r, was containing antigen, urine, serum, etc. The latex will become agglutinated and visibly on method of PS. They found that the percentage of grafting PS to the PBA was greatest for the batch reaction, and the PBA-PS core-shell particles with a high degree grafting remained spherical upon aging test because of the emulsifying abilcopolymer. 2.6.7 Various Applications of Latex Particles Latex particles are applicable to a wide range of areas such as biomedical applications [Piskin et al. 1994], especially as the solid phase such as in immunoassays[Chern et al. 2003; Radomske-Galant et al. 2003], DNA diagnostic, drug delivery carriers[Luck et al. 1998; Kurisawa et al. 1995; Yang et al. 2000], blood cell separations, and column pack become a center of attention. Of particular intere er particles having diameters in the range of 0.1 to several microns, whichused as functional tools by themselves or by coupling with biocompounds. Singer and Plotz [1956] firstly studied microsphere, or latex agglutination test (LATs), by using monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support owhich the biomolecules were going to adsorb. The biomolecule adsorption, howevelimited by possible desorption of the adsorbed species or loss of specific activity of the complex formed. Since this work was published, the latex particle applications for immunoassay have been rapidly and widely studied and developed. Latex agglutination test, or latex immunoassays, start with tiny, spherical latex particles with a uniform diameter and similar surface properties. The particles are coated with antibodies (sensitized) through the hydrophobic interaction of portions of the protein with the PS surface of the particles. If sensitized particles are mixed with a sample

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29 agglomerate. Latex tests are inexpensive as compared with the other techniques [Ba1988]. ngs sed tion. Rheumatoid factor (RF) in different age subpopulations has also been evalu studied a latex Unipath [Percival 1996] has manufactured a range of immunoassay materials baon a chromatographic principle and deeply colored latex particles for use in the home andclinical environments. The latex particles, which are already sensitized with a monoclonal antibody, can detect any antigens that bound to the surface of the latex particles. Some detectable pollutants include estrogen mimics, which induce abnormalities in the reproductive system of male fishes and lead to a total or partial malefeminiza ated according to a patients clinical status by using a rapid slide latex agglutination test for qualitative and semiquantitative measurement in human serum along with latex immunoassay method [Onen et al. 1998]. Magalhaes and his coworkers [2004]diagnostic method of contamination of male fishes by estrogen mimics, using the production of vitellogenin (VTG) as a biomarker. This was based on a reverseagglutination test, developed with monoclonal antibodies specific to this biomarker. Premstaller and his coworkers [Premstaller et al. 2000; 2001] have prepared a porouspoly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient chromatographic separation of biomelecules such as proteins and nucleic acids. They used a porogen, a mixture of tetrahydrofuran and decanol, to fabricate a micropellicular PS-DVB backbone. Legido-Quigley and his colleagues [2004] have developed themonolith column to obtain further chromatographic functionality to the column by introducing chloromethylstyrene in place of styrene into the polymer mixture.

PAGE 45

30 Core shell type monodisperse polymer colloids have been synthesized by Sarobeand Forcada [1998] with chloromethyl functionality in order to improve the biomolecuadsorption through a two-step emulsion polymerization process. They investigated the functonalized particles by optimizing the experimental parameters of the functionalmonomer including reaction temperature, the amount and type of redox initiator sysused, the type of addition of the initiator system, and the use of washing. They c le tem oncluded that thsis serve as separation chann of usually along the polypeptide chain which contain positive or negative charges [Norde 1998]. e relation between the amount of iron sulfate and the persulfate/bisulfite system added should be controlled to obtain monodisperse particles and prevent the premature coagulation of the polymer particles during the polymerization. A semi-continuous emulsion polymerization technique for latex particle synthewas performed by McDonald and other researchers [Ramakrishnan et al. 2004: Steve et al. 1999]. They introduced a variety of particles sizes, compositions, morphologies, and surface modifications to fabricate latex composite membranes (LCMs). Arraying and stabilizing latex particles on the surface of a microporous substrate form narrowly distributed interstitial pores formed between the particles, which els. They investigated the membrane performance using gas fluxes, water permeability, and the retention characterization of dextran molecules. From these tests they concluded that the narrow, discriminating layer made of the latex particles leads to ahighly efficient composite membrane. 2.7 Proteins Proteins are natural polyamides comprised of about 20 different -amino acids varying hydrophobicity [Norde 1998]. Proteins are more or less amphiphilic andhighly surface active because of the number of amino acid residues in the side groups

PAGE 46

31 Proteins are polymers of L--amino acids [Solomon and Fryhle 2000]. The refers to a carbon with a primary amine, a carboxy lic acid, a hydrogen and a variable side-chain group designated as R. Carbon atoms rent groups are asymmetric and can exhibl s e, on with four diffe it two different spatial arrangements (L and D configurations) due to the tetrahedranature of the bonds. The L refers to one of these two possible configurations. Amino acids of the D-configuration are not found in natural proteins and do not participate in biological reactions. Figure 2-8 shows the chiral carbon in 3-D as the L isomer. Proteinconsist of twenty different amino acids differentiated by their side-chain groups [Norde 1998]. The side-chain groups have different chemical properties such as polarity, chargand size, and influence the chemical properties of proteins as well as determine the overall structure of the protein. For instance, the polar amino acids tend to be on the outside of the protein when they interact with water and the nonpolar amino acids are the inside forming a hydrophobic core. Figure 2-8. L--amino acid. The covalent linkage between two amino acids is known as a peptide bond. A peptide bond is formed when the amino group of one amino acid reacts with the carboxyl group of another amino acid to form an amide bond through the elimination of water. This arrangement gives the protein chain a polarity such that one end will have a free amino group, called the N-terminus, and the other end will have a free carboxyl group, called the C-terminus [Solomon and Fryhle 2000]. Peptide bonds tend to be planar and

PAGE 47

32 give the polypeptide backbone rigidity. Rotation can still occur around both of the -carbon bonds resulting in a polypeptide backbone with different potential conformations relative to the positions of the R groups. Although many conformations are theoretically possible, interactions between the R-groups will limit the number of potential nd to form a single functional conformation. In other words, the conformation, or shape of the protein, is -helix can interact with other secondary structures within the sam conformations and proteins te due to the interactions of the chain side groups with one another and with the polypeptide backbone. The interactions can be between amino acids that are close together, as in a poly-peptide; between groups that are further apart, as in amino acids; or even on between groups on different polypeptides all together. These different types of interactions are often discussed in terms of primary, secondary, tertiary and quaternary protein structure. The primary amino acid sequence and positions of disulfide bonds strongly influence the overall structure of protein [Norde 1986]. For example, certain side-chains will promote hydrogen-bonding between neighboring amino acids of the polypeptide backbone resulting in secondary structures such as -sheets or -helices. In the conformation, the peptide backbone takes on a 'spiral staircase' shape that is stabilized by H-bonds between carbonyl and amide groups of every fourth amino acid residue. This restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure. Certain amino acids promote the formation of either -helices or -sheets due to the nature of the side-chain groups. Some side chain groups may prevent the formation of secondary structures and result in a more flexible polypeptide backbone, which is often called the random coil conformation. These secondary structures e polypeptide to form motifs or domains (i.e., a

PAGE 48

33 tertiary structure). A motif is a common combination of secondary structures and a domain is a portion of a protein that folds independently. Many proteins are composed omultiple subunits and therefore exhibit quaternary structures. 2.7.1 Interaction Forces between Proteins Proteins in aqueous solution acquire compact, ordered conformations. In such a compact conformation, the movement along the polypeptide chain is severely restrictimplying a low conformational entropy. The compact structure is possible only if interactions within the protein molecule and interactions between the protein molecule and its environment are sufficiently favorable to compensate for the low conformational entropy [Malmsten 1998]. Protein adsorption study is often focused on structural rearrangements in the protein molecules because of its significance to the biologicalfunctioning of the molecules and the important role such rearrangements play in the mechanism of the adsorption process. Knowledge of the majo f ed, r interaction forces that act structures helps to understand the behavior of proarge a ded between protein chains and control the protein teins at interface. These forces include Coulomb interaction, hydrogen bonds, hydrophobic interaction, and van der Waals interactions. Coulombic interaction. Most of the amino acid residues carrying electric chare located at the aqueous boundary of the protein molecule. An the isoelectric point (IEP) of the protein, where the positive and negative charges are more or less evenly distributed over the protein molecule, intramolecular electrostatic attraction makescompact structure favorable to proteins. Deviation to either more positive or more negative charge, however, leads to intramolecular repulsion and encourages an expanstructure. Tanford [1967] calculated the electrostatic Gibbs energy for both a compact impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

PAGE 49

34 molecule (protein) over which the charge is spread out. From the results, he found ththe repulsion force was reduced at higher ionic strength d at ue to the screening action of ion. ble e the d the tween dehyde, ent. oles tein Hydrogen bond. Most hydrogen bonds in proteins form between amide and carbonyl groups of the polypeptide backbone [Malmsten 1998]. The number of availahydrogen bonds involving peptide units is therefore far greater than that involving sidchains. Because -helices and -sheets are aligned more or less parallel to each other,interchain hydrogen bonds enforce each other. Kresheck and Klotz [1969] examinerole of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between peptide units do not stabilize a compact structure of protein. However, because the peptide chain is shielded from water due to other interactions, hydrogen bonding bepeptide groups do stabilize -helical and -sheet structures. Hydrophobic interaction. Hydrophobic interaction refers to the spontaneous ration and subsequent aggregation of non-polar components in an aqueous environment. In aqueous solutions of proteins, the various non-polar amino acid residues will be found in the interior of the molecule, thus shielded from water. The intermolecular hydrophobic interaction for the stability of a compact protein structure was first recognized by Kauzmann [1959]. If all the hydrophobic residues are buried in the interior and all the hydrophilic residues are at the outermost border of the moleculintramolecular hydrophobic interaction would cause a compact protein structure. However, geometrical and other types of interactions generally cause a fraction of the hydrophobic residues to be exposed to the aqueous environm Van der Waals interaction. The mutual interaction between ionic groups, dipand induced dipoles in a protein molecule cannot be established as long as the pro

PAGE 50

35 structure is not known in great detail. Moreover, the surrounding aqueous medium also contains dipoles and ions that compete for participation in the interactions wthe protein molecule. Dispersion interactions favor a compact structure. However, because the Hamaker constant for proteins is only a little larger than that of water, the resulting effect is relatively small [Nir 1977]. 2.7.2 ith groups of rgiles et al. 1996; Floeg %) is degraded [Floege 2001]. Renal 2M from the serum, resulting in an increase in 2M conce well the ) a al 2 -Microglobulin ( 2 M) The protein 2 M is of particular interest because it is involved in the human disorder dialysis-related amyloidosis (DRA) [Geiyo et al. 1985; A e 2001]. DRA is a complication in end stage renal failure patients who have been ondialysis for more than 5 years [Bardin et al. 1986; Drueke 2000]. DRA develops when proteins in the blood deposit on joints and tendons, causing pain, stiffness, and fluid in the joints, as is the case with arthritis. In vivo, 2 M is present as the non-polymorphic light chain of the class I major histocompatibility complex (MHC-I). As part of its normal catabolic cycle, 2 M dissociates from the MHC-I complex and is transported inthe serum to the kidney where the majority (95 failure disrupts the clearance of ntration by up to 60-fold [Floege 2001]. By a mechanism that is currently notunderstood, 2 M then self-associates into amyloid fibrils and typically accumulates inmusculoskeletal system [Homma et al. 1989]. Analysis of ex vivo material has shown that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRAis present as of full-length wild-type 2 M, although significant amounts (~20-30%) of truncated or modified forms of the protein are also present [Floege 2001; Bellotti et al.1998]. Figure 2-9 shows the ribbon diagram of human 2 M. Native 2 M consists of single chain of 100 amino acid residues and has a seven stranded -sandwich fold, typic

PAGE 51

36 of the immunoglobulin superfamily [Saper et al. 1991; Trinh et al. 2002]. 2 M was fiisolated from human urine and characterized by Berggard et al. [1980] in1968. The normal serum concentration of rst re f e 2 M is 1.0 to 2.5 mg/L. It is a small globular protein with a molecular weight of 11.8 kDa, a Strokes radius of 16, and a negative charge under physiological conditions (isoelectric point, IEP = 5.7). 2 M contains two -sheets that aheld together by a single disulphide bridge between the cysteines in positions 25 and 81 [Berggard et al. 1980; Parker et al. 1982; Cunningham et al. 1973]. 2 M cannot be removed completely by current dialysis techniques but through a better understanding othe structure and interaction forces that lead to this structure, it will be possible to morefficiently remove this problematic protein. Figure 2-9. Ribbon diagram of human M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal. 2000] 2.7.3 Serum Albumin Serum albumin is the most abundant protein found in plasma and is typically present in the blood at a concentration of 35~ 2an et 50g/L. According to extensive studies about its physiological and pharmacological properties, albumin has a high affinity to a very wide range of materials such as electrolytes (Cu+2, Zn+2), fatty acids, amino acids, metabolites, and many drug compounds [Fehske et al. 1981; Kraghhansen 1981; Putnam

PAGE 52

37 1984; Peters 1985]. The most important physiological role of the protein is therefore to bring such solutes in the bloodstream to their target organs, as well as to maintain the pH and osmotic pressure of the plasma. Bovine serum albumin (BSA) is an ellipsoidal protein with the dimensions of 140 X 40 X 40 [Peter 1985]. The primary structure is a single helical polypeptide of 66 kDa (IEP = 4.7) with 585 residues containing 17 pairs of as a soft anrfaces [Kondo et al. 1991; Norde et al. 1992; Soderquist et al. 1980;ikely is disulfide bridges and one free cysteine [Dugaiczyk et al. 1982]. BSA has been classified d flexible protein because it has a great tendency to change its conformation on adsorption to solid su Carter and Ho, 1994] and consists of three homologous domains (I-III) most lderived through gene multiplication [Brown 1976]. Each domain is composed of A and B sub-domains [He et al. 1992]. The secondary structure of human serum albumin (HSA)shown in Figure 2-10. Figure 2-10. Secondary structure of human serum albumin (HSA) with sub-domains [Zunszain et al. 2003]. HSA has the same structure domains with the serum albumin from other species such as BSA [Brown 1976]. Although all three domains of the albumin molecule have similar three-dimensional structures, their assembly is highly asymmetric [Sugio et al.

PAGE 53

38 1999]. Domains I and II are almost perpendicular to each other to form a T-shaped assembly in which the t ail of subdomain IIA is attached to the interface region between sub-domains IA and IB by hydrophobic interactions and hydrogen bonds. In contrast, domain III protrudes from sub-domain IIB at a 45 angle to form the Y-shaped assembly for domains II and III. Domain III interacts only with sub-domain IIB. These features make the albumin molecule heart-shaped. 2.8 Protein Adsorption Protein adsorption studies date back to the 1930s. At the beginning, these studies mainly focused on the determination of the molecular weight, electrophoretic and ral rearrangemadsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]. There are interaction forces at the interfaces between protein molecules and latex particles. These forces are mainly divided into the following groups; hydrophobic interaction, ionic interaction, hydrogen bonding, and van der Waals interaction [Andrade, 1985]. Hydrophobic interaction. It is known that hydrophobic interaction has a major role in protein adsorption phenomena. The adsorption of proteins on the low charged latex particles occurs by this interaction force. Generally, monomers such as styrene offer hydrophobic surfaces thby this interaction force is maximum at the isoelectric point (IEP) of the protein, and the strength [Suzawa et al. 1980; 1982; Shirahama et al. 1989; Kondo et al. 1992]. By the chromatographic applications. Later, the adsorption mechanism, especially the structu ents was studied. Recently, the studies of the relation between protein 2.8.1 Interaction between Protein Molecule and Latex Particle at protein molecules adsorb to. The amount of adsorbed protein pH at maximum adsorption shifts to a more acidic region with an increase in ionic

PAGE 54

39 reports [Suzawa et al. 1980; 1982; Lee et al. 1988], protein adsorption was greaterhydrophobic surface than on a hydrophilic one, implying that hydrophobic interaction is on a s ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA). Ionic ely t ces n force is operative over small distane ble n nt dialysis membrane therapy for end stage renal disease (ESRD) one of most dominant forces in protein adsorption. Ionic interaction. Negatively charged latex particles have ionic functional groups on their surfaces, such as salts of sulfonic and carboxylic acid. Sulfate grouporiginate from an initiator such as sodium persulfate, and carboxylic groups originate from a h bonds are formed between the negative charges of these latex particles and positive surface charges of protein molecules. The conventional low-charged latex particles rarform these ionic bonds. Hydrogen bonding. Hydrogen bond is a strong secondary interatomic bond thaexists between a bound hydrogen atom (its unscreened proton) and the electrons of adjacent atoms [Callister 1999]. Protein can be adsorbed on hydrophilic polar surfathrough hydrogen bonding. Hydrogen bonds are frequently formed between hydroxyl-carbonyl or amide-hydroxyl. Hydroxyl-hydroxyl or amide-hydroxyl bonds are also formed in protein adsorption. Van der Waals interaction. This interactio ces, only when water has been excluded and the two non-polar groups come closto each other. Lewins calculation showed that the van der Waals interaction is negligicompared with the forces involved in the entropy increases, i.e. hydrophobic interactio[Lewin 1974]. 2.9 Hypothesis for Toxin Removal As mentioned earlier, insufficient removal of middle molecular range toxins is a major drawback of curre

PAGE 55

40 patienis y ese haracteristics over the last decade. However, removal efficiing a novel membrane design composed of an assembly of engineered polymeric latex particles sions. Important factors will ie ith a is established, it is expece ts. This may cause destructive arthritis and carpal tunnel syndrome, inhibit the immune system, and accelerate cardiovascular disease leading to death among dialyspatients. In order to overcome these complications, artificial dialysis membranes have been developed to increase the capacity for removing middle molecular weight toxins bchanging the pore size of dialysis membranes and using new materials that adsorb thtoxins for improved removal c ency is not as high as those achieved by a normal healthy kidney. With the knowledge obtained from all above literatures, the following hypothesis has been established for the development of a membrane for the successful removal of the target protein, 2 -Microglobulin ( 2 M), without the removal of serum albumin. This will be done us ynthesized to predetermined specificat nclude pore size and surface chemistry. 2.9.1 Toxin Removal by Size Sieving Based on Monodispersed Pore Size The packing of monodispersed spherical particles can lead to the formation of porous layers suitable for use as filters and membranes [Hsieh et al. 1991]. The pore sizis defined as the largest spherical particles that can pass through the interstitial spaces. It is well known that monodispersed spherical particles can be obtained from the seed emulsion polymerization method. Many commercially available latex particles are synthesized by this technique and are inexpensive. When the defect free membrane wmonodisperse pore size distribution based on a particular particle array ted to outperform the traditional hemodialysis membrane limitation, which is a broad range of pore size distribution. When spherical particles pack in regular crystallinarrays, a number of packing geometries, such as hexagonal closest packing, cubic closest

PAGE 56

41 packing, and body-centered cubic packing, are possible. The pore size of the array depends on these packing geometries as well as particle size. Theoretical pore size can becalculated from the simple geometry of the arr ay. Figure 2-11 shows the relationship between particle size and pore size. 0.001 0.010.10.010.1110Pore 110m) Size (Particle Diameter(m) Body-Centered Cubic ArrayClosest Packed Array 0.001 Size (Particle Diameter(m) 0.010.10.010.1110Pore 110m) Body-Centered Cubic ArrayClosest Packed Array Body-Centered Cubic ArrayClosest Packed Array and closed packed arrays [Steve et al. 1999]. McDonald and his coworkers [Ramakrishnan et al. 2004: Steve et al. 1999] have described the fabrication of latex composite membranes and their performance in previous papers. It is from this prior research, and knowledge gained from conversations with Dr. McDonald, that this current research has been initiated [McDonald 2003]. The focus of this thesis is the extension of McDonalds research in the area of composite membranes to medical application such as dialysis membranes. 2.9.2 Toxin Removal by Selective Adsorption on Engineered Latex Particles The flexibility in latex surface properties is a significant advantage to this materials ability to separate proteins since the selectivity is strongly dependent on the charge interactions of both the protein and the latex particles under the conditions of Figure 2-11. The relationship between pore size and particle size in body-centered cubic

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42 separation [Menon et al. 1999; Chun et al. 2002]. First, all sorbents were designed to have negative surface properties because most plasma proteins in blood are negative and should not be removed with charge interaction between proteins and sorbents at physiological condition (pH7.4). It was reported that a negatively charged surface was more blood compatible than a positive one [Srinivasan et al. 1971]. The toxin protein, 2-Microglobulin (2M) can be selectively adsorbed on the surface of latex particles by the design of a suitable hydrophobic portioned surface where only 2M protein can be anchored with hydrophobic interactions. Albumin adsorption on the latex particle is not side-on madsorption of M protein on the engineered latex particles. allowed because charge repulsion is more dominant than hydrophobic interaction with ode. The figure 2-12 shows the schematic representation of the selective 2 PMMA/PAAShellCOOOSO3OSO3-PSCore COO------Albumin Charge repulsion -------Hydrophobic2-Microglobulin2 Interaction-Microglobulin PMMA/PAAShellCOO-----------Albumin Charge repulsion OSO3OSO3-PSCore COO--------------Hydrophobic2-Microglobulin2 particles at pH 7.4 Interaction-MicroglobulinFigure 2-12. Schematic representation of the protein adsorption on the core shell latex

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43 Hydrophilic/hydrophobic microdomain structures were proven to be more blood compatible [Mori et al. 1982; Higuchi et al. 1993; Deppisch et al. 1998]. The optimization of suitable hydrophobic to hydrophilic ratio is also important for the biocompatibility. The monomers, such as styrene (St), methyl methacrylate (MMA) and acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in emulsion polymerization process. In summary, the background and fundamental literatur e survey about the history, material properties and limitation of hemodialysis membrane; latex particle preparation, surface chemistry, and manufacturing process; target proteins and protein adsorption; and finally the hypothesis for a toxin removal with high separation efficiency have been suggested. This research focuses on such hypothesis and the materials and characterization methodology for achievement of suggested hypotheses are described in the next chapter.

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CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY As mentioned earlier, the goal of this study is to prepare polymeric latex particles with tailored properties to maximize separation of toxin molecules and to investigatefundamental interactions between the applied particles and molecules in thesystem to optimize the membrane performance for hemodialysis applications. Polymeric latex particles wit the biological h monodisperse size distribution to obtain uniform pore size and various size ranges to utilize membrane construction are necessary. Surface engineering with various combination of hydrophobic/hydrophilic domain on the surface of latex particles is expected to affect the removal of target protein by selective adsorption, and to improve the biocompatibility of membrane. Therefore, the materials and characterization methodology are addressed in this chapter. 3.1 Materials Styrene (St) monomer used for a seed and a core particle, was purchased from Fisher Scientific and used without any other purification process. Acrylic acid (AA), and methyl methacrylate (MMA) monomers introduced for shell formation, were purchased from Fisher Scientific and used without any other purification process. Sodium persulfate (SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as received. Divinylbenzene (DVB) crossliking agent was purchased from Aldrich. The anionic surfactant, Aerosol MA80-I [sodium di(1,3-dimethylbutyl) sulfosuccinate], was kindly donated by Cytec. 78-80% Aerosol MA80-I is mixed with isopropanol and water. Its critical micelle concentration (CMC) is 24.5 mM (1.19%). Ion exchange resin, 44

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45 AG 501-x8 Resin (20-50 mesh), was purchased from Bio-Rad Laboratories, Inc. It is a mixed-bed resin with H+ and OHas the ionic forms and is 8% crosslinked. Bovine serum albumin (BSA) (heat shock treated) was purchas Fisher Scientific. The isoelectric point of tlin (2M)as gma ed from he BSA is 4.7-4.9 and the molecular weight is 66,300Da. 2 -Microglobu was purchase from ICN Biomedicals, Inc., and was separated from patients with chronic renal disease, and lyophilized from ammonium bicarbonate. The molecular weight by SDS-PAGE was approximately 12,000Da. BSA and 2 M proteins were used received without further purification process. In order to investigate the protein adsorption evaluation, bicinchoninic acid protein assay kits were purchased from Si(Cat # BCA-1) and Pierce Biotechnology (Cat # 23225). The chemical structures for the main chemicals are shown in Figure 3-1. CH2CH Styren e (Mw = 104.15) CH2CH CH2CH Styren e (Mw = 104.15) CH2CH Acrylic acid (Mw = 72.06) C OH O CH2CH Acrylic acid (Mw = 72.06) C OH O CHCH 2 COCH3 O CHCHCH 32 COCH3 O CH 3 Methyl methacrylate(Mw = 100.12) Methyl methacrylate(Mw = 100.12) CH2CH Divinylbenzene(Mw = 130.2) CHCH 2 CH2CH Divinylbenzene(Mw = 130.2) CHCH 2 CHCH +(Na) -(O3S)CH-C-O-CH-CH-CH-CH3O 223OCH CH 3CH3CH3 CH-C-O-CH-CH2-CH-CH3 Anionic surfactant (MA80-I) (Mw = 388.5) +(Na) -(O3S)CH-C-O-CH-CH-CH-CH3O 223OCH CH 3CH3CH3 CH-C-O-CH-CH2-CH-CH3 Anionic surfactant (MA80-I) (Mw = 388.5) 2 +(Na)-(O) SOO OO-+Initiator, sodium persulfate(Mw = 238.11) OSO(O)(Na) +-OO-+ (Na)(O) SOOOSO(O)(Na) +-OO-+Initiator, sodium persulfate(Mw = 238.11) Figure 3-1. Chemical structure of main chemicals. (Na)(O) SOOOSO(O)(Na)

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46 3.2 Latex Particle Preparation The experimental set up and the particle preparation schemes for various types and size ranges of latex particles are shown in Figure 3-2 and 3-3. In the experimental setup, a mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at end of glass stirrer. The agitation rate is precisely adjusted by controller motor. Paar glasreactor with 1L volume is partially in silicon oil bath on hot plate. The reactor has foopenings for a stirrer, a thermometer, a nitrogen gas inlet, and a reactant feeding lnlet. The reactants for latex polymerization are fed by precisely controlled by meterinThe particle preparation is described in each latex preparation section. the s ur g pump. Figure 3-2. Experimental setup for semi-continuous emulsion polymerization. 1. Motor 2. Mechanical stirrer bar 3. Thermometer 4. Inlet of N2 5. Inlet for initiator and monomer 6. Glass reactor (l L) 7. Oil bath 8. Hot plate 9. Pump for initiator 10. Pump for monomer 11. Tubes for reactant feed

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47 Styrene monomer Bare PS PS/PMMA100PS/PMMA90PAA10 PS/PMMA/P 75AA25 Grwinticle sizePS seedparticles og par Styrene monomer Bare PS PS/PMMA100PS/PMMA90PAA10 PS/PMMA/P 75AA25 Grwinticle sizePS seedparticles og par Figure 3-3. The particle preparation scheme in various types and size ranges of latex particles. 3.2.1 Preparation of Seed Latex Particles Polystyrene seed particles were synthesized through a typical semi-continuous emulsion polymerization. Polystyrene latex particles were prepared in a four necked Paar glass vessel equipped with a mechanical glass stirrer, thermometer, glass funnel for nitrogen gas purge, and a tube for monomer feeding. The reactor was placed in a silicon oil bath on a hot plate for homogenous and stable tempehe vessel was firstly charged with de-ionized water, the emulsifier, soitiator, sodium bicarbonate (SBC), styrene (St) monomer, divinylbenzene (DVB) monomer, under nitrogen gas atmosphere, and then heated to 722as llowed to continue for another hour. Second, reactants St and DVB monomers, SPS initiator, and SBC were fed separately using fine fluid metering pumps (Model RHSY, Fluid metering INC, NY) under a nitrogen gas atmosphere for 2h. After complete feeding rature control. T dium persulfate (SPS) in o C for 1h. The reaction w a

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48 of all reactants, the reaction was continued for another hour at elevated temperature, 80 oC and then cooled to room temperature by removal of the oil bath. 3.2.2 Preparation of Seeded Latex Particles The particle growth was achieved by seeded continuous emulsion polymerization. The vessel was charged with the polystyrene seeds and de-ionized water to make desired solid content, and heated up to 722 oC for 1h under nitrogen gas atmosphere. Then, St and DVB monomers and solution of SPS and SBC in de-ionized water were continuously added using metering pump with a precisely controlled feed rate. After feeding all reactants, the reaction was continued for three hours keeping temperature of 802 oC and then the reaction vessel was cooled down to room temperature. Monodisperse, spherical polystyrene seeds, with a 280nm mean diameter were used e-polymerized polystyrene seeded emulsion was charged in a 500m 3.2.3 Preparation of Core Shell Latex Particles for core shell structured latex particle. The pr l glass flask under a nitrogen gas atmosphere. De-ionized water was added to achieve the desired solid content. The emulsion was heated to 80 o C for 1.0 hour. Monomers, initiator, and SBC, to form the shell, were added by fluid metering pumps (LAB PUMP JR.,RHSY model, Fluid metering Inc. USA) with fine controlled feed rate. Flow rate can be adjusted by control ring graduated in 450 division from 0 to 100% flow which is in the range of 10ml/min respectively. The reaction was sustained for additional 3.0 hours at 90 o C, then cooled down to room temperature. 3.2.4 Purification of Synthesized Latex Particles Synthesized latex particles were purified with an ion exchange resin. 200g (10%w/w polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin and stirred for 40 minute to remove unreacted monomers, initiators and other impurities.

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49 Then the emulsion was diluted with DI water to a specific degree of solid content as needed. 3.3 Characte rization 3.3.1 solid termined. oscopy d hours Dried latex (10mg) was mixed with 250 mon Degree of Conversion The degree of monomer to polymer conversion was determined gravimetrically following the procedure described by Lee et al. [1995] before the latex cleaning process.The synthesized emulsion raw materials were weighed and dried in a conventional oven at 120 o C for 30min to evaporate any unreacted monomers and water. The remainingwas weighed and the degree of conversion was determined. This process was repeated three times and a mean value of the degree of conversion was de 3.3.2 Fourier Transform Infrared (FTIR) Spectr A molecule can absorb only select frequencies (energy) of infrared radiation which match the natural vibration frequencies. The energy absorbed serves to increase the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al. 1996]. Only those bonds which have a dipole moment that changes as a function of time can absorb the infrared radiation. The range of wavelengths for infrared radiation is between 2.5 m (4000 cm -1 ) and 25 m (400cm -1 ). The synthesis of the latex particles such as polystyrene (PS) homopolymer and PS/PMMA, PS/PMMA 90 PAA 10 PS/PMMA 75 PAA 25 core-shell copolymers, were verifiewith FTIR. Purified latex particles were dried in vacuum oven at 40 o C for 24 before analysis in the FTIR (Nicolet Magma, USA). g of KBr, which had also been dried in vacuum oven at 120 o C for 3h. Transmissispectra using the drift mode were plotted with 128 scans and 4cm -1 resolution.

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50 3.3.3 Quasielastic Light Scattering (QELS) Lat ex particle size was measured by the Brookhaven ZetaPlus particle size analyzer. The solid concentration as 0.001% (w/v). This ation spectroscopy (PCS) of quasielastically scattered light. For ths ed using a FE-SEM (JEOL JSM-ith ent. 01 us zeta-potential analyzer. It measures the electrophoretic mobility, velocity of charged, colloidal of latex particles used w instrument uses photon correl is technique, time dependent interference patterns of light scattered from particlein the sample cell are analyzed. The interference pattern changes due to Brownian motion, giving rise to fluctuations in scattering intensity. The frequency of these fluctuations depends on the particles diffusion constant that is inversely proportional to the particle diameter. 3.3.4 Field Emission-Scanning Electron Microscopy (FE-SEM) Particle size and surface morphology were characteriz 6335F, Japan). A diluted suspension of latex particles in water was dropped onto a silicon wafer at room temperature and allowed to dry. The sample was then coated wthe thinnest layer of carbon needed to obtain the required conductivity for this instrumSecondary electron image mode was used with a 15KV of accelerating voltage. The magnification range used was between 10,000X and 70,000X. 3.3.5 Zeta Potential Measurement Synthesized and purified latex particles were diluted with de-ionized water to 0.wt % and adjusted six different pH values: 2.05, 3.45, 4.81, 5.65, 6.43, and 7.47. These particle suspensions were then transferred to standard cuvettes for zeta potential measurement. At least two runs of ten measurements were taken for each sample and averaged. The zeta potential measurement was carried out using Brookhaven ZetaPl

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51 particles in solution, and calculate zeta-potenti al by using the Smoluchowski equation. The fquency gnitude umin (BSA) as a standard model protein and 2-Microglobulin uffer (PB) and phosp .5% h the selected protein. e 0.05, 0.1, 0.3, 0.5, and 0.7 mg/ml and those for 2M d at teins in the supernatant after the centrifugation process using the bicinct requency of laser light passing through the sample and is compared to the freof a reference beam. The shift in the frequency, called a Doppler shift, and the maof the shift correspond to the polarity and the magnitude of the electrophoretic mobility, respectively. The zeta potential is calculated from the solution conditions and the measured mobility. 3.3.6 Protein Adsorption Protein adsorption experiments of the synthesized latex particles were performed with bovine serum alb ( 2 M) as a target protein in two types of buffer solution, phosphate b hate buffered saline (PBS). To make 5 mM of PB solution, 0.345g of sodium phosphate monobasic (NaH 2 PO 4 H 2 O) was dissolve in 500ml of de-ionized water. To make PBS solution, 0.345g of sodium phosphate monobasic (NaH 2 PO 4 H 2 O) and 4.178g(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water. The synthesized latex particles were diluted with each buffer to have a solids content of 0(w/w), adjusted to pH values of 3.2, 4.8, and 7.4, and mixed wit BSA concentrations were chosen to b were 0.015, 0.030, 0.045, and 0.060mg/ml. The mixture was gently rotated using the shaker, Labquake (Barnstead/Thermolyne, Model #4002110, USA), with 8 RPM inthe incubator at 37 o C for 12 hours before the latex-protein mixture was centrifuge13,000 rpm for 15min. The amount of protein adsorbed was determined by quantifyingthe free pro honinic acid (BCA) assay method [Lowry et al. 1951; Smith et al. 1985; Baptista eal. 2003; Wiehelman et al. 1988; Brown et al. 1989]. BCA assay kits consist of Reagent

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52 A, containing bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1N NaOH with pH=11.25 and Reagent B containing 4% (w/v) copper (II) sulfate pentahydrate. The BCA working reagent was prepared by mixing 50 parts of Reagent A with 1 part of Reagent B. 100l of protein supernatant was then mixed with 2ml of BCA working reagent in a UV cuvette. Incubation was allowed to continueat room temperature further until color developed about 2h. The absorbance at 562nm was pectrophotometer (Perkin-Elmer Lambda 800, USA). UnknSeparate the prticles in centrifuge, mix supernatant with BCA reagents, measured using a UV-VIS s own concentration of sample protein was determined by comparison to a standard of known protein concentrations. The adsorbed amount per unit surface area was determined by the mass balance of the protein after adsorption process. The simple schematic of the procedure for a protein adsorption test is shown in Figure 3-4. Incubate mixture with agitation for 12hrs at 37oC aand develop the color by incubation for 2hrs at room temperature Suspend latex particles in aqueous buffer mediaAdd the dissolved target protein Measure color intensity using UV/Visspectroscopy at 562nmSuspend latex particles in aqueous buffer mediaSeparate the prticles in centrifuge, mix supernatant with BCA reagents, Add the dissolved target proteinIncubate mixture with agitation for 12hrs at 37oC aand develop the color by incubation for 2hrs at room temperature Measure color intensity using UV/Visspectroscopy at 562nm Figure 3-4. Schematic of the procedure for a protein adsorption test.

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53 3.3.7 Blood Biocompatibility by Hemolysis Test Hemolysis is the destruction of red blood cells, which leads to the release of hemoglobin from within the red blood cells into the blood plasma. Hemolysis testing is used to evaluate blood compatibility of the latex particles and the damaged to the red blood cells can be determined by monitoring amount of hemoglobin released. The red blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutesFigure 3-5 shows the phase separated blood with plasma, white blood cells, and redcells after centrifugation process. Separated RBSs were then washed with isotonic phosphate buffer solution (PBS) at pH 7.4 to remove debris and serum protein. Thprocess was repeated 3 times. blood is Figure 3-5. Separation of RBC from whole blood by centrifuge process. Prepared latex particles were re-dispersed in PBS by sonification to obtain homogeneously dispersed latex particles. 100l of the mixture of red blood cell (3 parts) and PBS (11parts) was added to 1ml of 0.5% (w/w) particle suspension. PBS was used as a negative control resulting 0% hemolysis and DI water used as a positive control to produce 100% hemoglobin released by completely destroyed RBCs. The mixture was incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged at 1500 rpm for 15 minutes. 100l of the supernatant was mixed with 2ml of the mixture %) (EtOH/HCl = 200/5, w/w) to prevent the White blood cells Plasma Red blood cells of ethanol (99%) and hydrochloric acid (37

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54 precipitation of hemoglobin. In order to remove rem aining particles, the mixture was centri fuged again with 13,000 RPM at room temperature. The supernatant was thentransferred into the UV cuvette. The amount of hemoglobin release was determined by monitoring the UV absorbance at a wavelength of 397nm. The schematic of the procedure for hemolysis test is shown in Figure 3-6. Separate red blood cells (RBCs) from blood Mix RBCswith phosphate buffer solution (PBS) Wash and re-susp end RBC with PBS; repeat three times Add lal of RBC and incuin at 37oC tex particle suspension into 100mbate sample with agitation for 30m Separate particleith EtOH/HClsolution s and mix the supernatant w Ev aluate free hemoglobin in the solution using UV/Visspectroscopy at 397nm Separate red blood cells (RBCs) from blood Separate red blood cells (RBCs) from blood Mix RBCswith phosphate buffer solution (PBS) Mix RBCswith phosphate buffer solution (PBS) Wash and re-susp end RBC with PBS; repeat three times Wash and re-susp end RBC with PBS; repeat three times Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC Separate particles and mix theith EtOH/HClsolution supernatant w Separate particles and mix theith EtOH/HClsolution supernatant w Ev aluate free hemoglobin in the solution using UV/Visspectroscopy at 397nm Ev aluate free hemoglobin in the solution using UV/Visspectroscopy at 397nm Figure 3-6. Schematic of the procedure for hemolysis test. In summary, a description of the experimental materials and characterization methodology has been explained. In the next chapter, the results and discussion of thedata obtained from these experiments is addressed.

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CHAPTER 4 RESULTS AND DISCUSSION 4.1 Polymerization of Late x Particles 4.1.1 Polystyrene Seed Latex Particles Polymer particles have many different applications including spacer particles, lubricants, packing materials for chromatography, standard particles and diagnostic drugs. All applications strongly require these particles to have a uniform particle size. Uniform particle size is the first requirement for a latex composite membrane formed by particle arrays with the interstitial spaces serving as pores for size discriminations [Jons et al. 1999; Ramakrishnan et al. 2004]. Although suspension polymerization has been mainly used as one of conventional polymerization methods to prepare uniform particles, the uniformity of the recovered particles is insufficient for membrane use. There is another method known as seed emulsion polymerization where a vinyl monomer is absorbed into fine monodiserse seed particles and polymerization causes the monomer to crease the sizes of the seed particles uniformly. In order to obtain monodisperse larger particles by this mparticles and polymerization of the monomer is repeated. In this study, cross-linked polystyrene seed latex particles were firstly synthesized in the presence of styrene monomer, divinylbenzene crosslink agent, sodium persulfate initiator, sodium bicarbonate buffer, and the Aerosol MA80-I [sodium di(1,3-dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion polymerization process, in which one or more components can be added continuously. in ethod, the procedure of absorption of monomer into fine polymer 55

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56 Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and buffer are continuously added to enlarge the seed latex particles. Various profiles of particle nucleation and growth can be gene different orders of component addition during polymerizatif this process such as easy control of the polymerizaparticle number, and particle morphology. The main goal in this process is to avoid secondary particle rated from on. There are several advantages o tion rate, monodisperse particle size and colloidal stability, copolymer composition formation leading to a monodisperse particle size distribution. Figure 4-1 shows the schematic representation of seed latex particle preparation and growth. Monomer droplet Seed particles : Amount of surfactantsis variable Enlarged final Add monomersPSPSPSs4 particles: Number of particlesare constantSeed codePSs1s2s3 Monomer droplet Seed particles : Amount of surfactantsis variable Enlarged final Add monomersPSPSPSs4 tion. The particles: Number of particlesare constantSeed codePSs1s2s3Figure 4-1. Schematic representation of semi-continuous seed latex particles preparation and growth. After the first synthesis of a small size of crosslinked polystyrene (PS) seed particles stabilized by surfactants, styrene, DVB, and initiator are continuously fed into the system to enlarge the seed latex particles maintaining a narrow size distriburecipes and reaction conditions for synthesizing these seed particles are summarized in

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57 Table 4-1. The original recipe for latex particle preparation from McDonald was modified for this emulsion system [McDonald 2003]. Table 4-1. The polymerization recipe of polystyrene (PS) seed particles. Latex label PS S2.59 PS S2.33 PS S2.07 PS S1.81 Temperature ( o C) 75 5 75 5 75 5 75 5Divinylbenzene (g) 0.52 0.52 0.52 0.52 MA 80 (g) 2.59 2.33 2.07 1.81 NaHCO Watera (g) 131.5 131.5 131.5 131.5 Styrene (g) 99.48 99.48 99.48 99.48 1.12 1.12 1.12 1.12 Na2S2O8 (g) 1.90 1.90 1.90 1.90 Waterb (g) 20.0 20.0 20.0 20.0 Reaction time (min) 140 140 140 140 % Solids content 39.6 39.6 39.6 39.6 Mean particle diameter (nm) 126.5 171.9 182.1 216.3 Conversion (%) 96.5 97.9 95.7 97.1 3 (g) Four different seed latex particles were synthesized based on surfactant amount in order to determine the optimum condition for stable and monodisperse latex particles. The emulsion polymerization of latex particles has to be carried out in a narrow range of surfactant causes flocculation or phase separation leading to broad particle size distribution and an unstable emulsion sy surfactant concentration, where particles are stable [Nestor et al. 2005]. A wide range of concentrations stem. Therefore, the range of surfactant amount was varied from 1.81g to 2.59g in increments of 0.26g in each latex label. Other reactants amounts were kept constant. Watera is the initial charge amount and waterb represents the amount of aqueous solution containing dissolved initiator and buffer,

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58 which are fed continuously after the seed particle creation. The extent of monomepolymer conversion was obtained by gravimetric calcu r to lation and was more than 95% at cripts, such as S2.07 and S1.81, ithe surfmount added for the seed latex nversion indi perce experi obtain content divided by the theoretically estimated solid content and was calculated by the all seed particles labeled PS S2.59 PS S2.33 PS S2.07 and PS S1.81 Labeled subs S2.59, S2.33 ndicate actant a preparation. The co cates the ntage of mentally ed solid following formula: 100 colid s (%) entsoliatedntentoalExperimentConversionn, the experimenta content is the weight ofolid remaafter mulsion at 120oC fin and the estimated solid content is thht lated from the recipe. rtias di eacht loadd mple of eapension iluted by % (w/w) deionized water and the mean particle diameter was determined using the Brookhaven ZetaPlus particle size analyzer. From these results, the seed latex particle size increased as the amount of surfactant decreased. The amount of emulsifier also affects the polymerization process by changing the number of micelles and their size. It is known that large amount of emulsifier produces larger numbers of smaller sized particles [Odian 1991] and was corroborated in the current work. These synthesized seed latex particles should be of narrow size distribution in order to obtain monodisperse larger particles. The SEM image showing the particle morphology and size of a sample of polystyrene seed particles can be seen in the Figure 4-2. contd Estim (4-1) In this equatio l solid the s ining evaporation of e or 30m e weig of monomers calcu As expected, the seed pa cle size w fferent for surfactan ing in see emulsion system. A sa ch sus was d 0.001 with

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59 Figure 4-2. Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS S2.59 (B) PS S2.33 (C) PS S2.07 (D) PS S1.81 (subscripts indicate the amount of surfactant added) (A) (B)

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60 (C) (D) Figure 4-2. Continued.

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61 s seen in the image, the seed particles are very smooth and spherical. The uniformity in the particle size distribution of these PS seed latex particles can also be seen in the SEM image. From this data, we concluded that seed particles synthesized in the presence of a surfactant with the chosen amounts are suitable for use to prepare larger particles with high size uniformity and smooth, spherical morphology. 4.1.2 The Growth of Polystyrene (PS) Seed Latex Particles Seeded emulsion polymerization has been conducted for several decades, and various mechanisms have been proposed. Gracio et al. [1970] suggested that the growing polymer particles consist of an expanding polymer-rich core surrounded by a monomer-rich shell, with the outer shell providing a major locus of polymerization. Seeded mulsion polymerization is commonly used for preparing latex particles less than 1m in size [Cha et al. 1995; Zou et al. 1990; 1992; Gandhi et al. 1990; Park et al. 1990]. Such latexes can be obtained from the growth of pre-prepared seed particles. The seeds introduced in this process serve as nucleation sites for particle growth. By first swelling the seed latex particles with additional monomer to the desired size, polymerization can then be initiated. Several size ranges of monodisperse polystyrene (PS) latex particles were prepared by this multistep seeded emulsion polymerization method. For the fabrication of particle-based membranes, monodisperse latex particles with a variety of size ranges are required. In this study, seed particles from 171nm to 470nm in mean diameter and with a highly uniform size distribution were prepared and used. Watera is the initial charge mount and waterb represents the amount aqueous solution containing dissolved initiator and buffer that is continuously fed into system. Feed time is precisely controlled by metering pump. The % solid contents were maintained between 19.9% and 30.0%. After A e a

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62 polymd as es ere 92.3, 84.02, n of these latex particles are shss erization under these conditions, the conversions of the latex particles labelePS 258 PS 320 PS 370 and PS 410 were 95.2, 95.7, 94.6 and 96.4%, respectively. The recipused in seeded emulsion polymerization to form the particles less than 500nm in mean diameter are shown at the Table 4-2. The conversions of the latex particles larger than 500nm in mean diameter and labeled as PS 525 PS 585 PS 640 and PS 790 w 72.3 and 96.9%, respectively. The recipes used in the preparatio own at the Table 4-3. A simple calculation can be used to predict the ultimate size that a latex particle can reach after ending the polymerization. Lets suppose that seed density ( s ) and grown particle density ( gp ) are the same. The following equation is obtained from the mabalance (m = V), where m and V are mass and volume of a particle, respectively. gpgpssVmVmIn this equation, m (4-2) be s and m gp are the weight of seed and total weight of seed and added monomers, respectively; and V s and V gp are the volume of seed and grown particle, respectively. If we know the mass of monomer to be added, the ultimate particle size can calculated from the equation 4-3. 3 2sD mwhere D 1sD m (4-3) the in s and D gp are the diameters for seed and grown particle, respectively, and isweight of the monomer added. Crosslinked PS latex particles with a size range from 258.2 ~ 410.7 in diameter, polymerized by the recipes in Table 4-2 are shownthe SEM images in Figure 4-3.

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63 Table 4-2. Continuous addition emulsion polymerization recipe for growing polystyreneLatex label PS (PS) latex particles less than 500nm in size. 258 PS 320 PS 370 PS 410 Initial charge *Seed latex (g) 100.0 171 100.0 216 100.0 216 100.0 320 Watera (g) 147.0 250.0 198.4 260.0 0.6 0.7 0.6 0.6 FeeInitiator stream 228Feed time (min) 0-167 0-140 0-100 0-92 % Solid content 28.4 30.0 30.0 20.0 Mean particle diameter (nm) 258.2 320.4 182.1 410.7 Conv Monomer continuous Styrene (g) 80.0 120.0 80.0 60.0 Divinylbenzene (g) d time (min) 0-163 0-110 0-75 0-82 NaSO (g) 0.7 0.65 0.5 0.5 NaHCO 3 (g) 0.7 0.65 0.5 0.5 Water b (g) 75.0 120.0 20.0 37.0 ersion (%) 95.2 95.7 94.6 96.4 The solid content of seed latex for PS 258 PS 320 PS 370 and PS 410 are 39.2, 39.9, 39.9, anrespectively. d 30.0%,

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64 Table 4-3. Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size Latex label PS 525 PS 585 PS 640 PS 790 Initial charge *Seed latex (g) 100.0 390 100.0 470 100.0 470 100.0 320 Water a (g) 100.0 52.0 120.0 36.0 Monomer continuous Styrene (g) 29.0 20.0 40.0 17.3 Divinylbenzene (g) 0.2 0.14 0.3 0.09 Feed time (min) 0-80 0-40 10-76 5-65 Initiator stream Na 2 S 2 O 8 (g) 0.2 0.14 0.3 0.12 NaHCO 3 (g) 0.2 0.14 0.3 0.09 Water b (g) 20.0 120 40.0 20.0 Feed time (min) 0-85 0-46 0-77 0-75 % Solids content 19.9 20.0 20.0 20.1 Mean particle diameter (nm) 525.1 585.6 64017.8 790.3 Conversion (%) 92.3 84.02 72.3 96.9 The solid content of seed latex for PS 525 PS 585 PS 640 and PS 790 are 20.0, 18.2, 19.3, and 17.3%, respectively.

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65 Figure 4-3. SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm (A) (B)

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66 (C) (D) Figure 4-3. Continued.

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67 article size was well matched to that estimated by the geometrical calculation. From this it can be concluded that the added monomer was consumed for polymer particle growth with a high degree of conversion. From the SEM characterization, newly nucleated particles were not seen, as indicated by the narrow particle size distribution. The particles less than 500nm in mean diameter were very spherical in shape with a highly uniform particle size distribution. As the particles were grown larger than 500nm mean diameter, however, they became of non-spherical shape with an uneven surface. This irregularity of particle surface can be attributed to the non-homogeneous monomer swelling into the shell of a growing polymer, which can be controlled by the factors such as temperature, agitation speed, initiator feeding rate, and surfactant amount required to stabilize the colloidal system. The SEM image of latex particles larger than 500nm mean iameter is shown in Figure 4-4. The particle size distribution is broader than that of the s particles. These particles can, however, still be used for membrane construction as a support layer rather than skin layer. The support layer does not necessarily have to be as highly monodisperse as the skin layer, which require pores of high uniformity for the selective removal of toxins. As mentioned earlier, the amount of surfactant as well as monomer in an emulsion system is the primary determinant of the particle diameter [Odian 1991]. Anionic surfactants are generally recommended at the level of 0.2-3.0 wt % based on the amount of water [Odian 1991]. The critical micelle concentration (CMC) of Aerosol MA 80-I anionic surfactant is 1.19 %.The surfactant loading in this emulsion system ranged from the 1.21 % for SS1.81 to 1.71% for SS2.59. P d maller

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68 Figure 4-4. SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm (A) (B)

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69 (C) (D) Figure 4-4. Continued.

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70 T he surfactant to monomer ratio refers to the value of the surfactant amount divided by monomer amount after polymerization ends. The surfactant amount is the weight of the surfactant contained in emulsion and the monomer amount is the total weight of the monomer added for polymerization to form the ultimate polymer particles in emulsion. As the surfactant to monomer ratio decreases, mean particle diameter increases, as described by Odian [1991]. Generally, lower surfactant concentration forms fewer micelles resulting in larger particle size [Evans et al. 1999]. However, there is a plateau in particle mean diameter between a surfactant to monomer ratio of 0.005 and 0.013. This is due to low polymerization conversion of latex particles from monomer leading to a smaller particle size than expected. This is not a factor of surfactant amount. The dependence of the particle size on the amount of surfactant is shown in Figure 4-5. 020040060080010000.0000.0050.0100.0150.0200.0250.030Surfactant to monomer ratio (w/w)Particle mean diameter (nm) Figure 4-5. Dependence of the particle size on the surfactant to monomer ratio.

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71 4.1.3 e f bare microdomain structures is important for biocompatibility [Mori et al. 1982; Higuchi et al. 1993; Deppisch et al. 1998]. Figure 4-6 shows the schematic of core shell latex particle structure. The recipe for core shell structures is shown in Table 4-4. Core Shell Latex Particles The latex particles shown in Figure 4-3 (A) and labeled as S 258 in Table 4-2 werused as seeds to prepare core-shell latex particles because of their high particle uniformity and smooth surface morphology. Methyl methacrylate (MMA) and acrylic acid (AA) monomers were introduced to increase surface hydrophilicity over that opolystyrene particles. AA is a more hydrophilic monomer than MMA because of the carboxyl acid group in AA is more favorable for hydrogen boding with water. The surface carboxyl groups on PAA may have many promising applications in biomedicaland biochemical fields [Kang et al. 2005]. MMA monomer is less hydrophobic than styrene. PMMA is known to more biocompatible than PS but still PMMA is hydrophobicThe optimization of suitable hydrophobic to hydrophilic ratios and control of Polystyreneseed particle Core shelllatex particles PS/PMMA100PS/PMMA90PAA10 PS/PMMA75PAA25 Polystyreneseed particle Core shelllatex particles PS/PMMA100PS/PMMA90PAA10 Figure 4-6. Schematic of core shell latex particle structures. PS/PMMA75PAA25

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72 Table 4-4. The preparation recipe o f PS core with various shell latex particles. Latex25 label PS/PMMA 100 PS/PMMA 90 PAA 10PS/PMMA 75 PAA Initial charge PS seed latex (g) 50.050.050.0aMonomer continuous Methly methacylate (g) 32.0 28.8 24.0 Acrylic acid (g) 0.0 3.2 8.0 Initiator stream Na 258258258 Water (g) 157.0 157.0 157.0 Feed time (min) 0-120 0-50 0-60 b20.0 Feed time (min) 0-122 0-60 0-65 95.2 2 S 2 O 8 (g) 0.2 0.2 0.2 NaHCO 3 (g) 0.2 0.2 0.2 Water (g) 20.0 20.0 % Solids content 18.3 18.3 18.3 Mean particle diameter (nm) 370.6 370.8 370.1 Conversion (%) 95.5 92.4 Three types of core shell structures were prepared. PMMA75PAA25 shell is a copolymer consisting of the PMMA to PAA ratio of 75% to 25% by weight. PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90% to 10% by weight. PMMA100 is the PMMA homopolymer shell on PS core. The particle size of these core-shell particles as well as PS latex particle was prepared to be about 370nm in mean diameter and are characterized to determine their zeta potential, protein adsorption, and biocompatibility. The image of SEM of PS and core shell particles is shown at Figure 4-7. The synthesized latex particles had a high uniformity in size and a smooth, spherical surface morphology.

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73 Figure 4-7. Scanning Electron Micrograph of latex particles (A) PS (B) PS/PMMA100 (C) PS/PMMA90PAA10 (D) PS/PMMA75PAA25 (A) (B)

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74 (C) (D) Figure 4-7. Continued.

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75 4.2 Characterization of Latex Particles 4.2.1 Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectroscopy has proven to be a powerful tool to characterize polymeric materials. A molecule can absorb only select frequencies (energy) of infrared radiation which match the natural vibration frequencies. The energy absorbed serves to increase the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al. 1996]. Only those bonds which have a dipole moment that changes as a function of time can absorb infrared radiation. The FTIR spectra of polymerized latex particles made during this work are shown in Figure 4-8. There are number of characteristic peaks for PS latex particles [Bhutto et al. 2003; Li at al. 2005]. Aromatic C-H stretching vibration is shown at 3002 cm-1 and 3103 cm-1, and aliphatic C-H asymmetrical and symmetrical stretching shown at 2900 cm-1and 2850 cm-1, respectively. There is an additional carbonyl (C=O) adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some amount of either or both PMMA and PAA. The broad OH group peak at 3400 cm-1 appear for particles containing some amount of PAA and has intensity dependent on the amount of PAA in the shell. For example, the peak intensity of OH groups for PS/PMMA75PAA25 is greater than that of PS/PMMA90PAA10 since there is a higher AA monomer content in PS/PMMA75PAA25 than in PS/PMMA90PAA10. This OH peak is not seen for PS and PS core PMMA shell particles because of the absence of OH groups on PS and PMMA polymer chains. 4.2.2 Zeta Potential Measurements The electrical surface property was characterized by zeta potential measurements of tex particles. Zeta potential is an important and useful indicator of surface charge which can be used to predict and control the stability of colloidal systems. The greater the zeta is la

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76 5001000150020002500 300035004000 Wavenumbers (cm-1)li Reative % Transmttance Figure 4-8. FTIR spectra of polymerized latex particles. (A) bare polystyrene (PS) (B) PS/PMMA 100 (C) PS/PMMA 90 PAA 10 (D) PS/PMMA 75 PAA 25 (B) PS/PMMA 100 ( A ) Bare PS (C) PS/PMMA 90 PAA 10 (D) PS/PMMA75PAA25 C C OCH 3 O CH 3 X CH 2 CH2 n CH CH2 C C OCH 3 O X CH2 CH O C CH 3 Y OH

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77 potential the more likely the suspension is to be stable because the charged particles repel one another and thus overcome the natural tendency to aggregate. The measurement of zeta potential is often the key ontrol of the interaction of proteins with solid surfaces such as latex particles. Zeta potential () is the elecat exists at the shear plane of a particle, which is some small distance from the surface [Myers 1999]. The zeta potential of the particles can be determined by measuring the mobility of the particles in an applied electric field, termed electr its response to an alternating electric field. Colloidal particles dispersed inluically charged due to their ionic characteristics and dipolar attributes. The development of a net charge at the particle surface affects the distribution of ions in the neighboringng in an increased concentration of counter ions to the surface. Each particle dispersed in a ns called fixed layer or Stern layer. Outside thcompositions of ions of opposite polarities, formble layer is formparticle-liquid interface. This double layer may be considered to consist of two parts: an inner region which inclund relatively strongly to the surface and an outer, or diffuse region in which the ion distribution is determined by a balance of electrostatic forces and random thermal motion. The potential in this region, therefore, decays with the distance from the surface, until at a certain distance it becomes zero. Figure 4-9 shows a schematic representation of a typical ion distribution near a positively charged surface. The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the to understanding and c trical potential th ophoretic mobility, or a so tion are electr interfacial region, resulti solution is surrounded by oppositely charged ioe Stern layer, there are varying ed in the region of the ing a cloud-like area. Thus an electrical dou des ions bou center of those ions, which are effectively adsorbed to the surface [Hunter 1981]. The

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78 extent of ion adsorption is determined by electrical and other long-range interactions between the individual ions and surface of particles. The ions outside of the Stern layer form the diffuse double layer, also referred to as the Gouy-Chapman layer [Burns and Zydney 2000]. Shear planeStern layer0s Shear planeStern layer0s t controlled electric field is applied via electrodes immersed in a sample suspension and Figure 4-9. Schematic representation of ion distribution near a positively charged surfaceZeta potential is a function of the surface charge of a particle, any adsorbed layer athe interface and the nature and composition of the surrounding medium in which the particle is suspended. The principle of determining zeta potential is very simple. A

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79 this causes the charged particles to move towards the electrode of opposite polarity. Viscous forces acting upon the moving particle tend to oppose this motion and the equilibrium is rapidly established between the effects of the electrostatic attraction andthe viscosity dra g. The particle therefore, reaches a constant terminal velocity. Figure 4-10. Schematic representation of zeta potential measurement.(source: http://nition.com/en/products/zeecom_s.htm) Because protein adsorption mechanisms are very complex and hard to explain in biological systems, protein adsorption is generally studied by using more ideal systems consisting of one or more well characterized proteins, a well-characterized adsorbent, and a well-defined aqueous solution. Even so, small changes in the experimental conditions, such as pH, ionic strength, and temperature generate totally different results. Therefore, two media systems, phosphate buffer (PB) and phosphate buffered saline (PBS), were chosen to be used for the zeta potential measurements and protein adsorption study. PB is a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to n a constant pH. PBS is used to keep the water concentration inside and outside of the cell balanced. If the water concentration is unbalanced, the cell risks bursting, or shriveling up because of a phenomenon called osmosis. PBS is a solution with the 5 mM of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl) in water. maintai

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80 The results of zeta potential measurement at room temperature (25 o C) as function of pH and ionic strength (different media, PB and PBS) for synthesized latparticles are seen in Figure 4-11. Zeta potential values of polystyrene (PS) latex particleat the Figure 4-9 are negative between -29.1 mV and -59.9 mV in PB and betwe ex s en -20.3 mV and -27.8 mV in PBS at all pH ranges. These negative zeta potential values of PS latex particles are due to the sulfate groups [Vandenhu et al. 1970] originated from persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer chain where polymerization was initiated. -100 -80-60-40-200pHZta ptentl, 2040123456789eoia (mV) PB (5mM) PBS (5mM + 143mM) en Figure 4-11. Zeta potential of PS latex particles at 25 o C. The zeta potential measurements of PS/PMMA 100 core shell latex particles, as seen in Figure 4-12, were also negative with a range of -28 and -50.5 mV in PB and betwe14.3 mV and -18.6 mV in PBS at the pH 2.1-7.8. These negative values are also due to sulfate groups on the particle surface.

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81 -100 -80-60-40-2002040123456789Zea potentil, (mV pHta) PB (5mM) PBS (5mM + 143mM) Figure 4-12. Zeta potential of PS/PMMA100 latex particles at 25oC. The zeta potential for PS/PMMA90PAA10 and PS/PMMA75PAA25 was also negative, as seen in Figure 4-13 and 4-14. The zeta potential value for the PS/PMMA90PAA10 were between -36.7 mV and -67.8 mV in PB medium and between -14.7 mV and -19.3 mV in PBS both at 25oC. In case of the PS/PMMA75PAA25 core shell particles, zeta potential values were between -29.1 mV and -52.0 mV in PB and between -11.5 mV and -21.0 mV in PBS media. Sulfate groups in PS/PMMA75PAA25 and PS/PMMA90PAA10 core shell particles contribute to the negative zeta potential values. participate in forming a negatcid is expressed by the term pKa, which is the pH at which an acid is 50% dissociated. However, the contribution of the carboxylic acid Carboxylic groups from acrylic acid monomer also would ive surface charge of the latex particles at a pH greater than 7.0 because the pK a value for the carboxylic acid is between 4.0 and 6.0 pH range [Ottewill et al. 1967]. This indicates that carboxylic acid dissociation begins at a pH between pH 4.6-6.0 and increases with pH. The strength of an a

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82 group to negative zeta potential values of PS/PMMA-PAA core shell particles was not detectable because there was no significant difference in zeta potential values for synthesized latex particles at a pH greater than 7.0. -60-40-2002040123456789Zeta potential, (mV) PB (5mM) PBS (5mM + 143mM) -80 -100pH Figure 4-13. Zeta potential of PS/PMMA 90 PAA 10 latex particles at 25 o C. -100-80-60-40-200 2040123456789pHZe ponti, V) tateal (m PB (5mM) PBS (5mM + 143mM) Figure 4-14. Zeta potential of PS/PMMA 75 PAA 25 latex particles at 25 o C.

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83 The zeta potential plots and values were similar for all latex particles. This may bedue to the similar initiator density on the latex surface. The initiator concentrationsmonomers for shell polymerization were the same fo to r all core shell latex particles (0.62% w/w from Table 4-4). A similar concentration (0.55% w/w) of initiator to monomer was added to prepare bare PS latex particles used in zeta potential measurements. Since the zeta potential values of all synthesized latex particles were negative between pH=2.0 and pH=7.8, the isoelecric point (IEP) of these latex particles would be less than pH=2.0. Sulfates have a pKa in the range of 1.0 to 2.0 [James 1985], indicating that the sulfate group, the conjugate base of a strong acid, is protonated (-C-OSO3H) at less than pH 2.0. This means that the synthesized latex particles are all negative at the physiological pH. Most serum proteins have a negative surface charge in blood, therefore, when the negative latex particles are applied to blood stream, fatal teinis avoided by charge repulsion in the blood. Table 4-5 shows the common blood proteins and their isoelectric points (IEP). Table 4-5. The isoelectric point (IEP) of common proteins. Serum proteins Isoelectric point (IEP) complication by coagulation between applied latex particles and serum pro s Albumin 4.8 1-Globulin 2.0 2-Microglobulin 5.4 1-Globulin 5.8 Heptoglobin 4.1 Hemoglobin 7.2 1-lipoprotein 5.4 Immunoglobulin G (Ig G) 7.3 Fibrinogen 5.8 (source: http://www.fleshandbones.com/readingroom/pdf/1085.pdf ; http://www.medal.org/visitor/www%5CActive%5Cch13%5Cch13.01%5Cch13.01.05.aspx )

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84 The absolute zeta potential values of the latex particles in phosphate buffer (PB) was higher than that in phosphate buffered saline (PBS). This is because high concentration of sodium and chloride electrolytes in PBS compressed the electrical double layer causing rapid decay of surface potential. Figure 4-15 shows that the thickness of the electrical double layer is inversely proportional to the concent ration of electr olyte in the system. Surface Potential, 1/ 3 1.01/e 1/1/(1)(3)(2) 21Z Surface Potential, 1/ 3 1.01/e 1/1/(1)(3)(2) Figure 4-15. The decay of surface potential with distance from surface in various electrolyte concentrations: (1) low (2) intermediate (3) high [Myers 1999]. 21ZThe electrical potential in the solution surrounding the particle surface falls off the Debye-Huckel yers 1999] exponentially with distance (Z) from the surface according to approximation [M z) (exp 0 (4-4) e reciprocal of the thickness of the electrical double layer, referred to as the The potential fallen off by a factor/e. The theoretical equation for the ickness, 1/, is where is th Debye length of 1 double layer th 21 22i0ekT 1iizc (4-5)

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85 wheretration of y was performed to measure the protein adsorption on the synthesized latex particles. Many physical and chemical processes occur at the boundary between two phases and adsorption is one of the fundamental surface phenomena [Oscik 1982]. This phenomenon is driven by the change in the concentration of the components at the interface with respect to their concentrations in the bulk phase. There are two adsorption processes, physical adsorption occurring when non-balanced physical forces appear at the boundary of the phases, and chemical adsorption or chemisorption t the interface [Jn leads ational entropy, hence, adsorption takes place only if the loss in conformational entropy is compensated ter adsorption, Cp. The initial part of the isotherm merges with the -axis because at low polymer concentration, the protein has a high affinity for the surface and all of the polymer is adsorbed until the solid surface is saturated. 0 is the permittivity of a vacuum of free space, is the permittivity of a medium, e is the relative permittivity or dielectric constant of the medium, c is the concenan ion (i), z is the valency of an ion (i), k is Boltzmanns constant, T is absolute temperature (K). 4.3 Protein Adsorption Study The adsorption stud occurring when atoms and molecules from adjacent phases form chemical bonds aaroniec et al. 1988]. Flexible proteins in solution possess high conformational entropy as a result of the various states each of the many segments in the protein chain can have. Adsorptioto a reduction of this conform by sufficient attraction between polymer segment and solid surface [Martin 1998]. Isotherm shape of high affinity adsorption between polymer and solid is shown in Figure 4-16 where the adsorbed mass, is plottedagainst the polymer concentration in solution af

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86 Figurde arboxyl e 4-16. High-affinity adsorption isotherm of typical flexible polymer on solid surface. In order to investigate the adsorption of blood proteins, latex particle properties such as electrical surface charge, hydrophobicity, and environmental conditions like pH, ionic strength, and temperature are considered as experimental factors [Arai and Nor1990]. Figure 4-17 shows the overall schematic representation of the protein adsorption on the synthesized latex particles with the functional groups such as sulfate and cgroups. CP PMMA/PAAShell PS or PMMAShell OSO3OSO3-OSO3 -OSO3-PS -O3SO PSCore HOOC-O3SOO3SO3HOOCHOOC --OSO Protein NH3+++H3N+COO-Hydrophobic interaction NH3COO-H3NCOO--OOC Ionic interaction Hydrophobic interactionHydrogen bonding Ionic interaction PMMA/PAAShell OSO3OSO3-OSO3 -OSO3PS or PMMAShell -O3SO OSO3PSCore HOOC-O3SOO3SO3HOOCHOOC --OSO -O3SO OSO3-OSO3 -OSO3-PS PSCore HOOC-O3SOO3SO3HOOCHOOC --OSO Protein NH3+++H3N+COONH3COO-H3NCOO--OOC NH3+++H3N+COO-Hydrophobic interaction NH3COO-H3NCOO--OOC Ionic interaction Hydrophobic interactionHydrogen bondingFigure 4-17. Overall schematic representation of the protein adsorption on the Ionic interaction synthesized latex particles. The surface functional groups as well as suitable surface hydrophobicity / hydrophilicity of latex particles are closely related to protein adsorption property. As indicated at Figure 4-17, various interaction forces such as hydrophobic interaction, ionic

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87 interaction, hydrogen interaction, and van der Waals interaction [Andrade 1985] exist between protein molecule and the solid surface of latex particle. 4.3.1 Adsorption Isotherm The protein adsorption isotherm is generally defined as the relationship between the amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a ine the amount of protein adsorbed onto latex particles. To evaluate the equilibrium conces x function of the concentration of protein. Adsorption isotherms were plotted to determ ntration of target proteins, a calibration curve is firstly needed. Figure 4-18 showthe UV absorbance as a factor of bovine serum albumin (BSA) concentrations. The UV absorbance of the known protein concentration was measured at 562nm, which is the mafor a protein treated by a bicinchoninic acid (BCA) assay. 00.20.40.60.8 500520540560580600Waveleng th (nm)Absorbance Figurg/ml (B) 0.6mg/ml (C) 0.2mg/ml (A) (C) (B) Max.@562nm e 4-18. The dependence of UV absorbance on BSA concentration. (A) 1.0mFigure 4-19 shows the standard calibration curve used in adsorption isotherm experiment of protein onto latex particles. The net absorbance of each known protein

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88 concentration was plotted and then a linear equation passing through a concentration ofzero protein concentration was obtained. This equation is applied to determine the orbed on the synthesized latex particles. The amount of free proteieported trations (0.05, 0.1, 0.3, 0.5, and 0.7mg/ml) were used to generate this curve as well as the adsorption isotherm. unknown protein amount ads n was also determined using UV light at the 280nm wavelength, however, a distinguishable UV intensity was too low to determine a precise value of protein adsorption. The bicinchninic acid (BCA) assay technique [Smith et al. 1985] has been rfor high efficiency of protein evaluation and was used for determination of protein adsorption [Tangpasuthadol et al. 2003; Williams et al. 2003]. This technique has many advantages, such as high sensitivity, easy of use, color complex stability, and less susceptibility to any detergents. Five different BSA concen y = 0.0R2 = 004741x 0.99390.00.10.20.30.40200400600800BSA Standard (g/ml)Net Absorbance at 562nm Figure 4-19. Standard curve of net absorbance vs BSA sample concentration.

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89 All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinearegression method. Two adsorption isotherm models, the Langmuir type isotherm aLangmuir-Freundlich combination type isotherm, were compared. As seen the Figure 4-20, The Langmuir isotherm model deviated further from the experimental data. Thall isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al. 1996;al. 1998] usi r nd the erefore, Lee et ng the nonlinear regression method with following equation, nneqeqmkCkC111where q is the amount of adsorbed BSA per unit surface area, q qq (4-6) stant, were between 0.45 and 0.55, and hence n was fixed to its average 0.5. Actual qm values were evaluated after fixing the n value. m is the adsorbed amount in equilibrium, C eq is the equilibrium concentration of BSA, k is the adsorption conand n is the exponential factor for a heterogeneous system. n values are empirical and usually from zero to no more than unity [Jaekel 2002]. The values of n in our system 0.01.02.03.0 00.20.40.60.8C (mg/ml) eqq (mg/m2) pH 3.2 Langmuir-Freundlich model Langmuir model Figure 4-20. Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37oC in phosphate buffer media.

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90 The BSA protein adsorption on the synthesized latex particles was performed at three pH levels, 3.2, 4.8, 7.4, in both phosphate buffer (PB) and phosphate buffered saline (PBS), at physiological temperature, 37 o C. These pH levels correspond to an acidic pH lower than the isoelectric point (IEP) of BSA, the pH at the IEP, and an alkaline pH higher than the IEP of BSA, respectively. The IEP corresponds to the pH at which the amino acid becomes a zwitterion. Here its overall electrostatic charge is ne utral and the charge expressed by the basic amino group (-NH2, -NH3+) of the amino acid is equal to the charge expressed by the acidic carboxyl group (-COOH, -COO). The IEP of BSA is pH 4.8, and indicates that the side chain of the amino acids of BSA contains a greater amount of acidic functional groups than that of basic functional groups. At pH 3.2 and 7.4, BSA has a positive and negative charge, respectively. Figure 4-21 and 4-22, show adsorption isotherms of BSA onto polystyrene (PS)latex particles. 0.01.02.03.0 4.0 00.20.40.60.8Ceq (mg/ml)q (mg/m2) pH=3.2 pH=4.8 pH=7.4 Model (pH 3.2) Model (pH 4.8) Model (pH 7.4) Figure 4-21. Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 o C.

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91 0.0 1.03.04.000.20.40.60.8Ceq (mg/ml)q (mg/m2 2.0) pH=3.2 pH=4.8 pH=7.4 Model (pH 3.2) Model (pH 4.8) Model (pH 7.4) (PBS) on PS latex particles. At the IEP of BSA, the adsorbed amount (q Figure 4-22. Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline onto PS at equilibrium was maximum at the IEP of BSA. This is because the BSA molecules form the most compact structures at the IEP eliminating the repulsion force between proteins. A higher number of BSA molecules can adsorb on a given surface area by hydrophobic interaction when in this compact structure. This observation agrees well with reported data by other researchers who also indicated that the maximum adsorption from aqueous protein solution was observed at the IEP [Mularen 1954; Watkins et al. 1977; Oh at el. 2000]. There was no significant difference of adsorbed BSA amount in PB and in PBS media at pH 4.8. The conformational alteration of BSA was not affected significantly by the ionic strength of the two solutions. There was evidence of the effect of the electrostatic interaction to some extent, even at the IEP, since the amount of BSA adsorbed in PBS is slightly hig m ) of BSA um, indicating that hydrophobic interaction between latex particle and BSA protein is maxim her than in PB at this pH.

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92 The adsorbed amount of protein is shown to decrease at a pH 3.2 and 7.4. The decrease in adsorption efficiency when deviating from IEP of BSA is a result of the increase in conformational size of the protein molecules and the lateral electrostatic repulsions between adjacent adsorbed BSA molecules. Figure 4-23 shows a schematic of possible conformational changes at each pH, where hydrophobic interaction always exists between non-polar portion of a protein and hydrophobic portion of solid surface. --------Latex surfaceZeta potent ialBSA(+) (-)4.87.43.2 pH(0)++++++-+++--------Latex surfaceZeta potenpH(0)++++++-tialBSA(+) (-)4.87.43.2 +++ming .4. s al blood. Table 4-6 shows the calculated results of Figure 4-23. Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 7.4). In addition, the adsorbed amount of BSA at pH 3.2 was larger than that at pH 7.4 and still less than that at 4.8. This is because there is the electrostatic interaction between the positive BSA molecules and negative PS latex particles at pH 3.2, and electrostatic repulsion is dominant between negative BSA and negative PS latex particles at pH 7The amount adsorbed increased with increasing ionic strength in pH 3.2 and 7.4. This ibecause the high concentration of electrolytes reduces the electrostatic repulsion between BSA and PS latex particle. It should be noted that the BSA concentration used in these experiments was lower than that found in normal human blood. This is because theequilibrium BSA adsorption onto the latex particles is actually reached at a much lower concentration than that found in norm

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93 adsorrm ein leading to a compact conformation of BSA. There are two main forces affecting adsorption amount at an acidic pH of 3.2. They include the ionic attraction between the negative latex particle and the positive protein, and lateral repulsion between positive proteins. The smallest amount of BSA adsorption was observed at a pH of 7.4. At this point the charge repulsion between negatively charged particles and protein molecules, exists the h of cha. bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isotheequation. In summary, the hydrophobic interaction between PS latex particle and BSA protein exists and is the dominant mechanism for the adsorption process. Conformational changes of BSA at each pH also affected the adsorption amount. Adsorption was highest at pH 4.8 because at this pH there is the least amount of ionic repulsion in the prot and the lateral repulsion between proteins dominant adsorption even though there always ydrophobic interaction between the solid latex particle and protein. The effect rge repulsion was reduced by the high concentration of electrolytes in PBS mediumTable 4-6. The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model. Media pH q m (mg/m 2 ) 3.2 1.74 4.8 2.99 Phosphate buffer 7.4 1.36 3.2 2.60 4.8 2.83 Phosphate buffered saline 7.4 2.07 The adsorption isotherms of BSA on PS/PMMA core shell particles are shown in Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline (PBS). The adsorbed BSA amount was calculated using the Langmuir-Freundlich 100

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94 adsorption isotherm equation and the amount listed in Table 4-7. The trend seen in the adsorption results was very similar to the case of PS latex. The overall adsorption process was drticle rs hat 0 ominated by hydrophobic interactions between the PS/PMMA 100 core shell paand BSA proteins. At pH 4.8, the adsorbed amount (q m ) of BSA on PS/PMMA 100 core shell particles was 2.21 mg/m 2 in PB and 2.37 mg/m 2 in PBS, and are the maximum values recorded among the three pH levels, 3.2, 4.8, and 7.4. Suzawa and his co-worke[1982] have obtained similar results. This can be also explained by the compact conformation of BSA molecules at the IEP of BSA, which lead to a high protein population on the solid surface. At a pH of 3.2 and 7.4, BSA adsorption was less than tat pH 4.8, because of the protein conformation change. There was a small increase in adsorbed BSA at pH 3.2 because of the charge attraction between negative PS/PMMA 10core shell particles and positive BSA. At pH 7.4, however, the adsorption of BSA was further reduced by charge repulsion between the negative PS/PMMA 100 core shell particles and the negative BSA proteins. 0 .000.20.40.60.8Ceq (mg/ml) 1.02.0q (mg/m 3.02) 4.0 5.0 pH=3.2 pH=4.8 pH=7.4 Mode8) l (pH 4. Mode2) l (pH 3. Mode4)Figure 4-24. Adsorption isotherm of BSA 37oC in PB on PS/PMMA core shell latex l (pH 7. 100 particles.

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95 The adsorbed amount of BSA increased more rapidly in PBS with increasing ionistrength at pH 7.4. This shows that the low concentration of electrolytes promotes electrostatic repulsion between negative BSA and negative PS/PMMA c on. x l. 1980; 100 core shell particle. As the ion strength of PBS is increased, however, the high concentration of electrolyte leads to less electrostatic repulsion, promoting more rapid protein adsorptiBy comparing the amount of adsorbed protein between bare PS latex particles and PS/PMMA 100 core shell particles, the amount of protein adsorption on the PS lateparticles is greater than that on the PS/PMMA 100 core-shell particles [Suzawa et a1982; Lee et al. 1988]. 0.01.02.0 3.04.0m2) 00.20.40.60.8Ceq (mg/ml)q (mg/ pH=3.2 pH=4.8 pH=7.4 Model (pH.2) 3 Model (pH 4.8) Model (pH 7.4) Figure 4-25. Adsorption isotherm of BSA at 37oC in PBS on PS/PMMA100 core shell latex particles.

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96 Table particles calculated the Langmuir-Freundlich isotherm model. 4-7. The equilibrium concentration values (q m ) of BSA adsorption on PS/PMMA 100Media pH q m (mg/m) 2 3.2 1.40 4.8 2.21 Phosphate buffer 7.4 1.30 3.2 1.69 4.8 2.37 Phosphate buffered saline 7.4 1.62 The BSA adsorption m echanism and trend on PS/PMMA core shell particles was very similar to that of the PS latex particle. Unlike the case of PS and PS/PMMA100 latex particles, the amount of BSA adsorbed onto PS/PMMA90PAA10 and PS/PMMA75PAA25 core shell particles was higher at pH 3.2 than pH 4.8 and 7.4. Figure 4-26 through 4-29 shows the adsorption isotherms of BSA on PS/PMMA90PAA10 and PS/PMMA75PAA25 core shell particles. The amount of BSA adsorbed increased at pH 3.2 and pH 4.8 as the hydrophilicity was developed by the addition of PAA. On the contrary, the adsorbed amount of BSA was dramatically decreased at pH 7.4. The adsorption amount of BSA on PS/PMMA90PAA10 and PS/PMMA75PAA25 core shell particles at equilibrium is listed in Table 4-8 and 4-9, respectively. Carboxylic acid as well as sulfate groups are distributed on the surface of PS/PMMA90PAA10 and PS/PMMA75PAA25 core shell particles and seem to largely affect the BSA adsorption. The isotherms showed very strong pH dependence to the BSA adsorption onto these core shell latex particles. The carboxylic groups (COOH) may especially enhance the BSA adsorption prominently at pH 3.2 by hydrogen bonding with protein molecules. The pKa value for the PAA is 4.0-6.0 and a good indication that the

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97 carboxylic acid groups are protonated at pH 3.2.[Gebhardt et al. 1983]. The main adsorpt ion interaction force at pH 3.2 was generated by hydrogen bonding as well as charge attraction between latex particle protein even th the conformational repulsion between proteins results in a reduction of the protein adsorption at this pH. The amount of BSA adsorbed also increased as the PAA amount in eased. The ation of BSA winimal, however, less than 0.7 mg/m2 at pH 7.4. Here the carboxylic groups are ionized, which increases the charge repulsion by ionized carboxylic groups (COO-) resulting in PS/PMMA-PAA latex particle and ough change and charge the core shell particles incr dsorp as m s with much lower protein adsorption. 0.0 2.04.0q (mg/2) 6.0m pH=3.2 pH=4.8 pH=7.4 Model (pH 3.2) Model (pH 4.8) 00.20.40.60.8Ceq (mg/ml) Model (pH 7.4)o shell Figure 4-26. Adsorption isotherm of BSA at 37C in PB on PS/PMMA 90 PAA 10 corelatex particles.

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98 0.04.06.08.000.20.4C (mg/mmm2) 2.00.60.8eql)q (g/ pH=3.2 pH=4.8 pH=7.4 Model (pH 3.2) Model (pH 4.8) Model (pH 7.4) Figure 4-27. Adsorption isotherm of BSA at 37oC in PBS on PS/PMMA90PAA10 core shell latex particles. Table 4-8. The equilibrium concentration values of BSA adsorption on PS/PMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model. Media pH qm (mg/m2) 3.2 3.61 4.8 2.83 Phosphate buffer 7.4 0.43 3.2 5.19 4.8 2.26 Phosphate buffered saline 7.4 0.43

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99 0.02.04.06.08.010.000.20.40.60.8Ceq (mg/ml)q (mg/m2) pH=3.2 pH=4.8 pH=7.4 Model (pH 3.2) Model (pH 4.8) Model (pH 7.4) o Figure 4-28. Adsorption isotherm of BSA 37C in PB on PS/PMMA75PAA25 latex particles. 0.0 10.0 2.0 4.06.08.000.20.40.60.8Ceq (mg/ml)q (m2) pH=3.2 pH=4.8 pH=7.4 mg/ Model (p H 3.2) Model (pH 4.8) Mod el (pH 7.4) Figure 4-29. Adsorption isotherm of BSA 37oC in PBS on PS/PMMA75PAA25 core shell latex particles.

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100 Table 4-9. The equilibrium concentration values of BSA adsorption on PS/PMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model. Media pH qm (mg/m2) 3.2 10.83 4.8 6.62 Phosphate buffer (PB) 7.4 0.75 3.2 10.77 4.8 6.81 Phosphate buffered saline (PBS)7.4 0.57 The adsorption of 2-microglobulin (2M) was determined under one set of onditions, in phosphate buffer at 37oC and pH 7.4 because of the limited amount of 2M protein. The adsorption isotherms of the 2M protein on the synthesized latex particles are shown in Figure 4-30, 4-31 and 4-32. The 2M protein concentrations for adsorption isotherm experiment were chosen to be 0.015, 0.030, 0.045, and 0.060mg/ml since the 2M concentration for the patients with renal failure can be elevated to similar concentrations, about 0.05mg/ml or more, up to 50 times the normal level [Nissenson et al. 1995]. There were steep initial slopes of the adsorption isotherm curves of 2M on latex particles in all isotherms, indicating a high affinity adsorption type. It seems that the hydrophobic interaction is enough for the adsorption process of 2M proteins to occur on the latex particles, even though charge repulsion exists between protein and latex particle at pH 7.4. The complete plateau regions were not seen for all isotherms, indicating that 2M proteins were not saturated at this level and there are still available sites for 2M c adsorption on latex particles. The initial slope of the 2 M isotherms for PS was steeper

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101 than that of any of the other latex particle, PS/PMMA 100 PS/PMMA 90 PA A10, and PS/PMMA. 75 PAA 25 0.0 0.200.0020.0040.0060.008 0.40.61.0Ceq (mg/ml)q (m2) 0.8 g/m PS PS/PMMA100 Model (PS) Model (PS/PMMA100) Figure 4-30. Adsorption isotherm of 2 M onto PS and PS/PMMA 100 latex particles in PB at 37C and pH 7.4. o 0.00.20.40.60.81.000.0020.0040.0060.008C (mg/ml)q (mm2) eqg/ PS PS/PMMA90PAA10 Model (PS) Model (PS/PMMA90PAA10) Figure 4-31. Adsorption isotherm of 2M onto PS and PS/PMMA90PAA10 latex particles in PB at 37oC and pH 7.4.

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102 0.00.20.40.60.81.000.0020.0040.0060.008Ceq (mg/ml)q (mg/m2) PS PS/PMMA75PAA25 Model (PS) Model (PS/PMMA75PAA25) Figure 4-32. Adsorption isotherm of 2M onto PS and PS/PMMA75PAA25 latex particles in PB at 37oC and pH 7.4. The equilibrium concentration values (qm) of 2M adsorption on latex particles are calculated and listed in Table 4-10. The difference between the maximum amounts of 2M adsorption on all the latex particles was not significantly different and their values were between 0.69 and 0.8mg/m2. Table 4-10. The equilibrium concentration values of 2M adsorption calculated the Langmuir-Freundlich isotherm model. Latex particles qm (mg/m2) PS 0.80 PS/PMMA1000.69 PS/PMMA90PAA100.72 PS/PMMA75PAA250.72

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103 4 .3.2 Adsorbed Layer Thickness There are several possible explanations for large amount of BSA adsorbed at acidic pH levels, including the end-on adsorption pattern of BSA, the flexibility of BSA molecules, the tilting of protein molecules due to the asymmetry of the charge distribution, or multilayer formation [Peula et al. 1993]. The adsorbed BSA thickness () can be calculated by the following equation [Chiu et al. 1978]. This equation was also cited again by other researchers [Shirahama et al. 1985] BSAmq33 (4-7) where 33 is the packing factor, qm is the plateau value of the adsorbed amBSA is thehich corresponds to the reciprocal of its known partial specific volume. The layer thickness () of BSA proteins on synthesized latex partic he IEP [Shirahama et al., 1985]. Compared to the hydrodynamic dimensions of BSA, 140 40 40 3 [Fair et al, 1980; Peter 1985], these nd ode. It is unnecessary ultilayering because thickness do exceed 140 The layer thickness () of BSA proteins on synthesized latex particles ered saline (PBS) was ran 28 ~ 83 at 37oC and pH 4.8. These lso listed in Table 4-1 ount, and density of BSA molecule w les in phosphate buffer (PB) ranged from 27 81 at 37 o C and pH 4.8. These calculated values are listed in Table 4-11. Only values at pH 4.8, the isoelectric point(IEP) of BSA are important, because intraand intermolecular electrostatic repulsion of protein molecules is minimized at t values of layer thickness indicate that the BSA molecules adsorb by side-on (40) a end-on (140) m to consider m not in phosphat buffe ged of calculated values are a 2.

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104 Table 4-11. Absorbed BSA layer th ickness () of BSA in phosphate buffer (PB) at 37oC. pH PS PS/PMMA 100 PS/PMMA 90 PAA 10 PS/PMMA 75 PAA 25 3.2 21 17 44 133 4.8 37 27 35 81 7.4 17 16 5 9 Table 4-12. Absorbed BSA layer thickness () of BSA in phosphate buffered saline (PBS) at 37oC. pH PS PS/PMMA 100 PS/PMM A90PAA10PS/PMMA75PAA25 3.2 32 21 64 132 4.8 35 29 7.4 26 20 5 7 28 83 1/n 1/nneqmSo, k is closely related to the initial slope of the isotherm showing the affinity between BSA and latex particles [Yoon et al. 1996]. A key point in the characterization 4.3.3 Gibbs Free Energy of Protein Adsorption In the Langmuir isotherm model, k represents the ratio of adsorption to the desorption rate constants. This definition is not applicable to the Langmuir-Freundlich isotherm model but its meaning is similar [Yoon et al. 1996]. In this sense, k is closely related to the affinity between protein molecules and latex particles. When the equilibrium concentration, Ceq, reaches zero, the value of Ceqwill be very small (1>> kCeq) and then the equation 4-6 becomes (4-8) of adsorption processes on solid-liquid interfaces is to determine the change in Gibbss free energy (G), one of the corresponding parameters for the thermodynamic driving forces, during adsorption. The traditional method to evaluate adsorption thermodynamic kCqq/1

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105 constants is based on measurement of adsorption isotherms under static conditions [Malmten 1998]. The Langmuir-Freundlich adslfeasily found from. Then, this enables calculation of the free-energy change, G, during adsorption expressed as: (4-9) where R is the general gas constant (R = 8.314JmolK) and T is the absolute temture. Gibbe energyA Plisted in Table 4-13 and 4-14, respectively. As the Gadsore negative proadsorption on a solid substraill be more favorable. e of BSA adsorption in phosphate o pH PS PS/PMMA100PS/PMMA90PAA10PS/PMMA75PAA25 s orption equilibrium constant, k can be a slope of the isotherm ads lfadskRTGln -1-1 pera s fre change of BS adsorption values in B and PBS are values are m e, th tein te w Table 4-13. The values of Gibbs free energy chang buffer (PB) at 37C. 3.2 -24.11 -27.26 -28.46 -28.05 4.8 -23.52 -23.83 -21.98 -19.72 G o.80 ads (kJ/mol) 7.4 -24.52 -8.88 -3.24 -2 Table 4-14. The values of Gibbs free energy change of BSA adsorption in phosphate pH PS PS/PMMA buffered saline (PBS) at 37 o C. 100 PS/PMMA 90 PAA 10 PS/PMMA 75 PAA 25 3.2 -23.15 -26.20 -23.12 -26.83 4.8 -21.49 -22.99 -20.90 -21.38 Go(kJ/m ads ol) 7.4 -24.30 -19.92 -2.58 -2.63 Table 4-15 shows the calculated values of Gibbs free energy change of M adsorption on synthesized latex particles. PS/PMMAPAA core shell latex particle has 27525

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106 largest negative Gibbs free energy of 22 M adsorption, indicating largest affinity betw een 2M adsorption in phosphate buffere (PB) at 37C and pH7.4. PS PS/PMMA100 M and latex particle. Table 4-15. The values of Gibbs free energy change of o PS/PMMA 90 PAA 10 PS/PMMA 75 PAA 25 G o.03 ads (kJ/mol) -36.26 -34.66 -36.59 -39 The Gibbs free energy of 2 M was much more negative in phosphate buffer (PB) than that of BSA in physiological condition (37C and pH 7.4) ofrom the Table 4-13 and the Figure 4-31. This indicates that 2M molecules more readily adsorb on latex particles BSA. This can be explained by the rate of diffusion and accessibility of 2M molecules to the latex particle ier and faster for 2M man BSA because of size (molecight) erical shape molecules. Ito be due to the higher hydrophobic force, and less electrostatic repulsion, that contribute to the adsorption of 2M molecules not as prevalent in the case of BSA; because the IEP of 2M (5.7) is cloH 7.4) than that of BSA (4.8), causing 2M to be less electrocallylled procan be seen that the 2M adsorption onto the syntharticleincreasing wi hyicity ox particles s the Goads of the BSA molecules is decreasing or constant. This phenomenon supports the theory that the selectlizing than BSA, and that the affinity of 2 M for the latex particle is much larger than that of surface. It is eas olecules th the smaller ular we and sph of 2 M may als ser to physiological condition (p stati repe from the latex articles than BSA. F m Figure 4-33, it G o ads of esized latex p s is th the drophil f the late wherea ive adsorption of 2 M molecules over BSA molecules can be enhanced by utimore hydrophilic PS/PMMA 90 PAA 10 and PS/PMMA 75 PAA 25 core shell latex particles.

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107 -60-501234 -40Gads 30100o (kJ/mo -20l) 2-Microglobulin (2M) Bovine serum albumin (BSA) Figure 4-33. Gibbs free energy of adsorption of proteins on latex particles in PB at 37 and pH 7.4. 4.3.4 Kinetics of Adsorption to o C Protein adsorption is a complex process, known to have a large biological impact and is currently not well understood quantitatively because of extreme sensitivity to pH, the concentration of other electrolytes and molecules present, temperature, and many other factors that can change in the physiological system. Accurate knowledge of the adsorption kinetics under a given set of conditions is a prerequisite for elucidating the mechanisms of many fundamental biological processes to be predicted at the molecular level [Regner 1987]. This adsorption kinetics approach is used in protein adsorption studies because of the uncertainty related to the time needed for the interfacial proteinsreach the equilibrium. It is generally accepted that the process of protein adsorption is comprised of the following steps: a) transport toward the interface, b) attachment at the interface, c) eventual structural rearrangements in the adsorbed state, d) detachment from PS PS/PMMA 100 PS/PMMA 90 PAA 10 PS/PMMA 75 PAA 25

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108 the interface and transport away from the interface. Each of these steps can affect the overall rate of the adsorption process. The quantitative analysis of the protein adsorption kinetics requires that the protein amount adsorbed is known as a function of time. The kinetics tests were carried out in phosphate buffer (PB) at 37oC and pH 7.4, a condition similar to physiological condition. The protein concentrations chosen were 0.7mg/ml for BSA and 0.06mg/ml for 2M, which are the maximal concentrations used in an adsorption isotherm test. A time-based evaluation of the adsorption was made by measuring the concentration of protein in solution at different incubation times. The results are shown below from Figure 4-34 to 4-37. The adsorption kinetics of proteins onto thconde latex particles i s expressed as a s e order model [zacar 2003] shown by the following equation: 2)(tetqqkdtdq (4-10) Integrating above equation and applying the boundary conditions, yields eettq1 (4-11) where q qkqt222the previous adsorption isotherm results representing good reproducibility of adsorption t and q e stand for the amount (mg/m) of protein adsorbed at time t and at equilibrium respectively, k is the equilibrium rate constant of second-order adsorption (m/mg*hr), and t is the incubation time (hr). The fitting parameters calculated by using the equation 4-11 are listed in table 4-16. From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than that of 2 M on PS and PS/PMMA 100 latex particles but less than that of 2 M on PS/PMMA 90 PAA 10 and PS/PMMA 75 PAA 25 latex particles. These results are similar to

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109 0.00.51.52.002468101214Time (hr)rotn adsorbd, ( 1.02.5Peiemg/m2 BSA 2nd order model (BSA) ) 2M 2nd order model (2M) Figure 4-34. The kinetics of protein adsorption in PB on PS latex particles at 37 o C and pH 7.4. 0.00.51.01.52.02.53.0 02468101214Time (hr) Prteindso o arbed, (mg/m2) BSA 2nd order model (BSA) 2M 2nd order model (2M) Figure 4-35. The kinetics of protein adsorption in PB on PS/PMMA 100 latex partic37 les at o C and pH 7.4.

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110 0.00.30.60.91.21.502468101214Time (hr)Protein adsorbed, (mg/m2) BSA 2nd order model (BSA) 2M 2nd order model (2M) Figure 4-36. The kinetics of protein adsorption in PB on PS/PMMA 90 PAA 10 latex particleso at 37C and pH 7.4. 0.00.30.60.91.21.502468101214Time (hr)BSA adsorbed, (mg/m2) BSA 2nd order model (BSA) 2M 2nd order model (2M) Figure 4-37. The kinetics of protein adsorption in PB on PS/PMMA 75 PAA 25 latex particles at 37 o C a nd pH 7.4.

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111 test. The variation of 2M equilibrium adsorption amount was nearly negligible for all latex particles. The plateau region is reached earlier for 2M than BSA on all latex particles and the K values (equilibrium rate constant of second order adsorption) for the 2M adsorption process onto latex particles are much larger than for BSA adsorption. These values indicate that the 2M molecules have a higher adsorption rate onto the latex particles than the BSA molecules. Table 4-16. Fitting parameters of second-order kinetic model. BSA 2M Latex particles qe (mg/m2) *K (hr-1) qe (mg/m2) K (-1) hr PS 1.44 6.7 0.74 754. 9 PS/PMMA1001.75 35.3 0.76 443.8 PS/PMMA90PAA100.46 9.7 0.75 569.5 PS/PMMA75PAA250.60 17.9 0.75 516.8 *K = (k*qe), hr-1There are several explanations for the phenomena observed in these experiments. First, the smaller sizes of 2M molecules allow easier diffusion and a close approach to the latex substrate. Second, a reduced number of hydrophobic domains on the latex particle surface, replaced by sulfate groups (-OSO3-) and ionized carboxylic groups (COO-) are not favorable for larger BSA molecules. Third, the conformational change by intraand inter-molecular repulsion would be faster in 2M due to smaller molecular weight. Finally, the ionic repulsion between 2M and the latex particles is less than that condition (pH 7.4) than IEP (4.8) of BSA. between BSA and latex particles because IEP (5.7) of 2 M is closer to the tested

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112 4.4 Blood Compatibility Blood compatibility can be defined as the property of a material or device thapermits it to function in contact with blood without inducing any adverse reactions. Successful functioning of blood contacting biomedical materials or devices in contact with blood strongly depends on the extent to which they damage blood during operation. High shear stresses, turbulence, rela t tively long contact times, and cellular impact with artificial surfaces are most common events experienced by blood in contact with artificial 1978; Winslow and et al. 1993]. Therefore, before any materiae placed into the human bbiocompatibility testing must be the first performed in the view of safety. The hemolysis s a method for prary screening of biomaterials to determine h blood aer circulating body fluid. The tests estimatoglobin released from thblood cell de inducedforeign material. There are several types of blood cells in our body, but the ones that make blood red are erythrocytes, or red blood cells (RBCs). Red blood cells (erythrocytes) are the most numerous type in the blood. These cells are average about 4.8-5.4 million per cubic millimeter (mm3 : same as a microliter (l)) of blood but these values can vary over a larger range depending on such factors as health and altitude. RBCs are red because they contain hemoglobin, which is a protein that contains iron. When blood circulates through lungs, oxygen binds to the hemoglobin and is then delivered to all the parts of body. RBCs are made in the bone marrow. They start out as a nucleus, like other cells, but when they fill up with hemoglobin the nucleus becomes smaller until it disappears. Without a nucleus, RBCs are fragile and live only about 120 days. In a normal healthy devises and can cause hemolysis or thrombosis [Bernhard et al. ls can b ody, test serves a elimin the biocompatibility wit nd oth e the concentration of hem e red amag by a

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113 body, about 2 million red bloodne marrow produces new ones jing dy. le cells die per second. But bo ust as fast. RBCs are about 7.5 micrometer across, and are responsible for carryoxygen, salts, and other organic substances to the cells and tissues throughout our boIn humans and most mammals the cells are concave (indented in the middle) and flexibenough to squeeze through even the tiniest of blood vessels, the capillaries. Figure 4-38. Imag e of red blood cells. (Source:http://www.pbrc.hawaii.edu/bemf/microangela/rbc1.htm) ch 10090107525f RBCs are among the first cells that come into contact with a foreign material injected in the blood. Therefore, a hemolysis assay gives information about the biocompatibility in the case of an in vivo application. The concentrations of prepared latex particles used for the tests in this research are 0.5, 2.5, and 5.0% (w/w) and the incubation time under moderate shaking was 30 minutes at 37o C. Test results are shown in Figure 4-39. The amount of blood cells that ruptured when in contact with the PS latex particles was largely increased from about 30% to 91% as the solid content of PS increased from 0.5% to 5.0%. However, hemolysis by the core shell latex particles, suas PS/PMMA, PS/PMMAPAA, and PS/PMMAPAA was less than 3.5%. O

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114 particular interest was that hemolysis caused by PS/PMMAPAA particles at 5.0% solid concentration was less than 0.2% hemolysis, the lowest value among the latex particles. The low hemolysis value indicates that the PS/PMMAPAA core shell latex particles are the most blood compatible at all solid contents. Other core shell particles such as PS/PMMAand PS/PMMAPAAalso had excellent blood comp 90 10 90 10 100 75 25 atibility with low RBS breakage. 01234 20406080100Hemolysis (%) 0.5% 2.5% 5.0% Figure 4-39. He PS PS/PMMA 75PAA25 PS/PMMA 100 PS/PMMAPAA 9010 molysis caused by latex particles, n=5.

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CHAPTER 5 SUMMARY OF RESULTS, CONCLUSIONS AND RECOMMENDATION FOR FUTURE WORK 5.1 Summary of Results The goal of thi s study was to synthesize monodisperse polymeric latex particles with tailored properties, and to investigate the fundamental interactions between the synthesized latex particles and target proteins. An understanding of the fundamental mechanism of selective adsorption is strongly required in order to maximize the separation performance of membranes made of these materials so that it may applicable to hemodialysis therapy for end stage renal disease (ESRD) patients. For the achievement of above research goal, several investigations have been performed. Analysis of the obtained data leads to several conclusions as discussed below. Monodisperse polystyrene seed latex particles were synthesized with the size crosslinking agent to make the latex particles more hydrodynamically stable. The conversion of monomer to polymer was over 95% and seed latex particles are spherical in shape and have smooth surface. From these small seeds, bigger latex particles as large as 800nm were synthesized using a semi-continuous seeded emulsion polymerization process. The particles with a mean diameter less than 500nm are very spherical in shape and highly uniform in particle size distribution. However, as the particles grow larger than 500nm in mean diameter, they became non-spherical with an uneven surface. This irregularity of particle surface can be attributed to the non-homogeneous monomer ranges between 126.5 and 216.3 nm. Divinylbenzene (DVB) was used as a 115

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116 swelling into the shell of a growing polymer, which can be controlled by the factors such as temperature, agitation speed, initiator feeding rate, and surfactant amount required to stabilize the colloidal system. It was determrfactant to monomer ratio decic acid (AA) monomers were introrticle surfaces more hydro to ting of a orm ration is seen between 3002 0 cm-0cm-1 -1 0.3 ined that as the su reased, the mean particle diameter increased. Methyl methacrylate (MMA) and acryl duced to make the latex pa philic than bare polystyrene particles. The prepared core shell latex particles weremade of PMMA 100, PMMA 90 PAA 10, and PMMA 75 PAA 25 PMMA 100 is the PMMA homopolymer shell on a PS core. PMMA 90 PAA 10 shell is a copolymer with a PMMAPAA ratio of 90% to 10% by weight. PMMA 75 PAA 25 shell is a copolymer consisPMMA to PAA ratio of 75% to 25% by weight. The particle size of these core-shell particles were prepared to be about 370nm in mean diameter. The successful synthesis of latex particles was confirmed using Fourier TransfInfrared spectroscopy (FTIR). The C-H aromatic stretching vib cm -1 and 3103 cm -1 for PS latex particles. The C-H asymmetrical stretching andsymmetrical stretching vibration peaks of CH 2 for all latex particles is seen at 2901 and 2850 cm -1 respectively. There is a carbonyl (C=O) characteristic band at 173wavenumber for PS/PMMA core-shell latex particles. The broad OH group peak at 3400 cm 100 can be seen in the spectroscopy of the particle with PMMA/PAA shell (PS/PMMAPAA and PS/PMMAPAA). The peak intensity of OH group of PS/PMMAPAA was larger than that of PS/PMMAPAA relatively due to more AAmonomer content in PS/PMMAPAA than in PS/PMMAPAA. In the zeta potential measurements, polystyrene (PS) latex particles had negative values between -29.1 mV and -59.9 mV in the phosphate buffer (PB) and between -2 90 10 75 25 75 25 90 10 75 25 90 10

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117 mV and -27.8 mV in the phosphate buffered saline (PBS) at 25 o C and pH 2.1-7.8. These negative zeta potential values for PS latex particles are due to ionization of the sulfate groups (-OSO 3 ) originated from the initiator. Zeta potential values of the PS/PMMA core shell latex particles were also negative, between -28 mV and -50.5 mV in PB and between -14.3 mV and -18.6 mV in PBS at pH 2.1-7.8. Negative values are alsogenerated by dissociation of sulfate groups on a particle surface. The zeta potential valueof PS/PMMAPAA and PS/PMMAPAA were of negative values between -36.and -67.8 mV in PB medium and -14.7 mV and -19.3 mV in PBS medium at 25 100 s 7 mV MMA75PAA25 and PS/PM otein g the l change of BSA at each w 90 10 75 25 o C. For PS/PMMAPAA core shell particles, zeta potential values were between -29.1 mV and -52.0 mV in PB media and between -11.5 mV and -21.0 mV in PBS media. Sulfate groups contributed to the negative zeta potential values in PS/P 75 25 MA 90 PAA 10 core shell particles. Carboxylic groups from polyacrylic acid also contribute to the negative surface charge for the latex particles at greater than pH 7.0, however, the degree of their contribution was not detectable from the zeta potential measurements. The absolute zeta potential values of the synthesized latex particles in PBwere higher than that in PBS. This is because of the high concentration of sodium chloride electrolyte in PBS that compress electrical double layer thickness. The bicinchoninic acid (BCA) assay technique was used to determine the pradsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usinnonlinear regression method. The hydrophobic interaction between PS latex particle and BSA protein was dominant for the adsorption process. Conformationa pH also affected the adsorption amount, which was highest at pH 4.8 because of loionic repulsion making compact conformation of BSA. The next larger amount of BSA

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118 adsorbed onto PS was at pH 3.2. There were two main forces affecting the amount of adsorption at acidic pH values. One is ionic attraction between negative latex particle apositive protein. The other is lateral repulsion between positive proteins. The smallest amount of BSA adsorption was at pH 7.4 where charge repulsion between negative particles and proteins is present at this pH. Lateral repulsion between proteins wadominant for protein adsorption even though there always exists the hydrophobic interaction between solid latex particle and protein. The charge repulsion was reduced by the high concentration of electrolytes in the PBS medium. The BSA adsorption process on PS/PMMA core shell particles was very similar to the case of PS latex particle where hydrophobic interaction between latex particle and protein was dominant. However tadsorbed BSA amount was less than that of PS in PBS. The adsorbed amount of BSPS/PMMA nd s he A on an ed n. The main adsor 90 PAA 10 and PS/PMMA 75 PAA 25 core shell particles was higher at pH 3.2 thpH 4.8 and 7.4. The adsorbed BSA amount became higher at pH 3.2 and pH 4.8 as thehydrophilicity was increased by the higher PAA amount. On the contrary, the adsorbamount of BSA was dramatically decreased at pH 7.4. Carboxylic acid groups as well as the sulfate groups are distributed on the surface of PS/PMMA 90 PAA 10 and PS/PMMA 75 PAA 25 core shell particles and largely affect the BSA adsorptio ption interaction forces at pH 3.2 was generated by hydrogen bonding as well as charge attraction between latex particle and protein even when the conformational changeand charge repulsion between proteins reduce the adsorption of protein at this pH. The adsorbed BSA amount was also increased as the amount of PAA increases. However, the adsorption of BSA was minimal (less than 0.7 mg/m 2 ) at pH 7.4, where the carboxylic groups are ionized leading to more enhancement of charge repulsion by ionization of the

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119 carboxylic groups (COO ). Because of this, the PS/PMMA-PAA latex particles have amuch lower protein adsorption. There were steep initial slopes for adsorption isotherms of in s there M r e es o 2 M on latex particlesall isotherms, indicating a high affinity type adsorption. The complete plateau regionwere not seen for all isotherms, indicating that 2 M proteins were not saturated and are still available sites for 2 M adsorption on latex particles. The initial slope of 2isotherms for PS was steeper than for the other latex particles, PS/PMMA 100 PS/PMMA 90 PAA 10 and PS/PMMA 75 PAA 25 The layer thickness () of BSA proteins on synthesized latex particles in phosphate buffer (PB) was in the range of 27 81 at 37C and pH 4.8. Only the values at pH 4.8, the isoelectric point (IEP) of BSA, are important because intraand inter-molecular electrostatic repulsion of the protein molecules is minimized at the IEP. Compared to the hydrodynamic dimensions of BSA, 140 40 40 these values of the adsorbed layethickness indicates that the BSA molecules adsorb by the side-on (40) and end-on mod(140). Consideration of the multi-layering is unnecessary because the adsorbed thickness of BSA do not exceed 140 The Gibbs free energy of o 3 2 M was more negative in phosphate buffer (PB) than that of BSA in physiological condition (37C and pH 7.4), indicating that o 2 M moleculare more favorably adsorbed on the latex particles than BSA, and that the affinity of 2 M was much larger than BSA. This may be explained by the rate of diffusion and accessibility of 2 M molecules being easier and faster than that for BSA because of smaller size (or weight) and spherical shape of 2 M molecules. It may also be possible texplain that more hydrophobic forces (less electrostatic repulsion) contributes to

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120 adsorption of 2 M than to that of BSA because the IEP of 2 M (5.7) is closer to physiological condition (pH 7.4) than that of BSA (pH 4.8). The selective adsorption of 2M min 2M larger than in BSA adsorption, indicating that fa an that between BSA and latex particles because IEP (ch as Other olecules over BSA molecules can be enhanced by developing more hydrophilic PS/PMMA 90 PAA 25 and PS/PMMA 75 PAA 25 core shell latex particles The adsorption kinetics of proteins on latex particles was expressed as a second-order model. The plateau region for 2 M was reached earlier than BSA on all latex particles and the K values (equilibrium rate constant of 2nd order adsorption) adsorption process on latex particles were much ster adsorption rate of 2 M molecules than that of the BSA molecules. There are several explanations for these phenomena. First, the smaller size of 2 M molecules canmore easily diffuse and approach to the latex substrate. Second, enhanced hydrophilic domain on the latex particle surface by sulfate groups (SO 4 ) and ionized carboxylic groups (COO ) is not favorable for larger BSA molecules but is for smaller 2 M to be anchored. Third, the conformational change by intraand inter-molecular repulsion would be faster in 2 M due to lower molecular weight. Finally, the ionic repulsionbetween 2 M and latex particles is less th 5.7) of 2 M is closer to the tested condition (pH 7.4) than IEP (4.8) of BSA. In the biocompatibility test, the number of red blood cells (RBCs) ruptured by PS latex particles ranged from 30% to 91% as the solid content of PS increased from 0.5% to 5.0%, respectively. However, hemolysis of the other core shell latex particles, suPS/PMMA 100 PS/PMMA 90 PAA 10 and PS/PMMA 75 PAA 25 were less than 3.5%. ThePS/PMMA 90 PAA 10 particles at all applied solid concentrations had less than 0.2% hemolysis and was the most blood compatible among the prepared latex particles.

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121 core shell particles such as PS/PMMA 100 and PS/PMMA 75 PAA 25 also had excellecompatibility with low RBC breakage. 5.2 Conclusions The major conclusions from this study include: nt blood microglobulin (2M) over that of the bovine serum albumin (BSA) at pH 7.4, was achieved by controlling the PMMA and PAA ratio, to optimize e particles posses the most desirable requirements for use in a human dialysis ns icles olecule Using engineered core shell latex particles, selective adsorption of 2 physiological condition, was demonstrated. The rate of adsorption of the 2 M was also much higher than that of BSA. This hydrophobic/hydrophilic microdomain size. The core shell particles also exhibited excellent biocompatibility with blood. Thesmembrane. 5.3 Recommendation for Future Work With the results and conclusions obtained through this study, several suggestiomay be given for future work as follows: Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition Preparation of latex particles with optimum hydrophobic/ hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules. Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers. Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF). Application for hemodialysis as well as other technologies such as a water remediation.

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LIST OF REFERENC ES usible draBaker Sons, Bangs, L. B. (1987). Un. Bang-Baptista, R. P., Santos, A. M., Fedorov, A., Martinho, J. M. G., Pichot, C., Elaissari, A., yl methacrylate) latex particles. Journal of BardiNephrology Dialysis Transplantation 1, 151-154. Beckeal structure of beta 2-microglobulin. Proceedings of the National Academy of Sciences of the United States of America Bellotti, V., Stoppini, M., Mangione, P., Sunde, M., Robinson, C., Asti, L., Brancaccio, state myloid fibrils. European Journal of Biochemistry 258, 61-67. Berge, A., Ellingsen, T., Skyeltorp, A. T., Ugelstad, J. (1990). Scientific methods for the study of polymer colloids and their applications, Kluwer Academic, Dordrecht. Bernhard, W. F., Lafarge, C. G., Liss, R. H., Szycher, M., Berger, R. L., and Poirier, V. (1978). Appraisal of blood trauma and blood prosthetic interface during left ventricular bypass in calf and humans. Annals of Thoracic Surgery 26, 427-437. Abel, J. J., Rowntree, L. G., and Turner, B. B. (1990). On the removal of diffsubstances from the circulating blood by means of dialysis (reprinted from thetransactions of the association of American physicians, 1913). Transfusion Science11, 164-165. Ande, J. D. (1985). Surface and interfacial aspects of biomedical polymer, Plenum, New York. Argiles, A. (1996). Beta 2-microglobulin amyloidosis. Nephrology 2, 373-386. R. W. (2004). Membrane technology and applications, 2 nd Ed. John Wiley &New York. iform latex particles. In Technical Bulletin s, L. B. (1988). Latex agglutination tests. American Clinical Laboratoty News 7, 2026. Cabral, J. M. S., and Taipa, M. A. (2003). Activity, conformation and dynamics of cutinase adsorbed on poly(meth Biotechnology 102, 241-249. n, T., Zingraff, J., Kuntz, D., Drueke, T. (1986). Dialysis related amyloidosis. r, J. W., and Reeke, G. N. (1985). 3 Dimension 82, 4225-4229. D., and Ferri, G. (1998). Beta 2-microglobulin can be refolded into a nativefrom ex vivo a 122

PAGE 138

123 Bhat, A. A., and Pangarkar, V. G. (2000). Methanol selective membranes for the pervaporative separation of methanol-toluene mixtures. Journal of Membrane Science 167, 187-201. Bhutto, A. A., Vesely, D., and Gabrys, B. J. (2003). Miscibility and interactions in polystyrene and sodium sulfonated polethyl ether) PVME blends. Part II. FT. Bosch, T., Lennertz, A., Kordes, B., Samtelben, W. (1999). Low density lipoprotein linical esperience from a single center. Therapeutic Apheresis 3, 209-odialysis la. New England Brink, L. E. S., Elbers, S. J. G., Robbertsen, T., and Both, P. (1993). The anti-fouling Broadthe branes formed from poly (acrylonitrile-Browumin. Federation Proceedings Browidelimination of interfering dubstances. Analytical Biochemistry Bryja ystyrene with poly(vinyl m IR. Polymer 44, 6627-6631 Blanco, J. F., Nguyen, Q. T., and Schaetzel, P. (2001). Novel hydrophilic membranematerials: sulfonated polyethersulfone cardo. Journal of Membrane Science 186267-279. hemoperfusion by direct adsorption of lipoproteins from whole blood (DALI apheresis): c 213. Bowry, S. K. (2002). Dialysis membranes today. International Journal of Artificial Organs 25, 447-460. Brescia, M. J., Cimino, J. E., Appel, K., and Hurwich, B. J. (1966). Chronic hemusing venipuncture and a surgically created arteriovenous fistu Journal of Medicine 275, 1089-&. action of polymers preadsorbed on ultrafiltration and microfiltration membranes. Journal of Membrane Science 76, 281-291. head, K. W., and Tresco, P. A. (1998). Effects of fabrication conditions on structure and function of mem vinylchloride). Journal of Membrane Science 147, 235-245. n, J. R. (1976). Structural origins of mammalian alb 35, 2141-2144. n, R. E., Jarvis, K. L., and Hyland, K. J. (1989). Protein measurement using bicinchoninic ac 180, 136-139. k, M., Hodge, H., and Dach, B. (1998). Modification of porous polyacrylonitrile membrane. Angewandte Makromolekulare Chemie 260, 25-29. Burns, D. B., and Zydney, A. L. (2000). Buffer effects on the zeta potential of ultrafiltration membranes. Journal of Membrane Science 172, 39-48. Carllister, W. D. (1999). Materials science and engineering: an introduction, 5 th Ed, JohnWiley & Sons, New York.

PAGE 139

124 Carter, D. C., and Ho, J. X. (1994). Structure of serum albumin. In advances in prochemistry, Vol. 45, 153-203. tein Casadevall, N., and Rossert, J. (2005). Importance of biologic follow-ons: Experience Castin Nose, Y. (1976). Microemboli free blood detoxification utilizing plasma filtration. Transactions Cha, Y. J., and Choe, S. (1995). Characterization of crosslinked polystyrene beads and their composite in SBR matrix. Journal of Applied Polymer Science 58, 147-157. Chenolling 1063. ite ogy. Journal of Applied Polymer Science 45, 487-499. g Chen, Y. C., Dimonie, V. L., Shaffer, O. L., and Elaasser, M. S. (1993). Development of c Chern, C. S., Lee, C. K.,Chern, C. S., and Poehlein, G. W. (1990). Polymerization in nonuniform latex particles Part Cheung, A. K., Agodoa, L. Y., Daugirdas, J. T., Depner, T. A., Gotch, F. A., Greene, T., f the American Society of Nephrology 10, 117-127. Chiu,ative protein foreign surface interactions .4. Calorimetric and microelectrophoretic study of l Organs 22, 498-513. with EPO. Best Practice & Research Clinical Haematology 18, 381-387. o, F., Scheucher, K., Malchesky, P. S., Koshino, I., and American Society for Artificial Internal Organs 22, 637-645. Y. C., Dimonie, V., and Elaasser, M. S. (1991a). Interfacial phenomena contrparticle morphology of composite latexes. Journal of Applied Polymer Science 42, 1049Chen, Y. C., Dimonie, V., and Elaasser, M. S. (1992a). Role of surfactant in composlatex particle morphol Chen, Y. C., Dimonie, V., and Elasser, M. S. (1991b). Effect of interfacial phenomena on the development of particle morphology in a polymer latex system. Macromolecules 24, 3779-3787. Chen, Y. C., Dimonie, V., and Elasser, M. S. (1992b). Theoretical aspects of developinlatex particle morphology. Pure and Applied Chemistry 64, 1691-1696. morphology in latex particlesthe interplay between thermodynamic and kinetiparameters. Polymer International 30, 185-194. and Chang, C. J. (2003). Synthesis and characterization of amphoteric latex particles. Colloid and Polymer Science 281, 1092-1098. .2. kinetics of 2 phase emulsion polymerization. Journal of Polymer Sciencea-Polymer Chemistry 28, 3055-3071. Levin, N. W., Leypoldt, J. K., and Beck, G. J. (1999). Effects of hemodialyzer reuse on clearances of urea and beta(2)-microglobulin. Journal o T. H., Nyilas, E., and Lederman, D. M. (1976). Thermodynamics of n human fibrinogen sorption onto glass and LTI-carbon. Transactions American Society for Artificial Interna

PAGE 140

125 Chun, K. Y., and Stroeve, P. (2002). Protein transport in nanoporous membranes modified with self-assembled monolayers of functionalized thiols. Langmuir 18, 4653-4658. Cousid microbeads possessing amine functionality. Journal of Applied Polymer Science Cowgem perfusion with or without dialysis in normal and uremic dogs. Abstr. Eur. Renal Assoc-Eur. Dial. Cristol, J. P., Canaud, B., Rabesandratana, H., Gaillard, I., Serre, A., and Mion, C. l er synthesis and characterization. Macromolecules 36, 2198-2205. de Fr, G., Ronco, etta, C. (2000). Hemodiafiltration with online Delaculation of the latex particle morphology. Journal of Polymer Science Part a-Polymer Depp. Blood material interactions at the Dhondt, A., Vanholder, R., Van Biesen, W., and Lameire, N. (2000). The removal of Druek and amyloidosis. Nephrology Dialysis Duga he United States of America-Biological Sciences Elaiss98). o nce 202, 251-260. n, P., and Smith, P. (1994). Synthesis and Characterization of styrene base 54, 1631-1641. ill, L. D., Francey, T. (2001). Biocompatibility of adsorptive h Transplant Assoc, 292. (1994). Enhancement of reactive oxygen species production and cell surface markers expression due to hemodialysis. Nephrology Dialysis Transplantation 9, 389-394. Dahman, Y., Puskas, J. E., Margaritis, A., Merali, Z., and Cunningham, M. (2003). Novethymine-functionalized polystyrenes for applications in biotechnology. Polym ancisco, A. L. M., Ghezzi, P. M., Brendolan, A., Fiorini, F., La GrecaC., Arias, M., Gervasio, R., and T regeneration of the ultrafiltrate. Kidney International 58, S66-S71. al, J. C., Urzay, R., Zamora, A., Forcada, J., and Asua, J. M. (1990). Sim Chemistry 28, 1011-1031. isch, R., Storr, M., Buck, R., and Gohl, H. (1998) surfaces of membranes in medical applications. Separation and Purification Technology 14, 241-254. uremic toxins. Kidney International 58, S47-S59. e, T. B. (2000). Beta(2)-microglobulin Transplantation 15, 17-24. iczyk, A., Law, S. W., and Dennison, O. E. (1982). Nucleotide sequence and theencoded amino acids of human serum albumin messenger RNA. Proceedings of tNational Academy of Sciences of the 79, 71-75. ari, A., Chauvet, J. P., Halle, M. A., Decavallas, O., Pichot, C., and Cros, P. (19Effect of charge nature on the adsorption of single-stranded DNA fragments ontlatex particles. Journal of Colloid and Interface Scie

PAGE 141

126 Evans, D. F., Wennerstron, H. (1999). The colloidal domain: where physics, chembiology, and technology meet, 2nd Ed, Wiley-VCH, New York. istry, Fair, B. D., and Jamieson, A. M. (1980). Studies of protein adsorption on polystyrene Fehske, K. J., Muller, W. E., and Wollert, U. (1981). The location of drug binding sites in human serum albumin. Biochemical Pharmacology 30, 687-692. Floegonal 59, S164-S171. a, R., Ganachaud, F., Mouterd latex particles of different aminated GandJ. J., and Salovey, R. (1990). Model filled polymers .2. Stability of polystyrene beads in a polystyrene Gebh Gejyo dosisintensive removal of beta2-microglobulin with 17, 240-243. evaluation of an adsorbent column (BM-O1) of direct hemoperfusion type for ka, ., Shirahama, T., Cohen, A. S., and Schmid, K. (1985). A new form of amyloid protein associated with chronic hemodialysis was identified Gilbert, R. G. (1995). Emulsion polymerization: a mechanistic approach, Academic Press, London. Godjelized for immobilization of glucose oxidase. Journal of Membrane Science 152, 235-240. latex surfaces. Journal of Colloid and Interface Science 77, 525-534. e, J., and Ketteler, M. (2001). Beta(2)-microglobulin derived amyloidosis: An update. Kidney Internati Furuyoshi, S., Kobayashi, A., Tamai, N., Yasuda, A., Takada, S., Tani, N., NakazawMimura, H., Gejyo, F., Arakava, M. (1991). Blood Purification 9, 9. e, G., Delair, T., Elaissari, A., and Pichot, C. (1995). Preparation and characterization of cationic polystyrene surface charges. Polymers for Advanced Technologies 6, 480-488. hi, K., Park, M., Sun, L., Zou, D., Li, C. X., Lee, Y. D., Aklonis, matrix. Journal of Polymer Science Part B-Polymer Physics 28, 2707-2714. ardt, J. E., and Fuerstenau, D. W. (1983). Adsorption of polyacrylic acid at oxidewater interfaces. Colloids and Surfaces 7, 221-231. F., Homma, N., Hasegawa, S., and Arakawa, M. (1993). A new therapeuticapproach to dialysis amyloiadsorbent column. Artificial Organs Gejyo, F., Teramura, T., Ei, I., Arakawa, M., Nakazawa, R., Azuma, N., Suzuki, M., Furuyoshi, S., Nankou, T., Takata, S., and Yasuda, A. (1995). Long-term clinical beta(2)-microglobulin on the treatment of dialysis-related amyloidosis. Artificial Organs 19, 1222-1226. Gejyo, F., Yamada, T., Odani, S., Nakagawa, Y., Arakawa, M., Kunitomo, T., KataoH., Suzuki, M., Hirasawa, Y as beta2 microglobulin. Biochemical and Biophysical Research Communications129, 701-706. vargova, T., Konsulov, V., and Dimov, A. (1999). Preparation of an ultrafiltration membrane from the copolymer of acrylonitrile-glycidylmethacrylate uti

PAGE 142

127 Godjevargova, T., Konsulov, V., Dimov, A., and Vasileva, N. (2000). Behavior of glucose oxidase immobilized on ultrafiltration membranes obtain ed by copolymerizing acrylonitrile and N-vinylimidazol. Journal of Membrane Science Godjevargova, Z., Dimov, A., and Petrov, S. (1992). Chemical modification of dialysis 43. bin ial. Thrombosis Research 20, 543-Grancer particle in Guiot, CRC, Boca Guiveer Haagical significance of Nephrology Dialysis Transplantation15Hancolysulfone block copolymer. Hanse. rnal Hariss, J. M. (1992). Poly(ethylene glycol) chemistry, Plemum, New york. HarkiHe, X. M., and Carter, D. C. (1992). Atomic structure and chemistry of human serum albumin. Nature 358, 209-215. Hergeh, H. J., Eichhorn, F., Schlenker, S., Schmutzler, K., and Steinau, U. J. (1989). Polymerizations in the presence of seeds .5. Core shell structure of 2 172, 279-285. membrane based on poly(scrylonitrile methylmethacrylate sodium vinylsulphonate). Journal of Applied Polymer Science 44, 2139-21 Goosen, M. F. A., Sefton, M. V., and Hatton, M. W. C. (1980). Inactivation of thromby anti-thrombin III on a heparinized biomater 554. io, M. R., and Williams, D. J. (1970). Morphology of monomer polym styrene emulsion polymerization. Journal of Polymer Science Part A1-Polymer Chemistry 8, 2617-&. P., Couvreur, P. (1986). Polymeric nanoparticles and microspheres Raton (FL). r, M. D., Black, P., Tam, C. M., and Deslandes, Y. (1993). Functionalized polysulfone membranes by heterogeneous lithiation. Journal of Applied Polym Science 48, 1597-1606. -Weber, M., Cohen, G., and Horl, W. H. (2000). Clin granulocyte-inhibiting proteins. 15-16. ock, L. F., Fagan, S. M., and Ziolo, M. S. (2000). Hydrophilic, semipermeable membranes fabricated with poly(ethylene oxide)-p Biomaterials 21, 725-733. n, F. K., and Ugelstad, J. (1979). Particle nucleation in emulsion polymerization .2Nucleation in emulsifier free systems investigated by seed polymerization. Jouof Polymer Science Part a-Polymer Chemistry 17, 3033-3045. ns, W. D. (1947). A general theory of the mechanism of emulsion polymerization. Journal of the American Chemical Society 69, 1428-&. th, W. D., Bittric stage emulsion polymers. Polymer 30, 1913-1917.

PAGE 143

128 Hicke, H. G., Lehmann, I., Malsch, G., Ulbricht, M., and Becker, M. (2002). Preparand characterization of a novel solvent-resistant and autoclavable polymmembrane. Journal of Membrane Science 198, 187-196. ation er Higuchi, A., Ishida, Y., and Nakagawa, T. (1993). Surface modified polysulfone Higuchi, A., Mishima, S., and Nakagawa, T. (1991). Separation of proteins by surface Higuchi, A., and Nakagawa, T. (1990). Surface modified polysulfone hollow fibers .3. fibers having a hydroxide group. Journal of Applied Polymer Science 41, 1973-Higucoon, B. O., Sakurai, M., Hara, M., Sumita, M., Sugawara, S., and Shirai, T. (2003). Serum protein adsorption and platelet adhesion on Hoenich, N. A., and Katopodis, K. P. (2002). Clinical characterization of a new 3853-Homma, N., Gejyo, F., Isemura, M., and Arakawa, M. (1989). Collagen binding affinity sis. nes. Hunter, R. J. (1981). Zeta potential in colloid science: principles and applications, Huys. E. (1998). Hypertension and accelerated atherosclerosis in endstage renal disease. Journal of groups. Journal of Biomedical Materials Research 24, 1069-1077. Izquierdo, M. P. S., Martin-Molina, A., Ramos, J., Rus, A., Borque, L., Forcada, J., and Galisteo-Gonzalez, F. (2004). Amino, chloromethyl and acetal-functionalized latex Jaekel, U., and Vereecken, H. (2002). Transport of solutes undergoing a Freundlich type nonlinear and nonequilibrium adsorption process. Physical Review E 65. membranesseparation of mixed proteins and optical resolution of tryptophan. Desalination 90, 127-136. modified polysulfone membranes. Journal of Membrane Science 57, 175-185. 1979. hi, A., Sugiyama, K., Y pluronic (TM)-adsorbed polysulfone membranes. Biomaterials 24, 3235-3245. polymeric membrane for use in renal replacement therapy. Biomaterials 233858. of beta2 microglobulin, a preprotein of hemodialysis associated amyloidoNephron 53, 37-40. Hsieh, H. P., Liu, P. K. T., and Dillman, T. R. (1991). Microporous ceramic membraPolymer Journal 23, 407-415. Academic Press, London. mans, K., Lins, R. L., Daelemans, R., Zachee, P., and De Broe, M Nephrology 11, 185-195. Ishihara, K., Aragaki, R., Ueda, T., Watenabe, A., and Nakabayashi, N. (1990). Reducedthrombogenicity of polymers having phospholipid polar particles for immunoassays: a comparative study. Journal of Immunological Methods 287, 159-167.

PAGE 144

129 James, R. O. (1985). Polymer colloids, Elsevier, Amsterdam. iec, M. and Madey, R. (1988). Physical adsorption on hete Jaronrogenous solids. Elsevier, New York. Jons, 155, 79-99. ane Science 246, 67-76. Kauzes in Protein Chemistry 14, 1-63. 2. Khan, A. R.,ith an 405. t a-, 3187-3199. ers. Desalination 70, 229-249. lf-ilic poly(ethylene glycol) derivative-modified polysulfone membranes. Biomaterials 26, 2867-2875. Kim, odified with poly(ethylene glycol) derivatives. Korean Journal of Chemical Engineering 20, 1158-1165. Ko, Mn of ins on ultrafiltration membranes. Journal of Membrane Science 76, 101-120. Kolar S., Ries, P., and McDonald, C. J. (1999). Porous latex composite membranes: fabrication and properties. Journal of Membrane Science Jung, B. S., Yoon, J. K., Kim, B., and Rhee, H. W. (2005). Effect of crystallization andannealing on polyacrylonitrile membranes for ultrafiltration. Journal of Membr mann, W. (1959). Some factors in the interpretation of protein denaturation. Advanc Keusch, P., and Williams, D. J. (1973). Equilibrium encapsulation of polystyrene latex particles. Journal of Polymer Science Part A. Polymer Chemistry 11, 143-16 Baker, B. M., Ghosh, P., Biddison, W. E., and Wiley, D. C. (2000). The structure and stability of an HLA-A*0201/octameric tax peptide complex wempty conserved peptide-N-terminal binding site. Journal of Immunology 164, 6398-6 Kim, J. H., Chainey, M., Elaasser, M. S., and Vanderhoff, J. W. (1989). Preparation of highly sulfonated polystyrene model colloids. Journal of Polymer Science ParPolymer Chemistry 27 Kim, K. J., Fane, A. G., and Fell, C. J. D. (1988). The performance of ultrafiltration membranes pretreated by polym Kim, Y. W., Ahn, W. S., Kim, J. J., and Kim, Y. H. (2005). In situ fabrication of setransformable and hydroph Y. W., Kim, J. J., and Kim, Y. H. (2003). Surface characterization of biocompatiblepolysulfone membranes m K., Pellegrino, J. J., Nassimbene, R., and Marko, P. (1993). Characterizatiothe adsorption fouling layer using globular prote z, B. N., and Jermakowiczbartkowiak, D. (1995). Hyper-crosslinked sorbents for hemoperfusion. Angewandte Makromolekulare Chemie 227, 57-68.

PAGE 145

130 Kolarz, B. N., Trochimczuk, A., Wojaczynska, M., Bartkowia k, D. (1989). Polymer sorbents for hemoperfusion. I. Preparation, structure, and sorption properties of tion nterface Kond silica particles. Journal of Colloid and Interface Science 143, Korshikhonova, L. A., Ryabov, A. V., Kresh an aqueous solution. Biochemistry 8, 8-&. 46-350. composed of Lee, Cymer latex. Effect of heating on the morphology and physical properties of PMMA/PS core-shell composite Lee, C Lee, Cn the poly(methyl methacrylate) Lee, C. F., Chiu, W. Y., and Chern, Y. C. (1995). Kinetic study on the poly(methyl methacrylate) seeded soapless emulsion polymerization of Styrene .2. kinetic-model. Journal of Applied Polymer Science 57, 591-603. chemically modified polymer sorbents for hemoperfusion. Polimery Medycynie 19, 29-35. Kondo, A., and Higashitani, K. (1992). Adsorption of model proteins with wide variain molecular properties on colloidal particles. Journal of Colloid and I Science 150, 344-351. o, A., Oku, S., and Higashitani, K. (1991). Structural changes in protein molecules adsorbed on ultrafine 214-221. ak, V. V., Leikin, Y. A., Neronov, A. Y., T Kabanov, O. V., Gorchakov, V. D., Evseev, N. G. (1978). Sorbents compatible with blood for extraction of exogenous and endogenous toxins, Demande. Kraghhansen, U. (1981). Molecular aspects of ligand binding to serum albumin. Pharmacological Reviews 33, 17-53. eck, G. C., and Klotz, I. M. (1969). Thermodynamics of transfer of amides fromapolar to an Kurbel, S., Radic, R., Kotromanovic, Z., Puseljic, Z., and Kratofil, B. (2003). A calciumhomeostasis model: orchestration of fast acting PTH and calcitonin with slow calcitriol. Medical Hypotheses 61, 3 Kurisawa, M., Terano, M., and Yui, N. (1995). Doublestimuli responsive degradablehydrogels for drug delivery Interpenetrating polymer networks oligopeptide-terminated poly(ethylene glycol) and dextran. Macromolecular Rapid Communications 16, 663-666. F. (2000). The properties of core-shell composite pol latex and the polymer blends. Polymer 41, 1337-1344. F. (2002). The morphology of composite polymer particles produced by multistage soapless seeded emulsion polymerization. Colloid and Polymer Science280, 116-123. F., and Chiu, W. Y. (1995). Kinetic study o seeded soapless emulsion polymerization of dtyrene .1. experimental investigation. Journal of Applied Polymer Science 56, 1263-1274.

PAGE 146

131 Lee, C. F., Young, T. H., Huang, Y. H., and Chiu, W. Y. (2000). Synthesis and propeof polymer latex with carboxylic acid functional groups for immunological studiPolymer 41, 8565-8571. rties es. Lee, J. H., Yoon, J. Y., and Kim, W. S. (1998). Continuous separation of serum proteins Lee, S. H., and Ruckenstein, E. (1988). Adsorption of proteins onto polymeric surfaces of Legido-Quigley, C., Marlin, N., and Smith, N. W. (2004). Comparison of styrene-mns Lewin, S. (1974). Displacement of water and its control of biochemical reactions, Leypoldt, J. K., Cheung, A. K., Deeter, R. B., (1998). Effect of hemodialysis resue: Lin, W. C., Liu, T. Y., and Yang, M. C. (2004). Hemocompatibility of polyacrylonitrile als Lonn02). Beta(2)-microglobulin amyloidosis: effects of ultrapure dialysate and type of dialyzer membrane. Journal of the American Society LowrJ. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, Lu, M., Keskkula, H., and Paul, D. R. (1996). Thermodynamics of solubilization of functional copolymers in the grafted shell of core-shell impact modifiers .2. Luck,er, R. H. (1998). Analysis of plasma protein adsorption on polymeric nanoparticles with different surface using a stirred cell charged with carboxylated and sulfonated microspheres. Biomedical Chromatography 12, 330-334. different hydrophilicitiesa case study with bovine serum albumin. Journal of Colloid and Interface Science 125, 365-379. divinylbenzene based monoliths and vydac nano-liquid chromatography colufor protein analysis. Journal of Chromatography A 1030, 195-200. Academic Press, New York. dissociation between clearances of small large solutes, American Journal of Kidney Diseases 32, 295-301. Li, J., Guo, S. Y., and Li, X. N. (2005). Degradation kinetics of polystyrene and EPDM melts under ultrasonic irradiation. Polymer Degradation and Stability 89, 6-14 dialysis membrane immobilized with chitosan and heparin conjugate. Biomateri25, 1947-1957. emann, G., and Koch, K. M. (20 of Nephrology 13, S72-S77. y, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. 265-275. experimental. Polymer 37, 125-135. M., Paulke, B. R., Schroder, W., Blunk, T., and Mull characteristics. Journal of Biomedical Materials Research 39, 478-485.

PAGE 147

132 Luo, Y., Rong, M. Z., Zhang, M. Q., and Friedrich, K. (2004). Surface grafting onto SiC nanoparticles with glycidyl methacrylate in emulsion. Journal of Polymer Science Part a-Polymer Chemistr y 42, 3842-3852. ed by nsed Matter 16, S2167-S2175. Malchesky, P. S., Piatkiewicz, W., Varnes, W. G., Ondercin, L., and Nose, Y. (1978). Malmsten, M. (1998). Biopolymers at interface, Marcel Dekker, New York. McLation and reactions of enzymes and proteins on daolinite Journal of Physical Chemistry 58, 129-137. McDoMcMurry, J. (2004). Organic chemistry, Thompson-Brooks/Cole, Belmont. Menon, M. K., and Zydney, A. L. (1999). Effect of ion binding on protein transport embranes. Biotechnology and Bioengineering 63, 298-307. Min, T. I., Klein, A., Elaasser, M. S., and Vanderhoff, J. W. (1983). Morphology and Miraballes-Martinez, I., and Forcada, J. (2000). Synthesis of latex particles with surface Miraba, J. (2001). Synthesis of amino-functionalized latex particles by a multistep method. Journal of Polymer Science Part a-Polymer Chemistry 39, 2929-2936. Magalhaes, I., Pihan, J. C., and Falla, J. (2004). A latex agglutination test for the field determination of abnormal vitellogenin production in male fishes contaminatestrogen mimics. Journal of Physics-Conde Malchesky, P. S. (2004). Extracorpeal artificial organs, Elsevier Academic Press, San Diego. Sorbent membranes-device designs, evaluations and potential applications. Artificial Organs 2, 367-371. ren, A. D. (1954). The adsorp nald, C. (2003). Personal communications. Meier, P., von Fliedner, V., Markert, M., van Melle, G., Deppisch, R., and Wauters, J. P(2000). One-year immunological evaluation of chronic hemodialysis in end-stage renal disease patients. Blood Purification 18, 128-137. through ultrafiltration m Mikhalovsky, S. V. (1987). The organism detoxification with bioselective carbon sorbents. Artificial Organs 11, 504-504. Mikhalovsky, S. V. (1989). The detoxication with bioselective carbon sorbents. Biomaterials Artificial Cells and Artificial Organs 17, 157-160. grafting in polybutylacrylate-polystyrene core-shell emulsion polymerization. Journal of Polymer Science Part a-Polymer Chemistry 21, 2845-2861. amino groups. Journal of Polymer Science Part A. Polymer Chemistry 38, 4230-4237. alles-Martinez, I., Martin-Molina, A., Galisteo-Gonzalez, F., and Forcad

PAGE 148

133 Mok, S., Worsfold, D. J., Fouda, A., and Matsuura, T. (1994). Surface modification of polyethersulfone hollow fiber membranes by gamma ray irradiation. Journal of Applied Polymer Science 51, 193-199. Musale, D. A., and Kulkarni, S. S. (1997). Relative rates of protein transmission througpoly(acrylonitrile) based ultrafiltration membranes. Journal of Membrane Science 136, 13-23. h iator-modified polystyrene particles. Langmuir 21, 2209-2217. Nabe, A., Staude, E., and Belfort, G. (1997). Surface modification of polysulfone mbrane Science 133, 57-72. NagaPreparation and applications of polymeric microspheres having active ester groups. Colloids and Surfaces a-53, 133-136. S., Yasuda, Nelliappan, V., ElAasser, M. S., Klein, A., Daniels, E. S., Roberts, J. E., and Pearson, R. Nesto Booten, K., and Tadros, T. F. (2005). ngmuir of llow fiber Niemts ls science. Angewandte Chemie-International Edition 40, 4128-4158. Musyanovych, A., and Adler, H. J. P. (2005). Grafting of amino functional monomer onto init Myers, D. (1999). Surfaces, interfaces, and colloids: princeples and applications, 2 nd EdWiley-VCH, New York. ultrafiltration membranes and fouling by BSA solutions. Journal of Me i, K., Ohashi, T., Kaneko, R., and Taniguchi, T. (1999). Physicochemical and Engineering Aspects 1 Nakazawa, R., Azuma, N., Suzuki, M., Nakatani, M., Nankou, T., Furuyoshi A., Takata, S., Tani, N., and Kobayashi, F. (1993). A new treatment for dialysis related amyloidosis with beta2-microglobulin adsorbent column. International Journal of Artificial Organs 16, 823-829. A. (1997). Effect of the core/shell latex particle interphase on the mechanical behavior of rubber-toughened poly(methyl methacrylate). Journal of Applied Polymer Science 65, 581-593. r, J., Esquena, J., Solans, C., Levecke, B., Emulsion polymerization of styrene and methyl methacrylate using a hydrophobically modified inulin and comparison with other surfactants. La21, 4837-4841. Nie, F. Q., Xu, Z. K., Ming, Y. Q., Kou, R. Q., Liu, Z. M., and Wang, S. Y. (2004). Preparation and characterization of polyacrylonitrile-based membranes: Effectsinternal coagulant on poly(acrylonitrile-co-maleic acid) ultrafiltration ho membranes. Desalination 160, 43-50. eyer, C. M. (2001). Nanoparticles, proteins, and nucleic acids: biotechnology meemateria Nilsson, J. L. (1990). Protein fouling of UF membranes causes and consequences. Journal of Membrane Science 52, 121-142.

PAGE 149

134 Nilsson, K. G. I. (1989). Preparation of nanoparticles conjugated with enzyme and antibody and their use in heterogeneous enzyme immunoassays. Journal of Immunological Methods 122, 273-277. Nir, S. (1977). Vanderwaals interactions between surfaces of biological interest. Progressin Surface Science 8, 1-58. Nissenson, A. R., Fine, R. N., Gentile, D. E. (1995). Clinical dialysis, 3rd Ed, Appleton NKF (2002). Kidney disease: Are you at increased risk for chronic kidney disease?. Norde, W. (1998). Biopolymers at interfaces: chap2. driving forces for protein adsorption Norde. (1992). Structure of adsorbed and desorbed proteins. Colloids and Surfaces 64, 87-93. Obrieis. ns 5, 77-77. Oh, JTechnology 27, 356-361. Okubmer Onen, es 28, 85-88. Ottewaracterisation of surface Ozacan & Lange, Connecticut. http://www.kidney.org/atoz/atozitem.cfm?id=134. at solid surface, Marcel Dekker, New York. W., and Favier, J. P n, T. F., Baxter, C. R., and Teschan, P. E. (1959). Prophylactic daily hemodialysTransactions American Society for Artificial Internal Orga Odian, G. (1991). Principles of polymerization, 3 rd Ed. John Wiley & Sons, New York. T., and Kim, J. H. (2000). Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres. Enzyme and Microbial o, M., and Nakagawa, T. (1992). Preparation of micron-size monodisperse polyparticles having highly crosslinked structures and vinyl groups by seeded polymerization of divinylbenzene using the dynamic swelling method. Colloid and Polymer Science 270, 853-858. F., Turkay, C., Meydan, A., Doknetas, H. S., Sumer, H., Hocaoglu, L.Icagasioglu, S., Bakici, M. Z. (1998). Prevalence of rheumatoid factor (RF) and anti-native-DNA antibodies (anti-n DNA) in different age subpopulations. Journal of Medical Scienc Oscik, J. (1982). Adsorption. John Wiley & Sons. New York. ill, R. H., and Shaw, J. N. (1967). Studies on preparation and characterisation of monodisperse polystyrene latices .2. electrophoretic ch groupings. Kolloid-Zeitschrift and Zeitschrift Fur Polymere 218, 34-&. r, M. (2003). Equilibrium and kinetic modelling of adsorption of phosphorus o calcined alunite. Adsorption-Journal of the International Adsorption Society 9, 125-132.

PAGE 150

135 Panichi, V., Bianchi, A. M., Andreini, B., Casarosa, L., Migliori, M., De Pietro, S., Taccola, D., Giovannini, L., and Palla, R. (1998). Biocompatibility evaluatiopolyamide hemofiltration. International n of Journal of Artificial Organs 21, 408-413. ymerization. Colloids and Surfaces a-Physicochemical and Engineering Aspects 191, 193-199. Park, M., Gandhi, K., Sun, L., Salovey, R., and Aklonis, J. J. (1990). Model filled polymers .3. rheological behavior of polystyrene containing cross-linked Pavin, D. L., Lampman, G. M., Kriz, G. S. (1996). Introduction to spectroscopy: A guide Percival, D. A. (1996). The measurement of hormones and bacterial antigens using rapid Peters, T. (1985). Serum albumin. Advances in Protein Chemistry 37, 161-245. Peulaerum albumin on sulfonated polystyrene model colloids .1. adsorption isotherms and Pierac ulfone) ultrafiltration membranes. Journal of Membrane Science 202, 1-16. Piskinbased on rials Science-Polymer Edition 5, 451-471. uid Zolla, L., Chervet, J. P., Cavusoglu, N., van Dorsselaer, A., and Huber, C. G. (2001). High-performance ic ndQuint hemodialysis. Transactions American Society for Artificial Internal Organs 6, 104-113. Park, J. G., Kim, J. W., and Suh, K. D. (2001). Chloromethyl functionalized polymer particles through seeded pol polystyrene beads. Polymer Engineering and Science 30, 1158-1164. for students of organic chemistry, 2 nd Ed. Harcourt Brace College, Fort Worth. particle-based immunoassays. Pure and Applied Chemistry 68, 1893-1895. J. M., and Delasnieves, F. J. (1993). Adsorption of monomeric bovine s effect of the surface charge density. Colloids and Surfaces a-Physicochemical and Engineering Aspects 77, 199-208. ci, J., Crivello, J. V., and Belfort, G. (2002). Increasing membrane permeability ofUV-modified poly(ether s E., Tuncel, A., Denizli, A., and Ayhan, H. (1994). Monosize microbeads polystyrene and their modified forms for some selected medical and biological applications. Journal of Biomate Premstaller, A., Oberacher, H., and Huber, C. G. (2000). High-performance liqchromatography-electrospray ionization mass spectrometry of singleand doublestranded nucleic acids using monolithic capillary columns. Analytical Chemistry 72, 4386-4393. Premstaller, A., Oberacher, H., Walcher, W., Timperio, A. M., liquid chromatography-electrospray ionization mass spectrometry using monolithcapillary columns for proteomic studies. Analytical Chemistry 73, 2390-+. Punam, F. W. (1984). The plasma proteins, 2 Ed, Academic Press, London. on, W., Dillard, D., and Scribner, B. H. (1960). Cannulation of blood vessels forprolonged

PAGE 151

136 Radomska-Galant, I., and Basinska, T. (2003). Poly(styrene/alpha-tert-butoxy-omegvinylbenzylpolyglycidol) microspheres for immunodiagnosticsprinciple of a novlatex test based on combined electrophoretic mobility and particle aggregation a-el measurements. Biomacromolecules 4, 1848-1855. Ramaperties of the discriminating layer. Journal of Membrane Science 231, 57-70. Ray, lective rvaporation. Journal of Membrane Science 154, 1-13. Reb, nonpolymerizable isophthalic acid derivatives as surfactants in emulsion Rembaum, A., Tokes, Z. A. (1988). Microspheres: medical and biological applications, ree Roe, ects of emulsion polymerization. Industrial and Engineering Chemistry 60, 20-&. Roe, neering Chemistry 60, 8-&. ith -microglobulin in hemodialysis. Blood Purification 19, 260-263. RoncRoselaar, S. E., Nazhat, N. B., Winyard, P. G., Jones, P., Cunningham, J., and Blake, D. Samtl6). Plasma therapy at klinikum grosshadern: a 15-year retrospective. Artificial Organs ge krishnan, S., McDonald, C. J., Prud'homme, R. K., and Carbeck, J. D. (2004). Latex composite membranes: structure and pro S. K., Sawant, S. B., Joshi, J. B., and Pangarkar, V. G. (1999). Methanol semembranes for separation of methanol-ethylene glycol mixtures by pe P., Margarit-Puri, K., Klapper, M., and Mullen, K. (2000). Polymerizable and polymerization. Macromolecules 33, 7718-7723. CRC, Baca Raton (FL). Riceevans, C. A., and Diplock, A. T. (1993). Current status of antioxidant therapy. FRadical Biology and Medicine 15, 77-96. C. P. (1968a). Surface chemistry asp C. P. (1968b). Surface chemistry relationships in emulsion polymerization. Industrial and Engi Ronco, C., Brendolan, A., Winchester, J. F., Golds, E., Clemmer, J., Polaschegg, H. D., Muller, T. E., La Greca, G., and Levin, N. W. (2001). First clinical experience wan adjunctive hemoperfusion device designed specifically to remove beta(2) o, C., Ghezzi, P. M., Hoenich, N. A., Delfino, P. G. (2001). Membranes and filters for hemodialysis: Database 2001, Karger, Basel. R. (1995). Detection of oxidants in uremic plasma by electron spin resonance spectroscopy. Kidney International 48, 199-206. eben, W., Blumenstein, M., Bosch, T., Lysaght, M. J., and Schmidt, B. (199 20, 408-413. Samtleben, W., Gurland, H. J., Lysaght, M. J., Winchester, J. F. (1996). Plasma exchanand hemoperfusion, Kluwer Academic, Dordrecht.

PAGE 152

137 Santos, R. M., and Forcada, J. (1997). Acetal-functionalized polymer particles useful fimmunoassays. Journal of Polymer Science Part A. Polymer Chemistry 35, 1605-1610. or Saper, M. A., Bjorkman, P. J., and Wiley, D. C. (1991). Refined structure of the human Sarobe, J., and Forcada, J. (1998). Synthesis of monodisperse polymer particles with Sarobe, J., Molina-Bolivar, J. A., Forcada, J., Galisteo, F., and Hidalgo-Alvarez, R. e Scharnagl, N., and Buschatz, H. (2001). Polyacrylonitrile (PAN) membranes for ultraSchmidt, E. (1972). Synthetic latex foam rubber and method of making same. US Patent Shimizu, N., Sugimoto, K., Tang, J. W., Nishi, T., Sato, I., Hiramoto, M., Aizawa, S., abe, H., and Handa, H. (2000). high-performance affinity beads for identifying drug receptors. Nature Biotechnology Shirahama, H., and Suzawa, T. (1985). Adsorption of bovine serum albumin onto styrene Shiranto 83-490. al of Medicine 21, 888-892. s ilure 25, 419-430. o, of protein using bicinchoninic acid. Analytical Biochemistry 150, 76-85. Smithon polymerization. Journal of Chemical Physics 16, 592-599. histocompatibility antigen HLA-A2 at 2.6 resolution. Journal of Molecular Biology 219, 277-319. chloromethyl functionality. Colloids and Surfaces a-Physicochemical and Engineering Aspects 135, 293-297. (1998). Functionalized monodisperse particles with chloromethyl groups for thcovalent coupling of proteins. Macromolecules 31, 4282-4287. and microfiltration. Desalination 139, 191-198. 3 673 133. Hatakeyama, M., Ohba, R., Hatori, H., Yoshikawa, T., Suzuki, F., Oomori, A., Tanaka, H., Kawaguchi, H., Watan 18, 877-881. acrylic acid copolymer latex. Colloid and Polymer Science 263, 141-146. hama, H., Suzuki, K., and Suzawa, T. (1989). Bovine hemoglobin adsorption opolymer lattices. Journal of Colloid and Interface Science 129, 4 Singer, J. M., and Plotz, C. M. (1956). Latex Fixation Test .1. application to the serologicdiagnosis of rheumatoid arthritis. American Journ Singh, N. P., Bansal, R., Thakur, A., Kohli, R., Bansal, R. C., and Agarwal, S. K. (2003).Effect of membrane composition on cytokine production and clinical symptomduring hemodialysis: a crossover study. Renal Fa Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., ProvenzanM. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985). Measurement W. V., and Ewart, R. H. (1948). Kinetics of emulsi

PAGE 153

138 Snyder, H. W., Cochran, S. K., Balint, J. P., Bertram, J. H., Mittelman, A., Guthrie, T. H.,and Jones, F. R. (1992). Experience with protein -immunoadsorption in treatmentresistan t adult immune thrombocytopenic purpura. Blood 79, 2237-2245. Solomon, T. W., Fryhle, C. B. (2000). Organic chemistry, 7th Ed. John Wiley & Sons, Song, Y. Q., Sheng, J., Wei, M., and Yuan, X. B. (2000). Surface modification of polysulfone membranes by low-temperature plasma-graft poly(ethylene glycol) Sriniv characterizations of polymer with thier antithrombogenic characteristics, Marcel Dekker, New York. Suzawovine serum albumin on synthetic polymer lattices. Journal of Colloid and Interface Science 78, 266-268. Suzawa, T., Shirahama, H., and Fujimoto, T. (1982). Adsorption of bovine serum albumin onto homo-polymer and co-polymer lattices. Journal of Colloid and Takenitness of 9, 10-14. 412. Tanfo New York. Tangpion of chitosan filmseffects of hydrophobicity on protein adsorption. Carbohydrate Trinh S. E. (2002). Crystal structure of monomeric human beta 2 microglobulin reveals clues to its Trotta, F., Drioli, E., Baggiani, C., and Lacopo, D. (2002). Molecular imprinted polymeric membrane for naringin recognition. Journal of Membrane Science 201, Soderquist, M. E., and Walton, A. G. (1980). Structural changes in proteins adsorbed on polymer surfaces. Journal of Colloid and Interface Science 75, 386-397. New York. onto polysulfone membranes. Journal of Applied Polymer Science 78, 979-985asan, S., Sawyer, P. N. (1971). Correlation of the surface charge a, T., and Murakami, T. (1980). Adsorption of b Interface Science 86, 144-150. aka, T., Itaya, Y., Tsuchiya, Y., Kobayashi, K., and Suzuki, H. (2001). Fbiocompatible high-flux hemodiafiltration for dialysis-related amyloidosis. BloodPurification 1 Tamai, H., Hasegawa, M., and Suzawa, T. (1989). Surface characterization of hydrophilic functional polymer latex particles. Journal of Applied Polymer Science 38, 403rd, C. (1967). Physical chemistry of macromolecules, John Wiley & Sons, asuthadol, V., Pongchaisirikul, N., and Hoven, V. P. (2003). Surface modificat Research 338, 937-942. C. H., Smith, D. P., Kalverda, A. P., Phillips, S. E. V., and Radford, amyloidogenic properties. Proceedings of the National Academy of Sciences of the United States of America 99, 9771-9776. 77-84.

PAGE 154

139 Tuncel, A., Tuncel, M., Ergun, B., Alagoz, C., and Bahar, T. (2002). Carboxyl carrying large uniform latex particles. Colloids and Surfaces a-Physicochemical and Engineering Aspects 197, 79-94. Ugelstad, J., Kaggerud, K. H., Fitch, R. M. (1980). Polymer colloid II, Plenum, New York. USRDS (2003). United State Renal Date System (USRDS) 2003 annual report. Van Noordwijk, J. (2001). Dialyzing for life: the development of the artificial kidney. Vandpolymerisation of styrene from characterisation of polymer end groups. Chemical Vanholder, R., Desmet, R., Vogeleere, P., and Ringoir, S. (1995). Middle molecules 1992). f the American Society of Nephrology 11, 2344-2350. 38. anes by low temperature plasma induced graft polymerization. Journal of Membrane Science 209, 255-269. Westhssing with renalin on serum beta2 microglobulin and complement activation in hemodialysis patients. American Journal of Nephrology Wiecformation. Analytical Biochemistry 175, 231-237. WilliaAn evaluation of protein assays for quantitative determination of drugs. Journal of Biochemical and Biophysical Methods 57, 45-55. Kluwer Academic, Dordrecht. enhu.H, and Vanderho.J (1970). Inferences on mechanism of emulsion and Process Engineering 51, 89. toxicity and removal by hemodialysis and related strategies. Artificial Organs 19, 1120-1125. Vincent, C., Chanard, J., Caudwell, V., Lavaud, S., Wong, T., and Revillard, J. P. (Kinetics of I-125 beta2 microglobulin turnover in dialyzed patients. Kidney International 42, 1434-1443. Ward, R. A., Schmidt, S., Hullin, J., Hillebrand, G. F., and Samtleben, W. (2000). A comparison of on-line hemodiafiltration and high-flux hemodialysis: A prospectiveclinical study. Journal o Watkins, R. W., and Robertson, C. R. (1977). Total internal reflection technique for examination of protein sdsorption. Journal of Biomedical Materials Research 11915-9 Wavhal, D. S., and Fisher, E. R. (2002). Hydrophilic modification of polyethersulfone membr uyzen, J., Foreman, K., Battistutta, D., Saltissi, D., and Fleming, S. J. (1992). Effect of dialyzer reproce 12, 29-36. helman, K. J., Braun, R. D., and Fitzpatrick, J. D. (1988). Investigation of the bicinchoninic acid protein assay. Identification of the groups responsible for color ms, K. M., Arthur, S. J., Burrell, G., Kelly, F., Phillips, D. W., and Marshall, T. (2003).

PAGE 155

140 Winchester, J. F., Ronco, C., Brady, J. A., Cowgill, L. D., Salsberg, J., Yousha, E., Choquette, M., Albright, R., Clemmer, J., Davankov, V., Tsyurupa, M., PavlL., Pavlov, M., Cohen, G., Horl, W ova, ., Gotch, F., and Levin, N. W. (2002). The next step from high-flux dialysis: application of sorbent technology. Blood Purification Winchester, J. P., Ronco, J. A., Brendolan, A., Davankov, V., Tsyurupa, M., Pavlova, L., 2001). oglobin Yang. Q., and Yang, C. Z. (2000). Formation of an. Yoon, J. Y., Park, H. Y., Kim, J. H., and Kim, W. S. (1996). Adsorption of BSA on highly carboxylated microspheresquantitative effects of surface functional groups Zollars, R. L. (1979). Kinetics of the emulsion polymerization of vinyl acetate. Journal of Zou, oss-Zou, D., Ma, S., Guan, R., Park, M., Sun, L., Aklonis, J. J., and Salovey, R. (1992). 20, 81-86. Clemme, J., Polaschegg, H. D., Muller, T. E., La Greca, G., Levin, N. W. ( Rationale for combined hemoperfusion/hemodialysis in uremia. Contribution of Nephrology 133, 174-179. Winslow, R. M., Vandergriff, K. D., Motterlini, R. (1993). Mechanism of hemtoxicity. Thrombosis and Haemostasis 70, 36-41. S. C., Ge, H. X., Hu, Y., Jiang, X positively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosColloid and Polymer Science 278, 285-292. and interaction forces. Journal of Colloid and Interface Science 177, 613-620. Applied Polymer Science 24, 1353-1370. D., Derlich, V., Gandhi, K., Park, M., Sun, L., Kriz, D., Lee, Y. D., Kim, G., Aklonis, J. J., and Salovey, R. (1990). Model filled polymers .1. synthesis of cr linked monodisperse polystyrene beads. Journal of Polymer Science Part A. Polymer Chemistry 28, 1909-1921. Model filled polymers .5. synthesis of cross-linked monodisperse polymethacrylate beads. Journal of Polymer Science Part A. Polymer Chemistry 30, 137-144.

PAGE 156

Korea, on August 7, 1968. He obtained a Bachelor of Science degree in Chemistry from Keimd graduate school and earned a researn Korea Institute of Science and Coatie ia Busin BIOGRAPHICAL SKETCH Sangyup Kim was born and raised in the small town of Sung-Joo, Kyungbook, yung University (Daegu, Korea) in 1993. He entere Master of Science degree in Chemical engineering in 1995. He worked as a student cher for 2 years at Textile Polymer Division i Technology (KIST) in Seoul Korea. Next he worked over a year at the Automobile ng Division in DongJoo-PPG in Korea. In August 2001, he began studying for th degree of Doctor of Philosophy in materials science and engineering at the University of Florida, in Gainesville. After getting his Ph.D., he plans to work at the Digital Medess Division of Samsung Electronics in Korea, starting in January 2006. 141


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SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR
MEDICAL APPLICATIONS












By

SANGYUP KIM


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005



























Copyright 2005

by

Sangyup Kim

































To all who made this work possible.















ACKNOWLEDGMENTS

I owe what I am today and this moment to many people, without whom I could not

have been here. First of all, I would like to express my sincere and deepest appreciation

to my parents, wife Mikyung, daughters Heehyung and Erin, whose love, patience, and

support helped me pursue my dream. I gratefully acknowledge my advisor, Dr. H. El-

Shall, who gave me an opportunity to become part of an exciting and beneficial research

project and exert myself in the research work. His guidance, knowledge and stimulating

scientific and critical thinking have contributed to my success to a great extent, and

inspired me to dedicate myself to research work. I especially appreciate Dr. R. Partch as a

co-advisor, who has helped me conduct experiments with valuable discussion for my

research from my first year. I am greatly thankful to Dr. C. McDonald, whose idea and

valuable experimental advice and discussion in the area of polymer chemistry and

membrane technology helped cultivate my interest in this area and were vital to

performing this research. Without his contribution this work could not have been

achieved. I also thank my doctoral committee members, Dr. R. Singh, Dr. A. Zaman, and

Dr. B. Koopman for appreciated comments.

Financial support and equipment use for research from the Particle Engineering

Research Center at the University of Florida were vital in continuing my study for more

than 4 years. My sincere thanks are due to my former advisor Dr. Kiryoung Ha, in

Keimyung University, who helped me open my eyes, and broaden my thought and

introduced me into a limitless polymer field. I would like to also thank to past and current









members in Dr. El-Shall's research group for their assistance, advice, and support. I

sincerely appreciate Stephen for the assistance of thesis writing.
















TABLE OF CONTENTS



ACKN OW LED GM EN T S ..................................................... iv

LIST OF TABLES ........... ................... ............. .................... viii

LIST OF FIGURES ................................................. ...............x

1 IN TR OD U CTION ............... ..1.................... .................. ..........

2 BACKGROUND AND LITERATURE SURVEY................................................5

2.1 The Significance of End Stage Renal Disease (ESRD)........................................5
2.2 Hemodialysis (HD) Treatment .................................. ...............6
2.3 Advances in Membrane Technology ........................................8
2.4 Sorbent Technology ............. .. ................ ...................................... 16
2.5 Limitation of Current Hemodialysis Treatment ...................................... 19
2.6 Latex Particles ....................... .. ..... ................... .. ....20
2.6.1 The Components for Emulsion Polymerization .......................................21
2.6.2 Particle Nucleation ................................. .....................21
2.6.3 Types of Processes for Emulsion Polymerization............... ...............23
2.6.4 Chemistry of Emulsion Polymerization ............. ................................... 24
2.6.5 Seed Emulsion Polymerization......................... ............. 25
2.6.6 Polystyrene Latex Particles .......................................... 26
2.6.7 Various Applications of Latex Particles...............................................28
2.7 Proteins ....................................... ... .... ......... 30
2.7.1 Interaction Forces between Proteins...................................................33
2.7.2 02-M icroglobulin ( 2M )................................... ................. 35
2.7.3 Serum A lbum in ............................... ........................... 36
2.8 Protein Adsorption...................................... .. ... ............38
2.8.1 Interaction between Protein Molecule and Latex Particle.......................38
2.9 Hypothesis for Toxin Removal..................................39
2.9.1 Toxin Removal by Size Sieving Based on Monodispersed Pore Size .......40
2.9.2 Toxin Removal by Selective Adsorption on Engineered Latex Particles ..41

3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY ...............44

3.1 M materials ...................................................... 44
3.2 Latex Particle Preparation.................... ............... ...............46
3.2.1 Preparation of Seed Latex Particles...................................... ......47









3.2.2 Preparation of Seeded Latex Particles.................................................48
3.2.3 Preparation of Core Shell Latex Particles ..............................................48
3.2.4 Purification of Synthesized Latex Particles....................................48
3.3 Characterization....................................... .. .. .... .............. .... 49
3.3.1 Degree of Conversion. ................................... .. ................. ............... 49
3.3.2 Fourier Transform Infrared (FTIR) Spectroscopy...............................49
3.3.3 Quasielastic Light Scattering (QELS) ............................... .....................50
3.3.4 Field Emission-Scanning Electron Microscopy (FE-SEM) .....................50
3.3.5 Zeta Potential M easurem ent ............................ ....................50
3.3.6 Protein Adsorption................ .............. ................... 51
3.3.7 Blood Biocompatibility by Hemolysis Test ............................................ 53

4 RESULTS AND DISCU SSION .......................................... ........................... 55

4.1 Polymerization of Latex Particles ........ .......................................... 55
4.1.1 Polystyrene Seed Latex Particles......................... ..... .............55
4.1.2 The Growth of Polystyrene (PS) Seed Latex Particles.............................61
4.1.3 Core Shell Latex Particles ........................................ ....... ............... 71
4.2 Characterization of Latex Particles ................ ........ .... ................. ............... 75
4.2.1 Fourier Transform Infrared Spectroscopy (FTIR)...............................75
4.2.2 Zeta Potential M easurem ents ........................................ ............... 75
4.3 Protein Adsorption Study .................................. .....................................85
4.3.1 Adsorption Isotherm ............................................ 87
4.3.2 A dsorbed Layer Thickness ............. .................. ........... ............... 103
4.3.3 Gibbs Free Energy of Protein Adsorption................... ... .............104
4.3.4 K inetics of A dsorption ................................................. .....................107
4.4 Blood Compatibility .............. ............... ........................ 112

5 SUMMARY OF RESULTS, CONCLUSION, AND RECOMMENDATION FOR
FUTURE W ORK ................... ...... .......... ..... .......... ................. 115

5.1 Sum m ary of R esults............. ................................................ ...... ............... 115
5.2 Conclusions........................................... 121
5.3 Recommendation for Future Work............................ ...............121

LIST OF REFEREN CES .................................................................. ............... 122

B IO G R A PH ICA L SK ETCH .................................... ........... ................. .....................141
















LIST OF TABLES


Table page

4-1. The polymerization recipe of polystyrene (PS) seed particles.............. ................57

4-2. Continuous addition emulsion polymerization recipe for growing polystyrene
(PS) latex particles less than 500nm in size. ................ .................. ............. 63

4-3. Continuous addition emulsion polymerization recipe for growing polystyrene
(PS) latex particles largar than 500nm in size.................. ................. .........64

4-4. The preparation recipe of PS core with various shell latex particles.......................72

4-5. The isoelectric point (IEP) of common proteins. ........................................... 83

4-6. The equilibrium concentration values of BSA adsorption on polystyrene (PS)
latex particles calculated the Langmuir-Freundlich isotherm model....................93

4-7. The equilibrium concentration values (qm) of BSA adsorption on PS/PMMAloo
particles calculated the Langmuir-Freundlich isotherm model...........................96

4-8. The equilibrium concentration values ofBSA adsorption on PS/PMMA90PAAlo
particles calculated the Langmuir-Freundlich isotherm model...........................98

4-9. The equilibrium concentration values ofBSA adsorption on PS/PMMA75PAA25
particles calculated the Langmuir-Freundlich isotherm model............................100

4-10. The equilibrium concentration values of p2M adsorption calculated the
Langmuir-Freundlich isotherm model. ..........................................102

4-11. Absorbed BSA layer thickness (A) of BSA in phosphate buffer (PB) at 370C......104

4-12. Absorbed BSA layer thickness (A) of BSA in phosphate buffered saline (PBS) at
37 C. ............................................................. 104

4-13. The values of Gibbs free energy change of BSA adsorption in phosphate buffer
(PB) at 37C. ..................................................... 105

4-14. The values of Gibbs free energy change of BSA adsorption in phosphate
buffered saline (PB S) at 370C. ......................................... ............... 105


e (PB S) at 37 C. ........................................ .......................... 105









4-15. The values of Gibbs free energy change of p2M adsorption in phosphate buffere
(PB ) at 37 C and pH 7.4 ......... ................. .................................. ............... 106

4-16. Fitting parameters of second-order kinetic model.............. ...... ...............111
















LIST OF FIGURES


Figure page

1-2. Stereo drawing of the a-carbon backbone of f2M...................................................2

2-1. An image of the location and cross section of a human kidney................................

2-2. A schematic draw of the hemodialysis route....................................................7

2-3. Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes........................11

2-4. The chemical structure of polysulfone (PSf)................................ ................12

2-5. The chem ical structure of polyam ide (PA)............................................................ 13

2-6. Chemical structure of polyacrylonitrile (PAN). ........................................................14

2-7. Emulsion polymerization system ...... ............................................................ 24

2 -8 L -a -am in o a cid ..................................................................................................... 3 1

2-9. Ribbon diagram of human 32M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ)...................36

2-10. Secondary structure of human serum albumin (HSA) with sub-domains................37

2-11. The relationship between pore size and particle size in body-centered cubic and
closed packed array s........... ...... .................................... ................ .. .... ..... .. 4 1

2-12. Schematic representation of the protein adsorption on the core shell latex
p article s at p H 7 .4 ........... ..... ............................................................ ........ ............. .. 4 2

3-1. Chemical structure of main chemicals.............. ........... .................................. 45

3-2. Experimental setup for semi-continuous emulsion polymerization .........................46

3-3. The particle preparation scheme in various types and size ranges of latex particles. 47

3-4. Schematic of the procedure for a protein adsorption test. ..................................52

3-5. Separation of RBC from whole blood by centrifuge process ...................................53









3-6. Schematic of the procedure for hemolysis test .......................................................54

4-1. Schematic representation of semi-continuous seed latex particles preparation and
g ro w th ...................................... ................................................... 5 6

4-2. Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS2.59 (B) PSS2.33 (C) PSS2.07 (D) PSs.1 .............. ........................................59

4-3. SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D ) 410nm ....................................................... ...................... 65

4-4. SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C)
640nm (D ) 790nm ....................... .. ...... .................. .. .... .... ............... 68

4-5. Dependence of the particle size on the surfactant to monomer ratio..........................70

4-6. Schematic of core shell latex particle structures. ........................................ ...........71

4-7. Scanning Electron Micrograph of latex particles (A) PS (B) PS/PMMA100oo (C)
PS/PMMA90PAAlo (D) PS/PMMA75PAA25 ................................. ................73

4-8. FTIR spectra of polymerized latex particles. (A) bare polystyrene (PS) (B)
PS/PMMA1oo (C) PS/PMMA90PAAlo (D) PS/PMMA75PAA25 ..............................76

4-9. Schematic representation of ion distribution near a positively charged surface. .......78

4-10. Schematic representation of zeta potential measurement.......................................79

4-11. Zeta potential of PS latex particles at 250C. ......................................................80

4-12. Zeta potential of PS/PMMA00oo latex particles at 25C ..........................................81

4-13. Zeta potential of PS/PMMA90PAAlo latex particles at 250C. ..................................82

4-14. Zeta potential of PS/PMMA75PAA25 latex particles at 250C. .................. ...............82

4-15. The decay of surface potential with distance from surface in various electrolyte
concentrations: (1) low (2) intermediate (3) high ....................................... ....... 84

4-16. High-affinity adsorption isotherm of typical flexible polymer on solid surface.....86

4-17. Overall schematic representation of the protein adsorption on the synthesized
latex p a rtic le s ................................................... ............... ................ 8 6

4-18. The dependence of UV absorbance on BSA concentration. (A) 1.0mg/ml (B)
0.6m g/m l (C ) 0.2m g/m l......... ................. ................... .................... ............... 87

4-19. Standard curve of net absorbance vs BSA sample concentration. ...........................88









4-20. Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 370C in phosphate buffer media. ............................89

4-21. Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate
buffer (PB ) at 37C .................. ..................................... .. ........ .... 90

4-22. Adsorption isotherm of BSA adsorption 37C in phosphate buffered saline
(PB S) on P S latex particles. ......................................................................... .. .... 9 1

4-23. Conformation of bovine serum albumin (BSA) on latex particles...........................92

4-24. Adsorption isotherm of BSA 37C in PB on PS/PMMA00oo core shell latex
p article s. .......................................................... ................ 9 4

4-25. Adsorption isotherm of BSA at 370C in PBS on PS/PMMA00oo core shell latex
p article s. .......................................................... ................ 9 5

4-26. Adsorption isotherm of BSA at 370C in PB on PS/PMMA90PAAlo core shell
latex particles................................... ................................. .........97

4-27. Adsorption isotherm of BSA at 370C in PBS on PS/PMMA90PAAlo core shell
latex particles................................... ................................. .........98

4-28. Adsorption isotherm of BSA 37C in PB on PS/PMMA75PAA25 latex particles.....99

4-29. Adsorption isotherm of BSA 37C in PBS on PS/PMMA75PAA25 core shell latex
p article s. .......................................................... ................ 9 9

4-30. Adsorption isotherm of 32M onto PS and PS/PMMA00oo latex particles in PB at
37C and pH 7.4 ................................................................... 10 1

4-31. Adsorption isotherm of p2M onto PS and PS/PMMA90PAAlo latex particles in
PB at 37C and pH 7.4. ......................... .... .............. ...................... 101

4-32. Adsorption isotherm of p2M onto PS and PS/PMMA75PAA25 latex particles in
PB at 37C and pH 7.4. ......................... .... .............. ...........................102

4-33. Gibbs free energy of adsorption of proteins on latex particles in PB at 370C and
p H 7 .4 .......................................................................... 10 7

4-34. The kinetics of protein adsorption in PB on PS latex particles at 370C and pH
7 .4 .................. ........................................................................ 1 0 9

4-35. The kinetics of protein adsorption in PB on PS/PMMA1oo latex particles at 37C
and pH 7.4. .......................................... ........................... 109

4-36. The kinetics of protein adsorption in PB on PS/PMMA90PAAlo latex particles at
37C an d pH 7 .4 ................................................................................ 110









4-37. The kinetics of protein adsorption in PB on PS/PMMA75PAA25 latex particles at
37C an d pH 7 .4 ................................................................................ 110

4-38. Im age of red blood cells ................................................................................ 113

4-39. Hemolysis caused by latex particles, n=5 ...... .............. ................................114
















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR
MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair: Hassan El-Shall
Major Department: Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas, especially as solid

phase supports in numerous biological applications such as immunoassay, DNA

diagnostic, cell separation, and drug delivery carrier. Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD). It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin. Thus, it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes. This

improvement will have significant economic and health impact.

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood. Semi-continuous seed emulsion polymerization was used to prepare monodisperse









polystyrene seed particles ranging from 1267.5 to 2165.3 nm in size, which are then

enlarged by about 800nm. Surfactant amount played a key role in controlling the latex

particle size. Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer. The prepared polymeric particles include bare polystyrene (PS) particles, less

hydrophobic PS core and PMMA shell particles, and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter. SEM, light scattering,

and zeta potential measurements were used to characterize particle size and surface

properties. Adsorption isotherms of two proteins, bovine serum albumin (BSA) and 32-

microglobulin (02M), on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method. The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium.

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode. Adsorption

kinetics experiments for both proteins and all latex particles were also performed. The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and 32M. Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles. PS/PMMA90PAAlo and

PS/PMMA75PAA25 core shell latex particles had desirable material properties with, not

only a large amount and high rate of selective 32M adsorption over BSA but also high

blood compatibility showing less than 3% hemolysis.














CHAPTER 1
INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patient's survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation. The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF]. The

number of people with ESRD is rapidly increasing in the United States with

approximately 96, 295 incidents and 406, 081 prevalent patients, including 292, 215 on

dialysis and 113, 866 with a functioning graft in 2001. It is projected that there will be

more than 2.2 million ESRD patients by 2030 [USRDS 2003]. The expenditure for the

ESRD treatment program had reached $22.8 billion, 6.4% of the Medicare budget in

2001. Due in part to the limited availability of kidneys for transplantation, hemodialysis

is the primary clinical treatment for the patients with ESRD.

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5,000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood. One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as /l2-Microglobulin (f2M) and

interleukin 6 (IL-6).

/l2M is a causative protein of dialysis-related amyloidosis (DRA), a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al. 1985]. l2M deposition in tissue is the primary cause









of destructive arthritis and carpal tunnel syndrome [Vincent et al. 1992; Drueke 2000].

The f2M structure is shown in Figure 1-2.


















Figure 1-2. Stereo drawing of the a-carbon backbone of l2M [Becker et al. 1985].

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al. 1996; Ronco et al. 2001], removal

efficiency is not as high as those achieved by a normal healthy kidney. Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al.

1992; Leypoldt et al. 1998], such as a restricted choice of surface chemistries and limited

control of porosity. The development of novel engineering membrane technology is

needed to remove middle molecule toxins.

Polymeric latex particles have received increasing attention in medical application

areas, especially as solid phase supports in biological applications [Piskin et al. 1994].

Examples of these applications include immunoassay [Chen et al. 2003; Radomske-

Galant et al. 2003], DNA diagnostic [Elaissari et al. 1998], cell separation, drug delivery

carrier [Luck et al. 1998; Kurisawa et al. 1995; Yang et al. 2000], etc. This is because of









the well-defined colloidal and surface characteristics of the particles. By using a seed

emulsion polymerization method, it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry. Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization. Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage. This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al. 1973]. The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al. 1996; Nelliappan

et al. 1997]. Through the development of a hemodialysis membrane using monodisperse

latex particles, improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized. This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin. Thus, an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology.

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into today's powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications. This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001].

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD). The

interdisciplinary nature of this approach enables a more complete understanding of the









phenomena of protein adsorption and the material properties necessary for selective

separation. This innovative approach to membrane fabrication has the potential of making

inexpensive, highly efficient membranes for both industrial and specialized separation

processes.

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications.

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation. Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes. Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method. Core shell

latex particles, PS/PMMA1oo, PS/PMMA90PAA1o, PS/PMMA75PAA25, were also

synthesized to differentiate the degree of hydrophobicity of particles. Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape, and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles. Adsorption isotherms of target proteins, bovine

serum albumin (BSA), and f/2M on latex particles were obtained as a function of pH,

ionic strength, and protein concentrations using the BCA assay method. Adsorption

kinetics for both proteins on the latex particles were also measured. Finally, hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood. This research will be described in more detail in chapters 3 and 4.















CHAPTER 2
BACKGROUND AND LITERATURE SURVEY

2.1 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid, minerals, and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production. They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005; Kurbel

et al. 2003]. EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes. Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food, and also bone to mobilize

calcium from the bone to the blood. When kidneys fail, harmful waste builds up, blood

pressure rises, and body retains excess fluid. The body also does not make enough red

blood cells. When this happens, treatment is needed to replace the function of failed

kidneys.

Kidney





Renat artery Renal
pelvis
Renal vein
SMedulla
Ureter

Cortex
Figure 2-1. An image of the location and cross section of a human kidney.









The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF]. Chronic renal disease is a gradual and progressive

loss of the function, unlike acute renal disease where sudden reversible failure of kidney

function occurs. Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged. In the early stages, there can be no

symptoms. In fact, progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney. If left untreated,

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD). ESRD is a rapidly growing heath-care problem in the United States. In

2001, approximately 96,295 incidents and 406,081 prevalent patients were diagnosed

with ESRD, including 292,215 patients on dialysis and 113,866 patients with a

functioning graft [USRDS 2003]. The projected number of patients with ESRD is

expected to exceed 2.2 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003]. A great extent of ESRD program cost and Medicare budget

have been spent in 2001. Due in part to a limited availability of kidneys for

transplantation, hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61% of U.S. ESRD patients.

2.2 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane, referred to as a dialyzer. The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane. The cleaned blood is then returned to the blood

stream. A surgically constructed vascular access connects the extracorporeal circuit to the










patient's system. Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment. Figure 2-2 shows

the scheme of the hemodialysis route. The critical element of a HD instrument is the

semipermeable membrane, which allows for selective transport of low molecular weight

biological metabolites from blood.

Dialyzer inflow Blood inlet
pressure monitor Vnous Header
HEin puf _pressure monilorf
(topreeit Tube sheet
clolling)
dolig 7Solution
I Iy er 1 II I>" -outlet

ai Fibers
Air detiRllE
clamp 11111- Jacket
Claan blod Solution
ApriassM body turnl
Blood modnlet
B pump for cleansing .
Blod punp

SBlood outlet
Figure 2-2. A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia, fatigue, bone problems, joint

problems, itching, sleep disorders, and restless legs. Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO), which

stimulates the bone marrow to produce red blood cells. Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells. This leads to bone disease, referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults.

Older patients and women who have gone through menopause are at greater risk for this

disease. Uremic toxins, which cannot be remove from the blood by the current dialyzer

membranes can lead to itching. This problem can also be related to high levels of


membranes can lead to itching. This problem can also be related to high levels of









parathyroid hormone (PTH). Dialysis patients can also experience day-night reversal, that

is, they have insomnia at night and sleep during the day. This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxins.

Oxidative stress is a problem for patients on maintenance dialysis. This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al. 1995; Cristol et al.

1994]. Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al. 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al. 1993]. In order to reduce

antioxidant defense, dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants. Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years. DRA develops when proteins in the blood deposit on joints and tendons, causing

pain, stiffness, and fluid in the joint, as is the case with arthritis. Normally the healthy

kidneys can filter out these proteins, but dialysis filters are not as effective.

2.3 Advances in Membrane Technology

Dialysis for blood purification is widely used in the treatment of ESRD.

Hemodialysis (HD) techniques use a semi-permeable membrane to replace the filtration

role of the kidney. The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers. These membranes come

in various shapes such as sheets, tubular structures or hollow fiber arrangements. The

hollow fiber type is the most popular and is incorporated into over 800 different devices

in world wide [Ronco et al. 2001].

The first attempt at blood dialysis using a cellulose based membrane occurred

1913. John Abel [1990] from the Johns Hopkins Medical School, described a method









whereby the blood of a living animal may be submitted to dialysis outside the body using

a membrane based on cellulose and returned to the natural circulation without exposure

to air, infection by microorganisms or any alteration that would necessarily be prejudicial

to life. This same technique is still used to today, however the device used has been

modified over the years as better membranes were developed and the anti-coagulant,

heparin, has become available.

Cellulose membranes have been widely used for the treatment of renal failure from

1928, when the first human dialysis was performed, until the mid 1960. The basic

molecular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups. The realization that such groups imparted undesirable qualities on the material in

respect to blood contact behavior was discovered in the early 1970s and since has been

the focus of development. These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect. The result is a molecular mosaic of hydrophobic benzyll)

and hydrophilic (hydroxyl and cellulose) regions.

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943. Cellophane tubing was used for the membrane with

heparin as the anticoagulant. For the next 17 years, hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure. Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein, and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage.









Initially, the need for dialysis in patients with acute renal failure was determined

mainly by the development of signs and symptoms of uremia. After dialysis, some time

might elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis. Many patients with acute renal failure, secondary to accidental or surgical

trauma were hypercatabolic, but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet. However, Teschan and his coworkers [Obrien et al.

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis, or administration of the treatment before the patient

again became sick with uremia. Their report in 1959 was the first description of daily

hemodialysis.

Development of membrane accessories such as a shunt has also been an area of

focus for treatment improvement. In 1960, the development of a shunt [Quinton et al.

1960], a flexible polytetrafluoroethylene (PTFE or Teflono) tubing, made many more

hemodialysis treatments possible for chronic kidney failure patients. PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt.

Synthetic membranes are prepared from engineered thermoplastics such as

polysulfone (PSf), polyamide (PA), and polyacrylonitrile (PAN) by phase inversion or

precipitation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures. Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA), polyacrylethersulfone (PAES), and polyvinylpyrrolidone

(PVP) with the integral three-layer structure. The skin layer on the inside fiber type

membrane contacts blood and has a very high surface porosity and a narrow pore size









distribution. This layer constitutes the discriminating barrier deciding the permeability

and solute retention properties of the membrane. The skin layer is supported by thick

sponge-type structure larger pores, providing mechanical strength and very low

hydrodynamic resistance.






















Figure 2-3. Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch
et al. 1998]

PSf is a widely used membrane material for the hemodialysis application

[Malchesky 2004], because of its thermal stability, mechanical strength, and chemical

inertness. According to a report from the National Surveillance of Dialysis-Associated

Disease (NSDAD) in the US, over 70% of hemodialysis membranes were PSf based

[Bowry 2002]. This is most likely because PSf has many advantages over other materials.

This synthetic polymer is one of few materials that can withstand sterilization by steam,

ethylene oxide, and y-radiation. PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow









fiber or flat sheet hemodialysis membranes. The material also has a high permeability to

low molecular weight proteins and solute, and high endotoxin retention. The chemical

structure of PSf is shown in Figure 2-4.


CH3 01


CH3 0 n
Figure 2-4. The chemical structure of polysulfone (PSf).

There is one major disadvantage to PSf. The hydrophobic nature of the PSf causes

serious complications through the activation of the complement alternative pathway

leading to the adsorption of serum proteins onto the membranes [Singh et al. 2003].

Anticoagulants are added during dialysis therapy to avoid blood clotting, but this does not

completely eliminate the problem. In order to overcome this disadvantage of the PSf

membrane, various studies have been performed to change the material's surface

properties. These investigations include hydrophilic polymer coating [Brink et al. 1993;

Kim et al. 1988; Higuchi et al. 2003], layer grafting onto PSf membrane [Wavhal et al.

2002; Song et al. 2000; Pieracci et al. 2002; Mok et al.1994], and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al.; 1990; 1991; 1993;

Blanco et al. 2001; Nabe et al. 1997; Guiver et al. 1993]. Hydrophilic monomers, 2-

hydroxy-ethylmethacrylate (HEMA), acrylic acid (AA), and methacrylic acid (MMA),

have also been grafted onto PSf membrane to increase flux and Bovine Serum Albumin

(BSA) retention [Ulbricht et al. 1996]. Hancock et al [2000] synthesized

polysulfone/poly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion. Kim et al. [2003] also studied blending a sulfonated PEO acrylate

diblock copolymer into PSf in order to reduce platelet adhesion and enhance









biocompatibility. PEO is a commonly used biomaterial due to its excellent resistance to

protein adsorption and inherent biocompatibility [Harris 1992]. Kim et al. [2005] studied

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer. The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-S03 acrylate

and a hydrophobic block of octadecylacrylate (OA). Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information. McMurry [2004] developed a strategy using an amphiphilic graft

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glycol).

When compared to unmodified PSf, these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications.

Polyamide (PA) membranes have also been used for hemodialysis because of thier

mechanical strength in both wet and dry conditions. Polyamide consists of aromatic

or/and aliphatic monomers with amide bonding (-CONH-), also known as a peptide bond.

The basic amide bond in polyamide is shown in Figure 2-5. Ri and R2 can be either

aromatic or aliphatic linkage group.

0

~ R- C N R2~

H
Figure 2-5. The chemical structure of polyamide (PA).

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

and concluded that PA hemofiltration was a highly biocompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of









the activated anaphylatoxins and 32-Microglobulin (02M). The PA based membrane,

Polyflux (manufactured by Gambro GmbH, Germany) blended with polyamide,

polyarylethersulfone and polyvinylpyrrolidone (PVP), was able to clean small molecules

such as urea, dreatinine, and phosphate, as well as decrease 32M amount by 50.2%

[Hoenich et al. 2002]. Due to the non-selectivity of the membrane removal of these

unwanted materials also led to the undesirable loss of beneficial proteins during therapy.

Meier et al. [2000] evaluated different immune parameters using a modified cellulose

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients. They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation.

Polyacrylonitrile (PAN) is another commonly used membrane material because it is

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltration

[Scharnagl et al. 2001]. PAN is a semi-crystalline polymer and the mechanical properties

strongly depend on the crystalline structures. The chemical structure of PAN is shown in

figure 2-6.


CH2- CH

CN
Figure 2-6. Chemical structure of polyacrylonitrile (PAN).

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent, gives PAN membranes more flexible processing parameters and increased

performance [Jung et al. 2005]. PAN membrane performance has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate









[Godjevargova et al. 1999; Hicke et al. 2002], N-vinylimidazole [Godjevargova et al.

2000], hydroxyl ethyl methacrylate [Ray et al. 1999; Bhat et al. 2000], methacrylic acid

[Ray et al. 1999], vinyl pyrrolidone [Ray et al. 1999], acrylic acid [Trotta et al. 2002],

acrylamide [Musale et al. 1997], and vinylchloride [Broadhead et al. 1998]. These

monomers provide a reactive group for enzyme immobilization, improved mechanical

strength, solvent-resistance, pervaporation, permeation flux, anti-fouling and bio-

compatibility. Because of this, PAN-based copolymer membranes have great potential for

the treatment of hemodialysis in an artificial kidney. This material can also be used for

other applications like the treatment of wastewater, the production of ultra-pure water,

biocatalysis together with separation, and methanol separation by pervaporation.

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane. When a foreign material is

exposed to blood, plasma proteins are adsorbed, clotting factors are activated, and a non-

soluble fibrin network, or thrombus, is formatted [Goosen et al. 1980]. The result of this

research was that the biocompatible chitosan polymer and a blood anticoagulant heparin

prevented blood clotting. They showed prolonged coagulation time, reduced platelet

adsorption, thrombus formation, and protein adsorption.

Nie et al. [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs). In order to improve the membrane performance, acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and a-allyl

glucoside. They found that the number and size of macrovoid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the









internal coagulant. The water flux of the UHFMs also decreased while the bovine serum

albumin rejection increased minutely. Godjevargova et al. [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties. Formed functional groups like primary amine, oxime, and

tertiary amine groups, provided the membrane with more hydrophilic properties and a

substantial increase in the permeability of the membranes.

The wide use of filtration in practice is limited by membrane fouling. Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane. Fouling occurs mostly in the filtration of

proteins. Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990]. The first step is the transfer of solute to the surface. The second step is the

transfer of solute into the membrane until it either finally adsorbs or passes through after

a set of adsorption-desorption events. The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al. 1993]. Bryjak et

al. [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins. The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process. This modified PAN membrane

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test. The pore

size, however, decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode.

2.4 Sorbent Technology

Over the last three decades, sorbent technology [Castino et al, 1976; Korshak et al.

1978; Malchesky et al. 1978] has been further developed to increase the efficiency of









dialysis, or replace it, for the treatment of ESRD. Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent. Specific adsorption contains tailored ligands, or antibodies, with high

selectivity for target molecules. Specific adsorbents have been used in autoimmune

disorders such as idiopathic thrombocytopenic purpura [Snyder et al. 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al. 1999]. Nonspecific

adsorbents, such as charcoal and resins, attract target molecules through various forces

including hydrophobic interactions, ionic (or electrostatic) attraction, hydrogen bonding,

and van der Waals interactions.

New dialysate with sorbents has become an accepted modification of dialysis, and

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis, especially as

new sorbents are developed [Winchester et al. 2001]. Hemoperfusion is defined as the

removal of toxins or metabolites from circulation by the passing of blood, within a

suitable extracorpoteal circuit, over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et al. 1996], various resins [Ronco et al. 2001],

albumin-conjugated agarose etc. Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989]. Newly recognized

uremic toxins [Dhondt et al. 2000; Haag-Weber et al. 2000] have resulted in several

investigations on alternatives to standard, or high-flux, hemodialysis to remove these

molecules. These methods include hemodiafilteration with [de Francisco et al. 2000] or

without [Ward et al. 2000; Takenaka et al. 2001] dialysate regeneration using sorbents, as

well as hemoperfusion using such adsorbents as charcoal and resins.









Kolarz et al. [1989; 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application. They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with a, a'-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a dichloroethane

solution. They also realized that the hemocompatibility was useful for nemoperfusion and

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 0.2mmol/g.

A special polymeric adsorbing material (BM-010 from Kaneka, Japan) has been

investigated by another group [Furuyoshi et al. 1991] for the selective removal of p2M

from the blood of dialysis patients. The adsorbent consists of porous cellulose beads

modified with hexadecyl groups that attract 32M through a hydrophobic interaction. The

adsorption capacity of this material is Img of p2M per lml of adsorbent. Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer, several small clinical trials were performed. During 4-5 hours of

treatment, about 210mg of p2M were removed, thus reducing the concentration in the

blood by 60-70% of the initial level [Nakazawa et al. 1993; Gejyo et al. 1993; 1995].

RenalTech developed a hemoperfusion device, BetaSorb, containing the hydrated

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al. 2002]. In this

case, solute molecules are separated according to their size based on their ability to

penetrate the porous network of the beaded sorbents. The resin beads were prepared with

a blood compatible coating, and confirmed to be biocompatible in vivo in animals

[Cowgill et al. 2001].









2.5 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD.

However, it does not replace all of the complex functions of a normal healthy kidney. As

a result, patients on dialysis still suffer from a range of problems including infection,

accelerated cardiovascular disease, high blood pressure, chronic malnutrition, anemia,

chronic joint and back pain, and a considerably shortened life span. One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules. This is mainly because of the broad pore size distribution reducing the

selective removal of toxins, and unsatisfied biocompatibility causing lots of

complications such as inflammation, blood clotting, calcification, infection, etc.

Dialysis purifies the patient's blood by efficiently removing small molecules, like

salts, urea, and excess water. However, as toxic molecules increase in size, their removal

rate by hemodialysis substantially declines. Typically, only 10% 40% of these middle

molecular weight toxins (300-15,000 Da) are removed from the blood during a dialysis

session [Vanholder et al. 1995]. These toxins then reach an abnormally high level and

begin to damage the body. One such toxin, 32M, causes destructive arthritis and carpal

tunnel syndrome, by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al. 2002]. This is

also a main cause of mortality for long-term dialysis patients. Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients. Still others are believed to impair the

functioning of several other body systems, such as the hematopoietic and other endocrine

systems. This may contribute to accelerated cardiovascular disease, the leading cause of









death among dialysis patients, as well as clinical malnutrition, which affects up to 50% of

this patient population.

Over the last decade, polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

improved removal characteristics. However, removal efficiency is not as high as those

achieved by a normal healthy kidney.

2.6 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 1,3-butadiene and styrene made during World War II in the United

States. The Dow chemical company has been a major manufacturer of polystyrene,

including latex, which they used in paint formulations. The theory of emulsion

polymerization, in which a surfactant is used, was established by Harkins [1948] and by

Smith and Ewart [1948]. By 1956 the technology was complete, including the method of

building larger diameter particles from smaller ones. The product by this emulsion

polymerization is referred to as latex, a colloidal dispersion of polymer particles in water

medium [Odian 1991]. Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives, inks, paints, coatings, drug

delivery systems, medical assay kits, gloves, paper coatings, floor polish, films, carpet

backing and foam mattresses to cosmetics. The relatively well known and easy control of

the emulsion process is one of main advantages for these applications. Therefore,

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required.









2.6.1 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer, a

dispersing medium, a surfactant, and an initiator. Available monomers are styrene,

butadiene, methylmethacylate, acryl acid, etc. The dispersing medium is usually water,

which will maintain a low solution viscosity, provide good heat transfer, and allow

transfer of the monomers from the monomer droplets into micelles and growing particles

surrounded by surfactants, respectively. The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments. Its main functions is to provide the nucleation

sites for particles and aid in the colloidal stability of the growing particles. Initiators are

water-soluble inorganic salts, which dissociate into radicals to initiate polymerization. To

control the molecular weight, a chain transfer agent such as mercaptan, may be present.

2.6.2 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase. The location of the

polymerization is not in the monomer droplets but in micelles because the initiators are

insoluble in the organic monomer droplets. Such initiators are referred to as oil-insoluble

initiators. This is one of the big differences between emulsion polymerization and

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets. Because the monomer droplets have a much smaller total surface area,

they do not compete effectively with micelles to capture the radicals produced in solution.

It is in the micelles that the oil soluble monomer and water soluble initiator meet, and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets. As polymerization proceeds, the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of









monomer from the monomer droplets. There are three types of particles in the emulsion

system: monomer droplets; inactive micelles in which polymerization is not occurring;

and active micelles in which polymerization is occurring, referred to as growing polymer

particles.

The mechanism of particle nucleation occurs by two simultaneous processes:

micellar nucleation and homogeneous nucleation. Micellar nucleation is the entry of

radicals, either primary or oligomeric radicals formed by solution polymerization, from

the aqueous phase into the micelles. In homogeneous nucleation, solution-polymerized

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto the

oligomers whose propagation has ended [Fitch et al. 1969]. The relative levels of micellar

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration. Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration. It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968]. A highly water insoluble monomer such as

styrene [Hansen et al. 1979; Ugelstad et al. 1979] has probably created by micellar

nucleation, while a water soluble monomer such as vinyl acetate [Zollars 1979] has been

formed by homogeneous nucleation.

A third latex formation reaction mechanism has been proposed, referred to as

coagulative nucleation. In this reaction, the major growth process for the first-formed

polymer particles (precursor particles) is coagulation with other particles rather than

polymerization of monomer. The driving force for coagulation of precursor particles,









several nanometers in size, is their relative instability compared to larger sized particles.

The small size of a precursor particle with its high curvature of the electrical double layer

permits the low surface charge density and high colloidal instability. Once the particles

become large enough in size, maintaining the high colloidal stability, there is no longer a

driving force for coagulation and further growth of particles takes place only by the

polymerization process.

2.6.3 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization:

batch, semi-continuous (or semi-batch), and continuous. In the batch type process, all

components are added at the beginning of the polymerization. As soon as the initiator is

added and the temperature is increased, polymerization begins with the formation and

growth of latex particles at the same time. There is no further process control possible

once the polymerization is started. In the semi-continuous emulsion polymerization

process, one or more components can be added continuously. Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization. There are advantages to this process such as control of the

polymerization rate, the particle number, colloidal stability, copolymer composition, and

particle morphology. In the continuous process, the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously removed

at the same rate. High production rate, steady heat removal, and uniform quality of

latexes are advantages of the continuous polymerization processes.

Other available methods include the intermittent addition and shot addition of one

or more of the components. In the shot addition process, the additional components are

added at one time, during the later stages of the polymerization, prior to complete










conversion of the main monomer. This method has been used successfully to develop

water-soluble functional monomers such as sodium styrene sulfonate [Kim et al. 1989].

2.6.4 Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages: initiation, propagation, and termination. The emulsion

polymerization system is shown in Figure 2-7.

Aqueous phase
Polymer particle swollen
With monomer


I R*



Monomer
droplet

I R*

Emulsifier Micelle with
monomer




Figure 2-7. Emulsion polymerization system. [Radicals (R-) are created from initiators
(I). Monomer is transferred from larger monomer droplet into micelles by
emulsifier. Initiated polymer particle by radicals is keep growing until
monomers are all consumed. The reaction is performed in aqueous media]

In the initiation stage, free radicals are created from an initiator by heat or an

ultraviolet radiation source. The initiator with either peroxides groups (-0-0-), such as

sodium persulfate, or azo groups (-N=N-) such as azobisisobutyronitrile, is commonly

used for emulsion polymerization. The primary free radicals created from initiator react

with the monomer for initiation of polymerization. In the propagation stage, the polymer

chain grows by monomer addition to the active center, a free radical reactive site. There

are two possible modes of propagations, head-to-head addition and head-to-tail addition.









The head-to-tail mode is the predominant configuration of the polymer chain, a result of

steric hindrance of the substitute bulky group. In the termination stage, polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules, one having a saturated end group and the other

with an unsaturated end-group.

2.6.5 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatility

of the many applications heterophase polymerization processes like emulsion, dispersion,

micro-emulsion, seeded emulsion, precipitation, etc. Especially, to prepare well-defined

microspheres having monodisperse and various particle sizes as well as surface group

functionalilties, it is necessary to use seed particles prepared by emulsion polymerization

and enlarge them to a desired size in the further stages of reactions.

Polymeric particles are required to have a uniform particle size in many

applications, such as chromatography, where they are used as a packing material.

Morphological control of latex particle is also important for many practical applications

[Schmidt 1972]. Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morphological design. In

seeded emulsion polymerization [Gilbert 1995], preformed 'seed' latex is used to control

the number of particles present in the final latex. The advantage of seeded emulsion

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed, so that the number concentration of particles is constant and known. Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al. 1980;

Okubo et al. 1992] using this polymerization technique. The initial seed particle









preparation step is well known, and relatively easy to perform because, at the relatively

small particle sizes (0.2 to 0.5 micron), the particle growth process can be readily

controlled by the use of an emulsifier. Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles. As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to maintain a

stable, uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex. Recently, there have been several reports concerning the

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al. 1990; Delacal, et al. 1990; Lee

et al. 1995; Lee 2000; 2002].

2.6.6 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particles

bearing various functional surface groups such as carboxyl [Lee et al. 2000; Tuncel et al.

2002; Reb et al. 2000], hydroxyl [Tamai et al. 1989], marcapto [Nilson 1989], epoxy

[Shimizu et al. 2000; Luo et al. 2004], acetal [Izquierdo et al. 2004; Santos et al. 1997],

thymine [Dahman et al. 2003], chlorometyl [Izquierdo et al. 2004; Park et al. 2001;

Sarobe et al. 1998], amine [Counsin et al. 1994; Ganachaud et al. 1997; Ganachaud et al.

1995; Miraballes-Martinez et al. 2000; 2001; Anna et al. 2005], ester [Nagai et al. 1999],

etc. In order to produce these functionalized particles, different methods for particle

preparation must be used. The polymer emulsion with core-shell morphology of latex

particles is one of them. This is a multistep emulsion polymerization process in which the

polystyrene "core" particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles, thus forming the functionalized









polymer "shell" on the "core" particle [Keusch et al. 1973]. There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area; low surfactant concentration to prevent formation of

micelles; and the semi-continuous addition of monomer to create a starved-feed condition

and keep the monomer concentration low. There are some advantages [Hergeth et al.

1989] of dispersions with polymeric core-shell particles: First, it is possible to modify the

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction. Thus,

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al. 1996; Nelliappan et al. 1997]. In this way, the improvement of

surface properties of such dispersions is straightforward and inexpensive. The other is

that polymers with a core-shell structure are perfect model systems for investigating the

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple spherical

geometry of the system.

Their properties usually depend on the structures of latex particles. Chen and his

coworkers [Chen et al. 1991; 1992; 1993] reported the morphological development of

core shell latex particles of polystyrene/poly(methyl methacrylate) during

polymerization. Before the research by Chen and his coworker, Min et al. [Min et al.

1983] reported the morphological development of core shell latex of polystyrene

(PS)/polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the









addition method of PS. They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction, and the PBA-PS core-shell particles with a high degree of

grafting remained spherical upon aging test because of the emulsifying ability of graft

copolymer.

2.6.7 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al. 1994], especially as the solid phase such as in immunoassays

[Chern et al. 2003; Radomske-Galant et al. 2003], DNA diagnostic, drug delivery carriers

[Luck et al. 1998; Kurisawa et al. 1995; Yang et al. 2000], blood cell separations, and

column packing reagents. Thus protein adsorption on polymeric solid surfaces has

become a center of attention. Of particular interest are microspheres, defined as "fine

polymer particles having diameters in the range of 0.1 to several microns", which can be

used as functional tools by themselves or by coupling with biocompounds. Singer and

Plotz [1956] firstly studied microsphere, or latex agglutination test (LAT's), by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support on

which the biomolecules were going to adsorb. The biomolecule adsorption, however, was

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed. Since this work was published, the latex particle applications for

immunoassay have been rapidly and widely studied and developed.

Latex agglutination test, or latex immunoassays, start with tiny, spherical latex

particles with a uniform diameter and similar surface properties. The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles. If sensitized particles are mixed with a sample

containing antigen, urine, serum, etc. The latex will become agglutinated and visibly









agglomerate. Latex tests are inexpensive as compared with the other techniques [Bangs

1988].

Unipath [Percival 1996] has manufactured a range of immunoassay materials based

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments. The latex particles, which are already sensitized with a

monoclonal antibody, can detect any antigens that bound to the surface of the latex

particles. Some detectable pollutants include estrogen mimics, which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminization. Rheumatoid factor (RF) in different age subpopulations has also been

evaluated according to a patient's clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al. 1998]. Magalhaes and his coworkers [2004] studied a

diagnostic method of contamination of male fishes by estrogen mimics, using the

production of vitellogenin (VTG) as a biomarker. This was based on a reverse latex

agglutination test, developed with monoclonal antibodies specific to this biomarker.

Premstaller and his coworkers [Premstaller et al. 2000; 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids. They

used a porogen, a mixture of tetrahydrofuran and decanol, to fabricate a micropellicular

PS-DVB backbone. Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture.









Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecule

adsorption through a two-step emulsion polymerization process. They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature, the amount and type of redox initiator system

used, the type of addition of the initiator system, and the use of washing. They concluded

that the relation between the amount of iron sulfate and the persulfate/bisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization.

A semi-continuous emulsion polymerization technique for latex particle synthesis

was performed by McDonald and other researchers [Ramakrishnan et al. 2004: Steve et

al. 1999]. They introduced a variety of particles sizes, compositions, morphologies, and

surface modifications to fabricate latex composite membranes (LCMs). Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles, which serve as separation

channels. They investigated the membrane performance using gas fluxes, water

permeability, and the retention characterization of dextran molecules. From these tests

they concluded that the narrow, discriminating layer made of the latex particles leads to a

highly efficient composite membrane.

2.7 Proteins

Proteins are natural polyamides comprised of about 20 different a-amino acids of

varying hydrophobicity [Norde 1998]. Proteins are more or less amphiphilic and usually

highly surface active because of the number of amino acid residues in the side groups

along the polypeptide chain which contain positive or negative charges [Norde 1998].









Proteins are polymers of L-a-amino acids [Solomon and Fryhle 2000]. The a refers to a

carbon with a primary amine, a carboxylic acid, a hydrogen and a variable side-chain

group designated as R. Carbon atoms with four different groups are asymmetric and can

exhibit two different spatial arrangements (L and D configurations) due to the tetrahedral

nature of the bonds. The L refers to one of these two possible configurations. Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions. Figure 2-8 shows the chiral carbon in 3-D as the L isomer. Proteins

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998]. The side-chain groups have different chemical properties such as polarity, charge,

and size, and influence the chemical properties of proteins as well as determine the

overall structure of the protein. For instance, the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are on

the inside forming a hydrophobic core.

0

C-OH

H2N-HC

R
Figure 2-8. L-a-amino acid.

The covalent linkage between two amino acids is known as a peptide bond. A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water.

This arrangement gives the protein chain a polarity such that one end will have a free

amino group, called the N-terminus, and the other end will have a free carboxyl group,

called the C-terminus [Solomon and Fryhle 2000]. Peptide bonds tend to be planar and









give the polypeptide backbone rigidity. Rotation can still occur around both of the a-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups. Although many conformations are theoretically

possible, interactions between the R-groups will limit the number of potential

conformations and proteins tend to form a single functional conformation. In other words,

the conformation, or shape of the protein, is due to the interactions of the chain side

groups with one another and with the polypeptide backbone. The interactions can be

between amino acids that are close together, as in a poly-peptide; between groups that are

further apart, as in amino acids; or even on between groups on different polypeptides all

together. These different types of interactions are often discussed in terms of primary,

secondary, tertiary and quaternary protein structure.

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986]. For example, certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as P-sheets or a-helices. In the a-helix

conformation, the peptide backbone takes on a 'spiral staircase' shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue. This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure.

Certain amino acids promote the formation of either a-helices or P-sheets due to the

nature of the side-chain groups. Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone, which is often

called the random coil conformation. These secondary structures can interact with other

secondary structures within the same polypeptide to form motifs or domains (i.e., a









tertiary structure). A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently. Many proteins are composed of

multiple subunits and therefore exhibit quaternary structures.

2.7.1 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact, ordered conformations. In such a

compact conformation, the movement along the polypeptide chain is severely restricted,

implying a low conformational entropy. The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998]. Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process. Knowledge of the major interaction forces that act

between protein chains and control the protein structures helps to understand the behavior

of proteins at interface. These forces include Coulomb interaction, hydrogen bonds,

hydrophobic interaction, and van der Waals interactions.

Coulombic interaction. Most of the amino acid residues carrying electric charge

are located at the aqueous boundary of the protein molecule. An the isoelectric point

(IEP) of the protein, where the positive and negative charges are more or less evenly

distributed over the protein molecule, intramolecular electrostatic attraction makes a

compact structure favorable to proteins. Deviation to either more positive or more

negative charge, however, leads to intramolecular repulsion and encourages an expanded

structure. Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical









molecule (protein) over which the charge is spread out. From the results, he found that

the repulsion force was reduced at higher ionic strength due to the screening action of ion.

Hydrogen bond. Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998]. The number of available

hydrogen bonds involving peptide units is therefore far greater than that involving side

chains. Because a-helices and P-sheets are aligned more or less parallel to each other, the

interchain hydrogen bonds enforce each other. Kresheck and Klotz [1969] examined the

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein. However, because the

peptide chain is shielded from water due to other interactions, hydrogen bonding between

peptide groups do stabilize a-helical and P-sheet structures.

Hydrophobic interaction. Hydrophobic interaction refers to the spontaneous

dehydration and subsequent aggregation of non-polar components in an aqueous

environment. In aqueous solutions of proteins, the various non-polar amino acid residues

will be found in the interior of the molecule, thus shielded from water. The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959]. If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecule,

intramolecular hydrophobic interaction would cause a compact protein structure.

However, geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environment.

Van der Waals interaction. The mutual interaction between ionic groups, dipoles

and induced dipoles in a protein molecule cannot be established as long as the protein









structure is not known in great detail. Moreover, the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions with groups of

the protein molecule. Dispersion interactions favor a compact structure. However,

because the Hamaker constant for proteins is only a little larger than that of water, the

resulting effect is relatively small [Nir 1977].

2.7.2 P2-Microglobulin (P2M)

The protein P2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al. 1985; Argiles et al. 1996;

Floege 2001]. DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al. 1986; Drueke 2000]. DRA develops when

proteins in the blood deposit on joints and tendons, causing pain, stiffness, and fluid in

the joints, as is the case with arthritis. In vivo, 02M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I). As part of its

normal catabolic cycle, 32M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95%) is degraded [Floege 2001]. Renal

failure disrupts the clearance of p2M from the serum, resulting in an increase in 32M

concentration by up to 60-fold [Floege 2001]. By a mechanism that is currently not well

understood, P2M then self-associates into amyloid fibrils and typically accumulates in the

musculoskeletal system [Homma et al. 1989]. Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA)

is present as of full-length wild-type 32M, although significant amounts (-20-30%) of

truncated or modified forms of the protein are also present [Floege 2001; Bellotti et al.

1998]. Figure 2-9 shows the ribbon diagram of human 32M. Native 32M consists of a

single chain of 100 amino acid residues and has a seven stranded P-sandwich fold, typical










of the immunoglobulin superfamily [Saper et al. 1991; Trinh et al. 2002]. 02M was first

isolated from human urine and characterized by Berggard et al. [1980] in1968. The

normal serum concentration of p2M is 1.0 to 2.5 mg/L. It is a small globular protein with

a molecular weight of 11.8 kDa, a Strokes radius of 16A, and a negative charge under

physiological conditions (isoelectric point, IEP = 5.7). 32M contains two P-sheets that are

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al. 1980; Parker et al. 1982; Cunningham et al. 1973]. 32M cannot be

removed completely by current dialysis techniques but through a better understanding of

the structure and interaction forces that lead to this structure, it will be possible to more

efficiently remove this problematic protein.





al'. 2000] "J ^tS^
R3A
-ili30% N661
N-lernlinus .... 7A
.370 % k1
H84\ "- .-% ,17l>
i '


Figure 2-9. Ribbon diagram of human 32M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khan et
al. 2000]

2.7.3 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35-50g/L. According to extensive studies about

its physiological and pharmacological properties, albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2, Zn+2), fatty acids, amino acids,

metabolites, and many drug compounds [Fehske et al. 1981; Kraghhansen 1981; Putnam









1984; Peters 1985]. The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs, as well as to maintain the pH

and osmotic pressure of the plasma. Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40A [Peter 1985]. The primary structure is a

single helical polypeptide of 66 kDa (IEP = 4.7) with 585 residues containing 17 pairs of

disulfide bridges and one free cysteine [Dugaiczyk et al. 1982]. BSA has been classified

as a soft and flexible protein because it has a great tendency to change its conformation

on adsorption to solid surfaces [Kondo et al. 1991; Norde et al. 1992; Soderquist et al.

1980; Carter and Ho, 1994] and consists of three homologous domains (I-III) most likely

derived through gene multiplication [Brown 1976]. Each domain is composed of A and B

sub-domains [He et al. 1992]. The secondary structure of human serum albumin (HSA) is

shown in Figure 2-10.




IliB IA







ib
7
IIIA
3 IIA



IIB

Figure 2-10. Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al. 2003].

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976]. Although all three domains of the albumin molecule have

similar three-dimensional structures, their assembly is highly asymmetric [Sugio et al.









1999]. Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds. In contrast,

domain III protrudes from sub-domain IIB at a 450 angle to form the Y-shaped assembly

for domains II and III. Domain III interacts only with sub-domain IIB. These features

make the albumin molecule heart-shaped.

2.8 Protein Adsorption

Protein adsorption studies date back to the 1930's. At the beginning, these studies

mainly focused on the determination of the molecular weight, electrophoretic and

chromatographic applications. Later, the adsorption mechanism, especially the structural

rearrangements was studied. Recently, the studies of the relation between protein

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986].

2.8.1 Interaction between Protein Molecule and Latex Particle

There are interaction forces at the interfaces between protein molecules and latex

particles. These forces are mainly divided into the following groups; hydrophobic

interaction, ionic interaction, hydrogen bonding, and van der Waals interaction [Andrade,

1985].

Hydrophobic interaction. It is known that hydrophobic interaction has a major

role in protein adsorption phenomena. The adsorption of proteins on the low charged

latex particles occurs by this interaction force. Generally, monomers such as styrene offer

hydrophobic surfaces that protein molecules adsorb to. The amount of adsorbed protein

by this interaction force is maximum at the isoelectric point (IEP) of the protein, and the

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

strength [Suzawa et al. 1980; 1982; Shirahama et al. 1989; Kondo et al. 1992]. By the









reports [Suzawa et al. 1980; 1982; Lee et al. 1988], protein adsorption was greater on a

hydrophobic surface than on a hydrophilic one, implying that hydrophobic interaction is

one of most dominant forces in protein adsorption.

Ionic interaction. Negatively charged latex particles have ionic functional

groups on their surfaces, such as salts of sulfonic and carboxylic acid. Sulfate groups

originate from an initiator such as sodium persulfate, and carboxylic groups originate

from a hydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA).

Ionic bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules. The conventional low-charged latex particles rarely

form these ionic bonds.

Hydrogen bonding. Hydrogen bond is a strong secondary interatomic bond that

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999]. Protein can be adsorbed on hydrophilic polar surfaces

through hydrogen bonding. Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl. Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption.

Van der Waals interaction. This interaction force is operative over small

distances, only when water has been excluded and the two non-polar groups come close

to each other. Lewin's calculation showed that the van der Waals interaction is negligible

compared with the forces involved in the entropy increases, i.e. hydrophobic interaction

[Lewin 1974].

2.9 Hypothesis for Toxin Removal

As mentioned earlier, insufficient removal of middle molecular range toxins is a

major drawback of current dialysis membrane therapy for end stage renal disease (ESRD)









patients. This may cause destructive arthritis and carpal tunnel syndrome, inhibit the

immune system, and accelerate cardiovascular disease leading to death among dialysis

patients. In order to overcome these complications, artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins by

changing the pore size of dialysis membranes and using new materials that adsorb these

toxins for improved removal characteristics over the last decade. However, removal

efficiency is not as high as those achieved by a normal healthy kidney.

With the knowledge obtained from all above literatures, the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein, 02-Microglobulin (02M), without the removal of serum albumin. This

will be done using a novel membrane design composed of an assembly of engineered

polymeric latex particles synthesized to predetermined specifications. Important factors

will include pore size and surface chemistry.

2.9.1 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al. 1991]. The pore size

is defined as the largest spherical particles that can pass through the interstitial spaces.

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method. Many commercially available latex particles are

synthesized by this technique and are inexpensive. When the defect free membrane with a

monodisperse pore size distribution based on a particular particle array is established, it is

expected to outperform the traditional hemodialysis membrane limitation, which is a

broad range of pore size distribution. When spherical particles pack in regular crystalline

arrays, a number of packing geometries, such as hexagonal closest packing, cubic closest






41


packing, and body-centered cubic packing, are possible. The pore size of the array

depends on these packing geometries as well as particle size. Theoretical pore size can be

calculated from the simple geometry of the array. Figure 2-11 shows the relationship

between particle size and pore size.

1 0 I ..
Body-Centered Cubic Array
......... Closest Packed Array






E 0.1 0.11 10
-- - ----









and closed packed arrays [Steve et al. 1999].
0.1 -------------- -- ---- --- --------------



o 0.1 ;^ -,,






and closed packed arrays [Steve et al. 1999].
0.001 '-------------






McDonald and his coworkers [Ramakrishnan et al. 2004: Steve et al. 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers. It is from this prior research, and knowledge gained from conversations

with Dr. McDonald, that this current research has been initiated [McDonald 2003]. The

focus of this thesis is the extension of McDonald's research in the area of composite

membranes to medical application such as dialysis membranes.

2.9.2 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

material's ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of










separation [Menon et al. 1999; Chun et al. 2002]. First, all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH7.4). It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al. 1971]. The toxin protein, 32-

Microglobulin (02M) can be selectively adsorbed on the surface of laxex particles by the

design of a suitable hydrophobic portioned surface where only 02M protein can be

anchored with hydrophobic interactions. Albumin adsorption on the latex particle is not

allowed because charge repulsion is more dominant than hydrophobic interaction with

side-on mode. The figure 2-12 shows the schematic representation of the selective

adsorption of 02M protein on the engineered latex particles.




PMMA/PAA

Coo-
Charge
repulsion


oso3-


PS l2-Microglobulin
Core

Hydrophobic
COO- Interaction



/ a-Microglobulin
OS03-
Figure 2-12. Schematic representation of the protein adsorption on the core shell latex
particles at pH 7.4


particles at pH 7.4









Hydrophilic/hydrophobic microdomain structures were proven to be more blood

compatible [Mori et al. 1982; Higuchi et al. 1993; Deppisch et al. 1998]. The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility. The monomers, such as styrene (St), methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process.

In summary, the background and fundamental literature survey about the history,

material properties and limitation of hemodialysis membrane; latex particle preparation,

surface chemistry, and manufacturing process; target proteins and protein adsorption; and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested. This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter.














CHAPTER 3
EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier, the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate the

fundamental interactions between the applied particles and molecules in the biological

system to optimize the membrane performance for hemodialysis applications. Polymeric

latex particles with monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary. Surface engineering

with various combination of hydrophobic/hydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption, and to

improve the biocompatibility of membrane. Therefore, the materials and characterization

methodology are addressed in this chapter.

3.1 Materials

Styrene (St) monomer used for a seed and a core particle, was purchased from

Fisher Scientific and used without any other purification process. Acrylic acid (AA), and

methyl methacrylate (MMA) monomers introduced for shell formation, were purchased

from Fisher Scientific and used without any other purification process. Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received. Divinylbenzene (DVB) crossliking agent was purchased from Aldrich. The

anionic surfactant, Aerosol MA80-I [sodium di(1,3-dimethylbutyl) sulfosuccinate], was

kindly donated by Cytec. 78-80% Aerosol MA80-I is mixed with isopropanol and

water. Its critical micelle concentration (CMC) is 24.5 mM (1.19%). Ion exchange resin,









AG 501-x8 Resin (20-50 mesh), was purchased from Bio-Rad Laboratories, Inc. It is a

mixed-bed resin with H and OH- as the ionic forms and is 8% crosslinked. Bovine serum

albumin (BSA) (heat shock treated) was purchased from Fisher Scientific. The isoelectric

point of the BSA is 4.7-4.9 and the molecular weight is 66,300Da. 32-Microglobulin

(02M) was purchase from ICN Biomedicals, Inc., and was separated from patients with

chronic renal disease, and lyophilized from ammonium bicarbonate. The molecular

weight by SDS-PAGE was approximately 12,000Da. BSA and 32M proteins were used as

received without further purification process. In order to investigate the protein

adsorption evaluation, bicinchoninic acid protein assay kits were purchased from Sigma

(Cat # BCA-1) and Pierce Biotechnology (Cat # 23225). The chemical structures for the

main chemicals are shown in Figure 3-1.


CH,= CH



Styrene (Mw = 104.15) Acry


CH2= CH
Divinylbenzene (Mw = 130.2)


CH3
I
CH2- CH CH2= CH
C=O C=O
I I
OH OCH3
lic acid (Mw = 72.06) Methyl methacrylate (Mw = 100.12)
O CH3 CH3
II I I
CH2-C-O-CH-CH2-CH-CH3
I
+(Na) -(O3S) CH-C-O-CH-CH2-CH-CH3
II I I
O CH3 CH3

Anionic surfactant (MA80-I) (Mw = 388.5)


O O
II II
+(Na)(O) S-O-O- S- (O)-(Na)+
II II
O O
Initiator, sodium persulfate (Mw = 238.11)

Figure 3-1. Chemical structure of main chemicals.









3.2 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3. In the experimental setup, a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at the

end of glass stirrer. The agitation rate is precisely adjusted by controller motor. Paar glass

reactor with 1L volume is partially in silicon oil bath on hot plate. The reactor has four

openings for a stirrer, a thermometer, a nitrogen gas inlet, and a reactant feeding Inlet.

The reactants for latex polymerization are fed by precisely controlled by metering pump.

The particle preparation is described in each latex preparation section.















S1. Motor
2. Mechanical stirrer bar
3. Thermometer
4. Inlet of N2
5. Inlet for initiator and monomer
6. Glass reactor (1 L)
7. Oil bath
8. Hot plate
9. Pump for initiator
10. Pump for monomer
Fi e.. ...3. .Expe a 11. Tubes for reactant feed

Figure 3-2. Experimental setup for semi-continuous emulsion polymerization.









Styrene monomer

I e 000 Bare PS
.*.. PS seed
.**
"*.* particles

S* PS/PMMA100




0 .: PS/PMMA90PAA10



0* PS/PMMA7/PAA25
0ee 0 00


Figure 3-3. The particle preparation scheme in various types and size ranges of latex
particles.

3.2.1 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization. Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer, thermometer, glass funnel for

nitrogen gas purge, and a tube for monomer feeding. The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable temperature control. The vessel was

firstly charged with de-ionized water, the emulsifier, sodium persulfate (SPS) initiator,

sodium bicarbonate (SBC), styrene (St) monomer, divinylbenzene (DVB) monomer,

under nitrogen gas atmosphere, and then heated to 72+2 C for Ih. The reaction was

allowed to continue for another hour. Second, reactants St and DVB monomers, SPS

initiator, and SBC were fed separately using fine fluid metering pumps (Model RHSY,

Fluid metering INC, NY) under a nitrogen gas atmosphere for 2h. After complete feeding









of all reactants, the reaction was continued for another hour at elevated temperature, 801

TC and then cooled to room temperature by removal of the oil bath.

3.2.2 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization.

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content, and heated up to 722 TC for Ih under nitrogen gas atmosphere. Then, St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate. After feeding all

reactants, the reaction was continued for three hours keeping temperature of 802 TC and

then the reaction vessel was cooled down to room temperature.

3.2.3 Preparation of Core Shell Latex Particles

Monodisperse, spherical polystyrene seeds, with a 280nm mean diameter were used

for core shell structured latex particle. The pre-polymerized polystyrene seeded emulsion

was charged in a 500ml glass flask under a nitrogen gas atmosphere. De-ionized water

was added to achieve the desired solid content. The emulsion was heated to 80+2C for

1.0 hour. Monomers, initiator, and SBC, to form the shell, were added by fluid metering

pumps (LAB PUMP JR.,RHSY model, Fluid metering Inc. USA) with fine controlled

feed rate. Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100% flow which is in the range of 10ml/min respectively. The reaction was sustained

for additional 3.0 hours at 9020C, then cooled down to room temperature.

3.2.4 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin. 200g (10%

w/w polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers, initiators and other impurities.









Then the emulsion was diluted with DI water to a specific degree of solid content as

needed.

3.3 Characterization

3.3.1 Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al. [1995] before the latex cleaning process.

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120C for 30min to evaporate any unreacted monomers and water. The remaining solid

was weighed and the degree of conversion was determined. This process was repeated

three times and a mean value of the degree of conversion was determined.

3.3.2 Fourier Transform Infrared (FTIR) Spectroscopy

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies. The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al. 1996].

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation. The range of wavelengths for infrared radiation is between

2.5 tm (4000 cm-1) and 25 itm (400cm-1).

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PS/PMMA, PS/PMMA90PAA1o, PS/PMMA75PAA25 core-shell copolymers, were verified

with FTIR. Purified latex particles were dried in vacuum oven at 40C for 24 hours

before analysis in the FTIR (Nicolet Magma, USA). Dried latex (10mg) was mixed with

250 mg of KBr, which had also been dried in vacuum oven at 120C for 3h. Transmission

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution.









3.3.3 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer. The solid concentration of latex particles used was 0.001% (w/v). This

instrument uses photon correlation spectroscopy (PCS) of quasielastically scattered light.

For this technique, time dependent interference patterns of light scattered from particles

in the sample cell are analyzed. The interference pattern changes due to Brownian

motion, giving rise to fluctuations in scattering intensity. The frequency of these

fluctuations depends on the particle's diffusion constant that is inversely proportional to

the particle diameter.

3.3.4 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characterized using a FE-SEM (JEOL

JSM-6335F, Japan). A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry. The sample was then coated with

the thinnest layer of carbon needed to obtain the required conductivity for this instrument.

Secondary electron image mode was used with a 15KV of accelerating voltage. The

magnification range used was between 10,OOOX and 70,OOOX.

3.3.5 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0.01

wt % and adjusted six different pH values: 2.05, 3.45, 4.81, 5.65, 6.43, and 7.47. These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement. At least two runs of ten measurements were taken for each sample and

averaged.

The zeta potential measurement was carried out using Brookhaven ZetaPlus zeta-

potential analyzer. It measures the electrophoretic mobility, velocity of charged, colloidal









particles in solution, and calculate zeta-potential by using the Smoluchowski equation.

The frequency of laser light passing through the sample and is compared to the frequency

of a reference beam. The shift in the frequency, called a Doppler shift, and the magnitude

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility,

respectively. The zeta potential is calculated from the solution conditions and the

measured mobility.

3.3.6 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum albumin (BSA) as a standard model protein and 32-Microglobulin

(P2M) as a target protein in two types of buffer solution, phosphate buffer (PB) and

phosphate buffered saline (PBS). To make 5 mM of PB solution, 0.345g of sodium

phosphate monobasic (NaH2PO4-H20) was dissolve in 500ml of de-ionized water. To

make PBS solution, 0.345g of sodium phosphate monobasic (NaH2PO4-H20) and 4.178g

(143mM) of sodium chloride (NaC1) were dissolved in 500ml of de-ionized water. The

synthesized latex particles were diluted with each buffer to have a solids content of 0.5%

(w/w), adjusted to pH values of 3.2, 4.8, and 7.4, and mixed with the selected protein.

BSA concentrations were chosen to be 0.05, 0.1, 0.3, 0.5, and 0.7 mg/ml and those for

P2M were 0.015, 0.030, 0.045, and 0.060mg/ml. The mixture was gently rotated using

the shaker, Labquake (Barnstead/Thermolyne, Model #4002110, USA), with 8 RPM in

the incubator at 370C for 12 hours before the latex-protein mixture was centrifuged at

13,000 rpm for 15min. The amount of protein adsorbed was determined by quantifying

the free proteins in the supernatant after the centrifugation process using the

bicinchoninic acid (BCA) assay method [Lowry et al. 1951; Smith et al. 1985; Baptista et

al. 2003; Wiehelman et al. 1988; Brown et al. 1989]. BCA assay kits consist of Reagent










A, containing bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium

bicarbonate in 0. IN NaOH with pH=l 1.25 and Reagent B containing 4% (w/v) copper

(II) sulfate pentahydrate. The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B. 1001 of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette. Incubation was allowed to continue further

at room temperature until color developed about 2h. The absorbance at 562nm was

measured using a UV-VIS spectrophotometer (Perkin-Elmer Lambda 800, USA).

Unknown concentration of sample protein was determined by comparison to a standard

of known protein concentrations. The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process. The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4.


Suspend latex particles in aqueous buffer media




SAdd the dissolved target protein




Fgr ncubate mixture with agitation for 12hrs at 37C




Separate the particles in centrifuge, mix supernatant with BCA reagents,
and develop the color by incubation for 2hrs at room temperature




Fiur Measure color intensity using UV/Vis spectroscopy at 562nm a


Figure 3-4. Schematic of the procedure for a protein adsorption test.









3.3.7 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells, which leads to the release of

hemoglobin from within the red blood cells into the blood plasma. Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released. The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes.

Figure 3-5 shows the phase separated blood with plasma, white blood cells, and red blood

cells after centrifugation process. Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 7.4 to remove debris and serum protein. This

process was repeated 3 times.

Plasma



White blood cells

Red blood cells

Figure 3-5. Separation of RBC from whole blood by centrifuge process.

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles. 100[l of the mixture of red blood cell (3 parts)

and PBS (1 Iparts) was added to lml of 0.5% (w/w) particle suspension. PBS was used as

a negative control resulting 0% hemolysis and DI water used as a positive control to

produce 100% hemoglobin released by completely destroyed RBCs. The mixture was

incubated in water bath with gentle shaking for 30 minutes at 370C and then centrifuged

at 1500 rpm for 15 minutes. 100[l of the supernatant was mixed with 2ml of the mixture

of ethanol (99%) and hydrochloric acid (37%) (EtOH/HCl = 200/5, w/w) to prevent the









precipitation of hemoglobin. In order to remove remaining particles, the mixture was

centrifuged again with 13,000 RPM at room temperature. The supernatant was then

transferred into the UV cuvette. The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm. The schematic of the

procedure for hemolysis test is shown in Figure 3-6.


Separate red blood cells (RBCs) from blood

-ZL

Mix RBCs with phosphate buffer solution (PBS)


Wash and re-suspend RBC with PBS; repeat three times



Add latex particle suspension into 100ml of RBC
and incubate sample with agitation for 30min at 370C



Separate particles and mix the supernatant with EtOH/HCl solution



Evaluate free hemoglobin in the solution using UV/Vis spectroscopy at 397nm


Figure 3-6. Schematic of the procedure for hemolysis test.

In summary, a description of the experimental materials and characterization

methodology has been explained. In the next chapter, the results and discussion of the

data obtained from these experiments is addressed.














CHAPTER 4
RESULTS AND DISCUSSION

4.1 Polymerization of Latex Particles

4.1.1 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles,

lubricants, packing materials for chromatography, standard particles and diagnostic

drugs. All applications strongly require these particles to have a uniform particle size.

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discrimination [Jons

et al. 1999; Ramakrishnan et al. 2004]. Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles,

the uniformity of the recovered particles is insufficient for membrane use. There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

increase the sizes of the seed particles uniformly. In order to obtain monodisperse larger

particles by this method, the procedure of absorption of monomer into fine polymer

particles and polymerization of the monomer is repeated.

In this study, cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer, divinylbenzene crosslink agent, sodium persulfate

initiator, sodium bicarbonate buffer, and the Aerosol MA80-I [sodium di(1,3-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process, in which one or more components can be added continuously.









Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles. Various profiles of

particle nucleation and growth can be generated from different orders of component

addition during polymerization. There are several advantages of this process such as easy

control of the polymerization rate, monodisperse particle size and particle number,

colloidal stability, copolymer composition, and particle morphology. The main goal in

this process is to avoid secondary particle formation leading to a monodisperse particle

size distribution. Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth.

Seed particles Add monomers Enlarged final
Seed code : Amount of surfactants : Number of particles particles
is variable are constant
PS, .5



PS, *0** 0*
Monomer droplet Ps2 )1 Ae 0 0



PS, .-.-.-..-
.. ** *









Figure 4-1. Schematic representation of semi-continuous seed latex particles preparation
and growth.

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants, styrene, DVB, and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribution. The

recipes and reaction conditions for synthesizing these seed particles are summarized in









Table 4-1. The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003].

Table 4-1. The polymerization recipe of polystyrene (PS) seed particles.
Latex label PSs2.59 PSs2.33 PSs2.o7 PSsi.81

Temperature (C) 75 5 75 5 75 5 75 5
Water (g) 131.5 131.5 131.5 131.5
Styrene (g) 99.48 99.48 99.48 99.48
Divinylbenzene (g) 0.52 0.52 0.52 0.52
MA 80 (g) 2.59 2.33 2.07 1.81
NaHCO3 (g) 1.12 1.12 1.12 1.12
Na2S20 (g) 1.90 1.90 1.90 1.90
Waterb (g) 20.0 20.0 20.0 20.0
Reaction time (min) 140 140 140 140
% Solids content 39.6 39.6 39.6 39.6
Mean particle diameter (nm) 1267.5 1713.9 1824.1 2165.3
Conversion (%) 96.5 97.9 95.7 97.1


Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles.

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant concentration, where particles are stable [Nestor et al. 2005]. A wide range of

surfactant concentrations causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion system. Therefore, the range of surfactant

amount was varied from 1.81g to 2.59g in increments of 0.26g in each latex label. Other

reactants amounts were kept constant. Water is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer,









which are fed continuously after the seed particle creation. The extent of monomer to

polymer conversion was obtained by gravimetric calculation and was more than 95% at

all seed particles labeled PSs2.59, PSs2.33, PSs2.07, and PSsl.81. Labeled subscripts, such as

S2.59, S2.33, S2.07 and S1.81, indicate the surfactant amount added for the seed latex

preparation. The conversion indicates the percentage of experimentally obtained solid

content divided by the theoretically estimated solid content and was calculated by the

following formula:

Cn () Experimental solid content
Conversion (%) = x100 (4-1)
Estimated solid content

In this equation, the experimental solid content is the weight of the solid remaining after

evaporation of emulsion at 120C for 30min and the estimated solid content is the weight

of monomers calculated from the recipe.

As expected, the seed particle size was different for each surfactant loading in seed

emulsion system. A sample of each suspension was diluted by 0.001% (w/w) with

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer. From these results, the seed latex particle size increased

as the amount of surfactant decreased. The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size. It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work.

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles. The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2.





















































Figure 4-2. Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSs2.59 (B) PSs2.33 (C) PSs2.07 (D) PSsi.81 (subscripts indicate the amount of
surfactant added)



























































Figure 4-2. Continued.









As seen in the image, the seed particles are very smooth and spherical. The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image. From this data, we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth, spherical morphology.

4.1.2 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades, and

various mechanisms have been proposed. Gracio et al. [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell, with the outer shell providing a major locus of polymerization. Seeded

emulsion polymerization is commonly used for preparing latex particles less than 1 lm in

size [Cha et al. 1995; Zou et al. 1990; 1992; Gandhi et al. 1990; Park et al. 1990]. Such

latexes can be obtained from the growth of pre-prepared seed particles. The seeds

introduced in this process serve as nucleation sites for particle growth. By first swelling

the seed latex particles with additional monomer to the desired size, polymerization can

then be initiated. Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method. For the

fabrication of particle-based membranes, monodisperse latex particles with a variety of

size ranges are required.

In this study, seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used. Water is the initial charge

amount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system. Feed time is precisely controlled by

metering pump. The % solid contents were maintained between 19.9% and 30.0%. After









polymerization under these conditions, the conversions of the latex particles labeled as

PS258, PS320, PS370, and PS410, were 95.2, 95.7, 94.6 and 96.4%, respectively. The recipes

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2. The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525, PS585, PS640, and PS790, were 92.3, 84.02,

72.3 and 96.9%, respectively. The recipes used in the preparation of these latex particles

are shown at the Table 4-3.

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization. Let's suppose that seed density (ps) and grown

particle density (pgp) are the same. The following equation is obtained from the mass

balance (m = pV), where m and V are mass and volume of a particle, respectively.

ms m
Smp (4-2)
Vs Vgp

In this equation, ms and mgp are the weight of seed and total weight of seed and

added monomers, respectively; and Vs and Vgp are the volume of seed and grown

particle, respectively.

If we know the mass of monomer to be added, the ultimate particle size can be

calculated from the equation 4-3.

ms D, (4-3)
ms +Z D2

where Ds and Dgp are the diameters for seed and grown particle, respectively, and x is the

weight of the monomer added. Crosslinked PS latex particles with a size range from

25811.2 410+11.7 in diameter, polymerized by the recipes in Table 4-2 are shown in

the SEM images in Figure 4-3.









Table 4-2. Continuous addition emulsion polymerization recipe for growing polystyrene
(PS) latex particles less than 500nm in size.
Latex label PS258 PS320 PS370 PS410


Initial charge

*Seed latex (g)

Water (g)

Monomer continuous

Styrene (g)

Divinylbenzene (g)

Feed time (min)


100.0171

147.0


80.0

0.6
0-163


100.0216

250.0


120.0

0.7

0-110


100.0216

198.4


80.0

0.6

0-75


100.0320

260.0


60.0

0.6

0-82


Initiator stream

Na2S208 (g) 0.7 0.65 0.5 0.5

NaHC3 (g) 0.7 0.65 0.5 0.5

Water (g) 75.0 120.0 20.0 37.0

Feed time (min) 0-167 0-140 0-100 0-92

% Solid content 28.4 30.0 30.0 20.0

Mean particle diameter (nm) 25811.2 32015.4 1827.1 410+11.7

Conversion (%) 95.2 95.7 94.6 96.4
* The solid content of seed latex for PS258, PS320, PS 370, and PS410 are 39.2, 39.9, 39.9, and 30.0%,
respectively.











Table 4-3. Continuous addition emulsion polymerization recipe for growing polystyrene
(PS) latex particles larger than 500nm in size.
Latex label PS525 PS585 PS640 PS790


Initial charge

*Seed latex (g)

Water (g)

Monomer continuous

Styrene (g)

Divinylbenzene (g)

Feed time (min)


100.0390

100.0


29.0

0.2

0-80


100.0470

52.0


20.0

0.14

0-40


100.0470

120.0


40.0

0.3

10-76


100.0320

36.0


17.3

0.09

5-65


Initiator streNa

Na2S2Os (g) 0.2 0.14 0.3 0.12

NaHC03(g) 0.2 0.14 0.3 0.09

Water (g) 20.0 120 40.0 20.0

Feed time (min) 0-85 0-46 0-77 0-75

% Solids content 19.9 20.0 20.0 20.1

Mean particle diameter (nm) 52515.1 58518.6 64017.8 79069.3

Conversion (%) 92.3 84.02 72.3 96.9
* The solid content of seed latex for PS525, PS585, PS 640, and PS790 are 20.0, 18.2, 19.3, and 17.3%,
respectively.






























N *1

aI 4




p. V
4 ..^ A4
-4' 3 -4.

b- < 'c

/4 Al -.^ 1


4 .


Figure 4-3. SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm


a (A A


L. 1







'.4
4



FF


4 1
4


Se*

'4 .


4

V-..*


4%
*0

ON -




-t




























































Figure 4-3. Continued.









Particle size was well matched to that estimated by the geometrical calculation.

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion. From the SEM characterization, newly

nucleated particles were not seen, as indicated by the narrow particle size distribution.

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution. As the particles were grown larger than 500nm

mean diameter, however, they became of non-spherical shape with an uneven surface.

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer, which can be controlled by the factors such

as temperature, agitation speed, initiator feeding rate, and surfactant amount required to

stabilize the colloidal system. The SEM image of latex particles larger than 500nm mean

diameter is shown in Figure 4-4. The particle size distribution is broader than that of the

smaller particles. These particles can, however, still be used for membrane construction

as a support layer rather than skin layer. The support layer does not necessarily have to be

as highly monodisperse as the skin layer, which require pores of high uniformity for the

selective removal of toxins.

As mentioned earlier, the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991]. Anionic

surfactants are generally recommended at the level of 0.2-3.0 wt % based on the amount

of water [Odian 1991]. The critical micelle concentration (CMC) of Aerosol' MA 80-I

anionic surfactant is 1.19 %.The surfactant loading in this emulsion system ranged from

the 1.21 % for Ss.81s to 1.71% for SS2.59.





















































Figure 4-4. SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm




























































Figure 4-4. Continued.









The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends. The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion. As the surfactant to monomer ratio decreases, mean particle diameter

increases, as described by Odian [1991]. Generally, lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al. 1999]. However, there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0.005 and

0.013. This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected. This is not a factor of surfactant amount.

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5.


1000


5 800


E 600
20 0
c
400
E

0 200


0
0.000 0.005 0.010 0.015 0.020 0.025 0.030

Surfactant to monomer ratio (w/w)


Figure 4-5. Dependence of the particle size on the surfactant to monomer ratio.








4.1.3 Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 were

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology. Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that of bare

polystyrene particles. AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water. The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al. 2005]. MMA monomer is less hydrophobic than

styrene. PMMA is known to more biocompatible than PS but still PMMA is hydrophobic.

The optimization of suitable hydrophobic to hydrophilic ratios and control of

microdomain structures is important for biocompatibility [Mori et al. 1982; Higuchi et al.

1993; Deppisch et al. 1998]. Figure 4-6 shows the schematic of core shell latex particle

structure. The recipe for core shell structures is shown in Table 4-4.

Core shell
latex particles

Polystyrene PS/PMMAo10
seed particle W



PS/PMMA90PAAIo




L PS/PMMA75PAA25


Figure 4-6. Schematic of core shell latex particle structures.









Table 4-4. The preparation recipe of PS core with various shell latex particles.
Latex label PS/PMMAloo PS/PMMA90PAAlo PS/PMMA75PAA25

Initial charge

PS seed latex (g) 50.0258 50.0258 50.0258
Water (g) 157.0 157.0 157.0
Monomer continuous

Methly methacylate (g) 32.0 28.8 24.0
Acrylic acid (g) 0.0 3.2 8.0

Feed time (min) 0-120 0-50 0-60
Initiator stream

Na2S2Os (g) 0.2 0.2 0.2
NaHC03 (g) 0.2 0.2 0.2
Water (g) 20.0 20.0 20.0
Feed time (min) 0-122 0-60 0-65
% Solids content 18.3 18.3 18.3

Mean particle diameter (nm) 37018.6 37017.8 37019.1
Conversion (%) 95.5 92.4 95.2



Three types of core shell structures were prepared. PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75% to 25% by weight.

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90% to 10% by

weight. PMMA100 is the PMMA homopolymer shell on PS core. The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential, protein adsorption,

and biocompatibility. The image of SEM of PS and core shell particles is shown at Figure

4-7. The synthesized latex particles had a high uniformity in size and a smooth, spherical

surface morphology.





















































Figure 4-7. Scanning Electron Micrograph of latex particles (A) PS (B) PS/PMMAloo (C)
PS/PMMA9oPAAlo (D) PS/PMMA75PAA25





























































Figure 4-7. Continued.









4.2 Characterization of Latex Particles

4.2.1 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials. A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies. The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al. 1996].

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation. The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8. There are number of characteristic peaks for PS latex

particles [Bhutto et al. 2003; Li at al. 2005]. Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1, and aliphatic C-H asymmetrical and symmetrical stretching

is shown at 2900 cm-land 2850 cm-1, respectively. There is an additional carbonyl (C=0)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA. The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell. For example, the peak intensity of OH groups for

PS/PMMA75PAA25 is greater than that of PS/PMMA90PAAlo since there is a higher AA

monomer content in PS/PMMA75PAA25 than in PS/PMMA90PAAio. This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains.

4.2.2 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

latex particles. Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems. The greater the zeta



















































4000 3500 3000 2500 2000


1500 1000


500


Wavenumbers (cm )


Figure 4-8. FTIR spectra of polymerized latex particles. (A) bare polystyrene (PS) (B)
PS/PMMAloo (C) PS/PMMA9oPAAlo (D) PS/PMMA75PAA25









potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate. The measurement of

zeta potential is often the key to understanding and control of the interaction of proteins

with solid surfaces such as latex particles.

Zeta potential () is the electrical potential that exists at the shear plane of a particle,

which is some small distance from the surface [Myers 1999]. The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field, termed electrophoretic mobility, or its response to an alternating electric

field. Colloidal particles dispersed in a solution are electrically charged due to their ionic

characteristics and dipolar attributes. The development of a net charge at the particle

surface affects the distribution of ions in the neighboring interfacial region, resulting in

an increased concentration of counter ions to the surface. Each particle dispersed in a

solution is surrounded by oppositely charged ions called fixed layer or Stem layer.

Outside the Stern layer, there are varying compositions of ions of opposite polarities,

forming a cloud-like area. Thus an electrical double layer is formed in the region of the

particle-liquid interface. This double layer may be considered to consist of two parts: an

inner region which includes ions bound relatively strongly to the surface and an outer, or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion. The potential in this region, therefore, decays with the

distance from the surface, until at a certain distance it becomes zero. Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface.

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

center of those ions, which are effectively adsorbed to the surface [Hunter 1981]. The








extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles. The ions outside of the Stern layer

form the diffuse double layer, also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000].


Slern surface
diffaue
double laytr




^G
.,. (+


0





0





sudrice of shear
Stem layer


Shear plane


Figure 4-9. Schematic representation of ion distribution near a positively charged surface.
Zeta potential is a function of the surface charge of a particle, any adsorbed layer at

the interface and the nature and composition of the surrounding medium in which the

particle is suspended. The principle of determining zeta potential is very simple. A

controlled electric field is applied via electrodes immersed in a sample suspension and


uspension and










this causes the charged particles to move towards the electrode of opposite polarity.

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity drag. The particle therefore, reaches a constant terminal velocity.

Inside of the cell measurementt container)
ElectWBde ft"SU wowsK EBorode


++* S kln


(tonic lowyhd lay) r

of eMasuMwnt
Figure 4-10. Schematic representation of zeta potential measurement. (source:
http://nition.com/en/products/zeecom_s.htm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems, protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins, a well-characterized adsorbent, and

a well-defined aqueous solution. Even so, small changes in the experimental conditions,

such as pH, ionic strength, and temperature generate totally different results. Therefore,

two media systems, phosphate buffer (PB) and phosphate buffered saline (PBS), were

chosen to be used for the zeta potential measurements and protein adsorption study. PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4-H20) to

maintain a constant pH. PBS is used to keep the water concentration inside and outside of

the cell balanced. If the water concentration is unbalanced, the cell risks bursting, or

shriveling up because of a phenomenon called osmosis. PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4-H20) and 143 mM of sodium chloride (NaC1)

in water.


led osmosis. PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4-H20) and 143 mM of sodium chloride (NaC1)

in water.










The results of zeta potential measurement at room temperature (25+1C) as

function of pH and ionic strength (different media, PB and PBS) for synthesized latex

particles are seen in Figure 4-11. Zeta potential values of polystyrene (PS) latex particles

at the Figure 4-9 are negative between -29.1 mV and -59.9 mV in PB and between -20.3

mV and -27.8 mV in PBS at all pH ranges. These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al. 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated.


40
--PB (5mM)
20
S-* PBS (5mM +143mM)

E 0.
&_P 2 3 4 5 6 7 8
-20

-40

S-60
N
-80

-100

pH

Figure 4-11. Zeta potential of PS latex particles at 250C.

The zeta potential measurements of PS/PMMA1oo core shell latex particles, as seen

in Figure 4-12, were also negative with a range of-28 and -50.5 mV in PB and between -

14.3 mV and -18.6 mV in PBS at the pH 2.1-7.8. These negative values are also due to

sulfate groups on the particle surface.










40
-- PB (5mM)
20
S- -PBS(5mM+143mM)
E 0o...

20

I -40
0
-60
N
-80

-100

pH

Figure 4-12. Zeta potential of PS/PMMA1oo latex particles at 250C.

The zeta potential for PS/PMMA90PAAlo and PS/PMMA75PAA25 was also

negative, as seen in Figure 4-13 and 4-14. The zeta potential value for the

PS/PMMA90PAAlo were between -36.7 mV and -67.8 mV in PB medium and between -

14.7 mV and -19.3 mV in PBS both at 25C. In case of the PS/PMMA75PAA25 core shell

particles, zeta potential values were between -29.1 mV and -52.0 mV in PB and between

-11.5 mV and -21.0 mV in PBS media. Sulfate groups in PS/PMMA75PAA25 and

PS/PMMA90PAAlo core shell particles contribute to the negative zeta potential values.

Carboxylic groups from acrylic acid monomer also would participate in forming a

negative surface charge of the latex particles at a pH greater than 7.0 because the pKa

value for the carboxylic acid is between 4.0 and 6.0 pH range [Ottewill et al. 1967]. This

indicates that carboxylic acid dissociation begins at a pH between pH 4.6-6.0 and

increases with pH. The strength of an acid is expressed by the term pKa, which is the pH

at which an acid is 50% dissociated. However, the contribution of the carboxylic acid







82


group to negative zeta potential values of PS/PMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 7.0.


40
-- PB (5mM)
20' 1-- PBS(5mM+143mM)

E 0o.
3 4 5 6 7 8
-20 -

-40 -



N
-80

-100

pH

Figure 4-13. Zeta potential of PS/PMMA90PAAlo latex particles at 250C.

40
-- PB (5mM)
20. --PBS(5mM+143mM)

E 0.
4- 5 6 7 8
-20

-40-

-60.
N
-80

-100

pH

Figure 4-14. Zeta potential of PS/PMMA75PAA25 latex particles at 250C.









The zeta potential plots and values were similar for all latex particles. This may be

due to the similar initiator density on the latex surface. The initiator concentrations to

monomers for shell polymerization were the same for all core shell latex particles (0.62%

w/w from Table 4-4). A similar concentration (0.55% w/w) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements.

Since the zeta potential values of all synthesized latex particles were negative

between pH=2.0 and pH=7.8, the isoelecric point (IEP) of these latex particles would be

less than pH=2.0. Sulfates have a pKa in the range of 1.0 to 2.0 [James 1985], indicating

that the sulfate group, the conjugate base of a strong acid, is protonated (-C-OS03H) at

less than pH 2.0. This means that the synthesized latex particles are all negative at the

physiological pH. Most serum proteins have a negative surface charge in blood,

therefore, when the negative latex particles are applied to blood stream, fatal

complication by coagulation between applied latex particles and serum proteins is

avoided by charge repulsion in the blood. Table 4-5 shows the common blood proteins

and their isoelectric points (IEP).

Table 4-5. The isoelectric point (IEP) of common proteins.
Serum proteins Isoelectric point (IEP)
Albumin 4.8
al-Globulin 2.0
a2-Microglobulin 5.4
yi-Globulin 5.8
Heptoglobin 4.1
Hemoglobin 7.2
Pi-lipoprotein 5.4
Immunoglobulin G (Ig G) 7.3
Fibrinogen 5.8
(source: http://www.fleshandbones.com/readingroom/pdf/1085.pdf;
http://www.medal.org/visitor/www%5CActive%5Cchl3%5Cchl3.01%5Cchl3.01.05.asp
x)









The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS). This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential. Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system.


1.0





(3) (2) (1)
)1/e



1/K3 1/K2 1/K1 Z
Figure 4-15. The decay of surface potential with distance from surface in various
electrolyte concentrations: (1) low (2) intermediate (3) high [Myers 1999].

The electrical potential in the solution surrounding the particle surface falls off

exponentially with distance (Z) from the surface according to the Debye-Huckel

approximation [Myers 1999]

V = V/ exp (-K z) (4-4)

where K is the reciprocal of the thickness of the electrical double layer, referred to as the

Debye length. The potential fallen off by a factor of 1/e. The theoretical equation for the

double layer thickness, 1/K, is


1 ( Ce kT
-= 2Ygcz2 (4-5)


2









where S0 is the permittivity of a vacuum of free space, P is the permittivity of a medium, e

is the relative permittivity or dielectric constant of the medium, c is the concentration of

an ion (i), z is the valency of an ion (i), k is Boltzmann's constant, T is absolute

temperature (K).

4.3 Protein Adsorption Study

The adsorption study was performed to measure the protein adsorption on the

synthesized latex particles. Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982]. This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase. There are two

adsorption processes, physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases, and chemical adsorption or chemisorption

occurring when atoms and molecules from adjacent phases form chemical bonds at the

interface [Jaroniec et al. 1988].

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have. Adsorption leads

to a reduction of this conformational entropy, hence, adsorption takes place only if the

loss in conformational entropy is compensated by sufficient attraction between polymer

segment and solid surface [Martin 1998]. Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass, F is plotted

against the polymer concentration in solution after adsorption, Cp. The initial part of the

isotherm merges with the F-axis because at low polymer concentration, the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated.