MACROPOROUS MONOLITHIC CRYOGELS

FOR EXTRACORPOREAL MEDICAL DEVICES

Wuraola Akande

A thesis submitted in partial fulfilment of the

requirement of the University of Brighton for the

degree of Doctor of Philosophy

December 2012

UNIVERSITY OF BRIGHTON

Abstract

Cytapheresis is an extracorporeal separation technique widely used in medicine for elimination of specific classes of blood cells from circulating blood. It has been shown recently to have clinical efficacy in various disease states, such as leukaemia, autoimmune disorders, rheumatoid arthritis, renal allograft rejection and sickle–cell anaemia. There are two major methods of extracorporeal leukocyte removal therapy in use in the clinical field, these are the centrifugal method, and the adsorptive method with fibre or beads. Leukocytapheresis using the leukocyte filter Cellsorba and granulocytapheresis using an Adacolumn has been proven to reduce leukocyte load in patients with rheumatoid arthritis and inflammatory bowel disease, but still has major limitations of specificity and selectivity. An ideal extracorporeal technique with non-thrombogenic materials and selective adsorption matrix is still in demand. Extracorporeal separation techniques can be improved by a combination of properties, such as mechanical properties of the column, an appropriate porosity of the matrix, biocompatibility of the polymer and chemical modification of the surface by immobilization of a ligand with an affinity towards target molecules or cells.

The current study was undertaken to produce an affinity-binding column, based upon a macroporous monolithic cryogel with a structure of interconnected pores, with pore size and low flow resistance potentially suitable for use in cytapheresis. This involved the characterization of polymer material, assessing haemocompatibility, and functionalization of polymer matrix with biological ligands.

The goals were pursued by (i) Synthesizing macroporous monolithic polymer cryogel columns from poly (2- hydroxyl ethyl methacrylate) (PHEMA), poly vinyl pyrrolidone (PVP) and poly (hydroxyl ethyl methacrylate-co-N-(3-aminopropyl) methacrylamide) (APMA). (ii) Structural and mechanical characterization of cryogels were evaluated, (iii) haemocompatibility of material was established by measuring free haemoglobin and platelet activation examination after blood filtration through the column; (iv) development of the method for attaching biological ligand to the matrix. The results collected in this study show that PHEMA monolithic cryogel column consists of ~13% of polymer phase and ~87% of the interconnected pores are within the range of 1-120 µm. The column can withstand a maximum pressure of 180mmHg without deviation in linearity, creep test and compression release cyclic test shows ~ 97% recovery after 70% compression of cryogel slice and has a storage modulus of ~ 0.012MPa. Approximately 100% of red blood cells passed through the column with no evidence of haemolysis found in blood eluted and platelet activation showed that cryogel is a material of low thrombogenicity. Amylase and anti-human albumin (IgG) were immobilized onto the cryogel matrix via the glutaraldehyde method, and affinity binding of human serum albumin to the cryogel matrix with immobilized anti-human albumin was evaluated. It was found that ~80% of human serum albumin was retained on the monolithic IgG anti-human albumin cryogel matrix.

In conclusion, the obtained results suggest that cryogel is a macroporous monolithic matrix with low flow resistance, mechanically stable, haemocompatible, and capable of functionalization with antibody and thus can be an appropriate matrix for use in extracorporeal apheresis system.

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Table of Contents

Abstract ...... i

Table of Contents ...... ii

Acknowledgements ...... xiii

Declaration ...... xiv

List of Abbreviations ...... xv

List of Figures ...... xviii

List of Tables...... xxii

Chapter One: INTRODUCTION ...... 1

1.1 Blood ...... 1

1.2 Diagnosis and treatment of blood disorders ...... 3

1.2.1 Diagnosis ...... 3

1.2.2 Treatments ...... 5

1.3 Apheresis ...... 6

1.3.1 Manual apheresis ...... 6

ii

1.3.2 Automated apheresis ...... 6

1.4 Plasmapheresis ...... 8

1.4.1 Immunoadsorption ...... 9

1.4.2 Low-density lipoprotein LDL-apheresis ...... 9

1.4.3 Direct adsorption of lipoprotein (DALI) ...... 12

1.5 Cytapheresis ...... 14

1.5.1 Leukocytapheresis ...... 15

1.5.2 Erythrocytapheresis ...... 15

1.5.3 Thrombocytapheresis ...... 16

1.6 The ideal column for extracorporeal cytapheresis ...... 16

1.7 Methods used in cell separation ...... 17

1.7.1 Centrifugation method ...... 17

1.7.2 Adsorptive method ...... 18

1.8 Polymer and hydrogels ...... 21

1.9 Cryogels ...... 23

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1.9.1 Preparation of cryogels ...... 25

1.9.1.1 Chemical cross linking of polymer ...... 25

1.9.1.2 Free radical polymerization ...... 26

1.9.1.3 Composite entrapment cryogelation ...... 28

1.9.2 Characterization of cryogels ...... 29

1.9.2.1 Pore structure of cryogels ...... 29

1.9.2.2 Flow rate and flow resistance of monolithic cryogels ...... 30

1.9.3 Functionalization of cryogel matrix surface ...... 30

1.9.3.1 Epoxy method ...... 31

1.9.3.2 Schiff base method ...... 32

1.9.3.3 Carbonyldiimidazole (CDI) method ...... 33

1.9.3.4 Disuccinimidyl carbonate (DSC) method ...... 34

1.9.3.5 Hydrazide method ...... 35

1.9.3.6 Cyanogen bromide (CNBr) method ...... 36

1.9.3.7 Bio-specific adsorption ...... 37

1.9.3.8 Entrapment ...... 38

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1.9.3.9 Coordination methods ...... 39

1.9.3.10 Biomimetic affinity chromatography ...... 39

1.10 Blood biocompatible ...... 40

1.11 Cryogels applications in biomedicine and biotechnology ...... 41

1.12 Specific aims and objectives...... 44

Chapter Two: Cryogel synthesis and pore structure characterization ...... 47

2.1 Introduction ...... 47

2.2 Aims ...... 50

2.3 Materials and methods ...... 51

2.3.1 Cryogel synthesis ...... 51

2.3.2 Scanning electron microscopy (SEM) preparation ...... 52

2.3.3 Confocal laser scanning microscopy (CLSM) preparation ...... 52

2.3.4 Measurement of flow rate of water passing through cryogels ...... 54

2.3.5 Flow rate of different concentrations of dextran ...... 54

2.3.6 Void volume calculation ...... 54

v

2.3.7 Statistical analysis ...... 55

2.4 Results and discussions ...... 55

2.4.1 Synthesis of monolithic cryogels ...... 55

2.4.2 Physical properties of monolithic cryogels obtained after cryopolymerization at

-12°C ...... 57

2.4.3 Structure of cryogel obtained after cryopolymerization at -12° C ...... 59

2.4.4 Flow rate of water passing through different concentrations of epoxy PHEMA

cryogels column ...... 66

2.4.5 Flow rate of different concentrations of dextran passing through 8% epoxy

PHEMA cryogels ...... 68

2.4.6 Void volume measurement ...... 70

2.5 Conclusions ...... 75

Chapter Three: Characterization of poly (2-hydroxylethyl methacrylate) macroporous cryogel ...... 77

3.1 Introduction ...... 77

3.2 Aims ...... 82

3.3 Materials and methods ...... 82 vi

3.3.1 Yield percentage and swelling degree of cryogels...... 83

3.3.2 Pressure difference and flow rate of water and plasma passing through cryogel

column ...... 84

3.3.3 Measurement of mechanical properties of cryogel by dynamic mechanical

analyser (DMA) ...... 85

3.3.3.1 Measurement of storage modulus of cryogels ...... 85

3.3.3.2 Measurement of compressive modulus of cryogels ...... 88

3.3.3.3 Creep test of cryogels ...... 88

3.3.3.4 Measurement of the strain percentage against time with a constant stress when the

cryogel is immersed in water ...... 88

3.3.4 Statistical analysis ...... 88

3.4 Results and discussions ...... 89

3.4.1 Polymerization yield in water ...... 89

3.4.2 Swelling degree of cryogel in water ...... 91

3.4.3 Pressure difference and flow rate of water and plasma ...... 92

3.4.4 Compressive modulus of cryogels ...... 95

3.4.5 Storage modulus of 6% and 8% epoxy PHEMA cryogel ...... 99 vii

3.4.6 Creep-recovery analysis of cryogels...... 102

3.4.7 Cyclic compression/stress-relaxation analysis of cryogels ...... 105

3.5 Conclusions ...... 110

Chapter Four: Haemocompatibility of poly (2-hydroxylethyl methacrylate) macroporous cryogels ...... 111

4.1 Introduction ...... 111

4.2 Aims ...... 116

4.3 Materials and methods ...... 116

4.3.1 Blocking of the epoxy reactive group ...... 117

4.3.2 Blood collection ...... 117

4.3.3 Blood cell count ...... 117

4.3.4 Plasma haemoglobin determination ...... 120

4.3.5 Platelet activation ...... 120

4.3.5.1 Immuno-staining ...... 120

4.3.5.2 Fixation ...... 121

4.3.5.3 Positive control ...... 121 viii

4.4 Results and discussions ...... 122

4.4.1 Flow of whole blood through cryogel column...... 122

4.4.2 Blood cell count by Sysmex cell counter...... 125

4.4.3 Haemolysis determination by Blankey and Dinewoodie ...... 126

4.4.4 Platelet adhesion and activation ...... 129

4.4.5 Effect of different anticoagulants on platelet percentage after incubation of

cryogel for 30 minutes using Sysmex cell counter ...... 133

4.4.6 Effect of different anticoagulants on platelet activation after incubation of cryogel

for 30 minutes using FACS analysis ...... 136

4.4.7 Effect of different anticoagulants on platelet after incubation of cryogel for 60

minutes ...... 138

4.4.8 Fluorescent activated cell sorter analysis of PAC-1 and P-selectin expression in

sodium citrate anticogulant after incubation of cryogel for 60 minutes ...... 142

4.5 Conclusions ...... 148

Chapter Five: Immobilization of protein and antibody onto macroporous monolithic cryogels...... 150

5.1 Introduction ...... 150

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5.1.1 Principles of affinity chromatography ...... 152

5.1.2 Types of ligands in bio affinity chromatography ...... 152

5.1.3 Monoliths and affinity chromatography ...... 154

5.1.3.1 Glutaraldehyde method...... 156

5.1.4 The ideal characteristics of a stationary phase in affinity chromatography ...... 157

5.1.5 Assays for determination of amount of protein bound to the column ...... 158

5.1.6 Cryogel and affinity chromatography ...... 159

5.2 Aims and objectives ...... 161

5.3 Materials and methods ...... 163

5.3.1 Preparation of solutions ...... 164

5.3.1.1 Sodium phosphate buffer ...... 164

5.3.1.2 Sodium carbonate buffer ...... 164

5.3.1.3 Sodium borohydride solution ...... 164

5.3.2 Immobilization of amylase onto monolithic epoxy PHEMA and poly HEMA-co-

APMA cryogels ...... 164

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5.3.3 Coupling of IgG ligand onto monolithic epoxy PHEMA and poly HEMA-co-APMA

cryogels ...... 166

5.3.4 Affinity binding of human serum albumin to cryogel matrix with immobilized

anti- human albumin (anti-HSA IgG) ligands...... 167

5.3.5 Determination of protein content by bicinchoninic acid (BCA) method ...... 167

5.4 Results and discussions ...... 168

5.4.1 Immobilization of amylase onto macroporous monolithic epoxy PHEMA and poly

HEMA-co-APMA cryogels ...... 168

5.4.2 Determination of amylase protein bound to cryogel matrix by bicinchoninic acid

method ...... 170

5.4.3 Coupling of anti-human albumin (IgG) ligand onto monolithic epoxy PHEMA and

poly HEMA-co-APMA cryogel ...... 172

5.4.4 Affinity binding of human albumin to cryogel with immobilized IgG ...... 174

5.5 Conclusions ...... 178

Chapter Six: Summary of conclusions, discussions and future works ...... 180

6.1 Novel contribution to knowledge ...... 186

6.2 Future work ...... 191

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APPENDIX I ...... 193

Disseminations and Abstracts ...... 193

APPENDIX II ...... 194

Ethical Approval ...... 194

References ...... 195

xii

Acknowledgements

This work has been carried out under the administration of my supervisors at University of

Brighton, School of Pharmacy and Biomolecular Sciences from 2008 to 2012. I owe my appreciation to my supervisors for their support and inspiring guidance over the years: Special thanks to Professor Sergey Mikhalovsky for his keen interest in this project and for putting this project together; Dr Stuart James for his help and overall guidance; Dr Lyuba Mikhalovska for her overall assessment and support; and Dr Irina Savina for her support at the initial stage of the project.

Special thanks to Prof Dieter Falkenhagen, Dr Viktoria Weber, Dr Anita Schildberger and

Stephan Harm from Krems Donau University (Austria) for their help with fluorescent activated cell sorter analysis, haemolysis test and pressure difference analysis, Prof Bo Mattiasson and Dr

Harald Kirsebom from Lund University (Sweden) for guidance with functionalization of the matrix, Andre Leistner and Dr Aniela Leistner from Polymerics (Germany) for support with the dynamic mechanical analyzer analysis.

My sincere thanks to Dr Lubinda Mbundi, Joe Lacey, Hild Bergin and Dr Rosa Busquets for their friendship and regular support.

I am mostly indebted to my parents Chief and Chief (Mrs) A.B. Akande for their continuous financial, moral and emotional support.

FP7 Marie Curie MONACO-EXTRA project (218242) and University of Brighton financially supported this research.

xiii

Declaration

I declare that the research contained in this thesis, unless otherwise stated within text, is the original work of the author. The thesis has not been previously submitted to this or any other university for a degree and does not incorporate any material already submitted for a degree.

W. Akande 10/12/2012

Wuraola Akande December 2012

xiv

List of Abbreviations

AC Affinity chromatography

ACE Angiotensin converting enzyme

ADP Adenosine diphosphate

AGE Ally glycidyl ether

AML Acute myeloid leukaemia

APMA 2-amino ethyl methacrylamide hydrochloride

APS Ammonium persulphate

ARA Angiotensin II-receptor 1 antagonist

ATP Adenosine triphosphate

BCA Bicinchoninic acid

BSA Bovine serum albumin

Camp Cyclic adenosine monophosphate

CIM Connective interaction media

CLSM Confocal laser scanning microscope

Con A Concanavalin A

CPB Cardiopulmonary bypass

DALI Direct adsorption of lipoprotein

DFPP Double filtration plasmapheresis

DMA Dynamic mechanical analyser

DNA Deoxyribonucleic acid

xv

DSA Dextran sulphate adsorption

E.Coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

ESEM Environmental scanning electron microscopy

FACS Fluorescent activated cell sorter

FDA Food and drug administration

FITC Fluorescein 5(6)-isothio-cyanate

GMA/EMDA Glycidyl methacrylate and ethylene

dimethacrylate

HDL High density lipoprotein

HELP Heparin-induced extracorporeal LDL

precipitation

HEMA 2-hydroxyethyl methacrylate

IBD Inflammatory bowel disease

IBM International business machines

IMA Immunoadsorption

IMAC Immobilized metal-ion affinity chromatography

LCAP Leukocytapheresis

LCD Liquid crystal display

LCP Lymphocytoplasmapheresis

LDL Low density lipoprotein

xvi

Lp Lipoprotein

MBAA N,N' Methylene bisacrylamide

MIP Molecular imprinted particle

NTA Nitrilotriacetic acid

PE Phycoerythrin

PerCp Peridinin chlorophyll protein

PHEMA Poly (hydroxylethyl methacrylate)

PVA Poly vinyl alcohol

PVP Poly vinyl pyrrolidone

RBC Red blood cells

SEM Scanning electron microscope

SFPP Single filtration plasmapheresis

SLE Systemic lupus erythematosus

TEMED N,N,N',N'-tetramethylenediamine

TFPP Thermofiltration plasmapheresis

USA United States of America

WBC White blood cells

WHO World health organisation

xvii

List of Figures

Figure 1.1. Image of DALI 750ml column from (Fresenius, 2012b) ...... 13

Figure 1.2. Cellsorba column from (Shibata et al., 2003) ...... 19

Figure 1.3. Adacolumn from (Jimro, 2012) ...... 21

Figure 1.4. Schematic outline of the formation of cryogel from (Plieva et al., 2004a) and SEM micrograph of the cryogel...... 28

Figure 1.5. Schematic representation of epoxy immobilization method ...... 31

Figure 1.6. Schematic representation of Schiff base immobilization method ...... 32

Figure 1.7. Schematic representation of carbonyldiimidazole immobilization method ... 33

Figure 1.8. Schematic representation of disuccinimidyl carbonate immobilization method ...... 35

Figure 1.9. Schematic representation of hydrazide immobilization method ...... 36

Figure 1.10. Schematic representation of cyanogen bromide immobilization method .... 37

Figure 2.1. Cryogel in glass column after freezing, thawing and washed with water ...... 57

Figure 2.2. Image of parameters affecting pore structure of monolithic macroporous cryogel (Plieva et al., 2005) ...... 59

Figure 2.3. SEM microphotography of (A-D) 8%, 10%, 13% and 16% of epoxy PHEMA cryogels ...... 61

Figure 2.4. CLSM image of 8% epoxy PHEMA cryogel ...... 63

Figure 2.5. Pore size distribution of 8% epoxy PHEMA cryogel matrix n=10 ...... 64

Figure 2.6. Wall thickness of 8% epoxy PHEMA cryogel matrix n=10 ...... 65

Figure 2.7. Flow rate of water passing through different concentrations of epoxy PHEMA cryogel column n = 3 results are given as mean ± standard deviation...... 67

Figure 2.8. Flow rate of dextran passing through 8% epoxy PHEMA cryogel column n=3 results are given as mean ± standard deviation...... 69 xviii

Figure 2.9. Absorbance of blue dextran at 620nm passing through column n= 3 ...... 70

Figure 2.10. Absorbance of blue dextran at 620 nm passing through column, n=3...... 71

Figure 2.11. Absorbance at 280nm against time of albumin passing through column, n=3 ...... 73

Figure 2.12. Absorbance at 280 nm against volume of albumin passing through column, n=3 ...... 74

Figure 3.1. Strain response as a function of time for elastic solid and a viscous liquid, following the application of a constant stress (Jones et al., 2012)...... 81

Figure 3.2. SP 200-syringe pump connected to pressure monitoring box and device ...... 85

Figure 3.3. Slice of cryogel immersed in water on a compression plate ...... 87

Figure 3.4. Polymer yield of cryogels from different monomer ratio. There is a significant difference in the polymer yield from HEMA: APMA 5:1, 10:1, 50:1 and HEMA: AGE 5:1 with P ≤ 0 .05. (Mean ± SD n=3) ...... 90

Figure 3.5. Swelling degree of cryogel from different monomer concentrations in water. There is a significant difference in the swelling degree from HEMA: APMA 5:1, 10:1, 50:1 and HEMA: AGE 5:1 with P ≤ 0 .05. (Mean ± SD n=3) ..... 92

Figure 3.6. Flow rate of water versus ∆pressure within 8% epoxy PHEMA cryogel column (Mean ± SD n=3) ...... 94

Figure 3.7. Effect of flow rate on ∆pressure of water and plasma in 8% epoxy PHEMA cryogel column (Mean ± SD n=3) ...... 95

Figure 3.8. Comparative study of mechanical strength of epoxy PHEMA cryogel. There is a significant difference in the compression modulus when comparing 6%,8%,10% and 16% PHEMA cryogel with P ≤ 0 .05. (Mean ± SD n=6) ...... 98

Figure 3.9. Storage modulus of epoxy PHEMA cryogel. There is a significant difference where comparing 6% and 8% PHEMA cryogel from the three different layers. P ≤ 0 .05. (Mean ± SD n=3)...... 100

Figure 3.10. Storage modulus of epoxy copolymer PVP and PHEMA cryogel. There is a significant difference where comparing the three different layers of 8% PVP and PHEMA cryogel. P ≤ 0 .05 (Mean ± SD n=3) ...... 101

xix

Figure 3.11. Creep test of 8% epoxy PHEMA cryogel ...... 103

Figure 3.12. Creep test of 10% epoxy PHEMA cryogel ...... 104

Figure 3.13. Creep test of 16% epoxy PHEMA cryogel ...... 105

Figure 3.14. Cyclic compression of 6% epoxy PHEMA cryogel ...... 107

Figure 3.15. Cyclic compression of 8% epoxy PHEMA cryogel ...... 108

Figure 3.16. Cyclic compression of 10% epoxy PHEMA cryogel ...... 109

Figure 3.17.Cyclic compression of 16% epoxy PHEMA cryogel ...... 110

Figure 4.1. Sysmex KX -21N cell counter ...... 118

Figure 4.2. SP 200 syringe pump ready to pump blood through the cryogel column connected to the pressure box and the pressure monitor system. The metal clamps are used to close the tubes leading to the pressure box while loading blood sample...... 119

Figure 4.3. Flow of blood passing through the cryogel matrix at different times. No side flow leakage within the column was observed...... 123

Figure 4.4. ∆Pressure against flow rate as blood passes through 8% PHEMA cryogel column at a speed of 1 ml/minute ...... 124

Figure 4.5. RBC counts after blood flow through the 8% PHEMA cryogel column. .... 125

Figure 4.6. PAC-1 activation with and without cryogel matrix at different time intervals ...... 130

Figure 4.7. P-selectin activation with and without cryogel matrix at different time intervals ...... 131

Figure 4.8. Platelet reading from Sysmex cell counter after 30 minutes incubation with and without cryogel in both polyethylene centrifuge and polyethylene centrifuge tube coated with Sigmacote ...... 134

Figure 4.9. Percentage of free platelet in FACS analysis after 30 minute incubation with and without cryogel, in both polyethylene centrifuge tube and polyethylene centrifuge tube coated with Sigmacote ...... 137

xx

Figure 4.10. Percentage of platelet in FACS analysis after 60 minutes incubation with or without cryogel in polyethylene centrifuge tube coated with Sigmacote ...... 139

Figure 4.11. Platelet reading from Sysmex cell counter after 60 minutes incubation with or without cryogel in polyethylene centrifuge tube coated with Sigmacote ... 140

Figure 4.12. (A-D) Time 0 control, after 60 minutes incubation with cryogel in polyethylene tube, after 60 minutes of blood in Sigmacote tube and after 60 minutes incubation in Sigmacote tube with cryogel ...... 143

Figure 4.13.PAC-1 activation after 60 minutes incubation with and without cryogel in both polyethylene centrifuge tube and polyethylene centrifuge tube coated with Sigmacote ...... 145

Figure 4.14. P-Selectin activation after 60 minutes incubation with and without cryogel in both polyethylene centrifuge tube and polyethylene centrifuge tube coated with Sigmacote ...... 146

Figure 5.1. Schematic representation of glutaraldehyde immobilization method ...... 157

Figure 5.2. General approach for covalent immobilization of a ligand within a monolithic column ...... 162

Figure 5.3. Diagrammatic expression of a cytapheresis column ...... 163

Figure 5.4. A-B cryogel sample freshly prepared plain white and orange after derivatization with glutaraldehyde, cryogel sample after reducing Schiff base with sodium borohydride (pale yellow) ...... 169

Figure 5.5. Bovine serum albumin standard curve for the determination of protein ...... 171

Figure 5.6. Standard curve of anti-human albumin for the determination of protein ..... 176

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List of Tables

Table 1.1. A list of some of the common LDL-apheresis systems ...... 11

Table 2.1. Summary of 8% epoxy PHEMA cryogel matrix structure measured using images obtained from optical confocal laser scanning microscopy with the software image J. n=10. The results are given as mean ± standard deviation ...... 66

Table 3.1. Polymerization yield and swelling degrees of cryogels...... 90

Table 4.1.Conversion table of haemolytic index ...... 127

Table 4.2. Haemolysis results using Blankey and Dinewoodie spectra method ...... 128

Table 4.3. Haemolysis results using Sysmex cell counter ...... 128

Table 5.1. Respective protein (amylase) contents in the HEMA: APMA 5:1, 10:1, 50:1 and HEMA: AGE 5:1 determined using bicinchoninic acid (BCA) method. Protein content expressed as milligrams of protein present in a single 0.5ml monolith and per weight (g) of the monolithic cryogels (Mean ± SD n=3) ...... 172

Table 5.2. Respective protein (IgG) contents in the HEMA: APMA 5:1, 10:1, 50:1, HEMA: AGE 5:1 and plain HEMA: APMA 5:1 and plain HEMA: AGE 5:1 determined using bicinchoninic acid (BCA) method. Protein content expressed as milligrams of protein present per weight (g) of the monolithic cryogels, while immobilization yield expressed as percentage of actual immobilized protein to the unit of fresh protein utilized for immobilization (Mean ± SD n=3) ...... 177

Table 5.3. Respective total protein (anti-human albumin (IgG) plus human serum albumin) protein (IgG) contents in the HEMA: APMA 5:1, 10:1, 50:1, HEMA: AGE 5:1 and plain HEMA: APMA 5:1 and plain HEMA: AGE 5:1 determined using bicinchoninic acid (BCA) method. Protein content expressed as milligrams of protein present per weight (g) of the monolithic cryogels, while immobilization yield expressed as percentage of actual immobilized protein to the unit of fresh protein utilized for immobilization (Mean ± SD n=3) ...... 178

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Chapter One: INTRODUCTION

1.1 Blood

Blood is a specialized fluid in mammals that transports necessary substances such as nutrients and oxygen to the cells, and metabolic waste products away from those cells.

Blood consists of formed components, namely red and white blood cells and platelets suspended in a liquid called plasma. Plasma is composed of mainly water (90-92%) and makes up approximately 55% of blood fluid, albumin is the main protein in plasma

(Pallister, 1994). Red blood cells (erythrocytes) make about 99% of the cellular constituent, with the remaining 1% being white blood cells and platelets (Pallister, 1994).

Red blood cells contain haemoglobin, an iron containing protein that carries oxygen throughout the body and gives blood its red colour. Their primary function is to carry oxygen from the lungs to other cells all around the body and carbon dioxide from cells to the lungs. On average, a red blood cell has a life span of approximately 120 days and is about 7-10 μm in size, (Pallister, 1994). Red blood cells contain no nucleus. Meanwhile, white blood cells, ‘leukocytes,’ are associated with the immune system and are involved mostly in fighting infection, defending the body against pathogens, disease producing bacteria, viruses and fungi. They are colourless and possess a nucleus. There are various types of white blood cells and these include the neutrophils (polymorphs) (they invade site of infection and kill bacteria and viruses), B-lymphocytes (secrete antibodies to

1

destroy pathogens), T-lymphocytes (secrete cytokines critical to the immune response) eosinophils, monocytes (performs phagocytosis) and basophils.

Platelets, also known as thrombocytes, are very small cell fragments, irregularly shaped, colourless with a sticky surface. They help in the clotting process by sticking to the lining of the blood vessel, where they clump together to form a plug that stops bleeding.

Platelets are produced in the bone marrow from megakaryocytes and survive for about 9 days in the circulatory system before removal from the body by the spleen (Pallister,

1994).

Despite the well-regulated nature of blood, numerous blood related diseases result from trauma, cancer or abnormal development and metabolism (i.e., platelet disorders, anaemia, leukaemia and myeloma, autoimmune disorder, hepatitis, HIV and bacterial infection). These blood-related diseases are common and present costly medical and social economical challenges. Anaemia, a well-established group of blood disorders globally, is mainly characterized by either the presence of too few red blood cells than normal or reduced haemoglobin in each red cell. In 2008, anaemia was estimated to cost the nation’s economy $6 billion with $5 billion in direct health expenditures and $1 billion in indirect cost of mortality; 5,000 deaths were attributed to anaemia in the year

2008 in the United States of America (USA) (NHLBI, 2011). In the USA, it is estimated that perhaps 100,000 people live with sickle cell disease (Grosse et al., 2011, Hassell,

2010). Cancer can be a blood-related disorder (e.g., leukaemia), where abnormal white cells divide without control and are able to invade other tissues. It was estimated that 12.7 2

million patients suffer from cancer worldwide, of which 1.3 million are in the United

States of America, with blood cancers such as leukaemia, lymphoma and myeloma accounting for approximately 9.5% in both the USA and UK (Cancer research UK, 2011,

Siegel et al., 2012). Human immunodeficiency virus (HIV) is a blood-borne pathogen that causes acquired immunodeficiency syndrome, a condition in humans that causes progressive failure of the immune system. HIV permits life-threatening opportunistic infections and cancers to progress, although its effects are seen mostly in the lymphatics.

It was estimated that about 34.2 million people are living with HIV virus and about 1.7 million died of AIDS in the year 2011 (World health organisation(WHO), 2011). An autoimmune disease such as systemic lupus erythematosus (SLE), characterized by the production of unusual antibodies in the blood, which affects any part of the body, is another blood-borne disorder. Hepatitis, which is described as inflammation of the liver, and characterized by the presence of inflammatory cells in the tissue of the organ, is also a blood disorder. Hepatitis, has been reported to affect about 2 billion people worldwide and about 600,000 people die every year (World health organisation(WHO), 2012a,

World health organisation(WHO), 2012b). Any one of these syndromes may be alleviated to some degree by extracorporeal adjustment of the blood components.

1.2 Diagnosis and treatment of blood disorders

1.2.1 Diagnosis

Over the years, several advances have been made in endeavours to diagnose and treat blood disorders. The diagnosis of blood disorders involves physical examination by

3

physician for signs and symptoms such as: occasional yellowing of the eyes and skin jaundice in hepatitis, vaso-occlusive episode blockage of the small blood vessels by abnormal blood cells leading to inflammation in hands and feet in sickle cell; sore throat, body rash and swollen glands in HIV; and pale skin, swollen liver, swollen spleen in leukaemia. However, the physical examination is limited in that it lacks specificity for physiological and biochemical mechanisms involved in the disorder. As such, several other biomedical methods, such as biochemical, immunocytochemical and haematological techniques, are employed to inform the specificity and nature of the pathophysiological mechanisms involved, thereby helping inform the process of treatment choice.

Some of the examples of biochemical markers that are analysed include characteristics

(antigens) on the blast cell surface such as CD34 in diagnosis of acute myeloid leukaemia and CD4+ T lymphocyte in HIV-1 infection (Thompson et al., 2012). Antibody test used to identify viral infections such as anti-HBC is used to diagnosis hepatitis B virus (HBV) infection. In haematology, information such as platelet count and complete blood counts are useful for almost all blood disorders. In the diagnosis of anaemia, serum ferritin and serum iron tests are essential (Alleyne et al., 2008). However, such diagnostic tests have lead to a wealth of knowledge about surface markers on cells, or tumour specific antigens, of use in deciding which ligands to attach to cytapheresis columns.

4

1.2.2 Treatments

The various treatments available for each of these diseases include pharmacological and non-pharmacological treatment. Examples of pharmacological treatment are that of the use of antiretroviral drugs e.g., (HAART) in HIV infection. However, these drugs are very effective but are associated with rare but serious side effects, such as bone loss and abnormal lipid and glucose metabolism (Thompson et al., 2012). Myeloma, leukaemia and lymphoma are treated by chemotherapy, which uses anticancer drugs (cytotoxic drugs) to destroy cancer cells. These drugs cause side effects such as hair loss, mouth ulcer, sore throat, nausea and vomiting and severe tiredness (Cancer research UK, 2011).

Where pharmacological treatments are not sufficient on their own or have failed, non- pharmacological techniques are used. A clear example of this is the use of bone marrow transplant in cancers and stem cell transplant in sickle cell. Other examples include blood sorting and filtration or transfusion in the treatment of anaemia.

The many different diagnostic and treatment approaches, each with its advantages and disadvantages, available to clinicians today are a clear indication of how much progress has been made in this regard. Indeed more innovative techniques will continue to be developed with increase in knowledge as long as there are still shortfalls with available approaches. One of the techniques that is highly advocated for this purpose is apheresis, which involves the physical isolation of the pathological elements in blood.

5

1.3 Apheresis

Apheresis, from Greek ‘taking away’, is a medical technique of sorting out some components of blood from others and returning the remainder into circulation and it is commonly referred to as an example of extracorporeal circulation (Gorlin, 1999). Due to the associated therapeutic advantage of being able to select and remove particular components of blood, apheresis has been highly regarded in the management of various diseases, such as renal haematological, rheumatological and neurological diseases

(Borberg, 2006). Apheresis can be performed either by manual apheresis or automated apheresis.

1.3.1 Manual apheresis

In a similar way to obtaining human whole blood and dividing it into components (blood cells and plasma) in the blood centre, a unit of whole blood can be drawn, centrifuged and a portion returned to the patient. For example, if antigen negative platelets from a mother in a case of suspected neonatal alloimmune thromobocytopenia are required, it is possible to draw a unit of blood, prepare and save the platelet rich plasma and return the red cells to the mother. However, manual apheresis procedures are mainly used for practical reasons, and now rarely used.

1.3.2 Automated apheresis

The introduction of mechanical apheresis devices was truly an individual inspiration. An

IBM Endicott engineer (George T. Judson), in 1962, whose son developed leukaemia recognized the need for frequent large quantity platelet transfusions to oncology patients 6

and developed the first mechanical blood cell separator. Now, decades later, automated units are found in most blood centres (Jones, 1988) .

An apheresis device may either be an automated (continuous) or manual (discontinuous) device. A discontinuous device, withdraws a given volume of blood, processes it, removes the selected component and then returns the remainder. The main advantage of a discontinuous device is that it requires only a single site of vascular access. Its disadvantages, in comparison to a continuous device include longer time to process the same volume of blood and in vivo volume shifts. Alternatively, continuous devices withdraw and return similar volumes simultaneously, minimizing acute changes in patient intra-vascular volume (McLeod, 1997).

Available devices that function in continuous mode include the Baxter CS 3000, Amicus and COBE Spectra, Trima, and Fresenius device. Mechanical separators generally work on one of the two principles: (a) centrifugation to establish a density gradient and; (b) filtration to remove pathogenic substances. Most filtration devices are limited to plasmapheresis (the separation of plasma from blood cells). They work by passing blood under pressure over a filter or through a hollow fibre column, whose mesh size is smaller than 1μm (the approximate diameter of a platelet), but sufficiently large enough to allow free flow of the liquid components (Grossman et al., 1983). Some of the areas where extracorporeal filtering devices are being used are in the treatment of autoimmune diseases, such as systemic lupus erythematosus (SLE), ulcerative colitis and Crohn’s disease where immune complexes are removed (Takenaka, 1996). These devices adsorb 7

white blood cells and components through the perfusion of peripheral blood by means of simple extracorporeal circulation and thus adjust the immune responses (Shibata et al.,

2003).

Conventional techniques in apheresis include plasmapheresis (performed by online centrifugation or plasma filtration) and cytapheresis, which includes blood cell separation by centrifugation or filtration/adsorption.

1.4 Plasmapheresis

Plasmapheresis is the separation of plasma from blood cells by settling or using a centrifuge and returning the remaining blood components, mostly red blood cells, back into the patients. The rationales include the removal of pathogenic antibodies, as well as removal of other toxic plasma constituents (Okafor et al., 2010). Blood component separation from whole blood depends on density differences (simple centrifugation), which constantly separates plasma from red cells. Size differences of cells in the blood allow filter devices to remove plasma and retain cellular components (Gorlin, 1999). The isolated components (such as platelets or peripheral blood mononuclear cells) may be collected for subsequent transfusion or for further separation of intra-vascular pathogenic components whose presence is pathological. This removal of pathogenic components has many practical applications, including either removal of cellular or acellular components.

Removal of plasma components may be necessary if for example, an autoantibody, particularly an IgM, proves to be the cause of the illness. IgG antibodies may also be removed, but with lower efficiency, as they distribute in the entire extravascular fluid 8

volume, whereas IgM antibodies are concentrated in the vascular space (Kleinman et al.,

1982, Ratner et al., 2004). Indeed, plasmapheresis has also been used in plasma purification such as immunoadsorption, low density lipoprotein-apheresis (LDL- apheresis) and filtration selective removal of pathogens (Okafor et al., 2010).

1.4.1 Immunoadsorption

This is a therapy procedure where specific antibodies and circulating immune complexes are removed from the plasma of patients (Hehmke et al., 2000). Immunoadsorption has proved to be an effective alternative treatment in immunologically caused diseases, such as autoimmune diseases, or antibody-mediated transplant rejections. It has been reported that the potential mechanism of action of immunoadsorption can also have a direct positive effect on the humoral and cellular immune system (immunomodulation) (Braun and Risler, 1999, Hehmke et al., 2000). The Excorim extracorporeal immunoadsorption system, using a column containing protein A immobilized on Sepharose, has been used in the treatment for renal transplantation, cancer-associated haemolytic uraemic syndrome and systemic lupus erythematosus (Samuelsson, 2001).

1.4.2 Low-density lipoprotein LDL-apheresis

Low-density lipoprotein is one of the major groups of lipoproteins that transport cholesterol in the blood and are composed of moderate amount of protein and large amount of cholesterol. A raised level of LDL can be implicated in syndromes such as familial hypercholesterolaemia and arteriosclerosis that are causes of cardiovascular diseases. Cardiovascular diseases are one of the leading cause of death in the world, with 9

coronary heart disease (CHD) accounting for the main share, approximately 29 million deaths worldwide are associated with cardiovascular diseases (World health organisation(WHO), 2012b). Elevated levels of LDL can be reduced by LDL-apheresis, a selective lipid-lowering extracorporeal treatment where LDL and other atherogenic apoB-lipoproteins are removed from circulation while high-density lipoprotein (HDL) remains virtually unchanged.

There are six LDL-apheresis systems currently in the world (Winters, 2012). Four of these systems (double filtration plasmaapheresis, immunoadsorption, dextran sulphate adsorption and heparin-induced extracorporeal LDL precipitation) involve the separation of plasma from the whole blood. The plasma is then perfused through the filter-columns that selectively bind and remove apoB-containing lipoproteins (Bambauer et al., 2003,

Thompsen and Thompson, 2006, Thompson, 2003) before it is returned into the patient.

Table 1.1 shows a list of some of the common LDL-apheresis systems. The Food and

Drug Administration (FDA) have licensed two of these systems (dextran sulphate adsorption and heparin-induced extracorporeal LDL precipitation) for use in the United

States (Winters, 2012) .

Of the above, the re-usable columns for immunoadsorption is the most economical technique, despite a higher bacterial contamination risk (Bambauer et al., 2003,

Thompson, 2003). However, due to the highly simplified extracorporeal circuit systems, those where LDL is directly adsorbed from whole blood are the easiest and most rapid

(Bambauer et al., 2003, Bosch et al., 2002, Mabuchi et al., 2004). The direct removal of 10

LDL from whole blood does not require plasma separation, which simplifies the extracorporeal circuit and markedly reduces staff and treatment time.

Table 1.1. A list of some of the common LDL-apheresis systems

System Manufacturer Anticoagulant used Double filtration Numerous Heparin or Citrate plasmaapheresis (DFPP) Immunoadsorption (IMA) PlasmaSelect, Teterow, Heparin or Citrate Germany Dextran sulfate adsorption Kaneka, Osaka, Japan Heparin Liposorber (DSA) Heparin-induced Braun-Melsungen, Germany Heparin extracorporeal LDL precipitation (HELP) Polyacrylate-coated Fresenius-Kabi AG,Bad Heparin and or Citrate polyacrylamide direct Homberg,Germany perfusion (DALI) Dextran sulfate direct Kaneka, Osaka, Japan Heparin and or Citrate perfusion (Liposorber D)

In contrast to the conventional LDL-apheresis techniques, direct adsorption of lipoprotein

(DALI) is the first LDL-apheresis technique that is able to adsorb LDL and lipoprotein

(Lp) directly from whole blood (Bosch et al., 1997). Direct adsorption of lipoprotein

(DALI) was developed by the Germany-based medical technology company Fresenius

11

Medical Care for the treatment of patients with severe dyslipidaemic conditions and homozygous familial hypercholesterolaemia (Archontakis et al., 2008).

1.4.3 Direct adsorption of lipoprotein (DALI)

The adsorber in DALI consists of negatively charged polyacrylate ligands linked to

Eupergit matrix (macroporous beads). The housing is made of sufficiently tough and stiff polycarbonate material from Bayer Material Science, which protects it from becoming easily damaged in an often-hectic hospital environment. This material also readily withstands pressurized steam sterilization of the adsorber, in the DALI system, where temperatures reach at least 120°C for more than 20 minutes (Fresenius, 2012b).

In this system, the blood is drawn from a vein in the patient’s at a flow rate of ~ 50 ml/minute and passed through the adsorber, where the LDL cholesterol or Lp is bound selectively and removed from the whole blood. The purified blood is returned through the brachial vein of the other arm. Citrate solution (ACD-A) is used as an anticoagulant throughout the procedure. The treatment frequency ranges between once a fortnight and twice a week. The availability of different adsorbers as shown in Fig1.1 (DALI 500ml,

DALI 750ml) helps to tailor the treatment to individual patient’s needs (Bosch et al.,

2000, Fresenius, 2012a). DALI adsorber beads activate bradykinin, which is normally rapidly degraded into inactive metabolites by angiotensin converting enzyme (ACE)

(Bosch et al., 2000). Bradykinin is a peptide that causes blood vessels to dilate (enlarge), thereby lowering blood pressure. A class of drug called ACE inhibitors, which are used to reduce blood pressure, increases bradykinin by inhibiting its degradation. Thus, in 12

order to avoid undue bradykinin increase, ACE-inhibitors are contraindicated in DALI-

LDL-apheresis. In line with this, another class of blood pressure lowering drug,

Angiotensin II-receptor 1 antagonist (ARA) Losartan, was also shown for the first time to be effective in DALI-LDL-apheresis (Bosch, 2004, Bosch and Wendler, 2004). Due to the fact that DALI LDL-apheresis is safe, efficient, rapid and user-friendly, the LDL- apheresis procedure has seen wider use evidenced by more than 80,000 successful DALI sessions (Bosch, 2004).

Figure 1.1. Image of DALI 750ml column from (Fresenius, 2012b)

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Liposorber D manufactured by (Kaneka Co. Japan) is a newer whole blood system, utilizing the dextran sulphate direct perfusion technology (Bambauer et al., 2003,

Mabuchi et al., 2004). Acute LDL-reduction ranges between 49-76% with all six methods (Archontakis et al., 2008, Bosch et al., 2002). These different LDL-apheresis have been reviewed by others (Okafor et al., 2010, Winters, 2012).

1.5 Cytapheresis

Cytapheresis, an extracorporeal separation technique widely used in clinical applications for elimination of different types of blood cells, has been shown recently to have clinical efficacy in various diseases, such as rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis (Yamaji et al., 2001). It has become an attractive notion to seek improvement in a wide range of clinical situations, through the specific removal of pathologically significant cells from the blood. Cytapheresis has been tested for the removal of leukocytes after coronary artery bypass, to reduce post-operative organ dysfunction (Baksaas et al., 1999). It succeeded in eliminating leukocytes, but with little clinical improvement (Baksaas et al., 1999). It has also been used in clinical management of neurological diseases, such as multiple sclerosis and experimental encephalomyelitis to relieve symptoms and remove immune complexes (Nakane et al., 2003).

Therapeutic cytapheresis procedures include red cell exchange (erythrocytapheresis), platelet reduction (thrombocytapheresis) and white cell removal (leukocytapheresis).

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1.5.1 Leukocytapheresis

Leukocytapheresis (LCAP) involves the removal of leukocytes during extracorporeal circulation. It has been widely used and found to be useful in treating diseases whose pathophysiology involves an immunological abnormality, such as rheumatoid arthritis, inflammatory bowel disease, and chronic demyelinating neuropathy (Hidaka et al.,

1999b, Kosaka et al., 1999, Sasaki et al., 1998, Sato et al., 1994, Sawada et al., 1995,

Yajima et al., 1998). In the US and Europe, methods involving automated centrifugation technology are used in the mainstream. Meanwhile, in Japan, LCAP using a leukocyte removal filter made of polyester was used to treat systemic lupus erythematosus (SLE) for the first time in 1985 (Yamaji et al., 2006), and the column LCAP has since come into clinical application.

1.5.2 Erythrocytapheresis

This therapeutic apheresis allows for the rapid removal of altered red blood cells from the circulation and replacement with normal cells (usually in < 2 hours), retains plasma and has little effect on platelet and leukocyte function (Ratner et al., 2004). This was carried out on (COBE Spectra system Lakewood, CO, USA) using the red blood cell exchange procedure (McLeod, 1997). Red blood cell apheresis is used sparingly for complications of sickle cell disease and other haemoglobinopathies, and rarely for severe complications of red cell infections such as malaria and babesiosis (Szczepiorkowski et al., 2007).

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1.5.3 Thrombocytapheresis

Thrombocytapheresis involves the removal of platelets during extracorporeal circulation.

Therapeutic plateletapheresis has been used to reduce platelet count rapidly in essential thrombocytaemia and relieves patients of acute symptoms, such as shortness of breath and chest pain (Das et al., 2011).

1.6 The ideal column for extracorporeal cytapheresis

The perfect characteristics needed for a routine extracorporeal bio–specific column-based filter device include: (i) control of the matrix pore size during manufacture; (ii) low leukocyte activation and low thrombogenicity; (iii) high mechanical and chemical stability; (iv) low flow resistance; (v) a large plane area-to-volume ratio, (vi) functionalization with ease and (vii) large interconnected pores which can permit free cell passage (Tetala and Beek, 2010).

Existing filter devices and previous apheresis techniques do not meet many of these criteria. A majority of chromatography matrices, which may be used in cytapheresis, include hydrogels, agarose gels and beaded matrices, such as Sepharose or natural products such as alginate. These matrices are inappropriate due to a number of reasons, such as too small or too variable pore sizes for the passage of whole cells, and in some gels the pores are not well interconnected yielding large flow resistance. Column packing, such as Sepharose beads, present both inter-and-intra-bead pores of very different diameters, ranging from nanometres to hundreds of microns. It has previously been suggested to use monolithic columns such as those used in chromatographic 16

separations (Svec and Frechet, 1996), this has not be successful due the challenge of achieving an appropriate pore size. As such it is necessary to develop novel adsorbents that would satisfy most demands for haemocompatibility, biocompatibility, low flow resistance, high mechanical and chemical stability, large surface area ratio, functionalization with ease and macroporous interconnected pores (Mikhalovsky, 2003).

1.7 Methods used in cell separation

1.7.1 Centrifugation method

Continuous flow centrifugation devices have been used for cell separation for several decades such as Cohn centrifuge spectra envisioned by Dr Cohn in early 1950’s. This device was used for the immediate on-line separation of whole blood into components in which citrated or decalcified whole blood flowed upward under pressure into the conical upper portion of a separation chamber rotating at about 2000rpm (McLeod, 1997).

Another centrifugation technique NCI-IBM blood cell separator, a simple plateletapheresis procedure, has been used to decrease morbidity and mortality from haemorrhage in patients with leukaemia and other malignant diseases (Freireich et al.,

1963). One of the procedures, used to decrease a patient’s leukocyte amount in acute myeloid leukaemia (AML) is extracorporeal continuous-flow centrifugation such as the

American Instrument Company (Aminco) Celltrifuge. However, this device has not been precise for it involves the mechanical removal of the entire buffy coat layer with a broad selection of cell types, several of which are not harmful (Wirman et al., 1975).

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1.7.2 Adsorptive method

Adsorptive method for cell separation as been explored with the use of a filter made out of fibre or beads. One of the commercially available filter devices made of fibre is shown in (Fig.1.2) Cellsorba. This is a leukocyte removal filter developed by Asahi Medical Co., which adsorbs white blood cells through the perfusion of peripheral blood by means of simple extracorporeal circulation. Cellsorba filter is made of a non-woven fabric of polyester fibre, wound into a cylindrical shape. The filter is spiralled, secured at its perimeter and sealed with polyurethane. As the main filter, non-woven fabric made of a very fine fibre of 0.8-2.8 μm in diameter is used inside the cylinder. An outer prefilter is provided using a layer of non-woven fabric made of 10-40 μm fibre. This gives the filter a tandem structure. Leukocyte removal is achieved by adherence of cells to fine fibre and mechanically sieving with pores of contained fibres. A session treatment last ~60 minutes at a flow rate of 50 ml/minutes (Shibata et al., 2003).

Cellsorba has been used, to treat rheumatoid arthritis, by removing leukocytes and a large number of immunoreactive lymphocytes (Shibata et al., 2003). This treatment is not specific as it involves removal of most white cells to enable the elimination of just a small number of T-helper and T-cytotoxic cells that are involved in the disease (Kondoh et al., 1991). Nonetheless, marked clinical development has been achieved and the device is subsequently being employed in other autoimmune diseases, such as systemic lupus erythematosus, ulcerative colitis and Crohn’s disease (Takenaka, 1996). Recently, it has

18

been accepted for clinical purpose in Japan, particularly for the treatment of active ulcerative colitis (Shibata et al., 2003) and rheumatoid arthritis (Hohtatsu et al., 2011).

Housing length Total length 160 mm 200mm

Housing diameter 45 mm Header diameter 62 mm

Blood flow Main filter Fiber diameter 0.8-2.8 µm

Pre filter Fiber diameter 10-40 µm

Figure 1.2. Cellsorba column from (Shibata et al., 2003)

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One of the commercially available apheresis columns packed with beads is shown in

(Fig.1.3) Adacolumn, which is an adsorptive type extracorporeal leukocyte apheresis device, developed by Jimro Co. Ltd Japan. The apheresis column is a polycarbonate column with a capacity of ~335ml filled with specially designed ~ 220g cellulose acetate beads as the adsorptive carriers. In this system, the blood is drawn from a vein in the patient at a flow rate of ~ 30 ml/minute and passed through the Adacolumn (small beads of cellulose acetate) where the white blood cell are bound selectively and removed from the whole blood. It is used for the treatment of active inflammatory bowel disease (IBD) such as, Crohn’s disease and ulcerative colitis. It is a straightforward treatment with selective adsorption of leukocytes, largely granulocytes and monocytes/macrophages and lymphocytes from the blood. It aims to decrease inflammation and relieve patients’ symptoms. Therefore, the Adacolumn is for selective leukocyte apheresis. Treatment comprises of a ~ 60 minutes procedure once a week for five weeks. (Yamaji et al., 2001).

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Figure 1.3. Adacolumn from (Jimro, 2012)

New improved adsorbents such as macroporous cryogel have been advocated as suitable material for cytapheresis due to large interconnected pore size, low flow resistance, mechanical and chemical stability, ease of functionalization of the matrix and their high permeability to cells. This apheresis technique is envisaged to provide alternative immunomodulation and offer alternative to toxic drug therapy.

1.8 Polymer and hydrogels

Polymers are naturally occurring or synthetically produced and consist of large molecules in a linked series of repetitive simple monomers. There are ranges of different types of polymers:

21

 Natural polymers, found in both plants (cellulose, sodium alginate and natural

rubber), and animals (collagen, heparin, glycosaminoglycan, chitosan).

 Semi-synthetic polymers such as dextran and nitrocellulose

 Synthetic polymers such as polypropylene, poly (methyl methacrylate), poly

(hydroxyl ethyl methacrylate, poly (ethylene glycol)

Hydrogels are prepared from a wide variety of starting materials, ranging from synthetic monomers to natural polymers such as proteins (Allan S, 2002). Hydrogels are formed either by covalent chemical cross-linkage or non-covalent bonding of polymers. These cross-linked materials, which absorb water, without dissolving, contain over 99% water

(Peppas et al., 2000). Softness and the capacity to store water make the hydrogel a unique material (Shibayama and Tanaka, 1993). These hydrogels can therefore be used for immobilization of enzymes or microbial cells or used as scaffolds for cell culture (Jen et al., 1996).

Hydrogels are unsuitable for cytapheresis for a number of reasons. First and foremost, the pore of the gels is either too small or too irregular for the flow of whole cells. Also, in some gels the pores are not well interconnected, leading to high flow resistance.

Hydrogels are not mechanically stable, with their structure collapsing after drying and re- hydrating. A monolith support consists of single continuous pieces of porous material.

Monolithic columns for chromatographic separation have not been used for cell separations because of the failures in achieving appropriate pore structure. These problems could be overcome by using supermacroporous polymeric cryogels rather than 22

hydrogels. The latter do not have appropriate mechanical stability and large interconnected pores, thus cannot provide low flow resistance through a monolithic and collapse under pressure. The production of supermacroporous cryogels in a continuous

(monolithic) form can be carried out using a cryopolymerization technology.

1.9 Cryogels

Ice formation both in nature and laboratory is related to exposure of water to temperatures at or below freezing point. As the ice crystals start forming, only the water is contained in the ice lattice and any solutes get expelled from ice crystals (Ishiguro and

Rubinsky, 1994). The formation of ice crystals acts as a porogen and leads to a supermacroporous cryogel, which are three dimensional polymeric gel materials formed under sub-zero conditions (Plieva et al., 2007).

Cryotropic gelation is a peculiar type of gelation, which occurs upon cryogenic treatment of the primary systems capable of producing a gel. This process takes place at temperatures below freezing point with water as the main solvent (Lozinsky, 2002).

While the water is frozen out into ice crystals, the dissolved substances are concentrated in the small non-frozen regions, called ' non-frozen liquid microphase’ and the ice crystals of frozen solvent serve as a porogen. The unfrozen phase then polymerizes into a solid. After melting the ice crystals by either (freeze drying or thawing), a system of large interconnected pores is formed. Macroporous monolithic cryogels have a large size of interconnected supermacropores with sizes as large as 200 µm (Plieva et al., 2011).

23

Factors that affect cryogel morphology and characteristics include the concentration of the original gel composition, and the temperature during cryotropic gelation. For example, an increase in comonomer concentration results in a decrease in the water flow rate through the cryogel column (Plieva et al., 2006b). This could probably be due to the lower amount of solvent, which is frozen out from the more concentrated primary solution, resulting in smaller size of ice crystals forming the smaller size macropores.

However, an increase in primary comonomer concentration results in an increase in both polymer concentration in pore walls and the general strength of the macroporous gel material (Dainiak et al., 2007a).

The mechanical stability of cryogels is as a result of dense polymer walls formed when monomer/polymer pre-concentration occurs during cryogelation (Dainiak et al., 2007a).

Cryogels have a relatively higher mechanical strength as compared to the hydrogels with the same bulk concentration of the polymer (Lozinsky et al., 2003). Cryogels are particularly different from hydrogels due to the system of interconnected macropores that gives the cryogels a sponge–like morphology (Lozinsky et al., 2003) and to the composition of pore walls produced when an increase in polymer concentration in non- frozen sections takes place. Cryogels have spongy-like structure with water located inside large macropores. This water could be mechanically removed by cryogel squeezing owing to the elasticity of the material (Dainiak et al., 2006b). In summary, pore size, shape and morphology in macroporous gels produced at sub zero temperatures can be controlled by controlling initial gel formation composition and solvent freezing

24

(Lozinsky, 2002). Furthermore, the large interconnected pores allow liquid to be pumped through cryogels at high flow rates with minimal flow resistance (Galaev et al., 2007).

1.9.1 Preparation of cryogels

Cryogels can be made from either monomeric or polymeric precursors dissolved in a solvent and later frozen to influence crystallization, resulting in cryoconcentration of the precursors in an unfrozen liquid phase (Arvidsson et al., 2003, Kirsebom et al., 2008,

Lozinsky, 2002, Lozinsky et al., 2003). The most common solvent used is water, this is an attractive option both for environmental friendly and biocompatibility reasons.

However, the use of water limits the choice of precursors to water-soluble compounds, and thus results in hydrophilic cryogels. It has been proven that hydrophobic cryogels can be prepared with the use of organic solvents such as dimethylsulphoxide, dioxane, formamide, nitrobenzene, benzene and cyclohexane (Ceylan and Okay, 2007, Dainiak et al., 2007a, Dogu and Okay, 2008, Lozinsky et al., 1984, Plieva et al., 2006).

Cryogels can be synthesized by: (i) covalent chemical cross-linking of polymers and free radical polymerization; and (ii) non-covalently by cryogelation through the non-covalent structure and composite entrapment cryogelation.

1.9.1.1 Chemical cross linking of polymer

Cryogels can be prepared through chemical cross-linking of the pre-existing polymer by the formation of inter-polymer chain cross-links. Such a widely used cross linker is glutaraldehyde for which the generation of a reactive species is not required, since the

25

crosslinker is sufficient to form stable bonds. One example of this is the cross-linking of polyvinyl alcohol (PVA) with glutaraldehyde in acidic pH to form covalently cross- linked cryogels (Plieva et al., 2006b). This method has been used to prepare cryogel from linear polyacrylamide cross-linked with glutaraldehyde (Ivanov et al., 2007). In another study, the cross-linking of chitosan (a derivative of chitin) with glutaraldehyde resulted in stable cryogels (Kirsebom et al., 2007). Cryogels have also been prepared from proteins such as gelatin, fibrinogen, collagen and bovine serum albumin, with glutaraldehyde or aldehyde containing starch as crosslinkers (Dainiak et al., 2010, Hedstrom et al., 2008,

Savina et al., 2009).

1.9.1.2 Free radical polymerization

The formation of a polymer through successive addition of monomers to a growing radical polymer chain is known as free radical polymerization. This process is divided into two steps. The first step is the formation of free radicals from an initiator system

(Chen et al., 1999, Sarac, 1999) which increases the polymer chain by activating the monomers present and causing propagation (Enrique Lopez, 2002). The next step is when a termination reaction occurs when two reactive radical species react, thus creating a non- reactive end of the polymer (Lozinsky, 2002). The connection of different growing polymer chains into a network requires the presence of a cross linker during polymerization (Lozinsky, 2002). In the absence of a cross linker, the formation of a linear polymer takes place, thus no macroporous gel is formed. Ammonium persulphate

(APS) and N, N, N’, N’- tetramethylethylenediamine (TEMED) comprise the commonly

26

used redox initator systems for initiating the reaction. This system is a catalyst-based system whereby TEMED acts as an accelerator by reacting with the persulphate group and cleaving into one anionic sulphate radical and one sulphate ion, while the TEMED forms a tertiary cationic radical (Lozinsky et al., 1989). The radical generation from

APS/TEMED system is temperature dependent; it is essential to keep pre-polymeric mixtures chilled before adding the initiators to prevent gel formation before freezing.

When most of the solvent is frozen at sub-zero temperatures, the dissolved substances

(monomers or polymer precursors of a cryogel) are concentrated in small non-frozen regions and most of the ice crystals will connect to each other. In small micro phases with non-frozen liquid, the soluble precursor polymerizes and crosslinks to form a network around the ice crystals. When the ice crystals melt, a system of large interconnected pores is formed in their place providing channels for the mobile phase to flow through

(Dainiak et al., 2006a). Indeed, free radical polymerization has been used widely for the synthesis of macroporous cryogels (Ceylan et al., 2006, Chalal et al., 2009, Kirsebom et al., 2008, Plieva et al., 2005). Fig.1.4 illustrates the process of cryogel formation.

27

Monomers Ice crystals and Cross-linked Supermacropore SEM micrograph of and initial forming polymer cryogel Supermacropore Initiators polymers cryogel cryogel

Figure 1.4. Schematic outline of the formation of cryogel from (Plieva et al., 2004a) and

SEM micrograph of the cryogel.

1.9.1.3 Composite entrapment cryogelation

Composite cryogels are prepared by incorporating particles into the polymeric matrix of the cryogel. These homogenous particles added before freezing the precursor solution behave in a similar manner to solutes, in that they are not incorporated into the ice crystals. Structured gels built from the particles are formed if the particles are aggregated in the frozen state to form a stable network. Simple thawing of the sample or freeze- drying the sample is required to obtain stabilized structures (Hajizadeh et al., 2010). This method has been used to prepare polyvinyl alcohol cryogels (Savina et al., 2005), poly

(N-isopropylacrylamide-co-allylamine) NIPA nanoparticle cryogels (Kirsebom et al.,

28

2009), activated carbon embedded into poly (N-isopropylacrylamide-co-allylamine)

(Hajizadeh et al., 2010) and poly vinyl alcohol particle cryogel (Hajizadeh et al., 2012).

1.9.2 Characterization of cryogels

1.9.2.1 Pore structure of cryogels

The porous structure of the monolithic cryogel depends on the concentration of monomers and the content of cross linker in the reaction feed and the freezing conditions

(Kumar et al., 2005). Temperature is also a major factor in controlling ice crystallisation

(Ivanov et al., 2007, Lozinsky et al., 1986, Plieva et al., 2006). A lower freezing temperature creates ice nucleation sites, therefore leading to formation of larger number of ice crystals resulting in smaller pores (Ivanov et al., 2007, Plieva et al., 2006). Larger pores are created with higher temperature. However, the temperature used must be sufficient to freeze the sample. It has been noted in our laboratory that supercooling is important in cryogel formation, with the gel being cooled below 0°C before ice crystallisation either takes place, or is initiated. This seems to be important in the production of interconnected pores (Ilsley M and James, SL unpublished data). Pore structure of cryogel can be observed using (i) scanning electron microscopy (SEM), which could be done in a dry state, (ii) size exclusion chromatography (iii) environmental scanning electron microscopy (ESEM) can be used to observe changes in the structure of the material whilst the sample dehydrates slowly (Rizzieri et al., 2003) (iv) confocal scanning microscopy and optical microscopy have also been used for analysis of pore 29

sizes (Adrados et al., 2001).. Distribution of pore size of rigid monoliths can be obtained by mercury intrusion porosimetry (Bryntesson, 2002, Josic et al., 2001, Svec and Frechet,

1995, Viklund et al., 1996, Wu et al., 2003, Zou et al., 2002).

1.9.2.2 Flow rate and flow resistance of monolithic cryogels

Supermacroporous monolithic cryogels comprise of large macropore of different sizes, leading to high flow through in these materials (Persson et al., 2004, Plieva et al., 2004a).

More than 70% of the water within the monolithic bed can be squeezed mechanically from the pores owing to the highly porous nature of the column. Such highly porous systems allow relatively free flow within the matrix. The flow rates of water passing through the columns have been measured at a constant hydrostatic pressure equal to

100cm of water-column corresponding to a pressure of circa 0.01MPa (Plieva et al.,

2004b). The flow resistance of cryogels has also been evaluated with a peristaltic pump

(Adrados et al., 2001).

1.9.3 Functionalization of cryogel matrix surface

Cryogels can be easily modified either through modifying a pre-existing cryogel or during the cryogelation step (Arvidsson et al., 2003, Arvidsson et al., 2002, Dainiak et al., 2005, Galaev et al., 2007, Yilmaz et al., 2009). The modification of a cryogel surface can be done via covalent bonding (i.e., epoxy method, glutaraldehyde method, Schiff base method, carbonyldiimidazole method, disuccinimidyl carbonate method, hydrazide method and cyanogen bromide method), entrapment or bio specific adsorption (Mallik and Hage, 2006). 30

1.9.3.1 Epoxy method

The epoxy method involves the nucleophilic attack of an epoxy group on the matrix by amine groups on a protein or ligand, leading to formation of a stable secondary amine linkage as illustrated in Fig.1.5 (Mallik et al., 2004). This coupling technique has been used with monolith discs to immobilize protein G (Berruex et al., 2000, Gupalova et al.,

2002), protein A (Berruex et al., 2000, Luo et al., 2002, Pan et al., 2002), protein L

(Berruex et al., 2000), BSA (Ostryanina et al., 2002) and bradykinnin (Ostryanina et al.,

2002). It has also been used with glycidyl methacrylate and ethylene dimethacrylate

(GMA/EMDA) columns to immobilize L-histidine (Luo et al., 2002), HSA (Jiang et al.,

2005, Mallik et al., 2004) and antibodies (Jiang et al., 2005). This method can be performed as a single step (Jiang et al., 2005, Mallik et al., 2004) but does have a slower reaction rate than other available methods. This can result in low amounts of immobilized ligand or long immobilization time (Mallik et al., 2004). Depending on the reaction conditions, this method can be used for ligands that contain amine, sulfhydryl, or hydroxyl groups (Kim and Hage, 2005).

OH O Protein-NH2 Matrix Matrix H N protein

Figure 1.5. Schematic representation of epoxy immobilization method

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1.9.3.2 Schiff base method

The Schiff base method involves conversion of epoxy groups on the matrix into diols.

These diol groups are then oxidized with periodic acid to give aldehyde groups, which can react with primary amines on proteins and other ligands to form Schiff base. This reaction is reversible; hence the Schiff base is further converted by reducing it with sodium cyanoborohydride to give a secondary amine. Combining them with smaller amine-containing ligand or reducing them to alcohols by adding sodium borohydride

(Kim and Hage, 2005) can further eliminate any aldehyde group that remains on the support as shown in Fig. 1.6. This Schiff base method has been used with GMA/EDMA monoliths to immobilize protein A (Luo et al., 2002), HSA (Mallik et al., 2004) and antibodies (Jiang et al., 2005).

OH

O H2SO4 OH Matrix Matrix

Protein-NH HIO CHO 2 4 Matrix protein Matrix N NaCNBH 3 H

Figure 1.6. Schematic representation of Schiff base immobilization method

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1.9.3.3 Carbonyldiimidazole (CDI) method

The carbonyldiimidazole method involves converting epoxy groups on matrix into diols.

These diol groups are then reacted with 1,1’-carbonyldiimidazole to produce imidazolyl carbamate groups. This activated support can be used in ligand immobilization by means of a nucleophilic substitution that can occur between the activated sites and primary amines on a protein or ligand, resulting in a stable amide linkage as represented in

Fig.1.7. The CDI method has been used with GMA/EDMA monoliths to immobilize L- histidine (Luo et al., 2002), HSA (Mallik et al., 2004) and antibodies (Jiang et al., 2005).

This method is faster than the epoxy method and involves fewer steps than the Schiff base or glutaraldehye methods. However, the CDI method also tends to give lower ligand activities than Schiff base technique (Jiang et al., 2005, Kim and Hage, 2005, Mallik et al., 2004).

OH O H SO 2 4 OH Matrix Matrix

N O N OH N N O N O O O N Matrix O

OH Protein-NH H 2 O N Matrix protein O

Figure 1.7. Schematic representation of carbonyldiimidazole immobilization method 33

1.9.3.4 Disuccinimidyl carbonate (DSC) method

The DSC technique involves covalent immobilization of ligands containing amine groups on monoliths (Calleri et al., 2004). It can be used on monoliths that contain epoxy group by first converting epoxy group to diol groups. The diol groups are then reacted with

DSC to place succinimidyl carbonate groups on the monolith’s surface; (Jiang et al.,

2005, Mallik et al., 2004). The activated monolith is then reacted with a ligand, such as protein, that contains primary amine groups to form a stable carbamate linkage as illustrated in Fig.1.8 (Kim and Hage, 2005). This method has been used with an amine- containing silica monolith to immobilize β-glucuronidase (Calleri et al., 2004). The disuccinimidyl carbonate method is fast and has been reported to be complete within 10 hours (Mallik et al., 2004). The stability of DSC and its activated monolith is low, hence requiring proper care to be taken to avoid side reactions due to hydrolysis (Hermanson,

1995, Kim and Hage, 2005).

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OH O H SO OH Matrix 2 4 Matrix

O O O N N O OH O O O O O O Matrix N O O

OH Protein-SH H 2 O N Matrix protein O

Figure 1.8. Schematic representation of disuccinimidyl carbonate immobilization method

1.9.3.5 Hydrazide method

The hydrazide technique can be used with glycoproteins and carbohydrate-containing ligands. This starts by producing an aldehyde–activated matrix in the same manner as described in Schiff base method. The matrix is activated with a reagent, such as adipic dihydrazide, with any remaining aldehyde groups later being reduced to alcohols with sodium borohydride. The activated support can be used to immobilize carbohydrate- containing ligands that have been oxidized under mild conditions to form aldehyde groups (Ruhn et al., 1994). The latter can react with hydrazide groups on the support to form stable hydrazine bond as shown in Fig. 1.9. This approach has been used to

35

immobilize antibodies within GMA/EDMA monoliths (Jiang et al., 2005). Although this method involves more steps than other techniques mentioned earlier, the ability to immobilize through carbohydrate chains is an attractive approach for the site-selective attachment of antibodies and other glycoproteins to solid supports. This typically results in a higher activity for such ligands when compared with amine-based coupling methods

(Xuan and Hage, 2005).

OH O 0.5 M H SO OH Matrix 2 4 protein

O H IO H 5 6 CHO Adipic N Matrix Matrix N NH

dihydrazide O NH2

NaBH4 NH2 Matrix NH

a Protein-CHO N Matrix NH

Figure 1.9. Schematic representation of hydrazide immobilization method

1.9.3.6 Cyanogen bromide (CNBr) method

CNBr is the most common immobilization technique in traditional affinity chromatography. This method involves mixing an ice cold, basic solution of CNBr with agarose or a polysaccharide-based gel as shown in Fig.1.10. The approach has been used 36

successfully on monoliths to immobilize amine-containing ligands, such as analogues of

NAD’ and L-chlorosuccinamic acid (Gustavsson and Larsson, 1999, Valle et al., 2003).

The immobilization of protein A onto the poly (hydroxyethyl methacrylate) PHEMA cryogel surface was carried out via cyanogen bromide activation, for the removal of IgM antibody in human plasma and antibody purification (Alkan et al., 2009, Alkan et al.,

2010). Indeed, CNBr immobilization technique is relatively simple and easy to perform.

CNBr is however toxic and a chemical hazard (Gustavsson and Larsson, 1999). It has been reported that CNBr method also tends to generate ion-exchange sites on the support that can lead to nonspecific binding (Hermanson, 1995, Kim and Hage, 2005).

OH CNBr O Matrix OH Matrix N

NH Protein-NH 2 protein Matrix O N H

Figure 1.10. Schematic representation of cyanogen bromide immobilization method

1.9.3.7 Bio-specific adsorption

Bio-specific adsorption involves adsorbing ligand to a support through non-covalent interactions. The common way for accomplishing this is to first covalently immobilize another substance to the support (known as secondary ligand). The covalently bound

37

substance can bind the ligand of interest in a way that does not interfere with the ligand’s ability to bind its target. Two binding agents that are often used as secondary ligands in affinity columns are protein A and protein G. These are useful as secondary ligands due to ability to bind Fc region of antibodies from various species. There are several examples in which protein A or Protein G has been used on GMA/EDMA discs or rods for the purification of antibodies (Berruex et al., 2000, Gupalova et al., 2002, Luo et al.,

2002, Ostryanina et al., 2002). In similar work, the lectin Concanavalin A has been immobilized in GMA/EDMA monolith and used as a secondary ligand for the immobilization of glucose oxidase through the carbohydrate regions of enzyme (Josic et al., 1998). In similar work, Concanavalin A was immobilized on cryogel monolith as a secondary ligand for affinity chromatography of glycoproteins (Hajizadeh et al., 2012).

1.9.3.8 Entrapment

In the entrapment process, the affinity ligand is incorporated as part of the polymerization mixture. As polymerization occurs, the support grows around the ligand and entraps or encapsulates it within the support. This procedure is attractive for use with sol-gel materials as they are formed in an aqueous solvent, which allows the ligands to be entrapped in a compatible solvent that should not lead to any significant denaturation

(Hodgson et al., 2004, Kato et al., 2002). This procedure has been used on cryogels for in-vitro removal of iron out of the human plasma with beta thalassemia (Asliyuce et al.,

2012).

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1.9.3.9 Coordination methods

The coordination method in affinity monoliths involves the use of coordination chemistry and metal-ligand complexes. This is employed in applications involving immobilized metal-ion affinity chromatography (IMAC). In IMAC the affinity ligand is actually an immobilized metal ion such as Cu 2+ or Ni2+. The metals are held on the column by their ability to form a coordination complex with a chelating agent like iminodiacetic acid

(IDA) or nitrilotriacetic acid (NTA), covalently linked to the support (Hermanson, 1995,

Kim and Hage, 2005). Cryogels containing IDA and Cu 2+ have been utilized to purify histidine-tagged lactate dehydrogenase (Arvidsson et al., 2003) and Escherichia coli cells

(Arvidsson et al., 2002). Work was performed with an acrylamide-based supermacroporous cryopolymer to generate an IMAC column based on IDA and Cu 2+

(Dainiak et al., 2004, Plieva et al., 2006a, Plieva et al., 2004b). Separation of different microbial cells using IMAC supermacroporous monolithic columns (Dainiak et al., 2005) and immobilized metal affinity monolithic cryogels for cytochrome C purification

(Çimen and Denizli, 2012) has also been reported.

1.9.3.10 Biomimetic affinity chromatography

The biomimetic affinity chromatography process involves the use of other ligands including, peptides, that have been selected from combinatorial libraries, which imitate some small biological substances (Labrou et al., 2005). As an example, one study used a cryogel that contained immobilized protein A or sulphamethiazine. These two affinity monoliths were used to bind and analyse inclusion bodies generated during the

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fermentation of recombinant proteins. Protein A and sulphamethiazine were utilized in the report to bind immunoglobulin G and immunoglobulin Y class antibodies, that were generated against a 33 kDa target protein (Ahlqvist et al., 2006). Phage display libraries have been explored in the past as a mean for generating peptide libraries for affinity selection (Labrou et al., 2005). These have also been used in recent years with monolithic supports (Noppe et al., 2006). An example of such chromatography column is cryogels where phages expressing a peptide with an affinity for lactoferrin were covalently immobilized on the monolith and used for purification of lactoferrin milk (Noppe et al.,

2006, Noppe et al., 2007).

1.10 Blood biocompatible

About 60 years ago the word ‘biomaterial’ did not exist. However, biomaterials are widely used throughout medicine, dentistry and biotechnology in the 21st century.

Medical devices such as external prosthetic – limbs, fracture fixation, glass eyes were the only ones available, other medical devices were not manufactured (Ratner et al., 2004).

A biomaterial is a nonviable material used in a medical device, intended to interact with biological systems (Williams, 1987). The majority of biomaterials available produce non- specific, stereotyped biological reactions. Today, efforts are directed towards the development of engineered surfaces that could have rapid and highly precise reactions with cells and proteins tailored to bio-specific application.

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Biomaterials can be sub grouped into four major classes: (i) polymers, (ii) metals,

(iii) ceramics including carbons, glass – ceramic and glasses, and (iv) natural material both from plants and animals (Black and Hastings, 2011, Hoffman, 2000).

Biocompatibility is the ability of a material to perform with an appropriate host response in a specific application (Williams, 1987). Appropriate host responses include the resistance to blood clotting, resistance to bacterial colonization, and normal, uncomplicated healing. For some applications such as haemodialysis membranes, which could be in contact with blood for 3 hours, urinary catheter that could be implanted for a week or a hip-joint replacement prosthesis, which could be implanted for a lifetime.

During the past decades, synthetic membranes have become widespread in medical applications, including haemodialysis, haemofiltration, plasmapheresis, plasma collection and oxygenation of blood during cardiac surgery (Deppisch et al., 1998). However, patients develop complications – adverse interactions with some implants and extracorporeal devices. These complications are mostly a consequence of biomaterial – tissue interactions. The inflammatory and potential immunological interactions that occur with biomaterials comprise non-specific inflammation. There is no synthetic or modified biological surface developed so far that is as thromboresistant as normal unperturbed endothelium (the cellular lining of the circulatory system (Bonetti et al., 2003).

1.11 Cryogels applications in biomedicine and biotechnology

There has been an increasing interest in using macroporous materials for a wide range of applications within the field of biomedicine and biotechnology (Allan S, 2002, Galaev 41

and Mattiasson, 1999). Cryogel morphology is characterized by macroporous structures, which have interconnected pores, and are spongy, elastic and compressible (Arvidsson et al., 2003, Lozinsky, 2002). These characteristics make them suitable to pass liquid containing particles without blocking the pores of cryogel (Lozinsky, 2002, Lozinsky et al., 2003, Plieva et al., 2007).

Indeed, cryogels have been used for the separation of biological molecules and cells. In particular, monolithic materials have gained wide interest as chromatography supports in biomolecules separation (Jungbauer and Hahn, 2008, Svec, 2010). In line with this, monolithic cryogels have been widely studied as a suitable matrix for chromatography of cells, proteins and particles (Dainiak et al., 2007b, Derazshamshir et al., 2008, Wang et al., 2008, Yilmaz et al., 2009). One of the many properties of cryogels, that is large pores between 10-200μm, makes a cryogel a more suitable material for chromatography of whole cells or large particles (Dainiak et al., 2007a). It has been reported that red blood cells of ~7 µm size have been conventionally transported by liquid flow through monolithic cryogel column without being trapped mechanically (Dainiak et al., 2007a,

Kumar et al., 2005).

Affinity chromatography of cells is a well-known method; nonetheless, it has never been generally accepted due to the complex nature of the process and low capability of conventional packed beads. However, a macroporous monolithic cryogel may apparently provide an improvement to this method. In this regard using a cryogel monolithic column functionalized with protein A in the chromatographic regime, affinity binding of cells to 42

cryogel was achieved (Kumar et al., 2005). The capture of human acute myeloid leukaemia KG-1 cells expressing the CD34 surface antigen and fractionation of human blood lymphocytes were evaluated on poly vinyl alcohol beads and dimethylacylamide

(DMAAm) monolithic cryogel with immobilized protein A. About 90-95% of the cells were retained on the monolithic protein A cryogel compared with cryogel beads 76%

(Kumar et al., 2005). On the other hand, affinity fractionation of lymphocytes was also performed (Kumar et al., 2003). The IgG-bearing B-lymphocyte from peripheral blood lymphocytes was separated using polyacrylamide cryogel with immobilized protein A, which selectively binds immunoglobuliins via their Fc-part. The lymphocytes were labelled with goat anti-human IgG and loaded on the protein A cryogel. The B–cells were selectively retained and recovered with a very high viability (Kumar et al., 2003).

Research showed that the protein A cryogel system bound explicitly more than 90% of

IgG–labelled B-lymphocytes, while non–labelled T-lymphocytes passed through the column. However, monolithic cryogels have also been used for isolation of microbial cells (Dainiak et al., 2005, Hanora et al., 2005, Hanora et al., 2006), inclusion bodies

(Ahlqvist et al., 2006) and mitochondria (Teilum et al., 2006). Cryogels have also been used for antibody purification on protein A modified column (Alkan et al., 2009) and for the selective removal of the auto antibodies from rheumatoid arthritis patient plasma using protein A carrying affinity cryogels (Alkan et al., 2010). Chelating metal ions in cryogel for immobilized metal affinity chromatography (IMAC) have been used for purification of cells (Dainiak et al., 2005), DNA (Odabaşı et al.) and viruses (Cheeks et al., 2009). 43

The fact that cryogels are easily modified using various ligands and the broad selection of polymers that can be synthesized offers them as attractive prospects for further research and applications at a preparative scale. Thus, this might be an appropriate matrix suitable for use in extracorporeal apheresis systems.

1.12 Specific aims and objectives

The aims of this PhD were to study the structural, mechanical and haemocompatibility features required to develop an affinity-binding cryogel column from a non-biological

(synthetic) monomer 2-hydroxyethyl methacrylate (HEMA), as an appropriate matrix for use in extracorporeal apheresis systems. Such a matrix would be of use in a wide range of clinical situations where specific removal of specific blood components would be beneficial to the patient. The main objectives were to:

(I) Produce macroporous monolithic polymer cryogels from different materials

and with a range of interconnected pore sizes

(II) Investigate the mechanical stability and flow properties of the cryogels

(III) Assess the haemocompatibility of the cryogel

(IV) Functionalize the surface of the cryogel matrix with a biological ligand.

The objectives were pursued by synthesizing shape memory macroporous monolithic polymer cryogels with interconnected pores ranging from 5 μm-200 μm from different synthetic polymers (epoxy containing 2-hydroxyethyl methacrylate (PHEMA), poly vinyl pyrrolidone (PVP) and co-polymer of 2-hydroxyethyl methacrylate, (PHEMA and PVP) 44

and poly (hydroxyl ethyl methacrylate-co-N-(3-aminopropyl) methacrylamide) (APMA) via free radical polymerization at -12°C.

The morphology of these cryogels was evaluated using confocal laser scanning microscopy and porosity, pore size and wall thickness of cryogels were measured with image J analysis based on confocal laser microscopy and scanning electron microscopy.

Flow properties of the produced macroporous monolith were measured using water and different concentrations of dextran. Void volume of the column was evaluated by passing bovine serum albumin and blue dextran through the column.

Mechanical properties, such as storage modulus, compressive modulus, and cyclic compression and creep test, were evaluated using dynamic mechanical analyser. The monolithic cryogel mechanical stability was also examined by measuring the pressure difference of water and plasma passing through the monolithic cryogel column with a pressure-monitoring device.

The haemocompatibility of PHEMA macroporous monolithic cryogel was evaluated in vitro using fluorescent activated cell sorter (FACS) and Sysmex cell counter to examine platelet activation and haemolysis of red blood cells after passing through the column.

To functionalize the cryogel matrix biological-ligands such as amylase (enzyme) and anti-human albumin were immobilized using the glutaraldehyde method. The covalent immobilization of ligand was performed after the cryogel column had been synthesised.

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Affinity binding of human serum albumin to the cryogel matrix with immobilized anti- human albumin was achieved using the glutaraldehyde method. The total amount of protein bound to the matrix was measured with bicinchoninic acid.

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Chapter Two: Cryogel synthesis and pore structure characterization

2.1 Introduction

Cryogels are macroporous polymeric materials that can be synthesized by free radical polymerization or covalent chemical crosslinking of polymer or non-covalently by

(entrapment of particles to form composite cryogel or cryogelation through the non- covalent structuring of polymers). These techniques have been discussed in chapter one section 1.9.1. Briefly, free radical polymerization is controlled by the composition of initiating system in the reaction mixture. Concentrations of the initiator ammonium persulphate (APS) and activator (TEMED) have a major impact on the polymerization rate as well as on molecular weight of the resulting polymers. These processes take place at sub-zero temperatures (Arvidsson et al., 2002). The solvent (water) is frozen while the dissolved chemicals (monomers, cross-linkers and initiators/activator system) are concentrated in small non-frozen regions, referred to as ‘non-frozen liquid micro phase’ where polymerization occurs. Formation of the gel occurs in the liquid phase and the ice crystals of frozen solvent serve as pore agent. After melting the ice crystals, the shape and size of the ice crystals determine the shape and size of the pores formed, hence a system of large interconnected pores is formed (Plieva et al., 2005). Macroporous monolithic cryogels with large interconnected pore size range between 10 µm and 200

µm are heterophase systems where solvent (water) is present both inside interconnected

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pores and bound to the polymer network (Arvidsson et al., 2003, Dainiak et al., 2004,

Plieva et al., 2011).

To produce a cryostructurated monolith with organized structures, there should be appropriate control of freezing conditions. Pore size, porosity and wall thickness, of macropores in both freeze dried (removal of solvent crystals through sublimation) and freeze thawed (removal of solvent crystals through thawing) are dependent mainly on freezing condition and composition of initial reaction mixture used. Cryogels have some unique properties, such as being highly porous, elastic and spongy. They have also shown shape memory as they can be repeatedly dried and re-swollen in the solvent, acquiring the same shape in which they were synthesized. Concentration of reagents in the initial reaction mixture and freezing system are the main parameters affecting the final porous properties of the cryogels. The lower the freezing rate, the larger the size of developing ice crystals and, as a result, leads to cryogel with larger pore sizes. Meanwhile the higher the freezing rate, a higher nucleation is promoted, and more number of small ice crystals is formed. These cryogels are designed as monolithic rods, discs and sheets.

Cryogels can be easily functionalized either through modifying a pre-existing cryogel or introduction during cryogelation step (Arvidsson et al., 2002, Dainiak et al., 2005).

During direct copolymerization with functional comonomers the required functionality is incorporated in situ during synthesis of cryogel with single freeze-thawing cycle. This functional comonomer is added to the reaction mixture at concentration of 5-10 mol% to

48

the total concentration of main monomers (Arvidsson et al., 2002, Ceylan et al., 2006,

Dainiak et al., 2007a, Ozmen and Okay, 2005).

The technique used mainly in the analysis of the porous structure of rigid cryogel monoliths (silica, ceramic and carbon cryogels prepared via freeze-drying) is mercury porosimetry (Kwon et al., 2009, Nishihara et al., 2006, Nishihara et al., 2010). Cryogel monoliths with soft and elastic morphology (e.g., pAAm,pHEMA,pNIPA-prepared via freeze-thawing can be analysed with confocal laser scanning microscopy (CLSM) (Plieva et al., 2008, Savina et al., 2007), scanning electron microscopy (SEM) (Demiryas et al.,

2007, Gun'ko et al., 2010, He et al., 2007, Plieva et al., 2006, Yao et al., 2006) and environmental SEM (ESEM) (Chen et al., 2008, Kirsebom et al., 2007, Perez et al.,

2008, Plieva et al., 2005). The removal of water from cryogel when preparing for scanning electron microscopy (SEM) could affect the cryogel structure. CLSM allows the possibility of measuring the structure of the material in its hydrated state. Also confocal microscopy of cryogels in wet state was found to be in good agreement with SEM, illustrating the presence of large, 10-200 µm interconnected pores with smooth pore walls and dense polymer walls in between the pores. The flow rate of liquid passing through the column can be measured at the constant hydrostatic pressure equal to 100cm of water column corresponding to a pressure of circa 0.01MPa (Plieva et al., 2004a).

Monolithic macroporous cryogel columns are considered as a new chromatography medium that allows carrying out of high-speed separation of proteins without losing the column efficiency (Josic and Buchacher, 2001, Peters et al., 1999). 49

The procedure for producing supermacroporous continuous cryogels is technically simple and the monomers and initiators cheap, this makes continuous supermacroporous cryogels a very interesting alternative to existing media for protein purification.

In this thesis hydrophilic monolithic cryogels (PHEMA and PVP) were prepared through a free radical copolymerization reaction of ally glycidyl ether (AGE) and N-3- aminopropyl methacrylamide (APMA) with a cross-linker, N,N’-methylene bisacrylamide, (MBAA) in aqueous media. AGE was selected to introduce the reactive a epoxy group and APMA was used to introduce the amino group as functionalities during synthesis of the cryogel. Biocompatible synthetic polymers, such as poly (2-hydroxyethyl methacrylate (PHEMA) (Horak et al., 2004, Kaufman et al., 2007, Kroupová et al., 2006,

Vijayasekaran et al., 2000) and poly (vinylpyrrolidone) (PVP) (Engstrom and Helgee,

2006), were chosen to synthesize cryogels material for biocompatible filter in extracorporeal apheresis devices.

2.2 Aims

In this chapter, supermacroporous cryogel from different synthetic polymers (epoxy containing 2-hydroxyethyl methacrylate (PHEMA), poly vinyl pyrrolidone (PVP) and copolymer of 2-hydroxyethyl methacrylate, (PHEMA and PVP) and poly (hydroxyl ethyl methacrylate-co-N-(3-Aminopropyl) methacrylamide) (APMA) were produced at -12°C by free radical polymerization. The aim of this study was to produce a range of macroporous cryogels with interconnected pores ranging from 10 µm–200 µm and subsequently, characterize the morphology of the materials. 50

2.3 Materials and methods

Poly vinyl pyrrolidone (PVP), 2- hydroxy ethyl methacrylate, (HEMA 98%, stabilized), allyl glycidyl ether (AGE 99%), and N,N’-methylene bisacrylamide, (MBAA 96%) were purchased from Acros Organics UK. N,N,N’,N’–tetramethyethylenediamine electrophoresis grade (TEMED 97% Fisher Bio reagents) and ammonium persulfate

(APS) were purchased from Fisher Scientific UK. Poly (hydroxyl ethyl methacrylate-co-

N-(3-Aminopropyl) methacrylamide) (APMA) was supplied by Polysciences Europe

Germany.

Dimethyl sulphoxide K 402 70531 933 and Fluorescein 5(6)-isothio-cyanate, mixed isomers (FITC 90%) from Sigma, Sodium hydrogen phosphate dihydrate K381118980

804 and Sodium bicarbonate were products of Merck. Conical flasks, vacuum pump, eppendorf tubes and glass columns were used. Confocal laser scanning microscope (Zeiss

LSM410), cryobath (Julabo Ltd UK), scanning electron microscope (JOEL JSM -

5600LV) and Image J software were also used.

2.3.1 Cryogel synthesis

Approximately 4ml cylindrical blocks of epoxy-PHEMA monolithic cryogels were synthesized from monomers of 2-hydroxyethyl methacrylate (HEMA 5.28 ml) and allyl glycidyl ether (AGE 1.08 ml), N, N’- methylenebisacrylamide (MBAA1.34 g) used as a cross linker. These were dissolved in 100 ml deionized water (final concentration of monomers were 8 w/v %); monomers ratio to MBAA was 6:1 and the resulting solutions were degassed for about 20 minutes by using N2 gas or water pipe vacuum. Free radical 51

polymerization was initiated by N, N, N, N-tetramethylethyl-enediamine (TEMED) and ammonium persulphate (APS) pair (1.2 w/w % TEMED and 1.2 w/w % APS of the total weight of monomers and MBAA). The mixture was then cooled under ice bath for about

30 minutes. APS was added to the solution for the onset of reaction and solution (4 ml) was aliquoted into glass columns, and was frozen at -12°C for 18 hours. The cryogels were allowed to thaw at room temperature while still in the glass columns before being washed with water; the gel matrix was kept in water and stored in the fridge till further use. The same (monomer final concentration 8 w/v % and monomers ratio to MBAA 6:1) was used for other cryogels synthesized from PVP and APMA respectively, and various final concentrations of 10, 13 and 16 w/v % were prepared in epoxy PHEMA samples.

2.3.2 Scanning electron microscopy (SEM) preparation

The cryogel samples for scanning electron microscopy (SEM) were fixed in 2.5% glutaraldehyde for 2 hours at room temperature. Then the samples were dehydrated in ethanol (0-50-75-99.5%) and air dried overnight in the fume cupboard. The dried sample was coated with palladium and gold (40/60) and examined using a JOEL JSM-5600LV scanning electron microscope.

2.3.3 Confocal laser scanning microscopy (CLSM) preparation

The cryogel was taken out of the column and sectioned with a scapel from the top, middle and bottom of the column. Each slice were ~1 mm in thickness, and stained with

FITC (Fluorescein 5(6) - isothio-cyanate) in 0.02 mg/ml in 0.1 M sodium phosphate buffer, pH 7.0 for 48 hours. Non-bound FITC was washed out with buffer and cryogel 52

slices were observed under confocal laser scanning microscope (CLSM). The fluorescent images were obtained using a confocal laser-scanning microscope, (Zeiss LSM410). A plant-Apochromat 20x/0.5 objective was used in all experiments. All images were taken at 512 X 512 pixels with pixel size of 0.999 μm (for x-plane) and 0.98 μm (for z-plane).

The number of sections was in the range of 54-73. Images collected by CLSM were used for the calculation of porosity, pore sizes and wall thickness using the software image J.

Image J is a public domain Java image-processing program inspired by NIH Image for

Macintosh. It runs, either as an online applet or as a downloadable application, on any computer with a Java 1.1 or later virtual machine. Downloadable distributions are available for windows, Mac Os, Mac Os X and Linux. It can display, edit, analyze, process, save, and print 8-bit, 16-bit and 32-bit images. It can read many image formats including TIFF, GIF, JPEG, BMP, DICOM, FITS and “raw”. It supports “stacks”, a series of images that share a single window. It is multithreaded, so time consuming operations such as image file reading can be performed in parallel with other operations.

It can calculate area and pixel value statistics of user-defined selections. It can create density histograms and line profile plots. It supports standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection and media filtering.

It does geometric transformations such as sealing, rotation and flips. Image can be zoomed up to 32:1 and down to 1:32. All analysis and processing functions are available at any magnification factor. The program supports any number of windows (images), simultaneously, limited only by available memory.

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2.3.4 Measurement of flow rate of water passing through cryogels

Monoliths of the same geometrical size and shape (approximately 1 cm diameter, 5 cm length) were prepared in the glass column. The flow rate of water passing through the monolithic column at constant hydrostatic pressure equal to a pressure of 0.01 MPa was determined by measuring the cubic (volume) flux (cm3/min) of water eluted through the monolith-packed glass columns in specific time interval (3 minutes). Epoxy PHEMA cryogels of 8%, 10%, 13% and 16% w/v were used for this experiment and for each sample the average of at least three measurements was taken.

2.3.5 Flow rate of different concentrations of dextran

The flow rate of aqueous dextran passing through the columns was also measured at the constant hydrostatic pressure equal to a pressure of 0.01 MPa. The concentration of dextran was 20%w/v (MW 12,000), 24 %w/v (MW 67,000), and 30 %w/v (MW 80,000).

For each sample, the average of at least three measurements was taken.

2.3.6 Void volume calculation

Void volume is the volume of mobile phase in a column and it is a valuable characteristic for any chromatographic column. 500 µl of Blue Dextran (MW 2,000 kDa) 25mg/ml and

500 µl of bovine serum albumin (BSA) (MW 69 kDa) 100mg/ml dissolved in sodium chloride pH 7.0 was injected into a 4 ml cryogel column.

54

(a) Time taken to travel down the column were observed and (b) volume was measured against absorbance of blue dextran set (at 620 nm) and absorbance of albumin set at (280 nm) using a visible spectrometer.

2.3.7 Statistical analysis

All statistical analyses were performed by first using one-way analysis of variance

(ANOVA, minitab version 15). Whenever ANOVA indicated the groups significantly different, a t-test for independent samples was performed. Samples were considered significantly different at P ≤ 0 .05.

2.4 Results and discussions

2.4.1 Synthesis of monolithic cryogels

Epoxy-PHEMA, epoxy PVP and HEMA-APMA monolithic cryogel synthesis success was very dependent on freezing gelation interaction. The most important step during the synthesis process was the rapid freezing of the reaction mixture; this takes place before the onset of gelation. Gels not different from traditional ones are formed when gelation starts before freezing. In order to prevent the production of poor quality monolithic macroporous cryogels, freezing was facilitated. Both empty glass columns and the reaction systems (monomer mixture and initator system) were ice cooled for at least 30 minutes before the reaction was initiated. The transfer of the reaction mixture into the empty glass columns was done rapidly to minimize gelation rate due to high temperatures. The cryogelation polymerization reaction began with the reaction between the initiator APS and the activator TEMED to form free radicals. The free radical initiates 55

the reaction of monomers HEMA, MBAAm and AGE (epoxy groups enhancer).

MBAAm also acts as a cross-linker. Fig 2.1 shows a monolithic cryogel after cryopolymerization in a glass column. The percentage recovery of monoliths per batch of

20 independent batches for 8%, 10%, 13% and 16% epoxy PHEMA was 99%, 98%, 96% and 97%, while in the epoxy PVP cryogel, percentage recovery was 90%, 95%, 94% and

97%. However in 8% HEMA-APMA (5:1, 10:1 and 50:1), the percentage recovery was approximately 99% from all monolithic cryogels. This proves that the cryogelation process is highly reproducible.

56

Figure 2.1. Cryogel in glass column after freezing, thawing and washed with water

2.4.2 Physical properties of monolithic cryogels obtained after cryopolymerization at

-12°C

Epoxy PHEMA, epoxy PVP and HEMA-AMPA cryogel produced were white, opaque, soft and spongy cylindrical blocks built up with systems of interconnected pore structures. The monoliths appear relatively more opaque as monomer concentration increases in their wet state. This could be linked to increase in thickness of polymer walls 57

as the monomer concentration increases. When washing the cryogels and certain volumes of water were poured from the top of the monolithic cryogel, you could observe the eluent from the cryogel with higher monomer concentration was slower than from the ones with lower monomer concentration. It was possible to mechanically squeeze out water from the monoliths and the monoliths re-swell immediately when in contact with water. Monolithic cryogels produced from epoxy PHEMA and HEMA-APMA were generally more mechanically stable than the one produced from epoxy PVP. The epoxy

PVP monolith cryogels were brittle, quite fragile and have poor ability to re-swell after drying. The spongy–like morphology, macropores interconnection and mechanical stability of both epoxy PHEMA and HEMA-APMA are typical characteristics of cryogels. However, the observed spongy and soft morphology was decreasing as monomer concentration increases. This probably could be attributed to the formation of thicker (denser) walls following increase of polymer content as monomer concentration in the reaction system was increased as shown in Fig 2.2. From observation, 16% epoxy

PHEMA cryogels appeared relatively rigid at their removal from the glass columns after thawing. On the other hand, 8% epoxy PHEMA cryogels appeared less rigid and softer.

In general, decreasing the initial monomer concentration below 6% would be associated with the production of fragile cryogels while increasing the initial monomer concentration above 16% could lead to the formation of rigid and less macroporous cryogels.

58

2.4.3 Structure of cryogel obtained after cryopolymerization at -12° C

When cryogels are produced, polymerization occurs in the non-frozen fluids containing dissolved monomers and initiator. The ice crystals formed during freezing works as a pore agent. Thus, the shape and size of the ice crystals formed determine the shape and size of the pores formed after defrosting the sample. The freezing rate is determined by the starting temperature, which was 0°C in this situation, as all solutions before mixing were kept in an ice bath, and the freezing temperature was -12°C.

Figure 2.2. Image of parameters affecting pore structure of monolithic macroporous cryogel (Plieva et al., 2005)

59

In this study, SEM and CLSM were used for pore structure analysis. Although SEM operates in dry states, the method is still appropriate for the macroporous epoxy PHEMA and HEMA-APMA cryogels. As shown from the SEM micrographs (Fig: 2.3), the produced epoxy PHEMA monolithic cryogels were porous material of micrometre scale.

Observation from SEM images (Fig: 2.3) shows that 8% epoxy PHEMA monolithic cryogel has larger pores compared to 10%, 13% and 16%. This findings correlates with the explanation that a decrease in pore volume percentage is related to the increase in monomer concentration in the reaction mixtures.

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Figure 2.3. SEM microphotography of (A-D) 8%, 10%, 13% and 16% of epoxy PHEMA cryogels

The macroporous structures of prepared epoxy PHEMA monolithic cryogels were analysed with CLSM in their natural wet state. The cryogels were stained with fluorescent dye (Fluorescein 5(6)-isothio-cyanate, mixed isomers (FITC)) to make them visible. The polymer network of epoxy PHEMA (green pore walls of cryogel shown in

Fig: 2.4) shows it consists of continuous interconnected macropores. Images obtained from CLSM were used to calculate the pore size distribution from different layers of the 61

monolithic cryogel. Pore size distribution analysis in this thesis will focus on 8% epoxy

PHEMA cryogel. The pore sizes produced in 8% epoxy PHEMA cryogel range from 10

μm -120 μm as shown in (Fig: 2.5), owing to the data obtained from the image j software analysis.

8% epoxy containing PHEMA macroporous monolithic cryogel columns were produced from the copolymerization of HEMA and AGE at a temperature of -12°C. The cryogel matrix has a porosity of ~87% and consists of ~13% of solid polymer, values were obtained from calculation made using image J software. The main pore sizes seen in cryogel matrix from SEM image Fig: 2.3 are below 100 µm, this correlates with image J values obtained while using images from CLSM. Porosity of the cryogel was calculated by software image J using CLSM image shown in Fig: 2.4, this showed approximately

80% of pores are distributed within the range of 41-81µm as shown in Fig: 2.5 and ~ 70% of the wall thickness are in the range of 9-21µm as shown in Fig: 2.6. The average porosity of 8% epoxy PHEMA cryogel was calculated between the top, middle and bottom layer data shown in Table: 2.1. These properties, large pore size and high porosity, illustrate 8 % epoxy containing PHEMA cryogel has a low resistance to flow of fluids.

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Figure 2.4. CLSM image of 8% epoxy PHEMA cryogel

63

0.20

0.15

t

h

g

i

l

f

o

y 0.10

t

i

s

n

e D 0.05

0.00 1 11 21 31 41 51 61 71 81 91 01 11 21 1 1 1 Pore size distribution (µm)

Figure 2.5. Pore size distribution of 8% epoxy PHEMA cryogel matrix n=10

64

0.15

t h

g 0.10

i

l

f

o

y

t

i

s n

e 0.05 D

0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33

Wall thickness (µm)

Figure 2.6. Wall thickness of 8% epoxy PHEMA cryogel matrix n=10

65

Table 2.1. Summary of 8% epoxy PHEMA cryogel matrix structure measured using images obtained from optical confocal laser scanning microscopy with the software image J. n=10. The results are given as mean ± standard deviation

cryogel matrix Average porosity Pore size Wall thickness layer through the column distribution (μm) (μm) (%)

Top 88 ~80% of the pores ~70% of the wall are within the thicknesses are in Middle 83 range of 41-81μm the range of 9-21 μm Bottom 90

Average 87

2.4.4 Flow rate of water passing through different concentrations of epoxy PHEMA

cryogels column

Epoxy containing PHEMA cryogels, with starting monomer concentration of 8%, 10%,

13% and 16%, were produced. Water was passed through the 1cm x 5cm epoxy PHEMA cryogel column at a hydrostatic pressure equal to 100cm. Fig: 2.7 shows the different flow rates of water passing through the column. From the obtained results, the flow rate of water passing through different concentration of monolithic cryogel decreases as the monomer concentrations increases, which might illustrate that, an increase in monomer

66

concentrations leads to a decrease in pore size. 8% epoxy PHEMA cryogel has been chosen for evaluation in most of the experiments because it has the lowest flow resistance to fluid and seems to be most appropriate for the purpose in this thesis.

Figure 2.7. Flow rate of water passing through different concentrations of epoxy PHEMA

cryogel column n = 3 results are given as mean ± standard deviation

67

2.4.5 Flow rate of different concentrations of dextran passing through 8% epoxy

PHEMA cryogels

Different concentrations of dextran in water were passed through the column at hydrostatic pressure equal to 100 cm and flow rates were measured. The flow rate reading obtained showed that different concentrations of dextran 20%w/v (MW 12,000),

24 %w/v (MW 67,000), and 30 %w/v (MW 80,000) passed through 8% epoxy PHEMA cryogel column, with the most viscous fluid having the highest flow resistance, as shown in Fig 2.8. The viscosity of 30%w/v (MW80,000) dextran solution is almost equivalent to the viscosity of human whole blood, which is between ~2.5 and 3.0mPas, hence the reason it has been chosen as most viscous fluid in this experiment. These data show that epoxy PHEMA monolithic cryogel has pores that are large and interconnected, which allow the flow of viscous fluid through the column.

68

Figure 2.8. Flow rate of dextran passing through 8% epoxy PHEMA cryogel column n=3 results are given as mean ± standard deviation.

69

2.4.6 Void volume measurement

Void volume is the volume of mobile phase that is held within a column and it is a

valuable characteristic for any chromatographic column. The void volume of 8% epoxy

PHEMA cryogel was calculated by running 500 µl of blue dextran (MW 2000 kDa) and

500 µl of bovine serum albumin (BSA MW 69 kDa) through the column. Fractions were

collected at flow rate 1ml/minute and absorbance of elutes was measured at 620 nm.

2.0 )

m 1.5

n

0

2

6

(

e

c 1.0

n

a

b

r

o s

b 0.5 A

0.0 0 10 20 30 40 Time (Minutes)

Figure 2.9. Absorbance of blue dextran at 620nm passing through column n= 3

70

Figure 2.10. Absorbance of blue dextran at 620 nm passing through column, n=3.

The first drop of blue dextran were observed at 3 minutes while a volume of appropriately 1.5 ml of liquid was eluted from the column as shown in Fig 2.9 and Fig

2.10; these 1.5 ml represent the void of the column. Equation 2.1 calculated the total volume of the column

Equation 2.1

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Where: Vt is total volume of the column R2 = square of the radius of the column; diameter of column is 1cm Π= constant 3.14 and h is the height of the column.

Total volume of the column Vt = 3.14 X 0.5 cm X 0.5 cm X 5 cm = 3.925 ml. This value correlates with the initial volume of reaction mixture 4 ml that was poured into each glass column before cryopolymerization. Therefore, the void volume of this column as percentage of total volume = 1.5 ml/3.925 ml x 100 = 38.21%.

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1.5

)

m n

0 1.0

8

2

(

e

c

n

a

b r

o 0.5

s

b A

0.0 0 10 20 30 40 Time (Minutes)

Figure 2.11. Absorbance at 280nm against time of albumin passing through column, n=3

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Figure 2.12. Absorbance at 280 nm against volume of albumin passing through column, n=3

The first drop of albumin was observed at appropriately 3minutes after 1.7 ml of liquid were eluted from the column as shown in Fig 2.11 and Fig 2.12; these 1.7 ml represent the void of the column.

Equation 2.2

74

Where: Vt is total volume of the column R2 = square of the radius of the column; diameter of column is 1cm Π= constant 3.14 and h is the height of the column.

Total volume of the column Vt = 3.14 X 0.5 cm X 0.5 cm X 5 cm = 3.925 ml. This value correlates with the initial volume of reaction mixture 4 ml that was poured into each glass column before cryopolymerization. Therefore, the void volume of this column as percentage from total volume = 1.7 ml/3.925 ml X 100 = 43.31 %.

From the above data, the void volume of 8% epoxy PHEMA cryogel is within the range of 1.5 ml to 1.7 ml or 38 % to 43 % of the total volume of the column after passing both

BSA and blue dextran through the column. These results show that 8% epoxy PHEMA cryogel allows the passage of molecules between the range of 69 kDa and 2000 kDa and allows unrestricted diffusion of solutes of various sizes due to its macroporous property.

2.5 Conclusions

The cryogelation technique has made it possible to produce monolithic materials with unique properties. The porosity of monoliths produced can be diverse in nature combined with flow through channels to structures with uniformly distributed interconnected macropores of (~10 µm-120 µm) and twisted paths (main characteristics for freeze–

75

thawed cryogels prepared via free radical polymerization). The cryogelation process was highly reproducible with over 90% recovery. Pore size and thickness of pore walls in porous epoxy PHEMA cryogels are regulated mainly by changing the monomer concentration in the reaction mixture and the type of cross-linker used. Direct copolymerization was used to introduce the required functionality in cryogels during preparation. Depending on the potential applications, within the same technique one could produce materials with macropores and porosity up to 90 %, which possess low flow resistance and good mechanical properties (e.g., materials attractive as potential chromatographic adsorbents). These monoliths have an obvious potential as flow-through filters for different applications in extracorporeal apheresis devices. This definitely provides the opportunity to investigate the mechanical properties of the monolithic cryogel.

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Chapter Three: Characterization of poly (2-hydroxylethyl methacrylate) macroporous cryogel

3.1 Introduction

Poly (2-hydroxyethyl methacrylate) (PHEMA) is a well studied polymer widely used for many biomedical applications, ranging from production of contact lenses (Bowers and

Tighe, 1987) and wound dressing (Rosiak et al., 1990) to controlled release and drug delivery devices (Dziubla et al., 2001) as well as for the development of blood compatible surfaces (Bajpai and Shrivastava, 2002) and surgical prostheses (Ferruti et al.,

2004).

HEMA have primary alcohol groups that permit substitution reactions with different reactants, therefore introducing other required functionalities in the final product.

PHEMA membranes exhibit certain appealing properties such as permeability to oxygen combined with sufficient mechanical and viscoelastic behaviour (Bajpai and Shrivastava,

2002). Presently, there is a rising interest in macroporous polymer cryogels due to their distinctive various open porous structure which considerably increases their equilibrium sorption properties and allows unrestricted diffusion of solutes, nano – and even microparticles (Lozinsky et al., 2003). Cryogels have a unique combination of properties such as interconnected pores of 10 – 200 µm size, porosity exceeding 90%, high mechanical and chemical stability, elasticity and sponginess, drying and rehydrating without impairing pore structures (Arvidsson et al., 2002, Plieva et al., 2006).

77

Mechanical properties of polymeric materials which are the strength of today’s drug delivery and biomedical devices (Jones et al., 2012) is of particular importance as these have been shown to directly affect clinical efficacy (Jones et al., 1997). Therefore the successful development of such products requires extensive characterization and also comprehensive understanding of the polymeric material. These properties may be understood using a range of techniques including tensile analysis, thermomechanical analysis, thermodilatometry and the dynamic mechanical analyser (DMA).

DMA is the most easily accessible, providing a rapid and non-destructive way of quantifying the rheological/mechanical properties of polymers. These techniques used to measure the mechanical properties of polymeric materials involve application of an external force whilst measuring the resulting deformation of the material. The application of DMA for drug delivery and biomedical devices characterization has gained increased attention over the last decade. Comprehensive mathematical theories of the dynamic mechanical analyser have been previously described (Ferry, 1980, Jones, 1999, Ward and Hardley 1993). The dynamic mechanical analyser is an analytical technique in which oscillatory stress (the force per unit area (Pascal) required to deform the sample) is applied to the sample and the resultant strain (the amount by which the sample is deformed) selected from within the linear viscoelastic region and the result is determined as a function of both temperature and frequency (Jones, 1999). Dynamic mechanical methods characterize the viscoelastic properties (simultaneous existence of viscous

‘fluid’ and elastic ‘solid’ properties in all materials) (Barnes et al., 1996), such as storage

78

and loss modulus ( the resistance of a material to deformation ‘Pascals’) (Ferry, 1980).

Common characteristics of viscoelastic systems include after application of a constant stress, viscoelastic solids do not maintain constant deformation, but continue to deform as a function of time. (Craig and Johnson, 1995, Ferry, 1980, Ward and Hardley 1993).

This occurrence is referred to as creep. In flow process, viscoelastic liquid could store some of the applied energy and use this energy to partially recover from the stress- induced deformation. Hooke’s law states that the ratio of stress (σ) to strain (γ) in an elastic material is constant, within a specific elastic limit. The constant of proportionality is defined as Young’s modulus (Ε), which is calculated by Equation 3.1

Equation 3.1

The behaviour of elastic solids may be represented using an extendable spring whereby the load applied to the spring represents the stress and the extension represents the strain, shown in (Fig: 3.1). The load applied to the spring is directly proportional to the extension, which in the case of elastic solid is instantaneous; and time independent. On the removal of the load, the stored elastic energy is used to return the spring to its original 79

position; this response is also instantaneous (Patfoort, 1974). Deviations from ideal elastic behaviour occur whenever the elastic limit is exceeded (Barnes et al., 1996). 2- hydroxyethyl methacrylate (HEMA) was chosen in this project as the basic component because of its chemical and biological stability and biocompatibility (Denizli et al.,

2003).

Despite the various studies involving the use of cryogel and its derivatives as biomaterials, information about the various sterilisation methods and their effects on the properties of the materials is still limited. Therefore, sterilization of cryogel must be thoroughly investigated to determine optimal sterilization conditions.

The dynamic mechanical analyser determined mechanical properties of epoxy monolithic

PHEMA cryogel.

80

Figure 3.1. Strain response as a function of time for elastic solid and a viscous liquid, following the application of a constant stress (Jones et al., 2012) 81

3.2 Aims

In this chapter, supermacroporous cryogel from poly HEMA co- APMA, PVP and

PHEMA and epoxy PHEMA were produced. The aim of this study was to analyse some properties of monolithic cryogel such as i) yield percentage and swelling degree of cryogel ii) storage modulus, compressive modulus, creep test, strain percentage with a constant stress when cryogel is immersed in water iii) pore interconnectivity and mechanical properties of macroporous cryogel were also investigated by measuring the pressure difference of water and plasma passing through the macroporous monolithic column. Poly HEMA-co-N-(3-Aminopropyl) methacrylamide cryogels were produced/introduced to have a comparison with epoxy containing cryogel in terms of yield percentage and swelling degree.

3.3 Materials and methods

An epoxy containing PHEMA cryogel was produced from monomers of 2-hydroxyethyl methacrylate (HEMA 0.528 ml) and allyl glycidyl ether (AGE 0.108 ml), N, N’- methylenebisacrylamide (MBAAm 0.1342 g) was used as a cross linker, these were dissolved in deionized water (final concentration of monomers 8 % w/v), monomers ratio to MBAAm 6:1. Poly HEMA-co-N-(3-Aminopropyl) methacrylamide cryogel was produced from monomers 2-hydroxyethyl methacrylate (HEMA 0.493 ml, 0.549 ml and

0.606 ml) and N-(3-Aminopropyl) methacrylamide (APMA 0.145 g, 0.081 g and

0.0179g), N, N’- methylenebisacrylamide (MBAAm 0.125 g, 0.128 g and 0.131 g) was used as a cross linker, these were dissolved in deionized water (final concentration of

82

monomers 8% w/v), monomers ratio to MBAAm 6:1. Free radical polymerization was initiated by N’, N’, N’, N-tetramethylethyl-enediamine (TEMED) as well as ammonium persulphate (APS) pair (1.2 w/w % TEMED and 1.2 w/w % APS of the total weight of monomers and MBAAm). The mixture was poured into glass columns (0.5ml) and was frozen at -12°C for 18 hours. Dynamic mechanical analyser DMA 2980 by TA

Instruments, Inc. (USA), Sample clamp: Compression plates (15 mm diameter) were used. SP 200 syringe pump connected to a pressure box connected to a pressure- monitoring device were also used.

3.3.1 Yield percentage and swelling degree of cryogels

The swollen cryogel samples (0.5 ml) were put in an oven at 60°C overnight for drying.

After drying until constant weight was reached, the mass of the dried sample was determined. The solid polymer fraction yield was calculated using equation 3.2

Equation 3.2

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The swelling degree was determined as follows. Dried cryogel samples were weighed and then immersed in distilled water at room temperature and an equilibrium uptake was reached. The surface of the cryogel was blotted with filter paper prior to weighing.

Therefore, the degree of swelling was calculated by taking the weight of the wet samples and samples when dried overnight in 60°C. Equation 3.3 shows the calculation for swelling degree.

For each sample, the average of at least three measurements was taken.

Equation 3.3

3.3.2 Pressure difference and flow rate of water and plasma passing through cryogel

column

The pressure difference within the cryogel column (1 cm diameter, 5 cm length) of water and plasma was measured using SP 200 syringe pump connected to a pressure box and pressure-monitoring device as shown in Fig. 3.2. For each sample, the average of at least three measurements was taken.

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Figure 3.2. SP 200-syringe pump connected to pressure monitoring box and device

3.3.3 Measurement of mechanical properties of cryogel by dynamic mechanical

analyser (DMA)

3.3.3.1 Measurement of storage modulus of cryogels

The cryogel sample was removed from the column and sectioned with a scapel from the top, middle and bottom of the cryogel, each slice was approximately 10 mm diameter and

9 mm in height and immersed in water on a compression plate, then heated to 30°C and kept isothermal. A static force of 0.01 N was applied to touch the sample surface and

85

then the upper plate was oscillated in a vertical direction with amplitude of 10 µm at a frequency of 1 Hz. The material response (storage modulus and loss modulus) was then measured by DMA. The amplitude was increased to 50 µm and then up to 1000 µm in steps of 50 µm, measuring the material response after each step.

The storage modulus is the measure of the sample’s elastic behaviour, while ratio of loss to storage is the tan delta also known as damping; this measures the ability of material to disperse applied mechanical energy into heat.

86

Figure 3.3. Slice of cryogel immersed in water on a compression plate

87

3.3.3.2 Measurement of compressive modulus of cryogels

The compression test on epoxy PHEMA cryogel was performed using the uniaxial compression test. The sample from top, middle and bottom section of the cryogel column, was sliced to approximately 10 mm diameter and 9 mm in height and placed on a compression plate as shown in Fig.3.3, then heated to 37° C and kept isothermal. An initial force of 0.01 N was applied to touch the sample surface and then increased gradually to 18 N. The compressive modulus was determined as the slope of the stress- strain curve in the linear elastic region between 0 – 20% strain.

3.3.3.3 Creep test of cryogels

A cryogel slice of approximately 10 mm diameter and 9 mm height was placed on the compression plate as shown in Fig. 3.3. at constant temperature of 37°C and constant load (stress) of 0.2 N was applied. The sample was left for 10 minutes to recover after the release of constant load. The recovery strain versus time was measured.

3.3.3.4 Measurement of the strain percentage against time with a constant stress when the cryogel is immersed in water

The sample was treated as mentioned above in section 3.3.3.3, a constant load of 0.2 N was applied on the cryogel slice for one minute and allowed to rehydrate for two minutes, and the cycle was repeated four times.

3.3.4 Statistical analysis

All statistical analysis were performed by first using one-way analysis of variance

(ANOVA, minitab version 15). Whenever ANOVA indicated the groups significantly 88

different, a t-test for independent samples was performed. Samples were considered significantly different at P ≤ 0 .05.

3.4 Results and discussions

3.4.1 Polymerization yield in water

Different types of HEMA cryogel were prepared with different comonomer concentrations from both amino group (APMA) and epoxy group (AGE) as described in section 3.3. Table 3.1 presents the summary of the swelling degree and yield of polymer from HEMA: APMA 5:1, HEMA: APMA 10:1, HEMA: APMA 50:1 and HEMA: AGE

5:1. The cryogels were produced with high gelation yield (about 92%) for all cryogel from HEMA: APMA and 72 % from cryogel prepared from HEMA: AGE. This result could suggest that the cryogel prepared from a combination of HEMA and comonomer

APMA has a higher polymerization yield when compared to HEMA: AGE comonomer.

The results of the polymerization yield are also represented in Fig. 3.4; the graph shows the yield at different concentration of comonomers used in cryogel preparation immersed in water.

89

Table 3.1. Polymerization yield and swelling degrees of cryogels

Sample Yield percentage in water Swelling degree

HEMA: APMA 5:1 92±2.9 24±0.6

HEMA: APMA 10:1 94±1.3 22±0.2

HEMA: APMA 50:1 90±2.7 14±0.4

HEMA: AGE 5:1 72±2.9 16±0.9

Monomer ratio

Figure 3.4. Polymer yield of cryogels from different monomer ratio. There is a significant difference in the polymer yield from HEMA: APMA 5:1, 10:1, 50:1 and HEMA: AGE

5:1 with P ≤ 0 .05. (Mean ± SD n=3)

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3.4.2 Swelling degree of cryogel in water

The swelling degree of the cryogel was analysed and data obtained as shown in Fig. 3.5, suggests that the swelling degree of cryogel increases with increase in comonomer concentrations for cryogel prepared from HEMA: APMA. The swelling of the supermacroporous structure depends upon the total monomer concentration, cross-linking density, pore wall thickness and temperature, at which the gels were prepared. The swelling degree in cryogel is mainly related to the pore wall thickness, this could be explained as the amount of water absorbed is related to comonomer concentration at the backbone of the cryogel. The swelling degree in cryogels increases as the comonomer concentration increases from HEMA: APMA 50:1 to HEMA: APMA 5:1, with HEMA:

APMA 5:1 gels having the highest swelling degree of 24 as shown in (Fig. 3.5). The increase in swelling degree with concentration can be explained on the basis that as the monomer concentration increases, wall thickness increases and more rigid and less porous cryogel is formed, thus exhibiting increased swelling. The connectivity of pores plays a crucial role and leads to faster swelling rates of gels. Moreover from these data, the swelling degree of cryogel prepared from HEMA: AGE 5:1 was lower than those prepared from HEMA: APMA 5:1 and HEMA: APMA 10:1, but similar to the swelling degree of HEMA: APMA 50:1 which has the lowest comonomer concentration among the APMA cryogel produced, this result can be explained on the basis that cryogel

91

prepared from HEMA: AGE 5:1 has similar wall structure to cryogel produced from

HEMA: APMA 50:1.

Monomer ratio

Figure 3.5. Swelling degree of cryogel from different monomer concentrations in water.

There is a significant difference in the swelling degree from HEMA: APMA 5:1, 10:1,

50:1 and HEMA: AGE 5:1 with P ≤ 0 .05. (Mean ± SD n=3)

3.4.3 Pressure difference and flow rate of water and plasma

The flow rate and pressure difference of water and plasma within the cryogel column are needed to provide necessary information about the column mechanical stability to pressure. The pressure within the column increases as the flow rate increases, and at a certain point the pressure drops, hence the column could not withstand the pressure

92

exerted. The pressure difference of water within the cryogel column (1cm diameter, 5cm length) was linear up until a flow rate of 15 ml/ minutes and the column could withstand a maximum pressure of 180 mmHg as shown in (Fig: 3.6). For comparison, the average normal blood pressure is 120/80 mmHg, the arteries are strong, elastic vessels, which are designed to carry blood away from the heart. For the arteries to withstand and adapt to the pressure within, varying thicknesses of smooth muscles, which have elastic and inelastic connective tissue, surrounds them. This could suggest that 8% epoxy PHEMA monolithic cryogel is an elastic material. The elasticity of the cryogel ensures the tight connection of cryogel monoliths with the column walls and the absence of by-pass of liquid in between the cryogel monolith and the column wall. Thus 8% epoxy PHEMA monolithic cryogel might be suitable for whole blood to pass through it. The pressure difference in the flow of water and plasma through the column demonstrated that the higher the viscosity of the fluid, the more pressure needed to pass through the column.

Water has a viscosity of 1.0019mPa.s and plasma has a viscosity range between 1.50-

1.75 mPa.s, this correlates with the graph showing that water exerts less pressure to pass through the column as shown in (Fig. 3.7).

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Figure 3.6. Flow rate of water versus ∆pressure within 8% epoxy PHEMA cryogel column (Mean ± SD n=3)

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Figure 3.7. Effect of flow rate on ∆pressure of water and plasma in 8% epoxy PHEMA cryogel column (Mean ± SD n=3)

3.4.4 Compressive modulus of cryogels

As a result of the presence of large pores surrounded by dense polymer-pore walls, epoxy

PHEMA cryogels are highly elastic and have spongy-like morphology. They can be easily compressed. The elastic and compression properties of the cryogel were determined by exerting physical stress on the gel, which was in turn used to calculate

Young’s modulus, which is a mathematical description of a substance’s tendency to be deformed when force is applied to it. Fig.3.8 shows the compressive modulus of each cryogel sample of same size as calculated by analysing the stress and strain values of 95

each cryogel by the DMA. The compressive modulus of epoxy PHEMA cryogel from 6,

8, 10 and 16% is approximately 1.8 kPa, 3.4 kPa, 5.1 kPa and 8.5 kPa. These data could indicate that an increase in monomer concentration results in an increase in both polymer concentration in pore walls and the general strength of the macroporous gel backbone.

Epoxy PHEMA cryogels are quite elastic materials with rather low value for the compressive modulus equal between 1.8-8.5 kPa. For comparison, the compressive modulus of soft tissues in human body is about 100-fold higher (Hollister, 2005). It is well demonstrated that the stress required for both 6 and 8% epoxy PHEMA cryogel to undergo compression is less than the stress required for 10 and 16%. This infers that the 6 and 8% epoxy PHEMA cryogel is more elastic and soft. It was also observed that with the change in total monomer concentration from 6 to 16%, the rigidity of cryogels was altered as shown in (Fig. 3.8). As the concentration increases, the sponginess and elasticity decrease, which in turn decreases compressibility and squeezability of the cryogel. It can be assumed that as the cross-linking agent concentration increases in total monomer concentration, it causes the formation of more compact rigid cryogels. The compressive modulus within the three different layers of cryogel for all samples from 6 to

16% showed they were almost similar within the layers. These results suggest that at all layers the gel has equivalent strength to withstand compression.

It was observed that 6 and 8% epoxy PHEMA cryogel is spongier than 10 and 16%. One of the potential applications of these cryogels that has been recently established is the detachment of bio particles, which are attached/adsorbed to the surface of cryogel

96

(Dainiak et al., 2006b). This detachment of bio particles is facilitated by elastic deformation of cryogels. Thus, it can be said that elasticity of cryogel is an important factor for such applications.

97

) 15000

a TOP

P

(

s MIDDLE

u l

u BOTTOM

d 10000

o

m

e

v

i s

s 5000

e

r

p

m o

C 0

A A

A A

M M M M

E E E E

H H H H

P P

P P

% %

% %

0 6

6 8

1 1

Monomer concentration

Figure 3.8. Comparative study of mechanical strength of epoxy PHEMA cryogel.

There is a significant difference in the compression modulus when comparing

6%,8%,10% and 16% PHEMA cryogel with P ≤ 0 .05. (Mean ± SD n=6)

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3.4.5 Storage modulus of 6% and 8% epoxy PHEMA cryogel

Macroporous gels called cryogel possess large pores surrounded by dense polymer walls; hence they are elastic and have a spongy-like morphology. The storage modulus of a material is the measure of elasticity of a material and measurement of maximum energy stored in a material during one cycle of oscillation, while loss modulus is the ability of material to lose energy or the amount of energy that was dissipated as heat by the sample.

The storage modulus of cryogel columns was measured with the use of DMA as mentioned in section 3.3.3.1. The maximum storage modulus of both 6% and 8% epoxy

PHEMA cryogel is approximately 0.012 MPa. The storage modulus within the three different layers of both 6% and 8% PHEMA cryogel is shown in (Fig.3.9). The 6%

PHEMA cryogel has a higher storage modulus compared to the 8% PHEMA cryogel. The loss modulus for the cryogel in both 6% and 8% within the three layers was 0.000MPa.

This result explains that cryogel is a spongy and highly elastic material. The wide variation in the storage modulus in 8% PHEMA top cryogel could be due to wide variable pore sizes and possibly experimental errors. However storage modulus of 8%

PVP + PHEMA cryogel is approximately 0.025 MPa, this suggests that copolymer of

PVP and PHEMA cryogel is more stiffer and rigid than epoxy PHEMA cryogel, the storage modulus of the different layers is shown in (Fig. 3.10). The addition of PVP as a comonomer during cryopolymerization increased stiffness of this cryogel. The storage modulus of 50 μm diameter of banana fibre is approximately 32 GPa (Pothan et al.,

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2003), and the storage modulus of oil palm fibre-linear low density polyethylene is 10000

MPa (Shinoj et al., 2011). These materials are viscoelastic and have a loss modulus has high as 800 MPa. They are not soft, spongy and elastic like cryogels.

The data in this study suggest that cryogels made from epoxy PHEMA and PVP +

PHEMA cryogels are elastic material.

Figure 3.9. Storage modulus of epoxy PHEMA cryogel. There is a significant difference where comparing 6% and 8% PHEMA cryogel from the three different layers. P ≤ 0 .05

(Mean ± SD n=3)

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Storage modulus 8% PVP and PHEMA cryogel

0.03

a

P

M

s 0.02

u

l

u

d

o

m

e

g 0.01

a

r

o

t S

0.00 TOP MIDDLE BOTTOM

Layer of cryogel

Figure 3.10. Storage modulus of epoxy copolymer PVP and PHEMA cryogel. There is a significant difference where comparing the three different layers of 8% PVP and PHEMA cryogel. P ≤ 0 .05 (Mean ± SD n=3)

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3.4.6 Creep-recovery analysis of cryogels

A creep recovery test involves the application of a finite stress to a sample whilst recording the strain response as a function of time. Following a defined period, the stress is removed and the strain recovery is recorded. In particular, the creep test provides information on the viscoelastic or elastic behaviour of materials. The strain recovery percentage of epoxy PHEMA cryogel 8, 10 and 16% after compression to 0% for approximately one minute and released to recover for about 10 minutes is as shown in

(Fig.3.11) 100%, (Fig.3.12) 97% and (Fig.3.13) 96%. The decrease in strain recovery as the monomer concentration increases demonstrates that increase in monomer concentration increases the cryogel stiffness, hence less elastic. The creep test shows cryogels have a spongy-like structure with most of the water located within large macropores. The mechanical removal of the water by squeezing the cryogel is due to the elasticity property of cryogel.

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Figure 3.11. Creep test of 8% epoxy PHEMA cryogel

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Figure 3.12. Creep test of 10% epoxy PHEMA cryogel

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Figure 3.13. Creep test of 16% epoxy PHEMA cryogel

3.4.7 Cyclic compression/stress-relaxation analysis of cryogels

Creep-recovery and stress-relaxation methods are in principle the inverse of one another.

During stress-relaxation testing, a sample is held at a constant deformation (strain) and the force required to maintain this strain is monitored as a function of time. Stress relaxation testing has been used extensively within the pharmaceutical industry. It has been used to probe the internal deformation of compressed materials and to gather information on the energy required for elastic and viscous deformation and hence to interpret the consolidation of different pharmaceutical compacts (Casahoursat et al., 105

1988). Macroporous cryogels are highly elastic and have a spongy-like morphology, they can be easily compressed up to 70% of their original size without been destroyed mechanically. Epoxy PHEMA cryogels 6,8,10 and16% had approximately 97% recovery after applying a constant load of 0.2 N for one minute and released for two minutes and the cycle was repeated four different times. The results prove that cryogels did not lose their mechanical structure, as there was less than 1% difference in the recovery from the first cycle to the fifth cycle. However, the increase in monomer concentration affected the percentage of compressibility, this could be explained by the higher the monomer concentration the thicker the polymer walls, the smaller the interconnected pores and the more rigid and less elastic the cryogel material. Fig.3.14 shows 6% epoxy PHEMA cryogel was compressed to approximately 70%, Fig. 3.15 shows 8% epoxy PHEMA cryogel was compressed to approximately 59%, Fig.3.16 shows 10% epoxy PHEMA cryogel was compressed to approximately 60% and Fig.3.17 shows 16% epoxy PHEMA cryogel was compressed to approximately 35% after applying a constant load of 0.2 N for one minute in a cyclic manner.

The results observed in this study suggest that cryogel is an elastic and spongy material, and monomer concentrations affect both physical and mechanical properties of cryogel.

Thus, this suggests that cryogel is a mechanically stable material.

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Figure 3.14. Cyclic compression of 6% epoxy PHEMA cryogel

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Figure 3.15. Cyclic compression of 8% epoxy PHEMA cryogel

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Figure 3.16. Cyclic compression of 10% epoxy PHEMA cryogel

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Figure 3.17.Cyclic compression of 16% epoxy PHEMA cryogel

3.5 Conclusions

Macroporous cryogels produced from epoxy PHEMA and HEMA: APMA using cryogelation have high polymerization yield and demonstrated degree of swelling. These cryogels have proved to be mechanically stable, elastic, and sponge-like materials that could undergo up to 70% compression and restore their properties (~ 97% strain recovery). HEMA cryogels are soft materials and have potential as materials for cell separation and bio-separation in extracorporeal apheresis systems. This however, provides the possibility that cryogels can be explored for clinical applications requiring haemocompatibility.

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Chapter Four: Haemocompatibility of poly (2-hydroxylethyl methacrylate) macroporous cryogels

4.1 Introduction

The contact of blood with foreign surfaces stimulates numerous cascade reactions and activation occurrences. These reactions generate clinically significant side effects in the application of medical devices (e.g., cardiovascular implants, extracorporeal circulation, catheters) and restrict the success of the medical treatments (Cazenave et al., 1986).

Platelets play a fundamental role in the haemostatic process, including recognizing the site of injury, recruiting additional platelets via intercellular signalling, adhering to one another, and interacting with the coagulation cascade to form a haemostatic plug

(McNicol and Israels, 2003). Consequently, reviewing the interactions between platelets and the surface of biomaterials is compulsory to overcome thrombogenic complications

(Hasebe et al., 2007). Thus, the compatibility of blood with biomaterials

(haemocompatibility) is a critical assessment when evaluating a potential biomaterial for blood filter in extracorporeal systems. In-vitro measures of haemocompatibility should be straightforward, sensitive and reproducible (Shankarraman et al., 2012).

Haemocompatibility test of medical materials aims to detect adverse interaction between artificial surface and blood, which can activate or destroy blood components (Sanak et al., 2010). International Organisation for Standardization (ISO) developed guidance on testing medical materials that have contact with circulating blood (ISO 10933-4). In brief, a haemocompatible material must not adversely interact with any blood components 111

(Seyfert et al., 2002). Consequently, the development of improved haemocompatible biomaterials is one of the most important challenges in materials science.

The thrombogenicity of a material, defined as the ability of the material to promote or induce the formation of thromboemboli (Bakker et al., 1991), has been a challenge to evaluate outside the body. Such testing is critical to identify materials of low thrombogenicity. While there is obvious agreement that in-vitro evaluation testing should evaluate the effect the material has on coagulation, platelets and immunology, there is far less agreement on what specific tests should be employed.

Platelet activation results in a complex series of changes, including physical redistribution of receptors, changes in the molecular conformation of receptors, secretion of granule contents, development of a procoagulant surface, generation of platelet derived micro-particles and formation of leukocyte platelet aggregation. Each of these changes can potentially be used as a marker of platelet activation. Whole blood fluorescent activated cell sorting is the method of choice for the measurement of all the complex series of changes (Michelson, 2004, Michelson et al., 2000).

The fluorescent activated cell sorter (FACS), instrument is used to characterize the alteration in the structure of platelets which could be due to platelet activation, haemostatic function or due to maturation process (Kamath et al., 2001). Before FACS, analysis, cells in suspension are labelled, typically with an activation-dependent fluorescent conjugated monoclonal antibody. In FACS the suspended cells pass through a

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flow chamber, at a rate of 1000 to 10,000 cells per minute and through a focus beam of a laser. After the laser light activates the fluorophore at the excitation wavelength, detectors process the emitted fluorescence and light scattering properties of cells (Givan, 2001).

The intensity of the emitted light is directly proportional to the antigen density or the characteristics of the cell being measured. Monoclonal antibodies are used, each conjugated with a different fluorophore, such as phycoerythrin (PE), peridinin chlorophyll protein (PerCp) and fluorescein isothiocynate (FITC) (Barnard et al., 2001,

Krueger et al., 2001). The test monoclonal antibodies recognizes the antigen specific antibodies measured, such as P-selectin (CD62P) and PAC-1; platelet identifier monoclonal antibody CD42a.

P -selectin is a component of the α-granule membrane of the resting platelets that is only expressed on the platelet surface membrane after α-granule secretion. Therefore, P- selectin specific monoclonal antibody only binds to the surface of activated platelets. The most widely studied type of antigen specific monoclonal antibodies directed against secretion of granule membrane protein is P-Selectin (CD62P) (Leytin et al., 2000,

Michelson et al., 1994, Ruf and Patscheke, 1995).

PAC-1 is a fibrinogen-binding site exposed by conformation change in GP IIb/IIIa complex (integrin αIIb β3). This fibrinogen-binding site is not exposed on resting platelets. Thrombin stimulation results in a conformational change in the GP IIb/IIIa complex that exposes the fibrinogen-binding site. Therefore, PAC-1 antibody only binds to the surface of activated platelets (Shattil et al., 1985). 113

Anticoagulant, a substance that prevents coagulation, stops blood from clotting.

Chelation of calcium using EDTA results in decreased platelet adhesion or retention to glass surfaces (White et al., 1983). EDTA inhibits the clotting process by binding calcium ions, which are essential in the coagulation cascade and cell-to-cell interaction

(Macey et al., 2003). EDTA is the anticoagulant currently recommended for making full blood cell counts (ICSH, 1993, NCCLS, 1992). Sodium citrate removes calcium ion by forming a soluble calcium citrate complex. Sodium citrate is used primarily as an anticoagulant for haemostasis test. However, its use has also been recommended for fluorescent activated cell sorter studies of platelet surface antigens and activation

(Matzdorff et al., 1998, Ritchie et al., 2000). Heparin inactivates thrombin through binding of antithrombin, therefore stopping the formation of fibrin from fibrinogen; this is mostly used in cell cultures and enzyme studies (Golanski et al., 1996).

Haemolysis represents the breakdown or disruption of the red blood cell membrane causing the release of haemoglobin. Haemolysis in blood products is usually expressed by the presence of free haemoglobin in red cell suspending media, such as plasma or additive solutions. The major factors that cause haemolysis include shear stress, interaction of RBC with leachables, chemical contamination and temperature

(Henkelman et al., 2009, Kempe et al., 2005). RBC can also be damaged if they are forced through leukocytes reduction filters, small bore needles, narrow openings, passing through narrow capillaries in vivo, kinked or twisted intravenous tubing, or partially obstructed or occluded blood storage bags (Brecher et al., 1991, Sweet et al., 1996,

114

Walker 1993). On the other hand, bacterial contamination may cause haemolysis. The presence of clots and change in colour of blood to purplish or brownish are indicators of the presence of contamination and haemolysis.

There are various methods of determining levels of haemolysis in blood. These include spectrophotometry at discrete wavelength (Wong et al., 1995) and spectra wavelength scan analysis (Blakney and Dinwoodie 1975). The plasma haemoglobin assays can also be measured based on oxyhaemoglobin absorbance at peaks 415, 541 or 576 nm. Another quantitative method for measuring haemolysis involves chemical techniques, where all forms of haemoglobin form a coloured reaction product, cyanmethaemoglobin, when mixed with chemicals such as potassium ferricyanide or tetramethylbenzidine. The cyanmethaemoglobin method is the standard recommended by international committee for standardisation in Haematology in whole blood (Zwart et al., 1996). However, this method is used by few organisations, the direct optical spectrophotometric methods are safer, easier, and more precise and accurate than the chemical additions method used to measure plasma haemoglobin concentration (Sowemimo-Coker, 2002).

In this chapter, the fluorophores used for antibody conjugation are phycoerythrin (PE), peridinin chlorophyll protein (PerCp) and fluorescein isothiocynate (FITC). The antigen specific monoclonal antibodies used in this study were P-Selectin (CD62P) and PAC-1; platelet identifier monoclonal antibody was CD42a. Adenosine diphosphate (ADP) was used to activate the platelets as a positive control. Samples are stabilized by fixation, with a final concentration of 1% paraformaldehyde. Three different types of anticoagulant 115

were used to evaluate the effect of anticoagulant on platelet adherence/activation. The anticoagulants used were sodium citrate, ethylenediaminetetraacetic acid (EDTA) and heparin. In this study, fluorescent activated cell sorter (FACS), an instrument capable of rapid quantitative analysis was used to explore the haemocompatibility of 8% PHEMA cryogel and the effect of different anticoagulants on platelet activation.

4.2 Aims

The aims of this study were to determine the effect of 8% epoxy PHEMA macroporous monolithic cryogel and different anticoagulants on platelet adherence/activation. The following steps were taken i) Evaluation of the activity of the IgG control monoclonal antibodies ii) Optimization of the procedure for staining and measuring platelet activation and comparison of platelet status after contact with 8% PHEMA cryogel and without contact with 8% PHEMA cryogel at different time intervals in different anticoagulants.

4.3 Materials and methods

8% epoxy PHEMA cryogels, fresh blood from healthy donor, 3.8% sodium citrate, heparin, and K3EDTA from BD vacutainer Systems, (Franklin Lakes, NY, USA),

Sigmacote purchased from Sigma, 16% formaldehyde methanol purchased from

Polysciences, inc, BD Bioscience FACScan.

PE Mouse Anti-Human CD62P, FITC Mouse IgG 1K isotype control, PAC-1 FITC, PE

Mouse IgG 1K isotype, CD42a PerCp and PE-CY5 Mouse IgG 1K isotype control purchased from BD Pharmingen, Sysmex KX-21N cell counter (Sysmex Co.,

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Mundelein,IL, USA) and SP syringe pump connected to pressure box and pressure monitor system were used.

4.3.1 Blocking of the epoxy reactive group

The epoxy PHEMA cryogel column was washed with 0.1 M sodium carbonate buffer, pH

9.0 at flow rate 0.5 ml/minute for 4 hours. Then 50 ml 0.1 M ethanolamine pH 9.0 in

0.1M sodium carbonate buffer were pumped in a recycling mode for 4 hours. Finally, the column was washed with 50 ml of 0.1 M sodium carbonate buffer at a flow rate 1 ml/minute to remove all non-reacted ethanolamine, and then stored at 4°C until further use.

4.3.2 Blood collection

Venous blood was collected by clean venepuncture into vacuum tubes containing anti- coagulant (sodium citrate, heparin and EDTA) and was used within 24 hours. The volunteers were healthy and had not taken aspirin or non-steroidal anti-inflammatory drugs within the last 24 hours.

4.3.3 Blood cell count

The whole blood was counted with Sysmex KX-21N, an automated multiparameter blood cell counter for in-vitro diagnostic use in clinical laboratories, shown in Fig: 4.1. This system processes approximately 60 samples an hour and displays on the liquid crystal display screen the particle distribution curves of white blood cells, red blood cells and platelets, along with data of 19 parameters. WBC and RBC use direct current detection

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method. The haemoglobin detector block measures haemoglobin concentration using the non–cyanide haemoglobin analysis method. Non-cyanide haemoglobin analysis method applies the advantages of both cyanmethaemoglobin and oxyhaemoglobin methods. Non- cyanide haemoglobin analysis method rapidly converts blood haemoglobin as the oxyhaemoglobin method and contains no poisonous substance, making it suitable for automated method. This method is capable of analysing methaemoglobin, hence it can accurately analyse blood, which contain methaemoglobin.

Figure 4.1. Sysmex KX -21N cell counter

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A 1ml aliquot of blood in an eppendorf tube was evaluated for the cell numbers by using a Sysmex cell counter. The cryogel column was washed with approximately 50 ml of

0.9% NaCl solution at a flow rate of 1 ml/minute with the use of SP 200 syringe pump, then approximately 15 ml of blood were passed through the pump at a speed of 1 ml/minute, as shown in Fig: 4.2 and the fractions of approximately 1 ml were collected into an eppendorf tube for ~ 12minutes. The fractions in each tube were then evaluated for the cell numbers.

Pressure box Pressure monitoring Device Syringe passing blood

SP 200 Syringe Pump

Figure 4.2. SP 200 syringe pump ready to pump blood through the cryogel column connected to the pressure box and the pressure monitor system. The metal clamps are used to close the tubes leading to the pressure box while loading blood sample. 119

4.3.4 Plasma haemoglobin determination

1 ml aliquots of blood from test samples were centrifuged at 1000 g for 10 minutes and the plasma was placed in another eppendorf tube. The absorbance of the plasma (300 µl) was read and recorded at 3 wavelengths (562, 578 and 598 nm). The plasma haemoglobin concentration was calculated according to (Blakney and Dinwoodie 1975). The total haemoglobin content (volume (gram) of haemoglobin in dL of whole blood) was measured with the Sysmex cell counter and was 139g/L. Control samples (negative control 0.9% NaCl in a 1:1 dilution with blood sample and positive control 1:1 dilution with deionized water) were incubated for 1 hour at room temperature. At the end of the incubation period, the samples were treated as described with test sample. Final plasma haemolysis was calculated as haemolytic index.

4.3.5 Platelet activation

A 20 gauge needle was used to collect blood from the donor into a sodium citrate, EDTA and heparin vacutainer, the first 2 ml of blood collected were placed in an EDTA bottle but not used for the platelet activation experiment because it contains activated platelets.

4.3.5.1 Immuno-staining

Single or multi-colour staining can be used in the assay. With multi-colour staining, one antibody conjugate can be used to threshold data acquisition to analyse only those blood cells that bind an activation-independent, platelet specific antibody, for example CD61 or

CD42a (Abrams et al., 1990, Abrams and Shattil, 1991). Another antibody conjugated to a different fluorochrome can be used to simultaneously assess the binding of platelet- 120

associated, activation-dependent antibodies, for example, CD62P or PAC-1 (Shattil et al.,

1987). 10 µl of P-selectin (CD62P), CD42a and PAC-1 antibodies were added to each

FACS tube, thereafter 5 µl of blood collected from each anticoagulated vacutainer were added to the FACS tube containing antibodies then incubated in the dark for 15-20 minutes at room temperature.

4.3.5.2 Fixation

Fixation of stained cells with paraformaldehyde inhibits spontaneous cell activation and makes the assay more manageable. 2 ml of cold 1% paraformaldehyde solution were added to the stained samples after incubation, thereafter samples were gently vortexed to mix the solution, and stored in the fridge with temperature between 2-8°C for 30 minutes before FACS analysis.

4.3.5.3 Positive control

To activate the platelets i.e., positive control, 10 µl of ADP were added to 90 µl of blood and placed in dark for 2 minutes, and then 5 µl of the activated ADP blood were added to the FACS tube with the antibodies for staining and placed in dark for 15 minutes before fixation. This positive control is used to test the effectiveness of both PAC-1 and P- selectin antibodies in the presence of activation.

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4.4 Results and discussions

4.4.1 Flow of whole blood through cryogel column

Haemolytic potential of PHEMA cryogels has been evaluated by the haemolysis test. The haemolysis test is a qualitative characteristic that measures the free haemoglobin present in supernatant of the test sample; this measurement can be done photo-metrically.

To determine the effect of monolithic PHEMA cryogels on blood cells after transiting through the column, the following steps were taken.

i) The number of various blood cells in whole blood before passage through the

column was estimated.

ii) The pressure difference through the column during whole blood passage was

measured.

iii) Estimation of blood cells number eluted after transition through the column was

done.

iv) Evaluation of red blood cells lysis was determined.

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nd rd 1st ml flow 2 ml flow 3 ml flow

4th ml flow 5th ml flow 6th ml flow

Figure 4.3. Flow of blood passing through the cryogel matrix at different times. No side flow leakage within the column was observed.

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90 80 70 60 50 40 30 20 10 ∆Pressure (mmHg) ∆Pressure 0 0 2 4 6 8 10 12 14

Flow rate ml/minutes

Figure 4.4. ∆Pressure against flow rate as blood passes through 8% PHEMA cryogel column at a speed of 1 ml/minute

Whole blood was passed through the column at a flow rate of 1 ml/minute and at approximately 6 ml or 6 minutes the column was filled with blood as shown in Fig 4.3, the maximum pressure of approximately 81mmHg was observed as shown in Fig.4.4.

The gel matrix did not compress or reduce in size, which indicates that the cryogel matrix comprises macroporous interconnected pores, which allow the passage of various cells in the blood at a pressure of approximately 81mmHg at 1 ml/minute flow rate. These data suggest the column is mechanically stable and comprises macroporous interconnected pores.

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4.4.2 Blood cell count by Sysmex cell counter

Red blood cell (RBC) count in blood was performed using a Sysmex cell counter and the initial value of 4.77 x 106 cells/µL was observed before passing blood through the column. The experiment was repeated four different times and both cell count and haemolysis test were evaluated from the blood eluted after passage through the cryogel column. Blood was pumped through the column at a flow rate of 1 ml/minute up until

12minutes. The first 5 ml eluted were discharged to make sure any dilution of blood samples with 0.9% NaCl used to wash the column did not occur. Therefore, blood samples collected after 5 ml contained whole blood only.

6

5

4 /µL) 6 3 2

1 RBC (X (X RBC 10 0 0 2 4 6 8 10 12 14 -1

Elution time, min

Figure 4.5. RBC counts after blood flow through the 8% PHEMA cryogel column.

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The results in Fig 4.5 show that ~ 95% of the red blood cells were eluted at 6 minutes and after 9 minutes full recovery of RBC was observed.

4.4.3 Haemolysis determination by Blankey and Dinewoodie

Multiple factors can induce haemolysis, such as shear stress, RBC interaction with leachable, chemicals and electrical forces (Kempe et al., 2005). The most common method to determine the haemocompatibility properties of biomaterials used for blood filter is haemolysis testing. The release of intracellular haemoglobin (haemolysis) can be caused by red blood cells (RBC) interaction with biomaterials. The haemolysis test has been used for decades to identify the biocompatibility properties of biomaterials (Blakney and Dinwoodie 1975, Dillingham et al., 1975, Henkelman et al., 2009, Streller et al.,

2003). Freshly collected whole blood were passed through 8% PHEMA cryogel at a flow rate of 1 ml/minute with SP200 syringe pump connected to a pressure monitor device.

Approximately 12 ml of blood was passed through the cryogel column and 1 ml fractions were collected into eppendorf and analysed for haemolysis. This experiment was repeated four different times as described in 4.3.4.

Calculation used for free plasma haemoglobin:

Free plasma Haemoglobin (mg/dl) = (A578 X 155) – (A562 X 86) – (A598 X 69) =

To convert FHb (g/l) = FHb( )

Evaluation of results:

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The haemolytic index is calculated according to Equation 4.1

Equation 4.1

Table 4.1.Conversion table of haemolytic index

Haemolytic index (%) Haemolytic grade

0-2 Non haemolytic

2-10 Slightly haemolytic

10-20 Moderately haemolytic

20-40 Markedly haemolytic

40 Severely haemolytic

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Table 4.2. Haemolysis results using Blankey and Dinewoodie spectra method

Samples Haemoglobin Haemolytic index Haemolytic grade released (g/l) (%)

Negative control 0.036 0.026 Non haemolytic

Positive control 3.6 2.6 Slightly haemolytic

First column 0.043 0.03 Non haemolytic

Second column 0.033 0.024 Non haemolytic

Third column 0.115 0.082 Non haemolytic

Fourth column 0.238 0.171 Non haemolytic

Table 4.3. Haemolysis results using Sysmex cell counter

Samples Haemoglobin Haemolytic index Haemolytic grade released (g/l) (%)

Negative control 1.28 0.92 Non haemolytic

Positive control 12.8 9.21 Moderately haemolytic

First column 1.07 0.76 Non haemolytic

Second column 1.23 0.88 Non haemolytic

Third column 1.04 0.75 Non haemolytic

Fourth column 1.29 0.93 Non haemolytic

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No haemolysis was observed in any samples eluted from the four 8% PHEMA cryogel columns as shown in Table 4.2 and 4.3, using the conversion haemolytic index in Table,

4.1. The 100% elution of RBC without haemolysis after 12 minutes could indicate that the 8% PHEMA cryogel column has interconnected pores large enough for the passage of cells as big as 7-10 µm without any hindrances or entrapment. The results from Sysmex cell counter correlate with data from Blankey and Dinewoodie spectra method.

4.4.4 Platelet adhesion and activation

Whole blood, freshly collected from a healthy donor into a sodium citrate vacutainer, was circulated through PHEMA monolithic cryogel (1 cm x 5 cm) at a flow rate of 1 ml/minute with the use of a SP200 syringe pump. This experiment was designed to examine the effect of cryogel contact with blood at different time intervals and to examine the effect of the duration of pumping blood through the cryogel column.

The fresh blood was passed through the column with a SP200 syringe pump at a set speed of 1ml/minute and at 7 minutes a 1 ml fraction sample was collected. The time 7 minutes was chosen because at this time the 1ml fraction would have had contact with the cryogel matrix and the blood fraction will be out with no dilution with 0.9% NaCl solution used to wash the column. Then the pump was stopped for 10 minutes, to allow longer contact of cryogel with blood. Thereafter the pump was started; the first 2 ml that is 17 and 18 minutes were not used for the platelet activation experiment because these fractions might be either at the tap/gap before the matrix and might not have had 10 minutes contact with the matrix. Therefore, the sample was used for the experiment as contact 129

with the cryogel matrix after 10 minutes incubation. For comparison, 1 ml fraction of whole blood was placed into an eppendorf tube with no cryogel matrix and no pumping effect and left to rest on the bench for 7 minutes and 19 minutes before immunostaining and fixing as described in 4.3.5.1-2. The negative control for all samples is platelet activation at time 0 after collecting blood sample from the same patient.

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n 40

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% 20

0 0 st p st p e e m e m r u r u im te p te p T u u l n te n te o i u i u tr m in m in n o 7 m 9 m 1 9 C 7 1 Figure 4.6. PAC-1 activation with and without cryogel matrix at different time

intervals

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30

n o

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0 0 st p st p e re m re m m u u i te p te p T u te u te ol in u in u tr m in m in n 7 9 o m 1 m C 7 19

Figure 4.7. P-selectin activation with and without cryogel matrix at different time intervals

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Haemocompatibility of 8% PHEMA cryogel was examined by testing for platelet activation. Activation of platelets comprises a change in platelet shape, platelet aggregation and the release of platelet constituents. P-selectin is only expressed on the platelet surface after α-granule secretion, while PAC-1 only binds to the surface of activated platelets due to thrombin stimulation, which results to a conformational change in platelet shape. In this study, the blood was tested for both PAC-1 and P-selectin platelet activation. In PAC-1 platelet activation test (PAC-1 is directed against the fibrinogen-binding site exposed to conformational change of activated platelets) there was 56.1% activation in the 7 minute rest (where there was no contact of cryogel matrix with blood and no pumping effect), compared with 7 minute pumping which had 39.57% activation (where cryogel had contact with blood and pumping effect), and 19 minute rest had 48.6% activation (where there was no contact of cryogel with blood and no pumping effect), while 19 minute pumping had 47.7% activation (where cryogel had contact with blood and pumping effect), the control at time 0 had 18.85% activation as shown in

Fig.4.6. Meanwhile in the P-selectin activation, there was 26.8% activation in the 7 minute rest (where there was no contact of cryogel matrix with blood and no pumping effect), compared with 7 minute pumping which had 28.04% activation (where cryogel had contact with blood and pumping effect), and 19 minute rest had 26.76% activation

(where there was no contact of cryogel with blood and no pumping effect), while 19 minute pumping had 25.87% activation (where cryogel had contact with blood and pumping effect), however the control at time 0 had 14.37% activation as shown in Fig.

4.7. 132

These preliminary data could suggest that PHEMA cryogel is a material of low thrombogenicity.

4.4.5 Effect of different anticoagulants on platelet percentage after incubation of

cryogel for 30 minutes using Sysmex cell counter

Approximately 25 ml of sterile 0.9% NaCl were passed through the column at a flow rate of 1 ml/minute to wash the column. The cryogel was taken out of the column and sliced, each slice weighing approximately 0.2 g was placed into 2 ml of blood in a 15 ml polyethylene centrifuge tube and a 15 ml polyethylene centrifuge tube coated with

Sigmacote. For comparison, 2 ml of blood were placed in a 15 ml polyethylene centrifuge tube with no cryogel slice and 2 ml of blood were placed in a 15 ml polyethylene centrifuge tube coated with Sigmacote with no cryogel slice. (Sigmacote is a special silicone solution in heptanes that covalently binds to a glass tube to prevent cell adherence onto the surface of the tube). The samples were incubated for 30 minutes placed on a horizontal shaker at 23° C. After 30 minutes of incubation, 1.8 ml blood sample from each sample were aliquoted into eppendorf tubes, then from the 1.8 ml a further 500 µl of blood were aliquoted into three different eppendorf tubes to have a triplicate reading from each sample. Sysmex analysis was then taken. Samples were immunostained and fixed for FACS analysis.

133

CITRATE HEPARIN

300 EDTA 3

0 200

1

x

s

t

e

l

e

t

a l P 100

0 Control (Time 0) + - - - - Cryogel - + - + - Sigmacote - - - + +

Figure 4.8. Platelet reading from Sysmex cell counter after 30 minutes incubation with and without cryogel in both polyethylene centrifuge and polyethylene centrifuge tube coated with Sigmacote

The above data in Fig.4.8 show a platelet reduction in sodium citrate, EDTA and heparin anticoagulated blood with and without the presence of cryogel compared with the control time 0.

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These data also show that in citrate anticoagulated blood there was approximately 43% reduction in platelets in the presence of cryogel compared to approximately 45% reduction in the sample without cryogel, while in heparin anticoagulated blood there was approximately 38% reduction in platelet in the presence of cryogel compared to sample without cryogel having approximately 41% reduction in platelet and in EDTA anticoagulated blood there was approximately 10% reduction in platelet in the presence of cryogel and approximately 3% reduction in the sample without cryogel in the polyethylene centrifuge tube as represented in Fig.4.8. However, there was approximately

40% reduction in platelets in the presence of cryogel compared to approximately 36% reduction in the sample without cryogel in citrate anticoagulated blood, while in heparin anticoagulated blood there was approximately 39% reduction in platelet in the presence of cryogel compared to the sample without cryogel having approximately 25% reduction in platelet and in EDTA anticoagulated blood there was approximately 11% reduction in platelet in the presence of cryogel and approximately 3% reduction in the sample without cryogel in the polyethylene centrifuge tube coated with Sigmacote as shown in Fig.4.8.

These data therefore might suggest that the reduction in platelets when comparing with and without the presence of cryogel could be due to the presence of cryogel (platelet adhering to the surface or absorbed in the pores of the PHEMA cryogel). However, the reduction in platelets after 30 minutes incubation of blood with no cryogel in both the polyethylene centrifuge tube and the polyethylene centrifuge tube coated with Sigmacote when compared with control time 0 suggest that platelets adhere onto the surface of both polyethylene centrifuge tubes with or without Sigmacote, EDTA seems to protect the 135

adherence of platelets to both the surface of the cryogel and polyethylene tube, when compared to other anticoagulants sodium citrate and heparin, as observed from Fig:4.8.

4.4.6 Effect of different anticoagulants on platelet activation after incubation of

cryogel for 30 minutes using FACS analysis

Blood was collected from healthy donors into three different anticoagulant vacutainers

(sodium citrate, heparin and EDTA).

Approximately 25 ml of sterile 0.9% NaCl were passed through the column at a flow rate of 1 ml/minute to wash the column. The cryogel was taken out of the column and sliced, each slice weighing approximately 0.2 g was placed into 2 ml of blood in a 15 ml polyethylene centrifuge tube and a 15 ml polyethylene centrifuge tube coated with

Sigmacote. For comparison, 2 ml of blood were placed in 15 ml polyethylene centrifuge tube with no cryogel slice and 2 ml of blood were placed in a 15 ml polyethylene centrifuge tube coated with Sigmacote with no cryogel slice. The samples were incubated for 30 minutes placed on a horizontal shaker at 23° C. After 30 minutes of incubation 1.8 ml blood sample from each sample were aliquoted into eppendorf tubes, then from the

1.8 ml a further 500 µl of blood were aliquoted into three different eppendorf tubes to have a triplicate reading from each sample. Samples were immunostained and fixed for

FACS analysis.

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10 CITRATE

HEPARIN s

t 8 EDTA

e

l

e

t a

l 6

p

e

e

r f

4

f

o

% 2

0 Control (Time 0) + - - - - Cryogel - + - + - Sigmacote - - - + +

Figure 4.9. Percentage of free platelet in FACS analysis after 30 minute incubation with and without cryogel, in both polyethylene centrifuge tube and polyethylene centrifuge tube coated with Sigmacote

The obtained results show that in citrate anticoagulated blood there was approximately

43% reduction in platelets in the presence of cryogel compared to approximately 42% reduction in sample without cryogel, while in heparin anticoagulated blood there was approximately 33% reduction in platelet in the presence of cryogel compared to sample without cryogel having approximately 43% reduction in platelet and in EDTA anticoagulated blood there was approximately 24% reduction in platelet in the presence

137

of cryogel and approximately 33% reduction in sample without cryogel in the polyethylene centrifuge tube as expressed in Fig.4.9. However, there was approximately

32% reduction in platelets in the presence of cryogel compared to approximately 34% reduction in the sample without cryogel in citrate anticoagulated blood, while in heparin anticoagulated blood there was approximately 41% reduction in platelets in the presence of cryogel compared to the sample without cryogel having approximately 30% reduction in platelets and in EDTA anticoagulated blood there was approximately 31% reduction in platelets in the presence of cryogel and approximately 28% reduction in the sample without cryogel in the polyethylene centrifuge tube coated with Sigmacote as shown in

Fig.4.9. These data therefore might suggest that the reduction in platelets when comparing with and without the presence of cryogel could be due to the presence of cryogel, that is platelets adhering to the surface or adsorbed in the pores of the PHEMA cryogel. These results also correlate with the Sysmex cell counter platelet count result obtained in Fig.4.8.

4.4.7 Effect of different anticoagulants on platelet after incubation of cryogel for 60

minutes

The fluorescent activated cell sorter was used to examine the haemocompatibility of 8% epoxy PHEMA cryogel and the effect of different anticoagulants on platelet activation was evaluated. Cryogel samples were prepared as described in 4.3.5, briefly cryogel sample was removed from the column and sliced, each slice weighing 0.2 g was placed 138

into 2 ml of blood in a 15 ml polyethylene centrifuge tube and a 15 ml polyethylene centrifuge tube coated with Sigmacote. The cryogel matrices were incubated in the blood for 60 minutes placed on a horizontal shaker at 23°C. After 60 minutes of incubation, blood samples were prepared as described in 4.3.5, then Sysmex analysis, immunostainning and fixation for FACS analysis was done on the blood samples.

8 CITRATE HEPARIN

s EDTA

t 6

e

l

e

t

a

l

p

e 4

e

r

f

f

o

% 2

0 Control (time 0) + - - Cryogel - + - Sigmacote - + +

Figure 4.10. Percentage of platelet in FACS analysis after 60 minutes incubation with or without cryogel in polyethylene centrifuge tube coated with Sigmacote

139

CITRATE HEPARIN 250 EDTA

200

3

0 1

x 150

s

t

e

l

e

t a

l 100 P

50

0 Control (time 0) + - - Cryogel - + - Sigmacote - + +

Figure 4.11. Platelet reading from Sysmex cell counter after 60 minutes incubation with or without cryogel in polyethylene centrifuge tube coated with Sigmacote

140

The data from Fig.4.10 FACS analysis show there was 40% reduction in free platelet population in the presence of cryogel when compared to 40% reduction without cryogel in sodium citrate anticoagulated blood. There was 51% reduction in the presence of cryogel when compared with 56% reduction without cryogel in EDTA anticoagulated blood. The reading from heparin seems irregular this might be due to human error when preparing the sample. Therefore the data from heparin will not be discussed on this graph.

Fig.4.11 shows results from the Sysmex counter. There was 32% reduction in platelet population in presence of cryogel when compared to 31% without cryogel in sodium citrate anticoagulated blood. There was 13% reduction in the presence of cryogel when compared with 11% reduction without cryogel in EDTA anticoagulated blood. While in heparin there was 37% reduction in the presence of cryogel when compared with 34% reduction without cryogel. From the obtained data, there was a reduction in the platelets population both in the presence of cryogel and without cryogel when compared to control time 0, this could suggest that platelets adhere on to the surface of the polyethylene centrifuge tube after 60 minutes of blood in the tube. The above data could suggest that the reduction in platelet population when comparing in the presence of cryogel and without cryogel could be due to the presence of the cryogel matrix, platelets adherence to the surface of the matrix or within the pores of the PHEMA cryogel.

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4.4.8 Fluorescent activated cell sorter analysis of PAC-1 and P-selectin expression in

sodium citrate anticogulant after incubation of cryogel for 60 minutes

This results are expressed in quadrants, when the cell population is on the left within the quadrant line then it is negative to activation but when on the right side of the quadrant it is positive to activation and each quadrant is divided into upper and lower quadrant.

CD42a is the platelet identifier monoclonal antibody, PAC-1 only binds to the surface of activated platelets due to thrombin stimulation, which results to a conformation change in platelet shape, while P-selectin is only expressed on the platelet surface after α-granule secretion.

Abbreviations used in analysis

UL-Upper left UR-Upper right LL-Lower left LR-Lower right

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Figure 4.12. (A-D) Time 0 control, after 60 minutes incubation with cryogel in polyethylene tube, after 60 minutes of blood in Sigmacote tube and after 60 minutes incubation in Sigmacote tube with cryogel

143

There was an increase in PAC-1 activation from both the polyethylene centrifuge tube and the polyethylene centrifuge tube coated with Sigmacote both in the presence of cryogel and without cryogel when compared to control time 0 in sodium citrate as shown in Fig. 4.12 and Fig. 4.13. The data observed in heparin seem irregular, this could be due to human error while handling the sample, and hence data from heparin will not be discussed further. The adverse events reported in association with heparin anticoagulation are hemorrhagic in nature, usually at the vascular access site of insertion (0.7%) (Basic-

Jukic et al., 2010). There are a few case reports of heparin-induced thrombocytopenia

(HIT), venous thrombosis and pulmonary emboli. The incidence of HIT in haemodialysis population is between 1 to 4% (Vucic and Davies, 1998). There was an increase from

12.11% activation in the polyethylene centrifuge tube without cryogel to 18.19% as shown in Fig. 4.12 and Fig. 4.13 in the presence of cryogel in sodium citrate anticoagulated blood. There was an increase from 13.88% activation in the polyethylene centrifuge tube without cryogel to 16.50% as shown in Fig. 4.12 and Fig. 4.13 in the presence of cryogel in sodium citrate anticoagulated blood. There was no difference between the PAC-1 activation in both the polyethylene centrifuge tube and the polyethylene centrifuge tube coated with Sigmacote in the presence of cryogel when compared with or without presence of cryogel in EDTA anticoagulated blood, as shown in Fig. 4.13.

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40 CITRATE

HEPARIN

n 30

o EDTA

i

t

a

v

i t

c 20

a

f

o

% 10

0 Control (Time 0) + - - - - Cryogel - + - + - Sigmacote - - - + +

Figure 4.13.PAC-1 activation after 60 minutes incubation with and without cryogel in both polyethylene centrifuge tube and polyethylene centrifuge tube coated with

Sigmacote

145

50 CITRATE HEPARIN 40

EDTA

n

o

i t

a 30

v

i

t

c

a

f o

20 %

10

0 Control (Time 0) + - - - - Cryogel - + - + - Sigmacote - - - + +

Figure 4.14. P-Selectin activation after 60 minutes incubation with and without cryogel in both polyethylene centrifuge tube and polyethylene centrifuge tube coated with

Sigmacote

There was an increase in P-selectin activation from both the polyethylene centrifuge tube and the polyethylene centrifuge tube coated with Sigmacote both in the presence of cryogel and without cryogel when compared to control time 0 in sodium citrate as shown in Fig. 4.12 and Fig. 4.14. There was an increase from 24.59% activation in the polyethylene centrifuge tube without cryogel to 30.12% in the presence of cryogel in sodium citrate anticoagulated blood as shown in Fig. 4.12 and Fig. 4.14. P-Selectin 146

activation in heparin anticogulated blood in the polyethylene centrifuge tube without cryogel was 31.68% compared to in the presence of cryogel which was 33.09% as shown in Fig. 4.14. In EDTA anticoagulated blood, there was an increase from 43.42% activation in the polyethylene centrifuge tube without cryogel to 32.23%, represented in

Fig. 4.14 in the presence of cryogel in the polyethylene centrifuge tube. In the polyethylene centrifuge tube coated with Sigmacote, there was an increase in P-selectin in sodium citrate 25.09% without cryogel and 28.31% in presence of cryogel as shown in

Fig. 4.12 and Fig. 4.14, in heparin anticoagulated blood 39.51% without cryogel and

43.11% in presence of cryogel and in EDTA anticoagulated blood the increase in P-

Selectin was 37.23% without cryogel and 47.42% in the presence of cryogel, represented in Fig. 4.14.

The obtained data could suggest that PAC-1 will not bind to EDTA treated blood and hence the reason for EDTA not being recommended for the platelet activation test, but recommended for full blood cell counts as reported by (ICSH, 1993). The increase in

PAC-1 activation in sodium citrate anticoagulated blood when comparing in the presence of the cryogel matrix and without cryogel matrix, could be due to the presence of cryogel matrix, such as platelets adhering to the surface of cryogel or in pores of the cryogel.

EDTA and heparin are not recommended for the platelet activation test, thus this could explain the irregularities in EDTA and heparin data in fluorescent activated cell sorter study in this chapter.

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4.5 Conclusions

The haemocompatibility test is a critical test when evaluating a potential biomaterial that will have contact with circulating blood. This haemocompatibility test is necessary to identify materials of low thrombogenicity. Haemolysis can be caused by red blood cells interaction with biomaterials; hence, haemolysis test has been used for biocompatibility of biomaterials. Data observed in this study show that ~100% of RBC eluted after passing through the cryogel matrix had no haemolysis and the results from Blankey and

Dinewoodie spectra method correlate with the haemoglobin release data obtained from the Sysmex cell counter. The finding in this study shows that the platelet reading population data obtained from Sysmex cell counter correlate with FACS analysis findings. The increase in both PAC-1 and P-selectin activation after incubation of blood with and without cryogel compared to control (time 0) correlates with the finding reported by (Bonetti et al., 2003). This finding states that there is no synthetic or modified biological surface developed so far that is as thromboresistant as normal unperturbed endothelium, which is the cellular lining of the circulatory system. The 2% increase in platelet activation after incubation of blood, when compared with and without the presence of cryogel is low and could be acceptable in biocompatible devices. Thus, this could explain the use of anticoagulants in all existing apheresis systems. For example, DALI apheresis system uses both heparin and citrate anticoagulation before and during the treatment session. From all the work carried out in this study, PHEMA cryogel can thus be classified as a material of low thrombogenicity. 2-hydroxyethyl methacrylate

(HEMA) have been reported to be chemically and biologically stable and biocompatible 148

(Denizli et al., 2003). This indeed offers the possibility to investigate further functionalization of PHEMA cryogel for biocompatible and bio-specific filter support in extracorporeal apheresis devices.

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Chapter Five: Immobilization of protein and antibody onto macroporous monolithic cryogels

5.1 Introduction

The recent need for constant faster and more sensitive analyses of biomarkers

(Cummings et al., 2009, Han, 2009, Theodoridis and Wilson, 2008), growth promoters

(Stolker and Nielen, 2009), food constituents (Cifuentes, 2009), plant constituents (Beek et al., 2009, Xie and Leung, 2009), toxins (Maragos, 2009, Shephard, 2009) and pollutants (Blasco and Pico, 2009, Mitrevski et al., 2009) in decreasing sample volumes

(Chiu et al., 2009) requires additional advanced analytical tools (Kovarik and Jacobson,

2009), such as affinity chromatography, a technique for separation of molecules.

Affinity chromatography (AC) is a liquid chromatographic technique that uses a biologically related ligand as the stationary phase (Hage, 2002, Walters, 1985). AC is highly suitable for selective purification of trace organic molecules in complicated matrices using small devices (Tetala and Beek, 2010). Although affinity chromatography has been in use as long ago as 1910 when used by Emily Starkenstein for analysing the interaction of starch with α-amylase (Starkenstein, 1986), modern affinity chromatography began with the development of the cyanogen bromide method for immobilization of ligands on agarose support (Axén et al., 1967). Subsequently, high performance affinity chromatography was developed by the use of rigid, microparticulate supports (Ohlson et al., 1989). Other related methods include affinity partitioning,

150

affinity filtration and affinity targeting of drugs. More recently, the wide acceptance of monolithic stationary phases in affinity chromatography has been enabled due to their porosity, mass transfer properties, and chemical flexibility for ligand attachment and ability to polymerize in small channels and capillaries (Haginaka, 2009, Vlakh and

Tennikova, 2009). All these complement and enhance the characteristic properties of AC.

Affinity monolithic chromatography is an area that has seen significant growth over the past few years. Affinity chromatography and monolithic columns have been used in affinity separations. The term affinity in biochemistry can be used to describe the interactions of biomolecules, i.e., proteins, carbohydrates, polynucleotides, with appropriate ligands. Protein interactions with their binding partners play a major role in various cellular events, such as cell proliferation. However, biomolecules do not interact exclusively with other biomolecules, but they can undergo highly specific binding to metal ions or artificial ligands, such as small–molecule drugs (Chen et al., 2009, Ewings and Doelle, 1981, Porath et al., 1975). Solute retention during AC is based, therefore, on the specific, reversible interactions found in biological systems, such as binding of an enzyme with a substrate, or an antibody with antigen. Affinity chromatography has exploited these interactions by immobilizing (or adsorbing) one of a pair of interacting substances onto a solid support and using this as a stationary phase. This immobilized binding agent is referred to as the affinity agent or ligand (Mallik and Hage, 2006). The termed immobilized substance denotes a substance that is physically/chemically confined to or localized within a support with retention of its activity, hence can be used repeatedly

151

and continuously (Worsfold, 1995). Specific interactions between analytes and their matrix bound ligands widely used for enrichment, purification, or separation of a target compound from complex mixtures, such as blood or plasma thus can be achieved with the use of affinity monolithic chromatography.

5.1.1 Principles of affinity chromatography

In affinity chromatography, one of two interacting molecules, arbituary called the ligand, is immobilized on a stationary phase either by covalent immobilization or physical adsorption. The raw sample in a suitable solution containing the analyte of interest (e.g., proteins, glycolipids, biomarkers, toxins, viruses, bacteria, cells) along with other compounds (impurities) is then passed through the affinity column. The analyte is captured in a highly selective manner via molecular recognition by the ligand present in the column whereas other compounds pass through the column with little or no retention.

After passing the raw sample in solution through column, the eluted (solution removed) analyte is obtained in pure and concentrated form. In most cases the affinity column can be reused. These affinity ligands can be antibodies, enzyme inhibitors, lectins, or other molecules that reversibly and bioselectively bind to the complementary analyte molecules in the sample. The separations exploit the “lock and key” or induced fit binding that is established in biological systems.

5.1.2 Types of ligands in bio affinity chromatography

There is a wide variety of ligands available for AC, both biological (e.g., antibodies, antigens, lectins) and non-biological (e.g., metal chelates, synthetic dyes) and a variety of 152

stationary phases (e.g., agarose, Sepharose, monoliths) that could be used for immobilization, all these contribute to the success of affinity chromatography. The use of biological compounds, such as immunoglobulin-binding proteins, enzymes, lectins, carbohydrates, avidin/biotin system and antibodies, as ligand in affinity chromatography is called bio-affinity chromatography (Hage, 2006, Mallik and Hage, 2006). The most commonly used ligands in bio affinity chromatography are immunoglobulin-binding proteins (protein A, produced by Staphylococcus aureus and protein G produced by

Streptococci). These proteins are found on bacterial cell walls and bind to the Fc region of antibodies. In general, protein A binds to IgG, IgM, IgA and IgD, whereas protein G has specificity only towards all IgG (IgG1-4) class antibodies. There are other proteins, such as protein B, a β-antigen protein, produced by peptostreptococcus magnus that also fall in the bio affinity chromatography category. Protein B binds specifically to all classes of human IgA but does not bind to any IgA of mammals, whereas protein L with four binding sites specifically binds to kappa light chains of antibodies (Sproß and Sinz,

2011).

When antibodies or antibody – related reagents are used as ligands for the purification of antigens, this can be classified as immuno-affinity chromatography. This can be considered as a subclass of bio affinity. The uses of monolithic stationary phases for immuno affinity chromatography were summarized by (Gunasena and El Rassi, 2012).

Antibodies are highly selective but their production can be time-consuming and expensive and can require animals. (Mallik and Hage, 2006, Tetala and Beek, 2010).

153

Enzymes and enzyme inhibitors are another group of biomolecules that can be used as affinity ligands. Immobilized enzymes are used as catalysts in pharmaceutical and food industries. They can also be used for the purification of enzyme inhibitors and can act as a ‘hunter’ to remove impurities from crude extracts. Similarly, enzyme inhibitors can also be used as affinity ligands to purify enzymes from crude extracts. Enzymes such as trypsin were the first ligands to be immobilized on monolithic stationary phases and contributed to the wider application of monolithic supports in bio affinity chromatography, (Petro et al., 1996).

There are other sub classes of affinity chromatography such as biomimetic affinity chromatography, immobilized metal ion affinity chromatography, histidine affinity chromatography, boronate affinity chromatography, DNA affinity chromatography and dye-ligand affinity chromatography, all of which ligands have been immobilized and various analytes have passed for purification, separation and total enrichment. These various affinity chromatography techniques have been discussed extensively in various reviews (Hage, 2002, Hajizadeh et al., 2012, Mallik and Hage, 2006, Sproß and Sinz,

2011, Tetala and Beek, 2010, Walters, 1985, Zachariou, 2008).

5.1.3 Monoliths and affinity chromatography

There are different types of monoliths, which have been successfully used in affinity chromatography; Hjerten and co-workers reported the first polymer monolithic material using soft polyacrylamide gel in 1989, but the process of preparation was rather complicated (Hjerten et al., 1989). Monoliths can be described as a “continuous 154

stationary phases that form as a homogenous column in a single piece and prepared in various dimensions with fibrous microstructures (Podgornik and Strancar, 2005). Svec and Frechet in 1992 further improved these successes with the development of a much easier process to produce rigid macroporous monoliths from glycidyl methacrylate

(GMA) and ethylene dimethacrylate (EMDA) as monomer and crosslinker (Svec and

Frechet, 1992, Svec and Frechet, 1996). These early innovative works triggered the improvement of a variety of monoliths, and the majority of which are discussed in review articles (Bedair and El Rassi, 2004, Eeltink and Svec, 2007, Guiochon, 2007, Smith and

Jiang, 2008, Svec, 2009, Svec, 2010, Vlakh and Tennikova, 2009). The monolith materials are made from either an “organic” or “inorganic” matrix. Preparation of monoliths from in situ polymerization of organic monomers, crosslinkers, porogens and initiators can be termed organic. Methacrylate and acrylamide (AAm) based polymers; poly (styrenedivinylbenzene), agarose and cryogels belong to the group of organic monoliths. Silica prepared by sol-gel method or from bare silica particles are classified as inorganic monoliths. These monoliths can be prepared in different formats, such as rods, convective interaction media (CIM) discs, capillaries and microfluid chips. Ligands are coupled onto the monolithic stationary phase by covalent immobilization (epoxy, Schiff base, glutaraldehyde, carbonyldiimidazole, disuccinimidyl, hydrazide and cyanogen bromide method), entrapment or biospecific adsorption, as described in chapter one section 1.9.3. The glutaraldehyde covalent immobilization method will be discussed briefly in this chapter.

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5.1.3.1 Glutaraldehyde method

The glutaraldehyde method involves converting an epoxy-activated matrix to an amine form by reacting the epoxy groups with agents such as ethylenediamine. This amine– activated matrix is then reacted with a dialdehyde (e.g., glutaraldehyde) to produce an aldehyde-activated matrix. This aldehyde-activated matrix can react with primary amines on proteins and other ligands to form Schiff base. The formation of Schiff base is reversible, thus further reduction of Schiff base with sodium cyanoborohydride

(Hermanson et al., 1992), which reduced the imine bond to a stable amine bond. Sodium borohydride is further added to reduce the amine bond to alcohol and eliminate any aldehyde group that remains on the matrix as illustrated in Fig. 5.1 (Kim and Hage,

2005). This method has been used for immobilization of protein A (Luo et al., 2002) and trypsin (Petro et al., 1996) within a GMA/EDMA monolith. It has also been used to immobilize protein A (Kumar et al., 2003) and trypsin (Johnson et al., 2011) onto a cryogel monolith. However, this technique involves a number of steps for immobilization, it results in a longer spacer being placed between the support and ligand, which can be useful in avoiding steric obstruction effects when dealing with small ligands.

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O H2N-CH2-NH2 Matrix NH Matrix NH2

OHC-(CH2)4-CHO Matrix CHO

H Protein-NH2 N Matrix CH2 protein NaCNBH 3

Figure 5.1. Schematic representation of glutaraldehyde immobilization method

5.1.4 The ideal characteristics of a stationary phase in affinity chromatography

Should comprise the following

1) Surface chemistry - (easy to modify functional groups “e.g., carboxyl, epoxide,

azalactone) on the stationary phases for ligand immobilization

2) Porosity – the matrix should be highly porous with a high surface area material

for immobilizing a large amount of ligand

3) Ligand accessibility– allows solutes to have rapid and unhindered access to

immobilized ligand

4) Low non-specific adsorption – possess highly hydrophilic features with low non-

specific adsorption of proteins

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5) Stability – matrix should be able to withstand a wide range of thermal,

mechanical, chemical and physical conditions

6) Permeability – be highly permeable i.e., generate relatively low back pressure at

high flow rates

5.1.5 Assays for determination of amount of protein bound to the column

There is no single protein assay method that yields absolutely accurate results. Each method has different advantages and limitations. Commonly used protein assay methods in biochemical laboratories include the Lowry assay method. The Lowry assay is relatively sensitive, but requires more time than other assays and is susceptible to many interfering compounds. The Bradford assay is another method; this is a popular protein assay because it is simple, rapid, inexpensive, and sensitive. The bicinchoninic (BCA) assay is another protein assay method and UV spectroscopy can also be used to determine protein concentration. The BCA method measures the formation of Cu+ from Cu2+ by the

Biuret complex in alkaline solutions of protein using BCA. The mechanism of the BCA reaction was originally thought to be similar to the Lowry assay, but it is now known that there are two distinct reactions that take place with the copper ions, which are unique to the BCA assay (Wiechelman et al., 1988). The initial reaction, which occurs at lower temperatures, is the result of the interaction of copper and BCA with cysteine, cystine, tryptophan, and tyrosine residues in protein. At higher temperature, the peptide bond is also responsible for the development of colour. The colour is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. The BCA

158

reagent forms a complex with Cu2+, which has a strong absorbance at 562 nm. The BCA is advantageous due to the stability of the reagent and resulting chromophore, which also allows for a simplified, one-step analysis and enhanced flexibility in the procedure selection. BCA method maintains the high sensitivity and a low protein–to-protein variation associated with the Lowry technique.

5.1.6 Cryogel and affinity chromatography

Cryogels are three-dimensional polymer networks formed from monomer/polymer solutions at sub-zero temperatures (Plieva et al., 2006b). Cryogels have exceptional properties such as macroporous structure (pore sizes up to 100 μm) and interconnected channel systems that allow efficient mass transfer and diffusion of solutes in the gel. The simplicity of production and functionalization of cryogels are the two important reasons why applications in bio separation and purification seem attractive (Dainiak et al., 2005,

Demiryas et al., 2007). Generally, cryogels have found different applications in the medical, biotechnological and pharmaceutical areas (Lozinsky et al., 2001, Plieva et al.,

2006, Plieva et al., 2005).

Recently, monolithic macroporous cryogels have been reported to be a suitable matrix for chromatography of cells, proteins and particles (Dainiak et al., 2007a, Derazshamshir et al., 2008, Wang et al., 2008, Yilmaz et al., 2009). Kumar and co-worker have reported that cryogels have been used for chromatography separation of fragile cells such as mammalian cells (Kumar et al., 2003, Kumar et al., 2005). Affinity interactions for capturing lysozymes (Bereli et al., 2008), haemoglobin (Derazshamshir et al., 2010) have 159

been prepared using the molecular imprinted particles (MIP) approach combined with cryogelation. Cryogel composites for capturing IgG and albumin have also been incorporated through affinity interaction (Bereli et al., 2010). Affinity chromatography of cells is a well-known method; nonetheless, it has never been generally accepted due to the complication of the process and low capability of conventional packed beads. Super porous monolithic cryogel may apparently provide an improvement to this method. Using a cryogel monolithic column functionalized with protein A in the chromatographic regime, affinity binding of human CD19+ B-lymphocytes and CD34+ KG-1 human tumour cells and affinity fractionation of antibody lymphocytes was performed (Kumar et al., 2003, Kumar et al., 2005). Monolithic cryogels have also been used for isolation of microbial cells (Dainiak et al., 2005, Hanora et al., 2005, Hanora et al., 2006), inclusion bodies (Ahlqvist et al., 2006) and mitochondria (Teilum et al., 2006). Chelating metal ions in cryogel for immobilized metal affinity chromatography (IMAC) have been used for purification of cells (Dainiak et al., 2005), DNA (Odabaşı et al.), viruses (Cheeks et al., 2009) and a cytochrome C purification (Çimen and Denizli, 2012). Cryogels have been used for antibody purification on protein A modified column (Alkan et al., 2009) and also for the selective removal of the auto antibodies from rheumatoid arthritis patient plasma using protein A carrying affinity cryogels (Alkan et al., 2010). All the above named techniques using cryogel for cell purification are all in the analytical state. Due to the often-conflicting requirements, there is no universally recommended support at present. This calls for more research focusing on developing suitable supports for some intended applications. Apparently, there is no report on macroporous monolithic cryogel 160

used in extracorporeal medical devices for separation of protein and cells. Consequently, in this work, amylase was immobilized on to epoxy and amino containing cryogels.

Subsequently, anti-human albumin antibody was immobilized onto cryogel matrix and affinity binding of human albumin to the ligand was demonstrated.

5.2 Aims and objectives

This chapter focuses on the preparation of the affinity column based on these two types of monolithic cryogels; Epoxy containing PHEMA cryogels and poly HEMA-co- N (3- aminopropyl) methacrylamide cryogels. Both type of cryogel were immobilized with biological ligand using the glutaraldehyde method. The glutaraldehyde method is a standard and established metholodolgy, which has been used to immobilise protein A

(Kumar et al., 2003, Kumar et al., 2005) and trypsin (Johnson et al., 2011) onto a cryogel monolith successfully. In this report, the covalent immobilization of ligand is performed after the cryogel column has been synthesized. The immobilizing procedure can be accomplished by circulating a solution of the ligand through the column as illustrated in

Fig. 5.2 or by dipping the column into a solution containing the ligands (batch immobilization). A disadvantage of the circulating of a ligand through the monolith versus batch immobilization is that a larger amount of ligand is generally required in the circulation method to compensate for the additional volume of ligand solution that is employed. Amylase was immobilized as a pilot study, as part of learning of the immobilization technique. On successful immobilization of amylase, antibodies, which are more technical, expensive and related to the main aim in this study, was immobilized

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onto cryogel monolith. In this study, concentration of protein attached to the cryogel matrix was measured by the bicinchoninic acid assay method (BCA) (Smith et al., 1985).

Figure 5.2. General approach for covalent immobilization of a ligand within a monolithic column

The aim of this study is to produce a range of macroporous monolithic cryogels, suitable for protein and cell separation (cytapheresis) in extracorporeal medical devices using the covalent immobilization method. Fig.5.3 shows a diagrammatic expression of a cytapheresis column.

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Figure 5.3. Diagrammatic expression of a cytapheresis column

5.3 Materials and methods

Different monomer concentrations of poly HEMA-co-APMA cryogels (HEMA: APMA

5:1, HEMA: APMA 10:1, HEMA: APMA 50:1) and epoxy PHEMA (8% prepared at -

12°C), as described in Chapter 2 (section 2.2.1), glutaraldehyde 50% v/v aqueous solution, bicinchoninic acid (BCA) protein assay reagent, copper II sulphate solution, anti-human albumin IgG 034K4816 (anti-HSA IgG) from sheep serum and human serum albumin (HSA) were purchased from Sigma (St Louis USA), ethylenediamine from

Aldrich, sodium borohydride from Fluka and alpha-Amylase (Bacillus Subtilis) from 163

Merck. Buffers prepared from sodium phosphate (monobasic and dibasic) and sodium carbonate (monobasic and dibasic) from Sigma. UV/Vis spectrophotometers from

Pharmacia Biotech and oven for drying cryogel samples were used.

5.3.1 Preparation of solutions

5.3.1.1 Sodium phosphate buffer

Dissolve 1.37 g of NaH2PO4 in 10 ml of distilled water and dissolve 1.77 g of Na2HPO4 in 10 ml of distilled water. Take 7 ml of Na2HPO4 solution and 3ml of NaH2PO4 and make volume up to 100 ml to obtain a 0.1 M sodium phosphate buffer pH 7.2.

5.3.1.2 Sodium carbonate buffer

Dissolve 10.59 g of Na2CO3 in 1000 ml of H20 and dissolve 8.4 g of NaHCO3 in 1000 ml of distilled water. Take 10 ml of Na2CO3 and add to 90 ml of NaHCO3 to obtain 0.1 M of sodium carbonate buffer pH 9.2.

5.3.1.3 Sodium borohydride solution

Dissolve 0.1895 g of sodium borohydride in 100 ml of 0.1 M sodium carbonate buffers to obtain a 0.05 M of sodium borohydride

5.3.2 Immobilization of amylase onto monolithic epoxy PHEMA and poly HEMA-co-

APMA cryogels

The epoxy-containing cryogels were connected to a pump and washed with about 50 ml deionized water, at a flow rate of 1ml/min and then with 0.1 M Na2CO3 solution (30 ml).

The monoliths were then incubated into about 30 ml of 0.5 M ethylenediamine in 0.1 M 164

sodium carbonate buffer pH 9.2 (prepared by mixing 1 ml of ethylenediamine to 30 ml of

0.1 M of sodium carbonate buffer pH 9.2) and left overnight under gentle shaking at room temperature 22-24°C. Then the monoliths were washed with 0.1 M of sodium carbonate buffer pH 9.2 to remove unreacted ethylenediamine. The monoliths were then incubated into a 100 ml of 5% v/v, glutaraldehyde solution (prepared by mixing 10 ml of

50% v/v glutaraldehyde with 90 ml of 0.1 M sodium phosphate pH 7.2 buffer) and left under gentle shaking at room temperature overnight. The derivatized cryogel matrices with functional aldehyde groups were washed with sodium phosphate buffer pH 7.2 about 50 ml each and then coupled with 0.025g of amylase (50 ml solution of 0.5 mg amylase/ml phosphate buffer) and left under gentle shaking for about 24 hours at room temperature. Schiff base was formed after immobilization of amylase with the aldehyde- activated monolith. Several runs of freshly prepared 0.05 M sodium borohydride solution in 0.1 M sodium carbonate buffer, pH 9.2, reduced this Schiff base. The reduction was carried out either by flow-through conditions with the monoliths packed in glass columns or in stationary (batch) mode until a fairly persistent final colour was observed after ~90 minutes. The monoliths were then washed with ~50 ml each of 0.1 M sodium phosphate pH 7.2 buffer and stored in refrigerator till further use.

The same procedure as described in 5.2.2 was used for poly HEMA-co-APMA cryogels with a difference that an amino containing cryogel poly HEMA-co-APMA, was not incubated with 0.5 M ethylenediamine overnight at the initial step.

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5.3.3 Coupling of IgG ligand onto monolithic epoxy PHEMA and poly HEMA-co-

APMA cryogels

The epoxy PHEMA monolithic cryogels were connected to a pump and washed with about 50 ml deionized water at a flow rate of 1ml/min, and then with 0.1 M sodium carbonate buffer pH 9.2 (30 ml). The monoliths were then incubated into about 30 ml of

0.5 M ethylenediamine in 0.1 M sodium carbonate buffer pH 9.2 (prepared by mixing

1ml of ethylenediamine to 30 ml of 0.1 M of sodium carbonate buffer pH 9.2) and left overnight under gentle shaking at room temperature 22-24°C. Then the monoliths were washed with 0.1 M sodium carbonate buffer pH 9.2 to remove unreacted ethylenediamine. The monoliths were then incubated in a 100 ml of 5% v/v, glutaraldehyde solution (prepared by mixing 10 ml of 50% glutaraldehyde with 90 ml of

0.1 M sodium phosphate pH 7.2 buffer) and left under gentle shaking at room temperature overnight. The derivatized cryogel matrices with functional aldehyde group were washed with 0.1 M sodium phosphate pH 7.2 buffer ~50ml each and then each cryogel was placed into 2 ml of IgG, 10.3mg/ml of IgG dissolved in 20 ml in 0.1 M sodium phosphate buffer pH 7.2 and samples were left under gentle shaking for about 24 hours incubation at room temperature. The monoliths were then washed with sodium phosphate buffer pH 7.2 ~50ml each. Finally the freshly prepared NaBH4 solution, 0.05M sodium borohydride solution, in 0.1M sodium carbonate buffer, pH 9.2 was applied to the cryogels to reduce Schiff’s base formed between the protein (IgG) and the aldehyde- containing matrix. The reduction was carried out in stationary (batch) mode until a fairly persistent final colour was observed after about 90 minutes. The monoliths were then 166

washed with 0.1M sodium phosphate buffer pH 7.2 ~50ml each and stored in the refrigerator till further use.

The same procedure as described in 5.3.3 was used for poly HEMA-co-APMA cryogels but without the initial step of adding 0.5M ethylenediamine.

5.3.4 Affinity binding of human serum albumin to cryogel matrix with immobilized

anti- human albumin (anti-HSA IgG) ligands

Cryogel monoliths with immobilized anti-HSA IgG were produced as described in 5.3.3.

The monoliths were then incubated in 2 ml of HSA solution (prepared by dissolving

37mg of human serum albumin in 45 ml of 0.1 M sodium phosphate buffers, pH 7.2) and left under gentle shaking in the cold room overnight. The monoliths were then washed with 0.1 M sodium phosphate buffers, pH 7.2 ~50ml each. These monoliths were then stored in the refrigerator till further use in the wet state.

5.3.5 Determination of protein content by bicinchoninic acid (BCA) method

The amount of protein content immobilized on both poly HEMA-co-APMA and epoxy

PHEMA monolithic cryogel matrix was determined by the bicinchoninic acid method

(Smith et al., 1985). The BCA stock solution was freshly prepared by mixing bicinchoninic acid solution and copper II sulphate at a ratio of 50:1 (v/v) respectively.

The cryogel matrix was dried in the oven at 60°C overnight, and was then ground finely into powder. A suitable amount of dried IgG cryogel were suspended in 1 ml of the BCA solution and the mixture was incubated at 37°C with thorough shaking for 30 minutes.

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The absorbance was measured at 562 nm both with and without centrifuging the samples.

As the appropriate controls, poly HEMA-co-APMA and epoxy PHEMA cryogel were used. For the standard curve, samples of diluted anti-human albumin solutions (IgG) were prepared by serial dilution of 4.675 mg/ml IgG solution in 0.1 M sodium phosphate buffers, pH 7.2 to yield a calibration curve between 0.07 mg/ml to 1 mg/ml. As a blank,

PBS solution was used. The analysis of each sample was done in triplicate. UV absorbance was read against the blanks at 562 nm and an average value was recorded.

Thereafter, the amount of protein in the unknown sample was obtained from the standard curve. The protein based immobilization yield was calculated as the percentage of the protein content of the cryogel immobilized with amylase and IgG to the respective amount of protein used for immobilization.

5.4 Results and discussions

5.4.1 Immobilization of amylase onto macroporous monolithic epoxy PHEMA and poly

HEMA-co-APMA cryogels

Monolithic macroporous cryogels produced from epoxy PHEMA and poly HEMA-co-

APMA were macroporous polymeric materials of interconnected pore structure as described in chapter two section 2.4.3, with pore size ranging from ~10μm to 120μm. The cryogels were white in colour after polymerization but turned orange on reaction with glutaraldehyde and turned pale-yellow after passing NaBH4 through the cryogel column 168

to reduce the Schiff base during the immobilization process as shown in Fig.5.4. The colour change to orange could be as a result of the reaction of glutaraldehyde with the amine group on the cryogel matrix. The reduction of Schiff base with NaBH4 after covalently immobilizing amylase to the matrix resulted in a colour change to pale yellow, this might be attributed to elimination of excess aldehyde left on the matrix which has not reacted with amylase immobilized. Amylase was covalently attached onto the surface of epoxy PHEMA cryogels using ethylenediamine-glutaraldehyde spacer arm and using glutaraldehyde spacer arm for poly HEMA-co-APMA. This glutaraldehyde-Schiff base covalent immobilization has been reported by (Johnson et al., 2011, Petro et al., 1996) for the immobilization of trypsin onto support.

Figure 5.4. A-B cryogel sample freshly prepared plain white and orange after derivatization with glutaraldehyde, cryogel sample after reducing Schiff base with sodium borohydride (pale yellow) 169

5.4.2 Determination of amylase protein bound to cryogel matrix by bicinchoninic acid

method

The quantity of amylase (protein content) immobilized on 8% w/v poly HEMA-co-

APMA (5:1, 10:1 and 50:1) and 8% epoxy PHEMA was estimated by the standard BCA method. Fig.5.5 shows a bovine serum albumin (BSA) standard curve for the determination of protein. The graph shows linear variation of absorbance with concentration of standard BSA up to 1.0mg/ml and forms the basis for the determination of protein content from absorbance measurements.

The results of amylase protein contents are summarized in Table.5.1. The results show that amounts of protein immobilized on the HEMA: APMA cryogels were almost similar regardless of APMA concentration. However, much higher amount of amylase protein was measured in HEMA: AGE cryogels. These results could suggest that the epoxy containing cryogel, which had an ethylenediamine-glutaraldehyde spacer arm of seven carbon atoms, placed between the support and the ligand, might have contributed to easy accessibility and attachment of amylase macromolecules compared to the amino containing cryogel with just glutaraldehyde group attached between the support and ligands. The longer extension arms could lead to higher binding efficiency. Both the amino and epoxy plain cryogels (cryogels with no surface derivatized with either ethylenediamine or glutaraldehyde, but incubated with protein) had no protein on the surface of the cryogel.

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The values of protein content in mg/g–carrier obtained in this study are much higher than those reported for immobilization of amylase on functionalized glass beads and onto a cyclic carbonate bearing hybrid material by covalent attachment (Kahraman et al., 2007,

Turunc et al., 2009). This results shows that amylase can be coupled onto cryogel surface via covalent immobilization and have a relatively high yield compared to other support.

Figure 5.5. Bovine serum albumin standard curve for the determination of protein

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Table 5.1. Respective protein (amylase) contents in the HEMA: APMA 5:1, 10:1, 50:1 and HEMA: AGE 5:1 determined using bicinchoninic acid (BCA) method. Protein content expressed as milligrams of protein present in a single 0.5ml monolith and per weight (g) of the monolithic cryogels (Mean ± SD n=3)

Cryogels total monomer Protein content per single Protein content per weight concentration monolith of monolith

(mg/monolith 0.5ml) (mg/g- monolith) HEMA: APMA 50:1 8.0 ± 0.02 85 ± 1.3 HEMA: APMA 10:1 8.0 ± 0.02 87 ± 1.3 HEMA: APMA 5:1 7.0 ± 0.01 78 ± 1.1 HEMA: AGE 5:1 11.0 ± 0.12 125 ± 1.9

5.4.3 Coupling of anti-human albumin (IgG) ligand onto monolithic epoxy PHEMA

and poly HEMA-co-APMA cryogel

Monolithic macroporous cryogels produced from epoxy PHEMA and poly HEMA-co-

APMA were macroporous polymeric materials with interconnected pore structure. The large pore size in the cryogel, in combination with the highly interconnected pore morphology and hydrophilic nature of pore walls formed from a cryogel, allow microbial cells (Arvidsson et al., 2002) as well as animal cells (Kumar et al., 2003) to pass

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unretained through the monolithic cryogel column in the absence of ionic or specific interactions between the cell surface and ligands coupled to the cryogel.

The quantity of IgG (protein content) immobilized on 8% w/v poly HEMA-co-APMA

(5:1, 10:1 and 50:1) and 8% epoxy PHEMA was estimated by the standard BCA method.

Fig.5.6 shows the anti-HSA standard curve for the determination of protein. The graph shows linear variation of absorbance with concentration of standard anti-human serum albumin up to 1.0 mg/ml and forms the basis for the determination of protein content from absorbance measurements.

The results of proteins and protein based immobilization yields are summarized in

Table.5.2. The result shows that amounts of protein immobilized on the HEMA: APMA cryogels were almost similar regardless of APMA concentration. However, much higher amount of IgG protein was measured in HEMA: AGE cryogels. Both the amino and epoxy plain cryogels (cryogels with no surface derivatized with either ethylenediamine or glutaraldehyde, but incubated with protein) had no protein on the surface of the cryogel.

Steric hindrance between immobilized ligand (antibody IgG) and large target molecules can occur during interaction with immobilized protein (Luo et al., 2002). To overcome this problem, the IgG was coupled to the epoxy-containing supermacroporous cryogel matrix through a spacer arm. The two-step derivatization includes reaction with ethylenediamine, followed by the reaction with glutaraldehyde giving a spacer arm of seven carbon atoms. Further improvement in the binding efficiency could be expected

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when using even longer extension arms, but this may also increase the non-specific binding in some cases. The results show at least 65% of the amount of IgG used was immobilized onto the cryogel surface. The plain no protein immobilization explains that protein cannot be adsorbed or bound easily onto the cryogel matrix because the cryogel possesses hydrophilic features with low non-specific adsorption of protein and also adsorption is difficult without a spacer arm.

5.4.4 Affinity binding of human albumin to cryogel with immobilized IgG

In order to evaluate the application of the separation strategy on another cell system, IgG cryogel matrix was used for capturing human serum albumin. Table.5.3 presents the binding of human serum albumin. Prior to affinity binding of the human serum albumin on the cryogel–protein surfaces, as a control, the human serum albumin was applied to

0.5 ml poly HEMA-co-APMA and epoxy PHEMA cryogel matrix (without affinity ligand). The human serum albumin capture performed on such control surfaces showed minimum non-specific binding (<10%). Non-specific absorption of proteins to the surface occurs naturally, whenever protein has contact with any surface. However, bio- specific affinity reaction occurs with the use of biological compounds such as immunoglobulin-binding proteins, enzymes, lectins and carbohydrates as ligands. The analyte is captured in a highly selective manner via molecular recognition by the ligand present on the column. About 80% of human serum albumin was retained on the monolithic IgG cryogel matrix. The results could suggest that direct immobilization of antibodies on the cryogel material and applying for affinity captures a lower binding

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when compared with about 95% specific cell binding on protein A monolithic cryogel matrix as reported in (Kumar et al., 2005) and about 94% of specific B-cells on protein

A column was reported in (Kumar et al., 2003). This may be because of the poor orientation and also some inactivation of the antibody molecules when coupled directly to the matrix. For better orientation of the immobilized antibody molecules, one of the most common methods utilized is the immobilization of antibodies at Fc region through the use of protein A (Anderson et al., 1997). Cryogel is a highly porous polymeric material, which possesses large pore size and hence results in a small area (per unit of column volume) available for ligand coupling and hence in low binding capacity for protein binding (Arvidsson et al., 2003), as compared with traditional packed-bed adsorbents.

However, low ligand density seems to be beneficial for chromatographic separation of cells having multiple interactions with the surface. High ligand densities result in multipoint cell-matrix interactions and hence problems with the recovery of bound cells

(Ujam et al., 2003). The potential of polymeric cryogels in bioseparation has been discussed earlier in a review (Lozinsky et al., 2001). This present work demonstrates that cryogels have promising potential, as separation media when dealing with macromolecules, thus might be a suitable filter matrix in extracorporeal apheresis devices.

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Figure 5.6. Standard curve of anti-human albumin for the determination of protein

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Table 5.2. Respective protein (IgG) contents in the HEMA: APMA 5:1, 10:1, 50:1,

HEMA: AGE 5:1 and plain HEMA: APMA 5:1 and plain HEMA: AGE 5:1 determined using bicinchoninic acid (BCA) method. Protein content expressed as milligrams of protein present per weight (g) of the monolithic cryogels, while immobilization yield expressed as percentage of actual immobilized protein to the unit of fresh protein utilized for immobilization (Mean ± SD n=3)

Cryogel total monomer Protein content per weight Immobilization yield concentration of monolith (%) (mg/g-monolith) HEMA: APMA 50:1 206.6 ± 20.8 65 HEMA: APMA 10:1 233 ± 23.2 73.5 HEMA: APMA 5:1 270 ± 24.5 85.1 Plain HEMA: APMA 5:1 10 ± 1.3 3.2 HEMA: AGE 5:1 290 ± 26.4 91.5 Plain HEMA: AGE 5:1 10 ± 1.5 3.2

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Table 5.3. Respective total protein (anti-human albumin (IgG) plus human serum albumin) protein (IgG) contents in the HEMA: APMA 5:1, 10:1, 50:1, HEMA: AGE 5:1 and plain HEMA: APMA 5:1 and plain HEMA: AGE 5:1 determined using bicinchoninic acid (BCA) method. Protein content expressed as milligrams of protein present per weight (g) of the monolithic cryogels, while immobilization yield expressed as percentage of actual immobilized protein to the unit of fresh protein utilized for immobilization (Mean ± SD n=3)

Cryogel total monomer Protein content per weight Immobilization yield concentration of monolith (%) (mg/g –monolith) HEMA: APMA 50:1 540 ± 24.5 60 HEMA: APMA 10:1 590 ± 25.4 87 HEMA: APMA 5:1 600 ± 26.7 83 Plain HEMA: APMA 5:1 15 ± 0.9 2.2 HEMA: AGE 5:1 556 ± 25.8 82 Plain HEMA: AGE 5:1 15 ± 1.3 2.2

5.5 Conclusions

Recently, a growing interest has been shown in using monolithic cryogels as an adsorbent for diverse applications including protein purification and biomedical therapy (Alkan et al., 2010, Baydemir et al., 2009a, Baydemir et al., 2009b, Bereli et al., 2008, Çimen and

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Denizli, 2012, Sun et al., 2008, Yilmaz et al., 2009). Previous studies have demonstrated macroporous cryogels can be successfully used for chromatography of microbial cells, for direct product recovery from fermentation media and crude cell homogenates. The potential of polymeric cryogels in bioseparation has been discussed in a previous review.

In this chapter, amylase was successfully immobilized onto the cryogel matrix.

Moreover, immobilization of anti-human albumin (IgG) and affinity binding of human serum albumin to the IgG immobilized matrix demonstrate the bio-affinity potential of antibody used as ligands for protein purification. Thus, monolithic cryogels have promising potential as a matrix for blood purification and can be a suitable matrix for extracorporeal apheresis medical devices.

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Chapter Six: Summary of conclusions, discussions and future works

Pheresis, or apheresis, from Greek ‘taking away’, means separating some components of blood from others. Plasmapheresis is the complete separation of plasma from blood cells by settling or using a centrifuge and returning the remaining blood components mostly red blood cells back into the patient. Cytapheresis is aimed at separation of certain types of cells from blood by either centrifugation or adsorption. It has long been acknowledged that specific removal of pathologically significant cells or formed elements is an attractive perception. Cytapheresis has been shown recently to have clinical efficacy in various disease states, such as leukaemia, autoimmune disorders, rheumatoid arthritis renal allograft rejection, and sickle–cell anaemia (Ratner et al., 2004). Various physicians have reported the efficacy of cytapheresis in inflammatory bowel disease such as ulcerative colitis and Crohn’s disease (Hidaka et al., 1999a, Morihiro et al., 1997,

Rembacken et al., 1998, Sawada et al., 1995, Shimoyama et al., 2001, Ueki et al., 2000).

The efficacy of LDL-apheresis in homozygous familial hypercholesterolemia and hyperlipidemia has been reported (Bosch et al., 2002, Bosch and Wendler, 2004).

There are mainly two methods of extracorporeal leukocyte removal therapy in use in the clinical field. These are the centrifugal method and the adsorptive method with fibre or beads. The centrifugal method is the mainstream of leukocyte removal in the United 180

States and Europe, while the adsorptive method is the mainstream in Japan because of ease of operation and cell removal efficiency (Yamaji et al., 2001). Leukocytapheresis using the leukocyte filter Cellsorba (Shibata et al., 2003) and granulocytapheresis using the Adacolumn (Saniabadi et al., 2003) have been proved to have reduced leukocyte load in patients with rheumatoid arthritis and inflammatory bowel disease, but still has major limitations of specificity and selectivity (Agishi, 2002).

The adsorptive method with beads have been used in LDL-apheresis with direct adsorption of lipoprotein and this has been proved to reduce cholesterol level in patients with severe dyslipidaemic and homozygous familial hypercholesterolaemia, but still has limitations due to side effects (Archontakis et al., 2008).

The problem of the non-selective nature of this technique has been addressed by the functionalization of the filter support and attachment of a biological ligand, such as an antibody against target antigen or protein A (an avid ligand for IgG antibody). A recognizable application has been the use of anti-CD4 antibody to remove CD4+ T- lymphocytes (Onodera et al., 2003) or the more widely applicable functionalization of filter support with protein A to capture antibody-coated cells (Kumar et al., 2003). This allows a more targeted means of cell isolation, and provides a flexible technological platform that could be applied to a wide range of diseases in clinical applications.

Despite its excellent potential, apheresis has not yet received wide clinical applications for a number of reasons. An ideal extracorporeal cell-specific filter device should have

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combined characteristics, such as low flow resistance, high mechanical and chemical stability, ease of functionalization, large interconnected pores which can allow unrestricted cell passage, a large material surface area and control of pore size during manufacture (Tetala and Beek, 2010). Current filter devices and other apheresis techniques do not meet all, or many of these requirements. It is also desirable to have a monolithic column for therapeutic cytapheresis, with continuous on-line cell separation.

Use of monolithic columns for chromatographic separations has already been suggested

(Svec and Frechet, 1996) but so far they have not been used for cell separations due to failure in effort to achieve an appropriate pore structure.

These problems can be overcome by employing macroporous monolithic cryogels prepared using cryopolymerization technology. Cryogels have been shown to have unique properties, most importantly mechanical, chemical and osmotic stability, and interconnected macro or super–macropores capable of allowing passage and separation of whole blood cells in a chromatographic regime (Dainiak et al., 2006a). They have also shown shape memory as they can be repeatedly dried and re-swollen in the solvent acquiring the same shape in which they were synthesized (Dainiak et al., 2006b). These findings suggest that cryogels might provide an ideal structure to act as supports for bio- specific extracorporeal apheresis systems.

The main aims of this study were to analysis the structural, mechanical, and haemocompatibility features required to develop an affinity-binding cryogel column from a non-biological (synthetic) monomer 2-hydroxyethyl methacrylate (HEMA), as an 182

appropriate matrix for use in extracorporeal apheresis systems. Such a matrix would be of use in a wide range of clinical situations where specific removal of specific blood components would be beneficial to the patient.

The first objective of this study was to produce a range of macroporous monolithic cryogels with interconnected pores ranging from 5 μm-200 μm from different synthetic polymers (epoxy containing 2-hydroxyethyl methacrylate (PHEMA), poly vinyl pyrrolidone (PVP) and copolymer of 2-hydroxyethyl methacrylate, (PHEMA and PVP) and poly (hydroxyl ethyl methacrylate-co-N-(3-aminopropyl) methacrylamide) (APMA) via free radical polymerization at -12°C. The morphology of these cryogels was evaluated using confocal laser scanning microscopy and porosity, pore size and wall thickness of cryogels was measured with image J analysis based on confocal laser microscopy and scanning electron microscopy. Cryogels with pore sizes within the range of 1μm-120μm and average porosity of 87% with ~ 13% polymer wall thickness were successfully produced via free radical polymerization; the cryogelation process was highly reproducible with over 90% recovery. This is in agreement with reports documented elsewhere (Arvidsson et al., 2003, Plieva et al., 2004a, Wilfred et al., 2011) in which it has been reported that macroporous monolithic cryogels had a large volume of interconnected supermacroporous pores with a size range between 10μm and 200μm.

Flow properties of epoxy containing 2-hydroxyethyl methacrylate (PHEMA) and poly vinyl pyrrolidone (PVP) and copolymer of 2-hydroxyethyl methacrylate, (PHEMA and

PVP) were measured using water and different concentrations of dextran passing through 183

the monolithic cryogel (approximately 1cm diameter and 5cm length) and a void volume of 8% epoxy PHEMA was evaluated by running blue dextran (2000 kDa) and bovine serum albumin (BSA 69 kDa) through the column. The flow rate of water passing through the different concentration of monolithic polymer cryogel decreases as the monomer concentration increases. This could suggest that an increase in monomer concentration leads to a decrease in pore size. The free passage of water and different concentrations of dextran through the macroporous monolithic cryogel for the first time demonstrated that the monolithic macroporous cryogels produced in this work allow flow of liquid passing through the monolithic cryogel. The passage of both BSA and blue dextran for the first time shows that the cryogel column has a void volume between 38 and 43% of the total volume of the cryogel column, and the pore sizes allow the passage of molecules between 69 kDa and 2000 kDa. These results correlate with reports documented (Dainiak et al., 2005, Lozinsky et al., 2001, Lozinsky, 2002) where it is reported that an increase in monomer concentrations leads to an increase in polymer wall thickness which results in a decrease in pore size and a resulting decrease in transit of large molecules. It was also documented that bioparticles and molecules as large as 10

μm can be transported conveniently through a macroporous monolithic cryogel without the risk of mechanical entrapment (Dainaik et al., 2005, Dainiak et al., 2007a).

Measuring of the flow resistance and pressure difference of water passing through the cryogel columns for the first time, demonstrated that 8% epoxy PHEMA cryogel (~ 1cm diameter and 5cm length) could withstand a linear flow rate up until 15 ml/minute and a

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maximum pressure difference of 180mmHg. From the obtained results it was observed that the monolithic cryogel column has a linear flow rate up to 15 ml/minute at

180mmHg. This suggests that the column can withstand the applied pressure without any compression of the matrix. Further increase in flow rate could possibly leads to compression of the gel and reduction in pore size.

Mechanical properties such as storage modulus, compression modulus, and cyclic compression and creep test of the polymer cryogel produced in this work were evaluated using the dynamic mechanical analyser for the first time. These demonstrated that epoxy

PHEMA cryogel is a very elastic, spongy-like material that could undergo up to 70% compression and restore its properties with no damage to the pore structure. Indeed, the elastic and spongy characteristics of cryogels have been reported by others (Lozinsky et al., 2001, Noppe et al., 2007, Persson et al., 2004, Plieva et al., 2004b, Plieva et al.,

2009, Yun et al., 2011) in which they successfully demonstrated that macroporous cryogels are elastic and spongy-like material.

Haemocompatibility of macroporous monolithic polymer cryogel was assessed by free haemoglobin measurement, blood cell count before and after blood passage through the column and platelet activation for the first time. The results obtained suggested that about

100% of red blood cell passed through the cryogel column successfully with no evidence of haemolysis found in blood eluted. Platelet activation studies showed that the cryogel produced in this work is a material of low thrombogenicity. In line with these findings, it

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has been documented elsewhere that red blood cells (7μm) are transported through the monolithic cryogel with no red blood cell lysis observed (Dainiak et al., 2007a).

Anti-human albumin (IgG antibody) and amylase were successfully immobilized onto the cryogel matrix and human albumin binding on to IgG antibody coupled cryogel was achieved for the first time. Crucially, more than 75% of the total protein passed through the column was immobilized and retained on the cryogel matrix. However, there is evidence showing that other proteins, such as trypsin and protein A have been successfully immobilized onto cryogel (Johnson et al., 2011, Kumar et al., 2003).

These findings provide a flexible scientific platform for the development of macroporous monolithic cryogel column, which may be used as a bio-specific filter device in extracorporeal apheresis medical devices. This will lead to achieving efficient improved adsorptive cytapheresis, which would potentially provide an efficient treatment of a wider range of medical conditions where removal of infective or defective or malignant cells from circulation is needed. These include hepatitis, autoimmune diseases, sickle cell anaemia, and certain types of cancer such as (acute myeloid leukaemia) and bacterial infections. For many of these diseases, from which many millions of people suffer worldwide, no remedy is available at present.

6.1 Novel contribution to knowledge

Cell separation has emerged as an important area in research due to its relevance in cell- based therapy. Cell separation has come a long way from a simple centrifugation 186

technique to the more advanced methods of flow cytometry and magnetic bead separations. However, these techniques have scale limitations and are only used at an analytical and diagnostic scale.

Over the past decade, cryogel technology has advanced rapidly and is being used in major fields of scientific research including cell separation. Through the use of affinity- based cryogel chromatographic columns, cell separation shows several advantages over conventional techniques. In this method, the technology is gaining recognition due to its cost-effectiveness, user- friendliness; it is non-damaging to blood cells, and efficient and can be optimized for use at a preparative scale. The fact that cryogels can be synthesized from a variety of polymers and are easily modified using various ligands offers attractive prospects for further research and applications at a preparative scale. Their reusability, non-toxic nature, shape-memory and easy storage offer added advantages.

Cryogels have been produced with unique combinations of properties, namely, interconnected pores, pore sizes of 10-100 μm, porosity exceeding 90%, high mechanical and chemical stability, elasticity, drying and re-hydration without impairing pore structure, and retention of large amounts of liquid in the cryogel. Overall, the technology looks promising and one hopes to develop cryogel bio-specific filter device to be used in extracorporeal medical devices that will be available commercially.

The amount of work/studies in the development and characterization of cryogels is evidence of how flexible this material is. Indeed this provides other avenues that could be

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explored for clinical applications, such as haemocompatibility studies and functionalization for biocompatibility and bio specific cell sorting.

Hereinafter a list of items that to the best of our knowledge, no work has been produced on and as such is our contribution to knowledge from this work

1. The passage of different concentration of dextran (20%w/v, 24%w/v and

30%w/v) through the monolith polymer cryogel at hydrostatic pressure equal to

0.01 MPa in the characterizing of the matrix structure. This provides a general

profile of flow properties of viscous fluid through the column. This profile can

help improve the understanding of the interconnected pores/porosity and stability

of the polymer walls in a monolithic polymer cryogel. The calculation of void

volume with the passage of blue dextran and bovine serum albumin through the

monolithic cryogel contributes additional knowledge to the understanding of

characteristics of the polymer cryogel as a stationary phase.

2. Within the context of developing macroporous monolithic polymer cryogels

suitable for use in extracorporeal medical devices, passing water, plasma and

whole blood through the cryogel column attached to a pressure monitoring device

and evaluating the pressure difference, improves the knowledge of monolithic

cryogel mechanical stability and the pressure differences the column can

withstand. The experiments demonstrated that the monolithic cryogel could

withstand a specific maximum pressure difference before collapsing. Such an

188

approach has not previously been used for evaluation of the mechanical stability

of cryogels.

3. The mechanical property test with dynamic mechanical analysis for storage

modulus, compressive modulus, creep test and cyclic compression analyser has

been demonstrated for the first time to be useful in the evaluation of the elastic

and spongy-like properties of macroporous polymeric cryogels. The data

produced in this report can provide a technological platform for development

and/or use of macroporous monolithic cryogel in an extracorporeal medical

device and help improve understanding on the mechanical properties of this

material.

4. The in-vitro studies on the haemocompatibility of the material produced in this

work reported that ~100% (4.77 x 106 cells/μL) of red blood was eluted after

passing whole blood through the monolithic cryogel column and that no

haemolysis was observed, as calculated by the Blakney and Dinewoodie (1975)

method. The data obtained from fluorescent activated cell sorting (FACS) and

Sysmex cell counter suggest that PHEMA monolithic cryogel is a material of low

thrombogenicity. These results provide support for the use of macroporous

monolithic cryogel in an extracorporeal medical device. According to the

international organization for standardization (ISO) developed guidance on testing

medical materials that have contact with circulating blood (ISO 10933-4), a

haemocompatible material must not cause any adverse interaction with any blood

components. Considering the fact that the development of improved 189

haemocompatible biomaterials is one of the most important challenges in material

science, this work demonstrated for the first time that PHEMA monolithic cryogel

is haemocompatible by passing whole blood through the column by mean of

evaluating the free haemoglobin, amount of red blood cells eluted and low or no

platelet activation with the use of Sysmex cell counter and fluorescence activated

cell sorter.

5. The immobilization of amylase (enzyme) and anti-human albumin (antibody)

onto a monolithic cryogel via covalent immobilization with extended linker

groups has been demonstrated for the first time. This result obtained further

confirms that both enzymes and anti-human albumin can be coupled onto cryogel

matrix surface via glutaraldehyde–Schiff base method. Affinity binding of human

albumin onto coupled anti-human albumin on the surface of the cryogel matrix

further confirms that if the binding structure in the form of antibodies is available

that recognizes the surface of the proteins or cells aggregates, a general binding

and separation system can be established for antibody binding cryogel affinity

matrices.

190

6.2 Future work

Following the investigations and findings described in this thesis, a number of projects could be undertaken, involving the in vitro biocompatibility of cryogel material in an extracorporeal circulation.

Commercially available direct adsorption of lipoprotein (DALI) made from a polycarbonate column with a capacity of ~500ml filled with negatively charged polyacrylate ligands linked to Eupergit matrix macroporous beads as the adsorption carrier is used in the treatment of severe dyslipidaemic and homozygous familial hypercholesterolaemia. In this work, macroporous monolith cryogel with a capacity of

~6ml was produced via free radical polymerization. As such, the upscale innovation process of this cryogel column to the size of an extracorporeal unit of a few hundred millilitres should be studied. However in this study, it was done in analytical scale and might pose a challenge during scale up, such as uniform temperature distribution in the material during cryopolymerization. Consequently, this could result in poor structural integrity within the monolith (i.e., irregular pore size and weak polymer wall backbone).

This problem could be solved producing small cryogel monolith layers that could be stack together in an up-scaled model.

In this work, haematological parameters such as free haemoglobin and platelet activation have been studied. As such, other parameters such as macrophage function, complement activation and biocompatibility of the macroporous monolithic cryogel columns in vitro

191

with whole blood in a closed loop pumped circuit should be studied. It will also be interesting to view the column material under environmental electron microscopy to detect column instability and the potential for loss of column material to blood.

In this work, anti-human albumin was coupled onto the cryogel matrix via glutaraldehyde-Schiff base method. However, a possible direction might be to functionalize the cryogel matrix with antibodies to CD34, a stem cell marker frequently seen in acute myeloid leukaemia (AML); this has already been shown to be appropriate for this technology (Kumar et al., 2005); prostrate specific antigen (PSA) to test the possibility of the removal of metastatic cells ; MHC2 molecules to test the possibility of removing highly immunoreactive antigen-presenting cells in a range of autoimmune diseases and antibodies to a number of bacteria-specific antigens found on bacteria important in septicaemia could all be studied. This technology can be used in particular for both the reduction of bacteria load and immune complex removal from the blood of patients with acute meningococcal meningitis.

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APPENDIX I

Disseminations and Abstracts

1) W.Akande, L.Mikhalovska, S.James, S.Mikhalovsky (2011), Affinity Bio- separation column. Medical devices and carbon material project meeting 20th- 21st September 2011 University of Brighton. Poster presentation 2) W.Akande (2011), Affinity Bio-separation column UK Society for Biomaterials 30thJune-1stJuly 2011 University of Greenwich. Oral presentation. 3) W.Akande, L.Mikhalovska, H.Kirsebom, I.Savina, S.James, B.Mattiasson, S.Mikhalovsky (2011), Macroporous monolithic cryogel as a matrix for cytapheresis. European Society for Artificial Organ and American Society for Artificial Organ ESAO Winter school January 26th-29th 2011 Semmering Austria. Poster presentation. 4) W.Akande, L.Mikhalovska, I.Savina, S.James, S.Mikhalovsky (2010), Characterization of macroporous monolithic Poly (2-Hydroxyl ethyl methacrylate) cryogels. UK Society for Biomaterials 1st-2nd July 2010 University of Glasgow. Poster presentation. 5) W.Akande, L.Mikhalovska, I.Savina, S.James, S.Mikhalovsky (2009) Characterization of macroporous monolithic Poly (2-Hydroxyl ethyl methacrylate) cryogels. MONACO-EXTRA Summer School, Porous Hydrogels for Biomedical Applications: from cytapheresis to Tissue Engineering September 29th-October 2nd, 2009 Antalya Turkey. Poster presentation. 6) W.Akande, L.Mikhalovska, I.Savina, S.James, S.Mikhalovsky (2009), Structure of macroporous monolithic PHEMA cryogels. UK Society for Biomaterials 2nd-3rd July 2009 University of Ulster Belfast. Poster presentation. 7) W.Akande (2009), Macroporous cryogel as a matrix for cytapheresis. 7th Postgraduate Annual Research Presentation (PARP) Day June 2009. University of Brighton Oral presentation.

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APPENDIX II

Ethical Approval

School of Pharmacy &

Biomolecular Sciences

Cockcroft Building

Moulsecoomb [email protected] Brighton BN2 4GJ

13 November 2009

PABSREC APPLICATION 0902

Project title: “Monolithic Adsorbent Column for Adsorptive Cytapheresis”

Investigator name(s): Dr. Lyuba Mikhalovska, Ms Wuraola Akande, Mr Peter Haworth, University of Brighton, UK. Dr Xinming from ZhongKai University of Agriculture and Engineering, China

Name of Supervisor (s): Professor Sergey Mikhalovsky, Dr Stuart James

The School Ethics Committee has approved the above application.

Please ensure that records of tissue processing and disposal are kept in such a way as to allow the Principal Investigator to make them available for inspection purposes.

Yours sincerely

Dr Anne Jackson

Chair, School of Pharmacy and Biomolecular Sciences Research Ethics Committee.

194

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