Physically Crosslinked Polyurethane Hydrogels For Biomedical Applications

PHYSICALLY CROSSLINKED POLYURETHANE HYDROGELS
FOR BIOMEDICAL APPLICATIONS
(Spine title: Linear Polyurethane Hydrogel Biomaterials)
(Thesis format: Integrated-Article)
by
Alpesh Patel
Graduate Program
In
Chemical and Biochemical Engineering
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
The School of Graduate and Postdoctoral Studies
The University of Western Ontario

London, Ontario, Canada
©Alpesh Patel 2010

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Carada
THE UNIVERSITY OF WESTERN ONTARIO
SCHOOL OF GRADUATE AND POSTDOCTORAL STUDIES
CERTIFICATE OF EXAMINATION
Supervisor Examiners
Dr. Kibret Mequanint Dr. Heather Sheardown
Advisory Committee Dr. Leo Lau
Dr. Amin S. Rizkalla Dr. Jose Herrera
Dr. Paul A. Charpentier Dr. Lars Rehmann
Thesis by
Alpesh Patel
Entitled:
PHYSICALLY CROSSLINKED POLYURETHANE HYDROGELS FOR

BIOMEDICAL APPLICATIONS
is accepted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Date
Chair of the Thesis Examination Board
11
ABSTRACT
The ability of macromolecules to undergo substantial swelling when exposed to

aqueous environment constitutes the generic definition of hydrogels. The biomedical
applications of hydrogels (the ability to release drugs in a sustained manner, the high
permeability to allow metabolic products to be removed, the slippery effect that reduces
frictional irritation to the surrounding tissues and low interfacial tension with body fluids
that can reduce the tendency of proteins to be adsorbed; and their ability to be fabricated
into a large number of shapes) are linked to their swelling ability. Because of these
properties, hydrogels find valuable applications for suture and catheter coatings, contact
lenses, drug delivery vehicles, scaffolds for tissue engineering and artificial organs.
Because linear hydrogels tend to dissolve rather than swell in water and possess poor
mechanical properties, most hydrogels are chemically crosslinked which results
processing difficulties. In view of this, it would be desirable to design linear hydrogel
biomaterials that cannot be dissolved in water but can be processed using organic
solvents. Such hydrogels are beneficial for ease of fabrication and post-process
modifications.
The main objective of the research documented herein was to synthesize novel
physically crosslinked polyurethane hydrogels for biomedical applications. In order to
accomplish this objective, the concept of iniferter (initiator, transfer agent and
terminator), which is a known controlled radical polymerization technique, was utilized.
One important advantage offered by polymeric iniferters is the possibility that
polycondensation polymers could be further reacted with vinyl monomers to produce
novel block copolymers with interesting properties. Using this approach, a series of linear
polyurethane block copolymer hydrogels incorporating hydrophilic monomers, «-vinyl
pyrrolidone, acrylamide and 2-hydroxyethyl methacrylate were synthesized and
characterized. First, two novel polyurethane macroiniferters based on tetraphenyl ethane
diol and A^jV'-diethyl-7ViA^'-bis(2-hydroxyethyl)thiuram disulfide were successfully
synthesized. These macroiniferters were then used to initiate the polymerization of
iii
hydrophilic vinyl monomers in order to obtain multi-block copolymers. A linear
molecular weight increase with copolymerization time and monomer conversion was
observed indicating that the process followed a controlled radical polymerization. The
linear polyurethane hydrogels were characterized using FTIR, 'H NMR, DSC, swelling
and cell interaction studies and; demonstrated that the developed technique is versatile for
preparing physically crosslinked polyurethane-based hydrogels for biomedical
applications. In order to control the segmental lengths of the hydrophilic block that tune
the swelling ability of the current hydrogels, detailed kinetic analysis of polyurethane
macroiniferter with respect to the comonomer addition have been conducted. Further, the
surface of the prepared hydrogels was modified using fibronectin in order to improve
their cell attachment for tissue engineering application. Such biomimetic polyurethane-
based hydrogels showed good cell interactions.
Keywords: physically crosslinked hydrogels, polyurethanes, macroiniferters, swelling

kinetics, macroiniferter polymerization kinetics, cell-material interaction, surface
modification.
IV
CO-AUTHORSHIP
The work documented in this dissertation was a joint effort between the student,
Alpesh Patel and the supervisor, Dr. Kibret Mequanint. Their specific contributions for
each Chapter are summarized below:
Chapter 1: Alpesh Patel - wrote the Chapter; Dr. Kibret Mequanint - reviewed
and edited the Chapter.
Chapter 3: Alpesh Patel - designed experiments, synthesized and characterized
materials, organized and analyzed data, wrote the Chapter; Dr. Kibret Mequanint -
guided on designing the experiments and analyzing data, reviewed and edited the
manuscript.
manuscript.
manuscript.
Chapter.
v
II «fr : II
fa^m ^ ? T ™ F T II
"It cannot be stolen by thieves

Nor can it be taken away by kings
It cannot be divided among brothers

It does not cause a load on your shoulders
The more we spend the more it increases

Such is the wealth of knowledge "
The Vedas
VI
DEDICATION
To my unconditional lovers, my angels, Zal and Zankhana

who always bring a smile on my face.
Vll
ACKNOWLEDGEMENTS
I, Alpesh Patel, take this opportunity to express my deepest gratitude for those
individuals who directly or indirectly helped me to make my vision realized.
First and foremost, I would like to sincerely thank my supervisor, Dr. Kibret
Mequanint for giving me the opportunity to work under his guidance and having a trust in
my abilities. I am also deeply thankful to him for offering his valuable advice, guidance
and support throughout this adventurous and very important journey of my life. I would
like to thank my Advisory Committee members, Drs. Paul Charpentier and Amin
Rizkalla, for their valuable suggestions and constructive criticism throughout my
research.
I also sincerely thank Dawit Seifu, Dawid Martyniak and Dr. Shigang Lin for
their help with cell work and Darryl Knight for his assistance with NMR and GPC
studies.
I am also grateful to the staff of the Department of Chemical and Biochemical
Engineering for providing me all necessary assistance during my studies. It would be
improper not to mention the facility rendered by the Western libraries staff for giving me
unlimited access to their library resources.
Vlll
I am very thankful to the Natural Sciences and Engineering Research Council of
Canada (NSERC) and Western Graduate Research Scholarships for offering financial
assistance during my doctoral studies.
This acknowledgement would not be complete without thanking my friends and
my family for their continuous support and encouragement.
Above all, I wholeheartedly thank my mighty God, Lord Yogeshwer, for always
being with me and giving me the vision, spirit, power and endurance to complete this
exciting research.
ix
TABLE OF CONTENT
CERTIFICATE OF EXAMINATION ii
ABSTRACT iii
CO-AUTHORSHIP v
DEDICATION vii
ACKNOWLEDGEMENTS viii
TABLE OF CONTENT x
LIST OF FIGURES xiv
LIST OF TABLES xvii
LIST OF ABBREVIATIONS xviii
1. Introduction 1
1.1 Overview 1
1.2 Research Outline 4
1.3 References 5
2. Literature survey 8
2.1 Hydrogels 8
2.1.1 Classifications of hydrogels 9
2.2 Stimuli responsive hydrogels 12
2.2.1 pH responsive hydrogels 14
2.2.2 Temperature responsive hydrogels 15
2.2.3 Glucose responsive hydrogels 17
2.2.4 Protein responsive hydrogels 17
2.2.5 Antigen-responsive hydrogels 18
2.3 Water in hydrogels 18
2.3.1 Thermodynamics of hydrogels swelling 19
2.3.1.1 Determination of A(j.mixUre 20
2.3.1.2 Determination of Augiastic 23
2.3.2 Kinetics of hydrogels swelling 27
2.4 Suitability of hydrogels as biomaterials 29
2.4.1 Hydrogels for drug delivery applications 30
2.4.2 Hydrogels for cell encapsulation 31
2.4.3 Hydrogels for tissue engineering scaffolds 31
2.4.4 Hydrogels for contact lens application 32
2.5 Polyurethanes 33
2.5.1 Polyurethane synthesis 33
2.5.2 Polyurethanes as biomaterials 36
2.6 Poly(vinyl pyrrolidones) 38
2.7 Polyacrylamides 39
2.8 Poly(2-hydroxyethyl methacrylates) 40
2.9 Iniferter method 41
x
2.10 Study rationale 44
2.11 References 45
3. Physically crosslinked polyurethane-6/oc£-poly(/V-vinyl pyrrolidone) hydrogels from

tetraphenyl ethane-based polyurethane macroiniferters 57
3.1 Introduction 57
3.2 Experimental part 60
3.2.1 Materials 60
3.2.2 Synthesis of TPED 60
3.2.3 Synthesis of PUMI 61
3.2.4 Synthesis of PU-6-PVP 61
3.2.5 Characterization methods 62
3.2.5.1 Spectrometric analyses 62
3.2.5.2 Gel permeation chromatography 62
3.2.5.3 Differential scanning calorimetry 63
3.2.5.4 Equilibrium water content and swelling kinetics 63
3.2.5.5 Preparation of cell culture specimens 64
3.2.5.6 Cell culture on hydrogel surfaces 64
3.3 Results and discussion 65
3.3.1 Syntheses of macroiniferter and block copolymers 65
3.3.2 The effect of copolymerization time on molecular weight 71
3.3.3 Water transport properties of PU-6-PVP hydrogels 72
3.3.4 Thermal behavior of macroiniferter and block copolymers 74
3.3.5 Vascular smooth muscle cells interaction with PU-6-PVP hydrogels 75
3.4 Conclusions 77
3.5 References 77
4. Physically crosslinked PU-6-PVP and PU-6-PAAm hydrogels from dithiocarbamate -

based polyurethane macroiniferters 81
4.1 Introduction 81
4.2.1 Materials 84
4.2.2 Synthesis of DHTD 84
4.2.4 Synthesis of PU-6-PAAm 85
4.2.5 Synthesis of PU-6-PVP 86
4.2.6.1 Spectroscopic analyses 87
4.2.6.3 Thermogravimetric analysis 88
4.2.6.4 Equilibrium water content and swelling kinetics 88
4.2.6.5 pH dependent swelling study on PU-6-PAAm hydrogels 90
4.3.1 Synthesis of macroiniferter and block copolymers 90
XI
4.3.2 Water transport properties of polyurethane hydrogels 99
4.3.3 The effect of pH on swelling of PU-Z>-PAAm hydrogels 104
4.4 Conclusions 108
4.5 References 108
5. The kinetics of dithiocarbamate-madiated polyurethane macroiniferter 113

5.1 Introduction 113
5.2.1 Materials 117
5.2.2 Synthesis of DHTD and PUMI 117
5.2.3 Synthesis of PU-6-PMMA 117
5.3.1 Syntheses of macroiniferter and block copolymers 120
5.3.2 Reaction kinetics of PUMI 123
5.4 Conclusions 135
5.5 References 136
6. Dicyclohexylmethane diisocyanate-based physically crosslinked polyurethane

hydrogels and their surface modification 140
6.1 Introduction 140
6.2.1 Materials 142
6.2.2 Synthesis of DHTD 143
6.2.4 Synthesis of PU-6-PHEMA 143
6.2.5 Modification of PU-6-PHEMA copolymer surface with fibronectin 144
6.2.6.1 Spectroscopic analysis 145
6.2.6.3 Swelling study 146
6.2.6.4 Scanning Electron Microscopy 147
6.2.6.5 Thermal analysis 147
6.2.6.6 Protein adsorption study 147
6.2.6.7 Preparation of cell culture samples 148
6.2.6.8 Cell culture study 148
6.3.1 Synthesis of macroiniferter and block copolymers 149
6.3.2 Thermal behaviour of macroiniferter and block copolymers 152
6.3.3 Surface analysis and swelling properties of macroiniferter and block
copolymers 154
6.3.4 Short term cell culture study 156
xii
6.4 Conclusions 161
6.5 References 161
7. General conclusions and future directions 165

7.1 Summary 165
7.2 Strengths and limitations 167
7.3 Future directions 169
7.4 References 169
APPENDIX 171
CURRICULUM VITAE 183
xiii
LIST OF FIGURES
List of schemes
SCHEME 2.1: Schematic diagram of segmented polyurethane synthesis using two-step
step growth polymerization process 35
SCHEME 2.2: The polymerization mechanism of iniferter system 42
SCHEME 3.1: Schematic diagram of TPED synthesis 61
SCHEME 3.2: Schematic diagram of PUMI and PU-6-PVP syntheses 66
SCHEME 4.1: Schematic diagram of DHTD synthesis 85
SCHEME 4.2: Schematic diagram of PUMI, PU-6-PAAm and PU-6-PVP syntheses.... 87
SCHEME 5.1: Chemical structure of PU-ZJ-PMMA 119
SCHEME 5.2: The polymerization mechanism of DC-based macroiniferter 120
SCHEME 6.1: Schematic diagram of PUMI and PU-6-PHEMA syntheses 144
SCHEME 6.2: Schematic diagram of the modification of PU-6-PHEMA copolymers with

bovine fibronectin using CDI conjugation method 145
List of figures
FIGURE 2.1: Classifications of hydrogels 10
FIGURE 2.2: Classifications of smart hydrogels 13
FIGURE 2.3: Classifications of iniferters 43
FIGURE 3.1: FTIR spectra of TPED, PUMI and PU-6-PVP 67
FIGURE 3.2: 'H-NMR spectrum of TPED 68
FIGURE 3.3: 'H-NMR spectrum of PUMI 68
FIGURE 3.4: 'H-NMR spectrum of PU-6-PVP 70
FIGURE 3.5: GPC curves of PU-Z?-PVPs for different copolymerization times 71
xiv
FIGURE 3.6: Swelling kinetics of physically crosslinked PU-6-PVP copolymer
hydrogels at different reaction time at 22°C 73
FIGURE 3.7: DSC curves of (a) PUMI and (b) PU-6-PVP xerogel 75
FIGURE 3.8: Vascular smooth muscle cells adhesion and spreading on PU-6-PVP
hydrogels 76
FIGURE 4.1: FTIR spectra of DHTD, PUMI, PU-6-PVP and PU-6-PAAm 91
FIGURE 4.2: (a) 'H-NMR spectrum of DHTD 93
FIGURE 4.2: (b) 13C-NMR spectrum of DHTD 93
FIGURE 4.3: (a)'H-NMR spectrum of PUMI 94
FIGURE 4.3: (b) 'H-NMR spectrum of PU-6-PVP 95
FIGURE 4.3: (c)'H-NMR spectrum of PU-6-PAAm 96
FIGURE 4.4: Average molecular weight and PDI of PU-6-PVP copolymers as a function
of copolymerization time 97
FIGURE 4.5: Swelling isotherms of physically crosslinked PU-6-PVP hydrogels as a

function of immersion time at22°C 100
FIGURE 4.6: Swelling isotherms of physically crosslinked PU-6-PAAm hydrogels as a

function of immersion time at 22°C 101
FIGURE 4.7: Swelling isotherms of physically crosslinked PU-6-PAAm hydrogels at

different pH at 22°C 105
FIGURE 4.8: FTIR spectra of physically crosslinked PU-6-PAAml2 hydrogels at

different pH 106
FIGURE 4.9: TGA thermographs and related derivative thermographs of PUMI; PU-b-
PVP and PU-6-PAAm 107
FIGURE 5.1: 'H-NMR spectrum of PU-6-PMMA 122
FIGURE 5.2: Ln(M0/M) versus time plots for the MMA copolymerization with different
concentrations of PUMI in DMF under the induction of UV light 124
FIGURE 5.3: Molecular weight and monomer conversion plots versus time for the
copolymerization of MMA with different PUMI concentrations in DMF.
(a) [PUMI] = 15 g/L, (b) [PUMI] = 30 g/L and (c) [PUMI] = 40 g/L 127
xv
FIGURE 5.4: GPC elution curves for the PU-6-PMMA with different concentrations of
PUMI. (a) [PUMI] =15 g/L, (b) [PUMI] =30 g/L, (c) [PUMI] =40 g/L.. 130
FIGURE 5.5: Rate of polymerization versus time curves for MMA copolymerization with
different PUMI concentrations in DMF 132
FIGURE 5.6: Plots of rate of polymerizations as a function of PUMI concentrations for

various reaction times 133
FIGURE 5.7: TGA - primary and derivative mass losses for PUMI and PU-6-PMMA 135
FIGURE 6.1: FTIR spectra of PUMI, PU-6-PHEMA and Fn-g-PU-6-PHEMA 150
FIGURE 6.2: 'H-NMR spectrum of PU-6-PHEMA 151
FIGURE 6.3: GPC elution curves of PUMI and PU-6-PHEMA 152
FIGURE 6.4: DSC thermographs of PUMI and PU-6-PHEMA 153
FIGURE 6.5: TGA thermographs of PUMI and PU-6-PHEMA 154
FIGURE 6.6: Scanning electron microscopic images of PUMI (A-B) and PU-6-PHEMA
(C-D) surfaces 155
FIGURE 6.7: Swelling isotherms of PUMI and PU-6-PHEMA 156
FIGURE 6.8: Flouroscent microscopic images of VSMC interaction with PUMI (A-B)
and PU-6-PHEMA (C-D) surfaces 157
FIGURE 6.9: Fibronectin adsorption data of PUMI and PU-6-PHEMA 158
FIGURE 6.10: Fluorescent microscopic images of VSMC interaction with PUMI (A-B),
PU-6-PHEMA (C-D) and Fn-g-PU-6-PHEMA (E-F) surfaces 160
xvi
LIST OF TABLES
Table 2.1: Some commercial polyurethanes used in biomedical applications 37
Table 2.2: Biomedical devices fabricated from polyurethanes 37
Table 3.1: EWC and swelling kinetic parameter («) of PU-6-PVP hydrogels 74
Table 4.1: Molecular weights of PU-6-PVP block copolymers 98
Table 4.2: Water transport properties of PU-6-PAAm and PU-6-PVP hydrogels 102
Table 4.3: TGA analysis of PUMI, PU-6-PAAm and PU-6-PVP xerogels 107
Table 5.1: Block copolymerization of MMA with PUMI under UV radiation in DMF. 123
xvn
LIST OF ABBREVIATIONS
2D Two-dimensional
3D Three-dimensional
AAm Acrylamide
CDI l,l'-Carbonyldiimidazole
DBTDL Dibutyltin dilaurate
DC Dithiocarbamate
CDCb Deuterated-chloroform
DHTD iV,7^'-Diethyl-A/,A^'-bis(2-hydroxyethyl)thiuram disulfide
DI Deionized
DMF Dimethylformamide
DMSO-offi Deuterated dimethyl sulfoxide
DSC Differential scanning calorimetry
DTC Dithiocarbamyl
EDTA Ethylenediaminetetraacetic acid
EWC Equilibrium water content
FTIR Fourier transform infrared
GPC Gel permeation chromatography
xvin
Hi2MDI 4,4'-Dicyclohexylmethane diisocyanate
HCASMC Human coronary artery smooth muscle cells
HEMA 2-Hydroxyethyl methacrylate
LiBr Lithium bromide
MDI 4,4'-Diphenylmethane diisocyanate
MEK Methyl ethyl ketone
MgS04 Magnesium sulfate
MMA Methyl methacrylate
NMP jV-methyl pyrrolidone
NMR Nuclear magnetic resonance
NVP N-v'myl pyrrolidone
PBS Phosphate buffered saline
PDI Polydispersity index
PEO Polyethylene oxide
PS Polystyrene
PTMO 1000 Poly(tetramethylene

1000
PU Polyurethane
PU-6-PAAm Polyurethane-6/oc£-t
XIX
PU-6-PHEMA Polyurethane-6/ocA:-poly(2-hydrxyethyl methacrylate)
PU-6-PMMA Polyurethane-Z>/oc£-poly(methyl methacrylate)
PU-6-PVP Polyurethane-6/c>cA:-poly(JV-vinyl pyrrolidone)
PUMI Polyurethane macroiniferter
RP Rate of polymerization
SDS Sodium dodecyl sulfate
SEM Scanning electron microscope
TGA Thermogravimetric analysis
THF Tetrahydrofuran
TPED 1,1,2,2-Tetrapheny 1-1,2-ethanediol
UV Ultraviolate
VSMC Vascular smooth muscle cells
xx
1
CHAPTER
1
Introduction
1.1 Overview
A staggering number of medical devices, diagnostic and therapeutic products that
are designed to improve the health of mankind have exploited biomaterials as platform
technologies1. Defined as natural or synthetic materials (other than drugs) to treat,
augment, or replace any tissues, organ, or function of living tissues, biomaterials design
requires both materials and biological considerations. In addition to the mechanical
requirements, biomaterials have to accomplish some specific requirements, such as non-
toxicity, desired functionality, sterilizability and biocompatibility2. Despite the
widespread use of biomaterials in medicine, most biomaterials do not provide all of the
desired requirements to interact with biological systems. Therefore, there is an increasing
need to redesign current biomaterials or to develop new materials in order to overcome
limitations associated with fulfilling the above-mentioned requirements. Although the
term biomaterial includes metals and ceramics, polymers account for the vast majority. In
this last group, hydrogels, having considerable biocompatibility and similarity with tissue
components of the body, have demonstrated great potential as one of the most promising
groups of biomaterials2'3.
2
Hydrogels are crosslinked macromolecular network structures, which have the
ability to absorb large amounts of water without dissolving. Lower interfacial tension,
soft and tissue-like physical properties, higher permeability to undersized molecules and
release of entrapped molecules in a controlled manner made hydrogels to be explored in
different biomedical fields4'5.
The presence of chemical or physical crosslinking points within the network
maintains the three-dimensional integrity of hydrogels in their swollen state. In
chemically crosslinked hydrogels, the linear polymer chains are covalently bonded with
each other via crosslinking agents. Their usage is limited as the resulting network cannot
be reshaped and/or resized since the polymer is no longer soluble in solvents and heating
to melt-process can only degrade the polymer once crosslinking takes place. Moreover,
the crosslinking agents applied to develop strong hydrogel network systems are mainly
toxic. Thus, any unreacted crosslinking agents have to be leached out before the final
application. However, partially reacted toxic crosslinking agents have no possibility to be
completely leached out. In contrast, physically crosslinked hydrogels possess physical
junction domains associated with chain entanglements, hydrophobic interaction,
hydrogen bonding, crystallinity, and/or ionic complexation6. The presence of these
reversible crosslinking points allows solvent casting and/or thermal processing. The
interest for physically crosslinked hydrogel is obvious since the use of crosslinking
agents is avoided and they are beneficial for post-process bulk modification and ease of
fabrication7"11. Hydrophobic - hydrophilic block copolymers are one of the well-explored
and applied physically crosslinked polymers for various biomedical applications. The
3
major disadvantage of physically crosslinked hydrogels, however, is their weak
mechanical properties in the swollen state. This can be improved by using polyurethane
as a hydrophobic segment into hydrophobic-hydrophilic block copolymers. Due to the

1 ~)
excellent mechanical properties of polyurethanes , hydrogels based on their chemistry
are appealing for biomedical applications. Polyurethane-based hydrogels can form
strongly hydrogen bonded structures, allowing linear polymer systems to be designed
with tunable swelling and mechanical properties. Thus, along with the hydrophobic
interaction and chain entanglements, the presence of strong H-bonding between the
ether/ester and urethane groups into polyurethane can help to improve mechanical
12 13
properties ' .
Due to its known inertness and biocompatibility, polyethyleneoxide (PEO)-based
linear polyurethane hydrogels have been investigated by Petrini and co-workers14.
However, to design segmented polyurethanes, the available soft segments except PEO,
are hydrophobic and thus the variation in their properties related to the end-use of these
polyurethane hydrogels is limited. Thus in order to overcome this limitation we have used
an iniferter polymerization approach13 to develop polyurethane-based physically
crosslinked hydrogels. The iniferter (initiator, transfer agent and terminator) technique is
one of the controlled radical polymerization methods that has been first reported by Ostu
and Yoshida in 198215'16. Briefly, the term iniferter represents the group of compounds
that thermally or photochemically dissociates into free radicals to initiate the
polymerization, transfers the monomers to the growing polymer chains and eventually
participate in the reversible termination of the growing polymer chains. The presence of
4
reinitiation, propagation and reversible termination controls the molecular weight
distribution effectively17. The term macroiniferter means polymeric iniferter. In this
study, polyurethane-based macroiniferter systems have been developed and utilized to
prepare polyurethane-based linear hydrogel systems.
1.2 Research Outline
The overall objective of this research was to synthesize and to characterize
physically crosslinked polyurethane hydrogels for biomedical applications using the
macroiniferter technique. Such polyurethane macroiniferters (PUMI) have been
synthesized using a two step condensation polymerization method. In the first step, either
4,4'-Diphenylmethane diisocyanate (MDI) or 4,4'-Dicyclohexylmethane diisocyanate
(H12MDI) has been used as a diisocyanate and reacted with poly(tetramethylene oxide)
glycol (PTMO) of molecular weight 1000 to develop the polyurethane prepolymers. In
order to incorporate iniferter segments within the polyurethane backbone,
tetraphenylethane-based (TPE) and dithiocarbamate-based (DC) diols have been
successfully synthesized. These iniferter-based diols were then used as a chain extender
and reacted with polyurethane prepolymers to get the polyurethane macroiniferters. In
this study, three PUMI systems have been prepared. These are TPE-based PUMI using
MDI, DC-based PUMI using MDI and DC-based PUMI using H12MDI. Among these
PUMI systems, TPE-based PUMI system was used as a thermal macroiniferter system
whereas DC-based PUMI systems were utilized as photochemical macroiniferter systems.
In order to obtain multiblock copolymers, these macroiniferters have been used to initiate
the polymerization of hydrophilic vinyl monomers n-vinyl pyrrolidone, acrylamide and

5
2-hydroxyethyl methacrylate. Thus different novel polyurethane-based linear hydrogel
systems have been prepared with tuned water uptake. The current hydrogels have been
extensively characterized using FTIR, *H NMR, DSC, and TGA. Further, swelling and
cell interaction studies have been conducted to demonstrate their utility as a new class of
physically crosslinked polyurethane-based hydrogels18'19. In order to control the
segmental lengths of different blocks that tune the swelling ability of the current
hydrogels, detailed kinetic analyses of DC-based polyurethane macroiniferter have been
conducted with respect to comonomer addition . Moreover, bovine fibronectin were
conjugated on the hydrogels surfaces in order to improve their cell attachment for
scaffolding application. Such biofuctionalized polyurethane-based hydrogels showed
improved cell attachment and uniform cell spreading in short term cell culture
experiments.
This thesis is divided into 7 Chapters. First, a detailed literature review is
presented in Chapter 2. The main research findings are presented in Chapters 3-6. Finally,
conclusions, general discussion and future directions for this research are presented in
Chapter 7.
1.3 References
1. Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R., Hydrogels in biology
and medicine: From molecular principles to bionanotechnology. Advanced
Materials 2006, 18,(11), 1345-1360.
2. Rosiak, J. M.; Yoshii, F., Hydrogels and their medical applications. Nuclear
Instruments & Methods in Physics Research Section B: Beam Interactions with
Materials and Atoms 1999, 151, (1-4), 56-64.
6
Rogero, S. O.; Malmonge, S. M.; Lugao, A. B.; Ikeda, T. I.; Miyamaru, L.; Cruz,
A. S., Biocompatibility study of polymeric biomaterials. Artificial Organs 2003,
27, (5), 424-427.
Yaszemski, M. J.; Trantolo, D. J.; Lewandrowski, K. U.; Hasirci, V.; Altobelli, D.

E.; Wise, D. L. Tissue Engineering and Novel Delivery Systems. 2004; p 423-461.
Slaughter, B. V. K., S.S.; Fisher, O.Z.; Khademhosseini, A.; Peppas, N.A.,

Hydrogels in regenerative medicine. Advanced Materials 2009, 21, 3307-3329.
Park, J. H.; Bae, Y. H., Hydrogels based on poly(ethylene oxide) and

poly(tetramethylene oxide) or poly(dimethyl siloxane): synthesis,
characterization, in vitro protein adsorption and platelet adhesion. Biomaterials
2002,23, (8), 1797-1808.
Li, S. M.; Molina, I.; Martinez, M. B.; Vert, M., Hydrolytic and enzymatic
degradations of physically crosslinked hydrogels prepared from PLA/PEO/PLA
triblock copolymers. Journal of Materials Science-Materials in Medicine 2002,
13, (1), 81-86.
Hennink, W. E.; van Nostrum, C. F., Novel crosslinking methods to design

hydrogels. Advanced Drug Delivery Reviews 2002, 54, (1), 13-36.
Adams, M. L.; Lavasanifar, A.; Kwon, G. S., Amphiphilic block copolymers for
drug delivery. Journal of Pharmaceutical Sciences 2003, 92, (7), 1343-1355.
Kubo, M.; Matsuura, T.; Morimoto, H.; Uno, T.; Itoh, T., Preparation and
polymerization of a water-soluble, nonbonding crosslinking agent for a
mechanically crosslinked hydrogel. Journal of Polymer Science Part A: Polymer
Chemistry 2005, 43, (21), 5032-5040.
Liu, Y.; Vrana, N. E.; Cahill, P. A.; McGuinness, G. B., Physically crosslinked
composite hydrogels of PVA with natural macromolecules: structure, mechanical
properties, and endothelial cell compatibility. Journal of Biomedical Materials
Research Part B: Applied Biomaterials 2009, 90B, (2), 492-502.
Lamba, N. M. K.; Woodhouse, K. A.; Cooper, S. L., Polyurethanes in Biomedical

Applications. CRC Press, New York, D.C.: 1998.
Mequanint, K.; Patel, A.; Bezuidenhout, D., Synthesis, swelling behavior, and
biocompatibility of novel physically cross-linked polyurethane-6/ocA:-
poly(glycerol methacrylate) hydrogels. Biomacromolecules 2006, 7, (3), 883-891.
Petrini, P.; Tanzi, M. C; Moran, C. R.; Graham, N. B., Linear poly(ethylene

oxide)-based polyurethane hydrogels: Polyurethane-ureas and polyurethane-
7
amides. Journal of Materials Science-Materials in Medicine 1999, 10, (10-11),

635-639.
15. Otsu, T., Iniferter concept and living radical polymerization. Journal of Polymer
Science Part A: Polymer Chemistry 2000, 38, (12), 2121-2136.
16. Otsu, T.; Yoshida, M., Role of initiator-transfer agent-terminator (iniferter) in

radical polymerizations - Polymer design by organic disulfides as iniferters.
Makromolekulare Chemie-Rapid Communications 1982, 3, (2), 127-132.
17. Otsu, T.; Matsumoto, A., Controlled synthesis of polymers using the iniferter
technique: Developments in living radical polymerization. Microencapsulation -
Microgels - Iniferters 1998,136, 75-137.
18. Patel, A.; Mequanint, K., Novel physically crosslinked polyurethane-WocA:-

poly(vinyl pyrrolidone) hydrogel biomaterials. Macromolecular Bioscience 2007,
7, (5), 727-737.
19. Patel, A.; Mequanint, K., Syntheses and characterization of physically crosslinked
hydrogels from dithiocarbamate-derived polyurethane macroiniferter. Journal of
Polymer Science Part A: Polymer Chemistry 2008, 46, (18), 6272-6284.
20. Patel, A.; Mequanint, K., The kinetics of dithiocarbamate-mediated polyurethane-

Z>/oc&-poly(methyl methacrylate) polymers. Polymer 2009, 50 (19), 4464-4470.
8
CHAPTER
2
Literature Survey
Overview: As discussed in the introduction Chapter, the overall objective of this work is
to synthesize physically crosslinked linear polyurethane hydrogels. Thus, detailed
literature survey on hydrogels is presented here. Since the area of hydrogel research is
very diverse, this Chapter specifically focuses on relevant hydrogel materials including
polyvinyl pyrrolidone), polyacrylamide andpoly(2-hydroxyethyl methacrylate). It is also
important to understand polyurethane biomaterials in common use and; a brief review of
polyurethane biomaterials is discussed. Finally, the iniferter polymerization method to be
used in this study is surveyed in brief at the end of the Chapter.
2.1 Hydrogels
Hydrogels are three-dimensional (3D) materials with the ability to absorb large
amounts of water while maintaining their dimensional stability. The 3D integrity of
hydrogels in their swollen state is maintained either by physical or chemical crosslinking.
In the absence of crosslinking points, hydrophilic linear polymer chains dissolve in water
due to the thermodynamic compatibility of the polymer chains and water. However, in
the presence of crosslinking points, this force is counter balanced by the retractive force
of elasticity, induced by the crosslinking points of the network. Swelling reaches at an
equilibrium point as these forces becomes equal1. The amount of water absorbed in
hydrogels is related to the presence of specific groups such as -COOH, -OH, -CONH2, -
CONH-, and -SO3H. Capillary effect and osmotic pressure are other variables that also
influence the equilibrium water uptake of hydrogels2.

9
Following the pioneering work of Wichtrle et al.3 on crosslinked PHEMA
hydrogels as contact lenses, hydrogels have been of great interest as potential biomaterial
for cell encapsulation, drug delivery system, contact lenses, wound dressing,
immunoisolation, tissue engineering scaffolds, soft tissue replacement and other related
applications4'5.
2.1.1 Classifications of hydrogels
Depending on the preparation methods, ionic charges, sources, nature of swelling
with changes in the environment, rate of biodegradation or the nature of crosslinking,
hydrogels can be classified in several ways. A detailed classification of hydrogels is
presented in Figure 2.1 " .

10
Nonionic Cationic Anionic Ampholytic

Hydrogel Hydrogels Hydrogels Hydrogels
Ionic charge Smart Hydrogels
Biodegradable
Hydrogels
Conventional
Simple hydrogels
_ Biodegrad- Physical
Non-biodegradable ability properties
Hydrogels
Natural
Hydrogels
Hydrogels
Physically Cross-
linked Hydrogels Hybrid
Hydrogels
Crosslinking Source
Chemically Cross-
linked Hydrogels Synthetic
Hydrogels
Preparation
method
Homopolymers Copolymers Interpenetrating

Polymers
Triblock Copolymers Multiblock Copolymers
FIGURE 2.1: Classifications of hydrogels.
Among all, one of the important classifications is based on their crosslinking
nature. The network stability of hydrogels in their swollen state is due to the presence of
either chemical or physical crosslinking. Chemically crosslinked hydrogels are also
known as thermosetting hydrogels or permanent gels. They cannot be dissolved in any

11
solvents unless the covalent crosslinks are cleaved. Moreover, they cannot be reshaped
through heat melting. They can be prepared using any of these methods:
• Copolymerizing hydrophilic monomers with crosslinkers.
• Crosslinking of water-soluble polymer segments with di and/or multi functional
crosslinkers or with irradiation method (UV, y-irradiation and electron beam).
The utility of chemically crosslinked hydrogels is often limited by the lack of
processibility and post-process modifications. Because of this, shaping is carried out
along with their polymerization reaction step. Moreover the crosslinking agents used to
prepare hydrogels are highly toxic and the residues must be completely removed before
their use as biomaterials.
Physically crosslinked hydrogels, on the other hand, maintain their physical
stability due to the presence of reversible physical junction domains associated with
hydrogen bonding, hydrophobic interaction, chain entanglements, crystallinity, and/or
ionic complexation9"11. Physically crosslinked hydrogels are also known as thermoplastic
hydrogels or temporary gels. Swelling of these hydrogels is mostly dependent on the
thermodynamic parameters such as temperature, pH, salt type and/or ionic strength.
Changes in such parameters may increase or decrease their swelling. The presence of
reversible crosslinking points in physically crosslinked hydrogels allows solvent casting
and/or thermal processing. In the preparation of these hydrogels, the use of toxic
crosslinkers can also be avoided. Physically crosslinked hydrogels possess higher
compressive strength compared with the corresponding chemically crosslinked hydrogels

12
since the mechanical load can be more uniformly distributed through the crystallites of
10
the three-dimensional structure .
2.2 Stimuli responsive hydrogels
Stimuli responsive hydrogels are defined as hydrogels that undergo relatively
large and abrupt changes in their swelling behavior, network structure, permeability
and/or mechanical strength in response to small environmental changes. Stimuli
responsive hydrogels are also called intelligent, smart, or environmentally sensitive
hydrogels13'14. Stimuli responsive hydrogels could be further classified as either physical
or chemical stimuli responsive hydrogels as shown in Figure 2.2.

13
pH Glucose Antigen Enzyme Ligand

responsive responsive responsive responsive responsive
hydrogels hydrogels hydrogels hydrogels hydrogels
Chemical Biochemical
responsive responsive
hydrogels hydrogels
I
Smart
Hydrogels
i'
Physical
responsive
hydrogels
i '
1' i ' i ' i ' w ir
Pressure Tempera- Ultrasound Magnetic Electric Light (UV,

responsive ture responsive field field IR)
hydrogels responsive hydrogels responsive responsive responsive
lugtia
FIGURE 2.2: Classifications of smart hydrogels1
Chemical stimuli, such as pH, ionic factors and chemical agents, will change the
interactions between polymer chains or between polymer chains and solvent at the
molecular level. The physical stimuli, such as temperature, electric or magnetic fields,
and mechanical stress, will affect the level of various energy sources and alter molecular
interactions at critical onset points. Some systems have been developed to combine two
stimuli-responsive mechanisms into one polymer system, in the so-called dual responsive
polymer systems. Polyacrylic acid-co-polyvinyl sulfonic acid is an example of dual
responsive polymer system1 . Recently, biochemical stimuli have been considered as

14
another category, which involves the responses to antigen, enzyme, ligand, and other
biochemical agents13'14. Thus stimuli-responsive hydrogels are appealing biomaterials for
pharmaceutical, biotechnological and biomedical applications15.
2.2.1 pH responsive hydrogels
pH responsive hydrogels are made of polymeric backbones with ionic pendant
groups that can accept and/or donate protons in response to an environmental pH
change17. As the environmental pH changes, the degree of ionization in pH responsive
hydrogel is dramatically changed at specific pH known as pKa or pKb. This rapid change
in the net charge of ionized pendant groups causes abrupt volume transition by generating
electrostatic repulsive forces between ionized groups, which creates large osmotic
swelling force. There are two types of pH responsive hydrogels: anionic and cationic
1 8 00
hydrogels. In anionic hydrogels having pendent groups such as carboxylic " or sulfonic
acid23, deprotonation occurs when the environmental pH is above the pKa leading to the
ionization of the pendent groups. This in turn increases swelling of the hydrogel. On the
other hand, in cationic hydrogels containing pendent groups such as amine groups 4,
ionization takes place below the pKb and this increases the swelling due to an increase in
electrostatic repulsions .
Two major factors control the degree of swelling of ionic hydrogels. The first
factor is the properties of the polymers such as ionic charge, concentration and pKa or
pKb of the ionizable groups, degree of ionization, crosslink density as well as

15
hydrophilicity or hydrophobicity. The second factor is the properties of the swelling
medium like pH, ionic strength and the counterion and its valence25.
Polyvinyl sulfonic acid (PVSA)25, polymethacrylic acid (PMAA),26'27
polydiethylaminoethyl methacrylate (PDEAEMA)28 and polydimethylaminoethyl
methacrylate (PDMAEMA)29'30 and their copolymers are other examples of pH
responsive hydrogels.
2.2.2 Temperature responsive hydrogels
Temperature responsive hydrogels have gained considerable attention in the
biomedical field. Numerous researchers studied various applications of these hydrogels,
in the area of smart drug delivery system, injectable scaffolds, biosensors and intelligent
cell culture dishes13'31'32.
Temperature responsive hydrogels can be classified as positive or negative
temperature responsive systems. Physically crosslinked thermo sensitive hydrogels may
undergo sol-gel phase transitions instead of volume change at a critical solution
temperature13'15. Positive temperature responsive hydrogels show phase transition at
critical temperature called the upper critical solution temperature (UCST). Hydrogels
made from UCST based polymers shrink upon cooling them below their UCST. Negative
temperature responsive hydrogels have a lower critical solution temperature (LCST).
These hydrogels shrink upon heating above their LCST. Chemically crosslinked thermo
sensitive hydrogels undergo volume change rather than sol-gel transitions. Certain
16
molecular interactions, such as hydrophobic associations and hydrogen bonds play vital
role in the abrupt volume change of these hydrogels at the critical solution temperature
(CST). In the swollen state, water molecules form hydrogen bonds with polar groups of
polymer backbone within the hydrogels and organize around hydrophobic groups as
iceberg water. At the CST, hydrogen bonding between the polymer and water, compared
to polymer-polymer and water-water interactions, becomes unfavorable. This forces the
quick dehydration of the system and water is released out of the hydrogel with a large
gain in entropy, resulting in shrinkage of the polymeric structure33'34. The mostly studied
temperature responsive hydrogels are methylcellulose , hydroxypropyl
methylcellulose, chitosan, iV-isopropylacrylamide (NIPAAm) based copolymers
and other iV-alkylacrylamide polymers 43, poly(vinyl methyl ether) (PVME),44"46 poly(/V-
vinylisobutyramide) (PNVIBA),47"50 poly(ethylene oxide-Zj-propylene oxide-6-ethylene
oxide) (PEO-PPO-PEO)51'5 and poly(ethylene oxide)/(D,L-lactic acid-co-glycolic acid)
(PEO-PLLA-PLGA)53 copolymers.
Poly(7V-isopropylacrylamide) (PNIPAAm) is the most popular temperature-
responsive polymer since it exhibits a sharp phase transition in water at 34.3°C which is
i ~i
close to physiological temperature . Its LCST can be controlled by copolymerizing with
other monomers. The LCST increases with the addition of hydrophilic monomers
whereas it decreases with the incorporation of hydrophobic monomers. Grafting of
hydrophilic or hydrophobic monomers does not show any significant changes in LCST33.
17
2.2.3 Glucose responsive hydrogels
For the treatment of diabetes, the most desirable insulin delivery systems could be
developed having glucose-sensing carrier to trigger the release of required amounts of
insulin. Glucose sensitive hydrogels have been attractive for this particular application.
Cationic hydrogels as a carrier for insulin and glucose oxidase mixture are the most
extensively studied glucose sensor systems54"57. In the presence of oxygen, glucose
oxidase converts glucose to gluconic acid and reduces the local pH; which increases the
swelling of cationic hydrogels and releases insulin. To improve controlled loading of
insulin, glucose oxidase has been covalently tethered on the hydrogel system that reduces
its fast diffusion out of the system58. Other mechanisms including the use of
concanavalin-A as a crosslinker,59 use of phenylboronic acid60 or glucose
dehydrogenase15 as a biosensor have been also investigated to fabricate glucose
responsive hydrogels.
2.2.4 Protein-based hydrogels
Recently, protein-based hydrogels have been studied for drug delivery and tissue
engineering applications61'6 . These protein-based hydrogels are precisely designed with
defined compositions, sequences, stereochemistry, and molecular weights using
recombinant DNA technology. Coiled-coil is an attractive approach for protein-based
hydrogels63. The hydrophobic amino acid residues of coiled-coil proteins are used as
physical crosslinkers in protein-based polymer hydrogels. Tri-block copolymers with
coiled-coil domains at the end and water-soluble polypeptide domain at the centre have
been designed as physically crosslinked protein-based hydrogels64. Consequently,

18
temperature and/or pH-responsiveness may be achieved by manipulating the amino acid
sequences of the coiled-coil domains34'61. Addition of RGD sequence within the
hydrophilic polypeptide sequence also improves cell interactions. Moreover, coiled-coil
proteins are used as crosslinkers with water soluble linear synthetic polymers to prepare
ftO
3D structure of hydrogels .
2.2.5 Antigen-responsive hydrogels
Antigen-responsive hydrogels have been designed to deliver biomolecules at a
specific targeted site65'66. In these hydrogels, antigens are grafted on hydrophilic
polymeric backbones. They can also be mixed with antibody-grafted crosslinked
hydrophilic polymeric backbones. In the absence of a free antigen, the hydrogel structure
shrinks due to the intra-chain antigen-antibody binding in the polymer network. Specific
molecular recognition is a remarkable feature of the antigen-sensitive hydrogels that
made them useful biomaterials to fabricate an antigen sensing device for biomolecules,
protein or drug delivery at desired sites67.
2.3 Water in hydrogels
Swelling behavior of hydrogel systems is an important parameter governing their
application specifically in pharmaceutical, in ophthalmology and in tissue engineering.
The presence of water at the surface of hydrogels reduces the interfacial free energy in a
physiological environment and thus improves their biological properties . The final
water content of hydrogels depends on both the kinetics and thermodynamics parameters.
During the swelling process, the first water molecules hydrate the most polar, hydrophilic
19
groups, and this portion of water is called 'primary bound water'. As the hydration of
polar and hydrophilic groups is completed, the network swells, and exposes hydrophobic
groups, which start interacting with water through hydrophobic interaction called
secondary bound water molecules. Together, primary and secondary bound water
molecules are often called the total bound water5. After the water has interacted with both
hydrophilic and hydrophobic sites, the osmotic driving force of the network chains allows
the network to absorb more water. This additional swelling is opposed by the presence of
covalent or physical crosslinking junctions through an elastic network retraction force.
Finally, the balance of the retraction force and the infinite dilution force establish an
equilibrium swelling level. The additional water absorbed beyond the total bound water
is defined as 'free water' or 'bulk water'5.
2.3.1 Thermodynamics of hydrogel swelling
Hydrophilic polymer networks show high affinity to water and, in the presence of
water, the polymer-water interaction is preferred to the inter-polymer chains interactions.
Thus, the hydrophilic network allows large water absorption and proceeds towards
infinite dilution. However, the presence of crosslinking junctions resists the infinite
dilution by the retractive force of elasticity. In the absence of ionic moieties in the
polymer chains, the counter balance of these forces decides the water uptake of
hydrogels. The Flory-Huggins theory can be used to calculate the thermodynamic
behavior of hydrogel swelling1'69. Considering an isotropic crosslinked structure of
hydrogel, the total Gibbs free energy change of the system, upon swelling, can be written
as:
20
&G = AGm„,ure+AGelasllc (2.3.1)
Where,
AGmalure = the free energy of mixing due to water affinity of hydrophilic polymers.
AGelasllc = the elastic free energy as a result of the network expansion.
In order to express the chemical potential change of water in terms of elastic and
mixing contributions at any time of swelling, differentiating equation (2.3.1) with respect
to the water molecules in the system gives:
Mwh ~ / V = immure + ^Melasnc (2-3.2)
Where,
ju„h = the chemical potential of water within the hydrogel.
/u = the chemical potential of pure water.
Ajumixlure = the change in chemical potential due to mixing.
Ajuelasllc = the change in chemical potential as a result of the network expansion.
The chemical potential change on mixing can be obtained using Flory-Huggins
theory that is applied to the fundamentals of the thermodynamics of polymer solution8.
2.3.1.1 Determination ofAîmixure
The entropy of mixing:
In the absence of crosslinkages, the ideal entropy of mixing can be given by the
Boltzmann relation, ASm = k * In Q.. Here k represents the Boltzmann constant and Q.
represents the probability of arrangements of polymer chains within the solvent. By

21
considering that the polymer molecules have the same size, the Lattice Model can be
used to find the possibility of such arrangements69. The formation of the polymer solution
can be thought to happen in two steps: disorientation of the polymer chains and mixing of
the disoriented polymer with solvent. The entropy change related to both steps and the
overall entropy change is given as:
ASm =-*(«, In v,+» 2 In v 2 ) (2.3.3)
Where,
v, = volume fraction of water
v2 = volume fraction of polymer
«,, n2 = moles of water and polymer respectively.
The heat of mixing:
According to the Lattice Model, three types of first neighbor contacts are
possible: [1,1], [2,2] and [1,2]. The solution is prepared by having the chemical reaction
in which bonds of [1,2] types are formed at the expense of an equal number of [1,1] and
[2,2] as per the following stoichiometric balance:
i [ l , l ] + i[2,2] = [l,2]
If wn, W22 and W12 are the energies associated with these respective bonds, the
change in energy due to the formation of unlike pairs is given as:
Aw12 = w12 - - ( w n + w 2 2 )
The overall heat of mixing is then given as:
&Hm = Aw i2 x Pn = 2Aw12x,«! v2 (2.3.4)

22
Where,
pn = probability that the sites adjacent to a polymer segment is occupied by a solvent
molecule =zx,n,v 2 .
z = the lattice coordination number which equals the number of cells which are first
neighbors to a given cell.
JCI = the segments of water molecule.
The quantity zAw12Xj represents the change in the internal energy of a solvent
molecule immersed in the pure polymer compared with the one surrounded by molecules
of its own kind, i.e., in the pure solvent. Another parameter is introduced to define this
energy difference and is called water-polymer interaction parameter (x). It is the
l
dimensionless quantity, which is defined as ^-^-. Using this interaction parameter
kT
into the equation (2.3.4) gives:
AHm=kTxnx-w2 (2.3.5)
The chemical potential change on mixing:
The Gibbs free energy of the mixing is simply given by combining equations
(2.3.3) and (2.3.5). That is,
AGm =AHm- TASm = kTXnxv2 + kT(n, In v, + n2 In v 2 )
AGm=AHm-TASm=kT(Xnxy2+nx\nv^n2\ny2) (2.3.6)
23
Now, in the case of hydrogels, the number ni of polymer molecules is to be
equated to zero owing to the absence of individual polymer molecules in the network
structure. Thus
AG„=*r(^« 1 v 2 +/i 1 lnv 1 ) (2.3.7)
Differentiation of the above equation with respect to the number water molecules,
n\, at constant temperature and pressure (bearing in mind that \\ and V2 are functions of
n\) into the system gives,
A / / „ =^ = *r(ln v, + v2 + %w\) (2.3.8)
2.3.1.2 Determination of Ajuelasllc
The presence of crosslinks induces a retractive force as the dry hydrogel expand
into the water. This retractive force can be explained by using the theories of rubber
elasticity. Rubbers are materials that respond to stresses with nearly instantaneous and
fully reversible deformation up to 1000% elongation. Rubbers are crosslinked networks
possessing large free volume that makes them capable to respond to external stresses by
rearranging the polymer chains. In the swollen state, most hydrogels satisfy this
phenomenon of rubber. To derive the relationship for the chemical potential change of
water during swelling, statistical thermodynamics have been used69. The expansion of
hydrogel is considered to be isotropic. So the developed strain related to the expansion of
structure (say as) can also be considered the same in all direction. The entropy change
24
involved in expansion of hydrogels, obtained by using statistical thermodynamics and by
applying the Boltzmann expression69, is
ASel=-^[ax2 +ay
2
+az2 - 3 - In (axayaz)] (2.3.9)
Using ai = ax = ay = az, for isotropic expansion, we get
ASe;=-^-[3a12-3-ln(a,3)] (2.3.10)
The extensibility of a rubber is driven by entropic change rather than enthalpic
changes. For ideal elastic behavior, the extension takes place due to the rearrangement of
polymer chains and bonds are not stretched with change in length. This behavior is not
true for most other materials (e.g. metals) where changes in length cause internal energy
driven retractive force. For elastomeric materials, an increase in length is counter-
balanced by decreasing the entropy only, which is due to the changes in the end-to-end
distances of the network chains. The extensibility of hydrogels during swelling can be
considered in the same way. The enthalpy change is ideally zero and practically very
small in the case of swelling. By neglecting the enthalpy change during swelling, the free
energy of elasticity for swelling is given as:
AGe/=Atfe/-rASe/=^[3a/-3-ln(a/)] (2.3.11)
Differentiation of the above equation with respect to the number water molecules,
n\, at constant temperature and pressure (having in mind that as is the function of n\) into
the system gives,

25
dAG^
= kTvt\l v 22 " 3 - ^ (2.3.12)
dn. 2
Where,
V, = molar volume of water
v2 = volume fraction of unswollen polymer in swollen hydrogel
ve = effective crosslinking density = ——
vn = effective number of polymer chains in the network
V0 = volume of unswollen polymer
This equation assumes that the network is ideal and all chains in the network are
elastically active to contribute to the elastic stress. In hydrogels, free chain ends represent
gel network "defects" that do not contribute to the elasticity of the network. Other
network defects are chain "loops" and entanglements, which also do not contribute to the
permanent network elasticity. These network imperfections such as chain entanglements,
and chain ends are not taken into account. The corrected equation for these imperfections
is
dAG^ ( v \ 2M„
kT 1- v21/3--2 (2.3.13)
dn. K^Mcj M,n A 2/.
Where,
va = specific volume of unswollen polymer
Mc = the number average molecular weight between cross-link points

26
Mn = the number average molecular weight of linear polymer chains prepared at the
same conditions without crosslinking.
/ = functionality of crosslinking agent.
Now, the overall chemical potential change of water in swollen hydrogel is
dAG„ dAG„,
AM = NA
dn, dn{
1/3 V2
Aju = NA kT (in v, + v2 + Xv\) + kTveVx v.
A^ = RT lnv,+v 2 +^+v e V, 2 (2.3.14)

2
Here, NA is the Avogadro's number and form the definition; k = R/NA .
At equilibrium, the chemical potential of the water within hydrogel must be the
same as that of pure water. The term at the right hand side of the equation (2.3.14) should
be zero at equilibrium. At the equilibrium we can write:
A/u = RT In^+Vj+^+v.V, v =0 (2.3.15)

2 -
vi can be eliminate in favor of V2 since v; = 1-v 2 . This equation is used to calculate the
number average molecular weight between crosslinks:
l n ( l - v 2 ) + v2+veV,
(2.3.16)
x=—
27
2.3.2 Kinetics of hydrogel swelling
The kinetic behavior of hydrogel swelling is mainly due to diffusion and capillary
rise of water into the hydrogel. Water uptake through capillary rise is much faster than
the diffusion process. 1-cm rise of fluid in a narrow capillary (-100 urn) takes place in
the order of milliseconds70. The presence of small pore size (100 urn to 300 urn), good
pore size distribution and extensive interconnected capillary channels in super porous
hydrogel systems, make them fast swelling systems that are advantageous for specific
applications such as sanitary adsorbants. Following capillary rise, diffusion of water into
the polymer network takes place. The network relaxation, limited by the water-polymer
interaction, plays a major role during the water diffusion process. To determine the nature
of water diffusion into the hydrogels, the swelling data over the time intervals has been
fitted into the Fickian diffusion equation71:
W
/ = —£- = At" (2.4.1)
W.
Where, / is the fractional water uptake at time t, Wt and WM are the mass of the
hydrogel at time t and at equilibrium swelling respectively, K is a characteristic rate
constant that rely on the hydrogel structure and, n is a transport number that indicates
whether diffusion and/or network relaxation controls the swelling. For one-dimensional
slab geometry, the swelling is diffusion controlled for n < 0.50, known as Fickian
diffusion, where the rate of network relaxation is faster than the rate of diffusion. For n =
1.00, water transport is controlled by the rate of relaxation of the polymer network where
the rate of diffusion is faster than rate of network relaxation and is known as non-Fickian
diffusion. For the value of n between 0.50 and 1.00, both rates affect considerably on the
28
swelling rate and none of their effect can be neglected. Such transport is called
anomalous diffusion.
For non-Fickian behavior of hydrogels, the deviation from Fickian behavior is due
to the finite rates at which the polymer structure may change or reorient in response to the
sorption or desorption of water molecules. In such polymers, a sorption process will be
affected through the segmental motion that occurs at about the same rate or slower than
the diffusion process. Thus, non-Fickian behavior is polymer structure dependant, and
based on the polymer composition, wide range of relaxation times associated with
structural changes can be observed. A number of mathematical models have been

10
proposed for non-Fickian behavior of polymers , however no single model successfully
predicts all experimental observations.
For diffusion controlled swelling kinetics, the diffusion coefficient (D) is used to
describe the rate of swelling. The flux, J, of a diffusing substance through the unit area of
a section can be expressed by Fick's first law72:
S= -Df8C~
ox
Where, | — is the concentration gradient which is the driving force for

8x
diffusion, and D is the diffusion coefficient. The diffusion coefficient is a constant and
independent of x, C, and time, t. When the concentration gradient varies with time, the
rate of change of concentration in one-dimension is given by Fick's second law72:

29
8C\ „fd2CN
=D 2 (2.4.3)
a vdx y
Several solutions for equation 2.4.3 that depend on the boundary conditions were
developed by Crank72. Using the time-dependent swelling data on thin films, the
following equation can be used to calculate diffusion coefficient of water considering

10
unsteady state diffusion and using planner geometry :
W,
^? xt 050 (2.4.4)
\7lL )
Where, L is the initial section thickness and D is the diffusion coefficient of
water. Thus, the slope of the plot of — L against t provides the diffusion coefficient of
CO
water for a given hydrogel system. Equation 2.4.4 is a good approximation for the
solution obtained when the surface concentration is constant at both sides of the film for
W W
values of — - less than 0.6. Thus, when fractional swelling,—-, is linear with the
W 00
W 00
square root of time, the swelling profiles fit Fick's law, allowing the determination of
diffusion coefficients.
2.4 Suitability of hydrogels as biomaterials
Certain important properties of hydrogels for their applications as biomaterials
can be tabulated as follows:
• Superior biocompatibility
• Good oxygen permeability
• Low protein adsorption and cell adhesion

30
• Aqueous surface environment to protect cells and therapeutic drugs (peptides,
proteins, oligonucleotides, DNA)
• Minimal frictional irritation within the surrounding tissues upon implantation
• Soft and tissue-like physical properties
• Micro-porous structure for additional transport channels
• Ease of surface modification with specific biomolecules
• Can be injected in vivo as a solution that gels at body temperature
These properties of hydrogels made them ideal biomaterials for applications in
drug delivery system, cell encapsulation, contact lenses, scaffolds for tissue engineering,
biosensors, intelligent cell culture substrates, wound dressing, soft tissue replacement and
many more.
2.4.1 Hydrogels for drug delivery applications
Well-designed drug delivery systems must control solute release over time.
Various biomaterials have been investigated to control drug release; however, among
them, hydrogels show two distinct advantages, (i) Drugs can easily diffuse out through
the hydrogels. The rate of drug release can be controlled in many ways such as by
changing the crosslinking density, preparing the hydrogel with monomers of controlled
hydrophilicity and/or controlling the ratio of hydrophilic to hydrophobic monomers, (ii)
Compared with hydrophobic materials, hydrogels may interact less strongly with drugs;
consequently, a larger fraction of active molecules of drug, especially proteins and

•IT
peptides, can be released through hydrogel carriers .

31
2.4.2 Hydrogels for cell encapsulation
Cell encapsulation technology provides a promising therapeutic modality for
diabetes, hemophilia, cancer and renal failure74'75. The selection of a suitable biomaterial
as a membrane for encapsulating cells is the major challenge towards the success of cell
encapsulation therapy. Biocompatibility, microporous structure and minimal surface
irritation within the surrounding tissues of hydrogels attracted them for this application.
They can be designed with required porosity that resists any entrance of immune cells
and allows stimuli, oxygen, nutrients and/or waste transfer through the pores. Genetically
modified alginates76 and polyethylene oxide based hydrogels77 have been studied as cell
encapsulation systems.
2.4.3 Hydrogels for tissue engineering scaffolds
Tissue engineering has emerged as a promising technology for the design of an
ideal, responsive, living substitute with properties similar to that of the native tissue78. To
date, it has focused mainly on restoration, maintenance and/or improvement of the
functions of bone, cartilage, tendon, ligament, skin, blood vessels and heart valves.
Scaffolds play an important role in scaffold-guided in vitro tissue engineering. Scaffolds
are basically 3D structural templates which support cell adhesion, migration,
differentiation, proliferation and provide guidance for neo tissue formation. The chosen
scaffold material should be biocompatible and reproducible without any batch-property
variation with high porosity and well organized inter-connectivity79. Hydrogels in
particular emerged as useful scaffolding biomaterials as they most closely resemble the
natural tissues. Moreover, an aqueous environment provided by hydrogels mimics those

32
of cells in the body. They are porous for nutrient and waste diffusion, and as discussed
before they are usually considered to be biocompatible. However, the possibility of batch
to batch variation is an issue with natural hydrogels which can be overcome using
biologically modified synthetic hydrogels. Both synthetic and natural hydrogels are used
as scaffolds for tissue engineering in order to repair cartilage, tendon, ligament, skin,
blood vessels and heart valves79,80. Synthetic hydrogels focused as scaffolds are
polyurethanes (PU), poly(ethylene oxide) (PEO), poly(7V-isopropylacrylamide)
(PNIPAAm), poly(vinyl alcohol) (PVA), poly(acrylic acid) (PAA) and poly(propylene
furmarate-co-ethylene glycol) (P(PF-co-EG)) whereas, naturally derived hydrogels are
agarose, alginate, chitosan, collagen, fibrin, gelatin, and hyaluronic acid (HA)81.
2.4.4 Hydrogels for contact lens application
The cornea of the eye is a precisely formed transparent structure of protein fibers
containing about 80% water and 20% formed materials making it a natural hydrogel82.
Synthetic hydrogels have found to be suitable in contact lens applications when the
refractive power of cornea is compromised. In addition to their biocompatibility and
softness, inter-connected microstructures of hydrogels help oxygen diffusitivity to the
epithelial layer of the cornea. Certain hydrogels possess high refractive index, modulus,
and transparency, required to fit for this application. Poly(HEMA) was the first hydrogel
used as a contact lens in 1960 . Since no single hydrophilic polymer structure provides
all required properties, copolymers developed from a group of hydrophilic monomers like
dimethylacrylamide (DMAAm), N-vinyl pyrrolidone (NVP) and methacrylic acid
(MAA) and hydrophobic monomers like perfluoro polyethers (PFPE), methyl

33
methacrylate (MMA) and silicon-containing monomers are utilized to design contact
lenses ' .
Moreover, hydrogels have also been studied as potential biomaterials for
biosensors67, intelligent cell culture dish31, wound-dressing85, injectable scaffolds86, and

0*7
soft tissue replacement .
2.5 Polyurethanes
Polyurethanes (PU) cover a large area of applications due to their tailor-made
properties that can be controlled by choosing their precursors: flexible macro polydiols,
short chain extenders and polyisocyanates. Within the wide range of molecular weight
and molar ratio possible for each type of segments of polyurethanes, a broad range of
physical and mechanical properties can be obtained. These materials can be very brittle
and hard, or they can be soft, tacky and viscous or anywhere in between. Thus, due to
their wide range of properties, polyurethane can be produced in flexible or rigid foams,
elastomers, coatings, adhesives and sealants88.
2.5.1 Polyurethane synthesis
Polyurethanes are composed of short, alternating blocks of soft- and hard-
segment units. The hard segment could be an aromatic, cyclic or aliphatic diisocyanate
that has been polymerized with a low molecular weight diol, diamine or dicarboxylic acid
termed as the chain extender; to produce an intermediate precursor of molecular weights
between 300 and 3000. The glass transition temperatures of these hard segments are
34
above ambient temperature. The soft segment could be a polyester, polyether or polyalkyl
diols with molecular weights between 500 and 5000. The glass transition temperatures of
these soft segments are below room temperature. These macroglycols and the chain-
extended diisocynate combine to form as (AB)n type block copolymer. Thus,
polyurethanes have broad physical, mechanical, thermal or biological properties as they
can be tailored by variation of their precursors: the flexible polyol, short chain extender,
and diisocynates. Polyurethane typically exhibits a two-phase microstructure. This phase
segregation results in the superior physical and mechanical properties of these materials
as shown below.
• The hard, rigid diisocyanate segments separate into glassy or semicrystalline
domains.
• The macroglycol soft segments form an amorphous or semicrystalline matrix in
which the hard domains are dispersed at low to moderate hard content.
• The hard domains act as multifunctional cross-linking sites and as reinforcing
fillers, thus, resulting in materials which possess both high modulus and
elastomeric behavior.
The driving force for the segregation into domains is provided by the chemical
incompatibility of the hard and soft segments. Factors affecting the degree of phase
separation in the polyurethanes include hydrogen bond formation between the urethane
linkages and the carbonyl or ether functionalities, segment length, segment polarity and
crystallizability, overall composition, and mechanical and thermal history.

35
,Rv
m]
2mOCN' ^NCO +
Diisocyanate Poly-diol
Stepl
m OCN -JLN
H
N^
I
.NCO
H
I
Prepolymer
Y
o
,R'.
Step 2 + m HO OH
| Small diol (chain extender)
o o o
.R. ^R'
v
O^ N N *0' O N
I I m
H H H
Segmented Polyurethane
SCHEME 2.1. Schematic diagram of segmented polyurethane synthesis using two-step step-growth
polymerization process.
Polyurethanes are usually prepared by two basic processes. The simplest and most
obvious method is to mix the polyol and diisocyanate with a chain extender and to cast
the mixture in a mold while still liquid. Curing of the cast mixture yields an elastomeric
product. This is called one shot process. But the most common route for the synthesis of
segmented polyurethanes is the prepolymer method (Scheme 2.1). In this method, the
polymer is formed in two stages. Initially, the diisocynate and polyol are reacted together
to form an intermediate oligomer of molecular weight 1000 to 5000. The prepolymer that
is formed is normally a viscous liquid or low melting point solid. This prepolymer can be
shelf-stabilized with 0.01 to 0.1% of an acid chloride. The prepolymer is then converted
36
into the final high molecular weight polymer by further reacting with a diol or diamine
chain extender. This step is usually called the chain-extension stage.
2.5.2 Polyurethanes as biomaterials
Polyurethane biomaterials were first explored to construct composite
polyurethane foam breast prosthesis in the 1950s. Currently, polyurethane elastomers are
regarded as some of the best biomaterials available, due to their superior mechanical
properties, particularly tensile strength and fatigue resistance; blood and tissue
compatibility; controlled biodegradation; controlled protein adsorption and less thrombus
formation . The properties of polyurethanes are determined, in part, by the reagents
selected for synthesis from the large range of possible precursors. The mechanical
properties are also influenced by the methods used to fabricate and process the specific
device. However, the final tuning of selected polyurethanes related to the particular
medical applications can be done by controlling the molecular weight of precursors,
tuning the synthetic path of polyurethane chemistry, processing it with crosslinking
agents and/or biomolecules and thus the results can be still improved throughout their
transition from biomaterial to medical device89"91. Some commercial polyurethanes, used
for biomedical applications, are listed in Table 2.1 and medical devices constructed from
polyurethanes are listed in Table 2.2.

37
Table 2.1: Some commercial polyurethanes used in biomedical applications92.
Type Trade Name Company Form

Segmented PU Solution in
elastomer solution Biomer Ethicon Inc. dimethylacetamide
Thermo
Segmented PU Electron
cast elastomers Tecoflex HR Corporation Liquid cast system
Thermoplastic PU
elastomers Pellethane 2363 Upjohn Thermoplastic resin
PU-silicon Avo Medical Copolymer of polyether urethane

copolymer Avcothane 51 Products and polydimethyl-siloxane
Table 2.2: Biomedical devices fabricated from polyurethanes92.
Endotracheal tubes.
Aortic grafts as well as arterial, venous and vascular tubing.
Heart assist and heart by-pass devices.
Dialysis membrane, tubing, and connectors.
Artificial heart chamber, pacemaker lead insulation and heart valves.
Bone adhesive.
Diagnostic catheters; e.g. sinus flow, nasal, oral, rectal, myocardial,
gastrointestinal, and urological.
Surgical drapes (body bandage) and tapes.
Cavity liner in dentistry.
Preventive and restorative dentistry, e.g. tubing, orthodontic ligatures, etc.
Splint and anti-shock devices.
Breast augmentation.
38
• Drug delivery system for controlled release.
• Roller pump tubing in artificial heart or blood pump.
2.6 Poly(vinyl pyrrolidones)
Poly(vinyl pyrrolidone) (PVP) was first reported by Watler Reppe in 1939 as an
interesting acetylene derivative . PVP was initially utilized as a blood plasma substitute
during World War II. ./V-Vinyl pyrrolidone (NVP), the monomer for PVP, holds an amide
group similar to acrylamides. However, the vinyl group is directly attached to the
nitrogen rather than to a carbonyl group as in acrylamides94. PVP is known for its
excellent biocompatibility, very low toxicity, high surface hydrophilicity, easy
processability and environmental stability ' . PVP also shows high complexation ability
with various drugs to render them disperse or solubilized in aqueous media. Moreover,
due to its special molecular structure, PVP possess similarities with protein properties
like precipitation in most protein precipitators97. Based on their applications, PVP is used
in three different forms: linear PVP, crosslinked PVP and its copolymers. In order to tune
their mechanical, physical and/or biological properties related to final applications, their
copolymers with different natural or synthetic biomaterials are of great interest in novel
biomaterials development. Mainly, their copolymers with polyurethanes, chitosan,
polyvinyl alcohol (PVA), PNIPAAm have been investigated for a variety of
pharmeceutical, cosmetics, and biomedical applications .

39
2.7 Polyacrylamides
n
H2N Ô
Polyacrylamides were first developed for the field of electrophoresis as a platform
matrix in 1959 and thereafter they have been explored as a biomaterials in a variety of
applications such as enzyme immobilization, drug delivery carriers, tissue engineering
scaffolds, reconstructive surgeries and cosmetic products98. Polyacrylamides are
attractive for their specific properties such as their easy and inexpensive preparation in
wide range of molecular weight, high resolving power, applicability and stability over
wide pH ranges (pH 3-11) and biocompatibility. Moreover, polyacrylamides deformed
linearly over wide ranges of stress followed by complete recovery after removal of the
applied stress. Their advantages as a biomaterial have been reviewed in detail by Yang99.
Polyacrylamides show a lack for protein binding and thus are attractive as electrophoresis
matrices. However, their incompatibility with protein binding and their inertness makes
them less attractive for scaffolding applications in tissue engineering. Fortunately,
numerous surface modification methods using the amide group of the polymer have been
developed100. Partial alkaline or acid hydrolysis of polyacrylamides to prepare
polyacrylamides-co-polyacrylic acids is one of the simplest and widely used modification
methods1 '. Such developed carboxylic groups can be utilized to conjugate drugs102'103 or
biomolecules using EDC/NHS conjugation104. Pelham and Wang105 have used
sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate (sulfo-SANPAH)
coupling for the biofunctionalization of polyacrylamides. Reinhart-King et al106
developed a process based on the application of 7V-succinimidyl ester of

40
acrylamidohexanoic acid to conjugate proteins on polyacrylamides. Copolymerization of
polyacrylamides with other monomers containing functional groups to utilize for
biofunctionalization is another approach. However, the limitation of copolymer solubility
is always a challenge with polyacrylamide-based copolymers.
2.8 PoIy(2-hydroxyethyl methacrylates)
CH3
OH
Poly(2-hydroxyethyl methacrylate) (PHEMA) has been first synthesized in
1936107, and it was the first synthetic hydrogel investigated as a biomaterial in I96083. It
has been known for its nontoxicity, superior biocompatibility, high hemocompatibility
and inertness towards biological processes. It is easy to polymerize and possess good
mechanical strength compared with the other hydrogels108. PHEMA also shows high
refractive index, good oxygen permeability, and transparency10 . Moreover, the presence
of hydroxyl group allows them to be modified easily with bioactive molecules for drug
delivery110 and tissue engineering applications111. The chemical modifications of
PHEMA and their copolymers have been reviewed in detail by Chappard et al 1 , 2 n 3 . In
order to improve specific properties related to final applications, copolymerization and
grafting of PHEMA have been extensively studied with wide range of natural polymers
such as cellulose114, dextran115, chitosan108 and starch116 as well as with synthetic

41
polymers such as polyethylene117, polyurethanes118, polyesters119, polyvinyl alchohols120,

I 0 1 1 00 \0"X
polystyrene and other acrylate polymers ' . PHEMA and its copolymers have been
extensively used for numerous biomedical applications such as cell culture substrates,
contact lenses, corneal implants, cardiovascular implants, breast implants, nervous tissue
repairs, denture lines, drug release devices and tissue repair platform124"130.
2.9 Iniferter method
Radical polymerization is the most versatile among all polymerization methods,
since more than 70% of polymers are made using this method131. Radial polymerization
can be carried out over a wide temperature range and is applicable to a number of vinyl
1 "\0
monomers . A variety of organic solvents can be used for radical polymerization. Even
though, radical polymerization provides flexibility over the choice of monomers and
solvents, difficulties in controlling the molecular weight, its distribution as well as
handling the end groups of the polymers limits its applications133. Moreover, the presence
of irreversible termination reactions, termination by disproportionation and termination
by coupling, prohibits the preparation of well-defined architecture with required
chemoselectivity, such as segmented block copolymers. To get control on the polymer
chain length in free radical polymerization; chain transfer to the initiator has to be very
high while irreversible termination reactions need to be at a minimum. The irreversible
coupling termination can be avoided either by stopping the physical contacts of the
growing chain ends using specific guard or by providing reversible termination and/or
chain transfer with specific free radical that does not involve in initiation132. Iniferter is
42
one method to provide controlled radical polymerization using reversible termination
(Scheme 2.2).
A-B A- + B
Initiation M
A-M-B * ^ A-M- + B
Monomer Transfer,
A-M- + B •> A—M—Mx- + B
xM
Termination
A-M,- B
SCHEME 2.2: The polymerization mechanism of iniferter system.
An iniferter molecule photochemically or thermally dissociates into transient
radicals (A*), which have high reactivity towards the unsaturated monomer, and
persistent radicals (B«), which have high reactivity towards the free radicals. The
transient radicals initiate the polymerization and incorporate a small number of
monomers, based on the kinetic constants. The growing polymer chains reversibly
terminate with the persistent radicals that reinitiate to incorporate further monomers into
the polymer chain before reversible termination with persistent radicals. Repetition of
43
these steps gives the polymer with lower polydespersity index. The molecular weight of
the polymer synthesized using iniferter increases with monomer conversion as well as
with time1 4. Such polymers always have iniferter fragments at the chain ends. The
termination constant for the reversible termination with persistent radicals has to be
higher than the irreversible termination constant at any conversion in order to provide
controlled polymerization. Thus, the chemical nature of the persistent radicals is the key
factor for the success of the polymerization to the desired direction.
Thermally Photochemically
Iniferter Macroiniferter initiated Iniferter initiated Iniferter
Classification based Classification based

on the size on initiating process
Iniferter
Classification based on
polymer architecture
Monofunctional Difunctional Trifunctional Polyfunctional

iniferter iniferter iniferter iniferter
FIGURE 2.3: Classifications of iniferters1

44
2.10 Study rationale
In this study, physically crosslinked linear polyurethane-based hydrogels for
biomedical applications are developed. In order to achieve controlled segmental lengths
and better chemoselectivity in the final products, an iniferter method is utilized. Thus the
objectives of the current study were:
• To synthesize short chain diols that has the ability to follow the iniferter chemistry.
Since the tetraphelyl ethane131'136 and thiuram disulfide137'138 based derivatives are
well known, l,l,2,2-tetraphenyl-l,2-ethanediol (TPED) and AÂ^'-diethyl-MA^'-bis(2-
hydroxyethyl)thiuram disulfide (DHTD) were selected and synthesized.
• To synthesize segmented polyurethane macroiniferters using the above-mentioned
diols as chain extender.
• To synthesize hydrophobic-hydrophilic segmented block copolymers by
polymerizing the hydrophilic vinyl monomers, such as N-vinyl pyrrolidone (NVP),
acrylamide ( M m ) and 2-hydroxyethyl methacrylate (HEMA), in the presence of
polyurethane macroiniferters.
• To study the physical, thermal and biological properties of the synthesized hydrogels.
• To investigate the swelling kinetics of the prepared hydrogels.
• To study the kinetic behavior of the polyurethane macroiniferters in order to
understand their ability as controlled radical polymerization systems.
In order to achieve these objectives, the processes to prepare TPED and DHTD
were optimized. MDI-based PUMI using TPED as a chain extender was then synthesized
and utilized as a thermal macroiniferter to prepare PU-6-PVP based hydrogels. Detailed

45
characterizations had been conducted and discussed in Chapter 3. Further, MDI-based
PUMI using DHTD as a chain extender was prepared and used as a photo macroiniferter
to synthesize PU-6-PVP and PU-6-PAAm hydrogels. This PUMI and related hydrogels
had been analysized thoroughly and discussed in Chapter 4. In order to understand the
copolymerization kinetics of DHTD-based PUMI, detailed kinetic studies using methyl
methacrylate (MMA) as a model monomer were carried out (Chapter 5). An extension of
this study was the development of HnMDI-based PUMI using DHTD as a chain extender
and using them to synthesize PU-6-PHEMA hydrogels. These hydrogels were further
modified with bovine fibronectin to test their potential as a scaffolding platform for tissue
engineering. Detailed characterizations were carried out and discussed in Chapter 6.
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57
CHAPTER
3
Physically Crosslinked Polyurethane-#/0c&-poly(iV-vinyl

pyrrolidone) Hydrogels from Tetraphenyl Ethane (TPED)-
based Polyurethane Macroiniferters*
Overview: In this Chapter the synthesis and characterization of TPED-based

polyurethane macroiniferter (PUMI) is first described. The use of this PUMI to prepare
physically crosslinked polyurethane-block-poly(N-vinyl pyrrolidone) (PU-b-PVP)
hydrogels is also described. The success of both PUMI and PU-b-PVP syntheses has
been characterized using spectroscopic analysis. During short term cell culture studies,
vascular smooth muscle cells attachment and spreading on the hydrogels indicated that
these materials have a potential for use as scaffolds in tissue engineering.
3.1 Introduction
Hydrogels are crosslinked macromolecular network structures, having the ability
to absorb large amounts of water without dissolving. Due to their soft and tissue-like
physical properties, they resemble the hydrodynamic properties of cells and tissues in
many ways'. Higher polymer-water interaction in hydrogels decreases their coefficient of
friction and thus improves the hydrodynamic properties such as lubrication at low load2.
Thus, hydrogels also minimize the frictional irritation within the surrounding tissue upon
*A version of this chapter has been published: Patel, A.; Mequanint, K. (2007) Novel Physically
Crosslinked Polyurethane-Woc£-Poly(Vinyl Pyrrolidone) Hydrogel Biomaterials. Macromolecular
Bioscience; 7: 5; 727-737.
58
implantation. In addition, their surface hydrophilicity and high mobility of the polymer
chains at their surface contribute to improved biocompatibility . The aforementioned
properties of hydrogels make them an ideal biomaterial for applications in drug delivery
system, cell encapsulation, contact lens, scaffolds for tissue engineering, biosensors,
intelligent cell culture substrates, wound dressing, and soft tissue replacements4"6. In spite
of the favorable properties of hydrogels for biomedical applications, their poor
mechanical properties often preclude their practical end-use. This drawback is not
unexpected since water, which does not provide any contribution to their mechanical
properties accounts a significant proportion of hydrogels at equilibrium swelling7. The
conventional wisdom for improving the mechanical strength of hydrogels relies on
crosslinking density provided by chemical crosslinkers. However, increasing the
crosslinking density always results in a reduction of the equilibrium water content.
Removal of unreacted crosslinker residue from the hydrogel is also problematic due to
the poor solubility of these residues in aqueous extraction media. In addition, the post-
synthesis fabrication of devices from crosslinked polymers is not possible due to the
insolubility of the polymer network in most solvents. As a result of these limitations, the
potential of physically crosslinked hydrogels for biomedical applications has received
considerable attention8"10.
Since polyurethanes generally have excellent mechanical properties, hydrogels
based on polyurethane chemistry are appealing for biomedical applications.
Polyurethane-based hydrogels can form strong hydrogen bonded structures, allowing
linear polymer systems to be designed with tunable swelling and mechanical properties.
59
Polyethyleneoxide (PEO) is known for biocompatibility and PEO-based linear
polyurethane hydrogels have been investigated by different researchers31112. However,
most of the available polyurethane soft segments, except PEO, are hydrophobic and the
chemoselectivity to prepare polyurethane hydrogels is limited. This limitation can be
improved by using hydrophilic monomers like 2-hydroxyethyl methacrylate and 2-
hydroxypropyl acrylate as crosslinkers along with other hydrophobic soft segments1314.
However, such crosslinked polyurethane hydrogels cannot be reshaped or resized since
the polymer is no longer soluble and heating to melt-process can only degrade them. The
design of linear polyurethane hydrogels that can be dissolved in an organic solvent, while
maintaining their swelling ability in the presence of water, is beneficial for ease of device
fabrication and post-process modification. One versatile yet unexplored approach to
prepare physically crosslinked polyurethane hydrogels is the macroiniferter
polymerization technique15. In this approach, a polyurethane macroiniferter is first
synthesized from a suitable chain extender that has the ability to generate controlled
radicals thermally or photochemically. In the presence of hydrophilic vinyl monomers,
the macroiniferter can initiate the polymerization reaction thus creating a block
copolymer.
In this Chapter, we explored the macroiniferter approach to synthesize novel
polyurethane-6/0cA>poly(7V-vinyl pyrrolidone) (PU-6-PVP) hydrogels and report their
characterizations and biological properties. The choice of JV-vinyl pyrollidone (NVP) was
based on its reported useful biological properties such as low toxicity, low antigenicity
and good biocompatibility ' . Moreover, PVP resembles protein properties such as
60
precipitation in most protein precipitators17. PVP has also been studied for different
biomedical applications such as blood contacting devices, stimuli responsive drug
carriers, wound dressings, surgical drainage devices and injectable scaffolds16"20.
3.2 Experimental part
3.2.1 Materials
All chemicals, unless stated otherwise, were purchased from Sigma-Aldrich
(Milwaukee, WI). Poly(tetramethylene oxide) glycol of molecular weight 1000 (PTMO
1000) was dried under reduced pressure of 200 mmHg and 90°C until no bubbling was
observed. 4,4'-Diphenylmethane diisocyanate (MDI) was purified by filtration from a
melt. The inhibitor (0.01% sodium hydroxide) in JV-vinyl pyrrolidone (NVP) was
removed by passing through an alumina column. Methyl ethyl ketone (MEK) was
distilled at reduced pressure and the middle portion was used. All other chemicals and
reagents were of the highest purity available and were used without further purification.
3.2.2 Synthesis of TPED
9.10 g (0.05 mol) of benzophenone was dissolved in 5-fold molar excess
isopropanol. After adding lmL glacial acetic acid, the solution was exposed to 365 nm
UV light (Model B100AP; UVP Inc., Upland, CA) where the TPED was formed as white
powder. The product was then filtered and recrystallized from acetic acid and stored at
4°C until used21. (M = 366.46 g/mol, 88% yield).

61
OH
hv (365 nm), 25 °C
1/2 H 0 - -OH _|_
H3C CH3 CHjCOOH H,C XH,
Benzophenone 2-propanol Acetone
TPED
SCHEME 3.1: Schematic diagram of TPED synthesis.
3.2.3 Synthesis of PUMI
A 500 mL glass reactor equipped with heating element, a mechanical stirrer, a
charging and sampling port, a condenser, a nitrogen inlet and outlet was charged with
10.00 g (0.01 mol) of PTMO 1000 and 5.00 g (0.02 mol) of MDI with 20 mL MEK. The
mixture was heated to 65°C and allowed to react for 2.5 h. The prepolymer solution was
then cooled to 22°C and 3.66 g (0.01 mol) TPED along with dibutyltin dilaurate
(DBTDL) (0.2%, based on the residual isocyanate content) were added. Chain extension
reaction was allowed to proceed until the peak related to the isocyanate (2265 cm"1)
disappeared from the FTIR spectrum. The polyurethane macroiniferter (PUMI) was
precipitated by pouring it into tenfold excess cold methanol and dried at 30°C in a
vacuum oven. The product was stored at 4°C until further use.
3.2.4 Synthesis of PU-b-PVP
To 6.50 g of a polyurethane macroiniferter (15% in MEK), 27.78 g (0.25 mol)
NVP was added and stirred at 70°C. Samples were taken at regular time intervals, chilled
in ice-cold water to terminate the polymerization, precipitated into tenfold excess hexane
and washed with diethyl ether. The samples were dried under vacuum at ambient
temperature and soaked into deionised water for one week to further extract any
62
unreacted monomer or PVP homopolymer before characterization. The detailed reaction
scheme for the synthesis of polyurethane-6/oc^-poly(7V-vinyl pyrrolidone) (PU-6-PVP)
copolymer xerogel is shown in Scheme 3.2.
Sample nomenclature is done according to the copolymerization reaction time.
For example, PU-6-PVP03 means a polyurethane-Woc£-poly(./V-vinyl pyrrolidone)
copolymer with copolymerization time of 3 h. The polyurethane macroiniferter was used
as a control (PU-control).
3.2.5 Characterization methods
3.2.5.1 Spectrometric analyses
Fourier Transform Infrared (FTIR) spectra were recorded by using Bruker Vector
22 spectrophotometer. For each specimen, 32 Scans at 4 cm"1 resolution were collected.
Nuclear Magnetic Resonance ('H-NMR) spectra in d-chloroform (CDCI3) were recorded
on Varian® INOVA 400 instrument operating at 400 MHz. ACD/2D NMR software was
used for integrations of the observed peaks.
3.2.5.2 Gel permeation chromatography (GPC)
The molecular weight of the xerogels was determined by a GPC Model VE2001
(Viscotek, Houston, TX) system equipped with a column (GMHHR-L, styragel), and triple
detector system (viscometer, light scattering and refractive index). Xerogel specimens
were dissolved in HPLC grade tetrahydrofuran (THF) at a concentration of 1 mg/mL and
THF was used as a mobile phase at a flow rate of lmL/min.

63
3.2.5.3 Differential scanning calorimetry (DSC)
The DSC study of the PUMI and the xerogels were carried out using DSC 2910
(TA instrument, New Castle, DE). The specimens were dried in a vacuum oven at 50°C
for 24 h before use. Specimens weighing between 5-15 mg were sealed in a DSC pan and
quenched to -70°C. They were then left to equilibrate for 15 min and heated to 200°C at a
rateof5°C/min.
3.2.5.4 Equilibrium water content (EWC) and swelling kinetics
Polymer specimens (n = 4) were cut into 5 mm diameter from cast films. The
specimens were dried in a vacuum oven at 50°C, cooled to room temperature and
weighed. They were then swollen at room temperature (22°C) in distilled water for 24 h.
Swollen specimens were taken out of the water and blotted lightly with a filter paper to
remove the excess surface water. The weights of swollen specimens were determined and
EWC of the specimens were calculated using22:
W -W
EWC = - * ^xlOO (3.1)
Wh
Where Ws is the weight of the swollen specimen and Wd is the dry weight of the
specimen before swelling. For the swelling kinetics study, specimens were taken off the
swelling medium at regular time intervals and weighed. Equation 3.1 was then used
except that the EWC was replaced by the % water content at time t. To determine the
nature of water diffusion into the hydrogels, the swelling data of each sample were fitted
into the Fickian diffusion equation23:
f = W,IWm=Ktn (3.2)
64
Where,/is the fractional water content at time t, Wt and Wx are the weights of the
water in hydrogel at time t and at equilibrium swelling respectively, AT is a characteristic
constant related to the hydrogel structure, n is a number that indicates whether diffusion
or relaxation controls the swelling.
3.2.5.5 Preparation of cell culture specimens
2D films of the hydrogels were prepared from cast films. Circular films having a
diameter of 0.64 cm were punched out using a one-hole paper punch. Individual discs
were affixed to 1.2 cm diameter glass coverslips using silicone grease, sterilized with
70% ethanol and allowed to dry over night. Coverslips containing polymer films were
placed into individual wells of a 24-well tissue culture plate and equilibrated with growth
media for cell seeding.
3.2.5.6 Cell culture on hydrogel surfaces
The cells examined in this study were primary human coronary artery smooth
muscle cells (HCASMC) purchased from Cambrex Biosciences (Walkersville, MD).
Before cell seeding, HCASMC were cultured on tissue culture dishes in smooth muscle
growth media (SmGM-2 smooth muscle growth medium-2 with Bullet Kit; Cambrex).
Cells were cultured at 37°C in a humidified incubator containing 5% CO2. Growth media
was exchanged every 2 days until approximately 80% confluent cell layer was obtained.
Cells were then trypsinized with 4 mL of 0.05% trypsin-EDTA solution, centrifuged at
1100 rpm for five minutes and resuspended with 3 mL of fresh growth media. Cells were
then seeded on the hydrogel films at an initial cell density of -7750 cells/cm2 and
65
incubated in growth media for 20 h before fixation and immunostaining.
3.3 Results and discussion
3.3.1 Syntheses of macroiniferter and block copolymers
Controlled free radical polymerizations are important methods for the syntheses
of block and graft copolymers with the desired molecular weight and chain length. In the
present work, first a polyurethane macroiniferter using l,l,2,2-tetraphenyl-l,2-ethanediol
(TPED) as a chain extender was synthesized. Then, the synthesized polyurethane
macroiniferter was used to initiate the polymerization of TV-vinyl pyrrolidone (NVP) in
order to prepare novel hydrogels. Accordingly, polyurethane-6/ocA:-poly(Ar-vinyl
pyrrolidone) (PU-6-PVP) copolymers were successfully synthesized. The reaction
scheme and structures of PUMI and PU-6-PVP copolymer are presented in Scheme 3.2.
66
NCO
N O \ C6H5o
I C6H5
H
PUMI
-CH,
70 °C
SCHEME 3.2: Schematic diagram of PUMI and PU-6-PVP syntheses.

67
Figure 3.1 shows the FTIR spectra of TPED, PUMI and PU-6-PVP xerogel,
which verified the presence of the expected functional groups.
4000
Wave numbers, cm"
Figure 3.1: FTIR spectra of TPED, PUMI and PU-6-PVP.
The FTIR spectrum of TPED shows a broad peak between 3600-3500 cm"1 related
to the OH stretching. The spectrum also shows peaks between 3100-3000 cm"1 related to
CH stretching and at 735 cm"1 and 690 cm"1 due to out of plane CH bending of mono
substituted phenyl groups. Moreover, the peaks related to C=C of aromatic rings were
observed at 1600 cm"1, 1580 cm"1, 1500 cm"1 and 1450 cm"1. To further confirm the
purity of the TPED, 'H-NMR spectrum is presented in Figure 3.2.

68
i
3,4,5 HO- -OH
2,6
CDCI,
y
J
Chemical shift, ppm
FIGURE 3.2: 'H-NMR spectrum of TPED.
O, y ^ O <l
C6H3
S
J J ^JL
Chemical shift, ppm
FIGURE 3.3: 'H-NMR spectrum of PUMI.

69
The OH protons from TPED appeared at 3.05 ppm and the peaks related to the
aromatic protons appeared between 7.00-7.50 ppm. The FTIR spectrum of PUMI (Figure
3.1) shows the carbonyl stretching at 1700 cm"1 and a peak between 3420-3220 cm"1
corresponding to the NH groups. Thus, the presence of the urethane amide bond along
with the disappearance of OH peak related to TPED indicates that the chain extension
reaction was completed. In the 'H-NMR spectrum of PUMI, (Figure 3.3), the aromatic
protons between 7.00-7.50 ppm are from TPED and MDI whereas the aliphatic protons
from MDI are identified at 3.75 ppm. The aliphatic protons of PTMO, namely CH2,
OCH2 and CH2 attached to the urethane amide groups are observed at 1.64, 3.41 and 4.14
ppm, respectively. In the FTIR spectrum of PU-6-PVP, a new peak between 3640-3410
cm"1 related to the NC stretching (from NVP) is observed whereas the peak between 1760
and 1670 cm" related to the carbonyl group is increased. This confirms the successful
incorporation of NVP to the polyurethane backbone during block copolymerization.
The 'H-NMR spectrum of the PU-6-PVP (Figure 3.4), also proved the NVP
polymerization into the polyurethane backbone. New peaks, which were not observed for
the PUMI, corresponding to the methylene protons adjacent to the ketone group of the
NVP and adjacent to -C(C6Hs)2 from TPED were also observed at 2.00 ppm and 1.35
ppm respectively. The methylene protons corresponding to the tertiary amine of NVP and
adjacent to aliphatic CH from NVP appeared at 3.25 ppm and 1.35 ppm respectively.
Other related peaks are labeled in Figure 3.4.

70
P. H,Q
H,Q
*i
I C6H>N
CDC1 3
V-^\_ JUL
U ^L_
Chemical shift, ppm
FIGURE 3.4: 'H-NMR spectrum of PU-fe-PVP.
Although TPED is a known high-temperature free radical initiator for the
polymerization of vinyl monomers, it cannot be used as an iniferter to prepare block
copolymers24. It has been suggested that reacting at least one of the hydroxyl groups of
•ye
TPED with another reactant is required for its utilization as an iniferter . In the
manuscript from our group, the synthesis of PUMI based on TPED to prepare novel
physically crosslinked polyurethane-6/oc£-poly(glycerol methacrylate) hydrogels for
biomedical applications was demonstrated21. The work described in this Chapter showed
that novel physically crosslinked PU-&-PVP copolymer hydrogels could also be

71
synthesized by this method. This is the first time that, the successful synthesis of
polyurethane-6/oc&-poly(./V-vinyl pyrrolidone) is reported.
3.3.2 The effect of copolymerization time on molecular weight
Figure 3.5 shows the GPC results of PU-6-PVP copolymer hydrogels at different
NVP copolymerization time in the presence of PUMI. As can be seen from the Figure,
the elution volumes of the hydrogels are shifted to lower values when the
copolymerization time was increased. The elution volume for PU-6-PVP24 was the
lowest indicating that block copolymerization increased the molecular weight in a time-
dependent manner. This was similar to a previous report from our group that both
monomer conversion and molecular weight linearly increased with reaction time for PU-
6-poly(solketal methacrylate)21. No PVP homopolymer was also detected.
/ \\
/
/
^ - ^ PU-b-PVP24
1
\ ""~ PU-b-PVP 18
.,' 1 N
\ ' -----
^ \
\ V. PU-b-PVP 12
Jir . \
\\ V
—^K
~ ^ PU-b-PVP06
// \ \ ~~~~
\ \
/
\ ^--^ PU-b-PVP03
X^^^ Control
i i i i i i . i . i i i i i i i i
3 4 5 6 7 8 9 10 11
Elution volume, mL
FIGURE 3.5: GPC curves of PU-6-PVP copolymer for different copolymerization times.
72
Similar to any other iniferter chemistry, the polyurethane macroiniferter thermally
dissociates at the weak tetraphenyl ethane bonds that initiate the insertion of monomer
molecules by propagation; followed by primary radical termination and/or chain transfer
to give the final PU-6-PVP copolymer15. As a result, the rate of polymerization is lower
than that of the conventional free radical polymerization. The moderate shifts of the GPC
peaks support the aforementioned statement.
3.3.3 Water transport properties of PU-b-PVP hydrogels
Figure 3.6 presents the swelling kinetics of the current hydrogels from which it is
seen that with increased swelling time, the water uptake also increases. The control
polyurethane, on the other hand, had a water uptake of only 3% that did not change with
time. For the different hydrogels prepared for this study, copolymerization time increased
the water uptake. It is also interesting to note that a copolymerization time beyond 6 h did
not affect the water uptake significantly presumably due to the slowdown in the
conversion of NVP. Although with an increased copolymerization time, the GPC peaks
(Figure 3.5) shifted only moderately; the swelling data indicated that hydrogels with
water uptake up to 37% were synthesized. This observation could be explained as
follows: Crosslinked PVP homopolymer is a highly hydrophilic polymer with a reported
EWC of 90%26. Therefore a small amount of PVP incorporation into the polyurethane
could render hydrophilic properties that results PU-6-PVP copolymers with reasonable
swelling.
73
100 200 300
Swelling time, min
FIGURE 3.6: Swelling kinetics of physically crosslinked PU-Z>-PVP copolymer hydrogels at different
reaction time. PUMI (•); PU-6-PVP03 (o); PU-6-PVP06 (T); PU-6-PVP12 (A); PU-6-PVP18 (•) and
PU-6-PVP24 (n).
The current hydrogels were soluble in most organic solvents so that films or other
devices can be cast from solutions. However, the hydrogels swell in water without any
mass loss indicating that they were physically crosslinked hydrogels.
The water up-take behavior of hydrogels was determined by linearizing equation
3.2:
ln/=lnA: + «lnf (3.3)
If n < 0.5, the rate of diffusion is slower than the rate of relaxation meaning that swelling
is diffusion limited. If n = 1, the diffusion is fast compared with the rate of relaxation, and
74
relaxation controls the swelling. If the value of n falls between 0.5 and 1, an anomalous
diffusion takes place23. The value of n for the present hydrogels is less than 0.5 (Table
3.1); hence water diffusion into the network controls the swelling behavior of the
hydrogels. Since the only hydrophilic component of the present hydrogels is PVP, the
swelling data indicates the successful incorporation of the monomer via the
macroiniferter chemistry.
Table 3.1: Equilibrium water content (% EWC) and swelling kinetic parameter («) of PU-A-PVP
hydrogels.
Sample % EWC n
PUMI 2.94+0.12 -
PU-6-PVP03 15.38±2.63 0.30+0.09
PU-6-PVP06 32.80±0.76 0.39+0.05
PU-6-PVP12 32.94+0.54 0.36+0.03
PU-6-PVP18 33.36+1.31 0.23+0.02
PU-6-PVP24 36.86+3.51 0.20+0.02
3.3.4 Thermal behavior of macroiniferter and block copolymers
The thermal transitions of PUMI and PU-6-PVP are shown in Figure 3.7. Both
PUMI and PU-6-PVP show two Tgs; the lower temperature represents the soft segment
Tg and the higher temperature represents the hard segment Tg. The lower Tg is related to
the PTMO soft segment and is higher than the Tg of pure PTMO (-83°C)27 suggesting the
presence of dissolved hard segment polyurethane chains. The hard segment Tg for the
control PUMI is observed at around 90°C but the incorporation of the PVP block
75
increased its Tg to 181°C. Moreover, the hard segment Tg in PU-6-PVP copolymer
(181°C) is higher than the Tg of pure PVP (167°C)28 presumably due to the presence of
hydrogen bonding between the polyurethane and PVP segments.
^\ T =-50°C
o
x Tg=181°C
W
PU-A-PVP^\
T =
\ , ^ g •54°C
o T
CI 8 = 90°C
W PUMI ^ \ ^
— ... — .
-50 0 50 100 150 200
Temperature, °C
FIGURE 3.7: DSC curves of (a) PUMI and (b) PU-6-PVP xerogel.
3.3.5 Vascular smooth muscle cell interactions with P U-b-P VP hydrogels
Figure 3.8 presents some preliminary data on vascular smooth muscle cell
interactions with the current hydrogels. Cells were stained either by crystal violet (Figure
3.8A, B) or by immunostaining for h-caldesmon (Figure 3.8C, D) which is a known
smooth muscle cell marker. As can be seen from the figure, cells were well spread on the
76
hydrogel surfaces and expressed the differentiated marker (h-caldesmon). The formation
of focal adhesion is evident for the immunostained images.
FIGURE 3.8: Vascular smooth muscle cells adhesion and spreading on PU-A-PVP hydrogels. Cells
morphology under optical microscopy following crystal violet staining (A, B); immunostaining for h-
caldesmon (C, D). A well-spread cell behavior wih focal adhesion points (C,D) is observed. Arrow in (D)
represents the hydrogel surface.
For biomaterials to be used in regenerative and reparative medicine, the
promotion of cell adhesion to their surfaces is an important step. Most hydrogel
biomaterials are not supportive of cell adhesion and spreading without any cell adhesive
protein modifications. Although this study was carried only for 20 h, it however suggests
77
that the present novel hydrogels could potentially support vascular cell attachment and
differentiation over longer periods of time. Cell death was not detected suggesting that
the current hydrogels were not cytotoxic to cells. This is an essential step for any
biomaterials to be accepted as biocompatible. Cell attachment on the material surface is
the prerequisite step to allow further cellular activities such as differentiation,
proliferation and extracellular matrix synthesis. Moreover, the interaction of cells with
different materials alters their capacity to migrate and proliferate, their biosynthetic
patterns and phenotype. The hydrogel biomaterials described in this Chapter were found
to support vascular smooth muscle cell attachment.
3.4 Conclusions
Novel physically crosslinked polyurethane-Z>/ocA>poly(7V-vinyl pyrrolidone) (PU-
6-PVP) hydrogels were synthesized using a macroiniferter technique. FTIR and NMR
spectroscopy showed that block copolymer hydrogels were successfully synthesized.
Overall, increasing copolymerization time increased the equilibrium swelling of the
hydrogels but the increase seems to level off after 12 h reaction. In the study documented
herein, hydrogels up to 37% EWC were synthesized and characterized. Initial vascular
smooth muscle cell adhesion and spreading showed that these hydrogels are potential
biomaterials for tissue engineering applications.
3.5 References
1. Yaszemski, M. J.; Trantolo, D. J.; Lewandrowski, K. U.; Hasirci, V.; Altobelli, D.

E.; Wise, D. L., Tissue Engineering and Novel Delivery Systems. Marcel Dekker,
Inc., New York: 2004;/? 423-461.
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2. Ishikawa, Y.; Hiratsuka, K.; Sasada, T., Role of water in the lubrication of
hydrogel. Wear 2006, 261, (5-6), 500-504.

2002,23,(8), 1797-1808.
4. Schmaljohann, D.; Oswald, J.; Jorgensen, B.; Nitschke, M.; Beyerlein, D.;
Werner, C , Thermo-responsive PNIPAAm-g-PEG films for controlled cell
detachment. Biomacromolecules 2003, 4, (6), 1733-1739.
5. Sen, M.; Avci, E. N., Radiation synthesis of poly(Af-vinyl-2-pyrrolidone)-kappa-

2005,74,(2), 187-196.
Materials 2006, 18, (11), 1345-1360.
7. Muratore, L. M.; Davis, T. P., Self-reinforcing hydrogels comprised of

hydrophobic methyl methacrylate macromers copolymerized with N,N-
dimethylacrylamide. Journal of Polymer Science Part A: Polymer Chemistry
2000,38, (5), 810-817.
8. Hennink, W. E.; van Nostrum, C. F., Novel crosslinking methods to design

hydrogels. Advanced Drug Delivery Reviews 2002, 54, (1), 13-36.
9. Adams, M. L.; Lavasanifar, A.; Kwon, G. S., Amphiphilic block copolymers for
drug delivery. Journal of Pharmaceutical Sciences 2003, 92, (7), 1343-1355.
10. Kubo, M.; Matsuura, T.; Morimoto, H.; Uno, T.; Itoh, T., Preparation and
polymerization of a water-soluble, nonbonding crosslinking agent for a
mechanically crosslinked hydrogel. Journal of Polymer Science Part A: Polymer
Chemistry 2005, 43, (21), 5032-5040.
11. Xiong, X. Y.; Tarn, K. C; Gan, L. H., Polymeric nanostructures for drug delivery
applications based on pluronic copolymer systems. Journal of Nanoscience and
Nanotechnology 2006, 6, (9-10), 2638-2650.
12. Petrini, P.; Tanzi, M. C; Moran, C. R.; Graham, N. B., Linear poly(ethylene
oxide)-based polyurethane hydrogels: polyurethane-ureas and polyurethane-
635-639.
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13. Kim, J. Y.; Suh, K. D., Swelling behavior of novel polyurethane hydro-xerogels.
Polymer Bulletin 1996, 36, (6), 737-744.
14. Ying, Y.; Gu, X. R.; Yang, C. Z., Abnormal pH sensitivity of polyacrylate-
polyurethane hydrogels. Journal of Applied Polymer Science 1998, 70, (6), 1047-
1052.
16. Zhao, L.; Xu, L.; Mitomo, H.; Yoshii, F., Synthesis of pH-sensitive PVP/CM-
chitosan hydrogels with improved surface property by irradiation. Carbohydrate
Polymers 2006, 64, (3), 473-480.
17. Jiao, Y. P.; Liu, Z. H.; Ding, S.; Li, L. H.; Zhou, C. R., Preparation of
biodegradable crosslinking agents and application in PVP hydrogel. Journal of
18. Edlich, R. F.; Haines, P. C ; Pearce, R. S. C ; Thacker, J. G.; Rodeheaver, G. T.,

Evaluation of a new, improved surgical drainage system. American Journal of
Surgery 1985, 149, (2), 295-298.
19. Geever, L. M.; Devine, D. M.; Nugent, M. J. D.; Kennedy, J. E.; Lyons, J. G.;
Higginbotham, C. L., The synthesis, characterisation, phase behaviour and
swelling of temperature sensitive physically crosslinked poly(l-vinyl-2-
pyrrolidinone)/poly(7V-isopropylacrylamide) hydrogels. European Polymer
Journal 2006, 42, (1), 69-80.
20. Ng, L. T.; Swami, S., Copolymers of acrylic acid with JV-vinylpyrrolidinone and
2-hydroxyethyl methacrylate as pH-responsive hydrogels synthesized through a
photoinitiator-free photopolymerization technique. Polymer International 2006,
55, (5), 535-544.
21. Mequanint, K.; Patel, A.; Bezuidenhout, D., Synthesis, swelling behavior, and
biocompatibility of novel physically cross-linked polyurethane-block-
22. Mequanint, K.; Sheardown, H., 2-methacryloyloxyethyl Af-butylcarbamate: a new

co-monomer for synthesis of polyurethane hydrogels with improved mechanical
properties for biomedical applications. Journal of Biomaterials Science-Polymer
Edition2005, 16, (10), 1303-1318.
23. Caykara, T.; Kiper, S.; Demirel, G., Thermosensitive poly(/V-

isopropylacrylamide-co-acrylamide) hydrogels: Synthesis, swelling and
interaction with ionic surfactants. European Polymer Journal 2006, 42, (2), 348-
355.
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24. Braun, D.; Becker, K. H., Aromatic Pinacols as Polymerization Initiators.

Industrial and Engineering Chemistry, Product Research and Development 1971,
10,(4), 386-388.
25. Chen, X. P.; Qiu, K. Y.; Swift, G.; Westmoreland, D. G.; Wu, S. Q., A novel
thermal iniferter for radical polymerization. European Polymer Journal 2000, 36,
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26. El-Din, H. M. M. N.; Maziad, N. A.; El-Naggar, A. W. M., Radiation crosslinking

and sorption properties of hydrogels based on l-vinyl-2-pyrrolidone,
hydroxyethyl methacrylate, and their copolymers. Journal of Applied Polymer
Science 2004, 91, (5), 3274-3280.
27. Wang, L. F., Effect of soft segment length on the thermal behaviors of fluorinated
polyurethanes. European Polymer Journal 2005,41, (2), 293-301.
28. Mayo-Pedrosa, M.; Alvarez-Lorenzo, C; Concheiro, A., Thermorheological and

glass transition properties of PNIPAAm/PVP and PNIPAAm/Carbopol blends.
Journal of Thermal Analysis and Calorimetry 2004, 77, (2), 681-693.
81
CHAPTER
4
Physically Crosslinked PU-A-PVP and PU-A-PAAm Hydrogels

from Dithiocarbamate (DC)-based Polyurethane
Macroiniferters *
Overview: In Chapter 3, the use of TPED-based polyurethane macroiniferter to prepare

block copolymer hydrogels was described. In this Chapter, we describe DC-based
polyurethane macroiniferter (PUMI) and its use to prepare physically crosslinked
polyurethane-block-poly(N-vinyl pyrrolidone) (PU-b-PVP) and polyurethane-block-
poly(acrylamide) (PU-b-PAAm) hydrogels. The success of the reactions has been
confirmed by spectroscopic analyses. The detailed water transport behavior of these
hydrogels has been discussed. At last, thermal analyses data of the present materials is
presented.
4.1 Introduction
As stated in Chapter 3, hydrogels are well suited as low-duty biomaterials for
applications such as drug delivery, cells encapsulation, soft tissue substitution, coatings
for biomedical devices, scaffolds for tissue engineering and hemodialysis membranes1"4.
Since hydrogels have inherently poor mechanical properties, most studies in the past
were conducted on those which are chemically crosslinked. Chemically crosslinked
hydrogels, however, cannot be processed in organic solvents.
*A version of this chapter has been published: Patel, A.; Mequanint, K. (2008) Synthesis and
Characterization of Physically Crosslinked Hydrogels from Dithiocarbamate-derived Polyurethane
Macroiniferter. Journal of Polymer Science Part A: Polymer Chemistry; 46: 6272-6284.
82
Unreacted crosslinking agents and monomers that are trapped because of the high
crosslink density could leach out of the medical device, causing toxicity to tissues and
cells. Since many hydrogels for biomedical applications are designed to deliver
therapeutic agents, loading of biologically active molecules before crosslinking has
limitations. This is because the crosslinking process (thermally or photochemically) could
potentially alter the biological activity5. Moreover, redissolving the polymer after the
network is formed, for reshaping, is another issue related to chemically crosslinked
hydrogels. In this regard, physically crosslinked hydrogels offer a clear advantage. The
presence of crystalline domains and hydrophobic/hydrophilic interactions in physically
crosslinked hydrogels may provide higher mechanical strength while overcoming
problems associated with chemically crosslinked hydrogels6.
In order to synthesize physically crosslinked hydrogels with well organized
crosslinked structures, controlled radical polymerization methods; such as atom transfer
radical polymerization ' , reversible addition-fragmentation chain transfer
polymerization910 and iniferter111 could be advantageous. The use of a DC-based
macroiniferter technique to synthesize novel polyurethane hydrogels is described in this
Chapter. This method is beneficial because a high purity of monomers is not critical. As
discussed before, the term iniferter (initiator, transfer agent and terminator), coined by
Otsu and Yoshida, represents a group of molecules that dissociate thermally or
photochemically, initiate and propagate the insertion of monomers, followed by primary
reversible radical terminations and/or chain transfers13. This mechanism provides a
means to synthesize high molecular weight polymers with relatively narrow molecular
83
weight distribution14. The term macroiniferter represents a polymeric iniferter. DC-based
iniferters have been studied for both thermally and photochemically induced
polymerizations to synthesize block, star, graft and/or functional polymers15' 16. For
example, the thermal polymerization of methyl methacrylate (MMA) using DHTD
showed that the reaction proceeded through "living" radical polymerization and
maintained its end-capped diol functionality17. DHTD has also been studied as a
photoiniferter to prepare polystyrene (PS)18. When DHTD is used as a photoiniferter,
wave lengths between 320 and 380 nm decompose the DC group to generate radicals
which initiate and propagate the polymerization. These radicals can also be reversibly
terminated to follow a controlled radical polymerization process19. In only one study,
DHTD was incorporated into poly(ethylene oxide) (PEO) segments and used as a
macroiniferter to prepare PEO-6-PMMA copolymers under irradiation with UV light. A
linear increase in molecular weight of the block copolymers with reaction time was
observed indicating that the DC-based macroiniferter also followed the "living" radical
polymerization20.
Because of their excellent mechanical properties, polyurethanes are attractive for
many practical applications21"23. In Chapter 3, the syntheses of physically crosslinked
hydrogels using a tetraphenyl ethane diol (TPED)-based polyurethane macroiniferters
was described24. In a separate study25 our group also reported that the mechanical
properties of these hydrogels were also significantly higher than conventional hydrogels
and the biological properties such as low protein adsorption showed their potential
biomedical applications. In this Chapter, a novel dithiocarbamate-based polyurethane

84
macroiniferter that was used to synthesize physically crosslinked polyurethane-based
hydrogels is reported. The polyurethane hydrogels were structurally and thermally
characterized, and examined for the kinetics of water transport.
4.2.1 Materials
Common reagents and chemicals were purchased from Sigma Aldrich
(Milwaukee, WI, USA) as described in Chapter 3. The additional materials used for the
study presented in this chapter were also obtained from the same source unless stated
otherwise. Acrylamide (AAm) was recrystallized twice from chloroform whereas iV-vinyl
pyrrolidone (NVP) was passed through an alumina column to remove the inhibitors.
Methyl ethyl ketone (MEK) and dimethyl formamide (DMF) were distilled at reduced
pressure and the middle portions were used. All other chemicals were used as received.
4.2.2 Synthesis of DHTD
3.56 g (0.04 mol) of 2-ethylaminoethanol and 8 mL of triethylamine were
dissolved in 40 mL of chloroform and the mixture was cooled below 10°C. To this
mixture, 3.05 g (0.04 mol) of carbon disulfide was added dropwise. After 3 h, iodine
dissolved in chloroform was added dropwise, until the light violet color of iodine
persisted. The product was washed repeatedly with ice water to remove the
aminohydroiodide. The mixture was dried over MgS04 overnight, filtered and the solvent
was evaporated under vacuum at ambient temperature. The yellow viscous product
85
(Scheme 4.1) was stored in a dark at 4°C (Yield: 85%) and was characterized by FTIR,
'H-NMR and 13C-NMR Spectroscopy.

CH2OH
/ CH2OH HOH2C , /H*N(C2H5), H0H
2C~ s s
/ 10°C
2 UN + 2CS2 •»• N.
N
CHCI,, (CJHJ)JN
2
IODINE ' >
-CH 3 H,C ' X
* H,C
"
>s / CH,
DHTD
SCHEME 4.1: Schematic diagram of DHTD synthesis.
10.00 g (0.01 mol) of PTMO1000 and 5.00 g (0.02 mol) of MDI were charged
into a 500 mL glass reactor equipped with a heating element, a magnetic stirrer, a
condenser and a nitrogen inlet. The reaction was carried out at 65°C for 2.5 h. The
reaction temperature was then reduced to 22°C and 3.28 g (0.01 mol) of DHTD, together
with dibutyltin dilaurate (DBTDL) (0.2% based on the isocyanate content), were added
into the reaction mixture. 80 mL MEK was added to reduce the viscosity of the reaction
mixture. Samples were frequently taken for FTIR analyses and the chain extension
reaction was allowed to proceed until the peak related to the isocyanate (2265 cm"1)
disappeared from the FTIR spectrum. The polyurethane macroiniferter (PUMI) was
precipitated using tenfold excess cold methanol and dried at 30°C in a vacuum oven. The
PUMI was characterized using FTIR and 'H-NMR Spectroscopy.
4.2.4 Synthesis of PU-b-PAAm
1.50 g of PUMI and 5.33 g (0.075 mol) of AAm were dissolved into 50 mL DMF
and the mixture was poured into a glass vial. The solution was purged with nitrogen for
10 min. The glass vial was then irradiated with UV light (model B100AP; UVP Inc.,
86
Upland, CA) at 365 nm and a distance of 20 cm. After a designated reaction time, the
glass vial was quenched with an ice-salt mixture and the PU-Z>-PAAm was precipitated
using tenfold excess cold diethyl ether. The product was dried at 30°C in a vacuum oven.
The residual monomer and homo-PAAm has been extensively extracted in deionized (DI)
water. Similar procedure was followed for different reaction times.
4.2.5 Synthesis of PU-b-PVP
A similar method, described for PU-6-PAAm was used to synthesize PU-&-PVP.
But in this case, the PU-6-PVP was precipitated using tenfold excess cold methanol.
FTIR and 'H-NMR Spectroscopy were used to characterize the block copolymers. The
detailed schematic diagram to synthesize PUMI, PU-6-PAAm and PU-6-PVP is shown in
Scheme 4.2.
Similar to Chapter 3, the sample nomenclature is based on the copolymerization
reaction time. For example, PU-6-PVP08 means the polyurethane-Woc£-poly(Af-vinyl
pyrrolidone) copolymer after an 8 h copolymerization time. For water transport studies,
PUMI was used as control.

87
PU-4-PAAm
SCHEME 4.2: Schematic diagram of PUMI, PU-6-PAAm and PU-6-PVP syntheses.
4.2.6.1 Spectroscopic analyses
Fourier Transform Infrared (FTIR) spectra were recorded by using Bruker Vector
22 spectrophotometer. Samples were directly mounted into the sample holder and 32
scans at 4 cm"1 resolution were collected for each specimen. Nuclear Magnetic
Resonance (!H-NMR and 13C-NMR) spectra were recorded on a Varian® INOVA 400
instrument operating at 400 MHz. CDCI3 was used as a solvent for DHTD whereas
88
DMSO-Jg was used as a solvent for other products. ACD/2D NMR software was used for
the integrations of observed peaks.
4.2.6.2 Gel permeation chromatography
The molecular weights were determined in DMF with 0.1 M LiBr at 85°C using a
Waters 2695 separations module equipped with a Waters 2414 differential refractometer
and two PLgel 5 um mixed-D (300x7.5 mm) columns from Polymer Laboratories. The
calibration was performed using polystyrene standards. The flow rate was 1 mL/min, and
Empower 2 software was used to examine the eluted peaks.
4.2.6.3 Thermogravimetric analysis
TGA was carried out using a TA Instruments Q-series TGA Q 500 analyzer. The
specimens, dried overnight at 50°C in a vacuum oven, were weighed in the range of 5 to
10 mg and heated from 25°C to 700°C at a heating rate of 20°C/min under nitrogen. TA
Instruments Universal Analysis 2000 software was used to analyze the data.
4.2.6.4 Equilibrium water content and swelling kinetics
Polymer specimens (n=4) were cut into 5 mm diameter discs from 0.2 mm thick
cast films. To cast the films; chloroform, THF and acetic acid were used as solvents for
PUMI, PU-6-PVP and PU-6-PAAm respectively. The specimens were dried overnight in
a vacuum oven at 50°C, cooled to room temperature and weighed (Wd). They were then
swollen at ambient temperature (22°C) in distilled water for 24 h. Swollen specimens
were taken out and blotted lightly using filter paper to remove the excess surface water.
89
The hydrated specimens were weighed (Wh) and EWC of the specimens were calculated
using26,
W -W
EWC = ^ ^xlOO (4.1)
Wh
For the swelling kinetics study, specimens were removed at specified time
intervals, blotted with filter paper and weighed. The water uptake of these hydrogels at
specified time interval was calculated using equation 4.1 where Wh was the weight of the
hydrogel specimen at that time interval. To determine the water transport mechanism into
the hydrogels, the water uptake data of each sample were fitted into an empirical
equation27,
f = W,/WK=Kt" (4.2)
Where,/is the fractional water uptake at time t, Wt and Wx are the weight of the
water absorbed in the hydrogels at time t and at equilibrium swelling respectively. A' is a
characteristic rate constant that rely on the hydrogel structure and n is a transport number
which indicates whether diffusion and/or relaxation controls the swelling. From the
swelling data, the diffusion coefficient (D) for planar geometry can be calculated using
OR
the equation ,
, 0 50
/ I K / I
wjwm = {ni}) (4-3)
Where, L is the initial film thickness. The slope of the plot of W/Wco against t050
provides the value of D.

90
4.2.6.5 pH dependent swelling study on P U-b-PAAm hydrogels
Britton-Robinson buffers of different pH were prepared using the method
described by Caykara et al29. Briefly, phosphoric acid (2.70 mL), glacial acetic acid (2.30
mL) and boric acid (2.47 g) were dissolved in distilled water (1 L) and 10.00 M NaOH
was added in required amount to get the buffers with different pH. The PU-6-PAAm
specimens (n=4) were swollen in different pH buffers for 72 h and their EWC were
calculated using equation 4.1.
4.3.1 Synthesis of macroiniferter and block copolymers
The iniferter method is a class of controlled radical polymerization that could be
used to synthesize a variety of block copolymers, including hydrogels with well defined
chain lengths that are easily controlled. Thus, polymers and copolymers of desired
molecular weight having low polydispersity index can be prepared using the iniferter
technique. In the present study, DHTD was used as a chain extender to synthesize novel
DC-based PUMI. Such a PUMI was capable of photochemically initiating the insertion of
vinyl monomers and, thus allowed us to prepare physically crosslinked PU-6-PVP and
PU-Z>-PAAm hydrogels (Scheme 4.2).
The FTIR spectra of DHTD, PUMI, PU-6-PVP and PU-6-PAAm xerogels are
presented in Figure 4.1.

91
PU-ft-PAAm " WÂr^ ^
v n xvr ^
PU-A-PVP v
Y V\ /
v
PUMI Arv\j\^ /^.
DHTD Wvvwr
i
f
4000 3000 2000 1000
Wave number, cm"
FIGURE 4.1: FTIR spectra of DHTD, PUMI, PU-A-PVP and PU-6-PAAm.
The peak related to the DHTD hydroxyl stretching was observed at around 3410
cm'1. The peaks observed at 1487 cm"1, 1185 cm"1 and 1040 cm"1 are assigned to C-N
stretching, C=S stretching and S=C-S stretching vibrations of the DC group respectively.
The S-S stretching of the DC group showed a strong peak at 745 cm"1. The spectrum
related to PUMI prepared from DHTD showed H-bonded N-H stretching at around 3305
cm"1 and carboxyl stretching at 1730 cm"1 related to the urethane group. It also showed a
peak at 1217 cm'1 due to the mixed vibrations involving C-N stretching along with N-H
bending related to the urethane group. The peak corresponding to the C-O-C stretching of
PTMO segments was observed at 1105 cm"1. All these peaks were also observed in the
block copolymers. The PU-6-PVP spectrum showed a broad peak at 1730 cm"1 related to
92
carboxyl stretching compared with the peak observed in PUMI. In the PU-6-PAAm
spectrum, due to the presence of primary amide; symmetric H-bonded N-H stretching
was also observed at 3180 cm"1 along with the asymmetric H-bonded N-H stretching at
3305 cm"1. An asymmetric NH2 deformation of the polyacrylamide block gives a further
peak at 1665 cm"1.
Figure 4.2a shows the 'H-NMR spectrum of the DHTD where, the OH proton
from DHTD appeared at 3.95 ppm. The protons associated to methyl and methylene
groups attached to -NCS2 as well as the methylene protons attached to OH appeared at
1.10-1.30 ppm, 3.50-3.80 ppm and 4.35 ppm respectively. In the 13C-NMR spectrum of
the DHTD (Figure 4.2b), the presence of the peak at 187 ppm related to the -C=S further
confirmed the formation of a dithiocarbamate linkage.

93
H3CV
5
2
,CH2 „CH2 .OH
1 3/ 1
HO" ~CH 2 3^CH2
2
H,CV
CH1
3,4
CDC1 3
W LIAL
Chemical shift, ppm
FIGURE 4.2: (a) 'H-NMR spectrum of DHTD.
1
H,r\
„CH 2 3 N 5 .sv CH, 4 -OH

HO' y*K*s CH2
CH,
CDClj
Ha**** »**»»>»)««»
-T- T- — 1 —
WWiil -1—
200 180 160 140 120 100 80 60 40 20
Chemical shift, ppm
FIGURE 4.2: (b) 13C-NMR spectrum of DHTD.

94
In the 'H-NMR spectrum of the PUMI, (Figure 4.3a), the aromatic and aliphatic
protons from MDI are observed between 7.00-7.50 ppm and 3.75 ppm respectively. The
aliphatic protons related to PTMO, namely CH2, OCH2 and CH2 attached to the urethane
amide groups were identified at 1.46 ppm, 3.34 ppm and 4.03 ppm, respectively. The
urethane NH peak was observed at 8.48 ppm.
T *i
c^^v,
f Y
DMSO-rf,
6,7
U-
6 4 2
Chemical shift, ppm
FIGURE 4.3: (a) 'H-NMR spectrum of PUMI.
In the H-NMR spectrum of the PU-6-PVP (Figure 4.3b), additional peaks related
to the methylene protons adjacent to the ketone group of the NVP and adjacent to -CS2
from DHTD were also observed at 2.04 ppm and 1.85 ppm respectively. The methylene
protons corresponding to the tertiary amine of NVP and adjacent to aliphatic CH from
95
NVP appeared at 3.10 ppm and 1.30 ppm respectively. In the 'H-NMR spectrum of the
PU-6-PAAm (Figure 4.3c), peak related to NH2 protons of the amide group were
identified at 6.83 ppm. The aliphatic protons related to the PAAm block namely,
methylene protons near the CH group and attached to the -CS2 of DHTD were observed
at 1.21 ppm and 2.19 ppm respectively.
Chemical shift, ppm
FIGURE 4.3: (b) 'H-NMR spectrum of PU-ft-PVP.

96
DMSO<
10
2,8
KJL
13
6,7
\ikpJ
Chemical shift, ppm
FIGURE 4.3: (c) 'H-NMR spectrum of PU-6-PAAm.
Figure 4.4 shows the plot of the number average molecular weight (Mn) of PU-b-
PVP xerogels at different copolymerization times. A linear increase in molecular weight
with reaction time indicates that the PUMI, having DC groups incorporated into the
polyurethane backbone, initiated the copolymerization via a "living" system. The
polydispersity indices (PDI) of the present copolymers remained at around 1.45±0.05 for
the reaction times investigated (Table 4.1). These values are generally lower than
reported PDI for other DC-based iniferter systems11'30' '. This could be attributed to the
selective precipitation of the higher molecular weight polymer during the purification
97
process, such that lower molecular weight block copolymers and homopolymers
remained in solution, decreasing the polydispersity indices of the products. Selective
precipitation can, in fact, lead to remarkably low PDI for some polycondensation
polymers32. From Figure 4.4 and Table 4.1, it is also evident that the change in the
molecular weight of the xerogels with reaction time is low. This was not anticipated but
we attributed this to the hydrophilic nature of the poly(/V-vinyl pyrrolidone) block, which
would affect the overall hydrodynamic volumes of the polymers. For example, it has
been shown that grafting of hydrophilic oligo(ethylene glycol)s onto hydrophobic
polymers leads to an unexpected decrease in hydrodynamic volume and thus an apparent
decrease in molecular weight33. Because of the partial solubility in common GPC
solvents, the molecular weights for PU-6-PAAm could not be accurately determined.
Reaction time, h
FIGURE 4.4: Average molecular weight and PDI of PU-6-PVP copolymers as a function of
copolymerization time. Molecular weight versus reaction time (•); PDI versus reaction time (•).
98
Table 4.1: Molecular weights of PU-A-PVP block copolymers.
Reaction
Sample time (h) M„ x 10 4 M w x 10"4 PDI
PUMI 0 3.45 4.85 1.41
PU-6-PVP06 6 3.55 5.00 1.41
PU-6-PVP08 8 3.58 5.01 1.40
PU-6-PVP10 10 3.59 5.19 1.44
PU-6-PVP12 12 3.67 5.18 1.41
PU-6-PVP14 14 3.69 5.33 1.44
PU-6-PVP24 24 3.83 5.68 1.48
Solvent: Dimethyl formamide; [PUMI]0 = 3.00 g/dL; [NVP]0 = 1.50 mol/L.
Previous studies have shown that dithiocarbamate derivatives can be used as an
effective initiator for the controlled polymerization of different vinyl monomers11' ' 4.
Literature data on the synthesis of multiblock copolymers using polymeric
dithiocarbamate iniferter, however, is scarce20'35. The work documented in this Chapter
showed, for the first time, the use of polyurethane macroiniferter chain extended with
DHTD as a novel method to prepare PU-fr-PVP and PU-6-PAAm multiblock copolymers
and, demonstrated the success of this approach. In addition, the current polymers have
significance as physically crosslinked hydrogels from an application standpoint (see later
section). The S-S linkages of the DHTD are known to be responsible for the linear
increase of the molecular weight as well as monomer conversion with time17'36. Thus,
during photopolymerization, the DC groups of DHTD not only initiate the
polymerization, but they also possess excellent chain transfer properties and actively
99
involve in the reversible primary radical termination. Owing to this reversible
termination, reported DHTD initiated homopolymers such as PS and PMMA were almost
end-capped with hydroxyl groups17'36; that makes DHTD an excellent vehicle to prepare
desired macrodiols.
4.3.2 Water transport properties ofpolyurethane hydrogels
Figure 4.5 shows the water transport behavior of PU-6-PVP hydrogels at 22°C as
a function of immersion time. The DHTD based PUMI, which was used as a control,
showed a higher equilibrium water content compared with the previously mentioned
(Chapter 3) TPED based PUMI (8% vs 3%)24. The presence of the tertiary amine into
DHTD might be the reason for higher equilibrium water content in the control PUMI. For
the block copolymer hydrogels, as the copolymerization time was increased, the water
uptake as well as equilibrium water content increased. An increase of hydrophilic
segment into the copolymer, as indicated by an increase in molecular weight, (Figure 4.4)
increased the EWC which was expected. Kim et al37 studied polyurethane-Z>/oc&-
polyacrylic acid (PU-fr-PAAc) using a TPED-based macroiniferter and showed that the
swelling of the block copolymers decreased with reaction time. In that study, PEO had
been utilized as a soft segment and the net swelling of the block copolymers would be
from the combined effect of PEO and PAAc segments, which decreased with increased
copolymerization time.
100
0 100 200 300 400 500

Swelling time, min
FIGURE 4.5: Swelling isotherms of physically crosslinked PU-ft-PVP hydrogels as a function of
immersion time at 22°C. PUMI (•); PU-6-PVP06 (o); PU-6-PVP08 (T); PU-6-PVP10 (A); PU-6-PVP12
(•); PU-6-PVP14 (D).
The water uptake and EWC for the PU-6-PAAm, presented in Figure 4.6,
followed a similar trend. Hydrogels having an EWC up to 40% were prepared from PU-
6-PAAm copolymers; whereas from PU-6-PVP copolymers, hydrogels containing an
EWC up to 60% were prepared.

101
50 n 1
Swelling time, min
FIGURE 4.6: Swelling isotherms of physically crosslinked PU-6-PAAm hydrogels as a function of

immersion time at 22°C. PUMI (•); PU-2>-PAAm04 (o); PU-6-PAAm06 (T); PU-6-PAAm08 (A); PU-b-
PAAmlO (•); PU-A-PAAml2 (D).
In aqueous media, the PU-6-PVP hydrogels were swollen without any mass loss
but dissolved in THF, DMF and CHCI3 confirming that they are physically crosslinked.
PU-6-PAAm hydrogels were found to be partially soluble in these solvents while soluble
in acetic acid.
The water transport properties of the current hydrogels were determined by fitting
the water uptake data to equation 4.2 and, the results are listed in Table 4.2. For one
dimensional planar geometry, the swelling is diffusion controlled if n<0.50 (Case I or
Fickian diffusion); where the rate of structural relaxation is faster than the rate of
102
diffusion. If n = 1.00, (Case II diffusion) water transport is controlled by the rate of
relaxation of the polymer network. If the value of n lies between 0.50 and 1.00,
(anomalous or non-Fickian diffusion) diffusion as well as relaxation rates have a
considerable effect on the swelling rate of the hydrogels.
Table 4.2: Water transport properties of physically crosslinked PU-6-PAAm and PU-6-PVP
hydrogels.
Sample n kxio2 D (cm2/s)x 107 D (cm2/s)x 10* EWC (%)
PUMI 08.30±1.02
PU-Z>-PAAm04 0.50±0.02 1.38 0.04 25.67±2.32
PU-6-PAAm06 0.51±0.02 3.23 0.47 34.26±2.46
PU-6-PAAm08 0.57±0.02 4.52 1.10 34.64+0.67
PU-6-PAAmlO 0.51±0.04 7.72 3.17 38.80+1.16
PU-6-PAAml2 0.49±0.02 10.95 4.70 40.06±2.25
PU-6-PVP06 0.57±0.06 3.70 01.11 15.04+2.32
PU-6-PVP08 0.56±0.02 1.06 01.89 35.67±0.62
PU-6-PVP10 0.53±0.02 0.91 02.70 46.36±2.38
PU-6-PVP12 0.53±0.01 0.95 03.76 50.74±2.96
PU-6-PVP14 0.57+0.01 0.78 03.01 59.11+2.11
Except for PU-6-PAAm08, the transport numbers («) for PU-Z>-PAAm hydrogels
are around 0.50, indicating the PU-6-PAAm hydrogels followed Fickian diffusion. On the
other hand, the PU-6-PVP hydrogels exhibited transport number values between 0.53 to
0.57 and followed non-Fickian diffusion. Strong interchain interaction due to hydrogen
103
bonding between PVP segments and PU segments may have led to a more compact
structure and moved the value of n towards the anomalous diffusion. We should point out
that the accuracy of equation 4.2 is up to / = W, jWm < 0.6.
The water diffusion coefficients of the present hydrogels were calculated using
equation 4.3 and, the results are shown in Table 4.2. For both PU-6-PVP and PU-6-
PAAm hydrogels, the diffusion coefficients increased with copolymerization time. Since
physical crosslinking points were provided by the hydrophobic polyurethane segments,
the role of hydrophilic segments is primarily for water transport through the polymer
network. Therefore, as copolymerization time increased more hydrophilic monomer units
were added and, these in turn increased the available volume fraction for the water
diffusion into the network. In the PU-6-PVP hydrogels, the calculated diffusion
coefficients were in the range of 1.1 lxlO"9 to 3.01 xlO"9 cm2/s. These values are one order
of magnitude lower than reported values for chemically crosslinked PVP-homopolymer38.
This difference is attributed to the lower amounts of PVP incorporated into the hydrogels
structure compared with the homopolymer. The presence of the polyurethane segments
that are hydrophobic could restrict water diffusion and ultimately reduce the diffusion
coefficient values even for small molecules like water. The anomalous transport number
presented in Table 4.2 indicates that diffusion is, in part, controlled by structural
relaxation. Therefore, it is not surprising that we found lower diffusion coefficients for
PU-6-PVP hydrogels. The inclusion of hydrophobic segments into hydrophilic polymers
is also known to significantly reduce the diffusion coefficients39.

104
On the other hand, the diffusion coefficients of PU-6-PAAm vary from 4.30 xlO"9
to 4.70x10"7 cm2/s. By using Quasi-Elastic Light Scattering method, Peters and Candau40
reported the water diffusion coefficient for chemically crosslinked PAAm to be 4xl0"7
cm2/s. Chemically crosslinked PMMA-co-PAAm hydrogels had also been reported to
have similar water diffusion coefficient41. The current PU-6-PAAm hydrogels had
comparable water diffusion coefficients only after 8 h of copolymerization. Between 0 h
and 8 h copolymerization time, the water diffusion coefficients were very low
presumably due to the slow propagation rate of the iniferter system12.
4.3.3 The effect ofpH on swelling of PU-b-PAAm hydrogels
The equilibrium water content study of PU-6-PAAm hydrogels at different pH is
shown in Figure 4.7, which reveals that the PU-6-PAAm hydrogels have higher swelling
in both acidic and basic media compared with the neutral media. The strong hydrogen
bonding between the tertiary amine groups of the DHTD segments and the ether groups
of the PTMO segments may have reduced the swelling between pH 4-11. In strong acidic
conditions, the protonation of tertiary amines induce interchain repulsions, which results
in an expanded configuration and allows more water solvation. In strong basic conditions,
the amide groups of polyacrylamide segments can partially hydrolyze to acrylic acid and
ionize to sodium acrylate '. The ionic repulsion between sodium ions expands the
structure further and allows higher swelling of the matrix.

105
pH
FIGURE 4.7: Swelling isotherms of physically crosslinked PU-fe-PAAm hydrogels at different pH at

22°C. PUMI (•); PU-6-PAAm04 (o); PU-M>AAm06 (T); PU-6-PAAm08 (A); PU-6-PAAml0 (•); PU-b-
PAAm 1 2 ( D ) .
The hydrolysis at higher pH was further confirmed using FTIR Spectroscopy. The
amide II band at 1665 cm"1 almost disappeared while the carboxylate ion stretching at
1450 cm" appeared at higher pH confirming the hydrolysis (Figure 4.8).

106
p H = 13.0
1 1 p 1
1700 1600 1500 1400

W a v e number (cm" )
FIGURE 4.8: FTIR spectra of physically crosslinked PU-fc-PAAml2 hydrogels at different pH. The
spectra show hydrolysis of PU-i-PAAml2 copolymer at higher pH.
The TGA thermographs of the PUMI and the block copolymers are presented in
Figure 4.9. Contrary to conventional polyurethanes, which are known to degrade in two
stages42, Figure 4.9 shows that the PUMI and the copolymer xerogels degraded in three
distinctive stages. The initial decomposition temperatures (T,) and the maximum
decomposition temperatures (Tmax) of the polymers are listed in Table 4.3. The thiuram
disulfide groups decomposed in the first stage and released CS235; whereas the second
and third stage weight losses are associated for the hard and soft segment decompositions
respectively.
107
100 - — PUMI - 14
PU-6-PAAm
^^_^*~*--. PU-6-PVP
^^\^\ - 1 2
80 -|
\ \ A
\ ' "1 0
a \ tf\ i (J
x
o 60 - \ f\i
o03 \ ^ / \i _0 8 <_r
X \/ \i
X i \'
-4-»
J3
WJ
u 40 -
\p1 \ w\ _06
>
'§
h\ \\ Q
£
20 - /
/
/
/ V / \\
m\ 04
//
•N
:
^^y^
/ /
w
\\\\ 02
0- i i i 1 =sa=f 00
100 200 300 400 500 600
Temperature, °C
FIGURE 4.9: TGA thermographs and related derivative thermographs of PUMI; PU-6-PVP and PU-
6-PAAm. PUMI and block copolymers degraded in three distinct stages
Table 4.3: TGA analysis of PUMI, PU-6-PAAm and PU-fc-PVP xerogels.
CS2 release Hard segment Soft segment
Sample decomposition decomposition
T,(°C) Tmax(°C) T,(°C) Tmax(°C) T,(°C) Tmax(°C)
PUMI 153 196 277 362 395 440
PU-6-PAAm 151 195 287 368 396 439
PU-6-PVP 155 192 269 347 387 436

108
4.4 Conclusions
Physically crosslinked polyurethane hydrogels were synthesized using a novel
dithiocarbamate based polyurethane macroiniferter. Spectroscopic studies confirmed that
the block copolymers had been successfully prepared. A linear increase in molecular
weight with copolymerization time revealed that the DC-based polyurethane
macroiniferter follows the "living" radical polymerization mechanism. The PDI also
remained below 1.50. The water uptake study showed that PU-fr-PAAm hydrogels having
EWC up to 40% and PU-6-PVP hydrogels having EWC up to 60% were obtained. Water
transport in polyacrylamide-based polyurethane hydrogels showed Fickian diffusion
whereas poly(./V-vinyl pyrrolidone)-based polyurethane hydrogels showed non-Fickian
diffusion. PU-6-PAAm hydrogels interestingly had higher swelling behavior in strong
acidic and basic environments.
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photopolyinerization of methyl methacrylate by diethyl dithiocarbamato-(l,2)-
propane diol. Journal of Applied Polymer Science 2005, 98, (5), 2320-2328.
20. Cakmak, I., Synthesis of multiblock copolymers via macroiniferter. European

Polymer Journal 1998, 34, (10), 1561-1563.
21. Lligadas, G.; Ronda, J. C; Galia, M.; Biermann, U.; Metzger, J. O., Synthesis and
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Chemistry 2006, 44, (1), 634-645.
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24. Patel, A.; Mequanint, K., Novel physically crosslinked polyurethane-6/oc&-

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28. Crank, J., The mathematics of diffusion. 2n ed.; Clarendon Press Oxford: London,
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dimethylacrylamide-co-acrylamide) hydrogels: Comparison of swelling degree
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polymerization of vinyl monomers in homogeneous system using N,N'-
diethyldithiocarbamate derivatives as photoiniferters. European Polymer Journal
1995, 31,(1), 67-78.
32. De Wit, M. A.; Wang, Z. X.; Atkins, K. M.; Mequanint, K.; Gillies, E. R.,
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34. Kannurpatti, A. R.; Lu, S. X.; Bunker, G. M.; Bowman, C. N., Kinetic and
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35. Nair, C. P. R.; Clouet, G., Functionalization of vinyl-polymers through polymeric

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poly(styrene-6-phosphonamide). Polymer 1988, 29, (10), 1909-1917.
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35, (4), 311-323.
113
CHAPTER
5
The Kinetics of Dithiocarbamate (DC)-mediated Polyurethane

Macroiniferter*
Overview: The objective of this study was to investigate the reaction kinetics of DC-based
polyurethane macroiniferter using methyl methacrylate as a model monomer. The
copolymerization was followed in a controlled manner and the results are explained
thoroughly. The effect of PUMI concentration on the reaction kinetics has been
investigated.
5.1 Introduction
Many important commercial polymers are manufactured using free radical
polymerization technique. Even though free radical polymerization is applicably simple
and versatile, the difficulties in controlling the molecular weight, polydispersity index
and polymer end groups limit the final properties of the polymers obtained. The
limitations are due to irreversible bimolecular and/or disproportionation terminations in
conventional free radical polymerization making it hard to achieve well-defined
architectures with required chemoselectivity such as segmented block copolymers with
controlled block lengths. Such irreversible terminations can be avoided by either of the
following: (i) by stopping the physical contacts of the growing chain ends using specific
*A version of this chapter has been published: Patel, A.; Mequanint, K. (2009) The Kinetics of
Dithiocarbamate-mediated Polyurethane-Woc£-Poly(methyl methacrylate) Polymers. Polymer; 50: 4464-
4470.
114
guards or; (ii) by providing a reversible termination and/or chain transfer agent with
specific free radicals that do not involve in the propagation reaction1. Based on either of
these two approaches, different controlled radical polymerization techniques such as
atom transfer radical polymerization (ATRP)2'3, reversible addition-fragmentation chain
transfer polymerization (RAFT)4, nitroxy-mediated polymerization (NMP)5 and iniferter6
have been developed. Iniferter (Initiator-chain transfer-terminator) is a controlled radical
polymerization method that utilizes the concept of reversible termination7.
Iniferter molecules photochemically or thermally dissociate into transient radicals,
with high reactivity towards unsaturated monomers, and persistent radicals, with high
reactivity towards free radicals. The transient radicals initiate the polymerization and
incorporate monomers to the polymer chain, based on their kinetic constants. The
growing polymer chains reversibly terminate with the persistent radicals that can
reinitiate to incorporate further monomers into the polymer chains. The controlled
repetition of re-initiation, monomer insertion and reversible termination gives high
molecular weight polymer with lower polydispersity index than conventional free radical
polymerization. In addition, both the molecular weight of the polymer and monomer
conversion increase linearly with reaction time. Since the polymerization is carried out
through monomer insertion into the iniferter bonds, such polymers always have iniferter
fragments at the chain ends8. In order to provide controlled radical polymerization, the
reversible termination constant of the growing chain with the persistent radicals has to be
higher than the irreversible termination constants at any conversion. In addition, the rate
of reversible termination reaction has to be greater than the rate of the propagation
115
reaction. Thus, in the case of iniferter, the chemical nature of the persistent radicals is the
key factor towards driving controlled radical polymerizations to the desired direction9.
The three main characteristics of iniferter radical polymerization method are: (i)
the end groups of the polymers are iniferter fragments; (ii) both the molecular weight of
the polymer and monomer conversion increase with reaction time and; (iii) the polymer
prepared through iniferter technique should, if needed, act as polymeric iniferter to
produce block copolymers. Since the seminal work conducted by Ostu7, considerable
progress was made on the mechanism of iniferter polymerization. Dithiocarbamate (DC)-
based photoiniferters were among the earliest to be investigated for the controlled
polymerization of vinyl monomers . Since then, DC derivatives such as benzy\-N,N'-
diethyldithiocarbamate (BEDC)10, 2-AfiV'-diethyldithiocarbamyl) isobutylic acid
(DTCA)"'12, (4-cyano-4-diethyldithiocarbamyl) pentanoic acid (CDPA)" 12 , p-
xylylenebis(Af,A^-diethyldithiocarbamate) (XDT)13, diethyl dithiocarbamato-(l,2)-
propane diol (DCPD)14, benzyl-iV.AT-dimethyl dithiocarbamate (BMDC)15, N,N'-
(tetramethyl) thiuram disulfide (TMTD)15, 0,0'-diisopropylxanthic disulfide (DPXD)15,
diethyl-2,3-dicyano-2,3-di(p-A',7vr'-diethyldithiocarbamymethyl) phenyl-succinate
(DDDCS)16, Ar,AT-(tetraethyl) thiuram disulfide (TETD)10, and l,2,4,5-tetrakis(AÂ^'-
diethyldithiocarbamylmethyl) benzene (DDCMB)17 had been studied as photoiniferters in
the syntheses of diblock14'15, triblock16, star-block10 and graft18"21 copolymers. Many of
these studies, however, focused on small dithiocarbamyl (DTC)-centered persistent
radicals at the end of the growing polymeric chains. In order to prepare multiblock
copolymers, DC-derivatives can also be incorporated in the polymer backbone to provide

116
long polymeric DTC-centered persistent radicals22. Since DC-based iniferters that are
part of the polymer backbone can be affected by diffusion, the long polymeric persistent
radicals may behave differently and potentially influence the overall rate of
polymerization compared with the small persistent radicals.
One important advantage offered by polymeric iniferters is the possibility that
polycondensation polymers could be reacted with vinyl monomers to produce novel
block copolymers with interesting properties. In this regard, our group has been
investigating polyurethane-based macroiniferters to synthesize novel hydrogels for
biomedical applications22"24. These hydrogels have a wide variety of physical,
mechanical, thermal and biological properties expanding the possibilities towards
emerging areas of applications. While a minimal kinetic work was conducted on
polyurethane macroiniferters derived from l,l,2,2-tetraphenyl-l,2-ethanediol , kinetic
data for polyurethane macroiniferters derived from DC is notably absent in the literature.
Detailed kinetic analysis of such macroiniferter provides advanced information to tune
the final properties of the block copolymers. In Chapter 4, the synthesis of DC-based
polyurethane macroiniferter (PUMI) and its application to prepare physically crosslinked
polyurethane hydrogels has been described. Our approach is one of only a few reports
describing the incorporation of DC groups into polycondensation polymers in order to
synthesize block copolymers26"28. In this Chapter, the kinetics of DC-based polyurethane
macroiniferter is examined using methyl methacrylate as a model monomer.

117
5.2.1 Materials
All chemicals were purchased from Sigma-Aldrich (Milwaukee, WI, USA) as
described in Chapter 3. Methyl methacrylate (MMA) was used after passing through an
inhibitor remover column (Sigma-Aldrich, WI). All other chemicals and reagents were of
the highest purity available and used without further purification.
5.2.2 Synthesis of DHTD and PUMI
DHTD and PUMI were prepared using the methods described in Chapter 4. The
final products were characterized using ! H NMR and FTIR spectroscopy.
'H NMR (CDC13) (1) DHTD: 6 = 1.10-1.30 (-CH2CH3), 3.50 (-OH), 3.60-4.00 (-
CH2CH3, -S2CNCH2-) and 4.35 (-CH2OH) ppm; (2) PUMI: 5 = 1.25 (-CH3CH2N-), 1.42
(-CH3CH2N-), 1.61 (-CH2CH2-), 1.71 (-CH2N), 3.40 (-OCH2-), 3.86 (-C6H4CH2C6H4-),
4.15 (-COOCH2-) and 7.00-7.25 (aromatic) ppm.
FTIR: (1) DHTD: 3400 (b, OH), 2975-2935 (b, aliphatic), 1180 (s, C=S), 1040 (s, C-S)
and 750 (s, S-S); (2) PUMI: 3295 (b, NH), 2940-2850 (b, aliphatic), 1725 (s, C=0), 1604
(s, aromatic ring, MDI), 1530 (s, NH), 1480-1445 (aliphatic deformation), 1410 (b, -CH2
in MDI), 1220 (s, C-N, urethane), 1090 (s, C-O-C) and 817 (aromatic, out of plane).
5.2.3 Synthesis ofPU-b-PMMA
A solution of known amounts of PUMI (as shown in Table 5.1) and 12.00 g (0.12
mol) of MMA in 80 mL DMF was purged with nitrogen for 10 min. The solution was
then sealed and irradiated with UV light (100 W, model BIOOAP; UVP Inc., CA) at the
118
•y
distance of 6 cm (« 20 mW/cm ). Samples were taken at stipulated times, poured into
pre-weighed Petri dishes and the solvent was evaporated at 60°C and at reduced pressure
of 200 mmHg for 2 h. The conversions were calculated using a gravimetric method. In
order to calculate the rate of polymerization (Rp) for kinetic studies, the molecular weight
of the repeating unit in PUMI (1828 g/mol) was used for molar concentration
calculations. For molecular weight analyses, samples were taken in intervals of 6 h and
the PU-6-PMMA (Scheme 5.1) was precipitated using tenfold excess cold methanol. The
product was dried at 30°C in a vacuum oven overnight. Unreacted MMA residues and
possible homo-PMMA were extracted using acetonitrile. Since precipitation and
purification of the block copolymers after 8 h reaction was very difficult for PUMI
concentrations below 10 g/L, kinetic data above 8 h reaction was not obtained for these
concentrations.
'H NMR (CDC13) PU-6-PMMA: 6 = 0.78 (-CH3CR3, syndiotactic), 0.95 (-CH3CR3,
atactic), 1.18 (-CH3CR3, isotactic), 1.37 (CH3CH2N-), 1.55 (-CH2CH2-), 1.81 (-CH2-
CR2-CH2-), 3.34 (-OCH2-), 3.53 (CH3OCO), 3.81 (-C6H4CH2C6H4-), 4.09 (-COOCH2-)
and 7.00-7.25 (aromatic) ppm.
FTIR of PU-6-PMMA: 3297 (b, NH), 2940-2860 (b, aliphatic), 1725 (s, C=0), 1598 (s,
aromatic ring, MDI), 1535 (s, NH), 1485-1440 (aliphatic deformation), 1410 (s, CH2 in
MDI), 1275 (s, C-O, PMMA), 1220 (s, C-N, urethane), 1100 (s, C-O-C) and 817
(aromatic, out of plane).

119
o if^* 1 5 !^^!!^*^ ° C2 s
" s H
/°^/\/\ A AJ LA A / \ A/sj-Pki A /\/°^/N^

O H H S J^. C,H5 O
O Ô
I
CH3
SCHEME 5.1: Chemical structure of PU-6-PMMA.
Fourier Transform Infrared (FTIR) spectra were recorded on solid samples by
using a Bruker Vector 22 spectrophotometer. For every specimen, 32 Scans at 4 cm"'
resolution were collected. Nuclear Magnetic Resonance ('H-NMR) spectra were recorded
on a Varian® INOVA 400 (400 MHz) in CDC13. ACD/2D NMR software was used to
analyze the peaks. The molecular weights of the PU-6-PMMA were determined using a
Waters 2695 separations module equipped with a Waters 2414 differential refractometer
and two PLgel 5 urn mixed-D (300x7.5 mm) columns from Polymer Laboratories. The
samples, dissolved in DMF with 0.1 M LiBr and 1% (v/v) triethylamine, were injected at
85°C to the PLgel column using a flow rate of 1 mL/min. Calibration was done using
polystyrene standards. Empower 2 software was used to integrate the eluted curves. TGA
was carried out using a TA Instruments Q-series TGA Q 500 analyzer. The specimens
were dried at 50°C in a vacuum oven overnight, weighed in the range of 5 to 10 mg and
heated from 25°C to 700°C at a heating rate of 10°C/min under nitrogen. TA Instruments
Universal Analysis 2000 software was used to analyze the data.

120
5.3.1 Syntheses of macroiniferter and block copolymers
The chemistry of dithiocarbamate-based photoiniferters was first proposed by
Ostu and coworkers7.
hv
Ml t/wwwwvQ — Q V V W A W W • 9 AWWWWJSJ
[2] WWAWS' + M ** AW*AAA/WVSM#
[31 vVWWV\AAA>V§^/[ + IlM ^" AVWVWWW O f
AVWWWWV/ S P "^" &*MW*HV*V> ./WWVWWW S P S , / W V V * A A A A A A ' '

[4]
[5] JW/WWWwSP, + p §«WWWWW ^" AWWVWAA«SP ] P 7 S' AVWVWVW1 '
[6] ^wwwwvwSP." + •Awwwwwgp • »» ^wwwwwvSPj + >wwvwwwSP2
[7] «AAAA«WwSP* + ^MWWS-SWM^W *. /«VWWWW S P S * W M W ' + S ""W«WW
[8] AIWWVWWV S P + M *" «VWW1W S P + M
[9] ^WkWVWlM/ S P + S ^ ««««WW S P + S
SCHEME 5.2: The polymerization mechanism of DC-based macroiniferter. Reactions 1-4 are the
idealized controlled iniferter polymerization; reactions 5 and 6 are possible irreversible terminations
whereas reactions 7-9 are possible chain transfer reactions.
As presented in Scheme 5.2, the S-S bond in the DC units of the polyurethane can
cleave easily in the presence of UV light between 320 and 380 nm wavelengths'7. Thus,
121
the photolysis of S-S bonds produce two stable DTC-centered radicals that have higher
than 20 p,S lifetime15 (reaction 1). These radicals barely react with MMA monomer (M)
to initiate the polymerization, since they are more receptive to terminate the
polymerization. The only driving force towards the initiation reaction is a high monomer
to DTC-centered radical ratio. Once the DTC-centered radicals initiate and propagate the
polymerization (reactions 2 and 3), the resulting carbon (C)-centered radicals recombine
either through reversible primary radical termination (reaction 4) or through irreversible
bimolecular termination (reaction 5). The C-S bond length (« 1.8 A) formed due to these
0Q
terminations , is also long enough to be cleaved upon further induction of UV light
(reaction 4). The re-initiation provides again C-centered radicals having short life time
and hence participate into the propagation reaction (reaction 3). The DTC-centered
radicals generated through this re-initiation, on the other hand are more stable and
proceed through subsequent primary radical termination reaction (reaction 4). During the
polymerization, C-DTC reversible termination is more preferable than C-C bimolecular
irreversible termination, since the rate constant of C-C termination is at least one order of
magnitude lower than that of C-DTC termination reaction13. Thus, in the absence of
irreversible terminations and other side reactions, the DC-based macroiniferter proceeds
through controlled radical polymerization mechanism. In scheme 5.2, reaction 6 shows
the irreversible termination by disproportionation, whereas reactions 8 and 9 show the
irreversible termination through transfer to monomer and solvent respectively. The extent
of these irreversible termination reactions compete with the controlled mechanism and
potentially increases the polydispersity. In this study, we first prepared high molecular
weight polyurethane macroiniferter containing DC segments. PU-6-PMMA copolymers

122
were then prepared by solution polymerization of MMA with PUMI. The DC segments
within the PUMI backbone followed the photoiniferter chemistry, added PMMA
segments in the polyurethane backbone and thus allowing multiblock PU-6-PMMA
copolymers to be facilely prepared (Scheme 5.1). Figure 5.1 shows the 'H NMR
spectrum of copolymer. All peaks related to the polyurethane and PMMA segments were
7,8
o C2H5 9a,b,c s
H S
o
^L
Ô
C2HS
Yi
I
CH3
11
9a
CDCl,
9b
10
9c
1,6,7
jJL_ JLKJ AJUV
Chemical shift, ppm
FIGURE 5.1: *H-NMR spectrum of PU-MPMMA.

123
Table 5.1: Block copolymerization of MMA using PUMI under UV radiation in DMF.
Run PUMI (g) MMA(g) DMF PUMI Iniferter MMA
(mL) (g/L) concentration* (mol/L)
(mol/L) x 103
1 0.05 12.00 80.00 0.625 0.342 1.50
2 0.10 12.00 80.00 1.25 0.684 1.50
3 0.20 12.00 80.00 2.50 1.368 1.50
4 0.40 12.00 80.00 5.00 2.735 1.50
5 0.80 12.00 80.00 10.00 5.470 1.50
6 1.20 12.00 80.00 15.00 8.206 1.50
7 2.40 12.00 80.00 30.00 16.411 1.50
8 3.20 12.00 80.00 40.00 21.882 1.50
* The PUMI used in the reaction in units of g/L has been converted to mol/L by dividing it with the
molecular weight of the repeating unit (1828 g/mol).
In order to minimize the possibility of irreversible terminations and other chain

•y
breaking side reactions, a low intensity («20 mW/cm ) UV light was used. Accordingly,
PU-6-PMMA copolymers at different PUMI concentrations were prepared and, the
influence of macroiniferter concentrations on the rate of copolymerization was studied.
5.3.2 Reaction kinetics ofP UMI
The first order dependency of the copolymerization reactions at different PUMI
concentrations are shown in Figure 5.2.

0.20
0.15 -
g o.io-
s
0.05 -
0.00
Reaction time, h
0.8
• [PUMI] = 10.00 g/L

O [PUMI] = 15.00 g/L
B T [PUMI] = 30.00 g/L
0.6 - A [PUMI] = 40.00 g/L
2° 0.4 H
B
0.5 1.0 1.5

Reaction time, h
0.2 -
1 1
10 15 20 25 30
Reaction time, h
FIGURE 5.2: Ln(M0/M) versus time plots for the MMA copolymerization with different
concentrations of PUMI in DMF under the induction of UV light.
125
Here, M0 represents the initial monomer concentration and M represents the
monomer concentration at a specific reaction time of interest. The semi-logarithmic plots
represent that the polymerization reactions followed first order reaction kinetics.
Instantaneous chain growth was not observed at almost all PUMI concentrations. Thus a
lag phase for the first hour of the reaction is evident in Figure 5.2. The possible reasons
are explained as follows. As mentioned earlier, initial macroiniferter decompositions
provide only DTC-centered radicals and these radicals hardly initiate the polymerization
reaction. Once the C-S bonds are formed; the re-initiation, monomer addition and
reversible combination proceeded through first order reaction kinetics. This mechanism is
not unique to polymeric iniferters as small molecule iniferters are also known to initiate
at a slower rate. However, the lag phase was not observed when small molecules were
used as iniferters10'111 . Therefore, it is believed that the size of the iniferter molecule
played a role and exacerbated the observed lag phase. The polyurethane macroiniferter
used in this study has a molecular weight of 13,000 g/mol and is very large to diffuse
through the reaction solution and initiate the polymerization instantaneously.
During the course of the polymerization, a linear increase in both molecular
weight and conversion with reaction time had been observed for different PUMI
concentrations (Figure 5.3). This allows controlling the length of PMMA segments
within the polyurethane backbone by adjusting the radiation time. At every PUMI
concentrations, a linear increase in molecular weight with respect to the conversion is
consistent with the monomer conversion that proceeds via a controlled radical
polymerization mechanism. The polydispersity indices (PDI) remained unaffected or

126
decreased slightly and supported the hypothesis that the monomer addition into
polyurethane backbone progress in a controlled manner. Interestingly Figure 5.3a-c also
highlights that as the PUMI concentration increased from 15 g/L to 40 g/L, both
molecular weight and conversion decreased for fixed reaction times. This observation is
in sharp contrast to conventional radical polymerization whereby an increase of initiator
concentration increases conversion.
0 10 20 30 40
Reaction time, h
127
a
_©
°5
x h.
a >
a
o
U
Reaction time, h
- 40
- 30
a
o
X u
a
- 20 >
2 a
o
U
- 10
Reaction time, h
FIGURE 5.3: Molecular weight and monomer conversion plots versus time for the copolymerization
of MMA with different PUMI concentrations in DMF. (a) [PUMI] = 15 g/L, (b) [PUMI] = 30 g/L and
(c) [PUMI] = 40 g/L. The insert shows the molecular weight and PDI as a function of monomer
conversion.
128
Figure 5.4 shows the GPC traces of PU-6-PMMA for different polymerization
times and at different PUMI concentrations. For all PUMI concentrations used, the peaks
obtained were unimodel and moved distinctively towards lower elution volumes as the
reaction time increases, indicating a molecular weight increase. Turner and Blevins30
reported that when dithiocarbamate end-capped poly(methyl methacrylate) was chain
extended with MMA, the molecular weight first increased and then decreased at higher
conversion. Using dithiocarbamate end-capped polystyrene and poly(methyl
methacrylate) macroiniferters for sequential block copolymerization, the same authors
also showed that the addition of MMA as a second monomer resulted higher
homopolymerization than styrene although studies carried out by Otsu and coworkers
-i i n-y
demonstrated the reverse effect ' . Because the current study used a dithiocarbamate-
containing diol for the synthesis of the polyurethane, there are 7-8 iniferter units per mole
of polyurethane as part of the polymer backbone. These iniferter units are part of the
polymer backbone and, the radicals generated at any time will have a polyurethane
backbone. In dithiocarbamate end-capped inferter systems, the main reason for
homopolymer formation is the recombination of the persistent radicals followed by re-
initiation . In the current study, such recombination results polyurethane segments with
iniferter units. Upon re-initiation, MMA can only be added into yet polyurethane bearing
PMMA radical making the likelihood of MMA homopolymer formation rare. Therefore
the use of polycondensation polymers as macroiniferter is advantageous over the end-
capped macroiniferter27'28. However, the propagating PMMA radical is known to undergo
predominantly termination by disproportionation that may lead to the formation of PU-6-
PMMA diblocks instead of the multiblock final product. Given that we started with a
129
high molecular weight macroiniferter, such diblock polymer chains should either reduce
the molecular weights of the final product or result in a bimodal distribution
corresponding to the diblock and multiblock polymers. GPC traces showed neither of
these effects (Figure 5.4) indicating the possibility that the PMMA propagating radicals
towards disproportionation termination is less pronounced in the presence of polymeric
DTC-centered radicals. Further, the photolysis of dithiocarbamate iniferters are shown to
release some CS2 leading to the loss of the living nature of the polymerization30'34. That
was the case when dithiocarbamate-terminated poly(methyl methacrylate) was chain
extended with methyl methacrylate30 and when «-butyl acrylate was polymerized by n-
butyl-2-(jV,iV-diethyldithiocarbamyl)propionate34. Contrary to these, when
dithiocarbamate-terminated poly(«-butyl acrylate) was chain extended with «-butyl
acrylate, it followed a (pseudo-linear) molecular weight increase with conversion despite
CS2 evolution. This suggests that the extent of CS2 evolution is highly system-dependent
rather than simply the monomer choice. Despite all these possibilities, the current study
showed a linear molecular weight increase with conversion (Figure 5.3a,b,c) and support
the notion that the system followed a predominantly controlled manner. On a final note,
we should point out that the kinetics of macroiniferters are likely to be more complicated
than the ideal living polymerization.

130
10 12 14 16 18 20
Elution volume, mL
FIGURE 5.4: GPC elution curves for the PU-6-PMMA with different concentrations of PUMI in
DMF. (a) [PUMI] = 15 g/L, (b) [PUMI] = 30 g/L and (c) [PUMI] = 40 g/L.
131
The change in the rate of copolymerization with reaction time is shown in Figure
5.5. Initially a lower rate of polymerization was observed due to the slow rate of initiation
(refer to Figure 5.2). After 4 h of reaction time, the rate of polymerization appeared to
plateau. For PUMI concentrations of 10 g/L and 15 g/L, the plateau is followed by a
decrease in the rate of polymerization. There are three possible reasons for this: (i) For
these two PUMI concentrations, high monomer conversions were observed specifically
above 18 h which in turn affects the monomer concentration. Usually, for an effective
iniferter, the reversible termination is always faster than the propagation and the only
factor that promotes the propagation reaction is the high ratio of monomer to DTC-
centered radicals. At high monomer conversion this ratio may decrease to the level where
the effect of reversible termination becomes the controlling factor compared with the
rates of propagation and; the rate of polymerization starts to fall, (ii) With increased
conversion, the viscosity of the reaction mixture increases which may also be a
OH
considerable factor in decreasing the rate of polymerization . (iii) The presence of
irreversible terminations may also reduce the free radical concentrations with reaction
time and hence Rp decreases. Such irreversible terminations should have contributed to
increase the PDI which was not observed in our study (Figure 5.3). Thus, the possibility
of irreversible terminations is rare. Overall, the rates of polymerization in this study are
much lower than those of conventional free radical polymerization which is consistent
with controlled polymerization methods35. As a final point, due to the significant
depletion of monomer concentration above 50% conversion, the first order kinetic seems
to deviate from linearity.

132
Reaction time, h
0 5 10 15 20 25 30
Reaction time, h
FIGURE 5.5: Rate of polymerization versus time curves for MMA copolymerization with different
PUMI concentrations in DMF.
133
The effect of PUMI concentrations on the rates of polymerization is shown in
Figure 5.6. The rates of polymerization initially increased with an increase of PUMI
concentrations, passes through a maxima and then falls off as a function of PUMI
concentrations. In this work the optimal PUMI concentration to achieve the highest Rp is
5g/L(2.74xlO_3mol/L).
10 T 1
FIGURE 5.6: Plots of rate of polymerizations as a function of PUMI concentrations for various
reaction times.
The iniferter to monomer ratio plays an important role in iniferter chemistry. At
lower PUMI to monomer ratio, the rate of polymerization increases with iniferter
concentration similar to the conventional radical polymerization. As the PUMI to
monomer ratio increases, the concentration of persistant radicals also increases which, in
turn, increases the rate of reversible termination and reduces the overall rate of
134
polymerization. Thus the rate of polymerization increases until certain critical iniferter
concentration and then decreases with increasing iniferter concentration36. Moreover, at
the optimal PUMI concentration of 5 g/L (2.74x10" mol/L), maximum conversion as
well as maximum molecular weight can be obtained at a fixed reaction time. On the other
hand, in conventional radical polymerization the rate of polymerization always increases
linearly with square root of initiator concentration .
The TGA thermographs of the PUMI and PU-6-PMMA copolymers are presented
in Figure 5.7. Conventional polyurethanes are known to degrade in two stages that are
related to the soft and hard segments degradations37. Figure 5.7 shows that the PUMI and
PU-6-PMMA degraded in three distinctive stages. The first degradation stage is related to
the DC decomposition and release of CS2, whereas the second and third stage
degradations are related to the hard and soft segment decompositions respectively. PU-6-
PMMA showed higher thermal stability than the PUMI especially in the early stages of
the degradation.
135
100 ^"~^™"""^^** - . 1.2

^^\N PUMI
PU-6-PMMA
ww
. 1.0
\ r\ \
80 •
\ /M
• 0.8
| 60 l U
o
• 0.6 %
J5 'l\ _>
'C
.£? 40 ' M1 i>
*\'/ n\1\ l
. 0.4 Q
/ )
rJ /
/ 1 w \\i\
20
- 0.2
\ \ \
i i 0.0
100 200 300 400 500 600 700
Temperature, °C
FIGURE 5.7: TGA - primary and derivative mass losses for PUMI and PU-A-PMMA.
5.4 Conclusions
Several PU-6-PMMA copolymers were prepared using a macroiniferter technique
which followed the controlled radical polymerization with first order kinetics. The linear
increase in molecular weight and conversion with reaction time supported that the DC-
derived PUMI is an effective photo-macro iniferter. Thus, the ratio of the block lengths of
PU/PMMA can be easily controlled by selecting the molecular weight of PTMO in PUMI
synthesis and by monomer conversion in the PU-6-PMMA synthesis. The dome shape
curves of the rates of polymerization versus square roots of PUMI concentrations showed
a typical controlled polymerization system. The three stage thermal degradations of both
PUMI and PU-6-PMMA were related to the CS2 release, hard segment and soft segment
degradations respectively. The studied DC-derived PUMI offers a facile way for the
136
preparation of polyurethane-based elastomeric block copolymers with tuned segmental
lengths of different blocks for many practical applications.
5.5 References
1. Sebenik, A., Living free-radical block copolymerization using thio-iniferters.

2. Tong, L.; Shen, Z.; Zhang, S.; Li, Y. J.; Lu, G. L.; Huang, X. Y., Synthesis and
characterization of perfluorocyclobutyl aryl ether-based amphiphilic diblock
copolymer. Polymer 2008, 49, (21), 4534-4540.
3. Tsarevsky, N. V.; Matyjaszewski, K., Environmentally benign atom transfer

radical polymerization: Towards "green" processes and materials. Journal of
4. Moad, G.; Rizzardo, E.; Thang, S. H., Radical addition-fragmentation chemistry

in polymer synthesis. Polymer 2008, 49, (5), 1079-1131.
5. Sciannamea, V.; Jerome, R.; Detrembleur, C, In-situ nitroxide-mediated radical

polymerization (NMP) processes: Their understanding and optimization.
Chemical Reviews 2008, 108, (3), 1104-1126.
7. Otsu, T.; Yoshida, M., Role of initiator-transfer agent-terminator (iniferter) in

radical polymerizations - Polymer design by organic disulfides as iniferters.
8. Liu, P., Modification of polymeric materials via surface-initiated

controlled/"living" radical polymerization. e-Polymers 062, 2007.
Microgels - Iniferters 1998, 136, 75-137.
10. Otsu, T.; Matsunaga, T.; Doi, T.; Matsumoto, A., Features of living radical
polymerization of vinyl monomers in homogeneous system using N,N'-
diethyldithiocarbamate derivatives as photoiniferters. European Polymer Journal
1995, 31,(1), 67-78.
137
11. Ishizu, K.; Katsuhara, H.; Kawauchi, S.; Furo, M., Controlled radical
polymerization of styrene initiated by diethyldithiocarbamate-mediated iniferters.
Journal of Applied Polymer Science 2005, 95, (2), 413-418.
12. Ishizu, K.; Katsuhara, H.; Itoya, K., Synthesis of amphiphilic star block
copolymers via diethyldithiocarbamate-mediated living radical polymerization.
Journal of Polymer Science Part A: Polymer Chemistry 2006, 44, (10), 3321-
3327.
13. Kannurpatti, A. R.; Lu, S. X.; Bunker, G. M.; Bowman, C. N., Kinetic and
mechanistic studies of iniferter photopolymerizations. Macromolecules 1996, 29,
(23), 7310-7315.
14. Bhuyan, P. K.; Kakati, D. K., Effect of the reaction conditions on the
photopolyinerization of methyl methacrylate by diethyl dithiocarbamato-(l,2)-
propane diol. Journal ofApplied Polymer Science 2005, 98, (5), 2320-2328.
15. Lalevee, J.; El-Roz, M.; Allonas, X.; Fouassier, J. P., Controlled
photopolymerization reactions: The reactivity of new photoiniferters. Journal of
16. Qin, S. H.; Qiu, K. Y., A facile method for synthesis of ABA triblock copolymers
with macro-iniferter technique. Polymer 2001, 42, (7), 3033-3042.
17. Nemoto, Y.; Nakayama, Y., Optimal irradiation wavelength in iniferter-based

photocontrolled radical polymerization. Journal of Polymer Science Part A:
Polymer Chemistry 2008,46, (13), 4505-4512.
18. Ward, J. H.; Bashir, R.; Peppas, N. A., Micropatterning of biomedical polymer
surfaces by novel UV polymerization techniques. Journal of Biomedical
Materials Research 2001, 56, (3), 351-60.
19. Luo, N.; Hutchison, J. B.; Anseth, K. S.; Bowman, C. N., Synthesis of a novel
methacrylic monomer iniferter and its application in surface photografting on
crosslinked polymer substrates. Journal of Polymer Science Part A: Polymer
Chemistry 2002, 40, (11), 1885-1891.
20. Al-Kaabi, K.; van Reenen, A. J., Controlled radical polymerization of

poly(methyl methacrylate-g-epichlorohydrin) using A/,7V'-dithiocarbamate-
mediated iniferters. Journal of Applied Polymer Science 2008, 108, (4), 2528-
2534.
21. Reddy, S. K.; Sebra, R. P.; Anseth, K. S.; Bowman, C. N., Living radical
photopolymerization induced grafting on thiol-ene based substrates. Journal of
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biocompatibility of novel physically crosslinked polyurethane-6/ocA:-
24. Patel, A.; Mequanint, K., Novel physically crosslinked polyurethane-6/ocA:-

7, (5), 727-737.
25. Tharanikkarasu, K.; Radhakrishnan, G., Tetraphenylethane iniferters - 9.

Diphenylmethane diisocyanate-based polyurethane-polystyrene block copolymers
through "living" radical mechanism. European Polymer Journal 1997, 33, (10-
12), 1779-1786.
26. Cakmak, I., Synthesis of multiblock copolymers via macroiniferter. European

Polymer Journal 1998, 34, (10), 1561-1563.
27. Nair, C. P. R.; Clouet, G., Functionalization of vinyl-polymers through polymeric

iniferters - synthesis of poly(methyl methacrylate-6-phosphonamide) and
poly(styrene-Z)-phosphonamide). Polymer 1988, 29, (10), 1909-1917.
28. Nair, C. P. R.; Clouet, G., Block copolymers via thermal polymeric iniferters -
synthesis of silicone vinyl block copolymers. Macromolecules 1990, 23, (5),
1361-1369.
29. Ishizu, K.; Katsuhara, H.; Itoya, K., Controlled radical polymerization of
methacrylic acid initiated by diethyldithiocarbamate-mediated iniferter. Journal of
30. Turner, S. R.; Blevins, R. W., Photoinitiated block copolymer formation using
dithiocarbamate free-radical chemistry. Macromolecules 1990, 23, (6), 1856-
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31. Otsu, T.; Kuriyama, A., Living monoradical and biradical polymerizations in
homogeneous system synthesis of AB and ABA type block copolymers. Polymer
Bulletin 1984, 11, (2), 135-142.
32. Otsu, T.; Kuriyama, A., Polymer design by iniferter technique in radical
polymerization - synthesis of AB and ABA block copolymers containing random
and alternating copolymer sequences. Polymer Journal 1985, 17, (1), 97-104.
139
33. Manga, J. D.; Polton, A.; Tardi, M.; Sigwalt, P., Mechanism of the polymerization
of w-butyl acrylate initiated by iV.iV'-diethyldithiocarbamate derivatives. Part 1.
Photolysis of butyl-2-(AîV'-diethyldithiocarbamyl)propionate and oligomerization
of butyl acrylate. Polymer International 1998, 45, (1), 14-21.
34. Manga, J. D.; Tardi, M.; Polton, A.; Sigwalt, P., Mechanism of the polymerization
of rc-butyl acrylate initiated by 7V,A/'-diethyldithiocarbamate derivatives. Part 2.
Investigation of the reaction mechanism. Polymer International 1998, 45, (3),
243-254.
th
35. Odian, J., Principles of Polymerization. 4 ed.; John Wiley & Sons, New York
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36. Kongkaew, A.; Wootthikanokkhan, J., Synthesis of polyisoprene-poly(methyl
methacrylate) block copolymers by using polyisoprene as a macro-iniferter.
Polymer Bulletin 1999, 43, (4-5), 327-332.
37. Bajsic, E. G.; Rek, V.; Agic, A., Thermal degradation of polyurethane elastomers:
Determination of kinetic parameters. Journal of Elastomers and Plastics 2003,
35,(4), 311-323.
140
CHAPTER
6
Dicyclohexylmethane Diisocyanate (H12MDI)-based Physically

Crosslinked Polyurethane Hydrogels and Their Surface
Modification
Overview: Dithiocarbamate (DC)-based polyurethane macroiniferter (PUMI) was

prepared using HnMDI. The PUMI was further used to synthesize physically crosslinked
polyurethane-block-poly(2-hydroxyethyl methacrylate) (PU-b-PHEMA) hydrogels. The
surface of the hydrogels has been modified with bovine fibronectin and a short term cell
culture study has been conducted. The structural, swelling, thermal, and biological
analyses of the current materials have been discussed thoroughly. Fibronectin modified
PU-b-PHEMA hydrogels have shown excellent cell attachment and spreading suggesting
its potential use for tissue engineering applications.
6.1 Introduction
Tissue engineering has emerged as an alternative approach to current treatment
modalities for failed and diseased tissues as well as to improve the healthcare
standards1'2. One of the approaches to engineer living tissues uses polymeric scaffold as a
platform for cell attachment and subsequent controlled cell proliferation while
maintaining the desired cells function3'4. To direct cell function in order to remodel the
scaffold by extracellular matrix structure that functions similar to the living tissue to be
replaced presents both a challenge and an opportunity. The scaffold platform underlying
the cell surface has significant impact on the behavior of seeded cells5. Thus, any
functional biomaterials designed for scaffolding applications should have biofunctionality

141
for cell attachment, migration and proliferation. Hydrogels, having many similarities in
their physiological properties with living tissues, have demonstrated great potential as
one of the most promising groups of scaffolding biomaterials. Properties of hydrogels
such as aqueous surface environment, lower frictional irritation due to lower surface
energy, soft and tissue-like physical properties, micro-porous channels for additional
nutrition transport, ease of surface modification with biomolecules and unique
biocompatibility are some of the attractions for tissue engineering applications4'6.
Naturally occurring hydrogels possess specific cell binding ligands and can be used for
scaffolds but inconsistency between batches is an issue with natural hydrogels7. These
issues can be resolved using synthetic hydrogels, but they generally do not support cell
attachment. However, they can be modified using short chain cell-adhesive peptides,
collagen, fibronectin, laminin or vitronectin.
PHEMA was the first synthetic hydrogel investigated by Wichterle and Lim8 and
is one of the most important hydrogel biomaterial due to its biocompatibility and
inertness towards most biological processes. Moreover, PHEMA can easily be modified
with bioactive molecules for tissue engineering applications9. Thus, PHEMA has been
extensively used for numerous biomedical applications such as contact lenses10, corneal
implants11, cardiovascular implants12, breast implants13, nervous tissue repair14, drug
release devices15 and tissue repair platform16.

142
The three-dimensional structural integrity of a swollen hydrogel is maintained
either by chemical or physical crosslinking. The conventional wisdom for improving the
mechanical strength of hydrogels relies on chemical crosslinking. However, difficulties in
post-synthesis device fabrications and crosslinker toxicity are drawbacks of chemically
crosslinked hydrogels17. In this regard, physically crosslinked hydrogels offer a clear
advantage and more interest have been taken to develop such hydrogels.
Polyurethanes are known to have excellent blood compatibility as well as
mechanical properties that brought them as useful biomaterials. Compared with the other
physically crosslinked hydrogels, polyurethane-based linear hydrogels are attractive for
biomedical applications since they are deemed biocompatible and the presence of strong
hydrogen bonding provides excellent mechanical properties. In previous Chapters 3 and
4, we have studied different physically crosslinked polyurethane hydrogels using a
macroiniferter technique18'19. The objective of the work contained in this Chapter was to
synthesize physically crosslinked polyurethane-WocA:-poly(2-hydroxyethyl methacrylate)
(PU-6-PHEMA) hydrogels using the macroiniferter approach, modify the hydrogels by
fibronectin conjugation and examine their potential utility for tissue engineering
application.
6.2.1 Materials
All chemicals were purchased from Sigma Aldrich (Milwaukee, WI, USA) as
described in Chapter 3. 2-hydroxyethyl methacrylate (HEMA) was passed through an

143
inhibitor removal column (Sigma Aldrich, Milwaukee, WI, USA) before use. 2-Butanone
and dimethyl formamide (DMF) were distilled at reduced pressure and the middle
portions were used.
6.2.2 Synthesis of DHTD
DHTD was synthesized from 2-ethylaminoethanol and carbon disulfide in
presence of iodine as described in Chapter 417.
'H-NMR (CDCb): 5 = 1.10-1.30 (-CH2CH3), 3.50 (-OH), 3.60-4.00 (-CH2CH3,-
S2CNCH2-) and 4.35 (-CH2OH) ppm.
A mixture of 10.00 g (0.01 mol) of PTMO 1000, 5.25 g (0.02 mol) of 4,4'-
dicyclohexylmethane diisocyanate (H12MDI) into 20 mL 2-butanone was heated to 65°C
for 3.5 h. The mixture was then cooled to 22°C and a solution of 3.28 g (0.01 mol) of
DHTD in 60 mL 2-butanone with 0.5 mol % (based on isocyanate content) dibutyltin
dilaurate (DBTDL) catalyst was added. The chain extension reaction was carried out until
the isocyanate peak (2265 cm'1) disappeared from the FTIR spectrum. The polyurethane
macroiniferter (PUMI) was then precipitated in tenfold excess cold methanol and dried at
30°C in a vacuum oven.
6.2.4 Synthesis of PU-b-PHEMA
A solution of 0.93 g of PUMI and 4.88 g (0.075 mol) of HEMA in 50 mL DMF
was purged with nitrogen for 10 min and irradiated with UV light (model BIOOAP; UVP
144
Inc., Upland, CA) at a distance of 6 cm. After 24 h, the PU-6-PHEMA copolymer was
precipitated using tenfold excess cold diethyl ether. The product was dried at 30°C in a
vacuum oven. Any PHEMA homopolymer and unreacted HEMA monomers were then
extracted in acetonitrile. The product was further kept in distilled water for a week in
order to leach out any unreacted monomers. Water was exchanged every other day. The
schematic diagram of the synthetic route is shown in Scheme 6.1.
SCHEME 6.1: Schematic diagram of PUMI and PU-fe-PHEMA synthesis.
6.2.5 Modification of PU-b-PHEMA copolymer surface with fibronectin
2D film of PU-6-PHEMA was prepared by a solvent casting method using DMF.
Circular discs having a diameter of 0.50 cm were punched out using a one-hole paper
145
punch. The discs were equilibrated with acetone in individual vials overnight. The discs
were then transferred to vials having 20 mM l,l'-carbonyldiimidazole (CDI) solution in
acetone. After 3 h, the unreacted CDI was removed by rinsing the discs in excess
anhydrous acetone three times. The discs were then sequentially washed with distilled
water through increasing ratios of water/acetone solutions (25/75, 50/50, 75/25; v/v) and
finally washed with water. Surface activated discs were placed into vials containing 600
pL of 50 pg/mL bovine fibronectin (Fn) (Trevigen®, Gaithersburg, MD) solution in 1%
phosphate buffered saline (PBS). Protein conjugation was allowed to proceed for 24 h at
4°C. Adsorbed protein was removed by washing in 2% sodium dodecyl sulphate (SDS)
for 1 h. Finally, the discs were rinsed with PBS and equilibrated overnight. The Fn
modified hydrogel discs (Fn-g-PU-6-PHEMA) were then stored in sterile PBS.
—OH -O —O
H2N—vAAA/1 ^NH-WvV
y-N\ ^ N
T O
in PBS (50 pg/mL) O
o
—OH -o h-O
Acetone N 4°C ^—NH-WW
> - !\ ^ N
O
25°C 24h
—OH 3h —O
)j— NH-yAAA/1
O O
PU-6-PHEMA CDI-PU-6-PHEMA Fn-g-PU-6-PHEMA

SCHEME 6.2: Schematic diagram of the modification of PU-6-PHEMA copolymers with bovine
fibronectin using CDI conjugation method.
6.2.6.1 Spectroscopic analysis
Fourier Transform Infrared (FTIR) spectra were recorded using Bruker Vector 22
spectrophotometer. Samples were mounted directly over the sample holder and 32 Scans
146
at 4 cm"1 resolution were collected for each sample. Nuclear Magnetic Resonance ('H-
NMR) spectra were recorded on Varian® Mercury 400 instrument operating at 400 MHz.
DHTD was dissolved in d-chloroform (CDCI3), whereas PU-6-PHEMA was dissolved in
dimethyl sulfoxide-^ (DMSO-J6) for 'H-NMR study. The observed peaks were
analyzed using ACD/2D NMR software.
6.2.6.2 Gel permeation chromatography
The molecular weights were determined using a Waters 2695 separations module
equipped with a Waters 2414 differential refractometer and two PLgel 5 pm mixed-D
(300x7.5 mm) columns from Polymer Laboratories. The samples, dissolved in DMF with
0.1 M LiBr and 1% (v/v) triethylamine, were injected at 85°C to the PLgel column using
flow rate of 1 mL/min. The calibration was performed using polystyrene standards.
Empower 2 software was used to examine the eluted peaks.
6.2.6.3 Swelling study
Polymer discs (n=4) were cut into 5 mm diameter from cast films. The discs were
dried in a vacuum oven at 50°C, cooled to room temperature and weighed. They were
then swollen at room temperature (22°C) in distilled water. Swollen discs were taken out
of the water at regular time intervals and blotted lightly with a filter paper to remove the
excess surface water. The weights of swollen discs were determined and water uptakes of
the discs were calculated using:
W -W
WU = -± ^xlOO (6.1)
wh
147
Where, Wh is the weight of the swollen disc at time t and Wd is the dry weight of the disc
before swelling.
6.2.6.4 Scanning Electron Microscopy (SEM)
Surface morphology of PUMI and PU-6-PHEMA discs were visualized using
SEM, (S-2600N, Hitachi, Japan). Discs were mounted on carbon-taped aluminum stubs
and gold-sputtered at 15 mA for 1.5 min prior to imaging.
6.2.6.5 Thermal analysis
Differential scanning calorimetric (DSC) study and thermogravimetric analysis
(TGA) of PUMI and PU-6-PHEMA were carried out using TA instruments Model Q20
V24.3 Build 115. The samples were dried in a vacuum oven at 50°C for 24 h before use.
For DSC analysis, 5 mg Specimens were sealed in a DSC pan and quenched to -110°C. It
was then left to equilibrate for 15 min and heated to 200°C at a rate of 20°C/min. For
TGA, samples were weighed in the range of 5 to 10 mg and heated from 25°C to 700°C at
a heating rate of 20°C/min under nitrogen. TA Instruments Universal Analysis 2000
software was used to analyze the data.
6.2.6.6 Prote in adsorption study
PUMI and PU-6-PHEMA discs (n=3) were incubated in 0.10 M phosphate
buffered saline (PBS) solution for 24 h in 24-well plate. The PBS solution (pH 7.4) was
replaced by 1 mL of bovine fibronectin solution (20 p.g/mL). The protein adsorption
study was conducted for 6 h in an incubator at 37°C. The discs were then rinsed three
times with 1 mL PBS solution, for 10 min each. In order to extract the adherent proteins,
148
the discs were treated with 1 mL aqueous solution of SDS (2%) at room temperature for 2
h. The extracted proteins were analyzed using Bicinchoninic Acid (BCA) protein assay
reagent kit (Pierce Biotechnology, Rockford, IL) and quantified using a Beckman Coulter
DU 520 UV-Vis Spectrophotometer (Mississauga, ON) at 562 nm. Similar experiments
were repeated at higher fibronectin concentration (50 pg/mL).
6.2.6.7 Preparation of cell culture samples
Circular 2D discs having a diameter of 0.5 cm were affixed to 1.2 cm diameter
glass coverslips using silicone grease, sterilized with 70% ethanol and allowed to dry
overnight. Coverslips containing polymer films were placed into individual wells of a 24-
well tissue culture plate for cell seeding.
6.2.6.8 Cell culture study
The cells examined in this study were primary human coronary artery smooth
muscle cells (HCASMC) purchased from Cambrex Biosciences (Walkersville, MD).
Before cell seeding, HCASMC were cultured on tissue culture dishes in smooth muscle
growth media with 5% FBS (SmGM-2 smooth muscle growth medium-2 with Bullet Kit;
Cambrex). Cells were cultured at 37°C in a humidified incubator containing 5% CO2.
Growth media was exchanged every 2 days until approximately 80% confluent cell layer
was obtained. Cells were then trypsinized with 4 mL of 0.05% trypsin-EDTA solution,
centrifuged at 1100 rpm for five minutes and resuspended with 3 mL of fresh growth
media. Cells (passages 4-6) were seeded onto PUMI and PU-6-PHEMA discs at an initial
/y
cells density of -40,000 cells/cm and incubated in growth media for specific time
149
intervals. Cells on the discs were then fixed at ambient temperature in 2%
paraformaldehyde in PBS for 10 min, and permeabilized in 0.5% Triton X-100 in PBS
for another 10 min. After washing three times with PBS at 5 min each, cell-seeded discs
were left for 1 h at ambient temperature in Alexa-Fluor-568 conjugated phalloidin in PBS
(1:50), washed with PBS, and incubated for 10 min in Hoechst 33342 (20 pg/mL in
PBS). Discs were mounted on glass slides in Trevigen mounting media, sealed and
analyzed with a Leica DM-IRB fluorescence microscope.
6.3.1 Synthesis of macroiniferter and block copolymers
In this study, we have synthesized H^MDI-based PUMI using DHTD as a chain
extender. Compared with MDI-based PUMI (as described in Chapters 3-5), the lower
reactivity of H12MDI increased the overall reaction time including the chain extension
step. The prepared PUMI was then used as a macroiniferter in order to synthesize
physically crosslinked PU-6-PHEMA hydrogels. The prepared hydrogels were soluble in
dimethyl sulfoxide (DMSO), dimethyl formamide and N-MethyW-pyrrolidone (NMP);
however, they swelled in the presence of water without dissolving which proved that they
are physically crosslinked hydrogels.
The PUMI, PU-6-PHEMA and Fn-g-PU-Z>-PHEMA have been characterized using
FTIR spectroscopy and the spectra are shown in Figure 6.1.

150
Fn-£-PU-£-PHEMA
O
PU-6-PHEMA
PUMI
4000 3000 2000 1000
Wave number, cm"
FIGURE 6.1: FTIR spectra of PUMI, PU-6-PHEMA and Fn-g-PU-6-PHEMA.
The PUMI spectrum showed H-bonded N-H stretching at around 3330 cm"1 and a
carbonyl stretching at 1710 cm"1 corresponding to the urethane group. A peak at 1226 cm"
1
was due to the mixed vibrations involving C-N stretching along with N-H bending of
the urethane group. The peak related to the C-O-C stretching of the PTMO segments was
observed at 1080 cm"1. All these peaks were also observed in the block copolymers.
Along with that, the PU-6-PHEMA spectrum showed a broad peak in between 3660-3060
cm"1 related to H-bonded N-H stretching of urethane linkage as well as O-H stretching of
HEMA segment. Moreover, peaks related to carbonyl stretching (C=0) and ester
stretching (CO-O) of the PHEMA segment was observed at 1660 cm"1 and 1160 cm"1
respectively. The surface modified Fn-g-PU-6-PHEMA spectrum showed broad two

151
peaks between 3700-3000 cm' related to the primary amine stretching of the conjugated
fibronectin. Moreover, the ester linkage of the CDI conjugation had been observed at
1770 cm"1. In the *H NMR spectrum of PU-6-PHEMA copolymer (Figure 6.2), the peaks
related to the PTMO, H12MDI and PHEMA segments support the success of the
copolymer synthesis. Mainly the peak related to the urethane proton, observed at 7.95
ppm, supports the PUMI synthesis and the hydroxyl proton peak, observed at 4.81 ppm,
supports the presence of PHEMA segment into polyurethane backbone. Other related
peaks were listed in the Figure 6.2.
6 4 2
Chemical shift, ppm
FIGURE 6.2: *H NMR spectrum of PU-6-PHEMA.

152
Figure 6.3 shows the GPC traces of PUMI and PU-ZJ-PHEMA copolymer. Both
peaks are unimodel and the peak related to PU-6-PHEMA moved distinctively towards
lower elution volume indicating a molecular weight increase and supports the block
copolymer synthesis. In both cases, the polydispersity index remained below 1.50.
/ ^v PU-6-PHEMA
/ \ . Mn = 13.64 x 104 g/mol;

/ X^X ^ ^ ^ PDI =1.46
/ \ PUMI
/ \ Mn = 9.54 x 104 g/mol;
/ ^ ^ ^ P D I = 1.37
i 1 1 1
10 12 14 16
Elution volume, mL
FIGURE 6.3: GPC elution curves of PUMI and PU-6-PHEMA.
6.3.2 Thermal behaviour of macroiniferter and block copolymers
The thermal transitions of PUMI and PU-6-PHEMA are shown in Figure 6.4.
Both PUMI and PU-6-PHEMA show Tg related to the PTMO soft segment that are higher
than the Tg of pure PTMO (-90°C) with molecular weight of 1000 g/mol. This could be
either due to strong interaction between soft and hard segments or due to contamination
of PTMO with higher Tg hard segments20. The PU-6-PHEMA provides additional Tg at

153
120°C that is related to PHEMA segment. Moreover, the Tg related to the PHEMA
segment is higher than the Tg of pure PHEMA (113°C)21. This is probably contributed by
the reduction in the available free volume due to the strong physical interaction between
polyurethane and PHEMA segments. Overall, the present PU-6-PHEMA can be
considered as a microphase separated elastomer. In both polymers the absence of peaks
related to crystalline phase indicates the amorphous nature of both polymers.
T 1 1 1 1 r
-100 -50 0 50 100 150
Temperature, °C
FIGURE 6.4: DSC thermographs of PUMI and PU-6-PHEMA.
The TGA results (Figure 6.5) revealed that the PUMI is more thermally stable
compared with the PU-6-PHEMA. Similar to the conventional polyurethanes22, both
polymers followed two stage degradation pathway in which the first stage mass losses
154
were related to the thiuram disulfide decomposition combined with the hard segment
degradation17, whereas the second stage weight losses were observed from the soft
segment decompositions.
'53
300
Temperature, °C
FIGURE 6.5: TGA thermographs of PUMI and PU-6-PHEMA.
6.3.3 Surface analysis and swelling properties of macroiniferter and block copolymers
For SEM and swelling studies, PUMI and PU-6-PHEMA films have been
prepared from their respective DMF solutions by solvent casting method and, discs of 5
mm in diameter have been punched.
In the SEM images (Figure 6.6), it has been observed that PUMI shows smooth
surfaces whereas the PU-6-PHEMA copolymer shows comparative roughness

155
presumably due to the morphological changes caused by the PHEMA block. Both
surfaces were, however, free of any pores, voids, large scale separations, micro-
contaminations and cracks related to fatigue or environmental stress.
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«•_•
FIGURE 6.6: Scanning electron microscopic images of PUMI (A-B) and PU-A-PHEMA (C •D)
surfaces.
Figure 6.7 shows the water uptake behavior of PUMI and PU-6-PHEMA at 22°C
with respect to immersion time. The DHTD/HnMDI based PUMI showed a higher
equilibrium water content (EWC) of 12.13+1.85% which is DHTD/MDI based PUMI
(08.30+1.02%; Chapter 4) as well as TPED/MDI based PUMI (2.94±0.12%, Chapter 3).
For PU-6-PHEMA hydrogels uptake water rapidly in first 10 min and thereafter swelled
slowly up to EWC of 42.93+0.52 %. Thus, the incorporation of hydrophilic segments

156
within PUMI, as indicated by an increase in molecular weight (Figure 6.3), increased the
EWC which was expected.
200 400
Swelling time, min

FIGURE 6.7: Swelling isotherms of PUMI and PU-6-PHEMA.
6.3.4 Short term cell culture study
For tissue engineering biomaterials, cell-material interaction study is the first step
that provides information about the cytotoxicity of the material. Figure 6.8 shows the
fluorescence images of cells cultured on PUMI and PU-6-PHEMA hydrogels following 2
days of culture.
157
FIGURE 6.8: Flouroscent microscopic images of VSMC interaction with PUMI (A-B) and PU-A-
PHEMA (C-D) surfaces.
As can be seen in Figure 6.8, better cell attachment and spreading was observed
for PUMI. However PU-&-PHEMA hydrogels showed poor cell attachment and
spreading. The lack of serum cell-adhesion protein adsorption onto hydrogel materials
has been implicated to their poor cell adhesive properties12'23. To test this implication, we
carried out serum fibronectin adsorption experiments on both PU and PU-6-PHEMA. The
data presented in Figure 6.9 indicates that fibronectin was adsorbed more readily on
PUMI than on PU-6-PHEMA.

158
6000
5000 -
B
o
1? 4000
O
£>
.§ 3000
o
a 2000
o
1000
20 50
Fibronectin concentration, pg/mL
FIGURE 6.9: Fibronectin adsorption data of PUMI and PU-6-PHEMA.
Plasma fibronectin is a known cell adhesive protein due to its cells binding RGD
domains that are responsible for focal adhesion by binding to cell surface receptors called
integrins24. The amount of fibronectin adsorption on synthetic biomaterials controls the
cell interaction. However, other factors such as adsorbed fibronectin distribution, its
conformation and the integrin binding with these adsorbed fibronectin are also
responsible for further cellular functions such as differentiation and proliferation25. As
shown in Figure 6.9, the hydrogel adsorbed lower fibronectin compared with the PUMI at
both fibronectin concentrations. The surface chemistry, polarity and topography of the
biomaterials may be responsible for surface fibronectin adsorption. PHEMA is known to
undergo dynamic exchange of its surface groups from methyl to hydroxyl in water during
159
its swelling process and eventually increases the surface polarity in hydrated
environment26. Higher surface polarity due to PHEMA segment ultimately decreases the
fibronectin adsorption. This could be one of the reasons for the lower cells attachment on
the PU-6-PHEMA surfaces.
In order to improve vascular cell attachment on polymer surfaces containing
hydroxyl groups, various methods have been employed to chemically couple biologically
active ligands "30. These methods activate the hydroxyl groups facilitating the coupling
of cells adhesive ligands mostly via the amino terminus31. In this study, the CDI
conjugation chemistry has been used to modify PU-6-PHEMA surfaces for improved cell
adhesion; since CDI is well understood for polypeptide attachments on PHEMA based
biomaterials32.
160
FIGURE 6.10: Fluorescent microscopic images of VSMC interaction with PUMI (A-B), PU-6-
PHEMA (C-D) and F„-#-PU-A-PHEMA (E-F) surfaces.
Figure 6.10 shows fluorescent microscopic images of cells seeded on PUMI (A,
B), PU-6-PHEMA (C,D) and Fn-g-PU-Z>-PHEMA (E,F) after 2 days (A,C,E) and 4 days
(B,D,F) of culture. Similar to Figure 6.8(C,D), the PU-6-PHEMA surfaces showed poor
cell attachment and induced cells clustering. When the fibronectin was conjugated,
161
fluorescent images of cultured cells indicated that cells were uniformly and densely
populated throughout the surface. This suggests that the current PU-6-PHEMA hydrogel
could have a potential for the vascular tissue engineering applications.
6.4 Conclusions
Linear PU-6-PHEMA hydrogels had been successfully synthesized using
Hi2MDI-based PUMI. Spectroscopic characterization supported the success of the
synthetic procedure. TGA study demonstrated that the polymers followed the
conventional two stage polyurethane degradation. The Water uptake study revealed that
hydrogels having equilibrium water content up to 43% have been synthesized, which
mimic the water content of many natural tissues (20-80%). Due to the surface polarity the
PU-6-PHEMA hydrogel surfaces showed lower fibronectin adsorption as well as poor
cells adhesion. To improve cell attachment, chemical coupling of fibronectin on PU-6-
PHEMA surface has been successfully carried out. Fibronectin conjugated hydrogels
promote vascular cell attachment and spreading in short term culture. Thus, PU-b-
PHEMA could be used as a potential scaffold material for tissue engineering
applications.
6.5 References
1. Drury, J. L.; Mooney, D. J., Hydrogels for tissue engineering: scaffold design
variables and applications. Biomaterials 2003, 24, (24), 4337-4351.
2. Slaughter, B. V. K., S.S.; Fisher, O.Z.; Khademhosseini, A.; Peppas, N.A.,

Hydrogels in regenerative medicine. Advanced Materials 2009,21, 3307-3329.
162
Park, H.; Kang, S. W.; Kim, B. S.; Mooney, D. J.; Lee, K. Y., Shear-reversibly
crosslinked alginate hydrogels for tissue engineering. Macromoecularl Bioscience
2009,9, (9), 895-901.
Barcili, B., Hydrogels for tissue engineering and delivery of tissue-inducing

substances. Journal of Pharmaceutical Sciences 2007, 96, (9), 2197-2223.
Tibbitt, M. W.; Anseth, K. S., Hydrogels as Extracellular Matrix Mimics for 3D

Cell Culture. Biotechnology andBioengineering 2009, 103, (4), 655-663.
Gong, Y. H.; Wang, C. M.; Lai, R. C; Su, K.; Zhang, F.; Wang, D. A., An
improved injectable polysaccharide hydrogel: modified gellan gum for long-term
cartilage regeneration in vitro. Journal of Materials Chemistry 2009, 19, (14),
1968-1977.
Patel, A.; Fine, B.; Sandig, M.; Mequanint, K., Elastin biosynthesis: The missing
link in tissue-engineered blood vessels. Cardiovascular Research 2006, 71, (1), 40-
49.
Wichterle, O.; Lim, D., Hydrophilic gels for biological use. Nature 1960, 185,
(4706), 117-118.
Lou, X.; Vijayasekaran, S.; Sugiharti, R.; Robertson, T., Morphological and
topographic effects on calcification tendency of pHEMA hydrogels. Biomaterials
2005,26,(29), 5808-5817.
Kidane, A.; Szabocsik, J. M.; Park, K., Accelerated study on lysozyme deposition
on poly(HEMA) contact lenses. Biomaterials 1998, 19, (22), 2051-2055.
Salamone, J. C, Concise polymeric materials encyclopedia. CRC press LLC,

Florida: 1999.
Klee, D.; Hocker, H., Polymers for biomedical applications: Improvement of the
interface compatibility. Biomedical Applications: Polymer Blends 1999, 149, 1-57.
Vacanti, F. X., PHEMA as a fibrous capsule-resistant breast prosthesis. Plastic and

Reconstructive Surgery 2004, 113, (3), 949-952.
Lesny, P.; De Croos, J.; Pradny, M.; Vacik, J.; Michalek, J.; Woerly, S.; Sykova,
E., Polymer hydrogels usable for nervous tissue repair. Journal of Chemical
Neuroanatomy 2002, 23, (4), 243-247.
Trigo, R. M.; Blanco, M. D.; Huerta, P.; Olmo, R.; Teijon, J. M., L-ascorbic-acid
release from pHEMA hydrogels. Polymer Bulletin 1993, 31, (5), 577-584.
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16. Bryant, S. J.; Cuy, J. L.; Hauch, K. D.; Ratner, B. D., Photo-patterning of porous
hydrogels for tissue engineering. Biomaterials 2007, 28, (19), 2978-2986.
biocompatibility of novel physically crosslinked polyurethane-6/ocA:-poly(glycerol
methacrylate) hydrogels. Biomacromolecules 2006, 7, (3), 883-891.
19. Patel, A.; Mequanint, K., Novel physically crosslinked polyurethane-block-

poly(vinyl pyrrolidone) hydrogel biomaterials. Macromoeularl Bioscience 2007, 7,
(5), 727-737.
20. CognetGeorjon, E.; Mechin, F.; Pascault, J. P., New polyurethanes based on 4,4'-
diphenylmethane diisocyanate and 1,4:3,6 dianhydrosorbitol .2. Synthesis and
properties of segmented polyurethane elastomers. Macromolecular Chemistry and
Physics 1996, 197, (11), 3593-3612.
21. Meakin, J. R.; Hukins, D. W. L.; Imrie, C. T.; Aspden, R. M., Thermal analysis of
poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels. Journal of Materials
Science-Materials in Medicine 2003, 14, (1), 9-15.
22. Wang, L. F., Effect of soft segment length on the thermal behaviors of fluorinated
polyurethanes. European Polymer Journal 2005, 41, (2), 293-301.
23. Yayapour, N.; Nygren, H., Interactions between whole blood and hydrophilic or
hydrophobic glass surfaces: kinetics of cell adhesion. Colloids and Surfaces B:
Biointerfaces 1999, 15, (2), 127-138.
24. Pitt, W. G.; Weaver, D. R.; Cooper, S. L., Fibronectin adsorption-kinetics on phase
segregated polyurethaneureas. Journal of Biomaterials Science-Polymer Edition
1993, 4, (4), 337-346.
25. Garcia, A. J.; Vega, M. D.; Boettiger, D., Modulation of cell proliferation and
differentiation through substrate-dependent changes in fibronectin conformation.
Molecular Biology of the Cell 1999, 10, (3), 785-798.
26. Velzenberger, E.; El Kirat, K.; Legeay, G.; Nagel, M. D.; Pezron, I.,
Characterization of biomaterials polar interactions in physiological conditions
using liquid-liquid contact angle measurements Relation to fibronectin adsorption.
Colloids and Surfaces B: Biointerfaces 2009, 68, (2), 238-244.
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27. Nuttelman, C. R.; Mortisen, D. J.; Henry, S. M.; Anseth, K. S., Attachment of
fibronectin to poly(vinyl alcohol) hydrogels promotes NIH3T3 cell adhesion,
proliferation, and migration. Journal of Biomedical Materials Research 2001, 57,
(2), 217-223.
28. Denizli, A.; Piskin, E.; Dixit, V.; Arthur, M.; Gitnick, G., Collagen and fibronectin
immobilization on pHEMA microcarriers for hepatocyte attachment. International
Journal of Artificial Organs 1995, 18, (2), 90-95.
29. Chase, H. A.; Yang, Y. H., Immobilization of enzymes on poly(vinyl alcohol-

coated perfluorocarbon supports: comparison of techniques for the immobilization
of trypsin and alpha-amylase on poly(vinyl alcohol)-coated solid and liquid
perfluorocarbons. Biotechnology and Applied Biochemistry 1998, 27, 205-216.
30. Nilsson, K.; Mosbach, K., Immobilization of enzymes and affinity ligands to
various hydroxyl group carrying supports using highly reactive sulfonyl chlorides.
Biochemical and Biophysical Research Communications 1981, 102, (1), 449-457.
31. Ojha, U.; Feng, D. S.; Chandekar, A.; Whitten, J. E.; Faust, R., Peptide surface
modification of P(HEMA-co-MMA)-Z>-PIB-Z>-P(HEMA-co-MMA) block
copolymers. Langmuir 2009, 25, (11), 6319-6327.
32. Valdes, T. I.; Ciridon, W.; Ratner, B. D.; Bryers, J. D., Surface modification of a
perfluorinated ionomer using a glow discharge deposition method to control
protein adsorption. Biomaterials 2008, 29, (10), 1356-1366.
165
CHAPTER
7
General Conclusions and Future Directions
7.1 Summary
The overall objective of this study was to develop physically crosslinked
polyurethane hydrogels that can be tuned with respect to swelling. An iniferter method
has been selected to develop linear polyurethane-based hydrogels. Thermally sensitive
polyurethane macroiniferter using TPED as a chain extender had been successfully
developed and its ability to prepare polyurethane-based linear hydrogels had been
confirmed by synthesizing PU-6-PVP and characterizing their swelling behavior. The
polyether-based macrodiol was selected to develop biostable hydrogels. However, the use
of selective polyester-based macrodiol could give biodegradable hydrogels. Since the
TPED has tertiary hydroxyl groups, they have lower reactivity towards isocyanates.
Owing to this, higher temperature (ca. >50°C) would ideally favor chain extension
reaction. TPED, however, is a thermally sensitive molecule and precludes the use of high
temperature for the chain extension reaction. Therefore, the chain extension reaction was
carried out at room temperature. In order to offset the low reactivity of TPED, MDI was
selected as a diisocyanate because aromatic diisocyanates have higher reactivity
compared with the aliphatic diisocyanates at ambient temperature. Moreover, the reaction
166
was carried out in the presence of DBTDL as a catalyst to ensure complete chain
extension. Novel physically crosslinked PU-6-PVP hydrogels were synthesized using
TPED-based PUMI. The developed PU-6-PVP were soluble in DMF, MEK and NMP.
However, films prepared by casting from solution swelled in water without dissolving
indicating that the hydrogels were physically crosslinked. Hydrogels having equilibrium
water content up to only 37% were synthesized. Vascular smooth muscle cells attachment
on the PU-6-PVP surface on short term culture study proved that the hydrogels are not
cytotoxic and have a potential to be used as scaffolds for tissue engineering (Chapter 3)1.
In order to overcome the low reactivity of TPED, UV sensitive polyurethane
macroiniferters were prepared using DHTD as a chain extender. Since DHTD possesses
primary hydroxyl groups, it has higher reactivity towards isocyanates compared with
TPED. Thus, both aromatic (MDI) as well as aliphatic (H12MDI) diisocyanates had been
selected for the PUMI synthesis. However, DHTD is also thermally sensitive and the
chain extension reaction had been conducted at ambient temperature in the presence of
DBTDL as a catalyst. Physically crosslinked PU-6-PVP as well as PU-Z>-PAAm
hydrogels had been developed using DHTD-based PUMI and their detailed swelling and
water diffusion studies had been conducted (Chapter 4)2. The developed hydrogels also
dissolved in selective solvents whereas their casted films swelled in the presence of
water.
In order to understand the kinetics of the DHTD-based PUMI systems, detailed
kinetics analysis had been conducted using MMA as a model monomer. The study
167
showed a linear increase of molecular weight and conversion with reaction time and,
supports the controlled polymerization of DC-based PUMI. Moreover, the plot of the rate
of polymerization versus the square root of PUMI concentration showed a dome shaped
curve and thus supported the presence of reversible termination (Chapter 5)3.
Finally, aliphatic diisocyanate (Hi2MDI)-based PUMI system was developed and
used to prepare linear PU-6-PHEMA hydrogels. The DHTD shows lower reactivity
towards H12MDI compared with MDI and a longer reaction time was required. In order to
improve cell adhesion, fibronectin was successfully conjugated. The modified hydrogels
showed improved and uniform cell spreading throughout the surface in short term culture
studies (Chapter 6). Overall, the approach taken in this study proved to be a great success
towards designing linear polyurethane hydrogels and showed potential for biomedical
applications.
7.2 Strengths and limitations
The availability of a wide range of precursors allows polyurethanes to provide a
wide variety of properties. Developing polyurethanes, based on particular property
requirements related to the final application, is similar to tailoring a suit with required
measurements4'5. However, when it comes to the design of physically crosslinked
polyurethanes, the flexibility vanishes due to the limitation of hydrophilic macrodiols
availability. Other than PEO, available macrodiols are often hydrophobic. In previous
studies, the change in mechanical properties and/or swelling behavior were obtained
either using mass ratio of PEO over different hydrophobic macrodiols as controlling
168
parameter or by varying the chain length of PEO " . However, the use of PUMI system in
the current research facilitated the inclusion of a family of hydrophilic free radical
polymers onto the polyurethane backbone expanding the chemoselectivity of macrodiols
to develop linear polyurethane hydrogels. Moreover, the copolymerization time controls
the swelling of the resulting hydrogels and thus the water uptake can be tuned based on
the system requirements. The use of hydrophilic monomers carrying -OH, -COOH or -
NH2 groups can further allow to modify the hydrogels for different biomedical
applications such as drug delivery and tissue engineering.
The polyurethane macroiniferter systems of the current study have some
limitations. From a synthesis point of view, longer reaction times were required.
Precipitation and purification steps are also laborious at times. Another limitation of these
systems is the lower yield attributed to the lack of precipitation solvents for amphiphilic
block copolymers. The proper selection of precipitation solvent or mixture of solvents to
precipitate amphiphilic block copolymers is always challenging. The hydrogels were
non-transparent and that limits their application where optical properties are required.
Moreover, the hydrogel systems in this study are likely to be non-biodegradable in
physiological conditions.
An additional limitation of this study was the absence of mechanical properties
data for the hydrogels. Although mechanical properties data was part of an initial
planning in this work, it was excluded later to focus on the kinetic mechanisms of the
iniferter polymerization.
169
7.3 Future directions
Physically crosslinked polyurethane hydrogels have unique control over their
swelling ability and thus can be used in numerous drug delivery and tissue engineering
applications. The developed hydrogels can also be applied as coating for heavy duty
biomaterials. Thus, different applications in these areas need to be studied. The
mechanical properties of physically crosslinked hydrogels are important and should be
considered on a separate study. Moreover, it could be beneficial to use the DHTD-based
PUMI system to develop biodegradable polyurethane hydrogels for related applications.
7.4 References
1. Patel, A.; Mequanint, K., Novel physically crosslinked polyurethane-Z>/oc£-

7, (5), 727-737.

3. Patel, A.; Mequanint, K., The kinetics of dithiocarbamate-mediated polyurethane-

Z?/oc£-poly(methyl methacrylate) polymers. Polymer 2009, 50 (19), 4464-4470.
4. Lamba, N. M. K.; Woodhouse, K. A.; Cooper, S. L., Polyurethanes in Biomedical

Applications. CRC Press, New York, D.C.: 1998.
5. Zdrahala, R. J.; Zdrahala, I. J., Biomedical applications of polyurethanes: A

review of past promises, present realities, and a vibrant future. Journal of
Biomaterials Applications 1999, 14, (1), 67-90.
6. Haschke, E.; Sendijarevic, V.; Wong, S.; Frisch, K. C; Hill, G., Clear nonionic
polyurethane hydrogels for biomedical applications. Journal of Elastomers and
Plastics 1994, 26, (1), 41-57.
7. Petrini, P.; Tanzi, M. C; Moran, C. R.; Graham, N. B., Linear poly(ethylene

oxide)-based polyurethane hydrogels: polyurethane-ureas and polyurethane-
635-639.
170

2002,23,(8), 1797-1808.
9. Yoo, H. J.; Kim, H. D., Synthesis and properties of waterborne polyurethane

hydrogels for wound healing dressings. Journal of Biomedical Materials
Research PartB: Applied Biomaterials 2008, 85B, (2), 326-333.
171
APPENDIX
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Poly(Vinyl Pyrrolidone) Hydrogel Biomaterials. Macromolecul Bioscience; 7: 5; 727-
737.
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Dear Publisher:
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B.Eng. (Chem. Eng.), M.Eng. (Poly. Tech.),

Ph.D. Candidate,
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The University of Western Ontario,
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London, ON N6A5B9
174
Patel, A.; Mequanint, K. (2008) Synthesis and Characterization of Physically

Crosslinked Hydrogels from Dithiocarbamate-derived Polyurethane Macroiniferter.
Journal of Polymer Science Part A: Polymer Chemistry; 46: 6272-6284.
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CURRICULUM VITAE
ALPESH PATEL
EDUCATION: The University of Western Ontario, London, Canada 2010

Ph.D. in Chemical and Biochemical Engineering
Maharaja Sayajirao University, Gujarat, India 2001

M.Eng. in Polymer Technology
Gujarat University, Gujarat, India 1998

B.Eng. in Chemical Engineering
TEACHING EXPERENCE:
Graduate Teaching Assistant
2004-2009 ES 050: Introductory Engineering Design and Innovation Studio

CBE 4423a: Tissue Engineering
CBE 2221b: Fluid Flow
CBE 3392a: Polymer Engineering
AWARDS:
• Preapproved to participate in the Natural Science and Engineering Research Council

of Canada - Industrial R&D Fellowships (IRDF) program until June 2011.
• Selected for the Natural Science and Engineering Research Council of Canada - PGS
D as well as for the Ontario Graduate Study scholarship during the PhD graduation.
• Selected for the best research presentation awards in the Chemical and Biochemical
Engineering Department for the term 2006/04-2006/08.
WORK HISTORY:
2004 - Present Research Assistant, Teaching Assistant

UWO, London, ON
1998 - 2001 Process Engineer

Nirma Ltd., India
184
PEER-REVIEWED MANUSCRIPTS:
• Johnson, T.; Bahrampourian, R.; Patel, A.; Mequanint, K. (2010) Fabrication of

Highly Porous Tissue Engineering Scaffolds using Selective Spherical Porogens.
Biomed Mater Eng. Accepted.
• Patel, A.; Mequanint, K. (2009) The Kinetics of Dithiocarbamate-Mediated
Polyurethane-&/oc£-Poly(Methyl Methacrylate) Polymers. Polymer; 50: 4464-4470.
• Patel, A.; Mequanint, K. (2008) Synthesis and Characterization of Physically
Crosslinked Hydrogels from Dithiocarbamate-Derived Polyurethane Macroiniferter. J
Polym Sci Part A: Polym Chem; 46: 6272-6284.
• Patel, A.; Mequanint, K. (2007) Novel Physically Crosslinked Polyurethane-6/oc&-
Poly(Vinyl Pyrrolidone) Hydrogel Biomaterials. Macromol Biosci; 7: 5; 727-737.
• Patel, A.; Fine, B.; Sandig, M.; Mequanint, K. (2006) Elastin Biosynthesis: The
Missing Link in Tissue-Engineered Blood Vessels. Cardiovascular Research; 71: 40-
49.
• Mequanint, K.; Patel, A.; Bezuidenhout, D. (2006) Synthesis, Swelling Behavior and
Biocompatibility of Novel Physically Crosslinked Polyurethane-Woc£-Poly(Glycerol
Methacrylate) Hydrogels. Biomacromolecules; 7: 883-891.
PEER-REVIEWED CONFERENCE PRESENTATIONS:
• Patel, A.; Mequanint, K. (2009) Polyurethane-based Physically Crosslinked

Hydrogels for Biomedical Applications. 8th World Congress of Chemical
Engineering, Montreal, Quebec. Abstract # 1173. Oral presentation.
• Patel, A.; Mequanint, K. (2009) Linear Polyurethane Hydrogel Biomaterials. 27th
Annual Canadian Biomaterial Society meeting, Laval University, Quebec City,
Quebec. Abstract # PI. 9. Poster presentation.
• Patel, A.; Mequanint, K. (2009) Swelling Kinetics of Physically Crosslinked
Polyurethane-Z>/ocA:-Polyacrylamide Hydrogels. 35th Annual Northeast
Bioengineering Conference, Boston, USA. Abstract # 8. Poster presentation.
• Patel, A.; Mequanint, K. (2008) Physically Crosslinked Polyurethane Hydrogels for
Biomedical Applications. 8th World Biomaterials Congress, Amsterdam, The
Netherlands. Abstract # 2288. Poster presentation.
• Patel, A.; Mequanint, K. (2007) Cell-Interaction of Physically Crosslinked PU-b-
PVP Hydrogels. 26th Annual Canadian Biomaterial Society meeting, University of
Western Ontario, London, Ontario. Abstract # 42. Oral presentation.
• Mequanint, K.; Patel, A.; Bezuidenhout, D. (2006) Biocompatibility of Physically
Crosslinked Novel Hydrogels. 25th Annual Canadian Biomaterial Society meeting,
University of Calgary, Alberta. Abstract # 6. Oral presentation.

Physically Crosslinked Polyurethane Hydrogels For Biomedical Applications

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PHYSICALLY CROSSLINKED POLYURETHANE HYDROGELS

FOR BIOMEDICAL APPLICATIONS

(Spine title: Linear Polyurethane Hydrogel Biomaterials)

(Thesis format: Integrated-Article)

Chemical and Biochemical Engineering

A thesis submitted in partial fulfillment of the requirements for the degree of

The School of Graduate and Postdoctoral Studies

The University of Western Ontario

©Alpesh Patel 2010

395 Wellington Street 395, rue Wellington

The author retains copyright L'auteur conserve la propriete du droit d'auteur

In compliance with the Canadian Conformement a la loi canadienne sur la

Dr. Kibret Mequanint Dr. Heather Sheardown

Advisory Committee Dr. Leo Lau

Dr. Amin S. Rizkalla Dr. Jose Herrera

Dr. Paul A. Charpentier Dr. Lars Rehmann

PHYSICALLY CROSSLINKED POLYURETHANE HYDROGELS FOR

is accepted in partial fulfillment of the requirements for the degree of

The ability of macromolecules to undergo substantial swelling when exposed to

Keywords: physically crosslinked hydrogels, polyurethanes, macroiniferters, swelling

"It cannot be stolen by thieves

It cannot be divided among brothers

The more we spend the more it increases

To my unconditional lovers, my angels, Zal and Zankhana

individuals who directly or indirectly helped me to make my vision realized.

Rizkalla, for their valuable suggestions and constructive criticism throughout my

I am also grateful to the staff of the Department of Chemical and Biochemical

Engineering for providing me all necessary assistance during my studies. It would be

unlimited access to their library resources.

assistance during my doctoral studies.

This acknowledgement would not be complete without thanking my friends and

my family for their continuous support and encouragement.

3. Physically crosslinked polyurethane-6/oc£-poly(/V-vinyl pyrrolidone) hydrogels from

4. Physically crosslinked PU-6-PVP and PU-6-PAAm hydrogels from dithiocarbamate -

5. The kinetics of dithiocarbamate-madiated polyurethane macroiniferter 113

6. Dicyclohexylmethane diisocyanate-based physically crosslinked polyurethane

7. General conclusions and future directions 165

SCHEME 2.2: The polymerization mechanism of iniferter system 42

SCHEME 3.1: Schematic diagram of TPED synthesis 61

SCHEME 3.2: Schematic diagram of PUMI and PU-6-PVP syntheses 66

SCHEME 4.1: Schematic diagram of DHTD synthesis 85

SCHEME 4.2: Schematic diagram of PUMI, PU-6-PAAm and PU-6-PVP syntheses.... 87

SCHEME 5.1: Chemical structure of PU-ZJ-PMMA 119

SCHEME 5.2: The polymerization mechanism of DC-based macroiniferter 120

SCHEME 6.1: Schematic diagram of PUMI and PU-6-PHEMA syntheses 144

SCHEME 6.2: Schematic diagram of the modification of PU-6-PHEMA copolymers with

FIGURE 2.2: Classifications of smart hydrogels 13

FIGURE 2.3: Classifications of iniferters 43

FIGURE 3.1: FTIR spectra of TPED, PUMI and PU-6-PVP 67

FIGURE 3.2: 'H-NMR spectrum of TPED 68

FIGURE 3.3: 'H-NMR spectrum of PUMI 68

FIGURE 3.4: 'H-NMR spectrum of PU-6-PVP 70

FIGURE 3.5: GPC curves of PU-Z?-PVPs for different copolymerization times 71

FIGURE 4.1: FTIR spectra of DHTD, PUMI, PU-6-PVP and PU-6-PAAm 91

FIGURE 4.2: (a) 'H-NMR spectrum of DHTD 93

FIGURE 4.2: (b) 13C-NMR spectrum of DHTD 93

FIGURE 4.3: (a)'H-NMR spectrum of PUMI 94

FIGURE 4.3: (b) 'H-NMR spectrum of PU-6-PVP 95

FIGURE 4.3: (c)'H-NMR spectrum of PU-6-PAAm 96

FIGURE 4.5: Swelling isotherms of physically crosslinked PU-6-PVP hydrogels as a

FIGURE 4.6: Swelling isotherms of physically crosslinked PU-6-PAAm hydrogels as a

FIGURE 4.7: Swelling isotherms of physically crosslinked PU-6-PAAm hydrogels at

FIGURE 4.8: FTIR spectra of physically crosslinked PU-6-PAAml2 hydrogels at