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THE EFFECTS OF SURFACE MODIFYING MACROMOLECULES ON THE BLOOD COMPATIBILITY OF POLYETHERSULFONE MEMBRANES INTENDED FOR BIOMEDICAL APPLICATIONS

Jeannette Yin Chun Ho

A thesis subrnitted in conformity with the requirements for the degree of

MASTER OF APPLIED SCIENCE

Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto

O Copyright by Jeannette Y .C. Ho October 1997

395 Wellington Street 395, rue Wellington Ottawa ON K I A ON4 Ottawa ON KI A ON4 Canada Canada

Your hle Votfe reférence

Our /de Notre rétérence

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1 I A J 2 u 1 - l . m L 1 u u m . u u l \ m - r a b W L V X V U r n X r n .- A.=A =-a.-*.*---- - --- - A . - -a-

BLOOD COMPATIBILITY OF POLYETHERSULFONE MEMBRANES

INTENDED FOR BIOMEDICAL APPLICATIONS

Jeannette Yin Chun Ho

MASTERS OF APPLIED SCIENCE 1997

Graduate Department of Chernical Engineering and Applied Chernistry

University of Toronto

ABSTRACT

This work further investigates the use of novel surface rnodifying macromolecules (SMMs) for

use in polyethersulfone (PES) as a component of membrane materials and for consideration in

the development of blood compatible membranes in biomedical ultrafiltration applications.

Synthesized SMMs containing varying chemical compositions were characterized and blended

with PES for fabrication into flat sheet and hollow fibre membranes. The bulk thermal

transitions of PES materials were not significantly altered by the addition of 4 wt% SMMs.

Through microscopy techniques, a mode1 of the morphology of the SMM-PES membranes was

developed. It was shown that the surface energy of the modified membranes was lower due to

the migration of SMM microdomains to the air-membrane interface. The potential blood

compatibility of the PES hollow fibre membranes was investigated. Fibrinogen adsorption was

shown to be reduced on SMM modified membranes. An in vitro whole blood study using a

rocking platform apparatus and fluorescence activated flow cytometry was inconclusive as to the

potential platelet/leukocyte activating properties of hollow fibre membranes. This resulted from

inconsistencies in the donor blood, the supply of antibody used, and the effects of the surface

roughness and contamination on the membranes.

1 would like to express my gratitude to my research supervisor, Dr. Paul Santerre, for his

guidance and support throughout this thesis and the preparation of this document. 1 especially

appreciate his encouraging words and devoted tirne.

1 also want to thank those who not only welcomed me into their laboratories, but took the time to

assist me in my research and offer their knowledge. These include Mr. Frank Gibbs for DSC

studies at the Brockhouse Institute for Materials Research, McMaster University, Dr. Wilhelmy

Neumann and Mr. Dan Kwok for undenvater contact angle experiments at the Laboratory of

Applied Surface Thermodynamics, University of Toronto, Dr. Xiajia Gu for confocal microscopy

work at the Ontario Light and Laser Research Centre, University of Toronto, Dr. Takeshi

Matsuura and Dr. S. Deng for the fabrication of hollow fibre membranes at the Industrial

Membrane Research Institute, University of Ottawa, Mr. Glenn McClung and Dr. John Brash for

fibrinogen adsorption studies at the Department of Pathology, McMaster University, and ?.."S.

Elaine Chung, Dr. Cynthia Gemmell, and Dr. Michael Sefton for whole blood studies ai the

Canadian Red Cross and Department of Chemical Engineering and Applied Chemi,t;y,

University of Toronto. The expertise of others are also acknowledged; Mr. Robert Chernecky for

SEM and polarized microscopy studies at the Faculty of Dentistry, University of Toronto, Dr.

Cynthia Goh for AFM studies at the Department of Chemistry, University of Toronto, and Dr.

Rana Sodhi for XPS studies at the Centre for Biomaterials, University of Toronto. 1 also want to

thank Mr. Jonathan Albrecht for his artistic talent in assisting with the production of selected

diagrarns in this thesis and his help in the final edits of this document.

1 want to Say thank you to everyone in Room 461 who shared my life with me over the past two

years. 1 am grateful for their kindness and friendships. Special thanks to Greg Woo for lending a

hand (and a coffee) in every crisis.

This work was financially supported by the Ontario Centre for Materials Research.

1 would like to thank rny family for their support, and understanding. My father has always

s h o w me the rewards of hard work. Thanks to Warren, Kevin, Julie, and Andrew for supporting

me in al1 my decisions and offering their words of advice. Special thanks to my mother and

Catherine for listening with an open mind. Many thanks to my friends for being my crutch. A

special thank you to Mr. Jonathan Albrecht for his support and encouragement. 1 will never

forget these years.

ADSA-P

AFM

BAL

DMAC

DMF

DSC

EDTA

FAFC

FITC

FSO 1

FS02

FS03

GPC

HTB

MD1

PBS

PCL

PE

PEO

PES

PMA

PPO

PVP

SEM

SFLLRN

SMM

T,

XPS

Axisymmetric Drop Shape Analysis - Profile

atomic force microscopy

ZonylTM BA-L fluoro-alcohol (low fraction)

dimethylacetarnide

dimethylformarnide

differential scanning caIorimetry

ethylenediaarninetetraacetic acid

fluorescence activated flow cytometry

fluorescein isothiocyanate

ZonylTM FSO- 100 fluoro-alcohol (low fraction)

ZonylTM FSO- 1 00 fluoro-alcohol (intemediate fraction)

ZonylTM FSO- 100 fluoro-alcohol (high fraction)

gel permeation chromatography

HEPES-Tyrode's buffer

methylene bis-p-phenyl diisocyanate

phosphate buffered saline

polycaprolactone di01

polyethylene

polyethylene oxide

polyethersulfone

phorbal 12-myristate 1 3 -acetate

polypropylene di01

polyvinylpyrrolidone

scanning electron microscopy

thrombin peptide (ser-phenyl-leu-leu-arg-asp)

surface modifying macromolecule

glass transition temperature

X-ray photoelectron spectroscopy

ABSTRACT

ACKNOWLEDGEMENTS

LIST OF ABBREVIATIONS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF APPENDICES

1.0 INTRODUCTION

1.1 SYNTHETIC MATERIALS FOR BIOMEDICAL MEMBRANES

1.2 BIOCOMPATIBILITY OF BLOOD CONTACTING MEMBRANES

1.3 SURFACE MODIFICATION FOR IMPROVED BLOOD COMPATIBILITY

1.4 SURFACE MODIFYING MACROMOLECULES

1.5 RESEARCH OBJECTIVES

2.0 THEORETICAL PRINCIPLES

2.1 BASIC PRINCIPLES IN POLYMER ENGINEERING

2.1.1 Molecular Weight Averages

2.1.2 Thermal Properties of Polymers

2.1.3 Polymer Blends

2.2 POLYUETHANE CHEMISTRY

2.2.1 Isocyanate Chemistry

2.2.2 Reagents Used for Polyurethane Synthesis

2.2.3 Reaction Conditions For Polyurethane Synthesis

2.3 SURFACE CHEMISTRY AND PHENOMENA

2.3.1 Properties of Membrane Surfaces

2.3.2 Surface Thermodynamics

2.3.3 Macroscopic Approach of the Equation of State

1

. . 11

iv

v

ix

X

xii

1

1

2

3

6

7

9

9

9

10

11

12

12

14

16

17

17

17

20

L . J . I I "A J AI*". V U A CU-.-' Y J "-""'

2.3.5 Characterization of Surfaces

2.4 BIOCOMPATIBILITY OF BIOMEDICAL MEMBRANES

2.4.1 Protein Adsorption

2.4.2 Blood Component Reactions

2.4.3 Blood Compatible Membranes

3.0 EXPERIMENTAL METHODS

3.1 SYNTHESIS OF SURFACE MODIFYING MACROMOLECULES

3.1.1 Materials Used for SMM Synthesis

3.1.2 SMM Synthesis

3.2 CHARACTERIZATION OF BULK POLYMEIUC PROPERTIES

3.2.1 Elemental Analysis

3.2.2 Molecular Weight Determination by Gel Permeation Chromatography

3.2.3 Differential Scanning Calorimetry

3.3 SURFACE CHARACTENZATION OF PES MEMBRANES

3.3.1 Preparation of PES Flat Sheet Membranes

3.3.2 Characterization of PES Membrane Morphology

3.3.2.1 Polarized Microscopy

3.3.2.2 Confocal Microscopy

3.3.2.3 Atomic Force Microscopy

3.3.3 Surface Analysis by X-Ray Photoelectron Spectroscopy

3.3.4 Study of Surface Energetics by Contact Angle Studies

3.3.4.1 Apparatus for Air Contact Angle Studies

3.3.4.2 Apparatus for Underwater Contact Angle Studies

3.4 BLOOD COMPATIBILITY STUDIES OF HOLLOW FIBRE MEMBRANES

3.4.1 Fabrication of Hollow Fibre Membranes

3.4.2 Fibrinogen Adsorption Studies

3.4.3 Whole Blood Studies for Determining Platelet and Leukocyte Activation

3.4.3.1 Blood-Material Contact

- - - - ,

3.4.3.3 Ce11 Count

4.0 EXPERIMENTAL RESULTS

4.1 SYNTHESIZED SURFACE MODIFYING MACROMOLECULES

4.2 POLYMERIC BULK PROPERTIES

4.2.1 Elemental Composition of SMMs

4.2.2 Molecular Weights of Synthesized SMMs

4.2.3 Thermal Transition Properties of Bulk Polymeric Materials

4.3 SURFACE PROPERTIES OF PES MEMBRANES

4.3.1 PES Flat Sheet Membrane

4.3.2 Characterization of PES Membrane Morphology

4.3.2.1 Polarized Microscopy

4.3.2.2 Confocal Microscopy

4.3.2.3 Atomic Force Microscopy

4.3.3 Elemental Composition of Membrane Surfaces Determined by XPS

4.3.4 Contact Angle Studies

4.3.4.1 Results From Air Contact Angle Analyses

4.3.4.2 Results Frorn Undenvater Contact Angle Analyses

4.4 BLOOD COMPATIBILITY OF SMM MODIFIED PES MEMBRANES

4.4.1 Hollow Fibre Membranes

4.4.2 Adsorption of Fibrinogen ont0 PES Hollow Fibres

4.4.3 Platelet and Leukocyte Activation

5.0 DISCUSSION OF EXPERIMENTAL RESULTS

5.1 SURFACE MODIFYING MACROMOLECULES

5.1.1 Synthesis of SMMs

5.1.2 Properties of SMM Polymers

5.1.2.1 Physical Appearance

5.1.2.2 Elemental Composition

3. I .L .S lvlolecular weignt Y L

5.1.2.4 Thermal Transitions 93

5.2 SMM-PES MEMBRANE COMPOSITION AND MORPHOLOGY 93

5.3 MEMBRANE SURFACE PROPERTIES 99

5.3.1 Review o f Contact Angle Methods 99

5 -3.1.1 Air Contact Angle Method Using a Goniometer 99

5 -3.1 -2 Undenvater Contact Angle Method 1 O0

5.3.2 Surfcace Energetics of PES and SMM Modified Membranes 1 03

5.4 EFFECTS OF SMMs ON THE BLOOD COMPATIBILITY OF PES MEMBRANES 105

5.4.1 Properties of the PES Hollow Fibre Membranes 105

5 .4.2 Fibrinogen Adsorption 1 06

5.4.3 Activation of Blood Components 108

6.0 CONCLUSIONS I l l

7.0 RECOMMENDATIONS 113

8.0 REFERENCES 116

APPENDICES 126

Table 3-1 : Materials for SMM Synthesis

Table 4-1: Physical Appearances of SMMs in the Final Dry Processed Form

Table 4-2: Weight Percent Elemental Compositions of Synthesized SMMs

Table 4-3: Polystyrene Equivalent Average Molecular Weights of SMMs

Table 4-4: Glass Transition Temperatures of Synthesized SMMs

Table 4-5: Glass Transition Temperatures of PES and SMM Modified PES Membranes

Table 4-6: Sizes Of Microdomains in 4 wt% SMM Modified PES Membranes

Table 4-7: XPS Results of Elemental Composition at the Surfaces of PES and SMM

Modified PES Membranes

Table 4-8: Results of Air Contact Angle Analyses on PES Membranes

Table 4-9: Results of Underwater Contact Angle Analyses on PES Membranes

Table 4-10: Fibrinogen Adsorption Results For 4 wt% SMM Modified PES Hollow

Fibre Membranes

Table 4-1 1 : Platelet Activation on PES Hollow Fibre Membranes

Table 5-1 : Elemental Composition of FSO- 100 Fractions

Table 5-2: Possible Chemical Structures of FSO Fractions

Table 5-3: Estimates of Average Molecular Structures of FSO Fractions as

Determined by Elemental Analysis

Table 5-4: Theoretical (Based on a 3:2:2 Stoichiometry) and Experimental Compositions

of Elemental Nitrogen and Fluorine in SMMs

Figure 1-1:

Figure 2-1:

Figure 2-2:

Figure 2-3:

Figure 2-4:

Figure 2-5:

Figure 2-6:

Figure 3-1 :

Figure 3-2:

Figure 3-3:

Figure 3-4:

Figure 3-5:

Figure 4-1:

Figure 4-2:

Figure 4-3:

Figure 4-4:

Figure 4-5:

Figure 4-6:

Figure 4-7:

Figure 4-8:

Figure 4-9:

Chernical Structure of Polyethersulfone 2

Commonly Used Isocyanates for Polyurethane Synthesis 14

Commonly Used Polyols for Polyurethane Synthesis 15

Surface Energy Diagram 19

Typical Langmuir Adsorption Isotherm 27

Cascade of Reactions Leading to the Process of Coagulation 29

PIatelet Reactions involved in the Formation of Thrombus 30

Schematic of Two Step Synthesis of Surface Modifying Macromolecule 36

Reactions in the Synthesis of MDI-PPO-BAL 38

Apparatus for Air Contact Angle Studies 45

Apparatus for Undenvater Contact Angle Studies 47

Rocking Platform Apparatus for Contacting Blood with Tube Materials 53

for Low Shear Conditions

First and Second Themograms of PES-PVP Membrane 59

Microdomain Structures of 4 wt% SMMs within PES Membranes 62

Observed Through a Polarized Microscope

Confocal Microscopy Images of PES Membranes Underwater 64

Confocal Microscopy Images of 4 wt% MDI-PPO-BAL in PES Dry Membrane 65

Confocal Microscopy Images of the Dispersion of Microdomain Structures 66

in a PES Membrane Modified with 4 wt% MDI-PPO-BAL

Atomic Force Microscopy Images of PES and SMM Modified PES 68

Membrane Topography

Profile of Air Advancing Contact Angles and Contact Radii for 74

4wt% MDI-PCL-BAL in PES Flat Sheet Membrane

Profile of Air Receding Contact Angles and Contact Radii for 74

4wt% MDI-PCL-BAL in PES Flat Sheet Membrane

Surface Tension (y,,) of PES Membranes Underwater 76

Figure 4-10: SEM Images of Hollow Fibre Membranes

X

- SMM Modifications Containing BAL

Figure 4-12: Fibrinogen Adsorption Onto PES Hollow Fibre Membranes

- SMM Modifications Containing PPO and FSO

Figure 4-13: Fibrinogen Adsorption Onto PES Hollow Fibre Membranes

- SMM Modifications Containing PCL and FSO

Figure 4-14: Leukocyte Activation by the Upregulation of CD1 1 b

Figure 5-1: Illustration of Hydrogen Bonding Capabilities of PCI, Components

Figure 5-2: Schematic of a Flat Sheet SMM Modified PES Membrane

Figure 5-3: Dependence of Air Advancing Contact Angle on Contact Radii for

PES Flat Sheet Membrane

Figure 5-4: Illustration of Slip-Stick Action of Air Advancing Contact Angles on

4 wt% MDI-PPO-FS02 in PES Flat Sheet Membrane

Figure 5-5: Illustration of Slip-Stick Action of Air Receding Contact Angles on

4 wt% MDI-PPO-FS03 in PES Flat Sheet Membrane

Appendix A: Supplier Narnes of chernical Reagents

Appendix B: Distillation and Degassing Apparatus

Appendix C: Apparatus for the Synthesis of SMMs

Appendix D: Instrumentaion for GPC

Appendix E: Basic Components of an XPS Spectrometer

Appendix F: Apparatus for the Fabrication of Holtow Fibre Membranes by

a Solution Spinning Process

Appendix G: Serial Dilutions Used for Preparing 1 2 5 ~ - ~ g Solutions

Appendix H: DSC Thermograms of SMM Polymer and SMM Modified

PES-PVP Membranes

Appendix 1: TGA Thermograrns of Polymers Used in the Fabrication of Membranes

Appendix J: Confocal Images of SMM Modified Membranes

Appendix K: Statistical Analysis of Regions of Constant Contact Angles

in Undenvater Measurements

Appendix L: Experimental Data from the In Vitro Whole Blood Study

xii

1.1 SYNTHETIC MATERIALS FOR BIOMEDICAL MEMBRANES

The field of biomaterials encompasses a variety of materials, a wide range of applications, and

draws upon many disciplines. Polymers in particular, have been used extensively in biomedical

devices such as catheters, heart valves, and dialysis membranes. Many of these polymers were

originally developed for industrial applications and were then adopted as biomaterials based on

their favourable characteristics. One area in which polymers have been important is in the

fabrication of biomedical membranes for artificial kidneys in haemodialysis. At the present time,

haemodialysis is one of the most important clinical applications of blood contacting materials.

In industry, devices used for blood purification represents almost 50% of the ultrafiltration

membrane market (1 994) [Dutre et al., 19941. Earlier artificial kidneys employed cellulosic

membranes due to their tensile strength and ability to be processed into flat sheets, large tubes or

hollow fibres. In the 1970s, synthetic membranes were introduced to improve upon the

efficiency of dialysis and its biocompatibility. However, 85% of haemodialyzers still use

cellulosic membranes due to their relatively low cost [Kazuhiko, 19921.

Several designs of the artificial kidney have been introduced with membrane configurations

including flat plates and hollow fibres. Hollow fibres are currently the preferred mode for

dialysis systems due to their compact design, large surface area, and low cost. The most widely

used polymers for the membranes include polyacrylonitrile, polysulphone,

polymethylmethacrylate, polycarbonate, polyamide, and polyvinyl alcohol. A less studied

material is polyethersulfone which has more recentiy been considered due to its relative strength

and biocompatibility [Maher, 7 9951.

Polyethersulfone (PES) is a high performance thermoplastic that has been used for a number of

membrane separation processes including ultrafiltration, pervaporation, and dialysis [Cheryan,

19861. This is due in part to its excellent oxidative, thermal and hydrolytic stability. In the

--------- , - - - - - - - - - - - -- - - - - - - - - - - - - . - - , - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . - - - - - - - - - - . - - - - -

of repeated sterilization techniques [Maher, 19951. The siiperior characteristics inherent of PES

materials are due to its basic chernical structure, illustrated in Figure 1-1. The sulfone unit gives

thermal stability and strength, while the ether linkage provides the chain mobility and ease of

processing [Maher, 19951. PES also dissolves in a variety of solvents which makes the material

easily processable into films or tubes by casting procedures.

Figure 1-1: Chernical Structure of Polyethersulfone

1.2 BIOCOMPATIBILITY OF BLOOD CONTACTING MEMBRANES

A major limitation of a biomaterial is that its surface is foreign to the blood and tends to promote

the destruction of blood cells, denaturation of proteins, and the formation of blood clots. These

problems are no more pronounced than in the biocompatibility of dialysis membranes which may

lead to a number of adverse immune responses, with acute and chronic impacts on the patient.

Upon contact with a foreign surface, several events may occur from blood-membrane

interactions. These include protein transformation at the membrane interface, ce11 adhesion,

aggregation, and activation, mechanical shearing, and leachiiig of materials [Cheung, 19901. The

alteration of many proteins at the tnembrane surface is probably responsible for the most severe

clinical consequences [Cheung, 19901.

The initial events of blood contact is the adsorption of proteins such as fibrinogen and albumin.

Certain proteins may undergo alterations in their structures and subsequently activate the

coagulation and complement systems. The adsorption of fibrinogen also enhances the adhesion

of cells such as platelets, erythrocytes, and leukocytes, ont0 its surface. Consequently, activated

~ U I I I ~ I C I I I I C I I I L ~ I U L ~ L I I D QIIU a b u v a L b u I ~ L L R U L ~ L G D LQII a i l i c a u LW b i l l i i b a i cvciim a u ~ i i as L U A I L ~ L ~ aiiu

allergic responses [Cheung, 19901. The pathways and mechanisms of activation are complex and

are further described in Section 2.4.

There have been many methods introduced for minimizing the interactions of blood at the

biomaterial surface. For example, anticoagulants, the most popular being heparin, are used in

dose administration or have been attached ont0 the surface of dialysis membranes to reduce

clotting on rnedical devices. However, anticoagulation has been associated with several side-

effects including a risk of haemorrhaging [Amiji, 19941. Research has now focused on more

biocompatible membranes with most of the efforts concentrated on modifjing the surfaces of

existing materials.

1.3 SURFACE MODIFICATION FOR IMPROVED BLOOD COMPATIBILITY

Many researchers have investigated different methods of modi@ing currently used materials for

improving biocompatibility. The goal is to take advantage of the mechanical properties of

synthetic polymers but improve upon the surface characteristics which will render them blood

compatible. The most common methods introduced for modifying surfaces of biomaterials are

briefly described in this section.

One method of surface modification for improving the biocompatiblity of dialysis membranes is

to ionically bond biological species ont0 the surface of the polymer in order to simulate the

surface of a biological system [Piskin, 19921. The most studied of the bioactive species has been

lieparin, a natural anticoagulant responsible for the fluidity of blood. However, for long term

applications, heparinization may fail because the heparin will eventually be depleted through

desorption processes [Silver et al., 19921.

The surface properties of membranes can also be altered by the addition of functional groups.

There 11as been numerous studies on halogenation [Mohr et al., 1991; Clarotti et al., 19921,

LU uunyiaiuii 11 i ~ i u i a y C L ai., i YYLJ ana suironauon Llviclvriirin, 1 YUYJ or polymers. bxamples

include the grafting of 2-methacryloyloxyetl~yI phosphorylcholine [Kazuhiko, 19921 and

poly(ethy1ene glycol) [Amiji, 19941 ont0 cellulose membrane surfaces. Such grafting techniques

can be achieved by either a coupling reaction of existing polymer chains or plasma deposition of

monomers ont0 the surface of the porous material [Wu et al., 921. The latter technique has also

been used to deposit thin fluorocarbon coatings from an argon plasma containing

perfluorohexane to give a smooth hydrophobic surface membrane [Clarotti et al., 19921. Several

studies have also modified membrane surfaces to prevent protein adsorption by adsorbing block

copolymers ont0 the surface [Schroen et al., 1993; Matsuda et al., 19943. Although surface

coating and grafting methods are effective in modifiing the surface properties of polymers, the

techniques may require special equipment and therefore may be costly.

Surfaces can also be modified by blending polymer additives into the base material. This method

has been less popular for biomedical applications because of the possibility for leaching of the

additives, and concern of their cytotoxicity [Ikada, 19941. However, studies over the past several

years have suggested that this may be a promising method for improving the biocompatibility of

biomaterials. In these techniques, small amounts of surface modifj4ng additives are blended with

a base polymer which has the desired physiological properties for the biomaterial. Surface

modification is effected by the migration of the additives to the surface of the base polymer

during fabrication of the device. Therefore, the final surface characteristics reflect those of the

additives concentrated at the surface, leaving the bulk of the material unchanged. The additives

are relatively high molecular weight copolymers which are compatible with the base polymer and

should be amphiphiIic, having both polar and nonpolar segments [Ward, 19891. The

reorientation of these blocks at the surface minirnizes the interfacial energy between the blood

and polyiner surface and therefore improves its biocompatibility.

Studies on surface modi@ing additives for polymer surfaces have included the work of Ward

119891 who synthesized arnphipathic multipolymers as surface-modibing additives (SMA) for

use in biomedical polymers to reduce thrombogenicity. It was found that the approach to

equilibrium of the SMAs at the surface was slow and hence their group further developed

puiyrricrs wiiri aurlace-lvioairying c n a uroupsLWA (sivrc) [wnite et al., 1YYbJ. Biomedical

devices would then be synthesized by this modified polymer. The latter technique is different

from blending a polymer additive with a base polymer since here, each base polymer molecule is

synthesized by incorporating the SMEs. Other studies have also shown reduced protein

adsorption ont0 poly(ether urethaneurea) films modified with methacrylate based surface

additives. These include Brundstedt et al. [1993] through the addition of amphiphilic or

hydrophobie polyacrylate or methacrylate, and Freij-Larsson et al. [1993] with a

polymethacrylate additive identified as poIy(diisopropylaminoethyI methacrylate-co-decyl

methacrylate). Another exarnple is Kasemwa et al. [1993] who blended fluorine-containing

block copolymers, composed of methyl methacrylate, and heptadecafluorodecyl acrylate, with an

epoxy resin to improve water repellency.

The haecompatibility of fluorinated materials has been widely investigated. It has been

suggested that commercial fluoropolymers have a low surface tension and as a result adsorb Iess

protein [Kaku et al., 1994; Clarotti et al., 92; Garbassi et al., 19943. However, most commercial

fluoropoIymers are difficult to process and due to their limited solubility in common solvents, are

not suitable for some biomedical devices, such as dialysis membranes. Therefore, surface

modification techniques offer many solutions. Surface fluorination c m create a surface with

blood compatibility and chernical stability, without affecting the physiological properties of the

base polymer. This was s h o w for polyetherurethanes coated with a surface of a fluorinated

block copolymer [Kaku et al., 19941 and polyurethane catheters deposited with a fluorinated film

[Pizzoferrato et al., 19951. In copolyrners, fluorinated segments are usually enriched at the

surface. For this reason, there have been studies to synthesize polyurethanes with fluorinated

chah extenders and soft segments [Edelman, 1990; Yoon et al., 19901. As well as

biocompatibility, surface fluorination has been shown to improve the permselectivity of some

polymeric membranes including polysulfone hollow fibres [Mohr et al., 19911. There are

obvious advantages in the use of surface modifying additives and surface fluorination. This is

thc basis for the development of surface inodifj4ng macromolecules.

Surface modi@ing macromolecules (SMMs) are oligomeric fluoropolymers synthesized by

polyurethane chemistry and tailored with fluorinated endgroups [Tang et al., 1996; Pham et al.,

1997 (l)]. When added to a base polymer such as polyethersulfone, it has been shown that low

molecular weight SMMs will migrate to the surface due to their immiscibility with the base

polymer, and the fluorine endgroups will orient towards the air-polymer interface [Tang et al.,

1996; Pham et al., f 997 (2)]. This reduces the surface energy of the cast base polymer, achieving

surface energies close to that of TeflonB [Tang et al., 19961. Because only a small amount of

SMMs (not more than 4 weight percent) is added, the bulk properties of the base polymer remain

relatively unchanged [Tang et al., 1996; Pham et al., 1997 (2)]. Currently, several formulations

of SMMs have been synthesized with varying reactant ratios, each characteristically different.

The bulk properties of the SMM polymers have been characterized, including molecular weights,

elemental analysis, and thermal transitions, and the SMMs have been blended with base

polyrners of polyurethanes [Tang, 1995; Weiler, 19973 and polyethersulfone [Pham, 19951.

SMMs have been tested for use in several industrial membrane applications such as ultrafiltration

and pervaporation, providing membranes with low surface energies, chernical resistance, and

improved surface Iubrication [Harnza et al., 1996; Fang et al., 1994; Pham, 19961. In

ultrafiltration applications, polyethersulfone membranes modified with SMMs also showed

reduced fouling and increased flux for treating oil/water emulsions [Hamza et al., 19961. Tang et

al. 11996, 1997 (l)] have suggested the use of these additives for surface modification of

biomaterials such as segmented polyurethanes. Such modifications can improve the blood

cornpatibility of polyurethanes by reducing protein adsorption [Tang et al., 1997 (2)] and

minimizing white blood ce11 activation [jahangir, 19961. As well, the presence of SMMs at the

surface of polyurethanes reduced the hydrolysis of the material by lysosomal enzymes, thereby

showing an increase in overall biostability [Tang et al., 1997 (1); Tang et al., 1997 (2)]. The

degree to wliicl-i SMMs improve these aspects of biocompatibility depends on the formulations

and reagents used in their synthesis.

From the above discussion, there is a clear need for improving the blood compatibility of

biomedical membranes for applications such as haemodialysis. Recent work has focused on the

surface properties of materials that are in direct contact with blood. Surface modification c m

improve upon the biocompatibility of a material by altering its surface properties without

significantly affecting the strength and dexterity of the material. While conventional methods of

surface modification require a second processing step that may translate into added equiprnent

costs, surface modification with SMMs has been proven to be a simpler method.

It is hypothesized that the surface properties of dialysis membranes can be tailored by the

addition of SMMs so as to improve the blood compatibility of the membrane without affecting

its bulk properties. The addition of SMMs will lower the surface tension of the base material

producing relatively more hydrophobic surfaces through the migration and concentration of

SMMs at the surface. By lowering the surface tension, this rnethod has the potential for

improving the blood compatibility of a material by reducing protein adsorption and platelet

activation. Because only a small percentage of SMMs is used, the surface properties will be

altered without significantly changing the bulk properties. The surface properties will be

expected to depend on the chemical nature of the SMMs used, and SMMs with varying chemical

compositions should exhibit varying degrees of miscibility in the base polymer.

The scope of this thesis was to study the surface characteristics (physical and biological) of SMM

rnodified PES membranes intended for possible biomedical ultrafiltration and microfiltration

applications such as dialysis. The SMMs were synthesized to study the effect of varying

chemical compositions on both the miscibility of the additives in the base polymer and the

surface properties induced. The synthesized SMMs were characterized for their bulk properties,

and the morphology and surface properties of the SMM modified membranes were assessed.

The surfaces of the membranes were characterized by air and underwater contact angle studies,

X-ray photoelectron spectroscopy and various microscopy techniques. By these methods, a

mode1 of the dispersion of SMMs in PES membranes can be developed. Aspects of blood

~ u l I l p U ~ ~ ~ ~ ~ ~ ~ J LUI IIIVUIII~U I LLI L I W ~ ~ U V V IIUI C, IIILIIIUI a 1 ~ 3 WLI L a 1 w I u v c a u g a i c u W ~ L N 1 C ~ ~ C L L LU

fibrinogen adsorption, platelet activation, and leukocyte activation. From these results, the

effects of surface morphology and surface properties induced by SMMs of differing chernical

compositions may be correlated to their effects on the blood compatibility of PES membranes.

2.1 BASIC PRINCIPLES IN POLYMER ENGINEERING

2.1.1 Molecular Weight Averages

A polymer is a molecule consisting of many repeat units of a monomer and as a result has a high

molecular weight which rnay Vary depending on the number of repeat units. The general

influence of molecular weight on a polymer's mechanical properties is such that the properties

increase with increasing molecular weight, and levels off at a maximum that varies from polymer

to polymer [Speckhard et al., 19861. Therefore low mechanical strength is often due to low

molecular weights. However, the higher the molecular weight the more difficult it may be to

process the polymer [Lelah et al., 19861.

Since the number of repeat units can Vary between molecules of the same polymer, the polymer

molecular weight is an average of the total number of molecular weights and its definition

depends on the weighting factor. The number average rnolecular weight is based on the number

of moles in the sample and is given by:

where xi is the mole fraction of species i (Le. the number of polymer chains of a specific

inolecular weight), and Mi is the molecular weight of species i. A second measure of molecular

weight is the weight average molecular weight which reflects the weight of the different fractions

within the polymer and is given by:

- -

molecular weight species. The ratio of MW to Mn is the polydispersity of the polymer which

defines the breadth of the molecular weight distribution. A narrow molecular weight distribution

comesponds to a polydispersity close to 1. Typical values for commercial polymers range from

1.5 to 50 [Lelah et al., 19861. The shape of the molecular weight distribution can have a

significant effect on the polymer's physical characteristics.

2.1.2 Thermal Properties of Polymers

The thermal properties of polyrners and polymer blends are important considerations for

industrial applications. One important property is the glass transition temperature, Tg , which is

the temperature, or range of temperatures, below which the polymer is in a glassy state and above

which it is rubbery. It is associated with the abrupt cessation of polymer backbone rotations

resulting from a decrease in temperature [Lelah et al., 19861 and varies with each polymer and

polymer mixture. This parameter is distinguished from the melting temperature, Tm , which is

the temperature above which the solid polymer becomes a liquid phase. The glass transition

temperature depends on five factors, the free volume of the polymer, the attractive forces

between the molecules, the interna1 mobility of the chains, the stiffness of the chains, and the

chain length [Rosen, 19931. In segmented polymers, such as polyurethanes, the Tg of the

polymer may be used as a measure of microphase separation [Wang et al., 19831. In copolymers,

it usually varies monotonically with composition between those of the homopolymers [Rosen,

19931. These transition temperatwes can be determined by observing the changes in

thermodynamic properties of the polymer, for example the specific volume or refractive index,

with changes in temperature. A conventional method of characterization is differential scanning

calorimetry (DSC) where the thermal property monitored is the change in enthalpy. The method

of DSC analysis is detailed in Section 3.2.3.

The processability and final properties of polymer blends, such as the SMM modified PES

membranes studied in this work, will depend on the amount of SMM added in PES and the

miscibility of the two polymers in the casting solution. This may also determine the phase

separation of the two during membrane formation as the solvent evaporates. Phase separation is

a phenomena which has been observed in additive modified polymer blends in the formation of

films [Brundstedt et al., 19931 due to the therrnodynamic incompatibility of the polymer blend,

causing demixing. These blends wili have phase domains which consist of regions of

concentrated pure components.

An immiscible blend will affect the homogeneity of a cast membrane and thus its surface

properties since surface roughness plays an important role in surface energetics. In previous

studies, it was observed that phase separation was achieved with 1 wt% SMM in the casting

solution and that an increased arnount of SMM added may cause perturbations in the surface

morphology of the PES substrate [Pham et al., 1997 (2)] . The surface composition of SMM in

base polymer can also vary with the choice of casting solvent, the procedures used for

precipitation and the external drying environment [Bergbreiter, 19%; Sharma et al., 19841. The

miscibility of polymer blends is often determined by the rneasure of a single Tg as a function of

composition [Couchman et al., 1978; Aubin et al., 19881. In general, for a binary polymer blend,

the Tg is observed to increase approximately monotonically with increasing composition of the

component with the highest Tg [Aubin et al., 19881.

The chemistry used for the synthesis of SMMs in this thesis is based on polyurethane chemistry.

Polyurethanes are linear alternating block copolymers consisting of long flexible soft segments

and highly polar and stiff hard segments. The synthesis can be performed by a one step or two

step process. The reactants are mixed simultaneously in the one step process with a suitable

catalyst and the result is a random copolymer polyurethane. The two step process produces

random or block copolyrners by the initial reaction of a diisocyanate and a polyol, and then a

coupling reaction effected by adding a di01 or diamine. The mechanism of the reaction follows

that of step growth polymerization in which the reaction is dependent on the end functional

groups of both molecules, and is independent of the rest of the molecule chain if there are more

than five or six repeat units [Rosen, 19931. Synthesis of polyurethanes are either performed as

bulk or solution polymerizations.

2.2.1 Isocyanate Chemistry

The isocyanate group in the diisocyanate reagent consists of three resonance structures:

The presence of these structures allows for different side reactions with themselves and chemical

groups with active hydrogens. The nucleophilic attack of urethane and urea linkages ont0

isocyanates is also a possible sequence of reactions, producing allophanates and biurets [Lelah et

al., 19861. These products can result in chemical crosslinking of the polyrner chains. Other side

reactions include the formation of dimers and trimers from aromatic isocyanates. Aromatic

isocyanates easily dimerize at low temperatures, especially in the presence of acidic or basic

catalysts [Gogolewski, 19891. Both the formation of dimers and trimers will cause an imbalance

in the reactant ratios and therefore, branching and crosslinking can occur.

'1 he synthesis 01 polyuretnanes 1s aepenaent on rne resonance siruuurcs o r LM I ~ U L ~ L U I ~ ~ L G g i u u p .

A prepolymer of molecular weight between 1,000 and 5,000 is formed by an addition reaction

between an isocyanate group and a hydroxyl group of an oligomeric diol, as shown in Equation

(2-4) [Lelah et al., 19861.

The bonding between the isocyanate and the hydroxyl group generates a urethane linkage. To

produce long chain polyurethanes with high molecular weights, the prepolymer can be chain

extended with either a low molecular weight diol, resulting in a polyurethane, or a low molecular

weight diamine, resulting in the formation of a polyurethane-urea. As well, low molecular

weight polyurethanes with varying characteristics can be produced by endcapping the products of

the prepolymer reaction in place of the chain extension step.

While water molecules may be desired, as in the production of polyurethane foarns, in most cases

they are considered a contaminant. Water molecules react with the isocyanate groups in a similar

manner as hydroxyl groups, producing carbon dioxide and amines. The amines then react rapidly

with additional isocyanates to form urea linkages [Lelah et al., 19861, written as:

Many other impurities can also be a source of active hydrogens, including monofunctional

alcohols, amines and carboxylic acids. The reactions of the impurities will cause chain

temination and unbalance the reaction by using up diisocyanate. The end result is low molecular

weight polyiners. Therefore, it is crucial that high purity reagents be used in the synthesis of

linear polyurethanes.

2.2.2 Keagents Used tor voiyuretnane synrnesis

There are many diisocyanates used for the synthesis of polyurethanes, including toluene

diisocyanate (TDI), 4,4-diphenylmethane diisocyanate (MDI), hexamethylene diisocyanate (HDI)

and methylene bis@-cycIohexy1 isocyanate) (H12MDI) [Lelah et al., 19861. The chernical

structures of these diisocyanates are illustrated in Figure 2-1. The most commonly used

isocyanates are MD1 and TDI. The use of MD1 yields a rigid hard segment which may crystallize

with other hard segments to provide strength to the polyrner [Hergenrother et al., 19931. This is

not seen in al1 polyurethanes. For example, polymers synthesized with H12MDI do not produce

crystallized domains since the isocyanate exists in various conformational States which do not

allow the hard segments to pack appropriately. However, these isocyanates have been found to

produce polyurethanes with comparable strength to those with MD1 [Hergenrother et al., 19931.

Figure 2-1: Commonly Used Isocyanates for Polyurethane Synthesis

NCO CH3\ ,

NCO

2,4 toluene diisocyanate (2,4 TDI) 2,6 toluene diisocyanate (2,6 TDI)

methylene bis(p-phenyldiisocyanate) (MDI)

OCNHzC -(CH2)4-CH2NC0

1,6 hexane diisocyanate (HDI)

- A - - - -

chemical structure of the polyol soft segment. The polyols are generally low molecular weight

polyesters, polyethers, hydrocarbon polymers or polydimethylsiloxanes, al1 containing hydroxyl

or amine end groups. Some commonly used soft segment reagents are shown in Figure 2-2.

Polyester based polyurethanes yield good material properties but are unstable towards hydrolytic

cleavage at the ester linkage [Lelah et al., 19861. Polyethylene oxide based polymers are

hydrophilic and have poor water resistance, while polypropylene oxide based polymer are very

sofl [Lelah et al., 19861. The polyether which has shown the best physical properties is

polytetramethylene (PTMO). It produces polymers with good mechanical strength, relatively

good hydrolytic stability, and is water resistant [Lelah et al., 19861.

Figure 2-2: Commonly Used Poiyols for Polyurethane Synthesis

polyethyleneoxide (PEO) polypropylene oxide (PPO)

polytetramethylene oxide (PTMO) polycaprolactone di01 (PCL)

Specific characteristics can be introduced into the polyurethane by endcapping the prepolymer

reaction with oligomers containing certain functional groups. The chemical group of interest in

this study is fluorine and the result is an oligomeric fluoro-polyurethane polyrner. The fluorine

"tails" are introduced by reacting a fluorinated surfactant with the urethane prepolymer. The

fluoro-intermediates used in this study are manufactured by DuPont Chernicals and sold under

the registered trademark of ZonylTM. These compounds can be anionic, cationic, nonionic and

amphoteric, and are soluble in many common solvents. They can provide low surface tensions at

- - - .

material is the fluorocarbon chain, F(CF2CF2); in the molecule. The fluoro-intermediates are

effective at lowering the surface tension, while providing excellent chemical and thermal

stability. It is estimated that surfaces made with ZonylTM can achieve a critical surface tension

three times lower than that of Teflona [Dupont].

2.2.3 Reaction Conditions For Polyurethane Synthesis

The conditions of the polymerization system will influence the molecular weight, composition

distribution, and morphology of the resulting polyurethane [Speckhard et al., 19861. For

example, the molar ratios of the reactants can vary the sequence length distribution of the hard

segments and the molecular weight of the polymer [Peebles, 19741. In solution polymerization,

the solvent serves as a nonreactive medium for the reactants and resulting product. Solvents with

active hydrogens may induce undesired side reactions and are therefore not used. The most

commonly used solvents for polyurethane synthesis are dimethylsulfoxide, N-methylpyrrolidone,

dimethylformamide, N,N-dimetliylacetarnide and tetrahydrofuran [Gogolewski, 19891. The

formation of polyurethanes is an exothermic process [Gogolewski, 19891. However, in order to

achieve polyurethanes with high molecular weights, thermal energy is applied in the forrn of

heat. The temperature will depend on the reactants used and the desired properties of the final

polymer. In previous studies describing the synthesis of SMMs, it was found that the reaction

temperature, solvent volume, reactant mole ratio, reactant concentration, and stir rate were

important in determining the size and distribution of the SMM synthesized [Tang, 1995; Pham,

19951. Because of this, these variables were held constant throughout this study.

2.3.1 Properties of Membrane Surfaces

The surface of any material is inherently different from that of the bulk material and is an

important aspect in the study of biocompatibility and biomedical membranes. Wettability,

surface charge, chernical and physical heterogeneity, surface energy, and surface dynamics are

considered important properties of biomaterial surfaces [Piskin, 19921. Biocompatibility,

particularly thrombogenicity, is affected by these surface properties which will subsequently

determine its interactions with blood components. The mechanisms for adsorption of proteins to

non wettable surfaces are relatively well understood and in agreement between different

researchers. The minimum interfacial free energy hypothesis states that if the polymer-water

interface has a low interfacial free energy, then protein adsorption should be low and reversible

[Andrade et al., 1979 (2) ] . Generally, surfaces will try to rninimize their interfacial energy,

Ieading to different surface atomic structures. This is the driving force which causes the

migration of low energy surface additives, such as SMMs, to the air-polymer interface, thereby

lowering the interfacial energy.

2.3.2 Surface Thermodynamics

There are three governing therrnodynarnic expressions used to describe the energy system of a

surface. The system can be defined either by an energy balance (E), (equation 2-6), or by the

Gibbs (G) and Helmholtz (H) free energy expressions, (equation 2-7) and (equation 2-8)

respectively [Morra, 1 9901 :

dE = TdS - PdV + ydA + CpdN

dG = - SdT + VdP + ydA + CpdN

dF = - SdT - PdV + ydA + CpdN

v v ~ ~ u i u 1 8 5 uiu r u ~ i i y u r u r u i v , u A U SA*- w i i ~ . i v y J , . LW +.a- . -.....A.- ? ' *" "" r * - - - - * - y 1 '- ""

interfacial tension, p is the chemical potential, N is the normality, and A is the area of the

interface. The Helmholtz free energy is used to describe the system of contact angles because the

condition of constant T, V, and N c m be easily achieved.

The purpose of contact angle measurements is to determine the equilibrium condition at a three

phase boundary, the three phases being that of solid-liquid-vapour, or solid-liquid-liquid. For

instance, a liquid drop on a solid surface will change its shape to reach equilibrium. By

performing a thermodynamic analysis on the Helmholtz free energy expression, we c m rewrite

equation (2-8) in the form:

where 8 is the angle between the solid-liquid interface and the liquid-vapour interface, and y is

the surface tension of the corresponding interface. At thermodynarnic equilibrium, dF/dA = O

and the equilibrium is expressed by the Young Equation [Morra, 19901:

Y,$, - Y,, = Ylv 0

The Young equation has never been proven experimentally and there is much controversy on the

validity of this equation and when it applies. The reason for this is because equation (2- 10) is

described for ideal surfaces at equilibrium. However, there are many factors which c m affect the

equilibrium contact angle. One problem is the effect of spreading pressure for small contact

angles. Some argue that this can be ignored for angles greater than IO0, while otliers insist

otherwise [Morra, 19901. Another factor, not accounted for by the Young equation, is the drop

size effect as related to line tension. A 1-D analog to surface tension, its effect is to minimize the

circumference of the drop meumann et al., 19963. Drops of different sizes on the same surface

should therefore have different contact angles. This effect may be insignificant when compared

to effects due to surface chernical or morphological heterogeneity. As well, the liquid must not

---- - r - -d -

be effected by numerous factors which include thermodynamic effects such as chernical

heterogeneity and surface roughness. Time dependent effects such as swelling, penetration, and

surface mobility can also cause deviations from ideality.

Thermodynamic effects such as surface heterogeneity and roughness create free energy barriers

on the surface as illustrated in Figure 2-3 [Andrade et al., 1979; Morra, 1990; Neumann, 19741.

As a result, the system c m equilibrate in one of many free energy local minima (or metastable

states of equilibrium) giving rise to contact angIe hysteresis. The largest contact angle is termed

the advancing contact angle, and the srnallest is the receding contact angle. In general, hysteresis

is negligible for a roughness lower than 0.5 - 0.1 Pm, or heterogeneity phases smaller than 0.1

pm [Morra, 19901.

Figure 2-3: Surface Energy Diagram peumann et al., 19961

0 (degrees)

liquid system orients toward a more stable state. These effects include penetration, swelling, and

polymer mobility. Water has a tendency to penetrate and saturate intermolecular pores of some

material surfaces, and the swelling of gels have been observed in a number of studies [Morra,

1990; Holly et al., 19751. The sarne holds not just for hydrogels but for rigid polymers as well.

The driving force for surface reorientation is the thermodynamic requirement to minimize the

surface tension so that low energy groups are exposed to the polymer-liquid interface while the

higher energy polar groups are reoriented towards the bulk. Andrade et al. [1979] obtained

results which showed that hydrophilic components dominate the polymer-water interfacial

properties, even at low concentrations. Therefore it is evident that the characterization of

polymer surface properties is dependent on the working environment.

2.3.3 Macroscopic Approach of the Equation of State

The purpose of measuring contact angles is to obtain the surface tension associated with a

material surface. The determination of the solid surface tension has led to some controversy and

the debates have centred around the meaning of surface tension components. Several

investigators have introduced differing rnodels to describe the surface tension of a two phase

system. In this study, surface tension values are determined fiom contact angle analysis

according to the macroscopic approach of the equation of state. Other models are briefly

discussed in a review by Morra [1990].

A balance between surface tension and external forces is given by the classical theory of

capillarity Ir\reumann, 19741. Minimizing the overall free energy of the system gives the Laplace

equation of capillarity (equation 2-1 1) which describes the shape of the water droplet, and the

Young equation (equation 2-1 2) which provides a relationship between the contact angle and the

surface tension.

where Ri and R2 are the principal radii of curvature at a point

Ap is the density difference between the liquid and vapour phases

g is the acceleration due to gravity

z is the ordinate of a point at the liquid surface

c is a constant

AP is the capillary pressure

The details of the development of the theory of capillarity and the equations are given elsewhere

Ir\leurnann, 19741. Whereas the Young equation relates the equilibrium contact angle to the

surface tensions for smooth, homogeneous, isotropic, nondeformable ideal surfaces, the Laplace

equation of capillarity is valid for the range of contact angles inherent in non-ideal surfaces. This

is because the equation describes the shape of the liquid vapour phase and is not a consequence

of the character of the solid surface.

In the Young equation, only y,, and 8, (the equilibriurn contact angle) are rneasurable. In order to

calculate ysv and y,! , the Young equation has to be combined with an equation of state relation in

the form:

Ysi = f ( YS, , YIV ) (2- 13)

We can consider the free energy of adhesion of a molecule per unit area to be:

WSI = Yiv + Ysv - Ysi (2- 14)

where WsI is the reversible work of adhesion. From this, mathematical analysis yields the

equation of state:

The formulation of the equation of state is given elsewhere [Neumann et al., 19961. Combining

this with Young's equation (2-12) then gives:

where 8 is the Young contact angle. Equation 2-16 has been applied to experimental data and a

value for p was found to be 0.0001247 (rn2/m~)* meumann et al., 19961. For the purpose of

calculations, equation (2-1 6) may be rearranged in the form:

If we let

and

The equation of state becomes

Therefore, mathematical manipulation will involve solving for the roots x of equation (2-20) by a

numerical methods analysis and relating this to y,, in equation (2-1 8) [Neumann et al., 19961.

surface tension of other materials.

2.3.4 Polymer Surface Dynamics

Polymers are generally thought of as rigid and relatively immobile materials. However, in

changing environments, polymers have been shown to respond by re-equilibrating its surface

composition. As a result, the surface properties of these materials are dependent on their

surrounding environment. The most drarnatic example is in an aqueous environment where the

interaction of the polymer with the water provides a strong driving force for surface components

to reorient in order to reduce interfacial tension. This is an important consideration for

biomaterials since the environment for fabrication may be very different from that for its

intended use.

In an aqueous environment, the polarity of the water molecules creates an interfacial free energy

driving force for the migration of polar blocks, segments, or side chains towards the liquid

interface, in order to reduce the surface free energy. This phenornena has been demonstrated in

nurnerous studies. For example, polyurethane surfaces have been shown to undergo

rearrangements when the environment was changed from air to water. The polar component was

observed to increase in time which was due to the increase of the more polar hard segment at the

surface [Herng et al., 19901. Another example is the polymer poly(hydroxyethylmethary1ate)

(pHEMA) which orients the hydrophobic methyl groups at the polymer-air interface and the

hydrophilic hydroxyl groups at the polymer-water interface [Garbassi et al., 19941.

The equilibration of polymer surface components have been found to be temperature and time

dependent [Andrade, 1988; Jhon et al., 19881. It may also depend on the properties of the

interfacing medium which provides the thermodynamic drive for reorientation. The nature of

polymer rearrangement is dependent on the chernical and morphological composition of the

polymer to provide sufficient mobility. This mobility can occur at several levels, rotation of

lunctional groups, morlon or cnain segments, ur niouuii ur ~ r i c C I ~ L I L G riiauuiriuicuuc LUCU uaam GL

al.. 19941. If the glass transition ternperature of the block, for example the soft segment in

polyurethanes, is below that of use, the segment is highly flexible and in motion. However, from

a study on the molecular dynamics of the free surface of a polypropylene film, it was suggested

that the mobility of surface chains and hnctional groups is not the sarne as bulk mobility, and

therefore should not be described by bulk properties such as the glass transition temperature

[Garbassi et al., 19941. This may hold true for the hornogeneous system in that study, but for

heterogeneous systems, bulk properties of different domains may very well determine its

mobility at the surface.

2.3.5 Characterization of Surfaces

The characterization of surface properties is limited by existing analytical instruments and

environmental conditions. Surfaces are difficult to study because they are readily contaminated

by their environment and possess mobility which allows the surface chemistry and structure to

thermodynarnically adjust to their surroundings. This may also be complicated by impurities

which may be present in some commercial or synthesized polymers and may be surface active.

These include polymerization surfactants, catalysts, solvents, plasticizers, interna1 lubricants, and

degradation products.

Currently, there are many techniques for assessing the chemistry and properties of polymeric

surfaces. The conventional method of determining surface energies consist of measuring the

contact angle that a drop of liquid makes on the surface [Morra, 19901. Surface analysis by

contact angle studies c m provide quantitative and qualitative information such as surface energy,

wettability, surface roughness, and heterogeneity weumann, 19741. It has also been used more

recently for studying the dynamics of polymer surfaces in studies which have shown the mobility

of surface groups [Andrade et al., 1979; Morra, 1990; Holly et al., 19751. Most contact angle

studies are performed on an apparatus known as a goniorneter. This simple niethod allows for

the determination of contact angles by visually observing a drop of liquid on the surface. More

- - r ---- --

parameters such as contact radius and phase volume [Cheng et al., 1990; Advanced Surface

Technology Inc., Billerica, MA].

Due to polymer surface dynamics, there has been some question regarding the relevance of this

method and whether the angles measured are the true equilibrium contact angles. An improved

method of surface energy characterization is the captive bubble technique or underwater contact

angle methods [Hamilton, 19721. In this method the polymer surface is placed in a controlled

atmosphere and the surface is allowed to equilibrate with the medium. The phase that is

introduced is air and the contact angle is measured on the solid-liquid interface. The greatest

advantage of the captive bubble method is that the medium is simulated as the environment with

which the materials are used and surfaces energetics are measured on fully hydrated surfaces. As

well, the composition of the environment can be controlled and fixed. The purpose of the contact

angle study in this thesis was to assess the surface energetics of PES and SMM modified PES

membranes. Changes in the wettability of the modified surfaces were compared by conventional

contact angle analyses in air using a goniorneter. To study the PES membranes in an aqueous

environment, an undenvater contact angle method was developed. This method was then used to

assess the nature and behaviour of the SMM modified PES membranes.

2.4 BIOCOMPATIBILITY OF BIOMEDICAL MEMBRANES

The concept of biocompatibility has been debated with no final conclusion as to its definition.

The reason is that biocompatibility encornpasses many diverse concepts which cannot be

cotlcisely defined. There are two main areas of biocompatibility, one concerns the bulk property

of the biomaterial, and the other is its surface property [Ratner, 19961. Bulk biocompatibility is

more relevant for implantable devices such as heart valves and artificial joints, whereas surface

biocornpatibility relates to events occurring at interfaces for devices such as catheters and

bioniedical membranes. In haemodialysis, surface interactions are most important and the issue

of biocompatibility focuses on the events which occur when the blood cornes into contact with

device, to acute and chronic events in the patients. Standard dialysis with some membranes is

known to stimulate the immune system and activate plasma proteins and blood cells, leading to

inflammatory reactions [Cheung, 1 9901.

In the late 1960s, blood compatibility studies of blood purification devices focused on blood-

surface interactions associated with coagulation and the elimination of thrombus [Colton et al.,

19941. It is now realized that there are a host of events which are relevant to biocompatibility.

The relevant blood-material interactions are protein adsorption, platelet reactions, intrinsic

coagulation, fibrinolytic activity, erythrocytes, leukocytes, and complement activation [Courtney

et al., 19941. Some of the most important factors deterrnining blood cornpatibility are described

in this section.

2.4.1 Protein Adsorption

As soon as blood contacts an artificial surface, such as a dialysis membrane, the activation of the

coagulation system occurs in just a few seconds [Hakim, 19941. The initial event is the coating

of the surface with a layer of plasma proteins, mainIy fibrinogen from the blood [Baier et al.,

19691. Because of this, it is believed that cells only see the adsorbed protein layer and not the

biomaterial surface [Horbett, 19961. Protein-surface interactions can subsequently influence the

biocompatibility of a material by several different mechanisms. This is by direct initiation of the

intrinsic pathway in the blood coagulation scheme via contact activation, by direct activation of

the alternative complement pathway, or by destabilizing an equilibrium state that creates a

localized depletion of proteins [Hlady, 19931. Either of these pathways may result in thrombus

formation with the entrapment of red cells and fibrin. Continuous growth of thrombus may cause

detachment and further embolisation into the circulatory system. The most frequently studied

protein has been fibrinogen. This is due to the direct link of this protein to platelet activities,

activation of the intrinsic coagulation pathway and interactions with leukocytes [Courtney et al.,

1 9941.

1 ilL U U 3 U l ~ L i W i i u, piv..rriiu -u ..-.--- ------- - - --

The arnount of protein adsorption on surfaces, typically around 1 pg/cm2, is srnall in cornparison

to the bulk concentration present in blood [Horbett, 19861. In typical adsorption studies, the

adsorption reaches a plateau with increasing protein solution concentration, thus achieving an

adsorption isotherm characteristic of a Langmuir relationship (illustrated in Figure 2-4) [Horbett,

19961. The proteins can exist as two populations, a loosely bound, relatively rapid exchanging

portion, and a more tightly bound, slowly or nonexchanging portion [Horbett, 19861. Once on

the surface, the proteins can be enzymatically degraded, replaced by other proteins, or can

undergo conformational changes, and denaturization [Gogolewski, 19891. In this marner, it can

expose functional sites to the blood depending on the characteristics of the blood flow and

composition [Missirlis, 19921.

Figure 2-4: Typical Langmuir Adsorption Isotherm [Horbett, 19961

fi brinogen adsorption W c m 9

protein concentration (mg/mL)

It has been suggested that hydrophobic surfaces interact more strongly to and adsorb more

proteins [Bantjes, 1978; Courtney et ai., 1994; Absolom et al., 19871 than hydrophilic surfaces.

It is also suggested that the adsorption is more reversible on hydrophilic surfaces [Brash, 19911.

On a surface containing hydrophilic and hydrophobic microdomains, it was shown that serum

albumin preferentially adsorbed onto the hydrophilic domains while y-globulin and fibrinogen

- -

shown that hydrophobic surfaces are non-thrombogenic because they preferentially adsorb

albumin which passivates the surface to further interactions with blood components [Lyman et

al., 1974; Freij-Larsson et al., 19931. These and other investigators have shown that the more

hydrophobic surfaces show comparatively low values of adsorption of plasma proteins

[Mandenius et al., 19911. The relationship between protein adhesion and the surface probably

depends more on the interfacial surface tension of the biomaterial in the biological environment,

as suggested by the minimum interfacial free energy hypothesis stated by Andrade et al. [1979

(I)]. It is also suggested that materials show minimum bio-adhesion and thrombus formation, if

the critical surface tension is in the range of 20-30 dynes/cm [Gogolewski, 1989; Baier et al.,

19691.

2.4.2 Blood Component Reactions

The clotting procedure occurs by surface mediated reactions in the intrinsic pathway or through

factors from the tissue in the extrinsic pathway [Hanson et al., 19961. There is a cascade of

reactions involving clotting factors, leading to coagulation as illustrated in Figure 2-5. The

clotting factors are enzymatically activated after contact with the surface, which acts to further

activate other factors, the end result being the formation of significant arnounts of thrombin.

Thrombin acts on fibrinogen to fonn a fibrin gel which acts fùrther with factor XIIIa to form the

fibrin polymer. Once coagulation has been initiated, there are several control mechanisms which

regulate general thrombus formation [Hanson et al., 19961. The fibrinolytic system then removes

unwanted fibrin deposits to facilitate the healing process [Hanson et al., 19961.

[Hanson et al., 19961

INTRlNSlC SYSTEM

Surface Contact

l v Factor XII ---b Xlla 1 +

Factor XI ---b Xla 1 + Fador IX IXa

Ca ++

Ca ++

; Fador Vlll Platelets

. . . . . . . . . . . . . . . . . . . . . . . - . . . . . . Factor X - --

COMMON PATHWAY

EXTRlNSlC SYSTEM

Vlla - - Factor Vll

Ca ++

Tissue i Factor

. . . . . - . . . . . . . . . - - . . . . . . . . . . . . . - . . . - - . - . . . . . . . . . .

., Xa 4 Factor X

, Ca++ ; Factor V i Platelets

Factor XI Il l

When the circulating platelets contact with the adsorbed protein layer of biomaterial surfaces,

they often become activated. Once activated, they change shape, adhere and spread ont0 the

surface. The adhesion of platelets may occur through interactions with glycoprotein Ib (GP Ib) or

connective tissue with von Willebrand factor (vWF) as an important cofactor. As well, adhesion

may be mediated through GP IIbIIIIa, a platelet receptor for plasma proteins such as fibrinogen,

vWF, fibronectin, and vitronectin [Hanson et al., 961. Platelet adhesion leads to the release of

adenosine diphosphate (ADP), small amounts of thrombin, and generation of thromboxane A2.

These function to activate other circulating platelets to adhere to the surface and form platelet

microaggregates by further activating GP IIb/IIIa which binds plasma proteins. Thrombin acts

further by activating more platelets which further produces thrombin, stimulates ADP and

[Hanson et al., 19961. The sequence of platelet reactions and relevant components are illustrated

in the schematic of Figure 2-6.

Figure 2-6: Platelet Reactions Involved in the Formation of Thrombus

[Hanson et al., 19961

The adsorption of proteins to surfaces also promotes the adhesion of leukocytes (which include

neutrophils and monocytes), cells norrnally responsible for fighting bacterial infections

[Vanholder, 19921. These cells can be attracted to thrombus, activate platelets and contribute CO

fibrin formation, thereby effecting fibrinolysis [Courtney et al., 19941. As well, leukocytes bind

to activated platelets via P-selectin [Gemmell et al., 19951. The response of leukocytes have also

been liiiked to complement activation [Courtney et al., 19941, and in some biornaterial

applications, trie aanesion or ieuKuc;ym c;ai I G ~ U I L I I I a 1 ~ 3 3 ul UVLllLY 1 1 5 1 1 L I , I I b ~ C i V I i . r i i vb i ivr

result of platelet activation is the formation of platelet-derived microparticles which have been

shown to involve fibrinogen binding to GPIIbAIIa receptors [Nomura et al., 1992; Gemmell et

al., 19951. It is suggested that the microparticles have a role in normal hemostatic response to

vascular injury by providing a stable procoagulant surface [Gemmell et al., 19951.

2.4.3 Blood Compatible Membranes

The different biological responses, such as coagulation, complement activation and platelet

adhesion, are affected differently by the morphological and chernical nature of the surface such

as hydrophocity, polarity and ionic nature [Wilson, 19861. Surfaces with increased roughness

have been shown to be associated with thrombus formation resulting from a higher degree of

adhesion of fibrin, platelets, and red cells in the contours of the surfaces [Hecker et al., 811. It is

suggested that surfaces with minimal surface interfacial free energy show relevance for improved

blood compatibility [Andrade et al., 1973; Nair et al., 19921. Surface charge has been found to

be an important factor as well [Bantjes, 19781. It is believed that positively charged surfaces are

generally more thrombogenic than negatively charged surfaces. However, not al1 negatively

charged surfaces are non-thrombogenic.

Many studies have related the blood compatibility of surfaces to its wettability and conflicting

results have given rise to much debate between researchers. A study by Brui1 et al [1994],

investigated leukocyte adhesion as a function of wettability on a chemically homologous series of

modified polyurethane surfaces. It was found that adhesion of leukocytes increased on surfaces

which were more hydrophilic. A number of studies have shown that the more hydrophobic

surfaces show low values of adsorption of plasma proteins [Mandenius et al., 19911 and found to

be weaker complement activators [Matsuda et al., 19941. In contrast, studies have also shown

that hydrophobic materials were found to be more platelet activatiiig than hydrophilic materials

[Matsuda et ai., 19941. As well, several studies have shown that hydrophilic surfaces have more

relevance for improving blood compatibility, particularly with the use of PEO segments or

i' V r

segments have been found [Takahara et al., 199 11.

Some commercial polymers have been found to be thrombogenic, including polyvinylchloride.

polytetrafluoroethylene, cellulosics, polyesters, and polyamides [Bantjes, 19781. Due to the low

surface energy inherent in fluoropolymers, fluorinated surfaces have been found to adsorb less

protein, lower the degree of platelet adhesion, and thereby improve blood compatibility [Kaku et

al., 1 994; Ward, 1 989; Clarotti et al., 1 992; Petersen et al., 1 9751. Commercial fluoropolymers

such as TeflonB are difficult to process and have been found to be relatively thrombogenic.

Therefore, for biomaterial surfaces, the focus has been to modiQ the surfaces with fluorocarbon

coatings [Clarotti et al., 19921 or fluorinated copolyrner additives [Kaku et al., 19941. Other

studies have incorporated fluorinated segments into the base polyrner to effect the reduction in

surface energy, for example fluorinated polyurethanes [Sbarbati et al., 19941.

There are a number of studies showing improved blood cornpatibility of surfaces through the use

of arnphiphilic additives or copolymers [Brundstedt et al., 1993; Ishihara at al., 1996; Ward,

1989; Okano et al., 1981; Wesslen, 19941. In these studies, some researchers believe that the

improved blood compatibility is due to the orientation of the hydrophilic portion, for example

hydrophilic PEG or PEO segments, of the additive at the surface [Brundstedt et al., 1993;

Ishihara et al., 1996; Okano et al., 1981; Wesslen, 19941. Other groups believe that the

orientation of hydrophobic components, specifically fluorinated endgroups [Ward, 1989; Kaku et

al., 19941 and poly(dimethy1siloxane)-based segments [Takahara et al., 19911, at the surface is

responsible for the reduced protein adsorption. Some synthetic polymers which exhibit

microphase separat ion with a good balance of hydrophilic and hydrophobic microdomains have

shown good blood compatibility with regard to in vitro platelet adhesion and aggregation [Okano

et al, 19811 and in vivo antithrombogenicity [Okano et al, 19861. These studies emphasize the

importance of balancing the degree of hydrophilicity/hydrophobicity in polymeric surfaces to

induce blood compatibility.

part due to a lack of information. Many researchers have not accounted for other factors which

may play a role in blood interaction and events, for example, surface charge, polarity, surface

roughness, surface heterogeneity, and mobility of surface molecules [Missirlis, l!WZ]. In most

cases, only one or two of these factors have been considered. The blood compatibility of a

surface cannot be defined by any one parameter such as wettability. More recent studies have

shown that a balance between hydrophilicity and hydrophobicity is actually required for a blood

compatible surface pa ir et al., 19921. The extent of the literature and the discussion above is

proof that other factors must be considered.

3.1 SYNTHESIS OF SURFACE MODIFYING MACROMOLECULES

3.1.1 Materials Used for SMM Synthesis

In this work, SMMs of differing chemistries were synthesized in order to study the influence of

SMM composition on its miscibility in PES and their effects on membrane surface properties.

The materials used for polymer synthesis are given in Table 3-1 and supplier narnes of al1

chemicals used in this study are listed in Appendix A. Because diisocyanates readily react with

water to form diamines [Gogolewski, 19891, it was important to distill or degas the reagents prior

to their use. This removed trace amounts of moisture and impurities. The apparatus used for

distillation and degassing of chemicals and solvents is shown in Appendix B.

N,N-Dimethylacetarnide (DMAC) solvent was distilled with low heat under a pressure of 1.0

torr, the day prior to polymer synthesis. The diisocyanate used for the synthesis of al1 SMMs in

this project was methylene bis-p-phenyl diisocyanate (MDI), which was distilled at 150°C under

0.5 torr to separate dimerized products and commercial impurities. Two different polyols were

used, polypropylene di01 (PPO) and polycaprolactone di01 (PCL). Both reagents were degassed

overnight at room temperature under a pressure of 1 .O torr. As illustrated in Table 3-1, PCL is a

polyester with six carbons and one ester link per repeat unit, while PPO is a polyether with three

carbons and one ether link per repeat unit. The relative solubility of the two oligomeric diols

were deterrnined by dissolving each in water at 50 O C . It was observed that PPO was more

miscible in water than PCL of similar molecular weight, suggesting PPO to be more hydrophilic.

The oligomeric fluoro-alcohols used were commercial products of BA-L and FSO-100. As

indicated by their chernical structures in Table 3-1, the molecular weight distribution of these

compounds may Vary, giving a range in fluorine content. For this work, only the low molecular

weight fraction of BA-L was used. In a study on the reproducibility of SMMs, those synthesized

with BA-L low gave a lower overall variation in characteristics such as molecular weights, final

chain fluorocarbon with an ethanol end group. FSO-100 is similar in structure with a short chain

fluorocarbon, but is a larger molecule containing polyethylene oxide (PEO) segments. The FSO-

100 fluoro-alchohol was distilled into three molecular weight fractions, low, intemediate, and

high, by varying the distillation conditions. The stock FSO-100 was distilled to obtain the

intermediate molecular weight distillate, which included both FSOl and FS02. This product

was then further distilled to obtain the low molecular weight distillate, FSO1. The difference

between the three different fractions of FSO (1, 2 and 3) is believed to be a corresponding

increase in PEO segments. Details of the conditions and method for separating fractions of the

fluoro-alcohols are given elsewhere [Pham, 1 995; Tang, 1995; Weiler, 19971.

Table 3-1: Materials for SMM Synthesis

Reagen t

Solvent:

N, N-Dimethylacetamide

Diisocyanate:

methylene bis-p-phenyl di isocyanate

Polyol:

polypropylene diol

polycaprolactone diol'

Oligomeric fluoroalcohol:

BAL low

FSOlOO low FSO 1 O0 intermediate FS0100 high

Chemical Structure

C l-1, I

HO-(C k12CHO)n-I-I

Molecular Weight

- 443

average 73

Acronym

DMAC

PPO

PCL

BAL

FSO l FS02 FSO?

' The chemical structure of PCL is taken from [Wang, 971.

The SMMs were synthesized using a two step solution polymerization method as illustrated in

Figure 3-1 below. The initial step involved the reaction of diisocyanate with polyol in a common

solvent of DMAC. This mixture formed a urethane prepolymer solution. The reaction was then

terrninated by the addition of oligomeric fluoro-alcohol resulting in a solution of surface

modifiing macromolecules.

Figure 3-1: Schematic of Two Step Synthesis of Surface Modifying Macromolecule

3 diisocyanates + 2 polyols

dimethylacetarnide 45 - 50 O C 3 hours

urethane prepolymer

2 oligomeric fluoro-alcohols 1 21 OC 24 hours

surface modifiing macromolecule

The apparatus used for SMM synthesis is illustrated in Appendix C. To eliminate the effects of

moisture, al1 glassware was dried overnight at 100 O C , and the polymerization reaction and

endcapping steps were performed in a controlled atmosphere of prepurified nitrogen (grade 4.8,

BOC Canada Ltd., Etobicoke) inside a glove box. The molar ratio of reagents

MDI:polyol:fluoro-alcohol was 3:2:2 and scaled to a mass of 7.5 g of MD1 for al1 SMMs. 'The

initial reaction began by dissolving 0.03 moles (7.5 g) of MD1 in 50 mL of DMAC and 0.02

moles of polyol in 100 mL of DMAC, and mixing both in a 1 L PyrexTM reaction kettle. A

temperature/stirrer controller (Series 400HPS, VWR Scientific, Canada) was used to maintain a

stir rate of 175 rpm and a temperature range of 45 - 50 OC. This was allowed to react for 3 hours

after which time, the reaction was terminated by the addition of 0.02 moles of fluoro-alcohol

OC, resulting in a solution of surface modifiing macromolecules.

The solution of SMMs was precipitated dropwise into a four litre beaker of stirring distilled

water. Some SMMs were elastomeric and able to be cut into smaller pieces while several of the

SMMs were in the form of a tacky powder. The poIymer was lefi stirring in distilled water for 24

hours in order to leach out residual solvent. The washing solution was changed to 30% acetone

for another 24 hour period to further remove residual unreacted monomers (specifically fluoro-

alcohols). The final polymer was then placed in an aluminum dish and dried in an air drying

oven at 50°C for 5 days. The SMMs were stored in either brown glass bottles or aluminum

dishes, and placed in a dessicator.

The SMMs are named by the nomenclature of the reactants used to synthesize them. The

reactions for the synthesis of MDI-PPO-BAL is detailed in Figure 3-2 as an example of the

chemistry involved in SMM synthesis. For a 3:2:2 stoichiometric ratio of MDI:PPO:BAL, the

values for m in the SMM molecular structure would be 2, and the final SMM polymer structure

would be in the form:

BAL - MD1 - PPO - MD1 - PPO - MD1 - BAL (3-1)

Although this is the theoretical structure based on stiochiometric ratios, the prepolymer chain

lengths do Vary, resulting in a distribution of molecular weights.

Figure 3-2: Reactions in the Synthesis of MDI-PPO-BAL

2 PPO

I DMAC 45-50 OC 3 hours

urethane prepolymer

I

2 BAL

25 O C

24 hours

surface modi@ing macromolecule

3.2.1 Elemental Analysis

Synthesized SMMs were sent to Guelph Chemical Laboratories, Guelph, Ontario, for elemental

analysis of nitrogen, carbon, hydrogen and fluorine. The procedures used for these tests are

detailed elsewhere [Tang, 1 9951.

3.2.2 Molecular Weight Determination by Gel Permeation Chromatography

Many properties of polymeric materials, such as strength and processability, depend on their

molecular weights. The most common method of detemining polymer molecular weight is size

exclusion chromatography (SEC), also known more descriptively as gel permeation

chromatography (GPC). The instrumentation of a GPC unit consists of a series of columns

packed with particles of a porous substrate. With the injection of a polymer sample, a solvent

carries the sample into the columns at a constant flowrate. The smaller molecules diffüse in and

out of the porous substrate easier than larger molecules, and therefore have a longer retention

time. This mode of separation allows for the detemination of the molecular weight distribution

based on size. Details of GPC instrumentation and methodology can be found in several

literature references [Young et al., 1991 ; Rosen, 19931. A schematic of the GPC apparatus used

in this study (including mode1 numbers and supplier names) is detailed in Appendix D. Using

MilleniumTM Software (Waters Chromatography, Milford, U.S.), the GPC unit is interfaced with

a cornputer for data acquisition and analysis.

The mobile phase was made by dissolving lithium bromide (LiBr) in HPLC grade N,N,-

dimethylformarnide (DMF) to a concentration of 0.05 M. This was prepared under a nitrogen

atmosphere inside a glove box to avoid moisture adsorption by hygroscopic LiBr and DMF, and

to minimize the interference of water/LiBr peaks in the SMM chromatograms. Prior to use, the

solution was filtered using vacuum filtration through a 0.45 pm TeflonO membrane (Millipore

LOF., uearora, u.3.). 1 rit: uuwrait: was sel ai 1 .v r r i u r r i i i i iüiu iiic; c;uiuriiri Lciiipciiiiurt: SGL ai OU

OC. SMM samples were dried overnight at 30°C under vacuum and further dissolved in mobile

phase at a concentration of 2 mg/mL. The solutions were then filtered through a 0.5 pm TeflonB

filter (Millipore Corp., Bedford, U.S.). To relate retention volumes to molecular weights, a

direct standard calibration curve was used. Polystyrene polymer standards (Tosoh Corp., Tokyo,

Japan) with narrow molecular weight distributions were analyzed in the GPC unit. The

molecular weight of these standards are known and were used to relate retention volumes

associated with the system.

3.2.3 Differential Scanning Calorimetry

The thermal transition properties of SMMs and PES membranes were studied by differential

scanning calorimetry (DSC). This technique provides quantitative and qualitative information on

the changes in thermal transitions associated with endothermic and exothermic heat flow. A

heating block is used to raise and lower the temperature of the sarnple linearly while the area

themiocouples are used to monitor the resultant differential heat flow between the sample and

reference cells. Studies were perforrned at the Brockhouse Institute for Materials Research,

McMaster University, Hamilton, ernploying a 2910 Differential Scanning Calorimeter (TA

Instruments Inc., New Castle, U.S.).

Thin films of SMM and PES materials (pure and SMM modified) were cast from 10 mL of

polymeric solutions. PES powder (Vitrex 4800P, ICI Chemicals) was dried and used without

further purification. SMM modified solutions were prepared by dissolving 10 weight percent

(wt%) SMM in DMAC, and PES solutions were prepared by dissolving 3 wt%

polyvinylpyrrolidone (PVP), of molecular weight 10,000, and 10 wt% PES in DMAC. PVP is

ofien added during the fabrication of PES membranes as a nonsolvent swelling agent to improve

pore size and pore size distribution [Lafreniere et al., 1987; Cabasso et al., 1 9761. PVP can also

act as a polymeric compatibilizer to increase the miscibility of the SMM-PES blend. For SMM

modified PES solutions, 4 wt% SMM:PES was also added. The solutions were filtered through a

"-4 A.,""."- ""'̂ '- ------ - - ---- r------- --- - m m - - - - - - . - - =--,'-----y ---- r- -----

aluminum plates. The solvent was evaporated in a 50°C air drying oven for 48 hours and further

dried in a vacuum oven for 48 hours.

Samples of cast films were cut into 8-10 mg pieces and sealed in small aluminum pans (TA

Instruments, New Castle). For analyses of SMMs, the sarnples were equilibrated to -50 OC using

liquid nitrogen and further heated at a rate of 15 "C/min. up to 60 OC. PES membrane samples

were equilibrated to -50 OC, further heated at a rate of 15 OC/min. up to 250 OC, held isothermal

at this temperature for 10 minutes, and then quickly quenched in methanol cooled with dry ice.

This provided a first scan of the thermal behaviour of the polymer. By heating the polymer to a

high temperature and holding the polymer isothermal, the processing history of the polyrner is

removed. By quenching the sarnple immediately in methanol, the rapid cooling provides a

thermodynarnic driving force for phase separation in the polymer blend [Hesketh et al., 19801. A

second thermal scan of the polymer in this state may reveal the effects of the additive in the base

material. To obtain a second scan, the PES samples were again equilibrated to -50 OC and heated

at a rate of 15 "C/min. up to 250 OC.

3.3 SURFACE CHARACTERIZATION OF PES MEMBRANES

3.3.1 Preparation of PES Flat Sheet Membranes

To characterize surface properties, thin films of PES and SMM modified PES membranes were

cast fiom polymeric solutions ont0 glass substrates. The polymer solutions were prepared by

dissolving 25 wt% PES and 3 wt% PVP, in DMAC. For casting SMM modified membranes, 4

wt% SMM (relative to PES) was also added to the solution. The solutions were allowed to stir

for 4-5 days and filtered through a 0.5 pm TeflonO filter at a pressure of 70 psi. Before casting,

the glass slides were soaked in chromic acid for 24 hours, rinsed thoroughly with distilled water,

and placed in a 100 O C oven for complete drying. ?'O cast a PES flat sheet membrane, a g las

slide was dipped into the polymer solution and hung for curing in a 110 OC oven for 8 minutes.

aiuminum foil, to remove residual solvent. Analysis was performed within two days of san~ple

preparation.

3.3.2 Characterization of PES Membrane Morphology

Several microscopy techniques were used to study the polymeric rnorphology of PES and SMM

modified PES flat sheet membranes. Al1 membranes used in these studies were cast according to

the procedure described in Section 3.3.1.

3.3.2.1 Polarized Microscopy

In one technique, the membranes were investigated by polarized light microscopy. This was

performed at the Faculty of Dentistry, University of Toronto, using an Orthoplan light

microscope (Leitz Wetzlar, Gerrnany) equipped with a 35 mm carnera. A rotating polarizer

selectively transmits light polarized in one specific plane and the interaction of the polymeric

materials with this polarized light provides a contrasted image [Sawyer et al., 19961.

3.3.2.2 Confocal Microscopy

In conventional light microscopes, the depth of field at high power is approximately 3 prn and

therefore structures less than this are often superimposed in the images [Herrnan et al., 19931.

An alternative technique is confocal microscopy which has a depth of field less than 1 pm, and

can image thin cross sections of micro-structures in a sarnple at different depths [Herman et al.,

19933. Confocal microscopy was used to determine the smoothness of the flat sheet membranes

underwater and to study the interna1 morphology of SMM modified PES membranes.

Experiments were performed on an MRC-600 Laser Scanning confocal microscope (Biorad

Laboratories, CA., U.S.) at the Ontario Laser and Lightwave Research Centre, University of

Toronto.

rn me unuerwaLer s~uuy, rriciiiuiiuic iiriiis uii giilss S I I U C ~ WCIG L ~ ~ ~ L U C L S C U 111 a ~ C L I I U1311 VI

distilled water and hydrated for 5 minutes. Undenvater, the membranes show small bumps at the

surface. To study these structures, the confocal microscope was used in reflectance mode with

an eye objective of 10x and an undenvater objective of 4Ox. The interference resulting fiom the

reflected light impinging ont0 the membrane surface morphology produced optical rings. Using

equation 3-2 [Hemsley, 19891, the number of rings may be related to the height of the area under

study.

nurnber of optical rings x wavelength of reflected light Height = (3 -212

refractive index of water

By this method, the amplitude of surface roughness can be estimated. In a separate study, dry

SMM modified PES membranes were studied in reflectance mode at varying magnifications.

Each interna1 layer of the membrane was analyzed by vertically advancing the stage in 1.2 pm

steps. This provided images of the intemal morphology and the distribution of phase separated

microdomains in both lateral and vertical planes.

3.3.2.3 Atomic Force Microscopy

The surface topography of the membranes was also studied using atomic force microscopy

(AFM) on a Nanoscope III (Digital Instruments, Santa Barbara, U.S.) located at the Department

of Chemistry, University of Toronto. In this technique, a probe is mounted on a thin cantilever

which drags over the surface of interest. The cantilever is especially sensitive to deflections, at

less than 10 nN, caused by the surface terrain and is therefore a high resolution technique

[Sawyer et al., 19961. Glass slides were cut into 1 cm2 pieces, soaked in chromic acid overnight,

and used for casting PES membranes following the procedure described in Section 3.3.1.

Membranes cast on glass slides were mounted inside a charnber and analyzed in contact mode (in

which the probe contacts the sample) by scanning the surface multiple times until a desired

resolution was achieved.

-

This equation accounts for microscopy studies performed with the undenvater objective lens subrnersed. Therefore, light is passing through a medium of water with a refractive index of 1.3.

In order to confirrn that the surfaces of the SMM modified membranes are enriched with fluorine

groups, the elemental composition at the surface of cast films were determined by X-ray

photoelectron spectroscopy (XPS). This method is widely used to provide quantitative and

qualitative chernical information of the top 10-200 A of surfaces which are stable in a vacuum

[Andrade, 19851. In this method, photons from an X-ray source are directed at the sample at an

angle depending on what depth from the surface is to be analyzed. The photons may interact

with atomic orbital electrons of elements at the surface and transfer the photon's energy to the

electron, ejecting them with a kinetic energy that is measured by the instrument. The kinetic

energies are then related to binding energies characteristic of specific atoms. A schematic of the

basic components of an XPS spectrophotometer is illustrated in Appendix E. XPS studies were

performed at the Centre for Biomaterials, University of Toronto using a Leybold Max 200 X-ray

photoelectron spectrophotometer. The X-ray photons were generated by a magnesium K a

source.

PES and PES membranes modified with SMMs containing PPO sofi segments were analyzed by

XPS. The film sarnples were prepared fiom 5 mL of polymeric solutions by the casting method

used for DSC studies (Section 3.2.3). For XPS analysis, it was important to avoid any

contamination of the film surfaces and therefore samples were covered by KimwipesTM

throughout the drying process. The day prior to analysis, the films were cleaned with 1,1,2

trichlorotrifluoroethane (TCTFE) to remove trace contarninants and dried at 50 OC under

vacuum. Two sarnples were cut from each film and analyzed at take off angles of 15" and 90".

The take off angle is the angle between the surface and electron patli to the detector. By varying

this angle, the electron will travel different depths in the surface and therefore, at low take off

angles, the depth of surface analyzed is smaller [Andrade, 19851. The spectra of photoelectron

intensity versus binding energy were processed using Matlab Software Ver. 4.2b and Escatools

Software (Surface Interface Inc., Mountain View, CA, U.S.).

3.3.4.1 Apparatus for Air Contact Angle Studies

The apparatus used for contact angle studies is commonly known as a goniorneter (Rame-Hart

Inc., New Jersey, U.S.) as illustrated in Figure 3-3. A micro-syringe introduces HPLC grade

ultrapure water ont0 the surface and an enlarged image is observed through a microscope. The

contact angle is defined as the angle created between the liquid-solid interface and the liquid-air

interface. Measurements were taken by adjusting two hair lines on the microscope objective and

reading angles off a marked scale. The volume of the liquid droplet is slowly increased until the

three phase line moves and the drop takes on a shape that will minimize the surface fiee energy

of the system. The contact angle at this point is called the advancing contact angle. If the surface

is hydrophilic and the probing liquid is water, there will be a strong interaction between the

surface and water droplet. The liquid will be drawn towards the surface and the contact angle

will be low. Inversely, hydrophobic surfaces will repel the water droplet and give high contact

angles.

Figure 3-3: Apparatus for Air Contact Angle Studies

n microsyringe -

light source I I i u A microscope 1 ()

( air ( liquid /'

V

nleasured by slowly withdrawing the water drop until the three phase line moves. The receding

angle is considered to represent the high energy surface cornponent. The difference between the

advancing and receding contact angles is the contact angle hysteresis which gives an indication

of the heterogeneity of the surface. Ideal homogeneous systerns will show low contact angle

hysteresis and heterogeneous surfaces will show high contact angle hysteresis. For each surface

modification, three surfaces were analyzed, each with five liquid drops on different areas of the

mernbïane. The contact angle for that surface was taken as the average of the five measurements,

and the final contact angle values for each type of surface was taken as the average of the three

membranes.

3.3.4.2 Apparatus For Underwater Contact Angle Studies

It was observed in early experiments that the PES membranes would readily be displaced from

glass when placed in an aqueous environment. This was believed to result from the penetration

of water into the membrane pores which subsequently lifted the membranes fiom the glass

surface. To prevent this fiom occurring, the edges of the glass slides were etched on both sides

using a glass cutter. This allowed polymer solution to enter the cracks of the etched surface

during membrane casting and therefore hold the membrane in place.

To facilitate underwater contact angle studies, a 0.7 mm diameter hole was carefully drilled in

the centre of the glass slide using a precision drill press. Afier the membranes had been cured

and dried ont0 the g l a s substrates, a needle was used to remove the polymer that had plugged the

hole on the cast slides.

The undenvater contact angle study was performed at the Laboratory of Applied Surface

Thermodynamics (directed by Dr. A.W. Neumann), Department of Mechanical Engineering,

University of Toronto, and the apparatus is illustrated in Figure 3-4. The studies were performed

in a clean room, and the apparatus placed on an air table to reduce the effects of vibrations on the

I - created inside a glass cuvette. The cast sarnpIes were held undenvater with a standing vice. In

this set up, air was introduced as the second phase through the hole in the middle of the slide. To

minimize the effects of vibrations due to the operation of the syringe, TeflonB tubing was used

as a needle to connect the syringe and hole of the sample slide by fitting the tube inside the hole.

Because the air bubble is introduced fiom the top surface of the slide, through the hole, the

TeflonB needle did not interfere with the shape the air bubble as the system reached equilibrium.

A motorized syringe equipped with a speed controller was used to deliver air flow at a constant

rate. The computer was programmed to simultaneously take a picture of the image every second

and store it in memory. These images of the air bubble were observed through a high resolution

microscope and scanned into a digitizer. The digitized images were then entered into a SUN

Workstation computer, as well as being displayed on a monitor.

Figure 3-4: Apparatus for Underwater Contact Angle Studies

motorized syringe

TeflonB needle

controller 1-1 diçplay

A monitor

cornputer

linuid ( air \

- -

and the air-liquid interface. A more hydrophobic surface would tend to draw the air bubble

closer to the surface and result in a lower contact angle, while a high contact angle would

indicate a more hydrophilic surface. For undenvater contact angle measurements, the two angles

of interest will be referred to as the air advancing angle and the air receding angle.

The air bubble images were analysed using a computer program called Axisymmetric Drop

Shape Analysis - Profile (ADSA-P) developed by Cheng et al. [1990]. This is an automated

experimental technique written in C computer language for determining liquid-fluid interfacial

tensions and contact angles from the shape of the axisymmetric menisci. The computer program

fits the measured profile of a drop to a cuve given by the Laplace equation of capillarity (Section

2.3.3). This is done by forming an objective function between the deviation of the experimental

and theoretical profiles. It then perforrns non-linear regression analysis (a non-linear least

squares fit and Newton Raphson method) to minimize the objective function and yield a

theoretical contact angle. The program is able to calculate and report the interfacial tension,

contact angle, drop volume, surface area, radius of curvature, and contact radius of the drop.

3.4 BLOOD COMPATIBILITY STUDIES OF HOLLOW FIBRE MEMBRANES

3.4.1 Fabrication of Hollow Fibre Membranes

Hollow fibre membranes have been popular for several separation technologies including reverse

osmosis, ultrafiltration, pervaporation, and dialysis [Liu et al., 199 I l . In this work, PES hollow

fibres were used for blood compatibility studies because they better reflect the form of membrane

materials used in biomedical applications such as haemodialysis and oxygenator systems. As

well, the blood evaluation studies in this thesis utilizes apparatus originally developed to

accommodate tubular forms. Hollow fibre membranes were fabricated by a solution spinning

process [Liu et al., 1991; Ma et al., 19951 performed at the Industrial Membrane Research

Institute (directed by Dr. T. Matsuura), University of Ottawa. Polymer solutions were prepared

-

and modified membranes respectively), in DMAC. The total mass of the solutions was 150 g.

After stirring for five days, the solutions were filtered through a 5 pm TeflonB filter (Millipore

Corp., Bedford, U.S.) at 20 psi to remove impurities and polymer gels.

The apparatus used to spin the hollow fibres is shown in Appendix F. The polymer solution was

housed in an air tight reservoir overnight to settle out any air bubbles which would cause defects

in the membranes. A nitrogen pressure of 1-2 psi was applied to force the polyrner solution

through a bal1 valve and into a spimeret. Simultaneously, an internal precipitant of distilled

water was introduced through the central tube of the spinneret at a controlled flowrate by a

motorized syringe. The spinneret has a tube-in-orifice structure (Appendix F) such that the water

passes through the tube, which passes through the polymer solution in the orifice. Therefore, the

configuration that exits the spinneret is one of a polymeric hollow tube containing water. This

polymeric fibre was spun into a large cylindrical gelation bath of distilled water. The dimensions

of the hollow fibre can be controlled by altering the rate of delivery for the polymer solution or

internal precipitant, or by adjusting the air gap between the spinneret and gelation bath. The

hollow fibre was stored in distilled water for three days, changing the washing solution each day

to leach out residual solvent. They were then cut into 30 cm pieces and stored in a solution of

30% ethanol to prevent microbial growth.

S c m i n g electron microscopy (SEM) was used to provide images of the surfaces of PES and

SMM modified PES flat sheet membranes. This technique can provide high resolution images

with a three dimensional perspective. Prior to analysis, the membrane samples were sputter

coated with 3 nrn of platinum using an SC515 SEM Coating Unit (Polaron Equipment Ltd.,

England). In the SEM technique, a high energy electron bearn is applied on the sample and low-

energy secondary electrons frorn the surface of the material are emitted. The intensity of the

emission is detected on a phosphor screen and is a function of the atomic composition [Ratner et

al., 19961. The measured intensity is then reconstructed into the image of the surface. Scanning

electron rnicroscopy was performed at the Faculty of Dentistry, University of Toronto, using an

Hitachi S 2500 scanning electron microscope (Hitachi LTD, Mito City, Japan).

The biood compatibility of PES hollow fibre membranes was assessed with respect to protein

adsorption using a radiotracer technique [Horbett, 19861. Although there are numerous methods

available for studying protein adsorption, the radiotracer technique is the method of choice

because it offers specificity, flexibility, and sensitivity [Horbett, 19861. Fibrinogen adsorption

studies were performed in Dr. John Brash's laboratory at the Department of Pathology,

McMaster University, Hamilton. On the day prior to the experiment, 0.05 M phosphate buffered

saline (PBS), pH 7.4, was made with MilliQTM filtered water, sealed, and stored cold. The

hollow fibres were cut into 8 mm pieces, rinsed five times in distilled water to leach out residual

ethanol, and stored in PBS buffer overnight.

In order to monitor the amount of fibrinogen adsorbed ont0 the hollow fibres, 2 % of the total

fibrinogen in solution was radiolabeled with 12'1 as the radioactive tracer. The method of

iodination was done by covalently bonding [Horbett, 19861 10 mglm1 hurnan plasma fibrinogen

(95% pure, Calbiochem, CA, U.S.) with 5 pL of N ~ ~ ~ ~ I (0.5 mCi) in the presence of Iodo-

BeadsB (Pierce Chemical Comp., Rockford, U.S.). The solution was dialyzed in a dialysis

cassette (Pierce Chemical Comp.) by stirring in 500 mL of PBS buffer in order to remove

unbound 123. The buffer solution was replaced three times over a period of at least 2 hours. The

extracted radiolabelled fibrinogen solution was diluted with unlabeled fibrinogen to make up the

rest of the 98 % stock protein solution used in the experiment. The final protein concentration

was measured by absorbance at 280 nrn with an extinction coefficient of 1.5 OD/mg/ml for

fibrinogen [Horbett, 19861. By precipitating the fibrinogen of a small sarnple of the stock

solution, the supernatant from this procedure was counted in a gamma counter to confirm the free

iodide concentration to be less than 1 %. Five different concentrations were prepared by a series

of dilutions (detailed in Appendix G) of the original stock fibrinogen solution with PBS. Small

samples of these latter solutions were further diluted (1 : 100) and placed in a gamma counter to

determine the radioactivity (counts per minute) corresponding to that concentration.

- -

each of the five concentrations of radiolabeled fibrinogen. Using tweezers. the 8 mm fibres,

hydrated with PBS buffer, were gently immersed in 250 pL of fibrinogen solution housed in 96

well polystyrene tissue culture plates, starting from the lowest fibrinogen concentration to the

highest. Afier a three hour adsorption period, the fibres were removed from the solution and

placed vertically on KimwipesTM to allow any excess solution to "wick" out of the hollow tube

and ont0 the tissue. Each fibre was then rinsed three times in PBS buffer to carefully remove al1

but the adsorbed proteins, and wicked again to remove any excess solution. The hollow fibre

was then placed in a counting via1 and monitored for radioactivity.

3.4.3 Whole Blood Studies for Determining Platelet and Leukocyte Activation

A preliminary study on the ability of the PES and SMM modified PES hollow fibre membranes

to activate platelets and leukocytes was conducted at the Canadian Red Cross, in conjunction

with Dr. M. Sefton's laboratory in the Department of ChemicaI Engineering and Applied

Chemistry, University of Toronto. This was done by contacting the membranes with whole

blood in vitro and assessing platelet and leukocyte activation using fluorescence activated flow

cytometry (FAFC). By FAFC, a number of activation events can be identified. These include a-

granule release (P-selectin expression), formation of platelet-derived microparticles, and

activated platelets binding to leukocytes as indicated by the up-regulation of antibody CD1 1 b.

3.4.3.1 Blood-Materiai Contact

The hollow fibres synthesized in Section 3.4.1 were cut into 15 cm pieces and each were inserted

into 55 cm lengths of polydimethyl siloxane (SilasticO, Dow Corning, Midland, U.S.) tubing of

0.78 in. inner diarneter. By injecting a 30% ethanol solution through the Silastic@, the flow of

the solution was used to carry the hollow fibres to the centre portion of the larger tubing. An

empty 55 cm SilasticO tubing was used as a negative control sarnple and an empty 5 5 cm

polyethylene ( P E ) tubing was used as a positive control. The fibres were further rinsed with 60

with the latter solution and incubated at 37 OC for 5 minutes.

Afier discarding the first millilitre, blood was collected fiom healthy volunteers into a syringe

preloaded with heparin anticoagulant (5 units/mL) and immediately used for experiments. For

each experiment, the negative controls included a resting blood sarnpie (500 yL of blood in a

sealed microcentrifuge tube) and an ethylenediaminetetraacetic acid (EDTA) blood sarnple (500

pL of blood with 10 pL of 200 mM EDTA, in a sealed microcentrifuge tube). EDTA is an

anticoagulant.

A rocking platform apparatus illustrated in Figure 3-5 was used to facilitate contact of the blood

with the membrane materials. This assembly was designed to simulate a near-physiological

systern for contacting materials and blood at low shear rates (less than 25 sec-') [Gemmell et al.,

19951. The test materials were gravity drained of the NaCl solution and the tube attached to one

ami of the rocking platfonn apparatus. At the loose end, 500 pL of blood was injected into the

SilasticB tubing using a 1 cc syringe. The loose end was then attached to the rocking platform

(as in Figure 3-5) and gently rocked for one hour in a 37 OC room.

At the end of the experiment, the blood from each test material was drained into separate

microcentrifuge tubes. The materials were then rinsed with 250 pL of HEPES-Tyrode's buffer

(HTB) into the respective microtubes. 250 pL of HTB was added to the resting and EDTA blood

samples. At room temperature, 10 pL of 200 mM EDTA was added to the blood samples as weIi

as the resting sarnples to allow for platelet counts and flow cytometric analysis of platelet

activation. For FAFC analysis of leukocyte activity, the blood samples were analyzed without

EDTA.

- -O--- - - - - Low Shear Conditions - Adapted frorn [~emrnel l et al., 19951

pipette tip

Silastic 43 tubing -

whole blood - -

hollow fibre membrane - -

Silastic 43 negative control.---

PE positive control

v rocking platform . .- - - -

3.4.3.2 Fluorescence Activated Flow Cytometry (FAFC)

Flow cytometry test tubes were prepared to receive blood samples to facilitate leukocyte and

platelet analysis. For the positive control of platelet activation, 50 p L of resting blood was

incubated with 5 pL of 4 mM ser-phenyl-leu-leu-arg-asp thrombin peptide (SFLLRN) for 15 min

at room temperature in a sealed microtube. Then 5 pL of this activated blood was added to the

receiving flow cytometry tube which also contain 5 pL of SFLLRN and the respective antibodies.

The tubes for analyzing test sarnples were prepared with 50 pL of HTB, 5 pL of fluorescein

isothiocyanate (F1TC)-CD61 (a IIb/IIIa), and 3 pL of phycoerythrin (PE)-CD62P solution

(dilution 1 : 10) (a P-sel). 20 pL of blood samples from the experiment was added to the prepared

flow cytometry tubes and incubated for 20 minutes at room temperature. Afier this time, the

solutions were diluted and fixed with 150 pL of HTB and 150 pL of 2% paraformaldehyde, and

stored at 4 OC until analysis.

por leuKocyre activation, int: pusiiivt- LUIILIUI S ~ I I I ~ I G wa3 ~ I G ~ Q I C U L u l l L a l l l l l i g LU PL U L L . L U

dilution of stock phorbal 12-myristate 13-acetate(PMA) solution and 20 pL of resting blood.

PMA activates leukocytes and therefore provides an activated positive control. The tubes for

analyzing test sarnples were prepared with 50 pL of HTB and 20 pL of FITC-CD1 1 b. 20 pL of

each biood sarnple from the experiment was added to the prepared flow cytometry tubes and

incubated for 20 minutes at room temperature. Afier this tirne, 500 PL of FACS lysis solution

was added and the solution was vortexed at medium speed. This solution was then incubated for

15 minutes at room temperature in the dark. Afier this time, the solution was centrifûged for 1.5

minutes at 1300 rpm. The solution was aspirated up to the line of the solid pellet, leaving 50 pL

of the solution. The solid was resuspended in 500 pL of HTB, centrifuged, and aspirated again.

The solid was resuspended in 50 pL of HTB and 50 pL of 2% paraformaldehyde. This was

stored at 4 OC until analysis.

The sarnples were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View,

US.). In flow cytometry, cells are delivered in a single file past a point of measurement at which

light is focused (usually by using a laser) [Ormerod, 19941. Several pararneters can be

deterrnined by implementing different optical filters to apply varying colour light excitations, at

different angles. As the blood sarnple is passed through a flow cell, their fluorescence or light

scattering is measured by a detector, and their fluorescence intensities are reported. Analysis of

the results are perfomed on a cornputer. The data from one or two pararneters are displayed, and

gates are used around the regions of interest, which are defined for certain types of events. The

method of analysis for activation markers including P-selectin expression, percent microparticles,

and up-regulation of leukocyte CD1 lb are detailed in the literature [Gemmell et al., 1995;

Gemmell et al., l996].

3.4.3.3 Ce11 Count

Without further treatment, the remaining blood samples from the blood contacting experiment

were injected into a multipararneter, automated Sysmex E-2500 hematology analyzer to record

red blood cell, white blood cell, and platelet counts in the bulk.

4.1 SYNTHESIZED SURFACE MODIFYING MACROMOLECULES

In this study, eight SMMs with varying reactants were synthesized and studied for their effects on

polyethersulfone membrane surfaces. The SMMs can be divided into two series, the first series

is four SMMs containing PPO as the sofl segment in the urethane prepolymer and the second is

four synthesized with PCL as the soft segment. For each series, four different fluoro-alcohols

were used as the endcapping reagent, BAL, FSOI, FS02, and FS03. The differences between

these fluoro-compounds were highlighted in the previous chapter (Table 3-1). The physical

appearances of the processed SMM polymers are described in Table 4-1. The properties of

SMMs containing FS03 are notably different fiom those containing the lower molecular weight

FSO fractions and even BAL. The former tended to be more tacky or elastomeric than the latter

which were more crystalline in nature. As well, SMMs containing PPO as the soft segment

tended to be more tacky and difficult to process than those containing PCL.

Table 4-1: Physical Appearances of SMMs in the Final Dry Processed Form

--

MDI-PPO-BAL 1 light yellow, crystalline ' polymer

SMM Polymer Physical Appearance

' Here, crystalline is a descriptive terrn for the appearance of the polyrner and does not refer to the ability of the polyrner to crystallize.

MDI-PPO-FSO 1

MDI-PPO-FS02

MDI-PPO-FS03

MDI-PCL-BAL

MDI-PCL-FSO 1

MDI-PCL-FSO2

MDI-PCL-FS03

light yellow, crystalline polymer

yellow crystalline polymer, slightly tacky

dark brown, tacky polymer

white, powdery polymer

yellow, crystalline polymer

yellow, crystalline polymer

dark brown, elastomeric polymer

4.2.1 Elernental Composition of SMMs

The elemental composition of the SMMs are shown in Table 4-2. The weight percentages of

carbon, hydrogen, nitrogen, and fluorine were determined at Guelph Chemical Laboratories, and

it was assumed that oxygen made up the balance of the SMM composition2. The fluorine content

of the SMMs ranged between 9 and 21 weight percent (wt%). PCL has a slightly higher

molecular weight (MW = 530) and therefore generally produced SMMs with a lower fluorine

content as opposed to those with PPO (MW = 425). There is an increase in fluorine content from

FS03 to FSOl due to a corresponding decrease in PEO segments within the FSO fractions. As

well, there is a smaller difference in the percent fluorine between FSOl and FS02 than that of

FS03. This suggests that the lower weight fractions of FSO are closer in molecular weights and

character than is the third fiaction.

Table 4-2: Weight Percent Elemental Compositions of Synthesized SMMs

MDI-PPO-BAL

MDI-PPO-FSO 1

"he Company was unable to provide oxygen values because of equipment failure.

MDi-PCL-BAL

MDI-PCL-FSO 1

57.97

54.19

59.87

54.48

4.50

6.46

6.1 O

5.74

3.28

3.90

3.49

2.88

22.89

14.44

1 1.36

21.01

21.96

19.75

8.58

17.15

Table 4-3 lists the polystyrene equivalent molecular weights of synthesized SMMs as determined

by GPC, and their standard deviations on at least three measurements. The weight average

molecular weight (M,") of the SMMs ranged between 1.8 x 1 o4 and 3.1 x 1 04. Synthesis with the

PCL soft segment produced SMMs of relatively higher molecular weights as compared to those

with PPO. The additional length of the PCL soft segment chain is suspected to have some

contribution to this change. As well, molecular weights generally increased fiom FSOl to FS03

polymers. This too was expected since FS03 is the distillation fraction with the highest

molecular weight. It is interesting to note that there is little difference in molecular weights

between SMMs made with FSOl and FS02 as compared to the FS03 material. The difference

between the lower molecular weight materials and that of FS03 is much greater in the PCL series

as cornpared to the PPO series. The polydispersity (PD) values for the SMMs are low, indicating

a relatively narrow molecular weight distribution. As well, there are no significant differences

for this measurement between any of the SMMs.

Table 4-3: Polystyrene Equivalent Average Molecular Weights of SMMs

1 SMM Polyrner 1 M. 110' 1 MW / lo4 1 PD

[- MDI-PPO-BAL 1 1.99 k 0.47 1 2.52 f 0.45 1 1.28 + 0.08

The thermal transitions of SMM and SMM modified PES materials were studied with respect to

the glass transition temperature (Tg), as analyzed by DSC, and are detailed in Table 4-4 and

Table 4-5 respectively. The complete thermograms for the SMMs are plotted in Appendix H.

The glass transition temperatures of synthesized SMMs ranged between -20 and -1 OC. The Tg

temperatures (onset, midpoint and final) for SMMs containing PCL sofl segments were generally

Iower than those of SMMs with PPO. FS03 produced SMMs with the lowest Tg temperatures

(onset, midpoint, and final) of the four fluoro-alcohols used. The Tg width of the SMMs ranged

from 9 to 15 OC, and is a measure of the heterogeneity of these materials. The heterogeneity in

the SMMs rnay be due either to the distribution of polymer chains or their copolymeric

composition. In general, SMMs in the PPO series showed slightly greater Tg widths than those

in the PCL series, and are therefore thought to be more heterogeneous.

The first thermal scans of PES membranes (with and without SMMs) produced large fluctuations

at temperatures above 170 OC, as illustrated in Figure 4-1. Because first suspicions fell on either

the bulk materiai or SMMs, thermal gravimetric analysis (TGA) was performed on these

polymers. The detailed thermograrns are given in Appendix 1. The decomposition temperatures

of pure SMM, PES and PVP materials were approximately 230 O C , 450 OC, and 250 OC

respectively. However, TGA analyses of a cast PES film showed a 20 % weight loss between

175 OC and 250 OC, the temperature range in which fluctuations were observed in DSC analyses.

Since the weight loss occurs at a temperature much iower than the decomposition temperature of

SMMs, it is suspected that there may be residual solvent left in the cast films which would have

been removed after the first DSC scan. Second thermal scans of the membranes showed no

fluctuation peaks within the temperature range of interest. Hence, the T,s from these second

thermograrn scans are reported in this thesis.

The glass transition temperature of a PES-PVP membrane was approximately 172 OC, lower than

the literature value of 225 OC for pure PES [Mills, 19931. The addition of 4 wt% SMMs to PES

films either did not change the midpoint Tg or reduced it by not more than 20 OC. Therefore, the

The addition of SMMs did not significantly increase the Tg width of the PES materiais, with the

exception of MDI-PCL-FS03. In general, SMMs with PCL lowered the Tg of the PES material

more as compared to SMMs containing PPO. This may be representative of the lower Tg values

for pure SMMs containing PCL soft segments (Table 4-4).

Figure 4-1: First and Second Thermograms of PES-PVP Membrane

-- - --- -- -- - ---- - --- -- - - --- - " - - - - - -. . - --

2nd scan

1st scan Y 11 III 11 111

1 O0 150

Temperature ( OC)

Table 4-4: Glass Transition Temperatures of Synthesized SMMs

SMM Polymer 1 Onset Tg (OC) 1 Final Tg ( O C ) 1 Tg Width ( O C ) 1 Midpoint Tg ( O C )

MDI-PPO-BAL 1 -7.7 1 1 . 8 1 3.9 f 1.7 1 11.6 f 3.5 1 -1.2 1 0 . 2

- -

MDI-PCL-BAL 1 -23.4 f 2.3 1 -13.5 f 1.7 1 9.9 f 0.7 1 -18.1 f 1.6

Table 4-5: Glass Transition Temperatures of PES and SMM Modified PES Membranes

Membrane Onset Tg Final Tg Tg Width 1 M i d g t Tg

PES-PVP 167.3 f 5.2 1 176.2 + 3.8 1 8.9 c 1.4 1 171.9 k 2.8

MDI-PPO-BAL in PES 1 161.9 f 9.9 1 173.6 f 7.8 1 1 1.6 f 2.1 1 167.2 f 8.9

MDI-PPO-FSO1 in PES 1 170.3 + 2.1 1 179.1 + 2.5 1 8.8 f 0.4 1 174.6 f 2.6

NIA - Standard deviations are not available for this sample because only one run was perforrned

. ...

MDI-PPO-FS02 in PES

MDI-PPO-FS03 in PES

MDI-PCL-BAL in PES

MDI-PCL-FSOI in PES

MDI-PCL-FS02 in PES

MDI-PCL-FS03 in PES

168.1 k 3.0

147.9 + 5.1

161.3 f 3.7

153.9 &NIA

145.8 + 1.6

157.4 3- 1.4

176.9 i 2.9

161.3 + 1.8

172.7 + 2.6

163.6 I N / A

156.7 1- 0.9

171.6 i 0 . 4

8.8 f 0.1

13.4 f 3.3

11.4 rt: 1.1

9.6 4 NIA

11.0 1 0 . 7

14.2 i 1.0

171.8 -t- 1.5

156.8 -t 0.6

166.7 f 4.3

159.1 ~ N / A

152.4 f 1.1

165.4 k 0.6

4.3.1 PES FIat Sheet Membranes

The casting method described in Section 3.3.1 allowed for the fabrication of optically smooth

membranes (approximately 40 Fm in thickness). PES membranes without SMM were

transparent films while SMM modified membranes were opaque. However, several SMM

modified membranes showed streaks of thicker polymer close to the edges of the slide. This was

more prominent in membranes with SMMs containing FSO as the fluoro-alcohol as compared to

materials with BA-L. This may be due to the more hydrophilic nature of the FSO component

which contains long PEO segments. Due to the relatively long hydrophilic segments, these

SMMs will be more miscible in the PES material. Instead of migrating to the air interface, some

segments may become entangled within the bulk rnaterial. This would increase the viscosity of

the bulk solution and therefore cause the observed streaks of cast polymer. Only the middle

portions of the surfaces, far from the glass edges, were used for surface analyses.

4.3.2 Characterization of PES Membrane Morphology

4.3.2.1 Polarized Microscopy

Several rnicroscopy techniques were used to study the morphology of cast PES flat sheet

membranes. Polarized rnicroscopy perhaps provided the clearest evidence of microdomains

resulting from SMM addition. PES membranes were clear and transparent, and no

microstructures could be observed through a polarized light microscope. Figure 4-2 illustrates

the microdomains within three SMM modified PES membranes as seen through a polarized

microscope. These pictures suggest that the opaqueness of the membranes is due to spherical

microstructures. There is a sense of depth as some spheres appear reflective, indicative of

microdomains wllich are closer to the surface, whife some which are deeper within the membrane

are black, and others which are even further in depth appear out of focus. The range of diarneters

of the microdomains for selected SMMs are given in Table 4-6. Of the three SMM modified

while materials containing MDI-PPO-BAL had the largest, suggesting that microdomain sizes

depend on individual SMMs.

Figure 4-2: Microdomain Structures of 4 wtOA SMMs within PES Membranes Obsewed Through a Polarized Microscope

(a) MDI-PPO-BAL in PES

(b) MDI-PCL-BAL in PES (c) MDI-PPO-FS03 in PES

When cast PES films were placed undenvater, small bumps were observed on the films, likely

due to the penetration of water through the membrane pores. Confocal microscopy was used to

determine the height of these bumps and to confirm that studies by undenvater contact angles

would not be significantly affected. Figure 4-3(a) is an example of a PES membrane as seen

underwater by confocal microscopy. The image shows optical rings which were caused by the

interference of the light passing through the membrane and reflected in the aqueous medium.

The nurnber of rings observed can be related to the height of these bumps by [Hemsley, 19891:

number of optical rings x wavelength of reflected light Height =

refractive index of water (4- 1 )

The wavelength of light used in this study was 488 nrn and the refractive index of water is 1.3

[Perry et al., 19841. In general, the height of these bumps ranged from 1 pm over a length of 250

p m for the smallest number of rings to 1 1 pm over a distance of 1500 pm for the largest number

of rings. Because the height of these bumps are gradua1 over a large distance, interference from

these bumps on contact angle studies is assumed to be negligible. However, it c m o t be

completely dismissed that some contribution to contact angle hysteresis may come fiom this

effect. Similar observations were made for SMM modified PES membranes undenvater, as

shown in Figure 4-4(b) for 4 wt% MDI-PCL-BAL in PES. In addition, the image in Figure 4-

4(b) indicates that there are microstructures within the SMM modified membranes which are

much smaller in size than the bumps.

The microstructures of the PES materials containing 4 wt% SMM were further studied on dry

membranes using confocal microscopy at a higher magnification. In reflectance mode, the

microdomains are distinct spheres as illustrated in Figure 4-4(a) for MDI-PPO-BAL in PES.

Using confocal microscopy in transmittance mode for the same membrane, the depth of these

microdomairis can be seen as darker patches in Figure 4-4(b). The reflectance mode was

F U I L w & I L I W U V I A ou v V L U A U A I A A V I A A I V U L I I + U l l l ~ l l 1 U L L U 1 b 3 L U l U Lllb 1 l L I ~ l W U W 1 1 1 U 1 1 1 3 111 L L 1 b 3 b 1lllllJ V Q l l C U 111

size for different SMM modification. These data are given in Table 4-6.

Using the confocal microscopy apparatus, it was possible to image different depths within the

membranes by stepping up the microscope stage in 1.2 pm increments. Figure 4-5 illustrates the

dispersion of the spherical microdomain structures at different depths within a PES membrane

containing 4 wt% MDI-PPO-BAL. These results suggest that the microdomain structures are

dispersed throughout the bulk of the membrane materials, penetrating approximately the top

quarter to one third of the membrane on the side of the membrane-air interface. As well, going

deeper within the membrane fiom the top surface, there is a gradua1 depletion in the number of

structures.

Figure 4-3: Confocal Microscopy images of PES Membrane Underwater

(a) PES-PVP Mern brane (b) 4 wt% MDI-PCL-FS03 in PES

Figure 4-4: Confocal Microscopy Images of 4 wt% MDI-PPO-BAL in PES Dry Membrane

(a) Reflectance Mode

(b) Transmittance Mode

Figure 4-5: Confocal Microscopy Images of the Dispersion of Microdomain Structures in a PES Membrane Modified with 4 wt% MDI-PPO-BAL

(a) top surface (b) 3.6 pm from the surface

(c) 6.0 pm from the surface (d) 8.4 pm from the surface

The topography of dry PES and selected SMM modified PES surfaces were fùrther analyzed

using atomic force microscopy in order to confirm the dimensions of domains on the surfaces.

Figure 4-6 illustrates the topography of PES and SMM modified PES membranes as imaged by

atomic force microscopy. While the PES membrane has a relatively srnooth surface, that of the

SMM modified surface consists of hills and valleys. It is suggested by these pictures that SMM

modification creates surfaces with greater texture. The sizes of the features Vary for substrates

with different SMMs (Table 4-6). The height of the hills in the terrrain for al1 of the SMM

modified surfaces studied varied between 4 to 20 nm while the width of these surface features

ranged between 0.2 to 3 .O Pm.

Table 4-6: Sizes Of Microdomains in 4 wt% SMM Modified PES Membranes

1 Dry Membrane ( Range of Diameters in Microdomain Structures (pm) 1

1 MDI-PPO-FS03 in PES 1 0.1 - 0.5 1 0.5 - 1.0 1 N/A 1

MDI-PPO-BAL in PES

MDI-PPO-FSO 1 in PES

MDI-PPO-FS02 in PES

Polarizing

0.5 - 1.5

N I A

N IA

Confocal

1.0 - 2.5

0.5 - 1.5

0.5 - 2.0

AFM

1 .O - 3.0

N I A

N I A

Modified PES ~ e r n b r a k ~ o ~ & a ~ h ~

(a) PES Membrane

(b) 4 wt% MDI-PPO-BAL in PES

(b) 4 wt% MDI-PCL-BAL in PES

The surfaces of selected PES and SMM modified PES films were analyzed by X-ray

photoelectron spectroscopy (XPS) in order to confirm the concentration of fluorine at the surface.

Each film was analyzed at two take off angles, 15" and 90°, corresponding to a depth of analysis

of 30 A and 100 A respectively. For each film, two samples were analyzed and the relative

atornic percentages of fluorine (F), oxygen (O), nitrogen (N), carbon (C), and silicon (Si) are

given in Table 4-7. For non-modified PES films, some contamination of silicon on the surface

was observed but were not present on SMM modified surfaces. The fluorine content on the

surfaces of PES membranes which contained no SMMs were negligible, at no more than two

atomic percent for both take off angles. In contrast, the fluorine content on the surfaces of PES

containing SMMs ranged between 33 and 47 atomic percent at a depth of 30 A, and between 1 7

and 26 atornic percent at a depth of 100 A. Therefore, it is clear that fluorine groups are

concentrated at these sdaces .

When comparing the two different analyzed sites for the sarne sarnple and a specific take off

angle, there are variations in the fluorine contents for some of the surfaces (as much as 10 percent

for MDI-PPO-BAL), while for others the variations are small. This may be due to the

heterogeneity of the surfaces. As shown by AFM analysis, the topography of the modified

membranes consist of microdomain structures ranging from 1 to 2 microns, depending on the

SMM (Table 4-6). The spatial resolution in the XPS analysis is approximately 4-6 mm, and

therefore a particular scan may encompass any fraction of the microdomains, giving variations in

the fluorine content.

Table 4-7: XPS Results of Elemental Composition at the Surfaces of PES and SMM Modified PES Membranes

Membrane Site Ta ke Off

Angle

Relative Atomic Percent

0 1 s 1 N l s

PES-PVP

MDI-PPO-BAL in PES

MDI-PPO-FSO 1 in PES

MDI-PPO-FSO2 in PES

MDI-PPO-FS03 in PES

4.3.4 Contact Angle sruaies

4.3.4.1 Results From Air Contact Angle Analyses

Air contact angle analysis was used to qualitatively assess the relative wettability of PES and

SMM modified PES flat sbeet membranes. The results are shown in Table 4-8. Pure PES shows

an advancing angle of approximately 67" and those for the SMM modified surfaces ranged

between 97" and 120". The addition of the SMMs increased the contact angles of the membrane

surfaces considerably, by as much as 53". Therefore, the SMM-PES surfaces are considerably

more hydrophobic. This is attributed to the presence of the SMMs at the surface of the PES

membranes, and more directly from the hydrophobic nature of the SMM fluorine tails. SMMs

containing PCL segments consistently showed larger advancing contact angles than those with

PPO segments, despite the fact that fluorine content was generally higher for the SMMs with

PPO segments (Table 4-2).

SMMs containing BAL as the fluoro-alcohol produced membranes with larger receding angles

than those with FSO. This may be due to the more hydrophobic nature of BAL, which does not

contain the hydrophilic PEO segments that are present in FSO. There were some difficulties in

measuring the receding angle and this gave some variability in repeated measurements on the

sarne sarnple and between sarnples. This is largely due to the influence of the needle on the

shape of the liquid drop at low volumes, and as well on the accuracy of reading small angles on

the goniorneter. Overall, the addition of SMMs caused an increase in contact angle hysteresis,

which is expected since the addition of SMM in PES creates a chemically heterogeneous system.

l Membrane Water Advancing

Contact Angle (O)

PES-PVP

MDI-PPO-BAL in PES

1 MDI-PPO-FS03 in PES / 97.5 f 1.6 1 12.4 $r 3.3 1 85.1 k 3.7 1

Water Receding Contact Angle (O)

MDI-PPO-FSO 1 in PES

MDI-PPO-FS02 in PES

1 MDI-PCL-BAL in PES ( 113.2 -t- 1.1 1 30.5 + 3.8 1 82.7 + 4.0 1

Contact Angle Hysteresis (O) 1

67.2 + 0.4

107.3 k1.8

1 MDI-PCL-FSOl in PES 1 119.4 k 0.8 1 17.5 f 1.5 1 101.9 f 1.6 1

102.9 + 2.0

106.3 f 2.3

26.0 + 3.7

58.2 zk 4.5

4.3.4.2 Results From Underwater Contact Angle Analyses

41.2 k3 .7

49.1 + 4.8

32.9 t 5.3

24.2 $r 4.6

MDI-PCL-FS02 in PES

MDI-PCL-FS03 in PES

An undenvater contact angle method was used to qualitatively assess the nature and behaviour of

PES and SMM modified PES membranes in an aqueous environment. The cast membranes

were, in general, optically smooth. However, when the films were placed in water, bumps were

observed on the surface which were suspected to be due to the penetration of liquid into the

membrane pores. Confocal microscopy was used to determine that the hydrated films were

indeed smooth undenvater (Section 4.3.2.2).

70.0 15 .7

82.1 -t- 5.1

The contact angle that an air bubble made on the material surface was measured as air was

introduced onto the membrane. Images were taken every second to give a profile of the

advancing and receding behaviour of the bubble on the surface. The field of view was scaled

according to a premeasured grid which was digitized into the memory of the cornputer. For each

picture taken, the cornputer chose ten points along the image of the air bubble and fitted these

points according to a Laplacian curve to determine theoretical values for the experimental image.

112.2 f 0.7

99.7 f 2.8

21.0 f 1 . 2

29.0 f 6.9

91.2 k1.4

70.7 f 7.4

-

angle, contact radius, surface area, and volume. Using these values, data for the contact angle

was plotted against contact radius for each dynarnic contact angle measurement. A typical plot is

shown in Figure 4-7.

For air advancing contact angles, as time (or air volume) increased, the initial contact radius

remained constant with increasing contact angle. Once the contact angle was large enough to

overcorne the surface tension of the membrane, the three phase line moved and the contact radius

increased as the contact angle remained constant. Figure 4-7 illustrates this ideal behaviour of air

advancing on a PES membrane containing 4 wt% MDI-PCL-BAL. For air receding, the opposite

trends were observed as shown in Figure 4-8. In both cases, the contact angle of interest is the

angle which is constant at increasing or decreasing contact radius for advancing and receding

contact angles respectively. For each membrane (with and without SMMs), at least 3 different

slides were prepared for that SMM and for each slide, the advancing and receding angles were

measured three times.

The phase at which the advancing and receding contact angles do not Vary with increasing or

decreasing contact radius was defined by determining the covariance between the contact radius

and contact angle. The covariance of two variables is defined as the expected value of the

product of their deviations fiom their mean [Olson, 19871 and is a measure of how two variables

change together. If x and y are independent, then the covariance is close to zero and the average

of the contact angle values in this region of the contact angle versus radius plot is taken. As well,

the covariance is similar to the linear correlation coefficient which c m be estimated by dividing

the covariance by the product of the individual standard deviations [Olson, 19871. Since there

are no general rules that can be stated as to what values give a strong or weak correlation [Olson,

19871, it was arbitrarily decided that onIy those experiments which gave a covariance of less than

0.01 and standard deviation of Iess than 2 degrees would be reported as reliable data. The data

from this analysis are tabulated in Appendix K and the final contact angles are reported in Table

4-9.

Figure 4-7: Profile of Air Advancing Contact Angles and Contact Radii for 4 wt% MDI-PCL-BAL in PES Flat Sheet Membrane

O 20 40 60 80 100 120 Time (sec)

Figure 4-8: Profile of Air Receding Contact Angles and Contact Radii for 4 wtOh MDI-PCL-BAL in PES Flat Sheet Membrane

120 , - . 0.25

NI.....

15 20 Time (sec)

j +Contact Angle Contact Radius

n y u a t c u I LO I ~ I U U U L U U W J LillV .. -. .... ..-, -ui-iiiC) - D - - - - -

in the PES membranes decreased the air advancing contact angle by as much as 14", therefore

indicating that these surfaces are more hydrophobic. However, this change in hydrophobicity

(between PES and SMM rnodified PES) for the hydrated membranes is not as great as the change

for the dry sarnples (in the air contact angle study) which was as rnuch as 5 2 O (Table 4-8). This

may lend evidence to the mobility of the SMMs in the PES base material, when place in an

aqueous environment.

There were some difficulties associated with the measurement of the air receding contact angles.

Once the three phase line had receded, only a small volume remained, therefore limiting the

number of images that could be taken. It was impossible in some cases to obtain an accurate

receding angle from the few pictures and therefore they are not reported in Table 4-9. However,

in order to provide the reader with some indication of possible receding contact angles for these

surfaces, the range of contact angles observed are included in Table 4-9. The air receding contact

angles were significantly lower in SMM modified membranes than that in pure PES. Again, this

is attributed to the hydrophobic nature of the fluorine containing SMMs. The contact angle

hysteresis values of the membranes undenvater were close to those observed in the air contact

angle study, for both PES and SMM modified PES samples. Using the undenvater air advancing

contact angle values in Table 4-9, the surface tension (y,,) of the PES and SMM modified PES

membrane surfaces were calculated using the macroscopic approach of the equation of state

(Section 2.3.3). The results are shown in Figure 4-9. Hydrated PES membranes have a surface

tension of 68 f 0.6 r n ~ / r n ~ and the addition of SMM reduced the surface tension by as much as 8

r n ~ / r n ~ for MDI-PPO-BAL.

1 Z1Ult: 9'7. I\C;JUiLï4 Ui U i i U G i rr n r c i b u i i r a b r A U ~ L ~ n i i u i j a b u vii i rrv A r = r i i i v i uiirii

1 1 Contact Angle (O) 1 Contact Angle (O)

1 ~ e r n b r a n e ~ Air Advancing Hysteresis (O)

1 PES-PVP 1 157.5 '1.8 1 115.1 11.2

Air Receding

1 MDI-PPO-BAL in PES 1 143.7 f 1.2 1 ~ a n g ' = 53 - 72

Contact Angle

Range = 91 - 72

MDI-PPO-FSO1 in PES

MDI-PPO-FS02 in PES

MDI-PPO-FS03 in PES

Figure 4-9:

MD1-PCL-FS03 in PES

Surface Tension (Y,,) of PES Membranes Underwater

r

148.4 f 0.4

147.3 k l . l

147.7 k1.4

PES MD1 MD1 MD1 MD1 MD1 MD1 PPO PPO P PO PPO PCL PCL BAL FSOI FS02 FS03 BAL FS03

149.0 f 1.9

- -

MDI-PCL-FSOI and MDI-PCL-FS02 were not available for study at the time the undenvater contact angle equipment was available for use and therefore undenvater studies were not performed for these materials. %ue to difficulties in measuring the air receding contact angles, it was not possible to obtain an accurate value for these surfaces. A range is given to provide the reader with an idea of possible values.

60.5 ir 1.1

71.6 f 3.5

Range = 61 - 91

87.9 f 1.2

75.7 I 3 . 7

Range = 87 - 57

Range = 63 - 91 Range = 86 - 58

4.4.1 Hollow Fibre Membranes

The solution casting rnethod (Section 3.4.1) produced PES hollow fibre membranes with

optically smooth surfaces. The hollow fibres for both PES and SMM modified PES were white

as a result of precipitation of the polymer in a gelation bath. The dimensions of the fibres were

determined by photographing magnified images observed through a light microscope and

measuring the circurnference of the fibre tube. The average dimensions of the hollow fibre are:

inner diameter 0.70 f 0.04 mm

outer diameter 0.98 * 0.03 mm

length of tube used 8 _+ 0.5 mm

The total surface area of an 8 mm piece of hollow fibre membrane was 44.98 +_ 2.3 mm2. The

surfaces of the hollow fibre membranes were analyzed by scanning electron microscopy and the

images of a PES and an SMM modified PES membrane are s h o w in Figure 4-10. These images

reveal the presence of contaminants on the surfaces of the membranes for both non modified and

modified membranes. These defects was probably produced during the manufacturing of the

hollow fibres and may ultimately affect the results of blood compatibility studies.

Figure 4-10: SEM Images of Hotlow Fibre Membranes

(a) PES (b) 4 wt% MDI-PPO-BAL in PES

Preliminary blood compatibility evaluation of PES hollow fibre membranes were assessed with

respect to fibrinogen adsorption. The details of the adsorption experiment were given in Section

3.4.2. The nurnber of counts per minute associated with adsorbed '2S~-labelled fibrinogen was

related to the actual adsorbed fibrinogen concentration by the following equation:

net surface counts (cpm) x protein concentration (mg 1 mL) amount adsorbed (mg / cm') = x 1000

surface area (cm2 ) x solution count (cpm / mL)

When the solution counts were analyzed for each experiment, the solution counts for the low

fibrinogen concentration solutions did not have a high degree of correlation to the expected

fibrinogen concentrations. In previous fibrinogen adsorption experiments performed at the

McMaster laboratory, solution counts were only taken for the highest and moderate

concentrations. For al1 other concentrations, solution count values were estimated by

interpolation. This is because solution counts for the low concentrations are too close to the

detection limit of the gamma counter and are therefore more likely to produce errors. Because of

this concern and the fact that solution counts did not relate directly to the expected protein

concentrations, only solution counts for the two highest concentrations were directly used for

calculations.

The different modified surfaces were divided into three sets, and each set was run with different

preparations of radiolabelled fibrinogen. Since different preparations resulted in different

radiolabel levels, a PES control fibre was used in each experiment. Figure 4-1 1 shows the

adsorption of fibrinogen onto PES membranes containing BAL as the fluoro-alcohol. The effect

of FSO fraction on the performance of the SMM with respect to protein adsorption is given in

Figure 4-12 (SMMs synthesized with PPO) and Figure 4-13 (SMMs synthesized with PCL). In

al1 experirnents, it is clear that surface modification with SMMs reduced the amount of protein

adsorption as compared to non-modified PES membranes.

- - - - - - - - - - - - - - - - - - - - 1 - 1 1

between experiments, there may have been slight differences in experimental technique which

caused deviations in the absolute level of fibrinogen adsorption. Evidence of the latter is pointed

out by the fact that the plateau levels for pure PES varied from one experiment to the next.

Therefore, in order to compare the effects of the eight different SMMs on fibrinogen adsorption

as compared to PES, two approaches were used for comparison. The first approach involved

calculating the ratio of adsorption slopes of SMM modified PES to pure non-modified PES,

between the concentrations of 0.01 rng/rnL and 0.05 mg/mL (representing the initial adsorption

phase). The second factor used for comparison is the determination of the percent reduction in

fibrinogen adsorption as compared to pure PES at the highest fibrinogen concentration, 0.5

mg/mL. These data are given in Table 4-10.

The ratio of the adsorption slopes for modified to non-modified membranes are either near unity

or significantly lower than unity. A lower ratio indicates that with increasing protein

concentration, the arnount of protein adsorption is less for the modified membranes. In general,

SMMs having PPO as the soft segment showed a more pronounced reduction in the ratio value at

low fibrinogen concentrations. As well, these SMMs also showed the greatest percent reduction

of fibrinogen adsorption at the highest concentration (0.5 mg/mL). The only exception is that of

PCL with FS02 which also showed a greater reduction in fibrinogen adsorption.

Figure 4-1 1: Fibrinogen Adsorption Ont0 PES Hollow Fibre Membranes -SMM Modifications Containing BAL

i

-+- PES

-D-MDIPPO-BAL in PES ~ --

O 0.1 0.2 0.3 0.4 0.5

Protein Concentration (mglml)

Figure 4-12: Fibrinogen Adsorption Onto PES Hollow Fibre Membranes SMM Modifications Containing PPO and FSO

; + PES I

1 -M-MDI-PPO-FS03 in PES ' 1 + MDI-PPO-FSO~ in PES 1 1 \C MDI-PPO-FSOI in PES /

O 0.1 0.2 0.3 0.4 0.5

Protein Concentration (mglml)

Figure 4-13: Fibrinogen Adsorption Onto PES Hollow Fibre Membranes SMM Modifications Containing PCL and FSO

1 l

--- 1 t ------p. -- t J

O O. 1 0.2 0.3 0.4 0.5

Protein Concentration (mglml)

Table 4-10: Summary of Fibrinogen Adsorption Results for 4 wt% SMM Modified PES Hollow Fibre Membranes

Membrane Ratio of Adsorption Slopes (SMM Modified PES):PES

- -

MDI-PCL-FSO1 in PES 1.04 + 0.20 8.1 t 8.3

Percent Reduction of Fg Adsorption at 0.5 mg/mL 1

-

MDI-PPO-FSO2 in PES

MDI-PPO-FS03 in PES

1 MDI-PCL-FSO2 in PES 1 0.75 -C- 0.18 1 27.2 & 6.7 1

0.92 -t. 0.15

0.83 k0.16

15.7 2 8 . 1

22.5 + 7.2

MDI-PCL-FS03 in PES 0.91 k0.16 5.0 k9.1

The membrane materials were contacted with whole blood in a rocking platform apparatus

(Figure 3-5) for one hour at 37 OC. The blood collected from the test materials were analyzed for

platelet count in the bulk, platelet activation (as indicated by % P-selectin expression and %

microparticles), and leukocyte activation (as indicated by the upregulation of CD1 1 b). Table 4-

11 details the ce11 count and flow cytometric results of platelet activation on these materials as

compared to control samples. The first set of samples are negative controls of resting blood

stored in sealed microcentrifuge tubes during the experiment. Resting and EDTA anticoagulated

sarnples provide an indication of the lower limit of activation. The presence of SFLLRN

thrombin peptide in the blood sarnple will activate platelets to breakdown and form

microparticles and P-selectin expression, and therefore defines the upper lirnit of platelet

activation in the blood. Three control materials were analyzed in the rocking platform apparatus,

relatively non-activating Silastic, relatively activating polyethylene (PE), and non-modified PES

hollow fibre membrane. The modified hollow fibres were separated into two series for these

studies, one group with PPO based SMM modifications and the other with PCL based SMM

modifications. Each series were analyzed in three separate experiments with six different

volunteer blood donors. For the control blood samples and materials, averages and standard

deviations were calculated fiom al1 six experiments and for the test materials (SMM modified

membranes) averages and standard deviations were calculated for three experiments.

In the resting sarnple, the number of platelets in the bulk of the blood is approximately 123 3- 14

(x Io9) per litre. Polyethylene (PE) positive control material showed the lowest platelet counts at

74 + 13 (x 109) per litre indicating the uptake of platelets from whole blood. The platelet counts

for the Silastic@ negative control and PES membrane are comparable, showing only a slight

decrease in the arnount of platelets present relative to resting samples after the rocking

experiment. PES membranes modified with PPO based SMMs also show comparable platelet

nuinbers to that of Silastic@ and PES. However, membranes containing PCL based SMMs

resulted in a reduction in the amount of platelets in blood, comparable to that of PE. There were

no significant differences in this marker between the four modifications within each series.

I I I G piaLcicL r-~CICLLIII C A ~ I ~ J J I V I I 13 a pubuIiagb ui p u a i ~ i v b b v b i i ~ a i u i ~ i i b ~licunu L U U ~

detected by flow cytometry analysis gated for P-selectin expression. A high percent expression is

indicative of activated platelets which bind to leukocytes via P-selectin as illustrated in the case

for the activated sample SFLLRN with 87 + 27 %. The resting blood sample showed P-selectin

expression at 5 k 2 %, and that of al1 materials analyzed by the rocking platform experirnent were

similar to resting blood with respect to this parameter.

Table 4-11: Platelet Activation on PES Hollow Fibre Membranes

Material

Resting

EDTA

SFLLRN

O h P-selectin Expression

Platelet Count ( x 1o9n)

Silastic

PE

PES

MDI-PPO-BAL in PES

MDI-PPO-FSO1 in PES

MDI-PPO-FS02 in PES

MDI-PPO-FS03 in PES

MDI-PCL-BAL in PES

MDI-PCL-FSOl in PES

MDI-PCL-FS02 in PES

MDI-PCL-FS03 in PES

% Microparticles

123.4 k 14.3

119.8 k 18.2

not applicable

102.2 + 14.9

73.8 + 13.3

92.2 k l l . l

96.7 f 15.6

102.0 + 19.0

97.3 k15.3

106.0 k 14.8

79.0 i 6.0

84.0 + 7.0

78.5 + 4.5

84.0 k 0.0

4.8 f 1.6

7.1 + 0.7

87.4 k 27

14.3 f 8.0

11.2 '8.8

34.2 k 20

4.9 k l . l

5.0 k1.3

5.1 f 1.1

6.0 k 0.9

5.6 k0.4

6.2 k 1.0

5.9 + 0.6

5.4 f 2 . 6

4.4 k1.5

5.8 52 .0

5.1 k2.0

21.4 L-12

27.1 '14

26.1 f 1 3

31.8 k 13

32.1 -411

35.0 + 11

31.1 k 13

18.6 -t 9.3

15.9 k 4.6

20.7 14 .9

20.0 + 6.2

-

marker CD61 detected by flow cytometry gated for %microparticles. The formation of

microparticles results fiom the fibrinogen binding to GPIIbAIIa in the activation of platelets

momura et al., 19921. The resting samples showed some degree of microparticle formation at 14

k 8 %. Silastic@ and membranes containing PCL based SMMs showed comparable values,

slightly above that of the resting sarnple, although not significant. Resting sample with SFLLRN

activator, PE, PES, and membranes containing PPO based SMMs al1 showed comparable

microparticle formation, at approximately 30%. However, with the variability between

experiments and the large standard deviations in the measurements, the levels of microparticle

formation for al1 materials analyzed by the rocking apparatus were not significantly different.

In the blood sarnples, leukocyte activation was determined by the upregulation of the antibody

CD1 1 b as determined by flow cytornetric analysis. These results are shown for'all controls and

materials in Figure 4-14. The degree of activation of leukocytes associated with platelets is

quantified in terms of a linearized fluorescence unit and are used as relative terms. Both the

relatively non activating Silastic@ and relatively activating PE materials showed the sarne degree

of leukocyte activation as the resting blood sample at approximately 170 units but less than the

PMA activated blood sample. All PES and SMM modified PES membranes showed an

increased amount of leukocyte activation during blood contact. Membranes modified with BAL-

based SMMs appear to lower the arnount of leukocyte activation of PES membranes by as much

as 80 units. In contrast, membranes containing FS02 and FS03 based SMMs showed increased

leukocyte activation as compared to non-modified PES membranes, by as much as 116 units.

Figure 4-14: Leukocyte Activation by the Upregulation of CD1 1 b

Resting EDTA PMA Silastic PE PES MD1 MD1 MD1 MD1 MD1 MD1 MD1 MD1 PPO PPO PPO PPO PCL PCL PCL PCL BAL FSOI FS02 FS03 BAL FSOl FSOZ FS03

5.1 SURFACE MODIFYING MACROMOLECULES (SMMs)

5.1.1 Synthesis of SMMs

Previous work investigating the synthesis of SMMs found that the properties of these oligomers

depended on the reactant mole ratio, reagents used, and environmental conditions [Pham, 1995;

Tang, 19953. It was assumed that by keeping the reaction conditions constant, such as the

reactant mole ratio, apparatus, temperature and stir rate, differences in polymerization between

SMMs would not vary significantly. In that case, the variations between the SMMs and its

effects on the base polymer would be due to their individual chernical composition. An

investigation on the reproducibility of the synthesized SMMs was beyond the scope of this thesis.

It is important to note that only one batch of each type of SMM was synthesized and analyzed,

and therefore conclusions drawn fiom the data presented should be confinned with M e r

studies on additional batches of SMMs.

In the synthesis of commercial polyurethanes, it is desirable to produce polymers with high

molecular weight through chain extension with a di01 or diamine. This is because the final

application of the polywethane is often one that requires mechanical strength and integrity.

However, in this study, this factor is not as important since SMMs are used as an additive in

small weight fractions relative to the base poIymer. The properties that are important are the

miscibility of the SMMs with the base polymer and the characteristics which may dictate the

SMM7s stability at the material surface. Therefore, the reagents used in synthesis were chosen

for the purpose of distinguishing the effect of different soft segments and fluoro-alcohol

endcapping reagents. The influence of these components on the nature of SMM interactions,

within the main chain of the base polymer, was of particular interest. For exarnple, a high

molecular weight SMM may result in entanglements within the base polymer and reduce the

driving force for the SMMs to migrate to the surface. At the same tirne, however, if the SMMs

possibility for leaching of the additives from the base material.

The fluoro-alchohols, BAL and FSO-100, were commercial products which contained unknown

impurities. The low molecular weight fractions of BAL, FSOl and FS02 were believed to be

relatively pure and free of contaminants because these were recovered products from distillation

of the stock materials. However, FS03 was the residual fraction from distillation and therefore

would have contained unknown contaminants. When PES solutions containing FS03 based

SMMs were filtered through a 0.5 pm filter, brown particulates were observed on the filter paper.

This may have been irnpurities from the original FS03 fraction which did not react to form SMM

polymer. Sirnilar particulates were also recovered from FS03 during the purification of FSO

fractions [Weiler, 19971. Approximately one third of FSO-100 was purified as FS02, and only

one tenth of FS02 was recovered as FSOl. Furthemore, the latter fraction was often difficult to

recover fiom the FS02 phase because the boiling point range was not clear and the products were

only differentiated by slight colour variations (clear for FSOl and faint yellow for FS02).

Considering the difficulties associated with the processing of FSO-100, future manufacturing

work with this materiai should use a combination of FS02/FSOl (the distillation product of

FSO-100) since it contains less contarninants and is relatively easy to recover. Characterization

results indicating that FSOl and FS02 produce SMMs with similar properties fürther support

this approach.

The SMMs differed in physical character and this defined their ease of handling during

processing. After the precipitation of the polymer solution in distilled water, SMMs containing

BAL tended to give polymers which were elastomeric while those containing FSO tended to be

tacky powders. During the final washing stage with 30% acetone, al1 SMMs became more tacky

because the polymers were partially soluble in acetone. Processing at this stage became difficult

because the polymers would stick to the sides of the container and the stir blade. After the final

wash, the polymers had to be scraped frorn the sides of the beaker and as a resuIt, the final

polymer yield was considerably low. The acetone washing step was implemented to leach out

residual unreacted monomers, specifically fluoro-alcohols, which are soluble in acetone.

V W I I O L U ~ L L L ~ ~ ulr ~11~1115~ ALI A I L L G ~ ~ I A L ~ VI CIIC, )JULJIIIC,I a I 1 u I U W YILIU, I L 13 I~~ U I I L I I I G I I U C U illa1 a

different washing solution be used. Other possible solvents include ethyl acetate and toluene

which also dissolves the fluoro-alcohol monomers [Dupont]. Further work is required in order to

determine the solubility of the SMM polymers in these solvents.

5.1.2 Properties of SMM Polymers

The properties of eight SMM polymers synthesized in this study were characterized by physical

appearance, elemental composition, molecular weights, and thermal properties. The effects of

using different polyol and fluoro-alcohol reagents on each of these properties are described in

this section.

5.1.2.1 Physical Appearance

The physical appearances of the final processed SMM polymers are dependent on both the nature

of the sofi segment and the fluorine tails. The PPO based SMM materials were more tacky as

compared to the PCL based polymers. Since the structure of the polymer is dependant on the

main urethane prepolymer chain, this observation may be due to the higher molecular weight of

the PCL segment (MW=530) which generally produced SMMs with larger molecular weights

than the PPO equivalents (MW=425). Since SMMs are oligomeric fluoro-polymers with a

relatively short main chain, one would expect the fluorinated endgroups to have an effect on the

main polymer structure. The low molecular weight fluoro-alcohols (FSOl, FS02 and BAL)

tended to be associated with less elastomeric SMMs having a crystalline appearance.

5.1.2.2 Elemental Composition

The structure of BAL is known from the literature [Dupont] and was confirmed in the work of

Pham 119951. However, the molecular weights of the three FSO fractions were not provided by

the supplier of the stock FSO-100. In a previous study, sainples were sent to Guelph Chernical

LaDoratories Tor eiemenrai anaiysis on r;aroon, i iuorinc, a i u uxygt;~ w GUCL, 1 77 1 1. 1 HG LCSUILS

are given in Table 5-1.

Table 5-1: Elemental Composition of FSO-100 Fractions [Weiler, 19971

The molecular structure of FSO-100 is:

FSO Fraction

FSO l

where m ranges from 3 to 8 [Dupont]. Different combinations of m and n values were used to

determine possible chemical structures for the three FSO fractions with elemental compositions

matching that given in Table 5-1. The possibilities are given in Table 5-2. The average

molecular weight of FSO-100 stock solution is 730 [Dupont]. Since two thirds of the stock

material were obtained as FS03, it is assumed that the average molecular weight of this fraction

must be higher than 730. Based on Table 5-2, this value would be estimated at 860. The lower

molecular weight fractions would most likely be lower than 730. For FS02, the average

molecular weight should be at least as low as 628. It is believed that the molecular weight of

FSOl is close to that of FS02 due to difficulties in separating the two fractions by distillation.

Therefore, the molecular weight of FSOl would likely be 596. From this reasoning, the

molecular structures of the three FSO fractions are given in Table 5-3. While these molecular

weights are not believed to be exact, they are probably good first estimates. Future work could

confirrn this by analyzing pure FSOl and FS02 by mass spectroscopy. They are discussed here

in order to give the reader an idea of their molecular structures and how they might influence the

structure of the different SMMs.

wt% Carbon

31.7

wt% Oxygen

1 1 . 1

wtOh FIuorine

54.0

FSO Fraction m n Molecular Weight

FSOl 3 2 452

4 3 596

Table 5-3: Estimates of Average Molecular Structures of FSO Fractions as Determined by Elemental Analysis

Theoretical elemental compositions of the SMMs were calculated using approximate molecular

weights of each reagent (based on Table 3-1 and Table 5-3) and assuming a perfect 3:2:2

stiochiometric ratio of MDI:polyol:fluoro-alcohol. The theoretical elemental compositions of

nitrogen and fluorine in the SMMs are given in Table 5-4, and the experimental values are also

tabulated for cornparison. The composition of nitrogen is indicative of the incorporation of MD1

in the main chaiil during the prepolymer reaction and the composition of fluorine corresponds to

the relative arnounts of fluoro-alcohol in the SMM.

FSO Fraction

FSOl

FS02

FS03

Molecular Structure

F(CF2CF2)4CH2CH20(CH2CH20)3H

F(CF2CF2)3CH2CH20(CH2CH20)6H

F(CF2CF2)4CH2CH20(CH2CH20)9H

Molecular Weight

596

628

860

of Elemental Nitrogen and Fluorine in SMMs

wt% Nitrogen 1 wt% Fluorine

1 Theoretical 1 Ex perimental ( Theoretical

MDI-PPO-FSO 1 1

Experimental

MDI-PCL-BAL

MDI-PCL-FSO 1

Comparing experimental theoretical compositions, the fluorine contents for SMMs containing

BAL and FS03 were lower than expected, with the BAL based SMMs showing the greatest

deviation. For the sarne SMMs, the composition of nitrogen was slightly greater than expected

(with the exception of MDI-PPO-BAL), suggesting that prepolymer content was relatively larger

than that predicted by theory. Interestingly, the SMMs which showed the greatest deviation from

theoretical values were the three SMMs (MDI-PPO-BAL, MDI-PCL-BAL, and MDI-PCL-FS03)

with the highest molecular weights (Table 4-3). This trend was also observed in previous studies

on the synthesis of SMMs [Tang, 19951. The results indicate that the urethane prepolymer has a

greater degree of polymerization, than the expected 3:2 for MD1:polyol.

the prepolymer stage, polyol molecules are still present in solution and

with the fluoroalcohols for the isocyanate groups of MDI.

3 .O

2.7

This may

therefore

occur if afier

will compete

3.5

2.9

The elemental compositions for synthesized SMMs containing FSOl and FS02 are close to

theoretical values suggesting that these SMMs exhibit the expected 3:2:2 stoichiometry of

reactants used. The differences between the experimental and theoretical fluorine content in

Table 5-4 suggest th.e relative reactivity of fluoro-alcohols with MD1 to be in the order of FS02 >

23.1

21.1

- - - A .

have a large effect on the endcapping reaction. This is because in step growth polymerization,

the reaction is dependent on the end functional groups of both molecules [Rosen, 19931. In this

case, these would be the hydroxyl group of the fluoro-alcohol and the isocyanate group of MDI.

5.1.2.3 Molecular Weight

The rnolecular weights of the SMMs ranged between 1.8 x 1 o4 and 3.1 x 1 o4 (Table 4-4). In the

previous section, it was suggested that the increase in molecular weights between the different

SMMs was due to an increase in the prepolymer chain. The polydispersity index, or breadth of

molecular weight distribution, of the SMMs are close to 1.3 indicating a very narrow distribution.

This index for most commercial polymers ranges between 2 and 50 [Rosen, 19931. The n m o w

distribution is likely attributed to the low degree of polymerization (degree of polymerization of

2 for a 3:2:2 stoichiometry) of the SMM polymers.

The rneasured polystyrene average molecular weights are much higher than expected values for

the actual SMM polymers having a 3:2:2 stoichiornetric ratio. This is because molecular weights

were determined by a polystyrene calibration curve. Different polymer molecules in the same

solvent exhibit varying configurations depending on their solubility characteristics in that solvent

[Rosen, 19931. Since polystyrene is a homopolymer, polystyrene of varying rnolecular weights

c m be readily synthesized. Because SMMs are block copolymers, it would be difficult to

produce a series of polymers with varying molecular weights in order to construct a calibration

curve. In this work, the SMMs are assumed to be linear polymers, similar to polystyrene, and

polystyrene equivalent moiecular weights are reported for comparison between the different

SMMs.

The midpoint glass transition temperatures of the SMMs ranged between -20 and -1 OC,

indicating that at room temperature the chain segments of the polymers are mobile and the

material is ductile. In general, glass transition temperatures of polymers increase with increasing

molecular weight [Turi, 19811. This relationship was not observed between the molecular

weights (Table 4-4) and Tg values (Table 4-5) for the different SMMs since no general trends

were noted. This may be due to the heterogeneity of the SMMs, whereby the Tg of each segment

contributed to the overall Tg of the polymer. The microheterogeneity in the composition of the

SMMs was also reflected in their Tg widths which ranged between 9 and 15 OC. No other

thermal transitions were detected in the thennograrns, and therefore the SMMs are amorphous in

this temperature range. In general, the T,s (onset, rnidpoint, and final) for SMMs containing

PCL soft segments were lower than those for SMMs containing PPO. This confirms the finding

of previous studies on SMMs showing that the Tg of SMM polyrners are dependent on the polyol

soft segment [Tang, 19951 and furthemore that the Tg decreases with increasing molecular

weights of the soft segment [Petrovic, 1985; Weiler, 19971.

5.2 SMM-PES MEMBRANE COMPOSITION AND MORPHOLOGY

DSC analysis of cast PES membranes showed large fluctuations above 170 OC in the first thermal

scan. Through TGA analysis of the pure components (SMM, PES and PVP), it was concluded

that the fluctuations were due to the evaporation of residual solvent from the membrane films.

This discovery was surprising since the drying procedure of the membranes involved an

evaporation step in a 50 OC oven for 2 days and further drying under vacuum at 50 OC for 2 days.

This finding highlights the difficulty in drying polymer sarnples and evaporating solvent from a

polymer matrix. It was observed by TGA analysis that films which were pour cast in aluminum

plates contained more solvent than films which were dip cast on glass slides (Appendix 1, Figure

1-5). This was probably due to a thinner membrane film obtained by the latter method. It is

important to consider the presence of residual solvent in regards to the outcome of the various

experimems i r i iriis LIICSIS. ru1 ~xal~iplt;, 111 L U I I L ~ ~ L L illlgl~ ~ L U U I C ~ , r i i c icauiiiig ur aul v c i i ~ i i i r u L ~ I G

water phase rnay cause perturbations in the observed contact angles. In blood studies, the

presence of residual solvent rnay cause the destruction of blood cells or activate platelets and

other biological factors. Therefore, care should be taken to ensure that al1 solvent has been

removed from the polymer films.

The addition of SMMs in PES lowered the midpoint Tg of the modified membranes by not more

than 20 OC. This was expected because the SMM additives, which have low Tg values, act to

decrease the Tg of the base polymer. Hence, for well mixed blends, the final Tg of the polyrneric

material would be expected to fa11 somewhere between the values of the two pure components.

However, the mixing of the blends rnay not be immediately obvious because while PES

membranes were originally transparent, they became opaque with the addition of SMMs. The

miscibility of polymer blends is ofien determined by the measure of a single Tg as a function of

composition [Couchman, 1978; Aubin, 19881. Since only one Tg was observed for the modified

membranes (Appendix H), one rnay conclude that the SMMs were miscible in the PES material.

However, the microscopy analysis of PES-SMM membranes reveal the existence of phase

separated structures in the form of spherical microdomains. The single observable Tg rnay result

from the low concentration of SMMs (4 wtO/o) in the bulk material and therefore a distinct Tg for

the SMM phases was not detected. Therefore, the addition of SMMs do not have a significant

effect on this bulk property of the PES material.

The Tg width of the bulk material can provide an indication of the heterogeneity of the

membranes. Non-modified PES membranes showed a Tg width of 8.9 t 1.4 OC. This rnay be

due to the distribution of molecular weight chains of the material and also to the presence of PVP

in the membranes. In general, the addition of SMMs did not significantly increase the Tg width,

again indicating that a low concentration of SMMs did not increase the bulk heterogeneity of the

PES membrane. The importance of concentration has been previously described for two phase

systems containing a low concentration of one component in another [Olabisi, 19791.

homogeneous. However, observations of cast SMM-PES films by polarizing and confocal

microscopy confirm the existence of phase separated microdomains within the PES material

(Section 4.3.2). The domains are spherical and evenly dispersed at several layers through the

bulk of the membranes, with a greater density of microspheres at the surface. The degree of

miscibility of the SMMs in PES will be expected to influence the size of these microstructures,

their dispersion throughout the bulk, and the heterogeneity of the membrane surface.

The immiscibility of two polymers in a polymer blend has been previously s h o w to produce a

system of small spherical microdomains of the low fraction polymer in the rich phase of the

second polymer [Piirma, 19921. The formation of these spherical domains is due to the

difference in the interfacial tension of the two polymers. Hence, it is possible that if one polymer

is considerably more hydrophobic than the other, the driving force for the more hydrophobic

material to phase separate into domains is greater. For exarnple, in a study by Okano et al.

[1981], spherical microdomain structures were observed in phase separated copolymers

composed of hydrophilic 2-hydroxyethyl methacrylate (HEMA) and hydrophobic styrene. In

their study, the microdomains were composed of concentrated hydrophobic styrene chains within

a continuous phase of HEMA chains, even at a mole fraction of 0.35 of the more hydrophilic

HEMA. This emphasizes the ability of hydrophobic molecules to aggregate and form

microdomains.

It is proposed that the observed microdomains in the modified PES materials are SMM

aggregates in the forrn of micelles, resulting from the arnphiphilic nature of the fluorine tail and

long chain backbone. This proposed structure is analogous to the micelle formation of

surfactants in solution. Based on bond lengths for C-C and C-O [Zumdahl, 19893 and the

theoretical structure of the SMMs, if the microdomains were one SMM molecule in dimension,

the size of the microstructures would be in the range of 0.05 - 0.1 Pm. However, the

microdomains obsei-ved in this study are larger, approximately ten times, and therefore the

domains must consist of many SMM molecules. The number of molecules would be difficult to

determine since the specific orientation of the SMMs in the domains is not known.

Lumucal mu p v ~ a r ~ ~ t ; ~ IIIILIUSGU~~ SLUUIGY 1t;vt;alcu LIK SILL VI L I I I L L U U V I I L Q I I I ~ 111 r CD LU v a y

between the different SMMs (Table 4-6). This phenornena rnay reflect the different surface

energies of the individual SMMs [Piirrna, 19921 and their relative miscibility of chain segments

in the base polymer. As well, the molecular weights of the additives may also be a factor

[Hwang, 19951. For exarnple, bIock copolymers with low surface energy segments of siloxane-

and perfiuoroalkane- modified blocks have been previously shown to segregate when mixed with

a styrene homopolymer. The behaviour of the macromolecules depended on both additive

molecular weight and the processing conditions of the blend [Hwang, 19951. Surface

modifications with SMMs containing PPO soft segments exhibited larger microdomain

structures than those with PCL (Table 4-6). Comparing the images of MDI-PPO-BAL and MDI-

PCL-BAL modified membranes (Figure 4-2), it appears that the PCL component increased the

miscibility of the SMMs in the PES material. It has been suggested that PCL is capable of

hydrogen bonding with molecules containing an electron donating portion [Olabisi, 19791 as

illustrated by PCL and PVC miscible blends. In this case, hydrogen bonding results from the

donating character of Cl-C-H groups as shown in Figure 5-1 (a). An analogy may be made for

possible hydrogen bonding between PCL and PVP rnolecules in PES material as illustrated in

Figure 5-1 (b). The XPS data (Table 4-7) showed the presence of nitrogen in the non-modified

PES sample. This suggests that PVP was available at the surface to interact with the SMMs and

the PES. Hence, this may help to explain the greater miscibility of PCL based SMMs in PES as

compared to PPO based SMMs, since PPO segments would not have the same capacity to

interact with PVP.

Figure 5-1: Illustration of Hydrogen Bonding Capabilities of PCL Components

PVC

-(CH 3)5-C -O - II

PCL coniponent O

with FSO (Table 4-6). This may be explained in terms of relative hydrophobic character. The

more hydrophobic BAL based SMMs (as indicated by the chemical data structures for BAL and

FSO Table 3-1) resulted in larger microdomains than the relativefy more hydrophilic FSO based

materials. It is betieved that the PEO segments present in the FSO chains may improve its

miscibility with the PES material, thereby reducing the driving force to form SMM domain

structures. This in tuni should effectively reduce the domain size. The influence of an increase in

PEO segments on irnproving the miscibility of the SMMs is further suggested by the observation

of smaller domain structures for the FS03 based materials relative to those containing FSOl and

FS02.

Confocal microscopy (Section 4.3.2.2) revealed that microdomains were dispersed in the top

third of the cast membrane (ie. top 12 pm) with a gradua1 depletion of the micro-structures from

the surface going into the bulk material. This observation supports the hypothesis that during

membrane casting, the SMMs migrate to the air-surface interface. Based on AFM studies

(Section 4.3.2.3), the topography of the SMM-PES films also indicated the presence of SMM

microdomains on the surface (Figure 4-7). This observation was fùrther confirrned by XPS

elemental studies which showed an elevated concentration of atomic fluorine at the SMM

modified membrane surfaces. The AFM studies also confirmed the size of the surface micro-

structures to be similar to that observed in the bulk of the membranes observed by confocal

microscopy (Table 4-6).

From the microscopy results, a simplified mode1 showing the hypothesized organization of

SMMs in PES membranes (dry surfaces) is illustrated in Figure 5-2. The SMMs are aggregated

into micelle structures dispersed in the bulk PES material. The ring structure is based on

micellar formations analogous to that of surfactants, but the true structure of the core of the

SMM micelles has not yet been determined. The SMM domains are also shown to be exposed at

the membrane surface, as revealed by AFM studies. It is apparent from the diagram that XPS

surface analysis (limit of resolution is 10- 150 prn [Cooke et al., 19961) would not have the lateral

sensitivity to measure the chemical composition of individual microstructures, since any fraction

PES membrane l

-- - . .-

SMM micelle SMM micelle at the surface

surface modifying rnacromolecule

fluorine tail

urethane prspolymer chain

fluorine tail

of the microdomains in PES can be detected at any particular site. At the surface, it is

hypothesized that there is a reorganization of some SMM molecules in which the fluorine tails

are exposed at the surface. This is suggested by XPS studies (Table 4-7) which showed a high

elemental fluorine content at the surface. XPS also revealed a low concentration of elemental

nitrogen at the membrane surfaces, with an increase going deeper in into the material. Recall that

nitrogen is specifically associated with the main cliain of the SMM. Therefore, this suggests that

at least part of the SMM backbone chain is exposed. This was observed in a previous study on

very little sulfur content at the surface which would have been associated with PES,

approximately 1 atomic percent. Therefore, the surface contains high concentrations of other

components which may include SMM and PVP. It is hypothesized that the matrix substrate at

the surface (shown as blank space on the surface of the membrane in Figure 5-2) may contain

SMM molecules and PVP at the top few angstroms. If this is the case, it would not have been

detected by AFM studies. Further studies using spectroscopy techniques capable of high lateral

resolutions will be required to confirm this.

The observed heterogeneity of the modified surfaces may explain observations in other studies

conducted in this thesis. For example, the observed hysteresis behveen the advancing and

receding contact angles is considered to be a measure of the degree of heterogeneity at the

surface. In XPS studies, variations in the relative atornic percent of fluorine are also suspected to

result from the presence of SMM domains concentrated on the surface. The variations were

greatest for membranes containing MDI-PPO-BAL which have exhibited the largest

microdomain structures. For membranes containing the smaller microdomains, elemental

compositions at the surface for different analyzed sites were less variable. The heterogeneity of

the SMM-PES surfaces is also expected to influence blood studies. This has been suggested by

Okano et al. [1981], whose studies showed that the hydrophobic/hydrophilic nature of a surface

affects the pattern of platelet spreading and deformation ont0 these surfaces.

5.3 MEMBRANE SURFACE PROPERTIES

5.3.1 Review of Contact Angle Methods

5.3.1.1 Air Contact Angle Method Using a Goniorneter

Difficulties associated with measuring contact angles using a goniorneter resulted from botli the

nature of the surfaces and the apparatus. The chemical heterogeneity of the membrane surfaces

resulted in a "slip-stick" action of the water droplet. That is, as the volume of the water drop was

slowly increased, the three phase line did not advance slowly, but moved abruptly. The kinetic

energy due to this abrupt motion would have ultimately affected the equilibrium of the system.

There were also sources of errors inherent in the goniometer method for measuring contact

angles. The needle used to advance the liquid rernained in the droplet during measurement and

therefore interfered with its shape. The water tended to one side of the needle, and both the lefi

and right contact angles did not necessarily give the same readings. To account for this, both the

left and right contact angles were measured and averaged if the differencce between the two was

less than five degrees. Measurements with greater than five degree differences were rejected. In

addition, the manually operated syringe caused slight vibrations which may have interfered with

the equilibrium of the liquid drop and therefore the equilibrium contact angle measurements. As

a result of these complications, subsequent work for undenivater contact angle studies used an

apparatus designed to alleviate some of these problems.

5.3.1.2 Underwater Contact Angle Method

(i) Advantages of the Underwater Apparatus

There were several advantages to the undenvater apparatus. First, the needle was placed on the

opposite side of the surface analyzed and therefore did not interfere with the shape of the air

bubble. This was important in order to allow the air bubbIe to advance and recede uniformly as

well as allowing the system to reach equilibrium. The use of a TeflonB needle also eliminated

any vibrations which may have been caused by the operation of the syringe. A constant flowrate

of air was delivered by the use of a motorized syringe so that the rate of the advancing and

receding angles could be controlled. In this manner, the air couid be introduced slowly so as to

minimize the effect of kinetic energy on the equilibration of the system. The behaviour of the air

bubble could be monitored at different time intervals by using the autoinated computer program,

ADSA-P. This program alIowed for accurate and quick calculations of the contact angles and

other parameters, and errors due to visual readings of the contact angles would be eliminated.

There were difficulties in measuring contact angles due to the chemical nature of the membrane

surfaces. The ideal relationship between contact angle and contact radius illustrated in Figure 4-

7 and Figure 4-8 were not always observed. It has been suggested that the contact angle is

dependent on the contact radius as a result of line tension, the one dimensional analog of surface

tension [Neumann, 19961. This effect is illustrated for a PES sample in Figure 5-3, and this

behaviour was also observed for some SMM modified samples. As the three phase line moved

(or contact radius increased), line tension would act in the opposite direction of the motion of the

three phase line. Therefore, the contact angle did not remain constant.

A second problem was the "slip-stick" behaviour of the three phase line which did not advance

slowly on the surface, but moved abruptly to a new position. This action is clearly illustrated in

Figures 5-4 and 5-5 for advancing and receding angles respectively. It was difficult to determine

the correct equilibriurn contact angle on these surfaces since the angle did not remain constant

and would be greatly affected by the kinetic energy of the moving three phase line. This "slip-

stick" behaviour may reflect the chernical heterogeneity of the SMM modified surfaces as

revealed by AFM and XPS studies.

There were mechanical problems inherent in the underwater contact angle method. It was

difficult to centre the air bubble around the hole since the bubble tended to float to one side. To

prevent this, an initial air bubble was introduced and moved to the centre using an inverted

stainless steel rod. The air bubble could then be advanced and receded using the motorized

syringe. For air receding angles, once the air had receded and the motor was stopped, buoyancy

effects created a back pressure which caused the complete withdrawal of the air. As a result, few

pictures of the receding angle were taken and the angle was affected by the kinetic energy of the

receding air bubble.

Figure 5-3: Dependence of Air Advancing Contact Angle on

O 50 100 150 200 250 Tirne (sec)

Figure 5-4: Illustration of Slip-Stick Action of Air Advancing Contact Angles on 4 wt% MDI-PPO-FSO2 in PES Flat Sheet Membrane

t i . 0.1 10 15 20 25 30 35 40 45 50

Time (sec)

1 +Contact Angle . Contact Radius 1

Figure 5-5: Illustration of Slip-Stick Action of Air Receding Contact Angles on 4 wt% MDI-PPO-FS03 in PES Flat Sheet Membrane

O 5 1 O 15 20 25 30 Time (sec)

5.3.2 Surface Energetics of PES and SMM Modified Membranes

Contact angle studies in air and underwater confirrned that the addition of SMMs to PES creates

a more hydrophobic surface than pure PES. Membranes containing SMMs synthesized with PCL

segments showed larger advancing contact angles in air than membranes with SMMs having

PPO based segments (Table 4-8), despite the greater fluorine content in PPO based SMMs (Table

4-2). This may be due to the greater hydrophobic character of PCL sofi segments (a polyester) as

compared to PPO sofi segments (a polyether). If some fraction of the SMM backbone is exposed

at the membrane surface, as suggested by XPS data, then PCL surface modifications may be

expected to yield more hydrophobic surfaces.

The SMM modified surfaces showed a greater contact angle hysteresis, indicating that the

modified surfaces are more heterogeneous than the pure PES. This observation is expected for a

two phase system [Morra, 19901. AFM analysis of the membranes indicated that the films were

- - - - - - - - - - -

surface roughness may be neglected. However, chernical heterogeneity was observed by both

polarizing and confocal microscopy. This was observed in the form of spherical microdomains,

0.1 to 3 pm in size, concentrated at the air interphase which would be anticipated to contribute to

a high degree of contact angle hysteresis [Morra, 19901.

In the aqueous environment, the degree of hydrophobicity decreased in the SMM-PES surfaces as

compared to surfaces in an air environment. This observation is hypothesized to be related to

two possible events, the penetration of water into the membrane pores and polymer surface

dynarnics, both of which are believed to have sorne contribution. The first hypothesis is that in

an aqueous environment, the water molecules are penetrating the interstitial areas of the PES

matrix and therefore, the water rnolecules on the surface would enhance the hydrophilic nature of

the membranes. The tendency of water to penetrate into the membrane pores has been observed

in several studies [Morra, 1990; Holly, 19751, and was confimed in this study by the observation

that membranes cast on glass slides would readily be displaced when immersed in water. As

well, confocal microscopy of membranes undenvater showed areas of swelling (Section 4.3.2.2)

which were suspected to result fiom water passing through the membranes. One can argue,

however that if the observed microdomain structures are concentrated SMMs, the water

molecules would be repelled from these regions and only enter regions of concentrated PES.

Therefore, the surface would be made up of regions of SMMs and water molecules and the

difference in surface energetics, or heterogeneity, between the two phases would be even greater

than that of SMMs and PES. In this scenario, the contact angle hysteresis should be greater in an

aqueous environment as compared to an air environment. However, this was not observed. The

contact angle hysteresis for air and undenvater contact angle studies were comparable (Table 4-8

and Table 4-9). Hence, other factors must be present.

Another factor to consider in regard to the observed decrease in hydrophobicity for hydrated is

related to the mobility of the SMMs in the PES base material. In an aqueous environment, the

hydrophobic fluorine tails would be expected to orient away from the water interphase and

rearrange into the material. Such mobility of chain segments and even whole macromolecules

- - -- - - - - - -

introduction of an air bubble on a hydrated membrane provided the driving force for the

hydrophobic SMM fluorine tails to orient towards the air-surface interface. Since there is a finite

time required for the polymer segments or molecules to rearrange in a changing environment,

then equilibrium contact angles will not be achieved. In this case, the true equilibriurn contact

angle cannot be obtained. Further undenvater contact angle studies should be conducted to

determine the effective hydration time of the SMM modified and non-modified porous

membranes. This factor has particular relevance in biornaterial applications. If the surface of a

biomedical device can undergo reorientation in an aqueous environment prior to and following

protein adsorption, this will have an impact on blood compatibility. As well, packaging and

storage conditions will be important parameters to consider for defining the initial surface

character of the product.

5.4 EFFECTS OF SMMs ON THE BLOOD COMPATIBILITY OF PES MEMBRANES

5.4.1 Properties of the PES Hollow Fibre Membranes

Hollow fibre membranes were fabricated and used for blood compatibility studies for two

reasons. The hollow fibre membranes are easier to handle in an aqueous medium because flat

sheet membranes tended to either displace from its substrate or float in water. As well, the

hollow fibre configuration resembles design components that are incorporated into devices used

for biomedical applications, such as the hollow fibres in a dialysis unit or the hollow tubing of

polymeric catheters.

The study of the membrane morphology (Section 4.3.2) was performed on flat sheet membranes

which were translucent and observable by light microscopes. Unfortunately, the hollow fibre

membranes had a much greater thickness and could not be studied by the same rnicroscopy

techniques because light could not pass through them. Therefore, the presence of rnicrodomai~~s

at the surface and in the bulk of the hollow fibre membranes have not been confirmed. The

behaviour of the SMMs are expected to be similar, although may not be identical, to that of flat

predominantly dependent on the polymer molecular weights and interfacial tensions of the

SMMs. The principle differences in the two configurations, flat sheet versus hollow tube, are

membrane thickness and processing conditions. The thickness of the membrane may affect

phase separation of the SMM and the ability of the SMM to migrate to the membrane interface.

Processing conditions include the precipitating medium (which will have different interfacial

tensions for air and water medium), the curing temperature (which will affect the degree of

phase separation), and the apparatus used to process the sample (which will affect the quality of

the membranes).

Analysis of the hollow fibres by SEM revealed irregular roughness features on the surfaces. The

membranes showed striations in the morphology which may have resulted frorn the machining of

the spinneret component (Appendix F). The grooves and crevices of these materials is expected

to influence the results of the fibrinogen and blood contact studies.

5.4.2 Fibrinogen Adsorption

Fibrinogen adsorption isotherm experiments were conducted to determine the qualitative nature

of protein adsorption ont0 PES fibres and to determine the effects of SMM surface modifications

on the arnount of protein adsorbed at different concentrations. The in vitro experiment was

simple in its implementation and allowed for a quick assessrnent of fibrinogen adsorption ont0

hollow fibre materiais. In the experiment, there was uncertainty about the amount of fibrinogen

that was effectively radiolabeled with 12? during the iodination step. To account for this, PES

samples were run in every experiment. In cornparing the amount of fibrinogen adsorbed onto

non-modified PES control membranes, the three experiments showed reproducible results, with a

variation of approximately 10%. While the experiment was designed to give only fibrinogen

adsorption values for a non-flow and single protein solution, the results do provide some

indication of the potential for the materials to induce interactions with procoagulant proteins.

However, it is noted that the experiment does not account for other factors involved with in vivo

in adsorption and desorption of proteins at the surfaces, as well as their interactions with cells.

The adsorption of fibrinogen proteins on solid surfaces generally follows the characteristics of a

Langmuir isotherm [Horbett, 19861. While the incrernental amount of fibrinogen adsorbed did

decrease with increasing solution concentration, the amount of fibrinogen adsorbed ont0 the

fibres did not level off for the concentration range studied (Figures 4- 1 2,4- 1 3,4- 14). The trends

do appear to approach saturation and hence, the amount of protein adsorbed is expected to level

off at higher concentrations. Future experiments should extend the study to higher protein

concentrations in order to determine the true saturation values. The arnount of fibrinogen

adsorbed by the PES materials (at the highest protein concentration) is higher, although on the

sarne order, as fibrinogen adsorption results reported on other polymer surfaces such as

polyethylene (at 1.4 pg/crn2) and polystyrene (at 1.7 pg/cm2) [Brash, 19691. The higher values

observed in the current study may be a reflection of the increased surface areas due to surface

defects, since this was not accounted for in the original estimate of membrane surface area.

The fibrinogen adsorption studies showed that in most cases, the surface modification of PES

hollow fibre membranes with SMMs reduced the arnount of fibrinogen adsorbed as compared to

non modified PES fibres. Reduced fibrinogen adsorption on more hydrophobic surfaces was

observed by Brash et al. [1969] in a study of the affinity of several plasma proteins on different

polymeric materials. The reduction may have resulted from a decrease in the surface energies of

the membranes which was related to the presence of SMMs at the surface, as indicated in Figure

4-10. This hypothesis is supported by other studies which have also shown increased adsorption

of human fibrinogen ont0 surfaces with increased surface energy [Sipehia, 19931. It has been

suggested that materials will show minimum bio-adhesion [Gogolewski, 19891 and minimal

platelet spreading [Baier, 19851 at a critical surface tension range of 20-30 rn~/m*. The surface

energy of SMM modified PES membranes ranged between 60-63 r n ~ / m ~ . Hence, it was expected

that the membranes in this study would show some degree of fibrinogen adsorption and blood

activation. Because fibrinogen adsorption has been directly linked to platelet activities and

activation of the intrinsic coagulation pathway [Courtney, 19941, these studies suggest that

polymer and therefore improving blood compatibility.

5.4.3 Activation of Blood Components

The preliminary in vitro whole blood study allowed for the determination of material induced

activation of blood platelets and leukocytes on PES and SMM modified PES hollow fibre

membranes. The advantage of the rocking platform apparatus is that it is simple and reveals

platelet activity that has taken place in the blood and not on the surface of the material. This is

because studies that have focused on platelet adhesion or granule release fiom adhered platelets

do not account for surfaces which do not promote adhesion but are still platelet activating

[Gemmell, 19951. For example, in the results of the present study (Section 4.4.3), the platelet

counts for al1 materials (except those with PCL based modifications) were comparable to that of

the resting blood sample (with the exception of SFLLRN), thus indicating low adhesion of

platelets. However, other platelet activating markers (%microparticles and upregulation of

CD1 1 b) suggest that al1 PES materials are blood activating.

Between the six separate experiments, there were large variabilities in the results for al1 measured

parameters as indicated by large standard deviations. Several factors may have influenced the

consistency of the experiments. The blood of different donors is inherently different fi-om each

other, and is likely to have caused variations in the blood ce11 counts and degree of activation.

As well, there appeared to be problems with the CD6 1 antibody (the marker for P-selectin and

%microparticles) used in the experiments. In the laboratory where the blood studies were

conducted, the supplier of CD61 had been changed. The new type of CD61 gave variable results

in the current studies and as well in experiments performed by other investigators. The

inconsistencies and unexpected results due to these factors were observed on resting blood

sarnples, negative and positive control materials. Another large source of error was likely to

have resulted from the irregular structures and defects on the hollow fibre membranes as revealed

-

proteins and cells, and resulted in the activation of platelets.

Another consideration is that activation of platelets on the membranes may have resulted fiorn

the design of the experimental apparatus used for the in vitro whole blood studies. The original

set up as introduced by Gemme11 et al [1995], was designed such that hollow tube materials were

attached directly to the rocking platform (Figure 3-5) by rneans of short pieces of SilasticB

tubing at either end. In this way, the blood is introduced only on the inside of the test material at

a maximum shear rate of 25 sec-' [Gemrnell, 19951. Initial trial experiments with PES hollow

fibre membranes were assembled in this manner but had failed. This was because the

membranes are porous, and water had evaporated from the blood through the membranes,

leaving dried blood in the materials. The apparatus was modified to insert the membrane

materials inside a SilasticB tubing and whole blood ran through both materials (i.e. the SilasticB

tubing and the PES fibres). The shear rate induced by this design would be much greater than 25

sec" since the average distance between the platelets in solution and the material wall was

smaller, and shear rate is a function of distance from the surface. As well, the control surfaces

did not have the additional surface area since these tubes were attached directly to the rocking

apparatus and not inserted in Silastic@ tubing. Therefore, the apparatus used for measuring the

activation of blood components in this preliminary study did not permit for an appropriate

assessment of the effect of SMM surface modification on the blood compatibility of PES

membranes.

In general, none of the analyzed materials (including the controls) showed activation with respect

to P-selectin expression, with al1 materials showing values comparable with the resting blood

sample and much Iower than the activated SFLLRN sampIe (Table 4-12). Platelet activation is

revealed by the platelet count and Ieukocyte activation. In terms of leukocyte activation, al1 PES

membrane surfaces showed higher levels of CD1 l b upregulation than control samples and

resting blood san~ples, with PES membranes showing twice as much activation as SilasticB

negative control and polyethylene positive control (Figure 4-15). Membranes modified with

FS02 and FS03 based SMMs (with larger PEO segments than BAL and FSOI) showed a

-

30%. At the same time, BAL based SMM modifications showed the lowest degree of leukocyte

activation. In this preliminary study, the results suggest that the chemistry of the SMMs may

differentiate the potential activation of blood components on the modified membranes. Further

studies using a more appropriate apparatus will have to be conducted to confirm the significance

of increased PEO segments for increasing leukocyte activation, and BAL segments for reducing

leukocyte activation.

Based on the leukocyte activity data, it would appear that the PES membranes which were

fabricated by the solution spinning technique at the Industrial Membrane Research Institute are

blood activating. However, due to the large variability between experiments and sources of

errors which affected the activation of platelets, a general conclusion cannot be made on the

overall blood compatibility of PES membranes and the effects of SMMs on these events.

However, this preliminary data has highlighted several technical issues which can be overcome

in future studies. Proposals for improvements c m be found in Section 7.0.

1. It was shown that the addition of 4 wt% SMM in PES reduced the glass transition

temperature of PES membranes by not more than 20 O C , indicating littie change in this bulk

property due to the SMMs. As well, the addition of SMM oligomers did not broaden the Tg

width of the PES bulk material, and therefore the presence of SMMs does not generate a

more heterogeneous system in terms of thermal properties.

From microscopy studies, a schematic of the morphology of SMMs in the PES membranes

was proposed. When SMM oligomers are added to the base material, during the curing stage

of membrane formation, phase separation occurs forming microdomains (approximately 1-2

pm in diameter), dispersed in the top 12 pm of the membrane. Therefore, the SMMs were

shown to migrate and concentrate in the surface region of the air-membrane interface. The

concentration of microdomains was gradually depleted from the surface going into the bulk

of the membrane. It is hypothesized that the microdomains are SMM polymer concentrated

in the form of micelles, due to the amphiphilic nature of the chain segments.

3. Contact angle analysis in air and aqueous environrnents showed that the addition of SMMs in

PES lowered the surface tension, creating more hydrophobic surfaces, accompanied by an

increase in surface heterogeneity. These results are evidence of the presence of fluorinated

SMMs at the surfaces. XPS studies further confirmed the presence of elemental fluorine at

the surface. When membranes were placed in an aqueous medium, the degree of

hydrophobicity decreased. This effect is hypothesized to be due to both the penetration of

water into the membrane pores and the surface dynarnics of the SMMs hydrophobic tails

which rearrange away from the water interface, thereby exposing its more hydrophilic

backbone.

4. The chernical composition of the different sofi segment components and the fluorine tails of

the SMMs was shown to produce SMMs with varying characteristics which ultimately

affected its physical state in PES membranes. The miscibility of SMMs in PES depended on

U U L l L L 1 L b L l U L U l b W 1 L L L b G V L L J b 6 L l A W L I L C U A U A l U W L l L L U C b U k I A U 6 L U U r J + U L I I I V I Q V W A l L U L L A L I A E ) I LL J U A L

segments as compared to PPO soft segments increased their miscibility in PES. As well,

FSO based SMMs showed increased miscibility compared to BAL based SMMs. This was

likely due to the hydrophilic PEO segments present in the FSO components. The surface

energy of PES materials was affected by the chemical composition of the SMMs. Surface

modifications with PCL sol? segments and BAL fluoro-tails showed a greater degree of

hydrophobicity at the membrane surfaces due to the greater hydrophobic nature of these

segments as compared to PPO and FSO respectively.

5. The addition of 4 wt% SMM to PES hollow fibre membranes reduced the adsorption of

fibrinogen by as much as 36% compared to pure PES. And SMMs with PPO soft segments

appear to be more effective in reducing fibrinogen adsorption compared to those with PCL

soft segments. Because fibrinogen is directly linked to platelet activities, activation of the

intrinsic coagulation pathway and interactions with leukocytes, this study suggests that

surface modification with SMMs may be an effective method for improving blood

compatibility by reducing the initial adsorption of fibrinogen proteins.

6. From preliminary results of the in vitro whole blood studies, it was not possible to draw

conclusions on the relative activation of platelets and leukocytes by PES hollow fibre

membranes. Nor was it possible to determine effects of SMM surface modification on these

markers of blood compatibility. This was due in part to donor variability, unreliable sources

of antibody and irregular roughness features and the presence of surface contarninants on the

membranes. While the apparatus used in this study was originally developed to assess the

blood cornpatibility of polymers in tube form, it was not well suited for the evaluation of

porous fibres. Hence, it did not manage to isolate conditions in order to test the ability of

SMMs to alter the blood activation events on PES membranes.

1. It has been suggested in this and other studies on SMMs that the bulk properties of the base

material will not be significantly altered by the presence of small arnounts (1 to 4 wt%) of

SMMs. This has been proven in the case of bulk thermal transition properties such as glass

transition temperatures and Tg width. In order to make a generalization of the effects of

SMMs on bulk properties, the blended membranes should be studied in terms of other bulk

parameters such as membrane strength and membrane permeability. In addition to blood

compatibility, the latter two properties have been identified to be important in the

development of membranes for biomedical applications [Cheung, 19901.

2. It was hypothesized that the observed microdomain structures in the bulk and surface of the

PES membranes are due to the formation of SMMs concentrated in the form of micelles.

XPS data also showed a high fluorine content on the surface of the membranes which rnay

result fiom both SMM micelles and SMM molecules at the top few angstroms of the matrix

substrate. To confirrn this hypothesis, the chemical composition of the microdomains and

that of the matrix substrate should be analyzed. This rnay be facilitated by transmission

electron microscopy which has a lateral resolution of up to 0.5 nm and cm provide chemical

analysis using x-ray and electron loss theory [Sawyer, 19961.

It was revealed that the microdomain structures were dispersed at approximately the top third

of the base membrane and that there was a gradua1 depletion frorn the surface going into the

membrane. By using confocal microscopy in step mode, the layers of the membrane film

rnay be studied to develop a more detailed mode1 on the distribution of microdomains as a

function of depth. This rnay be performed for membranes at slightly larger thickness, at

different stages of the curing process, and hydrated in water. This rnay provide information

on the kinetics of the microdomain dispersion and on the mobility of these structures in its

arnorphous state with time. This rnay also have relevance in regards to estirnating the depth

of the SMM dispersion for thicker membranes.

much is known about the effects of the presence of PVP on the surface properties and

miscibility of the SMM modified PES membranes. Studies should be performed to

determine if and how PVP acts as a cornpatibiliser for SMMs in PES, and differences

between the surface chemistry and surface energies of membranes with and without PVP.

5. In undenvater contact angle studies, the membranes were allowed to hydrate for 5 minutes,

and were assurned to have reached equilibrium before measurement. Because surface

dynarnics is time dependent, a time dependent hydration study is recommended in order to

determine an optimal hydration period in which the water-membrane interface has reached

equilibrium. This may be performed by observing changes in contact angles with varying

hydration times. As well, information on the surface rearrangement of SMM micelles may be

obtained by perfonning AFM analysis on membranes undenvater.

6. The results fiom the fibrinogen adsorption study and the in vitro whole blood study may have

been significantly affected by defects present on the hollow fibre membranes. These defects

would likely have caused increased fibrinogen adsorption and elevated levels of platelet and

Ieukocyte activation. Before further studies are to be conducted on the blood compatibility of

the membranes, the spinneret apparatus should be manufactured in a rnanner to minimize the

introduction of irregular features on the final fabricated membranes. The quality of the

membranes can be evaluated by SEM analysis prior to fibrinogen or blood studies.

7. It was assurned that the migration and formation of SMM microdomains in the hollow fibres

would be similar to that present in flat sheet membranes. In order to provide evidence of this,

the surface chemistry and morphology of the hollow fibre membranes should be

characterized. This will be particularly important for the interface inside the hollow fibre

tube which is the surface which cornes into contact with blood components in applications

such as haemodialysis. This c m be can be carried out by XPS studies and microscopy

techniques such as AFM.

not observed, although the trends in the experiments do suggest that a plateau value would be

reached at concentrations greater than the highest concentration (0.5 mgjml) used in this

study. Therefore, similar experiments should be conducted for higher protein solution

concentrations in order to determine the saturation values. Once a protocol for single protein

adsorption has been established, future studies could examine the adsorption patterns for

multiple protein solutions.

9. The rocking platform apparatus introduced by Gemme11 et al. (1995) was shown to be

successfûl for blood contacting studies with poIymeric tube materials, but has failed for

studies of porous membranes such as those used in this thesis. The apparatus for future blood

studies should be modified to facilitate the study of porous structures. A flat sheet design in

which membranes cast on glass slides is contacted with whole blood, may be able to better

determine the effect of SMMs in the PES membranes. However, this would not account for

physiological conditions such as blood fiow. An alternative method would be to manufacture

a hollow fibre membrane bundle inside a plexiglas encasing (similar to an artificial kidney),

whereby water is introduced in the shell side and blood is introduced in the tube side. A

rocking motion similar to that used in this study can be implemented to simulate blood flow,

and analysis of drained blood from this experiment can foilow procedures used in this thesis.

10. It was suspected that platelets would readily adhere to the grooves and crevices of the hollow

fibre membranes used the whole blood studies. To confirrn the effect of surface roughness

and evaluate the degree of platelet adhesion, SEM analysis cm be performed on the hollow

fibres after blood contact.

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APPENDICES

Chernical Supplier -

Acetone Aldrich Chernical Company, Milwaukee, U.S.

DuPont Chemicals, Supplied by Van Waters & Rogers, Montreal, Cm.

Aldrich Chernical Company, Milwaukee, U.S.

DMF

F S 0 100

LiBr

MD1

PCL

PES

PPO

PVP

Aldrich Chernical Company, Milwaukee, U.S.

DuPont Chernicals, Supplied by Van Waters & Rogers, Montreal, Cm.

Aldrich Chernical Company, Milwaukee, U. S.

Eastman Kodak, Rochester, U.S.

Aldrich Chernical Company, Milwaukee, U.S.

Victrex 4800P, ICI Chernicals

Aldrich Chernical Company, Milwaukee, U.S.

Aldrich Chemical Company, Milwaukee, U.S.

jjj

j thermometer

condenser

1 I l:I cooling water 1 ; 1

: 1 reflux column I l 1 I ! !

- ! I I I l

, solution flask

\ ,/'

heating mantle

U stopcock

collection flask

liquid nitrogen

mobile phase reservoir 0.05 M LiBr in DMF

;z

injector

samp'e $I T

1 I,

computer interfaced with Millenium Software

-1

waters 410 1 - , 3 HPLC pump

. _ .-. - I

I

Waters 200 uL ID loop

heating jacket interna1 temperature of 80 OC

- . - . - -

HT3 HR2 HRI Waters StyragelTM SEC columns

Waters 41 O Differential

Refractometer

1 waste solvent reservoir

Adapted from [Ratner, 19931

Electrostatic hemispherical

eledron energy analyzer

Acceptance lense Retardation lense

...m.............

Vacuum PumP

(10 torr) Ultrahigh vacuum

pumping system (10 torr)

Multichannel detector

Instrument control

and data analysis - binding energy, eV 1

MEMBRANES BY A SOLUTION SPINNING PROCESS

interna1 precipitant (distilled water)

SPINNERET polymer solution

$. hollow fibre membrane

100 cc syringe r? with DH1 0

high precision pump controller

hollow fibre membrane

prepuried nitrogen 7 -2 psi

stainless steel reservoir for polymer solution

stainless steel

1

\

t

large container with DH2 O

Experiment 1: Fibrinogen adsorption on t0 PES Iiollow fibre membranes SMM modifications containing BAL

Fg concentration: 0.5 mg/mL 0.1 mg/mL 0.05 mg/mL 0.0 1 rng/mL 0.001 mg/mL

total volume: 7.93 mL 9.66 m L 7.32 m L 6.60 mL 6.00 mL

solution counts: experimental 30,800,000 5,250,000 1,750,000 240,000 14,000

expected 30,800,000 5,250,000 3,000,000 620,000 60,000

Experiment 2: Fibrinogen adsorption o n t 0 PES hollow fibre membranes SMM modifications containing PPO and FSO

Fg concentration: 0.5 mg/mL 0.3 mg/mL 0.1 mdmL 0.05 mg/mL 0.0 1 mg/mL

total volume: 9.60 mL 7.67 mL 8.00 mL 6.00 mL 5.00 mL solution counts: experimental not counted 1,500,000 700,000 250,000 20,000

expected 1 1,000,000 6,500,000 2,000,000 1,000,000 200,000

Experiment 3: Fibrinogen adsorption o n t 0 PES hollow fibre membranes SMM modifications containing PCL and FSO

Fg concentration: 0.5 mg/mL 0.3 mg/mL 0.1 mglmL 0.05 mg/mL 0.01 mg/mL

total volume: 9.60 mL 7.67 mL 8.00 mL 6.00 mL 5.00 mL

solution counts: experimental 1 1,000,000 6,500,000 2,000,000 600,000 55,000

expected 1 1,000,000 6,500,000 2,000,000 1,000,000 200,000

-

MODIFIED PES-PVP MEMBRANES

Figure H-1: Thermograms for MDI-PPO-BAL Materials

l

I

I PES with MDI-PPO-BAL

MDI-PPO-BAL

-50 O 50 1 O0 150 200 250 300

Temperature ( OC)

Figure H-2: Therrnograms for MDI-PPO-FSOI Materials

-6 1 -100 -50 O 50 100 150 200 250 300

Temperature ( OC)

Figure H-3: Therrnograms for MDI-PPO-FSO2 Materials

\ PES with MDI-PPO-FSOZ 1

-1 O0 -50 O 50 1 O0 150 200 250 300

Temperature ( OC)

Figure H-4: Thermograms for MDI-PPO-FS03 Materials -

Temperature ( OC)

Figure H-5: Thennograms for MDI-PCL-BAL Materials

PES with MDI-PCL-BAL

MDI-PCL-BAL

-50 O 50 1 00 150 200 250 300

Temperature ( OC)

Figure H-6: Thermograms for MDI-PCL-FSOI Materials - - - * - a -- - -. - - .-- -- - - . -

- PES with MDI-PCL-FSOI ,

-50 O 50 1 00 150 200 250

Temperature ( OC)

Figure H-7: Thennograms for MDI-PCL-FSO2 Materials

PES with MDI-PCL-FSOZ MDI-PCL-FSO2

-50 O 50 1 O0 150 200 250

Temperature ( OC)

Figure H-8: Thermograms for MDI-PCL-FS03 Materials

Table 1-1 : PES Resin Powder

APPENDIX 1: (Cont'd)

Table 1-2: PVP Resin

APPENDIX 1: (Cont'd)

Table 1-3: SMM (MDI-PPO-BAL)

TEMPERATURE, deg C

Table 1-5: PES-PVP Film Cast on a Glass Slide

(a) 4 wt% MDI-PPO-FSO1 in PES (b) 4 wt% MDI-PPO-FS02 in PES

(c) 4 wt% MDI-PPO-FS03 in PES

CONTACT ANGLES IN UNDERWATER MEASUREMENTS

Table K-1 : PES

Angle Measured

air advancing

Sample #

- -- -- - --

Range of Ave. Contact Stand. Covariance Contact Radius Angle Deviation

(mm) (degree) (degree)

1.67 - 2.12 150.8 1.7 -0.0002

2.02 - 2.07 154.2 0.5 0.003

1.92 - 2.13 157.0 0.8 0.004

1.19 -2.09 157.3 0.5 0.0009

1.42 - 1.65 158.5 0.7 0.002

1.39 - 1.62 158.6 0.5 0.0007

1.37 - 1.67 159.0 0.4 0.0006

- - --

air receding 1 a2 1 2.47 - 1.57 1 107.8 1 1.6 1 0.03

Table K-2: PES with 4wt% MDI-PPO-BAL

Angle Measured

air advancing

air receding

Sample #

a 1

a5

b 1

b 3

. b5

a2

a6

Range of Contact Radius

(mm)

2.06 - 2.57

1.97 - 2.33

2.01 - 2.27

2.04 - 2.26

. 1.79-2.23

1.68 - 1.30

1.76 - 1.48

Ave. Contact Angle

(degree)

133.9

139.1

143.3

144.2

, 144.2

67.3

Stand. Deviation (degree)

0.5

0.4

1 .O

0.4

0.8

Covariance

0.003

0.003

0.007

-0.00 1

-0.0 I

1 .O --

0.05

0.00 72.5 1.5

Angle Measured 1 Sample

air advancing 1 al

air receding 1 a2

Range of Contact Radius

(mm)

Ave. Contact Stand. Covariance Angle Deviation

(degree)

Table K-4: PES with 4 wt% MDI-PPO-FSO2

Angle Measured

air advancing

air receding

Sarnple #

a3

a5

a7

b 1

b3

b5

a4

a6

a8

b4

Range of Contact Radius

(mm)

1.49 - 1.65 1.40 - 1.63 1.90 - 2.1 1 2.00 - 2.05 1.80 - 2.3 1

1.40 - 1.69 1.73 - 1.44 1.80 - 1.42 1.69 - 1 .O8 2.0 1 - 1.75

Ave. Contact Angle

(degree)

153.0

145.8

143.1

146.0

147.8

148.1

67.97

72.0

74.8

70.4

Stand. Deviation (degree)

0.7

1.4

1.2

0.9

1.2

0.9

1.3

1.7

0.8

0.3

Covariance

0.0003

0.007

0.007

0.0002

0.006

0.007

-0.0 1

0.0 1

-0.0 1

0.003

Angle Measured

air advancing l---

1 air receding

Table K-6: PES with 4 wt% MDI-PCL-BAL

Angle Measured

Covariance

-0.00 1

-0.006

0.0004

0.0003

0.00 1

Sarnple #

a 1

a3

a5

b5

c 1

NIA

Sarnple Range of

air advancing 1 al 1 2.10 - 2.47

Stand. Deviation (degree)

0.6

0.8

0.3

0.5

0.5

Range of Contact Radius

(mm)

1.56 - 1.71

1.35 - 1.64

1.71 - 1.80

1.69 - 1.84

1.64 - 1.83

Ave. Contact Angle

(degree)

146.1

148.5

148.6

148.7

149.6

Ave. Contact Stand. Covariance Angle Deviation

(degree) (degree)

136.0

136.1

134.7

air receding

O .4

0.2

0.4

- -

b5

c 1

~3

~5

d 1

d3

d5

a2

a4

-0.003

-0.0003

0.003

1.41 - 1.69

1.50 - 1.73

1.49- 1.81

1.49 - 1.90

1.47 - 1.79

1.42 - 1.88

1.59 - 1.90

2.51 -2.19

2.41 - 2.14

144.2

152.1

151.6

151.8

152.8

151.8

150.7

63.2

65.5

0.7

0.7

1 .O

0.005

-0,004

-0.009

0.8

0.7

0.8

O -6

1.1

0.5

-0.008

0.0007

-0.006

-0.0007

-0.002

0.00 1

air receding 1 NIA 1

Angle Measured

air advancing

Sample #

al

Range of Contact Radius

(mm)

1 .72 - 1.85

Ave. Contact Angle

(degree)

15 I .O

Stand. Deviation (degree)

0.4

Covariance

0.00 1

Table L-1 : Percent P-Selectin Expression for SMM Modifications Containing PPO

Material Experiment 1 Experiment 2 Experiment 3 Average (% P-Selectin) ('/O P-Selectin) (% P-Selectin)

Resting Blood 3.94 7.20 5.17 5.44

EDTA 7.59 8.0 1 6.82 7.47

Standard Deviation

SFLLRN 1 142.49 f 71.21 1 73.67 1 9 5 . 7 9

PES 5.32 5.57 6.3 1 5.73

MDI-PPO-BAL in PES 5.34 7.30 5.4 1 6.02

MDI-PPO-FSOI in PES 5.45 6.15 5.33 5 -64

MDI-PPO-FS02 in PES 5.32 5.62 7.65 6.20

MDI-PPO-FS03 in PES 5.65 6.74 5.38 5.92

Table L-2: Percent P-Selectin Expression for SMM Modifications Containing PCL

Material Experiment 1 Experiment 2 Experiment 3 ( O h P-Selectin) ( O h P-Selectin) ( O h P-Selectin)

5.10 5.27 1.94

5.7 1 7.54 6.94

83.73 59.62 93.49

5.23 4.67 2.56

Standard Deviation

Average

Resting Blood

EDTA

SFLLRN

PES 1 5.97 1 4.09 1 3.06 1 4 . 3 7

MDI-PCL-BAL in PES 1 8.70 1 5.12 1 2.49 1 5.44

MDI-PCL-FSOZ in PES 1 8.39 1 5.23 1 3.71 1 5.78

MDI-PCL-FS03 in PES 1 7.74 1 4.42 1 3.08

Table L-3: Percent Microparticles for SMM Modifications Containing PPO

Table L-4: Percent Microparticles for SMM Modifications Containing PCL

Experiment 3 (% MPs)

8.00

3.50

14.59

13.33

15.04

19.01

14.33

I6.17

19.92

13.50

Average

19.32

16.44

43.3 1

28.69

34.62

35.37

3 1.80

32.06

34.99

3 1 .O9

Material

Resting Blood

EDTA

SFLLRN

Silastic@

PE

PES

MDI-PPO-BAL in PES

MDI-PPO-FSOI in PES

MDI-PPO-FSO2 in PES

MDI-PPO-FS03 in PES

Standard Deviation

8.12

9.63

20.68

12.70

14.92

1 1.76

12.45

1 1.30

10.72

12.61

Material

Resting Blood

EDTA

SFLLRN

SilasticB

PE

PES

MDI-PCL-BAL in PES

MDI-PCL-FSOI in PES

MDI-PCL-FS02 in PES

MDI-PCL-FS03 in PES

Experiment 1 (% MPs)

26.64

26.60

62.46

44.44

5 1.23

46.13

42.47

41.50

44.00

42.44

Experiment 3 (% MPs)

4.70

3 -30

18.96

10.17

10.82

13.71

8.86

10.69

18.96

1 7.29

Experirnent 2 (% MPs)

23.3 1

19.22

52.87

28.29

37.59

40.96

38.60

38.5 1

41 .O5

37.32

Experiment 1 (% MPs)

12.34

8.95

43.48

16.46

28.89

2 1.57

3 1 .O8

21.91

27.36

28.52

Average

9.20

5.86

25 .O0

14.04

19.49

16.77

18.55

15.93

20.70

19.99

Experiment 2 ('10 MPs)

10.56

5.32

12.55

15.48

18.76

15.04

15.70

15.19

15.77

14.17

Standard Deviation

3.26

2.34

13.33

2.76

7.40

3.43

9.29

4.6 1

4.89

6.16

Table L-5 : CD 1 1 b Upregulation for SMM Modifications Containing PPO

Material Experiment 1 Experiment 2 (units) (units)

Resting Blood 187.57 144.40

EDTA 60.43 39.06

PMA 762.92 422.1 1

Silastic@ 100.97 78.17

Experiment 3 Average (units)

294.84 208.94

39.24 46.24

393.43 526. f 5

234.40 137.85

I -

PES 1 404.15 1 269.97 1 382.59 1 352.24 -- - -- - - - -- -

MDI-PPO-BAL in PES 3 10.81 238.12 272.20 273.7 1

MDI-PPO-FSO 1 in PES 437.49 192.58 408.72 346.26

MDI-PPO-FSO2 in PES 59 1 .O0 288.09 403.93 427.67

MDI-PPO-FS03 in PES 597.1 1 325.62 440.15 454.29

Table L-6: CD 1 1 b Upregulation for SMM Modifications Containing PCL - --- -- - - -

Material Experiment 1 Experiment 2 Experiment 3 (units) (units) (units)

Resting Blood 155.37 183.15 140.1 1

EDTA 32.08 3 1.30 32.24

PMA 502.62 151.68 486.24

PES 1 307.50 1 356.98 1 372.44

MDI-PCL-EAL in PES 244.25 267.33 286.44

MDI-PCL-FSO 1 in PES 250.44 263.03 377.46

MDI-PCL-FS02 in PES 437.14 4 16.08 539.68

1 MDI-PCL-FS03 in PES 1 424.81 1 363.97 1 506.48

Standard Deviation

Average 1 Standard 1 Deviation

Table L-7: Platelet Counts for SMM Modifications Containing PPO

PES 1 10.00 1 86.00 1 85.00 193.67 ( 11.56 1

Standard Deviation

12.81

20.70

Table L-8: Platelet Counts for SMM Modifications Containing PCL

Material

Resting Blood

EDTA

. - -

MDI-PPO-BAL in PES

MDI-PPO-FSOI in PES

Standard Deviation

1 1 .O0

Experiment 2 (#)

1 12.00

108.00

Experiment 1 (#)

142.00

155.00

Material ( Erperiient 1

- - -

1 1 1.00

12 1 .O0

Average

Experiment 3 (#)

135.00

115.00

R e s t i n ~ Blood 1 125.00

Average

129.67

126.00

-

75 .O0

76.00

EDTA 1 117.00

- - -

PES 1 100.00

-

104.00

109.00

- - -

MDI-PCL-FS03 in PES 84.00

-

96.67

102.00

-

MDI-PCL-BAL in PES

MDI-PCL-FSOI in PES

--

15.58

19.03

85 .O0

9 1 .O0

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