Temperature-responsive Biocompatible Block Copolymers PhD JMadsen

Temperature-responsive Biocompatible Block
Copolymers based on
2-(Methacryloyloxy)ethyl Phosphorylcholine
Peter Jeppe Madsen
Department of Chemistry
The University of Sheffield
Submitted to the University of Sheffield
In fulfillment of the requirements for the award of
Doctor of Philosophy
March 2009

I
Declaration
The work described in this thesis was carried out at the University of Sheffield
between May 2005 and March 2009 and has not been submitted, either wholly or
in part, for this or any other degree. All the work is the original work of the
author, except where acknowledged by references.
Signature:
Peter Jeppe Madsen March 2009

II
Acknowledgements
I would like to thank my supervisor Steve Armes for the opportunity to work in his
group. The wealth of ideas and suggestions is truly amazing (and sometimes quite
exhausting!)
So many people around University of Sheffield have been helpful with so many
aspects of science and life in general.
Special thanks to Karima Bertal who has had the (sometimes quite frustrating)
task of evaluating these polymers for biomedical applications. At least I have
learnt a lot during the process about what you can and in particular what you
cannot do to cells.
Also a special thanks to past and present members in the Armes group. It is a
great group and never boring! (Fortunately they are all great chemists because
nobody would be able to make a career from singing). Everybody’s been very
kind and helpful and I hope they will forgive me for not mentioning their names
here.
I would also like to thank the skilled technicians at the university without whom
many of the data presented in this work would not be.
In addition I would like to thank everybody at Kroto Institute who have found
interesting uses for my polymers and for introducing me to the marvels (and
horrors) of biochemistry.
Thanks to Biocompatibles UK Ltd. who have sponsored part of my project and
especially to Andy Lewis who allowed a lot of freedom in the various projects
presented here.
I should also thank Kjeld Schaumburg for advice on almost any aspect of
chemistry imaginable and for his help in pursuing a career in this fascinating
discipline. In addition I would like to thank Søren Hvidt for introducing me to
rheology and for help and discussions.
My various housemates who have been kind enough to involve me in the wicked
ways of the natives, especially student life and the marvels of a good ‘brew’
deserve my gratitude. Life in the UK would have been a lot less interesting
without them.
Thanks to some wonderful friends who are always ready for a beer (or ten)
whenever I get around. Special thanks to Henrik for helping me to obtain a lot of
literature.
Finally, I would like to thank my family for accepting my absence from so many
celebrations and special days. Special thanks to my mum for always caring and
accepting my occasional bad moods in such good spirit.

III
Publications arising from work described in this thesis
1. J. Madsen, S. P. Armes, A. L. Lewis “Preparation and Aqueous Solution
Properties of New Thermoresponsive Biocompatible ABA Triblock
Copolymer Gelators”, Macromolecules 2006, 39, 7455-7457
2. C. Li, J. Madsen, S. P. Armes, A. L. Lewis: “A New Class of Biochemically
Degradable, Stimulus-Responsive Triblock Copolymer Gelators”,
Angewandte Chemie International Edition 2006, 45, 3510-3513
3. J. Madsen, S. P. Armes, K. Bertal, H. Lomas, S. MacNeil, A. L. Lewis: “New
biocompatible wound dressings based on chemically degradable triblock
copolymer hydrogels”, Biomacromolecules 2008, 9, 2265-2275
4. H. Lomas, M. Massignani, K. A. Abdullah, I. Canton, C. L. Presti, S.
MacNeil, J. Du, A. Blanazs, J. Madsen, S. P. Armes, A. L. Lewis, G.
Battaglia: “Non-cytotoxic polymer vesicles for rapid and efficient intracellular
delivery”, Faraday Discussions 2008, 139, 143-159
5. P. Topham, N. Sandon, E. Read, J. Madsen, A. Ryan, S. Armes: “Facile
Synthesis of Well-Defined Hydrophilic Methacrylic Macromonomers using
ATRP and Click Chemistry”, Macromolecules 2008, 41, 9542-9547
6. V. Hearnden, S. MacNeil, M. Thornhill, C. Murdoch, A. Lewis , J. Madsen,
A. Blanazs, S. Armes, G. Battaglia: “Diffusion studies of nanometer
polymersomes across tissue engineered human oral mucosa”, accepted for
Pharmaceutical Research 2009
Manuscripts in preparation arising from work described in this
thesis
1. J. Madsen, K. Bertal, S. MacNeil, A. L. Lewis, S. P. Armes: “Preparation and
Aqueous Solution Properties of Thermoresponsive Biocompatible AB
Diblock Copolymers”, submitted to Biomacromolecules 2009

IV
2. M. Massignani, C. LoPresti, A. Blanazs, J. Madsen, S. P. Armes, A. L. Lewis,
G. Battaglia: “Controlling cellular uptake by surface chemistry, size and
surface topology at the nanoscale”, submitted to Nano Letters 2009
3. K. Yoshimoto, T. Hirase, J. Madsen, S. P. Armes, Y. Nagasaki: “Construction
of Poly[2-(methacryloyloxy)ethyl Phosphorylcholine] Modified Gold
Surfaces by the “Grafting to” Method: Comparison of its Protein Resistance
with Poly(ethylene glycol) Modified Gold Surfaces”, submitted to Chemical
Communications 2009
4. J. Madsen, N. J. Warren, M. Massignani, G. Battaglia, A. L. Lewis, S. P.
Armes: “Synthesis of Fluorescently-Labelled Biocompatible Polymers Based
on 2-(Methacryloyloxy)ethyl phosphorylcholine”, manuscript in preparation
5. K. Bertal, J. Shepherd, I. Douglas, J. Madsen, S. P. Armes, A. L. Lewis,
Sheila MacNeil: “Antimicrobial activity of novel biocompatible wound
dressings based on triblock copolymer hydrogels”, manuscript in preparation
Presentations at conferences
2008 The 82nd
ACS Colloid & Surface Science Symposium, June 15-18, 2008,
North Carolina State University, Raleigh, North Carolina, United States of
America
Oral presentation: “Thermoresponsive Biocompatible Chemically
Degradable Triblock Copolymer Hydrogels”
Frontiers of Research and Young Researchers Meeting, April 17-18, 2008.
University of Warvick, United Kingdom.
Poster presentation: “Synthesis of Novel Rhodamine-Based Fluorescent
ATRP Initiators and Their Use in Preparing Responsive Biocompatible
Block Copolymers”
2007 MRS 2007 Fall Meeting, November 26-30, 2007, Boston, Massachusetts,
United States of America.

V
Oral presentation: “Synthesis and Characterization of Stimulus-
Responsive Biocompatible Triblock Copolymer Gelators”
Controlled/Living Polymerisation, October 25-29, 2007, Antalya, Turkey.
Responsive Biocompatible Triblock Copolymer Gelators”
RSC Biomaterials Chemistry Group 2nd Annual Meeting, January 16,
2007, University of Nottingham, United Kingdom.
Responsive Biocompatible Block Copolymers”
2006 Functional and Biological Gels and Networks: Theory and Experiment,
September 3-7, 2006, University of Sheffield, United Kingdom.
Poster presentation: “Synthesis and Characterization of Stimulus-
Responsive Biocompatible Block Copolymers”
Macro Group UK International Conference on Polymer Synthesis, July
31- August 3, 2006, University of Warvick, United Kingdom.
Poster presentation: “Synthesis and Characterisation of New
Biocompatible ABA Triblock Copolymers”
RSC Biomaterials Chemistry, January 18, 2006, University of Sheffield,
United Kingdom.
Oral presentation: “Synthesis and Characterisation of New Biocompatible
ABA Triblock Copolymers”

VI
Abstract
The synthesis of novel thermo-responsive ABA triblock copolymers in which the
outer A blocks comprise poly(2-hydroxypropyl methacrylate) (PHPMA) and the
central B block comprise highly biocompatible poly(2-(methacryloyloxy)ethyl
phosphorylcholine) (PMPC) was achieved using atom transfer radical
polymerization (ATRP) by sequential monomer addition using various
bifunctional initiators in methanol at 20 °C. These novel triblock copolymers
form thermo-reversible, free-standing physical gels in aqueous solutions with
critical gelation temperatures and mechanical properties that are highly dependent
on the copolymer composition and concentration. Incorporating a central disulfide
bond into the triblock copolymers led to thermoresponsive gels that were readily
degradable using mild reduction agents such as dithiothreitol (DTT).
The synthesis of a series of amphiphilic PMPC-PHPMA diblock copolymers was
achieved by ATRP. The aqueous solution properties of these new diblock
copolymers were examined using dynamic light scattering and temperature-
dependent 1
H NMR spectroscopy. Copolymers with shorter thermo-responsive
PHPMA blocks formed relatively large aggregates, while copolymers with longer
PHPMA blocks formed smaller aggregates. This apparently ‘anomalous’ self-
assembly behavior occurs because the PHPMA block becomes more hydrophobic
as its degree of polymerization is increased. Therefore, shorter PHPMA blocks
lead to the formation of highly hydrated aggregates, whereas longer blocks
formed relatively dehydrated aggregates.
A facile route to derivatize rhodamine 6G was used to prepare fluorescent ATRP
initiators based on 2-bromoisobutyryl esters, as well as a fluorescent monomer.
The synthesis of several monofunctional initiators and one bifunctional initiator
was achieved. Depending on the initiator structure, either pH-dependent or pH-
independent derivatives could be prepared. Thus, one example of an ATRP
initiator was only fluorescent below pH 5-6, whereas the majority of the
derivatives were always fluorescent from pH 1 to pH 10. These initiators were
used to prepare well-defined PMPC homopolymers by ATRP. However,
spectroscopic analysis showed that the end group content of these polymers was
lower than targeted. Several causes for this observation were identified. Most
notably it was found that the ATRP catalyst could also act as a transesterification
catalyst, leading to of the 2-bromoisobutyryl ester with the methanol solvent.
Nevertheless, these initiators could be used to prepare well-defined fluorescently-
labelled diblock copolymers with end-groups that were hydrolytically stable
under physiological conditions over a period of at least one week. Such
copolymers have been shown by our collaborators to be useful for various
biomedical studies based on confocal microscopy.

VII
Table of contents
Declaration ................................................................................................................................. I
Acknowledgements ...................................................................................................................II
Publications arising from work described in this thesis...................................................... III
Manuscripts in preparation arising from work described in this thesis............................ III
Presentations at conferences...................................................................................................IV
Abstract ................................................................................................................................VI
Table of contents....................................................................................................................VII
List of Tables........................................................................................................................ XIII
List of Figures .......................................................................................................................XVI
List of Schemes.................................................................................................................... XXV
Abbreviations................................................................................................................... XXVII
Chapter 1: Introduction ...............................................................................1
1.1 Macromolecules and polymer science...................................................................2
1.2 Preparation of macromolecules.............................................................................2
1.2.1 Free-radical polymerization......................................................................................3
1.2.2 Controlled / “Living” polymerization.......................................................................7
1.2.3 Controlled radical polymerization methods..............................................................9
1.2.4 Reversible Addition-Fragmentation Chain Transfer (RAFT) Polymerization........11
1.2.5 Atom Transfer Radical Polymerization (ATRP) ....................................................13
1.3 Aggregation of amphiphilic diblock copolymers in selective solvents..............16
1.4 Network formation of triblock copolymers in selective solvents ......................19
1.5 Gel structure of amphiphilic block copolymers .................................................23
1.6 Preparation of thiol-functional polymers ...........................................................24
1.6.1 Why thiols?.............................................................................................................24
1.6.2 Thiols from disulfides.............................................................................................25
1.6.3 Thiols from double bonds.......................................................................................26
1.6.4 Thiols from alcohols...............................................................................................26
1.6.5 Thiols from alkyl halides........................................................................................27
1.6.6 Hydrolysis of thiol esters and related compounds ..................................................28
1.6.7 Thiolated macromolecules......................................................................................28
1.7 Reactions of thiol-functional polymers ...............................................................28
1.7.1 Direct oxidation of thiols, formation of symmetrical disulfides.............................28

VIII
1.7.2 Formation of asymmetric disulfides .......................................................................31
1.7.3 Free-radical mediated coupling of thiols to double bonds......................................32
1.7.4 Michael-type addition of thiols to electron-deficient double-bonds.......................35
1.7.5 Reaction between thiols and alkyl halides..............................................................40
1.7.6 Reactions of thiols and disulfides with metal surfaces ...........................................40
1.8 Phospholipids and phosphorylcholine-based polymers.....................................40
1.8.1 2-(methacryloyloxy)ethyl phosphorylcholine, MPC ..............................................41
1.8.2 Hydrogels based on random copolymers of PMPC................................................42
1.8.3 Controlled Polymerization of MPC ........................................................................43
1.8.4 Well-defined PMPC-based block copolymers........................................................47
1.8.5 PMPC-based pH-responsive block copolymers......................................................49
1.9 PMPC-based temperature-responsive block copolymers..................................52
1.10 References .............................................................................................................55
Chapter 2: Preparation and Aqueous Solution Properties of New
Thermo-responsive Biocompatible ABA Triblock
Copolymer Gelators ................................................................64
2.1 Introduction ..........................................................................................................65
2.2 Experimental.........................................................................................................65
2.2.1 Materials.................................................................................................................65
2.2.2 Triblock copolymer syntheses using the diethyl meso-2,5-dibromoadipate initiator
66
2.2.3 1
H NMR spectroscopy............................................................................................67
2.2.4 Molecular weight determination.............................................................................67
2.2.5 HPMA composition assessed by HPLC .................................................................68
2.2.6 Preparation of copolymer solutions for rheology studies .......................................68
2.3 Results and discussion..........................................................................................69
2.3.1 NMR characterization of triblock copolymers........................................................69
2.3.2 Gel Permeation Chromatography (GPC) in chloroform:methanol mixture............70
2.3.3 Hydroxypropyl methacrylate..................................................................................74
2.3.4 Characterization of commercially available grades of hydroxypropyl methacrylate..
.......................................................................................................................75
2.3.5 Copolymer synthesis...............................................................................................79
2.3.6 Aqueous solution behavior .....................................................................................81
2.4 Summary and conclusions ...................................................................................86
2.5 References .............................................................................................................87

IX
Chapter 3: New Biocompatible Wound Dressings based on Chemically
Degradable Triblock Copolymer Hydrogels.........................89
3.1 Introduction ..........................................................................................................90
3.2 Experimental Section............................................................................................91
3.2.1 Materials.................................................................................................................91
3.2.2 Synthesis of the disulfide-based bifunctional ATRP initiator, bis[2-(2-
bromoisobutyryloxy)ethyl] disulfide, (BiBOE)2S2 .................................................92
3.2.3 Synthesis of the propanediol-based bifunctional ATRP initiator, 1,3-bis (2-
bromoisobutyryloxy) propane (BiB)2P...................................................................93
3.2.4 Copolymer Synthesis and Purification....................................................................94
3.2.5 Bipyridine content assessed by HPLC....................................................................94
3.2.6 1
H NMR Spectroscopy ...........................................................................................96
3.2.7 Molecular Weight Determination ...........................................................................97
3.2.8 Dynamic Light Scattering.......................................................................................97
3.2.9 Transmission Electron Microscopy ........................................................................98
3.2.10 Gel Rheology Studies .............................................................................................98
3.2.11 Disulfide Gel Cleavage Experiments with Dithiothreitol (DTT)............................98
3.2.12 Disulfide Cleavage Experiments with Glutathione.................................................99
3.3 Results and Discussion .........................................................................................99
3.3.1 Synthesis of bifunctional initiators with and without disulfide ..............................99
3.3.2 Copolymer Synthesis............................................................................................101
3.3.3 Purification of copolymers ...................................................................................104
3.3.4 Aqueous Solution Properties ................................................................................108
3.3.5 Cleavage of disulfide bonds in disulfide-based triblock copolymer gels with
dithiothreitol (DTT)..............................................................................................123
3.3.6 Cleavage of disulfide bonds in disulfide-based triblock copolymer gels with
glutathione ............................................................................................................125
3.3.7 Properties of thiol-terminated diblock copolymers...............................................127
3.4 Conclusions .........................................................................................................127
3.5 References ...........................................................................................................128
Chapter 4: Preparation and Aqueous Solution Properties of
Thermoresponsive Biocompatible AB Diblock Copolymers
.................................................................................................130
4.1 Introduction ........................................................................................................131
4.2 Experimental Section..........................................................................................132
4.2.1 Materials...............................................................................................................132

X
4.2.2 Synthesis of the 2-phenoxyethyl 2-bromoisobutyrate initiator, PhOBr................133
4.2.3 Copolymer Synthesis and Purification..................................................................133
4.2.4 1
H NMR Spectroscopy .........................................................................................134
4.2.5 Molecular Weight Determination .........................................................................135
4.2.6 Dynamic Light Scattering.....................................................................................135
4.3 Results and Discussion .......................................................................................136
4.3.1 Initiators................................................................................................................136
4.3.2 Copolymer Synthesis............................................................................................136
4.3.3 Temperature-dependent dynamic light scattering studies.....................................139
4.3.4 Concentration-dependent dynamic light scattering...............................................143
4.3.5 Temperature-dependent 1
H NMR studies.............................................................150
4.3.6 Aggregation mechanism.......................................................................................157
4.4 Conclusions .........................................................................................................160
4.5 References ...........................................................................................................161
Chapter 5: Derivatization of Rhodamine 6G and Preparation of
Fluorescent PMPC-based (co)polymers..............................163
5.1 Introduction ........................................................................................................164
5.2 Experimental Section..........................................................................................169
5.2.1 Materials...............................................................................................................169
5.2.2 Preparation of 2-bromoisobutyric anhydride........................................................170
5.2.3 Reaction between rhodamine 6G and 3-aminopropan-1-ol to give rhodamine 6G N-
(3-hydroxypropyl)amide, 1...................................................................................170
5.2.4 Esterification of 1 with 2-bromoisobutyryl bromide to give rhodamine 6G N-(3-(2-
bromoisobutyryl)propyl)amide, 2.........................................................................171
5.2.5 General reaction between rhodamine 6G and a secondary amine ........................172
5.2.6 Reaction between rhodamine 6G and 2-(methylamino)ethanol to give rhodamine
6G N-(2-hydroxyethyl)-N-methyl amide, 3..........................................................172
5.2.7 Reaction between rhodamine 6G and diethanolamine to give rhodamine 6G N-
(bis(2-hydroxyethyl))amide, 4..............................................................................172
5.2.8 Reaction between rhodamine 6G and N-(2-hydroxyethyl)piperazine to give
rhodamine 6G N-(4-(2-hydroxyethyl)piperazine) amide, 5..................................173
5.2.9 Reaction between rhodamine 6G and 2-(butylamino)ethanol to give rhodamine 6G
N-(4-hydroxy butyl)-N-methyl amide, 6 ..............................................................173
5.2.10 Reaction between rhodamine 6G and morpholine to give rhodamine 6G N-
morpholinamide, 11..............................................................................................174

XI
5.2.11 Reaction between hydroxy-functional rhodamine derivatives and 2-
bromoisobutyric anhydride to give a monofunctional ATRP initiator using 2-
bromoisobutyric acid as solvent. ..........................................................................174
5.2.12 Reaction between 3 and 2-bromoisobutyric anhydride to give a monofunctional
initiator, rhodamine 6G N-(2-(2-bromoisobutyryl)- ethyl)-N-methyl amide, 7....175
5.2.13 Reaction between 5 and 2-bromoisobutyric anhydride to give a monofunctional
initiator, rhodamine 6G N-(4-(2-(2-bromoisobutyryloxy)ethyl))piperazine amide, 8
.....................................................................................................................175
5.2.14 Reaction between 5 and methacrylic anhydride to give a monofunctional monomer,
rhodamine 6G N-(4-(2-(methacryloyloxy)ethyl))piperazine amide, 9 .................176
5.2.15 Reaction between 4 and 2-bromoisobutyric anhydride to give a bi-functional
initiator, rhodamine 6G N-(bis((2-bromoisobutyryloxy)ethyl))amide, 10 using
phase-transfer conditions......................................................................................177
5.2.16 Preparation of PMPC homopolymers using a rhodamine-based initiator.............178
5.2.17 Preparation of pH-responsive PMPC-PDPA diblock copolymers using a
rhodamine-based ATRP initiator..........................................................................179
5.2.18 Preparation of a temperature responsive PMPC-PHPMA diblock copolymer using a
rhodamine-based ATRP initiator..........................................................................179
5.2.19 Preparation of temperature responsive PHPMA-PMPC-10-PMPC-PHPMA triblock
copolymer gelators using a bifunctional rhodamine-based initiator .....................180
5.2.20 Synthesis of deuterated methyl 2-bromoisobutyrate.............................................180
5.2.21 General protocol for examining transesterification of 2-bromoisobutyryl esters in
methanol in the presence of the CuBr/2 bpy ATRP catalyst. ...............................181
5.2.22 Calculation of the fraction of remaining 2-bromoisobutyryl ester initiator in the
presence of the ATRP catalyst..............................................................................181
5.2.23 Gel permeation chromatography ..........................................................................181
5.2.24 Reverse-phase high performance liquid chromatography.....................................182
5.2.25 Molar absorption coefficient determination..........................................................182
5.2.26 pH-dependent absorption and emission of 1 and 3...............................................183
5.2.27 pH-dependent absorption, emission and dynamic light scattering of PMPC-PDPA
diblock copolymers...............................................................................................183
5.2.28 Temperature-dependent absorption and fluorescence emission of 7-PMPC30-
PHPMA60..............................................................................................................184
5.2.29 Thermogravimetric analysis .................................................................................184
5.2.30 Gel Rheology Studies ...........................................................................................184
5.2.31 Evaluation of the extent of hydrolysis of the initiator end-groups........................185
5.3 Results and discussion........................................................................................185
5.3.1 Reaction between rhodamine 6G and 3-aminopropan-1-ol ..................................185
5.3.2 Direct reaction between secondary amines and rhodamine 6G ............................186
5.3.3 Esterification of hydroxy-functional rhodamine derivatives ................................188

XII
5.3.4 Elemental analyses of rhodamine 6G derivatives.................................................193
5.3.5 Absorption maxima and molar absorption coefficients obtained for various
rhodamine derivatives...........................................................................................193
5.3.6 pH-dependence of absorption and emission behavior ..........................................196
5.3.7 Use of rhodamine-based ATRP initiators to prepare PMPC homopolymers........198
5.3.8 Ethyl 2-bromoisobutyrate (EtOBr) under ATRP conditions ................................202
5.3.9 Chemical stability of the 2-phenoxyethyl 2-bromoisobutyrate (PhOBr) initiator
under ATRP conditions ........................................................................................206
5.3.10 Chemical stability of rhodamine 6G-based initiators under ATRP conditions.....209
5.3.11 Copper(I)bromide:2,2’-bipyridine as a transesterification catalyst.......................211
5.3.12 Use of rhodamine-based ATRP initiators to prepare pH-responsive PMPC-PDPA
diblock copolymers and PMPC-PHPMA di- and triblock copolymers ................212
5.3.13 pH-dependent self-assembly behavior of rhodamine-labelled PMPC-PDA diblock
copolymers ...........................................................................................................216
5.3.14 Temperature-dependent self-assembly of rhodamine-labelled PMPC-PHPMA
block copolymers..................................................................................................219
5.3.15 Temperature-dependent gelation of thermo-responsive triblock copolymers.......220
5.3.16 Stability of initiator group in aqueous solution.....................................................222
5.4 Conclusions .........................................................................................................224
5.5 References ...........................................................................................................226
Chapter 6: Conclusions and Future Work.............................................229
Chapter 2: Preparation and Aqueous Solution Properties of New Thermo-responsive
Biocompatible ABA Triblock Copolymer Gelators.........................................230
Chapter 3: New Biocompatible Wound Dressings based on Chemically Degradable
Triblock Copolymer Hydrogels.........................................................................230
Chapter 4: Preparation and Aqueous Solution Properties of Thermoresponsive
Biocompatible AB Diblock Copolymers ...........................................................231
Chapter 5: Derivatization of Rhodamine 6G and Preparation of Fluorescent PMPC-based
(co)polymers........................................................................................................232

XIII
List of Tables
Table 1.1: Common synthetic routes to aliphatic thiols. Typical conditions: (i) Zinc in dilute
acid,115,116
sodium boronhydride, NaBH4 in ethanol,117
triphenylphosphine and water
in methanol and dimethoxyethane,118
dithiothreitol, DTT, in various solvents119-122
or
trialkylphosphines and water in various solvents.123-126
In aqueous solutions, tris(2-
carboxyethyl)phosphine, TCEP, is frequently used due to its solubility and high
efficiency.127
(ii) The addition of hydrogen sulfide to double bonds is efficient in the
presence of free-radical initiators. The reaction can also be catalyzed by proton or
Lewis acids but only nucleophilic substrates undergo base-catalyzed addition. Since
the resulting thiol is capable of adding to a second double bond, sulfides are often by-
products.128
(iii) Various reagents have been employed for this reaction.129
(iv) Alkyl
halides can be reacted directly with hydrogen sulfide or sodium hydrogen sulfide
although sulfides are often by-products.130
(v) Indirect methods include reaction of
the alkyl halide with either thiourea or thiosulfate followed by hydrolysis of the thiol
esters or dithioesters under (vi) acidic or (viii) basic conditions.131
..........................25
Table 1.2: Common reactions of thiols applied to macromolecules and/or biomacromolecules.30
Table 2.1: GPC-data for OEG-MPC polymers using two different eluents. All polymers were
prepared using an oligo(ethylene glycol) initiator with DP~7. Entries 1-4 are the
same polymers as entries 5-8, analyzed with different amount of LiBr in the eluent.
The target DPs and calculated molecular weights of the samples are given in column
3 and 4. In column 5 and 6, the measured number-average molecular weights and
polydispersities are given. Columns 7 and 8 give the corresponding numbers for the
same polymers in an aqueous eluent at pH 7.0 The percentage deviation of the
number-average molecular weight in the non-aqueous eluent vs. the aqueous eluent
and vs. the theoretical value is given in columns 9 and 10 respectively. Details of
preparation of the polymers are given in reference 15...............................................73
Table 2.2: Mole fractions of 2-hydroxyisopropyl methacrylate (HIPMA) measured by 1
H NMR
(400 MHz in CDCl3) and HPLC (15-40 % CH3CN in 0.1 % aqueous TFA, 254 nm,
Column: GraceSmart R.P.18 5 m 150 mm x 4.6mm). The mole fractions from the 1
H
NMR measurements were obtained by calculating the ratio between well-separated
peaks assigned to on isomer (peaks h, c+d and j in Figure 2.3A respectively) and
peaks assigned to both isomers (peaks b+g, a+f and e+i in Figure 2.3A). These were
averaged and the error is the standard error. The mole fractions from HPLC were
obtained by calculating the ratio between the area of the minor peak at 10-11 min in
the chromatograms (Figure 2.4B) and the sum of the areas of both peaks. ...............78
Table 2.3: Summary of the 1
H NMR and GPC data for the three ABA triblock copolymers
examined in this chapter. a)
Subscripts indicate the mean degrees of polymerization

XIV
(DP) of each block. b)
As determined by 1
H NMR. c)
As determined by GPC
conducted in a 3:1 chloroform/methanol mixed eluent using poly(methyl
methacrylate) calibration standards. ..........................................................................80
Table 3.1: Summary of block compositions and molecular weight data obtained from 1
H NMR
and GPC studies of the triblock copolymers. All copolymers were prepared using the
disulfide initiator, except for the first entry, which was prepared using the
commercially available DEDBA initiator. 1
H NMR were recorded at 400 MHz. GPC
data were obtained using a 3:1 v/v chloroform/methanol eluent and a series of
PMMA calibration standards...................................................................................103
Table 3.2: Steps used in purification of copolymers for cytotoxicity studies ...........................104
Table 3.3: 2,2’-Bipyridine content and measured 2D viability for a 10.0 % gel of a series of
copolymer batches with composition PHPMA~90PMPC200-S-S-PMPC200-PHPMA~90.
Primary human dermal fibroblast viability was assessed using a MTT assay and
ThinCert inserts. Cell viability studies were performed by K. Bertal and details of the
assay can be found in reference 29. a
This sample was only precipitated once into
THF. The result of two further precipitations is shown in entry 1. b
This measurement
was only repeated once............................................................................................106
H NMR
and GPC studies of the diblock copolymers. 1
H NMR spectra were recorded at 400
MHz. GPC data were obtained using a 3:1 v/v chloroform/methanol eluent and a
series of PMMA calibration standards.....................................................................137
Table 5.1: Maximum wavelength and corresponding molar absorption coefficients in MeOH
and 0.10 M HCl for various rhodamine 6G derivatives. a)
These measurements were
performed in methanol containing 0.1 % v/v trifluoroacetic acid. b)
The dye was
dissolved in 25.0 mL methanol containing 0.1 % v/v trifluoroacetic acid and diluted
with 0.1 M aqueous HCl. The error is the standard error for the three measurements.
Each uncertainty is the standard error of three measurements at three different
concentrations. ‘N/M’ simply means not measured.................................................194
Table 5.2: Summary of 1
H NMR, GPC and absorption data for homopolymers prepared using
two rhodamine-based ATRP initiators, 7 and 8. a) 1
H NMR spectra recorded in
CD3OD. b)
GPC in 3:1 CHCl3:CH3OH using poly(methyl methacrylate) calibration
standards. c)
Mn Calculated from the εmax value of the initiator (see Table 5.1) in
methanol. The uncertainty values are the standard error of three measurements at
three different concentrations. d)
Signals from the rhodamine monomer could not be
integrated due to their low intensity.........................................................................201

XV
H NMR, GPC and absorption data for PMPC-based block copolymers
prepared using three rhodamine-based ATRP initiators, 2, 7 and 10. a) 1
H NMR
spectra recorded in CD3OD for PMPC homopolymers and in a 3:1 CDCl3:CD3OD
mixture for PMPC-PDPA diblock copolymers. The initiator end-group could be used
for assessing the degree of polymerization for the PMPC homopolymers, which in
turn allowed assessment of the block composition (N.B. the initiator end-groups were
not quantifiable in the block copolymer spectra. b)
GPC data obtained for a 3:1
CHCl3:CH3OH eluent using poly(methyl methacrylate) calibration standards. c)
Mn
calculated from the εmax value of the initiator (see Table 5.1) in methanol for PMPC
homopolymers, PMPC-PHPMA diblock copolymers and PHPMA-PMPC-PHPMA
triblock copolymers and in 0.10 M HCl for PMPC-PDPA diblock copolymers. The
uncertainties are the standard error of three measurements recorded at three different
concentrations..........................................................................................................215

XVI
List of Figures
Figure 1.1: Examples of polymer architectures obtained using controlled polymerization
techniques....................................................................................................................8
Figure 1.2: A) Definition of packing parameter p on geometric parameters.62
B) Definition of
hydrophobic mass ratio, fhydrophobic of block copolymer.63
C) Typical aggregate
structures and their dependence on p62
and fhydrophobic.63
.............................................17
Figure 1.3: Two pathways to micellar network formation: A) If the end-blocks are highly
incompatible with the solvent, ‘flower micelles’ are formed at relatively low
copolymer concentration. Increasing the copolymer concentration leads eventually to
overlap where bridging is facilitated. B) If the end-blocks are more compatible with
the solvent, a looser structure is formed at intermediate concentrations as the penalty
of ‘dangling ends’ is lower. This eventually leads to a network structure on
increasing the copolymer concentration. ...................................................................20
Figure 1.4: The Lawesson reagent, 4-Methoxyphenylthiophosphoric cyclic di(thioanhydride)..27
Figure 1.5: Schematic of the Tetronics® T1107-based gels described by Cellesi et al.113
The
system was optimized to give gels with alginate-mimetic viscosity in one step from
the acrylated T1107 and a protected form of the thiolated T1107. The gels degraded
due to hydrolysis of the acrylate ester over several days, this is not shown. .............39
Figure 1.6: Schematic representation of the gelation mixture described in reference 195. The two
MPC-based statistical copolymers are made up as 5 % aqueous solutions. On mixing
these solutions the acid groups form dimers in the hydrophobic domains created by
the BMA groups and these serve as physical crosslinks............................................43
Figure 1.7: Monomers that form well-defined block copolymers with MPC. DMA: 2-
(dimethylamino)ethyl methacrylate. DEA: 2-(diethylamino)ethyl methacrylate. DPA:
2-(diisopropylamino)ethyl methacrylate. HEMA: 2-hydroxyethyl methacrylate.
HPMA: 2-hydroxypropyl methacrylate. GMA: glycerol monomethacrylate. Me-
DMA: 2-(trimethylammonium)ethyl methacrylate hydrochloride. Bz-DMA: benzyl
dimethyl 2-(methacryloyloxy)ethyl ammonium chloride. CBMA: N-
(carboxymethyl)-N-(methacryloyloxy)ethyl-N,N-dimethylammonium betaine.
SBMA: N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine.
MMA: methyl methacrylate. OEGMA: monomethoxy-capped oligo(ethylene glycol)
methacrylate...............................................................................................................48
Figure 1.8: A) Formation of PMPC-PDMA/DNA complexes. B) TEM images of PMPC-PDMA
/ DNA complexes formed at a 2:1 DMA/nucleotide molar ratio. Scale bar is 500 nm.
207,208
...........................................................................................................................50

XVII
Figure 1.9: Schematic of thermoresponsive gelation of PNIPAM-PMPC based copolymers. A)
Gelation of PNIPAM-PMPC-PNIPAM copolymers202
B) Gelation of PPO-PMPC-
PNIPAM copolymer.203
.............................................................................................54
Figure 2.1: Apparent molar ratio between pendent methylene group of PMPC and the pendent
methoxy group of PMMA in a PMMA55-PMPC240-PMMA55 triblock copolymer as a
function of the volume fraction of CDCl3. The inset shows 250 MHz 1
H NMR
spectra in pure CD3OD and at a CDCl3:CD3OD volume fraction of 0.6. ..................70
Figure 2.2: Gel permeation chromatograms of a OEG-PMPC150 polymer in chloroform:methanol
3:1 v:v with different concentrations of LiBr. Flow rate: 1.0 mL / min. Temperature:
40 °C. Columns: Two Polymer Laboratories PL Gel 5 µm Mixed-C (7.5 x 300 mm)
columns in series with a guard column......................................................................71
Figure 2.3: (A) Assigned 1H-NMR spectra in CDCl3 at 400 MHz of HPMA from Aldrich and
Cognis respectively. The inset shows the region from 1-2 ppm enlarged. (B)
Assigned 13C JMOD spectrum of the Cognis product in CDCl3 at 400 MHz (1
H-
frequency). C=O, CH2 positive, CH, CH3 negative...................................................76
Figure 2.4: (A) ATR-FTIR spectra of HPMA from Aldrich and Cognis respectively. The (B)
HPLC chromatograms of HPMA from Aldrich, Cognis and a 1:1 V/V mixture of the
two. Conditions: 15-40 % acetonitrile/0.1 % aqueous trifluoroacetic acid in 20
minutes, 1 mL/min, detection at 254 nm, Column: Alltima HP C18 HL 5µ 150 x 4.6
mm.............................................................................................................................77
Figure 2.5: GPC traces obtained for the various PMPC-based triblock copolymers in 3:1
CHCl3:CH3OH with 2 mM LiBr................................................................................81
Figure 2.6: Temperature dependence of the solution viscosity for 10 w/v % aqueous solutions of
the three PMPC-based triblock copolymers shown in Table 2.3...............................82
Figure 2.7: Top: From left to right: (A) a free-flowing 10 % PHPMA55-PMPC250-PHPMA55
solution at 50 °C; (B) an opaque 10 % PMMA55-PMPC240-PMMA55 gel at 50 °C; (C)
7.5 % PHPMA44-PMPC250-PHPMA44 at 4°C (free-flowing solution) and (D) the
same copolymer solution at 50 °C (now a transparent, free-standing gel). Bottom:
Consequence of heating a PHPMA44-PMPC250-PHPMA44 solution: At low
temperature, the copolymer is molecularly dissolved. Increasing the temperature
leads to formation of ‘flower-micelles’. At sufficiently high concentration, bridges
between individual micelles may form, leading to a micellar gel network................83
Figure 2.8: (A) Storage (G’) and loss (G’’) moduli obtained for 5 and 10 % aqueous solutions of
the PHPMA44-PMPC250-PHPMA44 triblock copolymer, respectively. (B) The G’ –
G’’ cross-over temperature as a function of concentration for the same PHPMA44-
PMPC250-PHPMA44 copolymer.................................................................................84

XVIII
Figure 2.9: Temperature dependence of the scattered light intensity count rate obtained for a 0.10
w/v % aqueous solution of PHPMA44-PMPC250-PHPMA44. Note the upturn at around
10 °C due to micellar self-assembly. The diameters are the calculated hydrodynamic
diameter from the correlation functions.....................................................................85
Figure 2.10: 500 MHz 1
H NMR spectra recorded for a 3.7 % w/V PHPMA44-PMPC250-PHPMA44
triblock copolymer solution in D2O at 5°C and 46°C. The pendent methyl groups and
part of the backbone signals are assigned. Spectral shifts are due to differences in
temperature................................................................................................................86
Figure 3.1: Calibration curve of mass of 2,2’-bipyridine versus detector count at λ=300 nm.
Conditions: 1 mL/ min, 5-100 % acetonitrile in 0.1 % aqueous TFA over 20 minutes.
Column: GraceSmart R.P.18, 5µm. 150 mm x 4.6 mm. A linear fit through (0,0) gave
a straight line with equation: m(bpy) [µg] =4.94·10-7
µg x Detector Count, R2
=0.999.
...................................................................................................................................95
Figure 3.2: Measured Count Rate versus Calculated Count Rate of a PHPMA90-PMPC200-S-S-
PMPC200-PHPMA90 triblock copolymer solution spiked with known concentrations
of 2,2’-bipyridine using the calibration constant derived from Figure 3.1. Conditions:
1 mL/ min, 5-100 % acetonitrile in 0.1 % aqueous TFA over 20 minutes. Column:
GraceSmart R.P.18, 5µm. 150 mm x 4.6 mm............................................................96
Figure 3.3: HPLC traces of a PHPMA90-PMPC200-S-S-PMPC200-PHPMA90 copolymer batch
(JMASh469) precipitated with tetrahydrofuran once (JMASh469 x 1) and thrice
(JMASh469 x 3). Conditions: 1 mL/ min, 5-100 % acetonitrile in 0.1 % aqueous TFA
over 20 minutes. Column: GraceSmart R.P.18, 5µm. 150 mm x 4.6 mm ...............105
Figure 3.4: Gel Permeation Chromatograms recorded for a PHPMA90-PMPC200-S-S-PMPC200-
PHPMA90 before and after being subjected to the heating protocol described in the
experimental section (80 °C for 48 h, followed by 90 °C for 5 h)...........................107
Figure 3.5: Storage and loss modulus as a function of strain at 1 Hz for a 10.0 % w/v aqueous gel
of PHPMA88-PMPC200-S-S- PMPC200-PHPMA88 at 37 °C. The graph shows three
consecutive measurements obtained for the same solution recorded directly after one
another. ....................................................................................................................108
Figure 3.6: Comparison of rheometer geometry and heat rate for 15.0 % PHPMA43-PMPC125-S-
S-PMPC125-PHPMA43 copolymer solution in PBS (pH 7.2) at 1 rad/s, 0.5 Pa. The
measurement with the concentric cylinder was covered with a layer of paraffin oil to
suppress water evaporation......................................................................................109
Figure 3.7: Temperature-corrected heating and cooling scans of a 10.0 % aqueous solution of
PHPMA88-PMPC200-S-S- PMPC200-PHPMA88 copolymer. Conditions: 0.5 °C/min,

XIX
1.0 Hz, 0.5 Pa, concentric cylinders. The solution was covered with a layer of
paraffin oil to suppress water evaporation...............................................................110
Figure 3.8: Temperature dependence of: (A) storage and (B) loss moduli of various 10.0 w/v %
PHPMA-PMPC-PHPMA copolymer solutions in PBS buffer (pH 7.2). Conditions: 1
rad/s, 3 °C/min, 0.5 Pa.............................................................................................111
Figure 3.9: (A) Critical gelation temperature (Tgel) as a function of copolymer concentration for
three PHPMA-PMPC-PHPMA triblock copolymers; (B) storage and loss moduli
determined at 37 °C as a function of copolymer concentration for the same three
copolymers. Vertical arrows indicate the critical copolymer concentration required
for gelation in each case...........................................................................................114
Figure 3.10: Two pathways to formation of physical networks: (A) If the end-blocks are highly
incompatible with the solvent, ‘flower micelles’ are formed at relatively low
concentration. Increasing the concentration leads eventually to overlap where
bridging is facilitated and this leads to a micellar gel network. (B) If the end-blocks
are more compatible with the solvent, a looser structure is formed at intermediate
concentrations as the penalty of ‘dangling ends’ is lower. This eventually leads to a
network structure on increasing the concentration, however, the constituents of this
network are less well-defined than in the case of the micellar gel. If the solvent
compatibility changes with temperature, this may cause formation of a well-defined
micellar network gel. ...............................................................................................116
Figure 3.11: Temperature dependence of the light scattering intensity at 173 ° for 0.10 % aqueous
solutions of six triblock copolymers in PBS buffer (pH 7.2)...................................117
Figure 3.12: Autocorrelation functions obtained from dynamic light scattering studies of six
triblock copolymers (0.10 w/v % aqueous solutions in PBS buffer, pH 7.2 at 4 °C, 19
°C and 37 °C. Scattering angle = 173 ° in each case. ..............................................119
Figure 3.13: Transmission electron microscopy images of dried ‘flower-like’ micelles obtained by
drying a 0.40 w/v % aqueous solution of PHPMA88-PMPC200-S-S-PMPC200-
PHPMA88, followed by staining with uranyl acetate. H. Lomas is acknowledged for
the requisition of the image. ....................................................................................121
Figure 3.14: Temperature dependence of the apparent PHPMA contents of 7.0 w/v % solutions of
four triblock copolymers in D2O normalized with respect to their corresponding
block compositions determined in CD3OD. The apparent reduction in PHPMA
content that occurs on increasing the temperature indicates poorer solvation and/or
lower mobility. Spectra recorded at 21 °C in D2O and CD3OD were obtained using a
400 MHz spectrometer, the remaining spectra were recorded at a 500 MHz
spectrometer.............................................................................................................122

XX
Figure 3.15: (A) Gel permeation chromatograms recorded for a PHPMA88-PMPC200-S-S-
PMPC200-PHPMA88 triblock copolymer before and after exposure to DTT.
Conditions: DTT/S-S molar ratio = 10, methanol, 25 °C, 12 h. (B) Kinetics of gel
dissolution caused by cleavage of the disulfide bonds in a 10.0 w/v % gel comprising
a PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 copolymer in PBS buffer (pH 7.2) at
37 °C using DTT/disulfide molar ratios of 10.0, 5.0, 2.0, 1.0 and zero...................125
Figure 3.16: Cleavage of a 1.0 % solution of a 1.5 % w/v PHPMA88-PMPC200-S-S-PMPC200-
PHPMA88 copolymer solution, 9 eq. glutathione, N2-purged PBS pH 7.2, 37 °C. GPC
conditions: 70 % 0.2 M NaNO3, 0.01 M NaH2PO4, adjusted to pH 7.0; 30 %
methanol. Calibrated with near-monodisperse poly(sodium 4-styrenesulfonate)
standards..................................................................................................................126
Figure 3.17: Temperature dependence of the light scattering intensity at 173 ° for 0.10 % aqueous
solutions of PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 and PHPMA88-PMPC200-
SH in PBS buffer (pH 7.2). PHPMA88-PMPC200-SH was prepared by adding 2000
equivalent of DTT to the 0.1 % PHPMA88-PMPC200-S-S-PMPC200-PHPMA88
solution, leaving this for 10 minutes at 25 °C followed by filtering through a 0.22 µm
nylon filter immediately before starting the measurement. .....................................127
Figure 4.1: Gel permeation chromatograms of the PMPC-PHPMA diblock copolymers obtained
using a 3:1 chloroform: methanol eluent and a series of near-monodisperse
poly(methyl methacrylate) calibration standards.....................................................138
Figure 4.2: (A,B) Scattering intensity vs. temperature plots for 1.0 w/v % PMPC-PHPMA
diblock copolymers in PBS (pH 7.2). (C,D) Hydrodynamic radius vs. temperature
plots for the same aqueous diblock copolymer solutions.........................................139
Figure 4.3: Temperature dependence of hydrodynamic radii determined from cumulants analyses
of 1.0 w/v % aqueous PMPC-PHPMA diblock copolymer solutions in PBS at pH 7.2.
.................................................................................................................................140
Figure 4.4: Concentration dependence of the apparent hydrodynamic radius of solutions of (A)
PMPC~25-PHPMAn diblock copolymers in PBS, pH 7.2 at 4 °C; (B) PMPC~25-
PHPMAn diblock copolymers in PBS, pH 7.2 at 37 °C; (C) PMPC49-PHPMAn
diblock copolymers in PBS, pH 7.2 at 4 °C; (D) PMPC49-PHPMAn diblock
copolymers in PBS, pH 7.2 at 37 °C. (E) Hydrodynamic radius as a function of
temperature for 1.0 w/v %, 2.0 w/v % and 5.0 w/v % solutions of PMPC23-
PHPMA24. Dotted lines indicate aggregation/precipitation. (F) Hydrodynamic radius
as a function of temperature for 1.0 w/v %, 2.0 w/v % and 5.0 w/v % solutions of
PMPC49-PHPMA49. Dotted lines indicate aggregation/precipitation.......................144

XXI
Figure 4.5: (A) Hydrodynamic radii from cumulants analyses of 0.1 w/v % , 2.0 w/v % and 5.0
w/v % solutions of the PMPC25-PHPMAn diblock copolymers at 4 °C, 22 °C and 37
°C. (B) Hydrodynamic radii from cumulants analyses of 0.1 w/v % , 2.0 w/v % and
5.0 w/v % solutions of the PMPC49-PHPMAn diblock copolymers at 4 °C, 22 °C and
37 °C........................................................................................................................147
H NMR spectra of PMPC25-PHPMA39 recorded in CD3OD at 21 °C and in
D2O at 4.6 °C, 25 °C and 37 °C. All spectra are normalized relative to peak ‘a’. The
arrows indicate those PHPMA signals that are significantly attenuated at elevated
temperature. .............................................................................................................151
Figure 4.7: Temperature dependence of the apparent PHPMA content of 1.0 w/v % aqueous
solutions of various PMPC-PHPMA diblock copolymers in D2O normalized with
respect to their actual block compositions (as determined in CD3OD). The monotonic
reduction in apparent PHPMA content on increasing the temperature indicates
progressively poorer solvation and/or lower mobility for this block; this is consistent
with the onset of micellar self-assembly. (A) Data set obtained for PMPC-PHPMA
diblock copolymers with a fixed PMPC DP of ~ 25; (B) data set obtained for PMPC-
PHPMA diblock copolymers with a fixed PMPC DP of ~ 49. Lines are guides for the
eye, rather than fits to the data.................................................................................153
Figure 4.8: (A) Apparent PHPMA content measured by 1
H NMR spectroscopy in 1.0 w/v %
solutions in D2O for PMPC25-PHPMAn (triangles) and PMPC50-PHPMAn (circles)
diblock copolymers as a function of the actual degree of polymerization of the
PHPMA block at 5 °C (open symbols) and 37 °C (closed symbols). (B) DLS
hydrodynamic radius obtained for 1.0 w/v % solutions in PBS at pH 7.2 containing
PMPC25-PHPMAn (triangles) and PMPC50-PHPMAn (circles) diblock copolymers as
a function of the actual degree of polymerization of the PHPMA block at 4 °C (open
symbols) and 37 °C (closed symbols). Lines are guides to the eye, rather than data
fits............................................................................................................................155
Figure 4.9: Angular dependence of the diffusion coefficient for two 1.00 w/v % copolymer
solutions in PBS at 4 °C and 38 °C..........................................................................157
Figure 4.10: Schematic representation of the effect of raising the solution temperature and
increasing the mean degree of polymerization of the PHPMA block on the colloidal
aggregates produced by self-assembly.....................................................................158
Figure 5.1: Kinetics of formation of rhodamine 6G-based initiator 7 versus time as determined
by reverse phase HPLC. ..........................................................................................190
Figure 5.2: Assigned 1
H-NMR spectrum of the pH-independent bifunctional rhodamine-based
ATRP initiator 10 ....................................................................................................192

XXII
Figure 5.3: Absorption spectra obtained for 7 in methanol and 0.1 M HCl. Scan speed: 240
nm/min.....................................................................................................................195
Figure 5.4: Normalized absorption and emission spectra of 3 in aqueous HCl at pH 2.0. The
emission spectrum was recorded with an excitation wavelength of 530 nm. ..........196
Figure 5.5: (A) Absorption spectra of 1 versus pH. (B) Absorption spectra of 3 versus pH......197
Figure 5.6: (A) Effect of increasing the solution pH on the maximum emission normalized with
respect to pH 1.0 and absorbance at 530 nm for a solution initially containing 5•10-5
M 1; (B) Effect of increasing the pH on the maximum emission and the relative
absorbance at 530 nm and 508 nm respectively for a solution initially containing
1•10-5
M 3. (C) Digital image of 5•10-5
M 1 at different pH (D) Digital image of
1•10-5
M 3 at different pH........................................................................................198
Figure 5.7: Weight loss as a function of heating in air of 7-PMPC20 and 7-PMPC100. J. Balmer is
acknowledged for the TGA experiments. ................................................................200
Figure 5.8: Kinetics of the reaction of ethyl 2-bromoisobutyrate:CuBr:bpy at a relative molar
ratio of 1:1:2 in CD3OD in the absence of any added monomer. (A) 400 MHz 1
H
NMR spectra recorded for ethyl 2-bromoisobutyrate, kinetic samples, deuterated
methyl 2-bromoisobutyrate and ethanol. (B) HPLC chromatograms recorded for ethyl
2-bromoisobutyrate, kinetic samples, deuterated methyl 2-bromoisobutyrate and 2-
bromoisobutyric acid. HPLC column: Thermo Hypersil Keystone 100 x 4.6 mm, 3µ
Betabasic-18 Detection: UV at 254 nm. ..................................................................204
Figure 5.9: 1
H NMR spectra recorded for: (A) EtOBr:CuBr2:bpy 1:1:2 reaction mixture in
CH3OH after 48 h; (B) EtOBr in CD3OD................................................................205
Figure 5.10: Kinetics of the reaction of PhOBr: CuBr: bpy at a relative molar ratio of 1:1:2 in
CH3OH. (A) 400 MHz 1
H NMR spectra recorded for phenoxyethanol, kinetic
samples and PhOBr. (B) HPLC chromatograms obtained for deuterated methyl 2-
bromoisobutyrate, phenoxyethanol, kinetic samples and PhOBr. Column: Thermo
Hypersil Keystone 100 x 4.6 mm, 3µ Betabasic-18 Detection: UV at 254 nm. ......207
Figure 5.11: (A) HPLC chromatograms recorded for a 1:2 PhOBr:bpy mixture after 120 min in
methanol at 22 °C. Column: Thermo Hypersil Keystone 100 x 4.6 mm, 3µ Betabasic-
18 Detection: UV at 254 nm. (B) 400 MHz 1
H NMR of a PhOBr: CuBr2: bpy
mixture at a relative molar ratio of 1:1:2 after 48 h in CH3OH compared to PhOBr.
.................................................................................................................................208
Figure 5.12: Analysis of the chemical degradation of rhodamine 6G-based initiators under ATRP
conditions. (A) HPLC chromatograms obtained for compound 5, kinetic samples of 8
with CuBr and bpy (8: CuBr: bpy = 1:1:2) and compound 8. (B) ESI-MS of selected

XXIII
kinetic samples of 8 with CuBr and bpy (8: CuBr: bpy = 1:1:2) and initiator 8. (C)
HPLC chromatograms obtained for compound 3, kinetic samples of 7 with CuBr and
bpy (7: CuBr: bpy = 1:1:2) and compound 7. (D) ESI-MS of selected kinetic samples
of 7 with CuBr and bpy (7: CuBr: bpy = 1:1:2) and initiator 7 under ATRP
conditions. (E) HPLC chromatograms obtained for compound 4, kinetic samples of
10 with CuBr and bpy (10: CuBr: bpy = 1:2:4) and compound 10. (F) ESI-MS of
selected kinetic samples of 10 with CuBr and bpy (10: CuBr: bpy = 1:2:4) and
initiator 10 under ATRP conditions.........................................................................210
Figure 5.13: Fraction of EtOBr, PhOBr and rhodamine initiators present as a function of time. For
EtOBr, the fraction was calculated by both 1
H NMR and HPLC. For the remaining
compounds, only the HPLC data were used. These calculations assumed no side-
reactions and identical absorption coefficients for both the initiator and its by-
product.....................................................................................................................211
Figure 5.14: Variation of hydrodynamic diameter with solution pH obtained by dynamic light
scattering at 25o
C for 0.20 % aqueous solutions of pH-responsive diblock
copolymers: (A) 2-PMPC28-PDPA56 and 2-PMPC24-PDPA115 and (B) 7-PMPC25-
PDPA90. ...................................................................................................................216
Figure 5.15: (A) Absorption and emission spectra recorded for dilute aqueous solutions of 2-
PMPC28-PDPA56 at pH 3.0 and pH 8.0. Note the logarithmic scale on the emission
spectra. (B) Fluorescence intensity versus pH normalized to pH 3.0. The initial
concentration was 0.20 % in 0.1 M HCl. Excitation wavelength = 530 nm, emission
slit = 5 nm................................................................................................................217
PMPC25-PDPA90 at pH 3.0 and pH 8.0. The initial concentration was 0.20 % in 0.1 M
HCl. Excitation wavelength = 530 nm (B) Ratio between the magnitude of the 530
nm and 508 nm bands compared to the maximum normalized fluorescence intensity
versus pH. (C) Digital photographs of a 0.20 % w/v solution of 7-PMPC22-PDPA84 at
increasing pH. Notice the color shift due to dimer formation above pH 6.5. ..........218
Figure 5.17: (A) Relative fluorescence intensity and hydrodynamic radius of the rhodamine-based
diblock copolymer, 7-PMPC30-PHPMA60. A 0.10 w/v % aqueous solution with
excitation at 530 nm was used for the fluorescence studies. Light scattering studies
were conducted using a 1.00 w/v % aqueous solution filtered through a 0.22 µm
Nylon filter prior to measurements. The average of three consecutive light scattering
measurements is shown. (B) Absorption spectra recorded at 5 °C (blue), 20 °C
(black) and 37 °C (red) for a 0.10 w/v % solution of 7-PMPC30-PHPMA60. The
arrows designate increasing temperature.................................................................219

XXIV
Figure 5.18: Temperature dependence of the loss and storage modulus for 10.0 w/v % and 20.0
w/v % PHPMA50-PMPC125-10-PMPC125-PHPMA50 aqueous solutions. Experimental
parameters: 1 rad/s, 0.5 Pa, 3 °C/min. Insert shows a digital picture of a 10.0 w/v %
solution of PHPMA50-PMPC125-10-PMPC125-PHPMA50 in water. .........................221
Figure 5.19: Visible absorption (λ = 530 nm) and refractive index detector GPC traces for 1.0 %
7-PMPC25 at zero time and after 7 days storage in PBS buffer at pH 7.2 and 37o
C.
Eluent: 0.2 M NaNO3 and 0.01 M NaH2PO4 adjusted to pH 7; flow rate = 1.0 mL
min-1
.........................................................................................................................222
Figure 5.20: Evolution of the rhodamine end-group functionality of 1.0 % aqueous solutions of 7-
PMPC25 and 7-PMPC100 in PBS buffer (pH 7.2) stored at 37 °C determined by
comparing the integrated absorbance signal at 530 nm with the integrated refractive
index signal and normalizing the ratio to the ratio at t=0.01 days (15 min). ...........224

XXV
List of Schemes
Scheme 1.1: Reactions and reaction rates of free-radical polymerization at low conversion
assuming steady-state kinetics according to references 7 and 8 ..................................5
Scheme 1.2: Two types of controlled radical polymerization. A) Reversible radical trapping. B)
Reversible transfer.....................................................................................................10
Scheme 1.3: Basic RAFT mechanism according to Rizzardo’s group.17
.......................................12
Scheme 1.4: Basic ATRP mechanism according to Matyjaszewski31
............................................13
Scheme 1.5: Possible reactions of thiol radicals and structure of the addition product according to
reference 114 .............................................................................................................34
Scheme 1.6: Preparation of 3-arm PNIPAM star copolymer according to reference 145..............36
Scheme 1.7: Mechanism for the PEGylation of protein thiols described by Brocchini and co-
workers.167
.................................................................................................................37
Scheme 1.8: Approaches to controlled polymerization of MPC by A: ATRP 38,49
, B: RAFT195
and
C: photoinduced living radical polymerization.196
.....................................................46
Scheme 2.1: Synthetic route to the HPMA monomer. The asterisk denotes a chiral center..........75
Scheme 2.2: ATRP synthesis of the PHPMA-PMPC-PHPMA triblock copolymer ......................79
Scheme 3.1: a) Preparation of bis[2-(2-bromooisobutyryloxy)ethyl] disulfide, BiBOE2S2 b)
Preparation of 1,3-bis (2-bromoisobutyryloxy) propane BiB2P...............................100
Scheme 3.2: Synthesis of PHPMA-PMPC-S-S-PMPC-PHPMA triblock copolymers via ATRP101
Scheme 3.3: Chemical degradation of the free-standing aqueous micellar gel formed by the
PHPMA–PMPC-S-S-PMPC–PHPMA triblock copolymer after cleavage of the
disulfide bonds by using dithiothreitol (DTT).........................................................124
Scheme 4.1: Synthesis of PMPCm-PHPMAn diblock copolymers via ATRP using sequential
monomer addition (MPC monomer polymerized first). ..........................................136
Scheme 5.1: Base-induced conversion of hydroquinone to spirolactone for 2’-substituted
rhodamine 6G ..........................................................................................................166
Scheme 5.2: a) Reaction of 2-bromoisobutyric esters with a Cu(II)(bpy)2 complex to form a
radical species.33
b) ATRP with a monomer according to Matyjaszewski.33
c) Radical
recombination.34,35
d) Transfer to solvent.34,35
e) Transesterification with methanol.36
.................................................................................................................................168

XXVI
Scheme 5.3: General reaction of rhodamine 6G with various secondary amines to form the
corresponding substituted amides. Numbers in parentheses are yields of isolated
purified compounds. ................................................................................................186
Scheme 5.4: Esterification of three hydroxyfunctional rhodamine derivatives to produce various
fluorescently-labelled ATRP initiators and a fluorescently-labelled methacrylic
monomer. Reaction conditions: a) (i) CH3CN, 32 % HCl, reflux. (ii) 2-
bromoisobutyryl bromide, 3h, reflux (iii) Aqueous NaHCO3:CH2Cl2. Yield: 89 % ; b)
(i) 2-bromoisobutyric acid, 70 °C, (ii) 2-bromoisobutyric anhydride, 70 °C. (iii)
Aqueous NaHCO3:CH2Cl2. Yield: 66 %; c) (i) methacrylic acid, CHCl3, 25 °C, (ii)
methacrylic anhydride, 25 °C, (iii) Aqueous NaHCO3:CH2Cl2. Yield: 76 % d) (i) 2-
bromoisobutyric anhydride in water:dichloromethane 5:3, 47 h, 25 °C (ii) Aqueous
NaHCO3:CH2Cl2. Yield: 14 %.................................................................................188
Scheme 5.5: Synthesis of PMPC homopolymers by ATRP using the rhodamine 6G-based
initiators...................................................................................................................199
Scheme 5.6: Synthesis of PMPCn-PDPAm and PMPCn-PHPMAm diblock copolymers by ATRP.
.................................................................................................................................213

XXVII
Abbreviations
(BiB)2P 1,3-bis (2-bromoisobutyryloxy) propane
(BiBOE)2S2 bis[2-(2-bromoisobutyryloxy)ethyl] disulfide
AIBN 2,2′-azobis(2-methylpropionitrile)
ATRA atom transfer radical addition
ATR-FTIR attenuated total reflection fourier transform infrared
ATRP atom transfer radical polymerization
BHT 2,6-di-tert-butyl-4-methylphenol
BMA n-butyl methacrylate
bpy 2,2’-bipyridyl
BSA bovine serum albumin
Bz-DMA benzyl dimethyl 2-(methacryloyloxy)ethyl ammonium chloride
c.a.c. critical aggregation temperature
c.g.c. critical gelation temperature
CBMA N-(carboxymethyl)-N-(methacryloyloxy)ethyl-N,N-
dimethylammonium betaine
CTA chain transfer agent
DEA 2-(diethylamino)ethyl methacrylate
DEAD diethyl azodicarboxylate
DEDBA diethyl meso-2,5-dibromoadipate
DLS dynamic Light Scattering
DMA 2-(dimethylamino)ethyl methacrylate
DMAP 4-(dimethylamino)pyridine
DMF N,N-Dimethylformamide
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DoMA n-dodecyl methacrylate
DP degree of polymerization
DPA 2-(diisopropylamino)ethyl methacrylate
DPPC dipalmitoylphosphatidylcholine
DTT DL-dithiothreitol
EOEOVE 2-(2-ethoxy)ethoxyethyl vinyl ether
ESI-MS electrospray ionization mass spectroscopy
Et3N triethylamine
EtOBr ethyl 2-bromoisobutyrate
FRP free-radical polymerization
FTIR fourier transform infrared
G’ shear storage modulus
G’’ shear loss modulus
GMA glycerol monomethacrylate
GPC gel permeation chromatography
GTP group transfer polymerization
HEMA 2-hydroxyethyl methacrylate
HIPMA hydroxyisopropyl methacrylate
HPLC high performance liquid chromatography
HPMA 2-hydroxypropyl methacrylate
HPMA 2-hydroxypropyl methacrylate
IUPAC International Union of Pure and Applied Chemistry
LCST lower critical solution temperature

XXVIII
Me-DMA 2-(trimethylammonium)ethyl methacrylate hydrochloride
MeOH methanol
MMA methyl methacrylate
MMA methyl methacrylate
Mn number-average molecular weight
MOVE 2-methoxyethyl vinyl ether
MPC 2-(methacryloyloxy)ethyl phosphorylcholine
Mt (transition) metal
Mw weight-average molecular weight
Mw/Mn polydispersity index
NIPAM N-isopropylacrylamide
NMR nuclear magnetic resonance
OEG oligo(ethylene glycol)
OEGMA monomethoxy-capped oligo(ethylene glycol) methacrylate
P2VP poly(2-vinylpyridine)
PBD 1,2-polybutadiene
PBMA poly(n-butyl methacrylate)
PBS phosphate buffered saline
PC phosphorylcholine
PCL poly(ε-caprolactone)
PDEA poly(2-(diethylamino)ethyl methacrylate)
PDMA poly(2-(dimethylamino)ethyl methacrylate)
PDMS poly(dimethylsiloxane)
PDPA poly(2-(diisopropylamino)ethyl methacrylate)
PEG poly(ethylene glycol)
PEO poly(ethylene oxide)
PEOEOVE poly(2-(2-ethoxy)ethoxyethyl vinyl ether)
PHEMA poly(2-hydroxyethyl methacrylate)
PhOBr 2-phenoxyethyl 2-bromoisobutyrate
PHPMA poly(2-hydroxypropyl methacrylate)
PI poly(isoprene)
PLGA poly(lactic-co-glycolic acid)
PMA poly(methacrylic acid)
PMMA poly(methyl methacrylate)
PMOVE poly(2-methoxyethyl vinyl ether)
PNaStS poly(sodium 4-styrenesulfonate)
PNIPAM poly(N-isopropylacrylamide)
PPO poly(propylene glycol)
PPS poly(propylene sulfide)
PS poly(styrene)
PSGMA poly(sulfonated glycidyl methacrylate)
RAFT reversible addition-fragmentation chain transfer
RP reverse phase
SBMA N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium
betaine
TCEP tris(2-carboxyethyl)phosphine
TEM transmission electron microscopy
TFA trifluoroacetic acid
TGA thermogravimetric analysis
THF tetrahydrofuran

XXIX
Trizma 2-amino-2-(hydroxymethyl)-1,3-propanediol
w/v weight/volume

Chapter 1: Introduction
1

2
1.1 Macromolecules and polymer science
The existence of macromolecules was only gradually accepted as an explanation
for the special properties of a range of natural and synthetic polymers due to
Staudinger’s pioneering work in the field.1
He was also the first to propose the use
of the word “macromolecule” (Makromoleküle). According to the IUPAC
recommendation, a macromolecule is defined as: “A molecule of high relative
molecular mass, the structure of which essentially comprises the multiple
repetition of units derived, actually or conceptually, from molecules of low
relative molecular mass.”2
Interestingly, the word “polymer” (literally: “many
parts”) pre-dates “macromolecule” by almost 100 years.3
The original introduction
of the former phrase is ascribed to Berzelius who used it to describe molecules
with the same chemical composition but different molecular weights. Modern
terminology makes no distinction between “polymer” and “macromolecule”.
1.2 Preparation of macromolecules
Polymers were originally classified according to the method by which they were
prepared. Carothers divided polymers into two types: Addition polymers and
condensation polymers. In case of addition polymers, the molecular formula of
the structural repeat unit of the polymer is identical to that of the monomer. In
contrast, the molecular formula of the structural repeat unit of condensation
polymers differs from that of the constituent monomer(s) because these are
typically prepared by loss of a (small) molecule such as water. Although most
polymers can be classified into one of these categories, there may be cases where
a polymer can be classified into either category depending on how it is prepared.
One such example is poly(ethylene glycol)/poly(ethylene oxide). In general, this
common polymer is prepared by the ring-opening polymerization of ethylene
oxide, in which case it should be classified as an addition polymer and be termed
poly(ethylene oxide) after the monomer.4,5
However, it is possible to prepare
polymers with identical composition using ethylene glycol and 1,2-dihaloethane
at elevated temperatures.6
In this case, the polymer should be classified as a
condensation polymer. Therefore, the current trend is to classify the

3
polymerization method rather than the final polymer. A more recent classification
of polymerization reactions is to divide them into step reactions and chain
reactions.7
In step reactions all monomers may react at any time and the chain
length increases slowly with conversion. This procedure includes
polycondensation reactions as well as some related polymerizations involving
cyclic monomers that do not give off any by-products. In chain reactions,
relatively few active centers add monomers one at a time, which results in a shift
in the active centre along the growing polymer chain. High molecular weight
chains can be achieved even at relatively low monomer conversion.
1.2.1 Free-radical polymerization
Around half of all commercially available synthetic polymers are prepared by
free-radical polymerization (FRP).8
The process is easy, relatively tolerant
towards impurities and long polymer chains are formed fast. In addition, a very
wide range of vinyl monomers amenable to FRP are commercially available and
this chemistry is compatible with a range of industrially acceptable methods. In
addition, random copolymers can be prepared relatively simple. However, this
method is not suitable for preparing near-monodisperse polymers and well-
defined block copolymers, primarily due to termination reactions during the
polymerization process.
The mechanism of free-radical polymerization is well known and consists of at
least three distinct steps: initiation, propagation and termination. Often, the
possibility of transfer to monomer, polymer, solvent, initiator or other additives
should also be considered (Scheme 1.1). Since radicals do not disappear during
the transfer reaction, this reaction has little influence on the kinetics provided that
re-initiation is fast.8
However, transfer may lead to branching or crosslinking and
will in general have an influence on the molecular weight.
Initiation by thermal- or light-induced fission of a covalent bond is normally a
relatively slow reaction. Once formed, the radicals may recombine,
disproportionate or undergo reactions with solvent or monomer.
Disproportionation or reactions with solvent will affect the initiator efficiency, f,
which is typically between 0.3 and 0.8. Although recombination will lead to

4
slower kinetics, this is not normally considered to lower the efficiency, since the
starting initiator is recovered.9
The reason for this phenomenon is that the
surrounding condensed phase forms a ‘cage’ around the fragments, hindering
their separation, which causes the fragments to preferentially react with one
another. This is known as the ‘cage effect’. In comparison to the initiator
decomposition, the reaction of primary or secondary radicals with monomer must
be relatively fast in order to efficiently initiate the polymerization.

5
Step Reaction Rate Description
⋅+⋅⎯→⎯− IIII dk
][
][
IIk
dt
IId
r dd −=
−
−= Initiator decomposition
Initiation
⋅=⋅−⎯→⎯+⋅ 1PMIMI ik ][22 IIfkfrr ddi −== f: initiator efficiency
Propagation ⋅⎯→⎯+⋅ +1n
k
n PMP p
]][[
][
MPk
dt
Md
r pp ⋅=−= Polymer formation
mn
k
mn PPP tr
+⎯→⎯⋅+⋅ ]][[ ⋅⋅−= mntrtr PPkr Recombination
H
mn
k
mn PPPP td
+⎯→⎯⋅+⋅ = ]][[ ⋅⋅−= mntdtd PPkr DisproportionationTermination
2
])[(2 ⋅+= Pkkr tdtrt Total termination
⋅+−⎯→⎯−+⋅ IIPIIP n
k
n
trI ]][[ IIPkr trItrI −⋅= Transfer to initiator
⋅+⎯⎯ →⎯+⋅ 1PPMP n
k
n
trM ]][[ MPkr trMtrM ⋅= Transfer to monomer
⋅+⎯⎯ →⎯+⋅ xn
k
xn PPPP trP ]][[ xtrPtrP PPkr ⋅= Transfer to polymer
⋅+−⎯⎯→⎯+⋅ SXPSXP n
k
n
trS ]][[ SXPkr ntrStrS ⋅= Transfer to solvent
⋅+−⎯⎯ →⎯+⋅ TAPTAP n
k
n
trTA ]][[ TAPkr trTAtrTA ⋅= Transfer to transfer agent
Transfer
⋅⎯⎯ →⎯+⋅ 1
'
PMT trTAk
]][['' MTkr trTAtrTA ⋅= Transfer from transfer agent
Total nPIMII −⎯→⎯+−2
1 ][])[)(/( 2
1
2
1
MIIfkkkr dtpp −= Total polymerization assuming transfer does not affect kinetics
Scheme 1.1: Reactions and reaction rates of free-radical polymerization at low conversion assuming steady-state kinetics according to references 7 and 8

6
Propagation continues until either all radicals are annihilated or there is no more
accessible monomer. Termination may occur by two different pathways (Scheme
1.1). Either two polymers may combine to form a single chain (recombination) or
a polymer can abstract a hydrogen atom from another chain, leading to an
unsaturated terminal group on this second chain (disproportionation). Rate
coefficients of reactions including growing polymer radicals such as propagation,
termination and several transfer reactions are not constant but depend on the chain
length and conversion.7
The main reason is that the diffusion of polymer radicals
becomes increasingly restricted as the chain grows. In addition, higher conversion
and longer chains increase the viscosity of the medium. Diffusion of small
molecules like monomer and solvent is proportional to the viscosity of the
continuous phased, which is why the rate coefficients of these reactions are
normally only suppressed significantly at very high conversions. Termination,
which involves the reaction of two growing polymer radicals, becomes
increasingly improbable as the chains grow longer due to slower diffusion and
increasing chain entanglements. Therefore, termination is suppressed and the rate
of polymerization increases.
The average length of a kinetic chain is given by the ratio of the rate of
propagation to the rate of initiation. The number-average degree of
polymerization depends on the kinetic chain length, as well as the chain transfer
and the mode of termination. In the absence of chain transfer, and if termination
occurs by both recombination and disproportionation, the number-average degree
of polymerization at low conversion is given by:7
Equation 1.1
2/])[(
][
2/ 2
1
2
12
1
0
trtd
trtd
dt
p
trtd
p
n
kk
kk
IIfk
M
k
k
rr
r
DP
+
+
⋅
−
=
+
=
with kt=ktr+ktd. In principle, transfer to essentially all species present may occur
and typically influences the molecular weight. Although transfer does not affect
the kinetics if initiation is effective from the new radical, it serves as a means of
terminating the growing polymer chains. Therefore, transfer generally leads to a
reduction of the mean polymer chain length. One exception is if the transfer to

7
polymer chains is high. This may lead to branching due to growth from the chain
or crosslinking due to recombination of two chains. If transfer is considered, the
number-average degree of polymerization becomes:
Equation 1.2
...2/ +++++
=
trStrMtrItrtd
p
rrrrr
r
DPn
which may be converted to:
Equation 1.3 ...
][
][
][
][11
0
+++
−
+=
M
SX
CC
M
II
C
DPDP
SMI
nn
where CX=ktrX/kp (with X=I, M, S…) are designated transfer constants which are
tabulated for a range of common monomers and solvents.10
From this relation, it
is clear that the degree of polymerization is reduced if transfer to initiator,
monomer or solvent occurs.
1.2.2 Controlled / “Living” polymerization
The concept of living polymerization was first defined by Szwarc as a
polymerization process that does not involve a termination step.11
This definition
is essentially the same as the current IUPAC recommendation, which defines a
living polymerization as: “A chain polymerization from which chain transfer and
chain termination are absent”.2
This recommendation was further refined by the
American Chemical Society to describe existing polymerization methods.12
In
addition, the term controlled polymerization was suggested to describe synthetic
methods to prepare well-defined polymers with respect to topology, end-group
functionality and composition. In addition, the target molecular weight should be
governed by the ratio between the (reacted) monomer and initiator concentrations.
This definition is broader than that for living polymerization and covers methods
where a small amount of irreversible termination occurs but which are still
capable of producing well-defined polymers.

8
These controlled systems with efficient initiation and suppressed termination may
lead to a wide range of copolymer architectures. Some of the more common
examples are shown in Figure 1.1.
AB diblock
ABA triblock
ABC triblock
Graft/comb
Star
Cyclic or ring
Figure 1.1: Examples of polymer architectures obtained using controlled polymerization
techniques
For a polymerization with efficient initiation in the absence of any termination,
the degree of polymerization is given by:5
Equation 1.4
][
][ 0
XR
M
pDPn
−
⋅=
where p designates the monomer conversion. The molecular weight distribution
of the resulting polymer follows a poisson distribution, where the polydispersity
is given as:

9
Equation 1.5
⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
−
⋅
+=+≈
+
+=
][
][
1
1
1
1
)1(
1
0
2
XR
M
p
DPDP
DP
M
M
nn
n
n
w
Thus the polydispersity decreases with increasing degree of polymerization.
The first example of a living polymerization was reported by Szwarc et al.11,13
who described the anionic polymerization of styrene. In the absence of proton
donors and oxygen, this reaction went to completion. Adding more monomer to
the reaction mixture led to a continuation of the polymerization, demonstrating its
living character. For the last 50 years or so, a large number of controlled
polymerization methods have emerged, the description of which is largely outside
the scope of this thesis.14
Here, only the basic principles of controlled radical
polymerization methods will be mentioned and two of the most versatile methods,
RAFT and ATRP will be dealt with in more detail. In particular, ATRP has been
used to prepare the various copolymers described in this thesis.
1.2.3 Controlled radical polymerization methods
Although free-radical polymerization has proven to be very versatile, it has
certain drawbacks. As it is characterized by slow initiation, leading to relatively
few initiating centers at any given time as well as a high degree of chain
termination, the resulting polymers are characterized by a relatively high
polydispersity. In addition, all chains are essentially ‘dead’ on any useful time-
scale which makes it impossible to prepare block copolymers.15
In order to
circumvent this, various approaches have been followed. In general, it is desirable
to achieve fast initiation and suppress termination, which effectively increases the
mean radical life-time. Since both initiation and termination kinetics are governed
by the radical concentration in FRP (Scheme 1.1), this apparently presents a
paradox. It has been solved by introducing a dynamic equilibrium between the
propagating radical species and a dormant species. If the radical only reacts with
relatively few monomers before it is deactivated, the number of reactive centers
increases, which leads to more chains being initiated. As the dormant radical may
be reactivated, the chain is not ‘dead’ and further propagation is possible. At the

10
same time, the concentration of active polymer radicals is kept relatively low,
which suppresses termination.
There are two main types of such equilibria. The first relies on reversible radical
trapping via the so-called persistent radical effect (PRE), where propagating
radicals are in dynamic equilibrium with deactivated species, which cannot self-
terminate. If the equilibrium is shifted towards the deactivated species, the
concentration of propagating polymer radicals is reduced. Since the rate of
termination is proportional to the square of the radical concentration whereas
propagation is directly proportional to the radical concentration, this leads to a
relative reduction in termination which effectively increases the radical life-time.
This process is shown in Scheme 1.2 A.
Pn
.
+ Mkp
kt
kda
ka
Pn-X+ X
A) Reversible radical trapping
Growing chain Dormant chain
B) Reversible transfer
Pn
.
+ Mkp
kt
+ Pm-X
Growing chain Dormant chain
Termination
Termination
kexchange
Pn-X
.-Pm
kexchange
Pm
.
+ Mkp
kt
+Pn-X
Growing chainDormant chain
Termination
Intermediate
kside
Side-reactions
( )
Scheme 1.2: Two types of controlled radical polymerization. A) Reversible radical trapping.
B) Reversible transfer.
The second is based on so-called reversible transfer mediated by a suitable
transfer agent. In this synthetic procedure, radicals are continuously transferred
between growing chains, typically via a reactive intermediate of polymer chains
and transfer agent (Scheme 1.2). In this polymerization method, each chain only
grows for a short period of time before the propagating radical is transferred to
another chain. Thus, the dormant species in this case is the adduct between
polymer and a fragment of the transfer agent. In this system, termination may
occur from the propagating polymer radicals similar to what is observed in FRP.
In addition, there are examples of termination through recombination from the

11
reactive intermediate (Scheme 1.2 B) leading to more complex polymer
architectures. 15
Probably the most common and versatile protocols for controlled radical
polymerization are Reversible Addition-Fragmentation Chain Transfer (RAFT)
Polymerization and Atom Transfer Radical Polymerization (ATRP), although
there are various related polymerization methods.8,15
1.2.4 Reversible Addition-Fragmentation Chain Transfer (RAFT)
Polymerization
RAFT polymerization is an example of a reversible transfer process. It was first
reported in 1998 by Rizzardo and co-workers.16
RAFT polymerization is
essentially a free-radical polymerization conducted in the presence of an efficient
chain transfer agent (CTA). The generally accepted mechanism is described as a
free-radical polymerization with two superimposed equilibria, as shown in
Scheme 1.3.17
Conventional free-radical initiators are used to initiate the
polymerization. The growing polymer chain reacts with the chain transfer agent to
give a dormant capped polymer chain in equilibrium with a radical from the chain
transfer agent, R·. This radical may re-initiate the polymerization upon reaction
with monomer to give a second growing polymer chain where one end is capped
with the chain transfer agent fragment. Propagation proceeds as in other radical
polymerization methods but, due to the main equilibrium, the concentration of
dormant species is on the order of a million times greater than the concentration
of active species at any given time.8

12
S S R
Z
S S R
Z
S S
Z
S S
Z
S S
Z
S S
Z
I - I I
.
I
.
+ M I - M
. = P1
.
Initiation
Pm
.
+
Pm . Pm
+ R
.Pre-equilibrium
Pm
.
+ M Pm+1
.
R
.
kp
ki'
R - M
. = P'1
.
+ M
Propagation
Pn
.
+
Pn . Pn
+
Pm
.Pm Pm
Main Equilibrium
Termination Pm
. Pn
.
+
kexchange
kexchange
kt
Pn+m or Pn
H + Pm
=
Re-initiation
ki
kd
Scheme 1.3: Basic RAFT mechanism according to Rizzardo’s group.17
Propagation may occur both from the free-radical initiator and the consumed
CTA. In general, the rate of polymerization in RAFT is dependent on the square
of the initiator concentration, as in conventional free-radical polymerization
(Scheme 1.1).18
The degree of polymerization is given by the ratio of consumed
monomer concentration to the sum of consumed chain transfer agent and
decomposed initiator:8
Equation 1.6
][
][
][][
][ 00
CTA
M
p
IIfCTA
M
pDPn ⋅≈
−∆+
⋅=
Both the contribution from the CTA and the initiator should be included in
determining the degree of polymerization, as both species may initiate
polymerization. Since the CTA/initiator molar ratio is normally 5-10 and the
initiator efficiency is typically less than unity, the approximation indicated in
Equation 1.6 is usually valid. When the contribution from the initiator can be
ignored and the transfer is sufficiently fast, the polydispersity can be given as:8
Equation 1.7 ⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
−+= 1
2
1
pk
k
M
M
exchange
p
n
w

13
From this relation, two points emerge. Firstly, the polydispersity decreases if
radical exchange is fast compared to propagation. In addition, the polydispersity
decreases with increasing conversion, which is characteristic of a living
polymerization (Equation 1.5).
RAFT is generally considered to be the most versatile controlled radical
polymerization mechanism. This is because it is very similar to conventional FRP
and in principle it should be possible to polymerize a wide range of vinyl
monomers.8
In some cases, certain monomers and CTAs may react, in which case
polymerization is unsuccessful. In addition, it is necessary to optimize the
polymerization with respect to the CTA. This may make block copolymerization
for different monomer classes problematic, as different CTAs may be necessary
for the individual blocks. In addition, relatively few CTAs are commercially
available these compounds are often highly colored and malodorous.
Nevertheless, RAFT has been successfully used to polymerize
(meth)acrylates,16,18
(meth)acrylamides,19-21
vinylpyridines,22
styrene and
substituted styrenes,16,23
(meth)acrylic acid,24,25
N-vinylpyrrolidone26
and vinyl
acetate.27
1.2.5 Atom Transfer Radical Polymerization (ATRP)
ATRP was developed independently by Sawamoto28
and Wang and
Matyjaszewski.29
It was named ATRP by Matyjaszewski due to its similarity to
metal-catalyzed atom transfer radical addition (ATRA).29,30
The proposed
mechanism according to Matyjaszewski is shown in Scheme 1.4.31
R - X
Initiation
Pm
.
+ M Pm+1
.kp
Propagation
Pn
.
+Equilibrium
Termination Pm
. Pn
.
+
kt
Pn+m or Pn
H + Pm
=
+ Mtn-Y:Ligand R
. + X-Mtn+1-Y:Ligand
R
. R - M
. = P'1
.
+ M
X-Mtn+1-Y:Ligand
ka
'
kda
'
kda
ka
Pn-Br Mtn-Y:Ligand+
Scheme 1.4: Basic ATRP mechanism according to Matyjaszewski31

14
ATRP is an example of a reversible radical trapping process (Scheme 1.2). The
polymerization formulation consists of monomer, an activated organic halide
initiator, R-X, a transition metal halide, Mtn
-Y, and a suitable ligand. The
transition metal must have two accessible oxidation states separated by one
electron. The ligand serves to complex and hence solubilizes the metal halide.
Complexation may also influence the relative stability of the two oxidation states
of the metal relative to the metal in the absence of the ligand. Thus, complexation
can also influence the redox potential and thereby the position of the ATRP
equilibrium.32-34
The transition metal complex and the organic halide initially react to form a
radical species and the oxidized metal halide complex. Preferentially, this reaction
should be fast and quantitative in order to ensure high initiation efficiency. The
formed radical may propagate (with rate constant kp), terminate (with rate
constant kt) or become deactivated by formation of the non-reactive (dormant)
halide-capped chain on reaction with the oxidized metal halide complex (kda) in a
reversible equilibrium (Scheme 1.4). This ATRP equilibrium is shifted strongly
towards the dormant chain (kda >> ka). Thus the instantaneous polymer radical
concentration is relatively low, which suppresses termination.
If initiation is efficient, the mean degree of polymerization of the chains is given
by the ratio between the monomer concentration and initiator concentration
multiplied by the fractional conversion, p:
Equation 1.8
][
][ 0
XR
M
pDPn
−
⋅=
Thus, the degree of polymerization is not dependent on the concentration of either
the transition metal or the ligand. The rate of polymerization is given by:15
Equation 1.9
]:[
]:[
][][]][[
][
10
LigandYMtX
LigandYMt
XR
k
k
MkPMk
dt
Md
R n
n
da
a
ppp
−−
−
−=⋅=−= +
Integration gives:

15
Equation 1.10 tkt
LigandYMtX
LigandYMt
XR
k
k
k
M
M eff
pn
n
da
a
p ⋅=⋅⎟
⎟
⎠
⎞
⎜
⎜
⎝
⎛
−−
−
−=⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
+
]:[
]:[
][
][
][
ln 10
0
Thus, if the effective rate constant does not change, a semilogarithmic plot of
[M]0/[M] versus time will give a straight line. The polymerization rate constants
and the equilibrium rate constants do not normally change significantly. The ratio
between the reduced and oxidized metal halide complex may decrease due to
termination. The termination reaction between two radicals will increase the
amount of oxidized metal halide complex. Frequently, deviations from linearity
are observed at high conversions, due to enhanced termination under monomer-
starved conditions.35
If initiation is inefficient, the equilibrium between the
reduced and oxidized species adjusts slowly and this may lead to a deviation from
linearity at short polymerization times (low conversions).
In the absence of significant termination and chain transfer, the polydispersity is
given by:15,32
Equation 1.11 ⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
−
⎟
⎟
⎠
⎞
⎜
⎜
⎝
⎛
−−
++= +
1
2
]:[
][1
1 1
0
pLigandYMtXk
RXk
DPM
M
n
da
p
nn
w
The polydispersity decreases if the rate of deactivation is fast relative to the rate
of propagation, or if the concentration of the oxidized metal halide complex is
increased. Both these factors will affect the polymerization rate according to
Equation 1.9. Hence in principle it is possible to get very low polydispersities by
increasing the concentration of the oxidized species, but at the expense of very
long reaction times. On such time-scales, termination cannot be neglected.
ATRP has been used to polymerize a wide range of monomers. The
polymerization rate is dependent on the product of the propagation constant kp
with the ka/kda ratio. Thus, if the radical is deactivated too fast, polymerization
will be very slow. In general, monomers that possess a radical stabilizing group
such as carbonyl or phenyl adjacent to the double bond may undergo ATRP.35
Successful ATRP has been reported for a range of vinylic monomers including
(meth)acrylates,36-38
(meth)acrylamides,39-41
styrene and substituted styrenes,42-44

16
vinylpyridines45-47
and acrylonitrile.48
The choice of transition metal halide,
ligand and initiator may have a large influence on the degree of control over the
polymerization.8,35
The polymerization may be conducted in bulk for liquid
monomers or in the presence of a solvent, which may also have an influence on
the degree of control. Furthermore it is possible to conduct ATRP under
heterogeneous conditions, which also can affect the degree of control of the
polymerization.8
Therefore the polymerization conditions should preferentially be
optimized for each monomer. Our group has found that the polymerization of
various methacrylic monomers is reasonably well-controlled in protic solvents
like methanol at ambient temperature when 2-bromoisobutyric esters are used as
initiators and CuBr:2,2’-bipyridyl is used as the catalyst system.38,43,44,49-56
In
most cases the final polydispersity is below 1.30 and the semi-logarithmic plot is
linear up to high conversion. This makes the preparation of block copolymers
relatively easy, since it is not necessary to isolate the macromonomer. The second
monomer is simply added at a suitable conversion (>95 %).
It should be noted that not all monomers can be used directly. Hydrophobic
monomers tend to phase-separate from methanol as the polymer chain grows. In
such cases increasing the temperature was found to increase the conversion,
although some loss of control may be observed.57
In other cases it is necessary to
isolate and purify the macroinitiator (e.g. if polymerization of acrylamides is
desired).58
1.3 Aggregation of amphiphilic diblock copolymers in selective
solvents.
One of the great advantages of controlled polymerization methods is that it is
possible to prepare well-defined block copolymers. In the bulk, block copolymers
tend to undergo microphase separation due to unfavorable enthalpic interactions
between different chains.59
Macroscopic phase-separation is prevented due to the
covalent bonds between the blocks. Instead, a range of different structures are
formed due to the formation of domains of each block. A large number of
different structures have been identified for AB diblock copolymers, where the
type of structure formed is highly dependent on the volume fraction of each
block.59,60
If the composition of an AB block copolymer is such that a solvent

17
exists which dissolves one of the constituent polymers but not the other, the block
copolymer is said to be amphiphilic. For biologically relevant systems, water is
the only solvent of significance and therefore the blocks are either hydrophilic or
hydrophobic. If such a copolymer is dissolved in water, the hydrophobic blocks
will adopt a conformation that minimizes its contact with water. On the other
hand, the hydrophilic blocks will tend to repel each other and extend into the
aqueous phase.61
p
V
L
A
LA
V
p
⋅
=
1/3 1/2 1
Bicontinuous and
inverted
structures
Vesicles
Membrane/bilayer
Cylindrical
micelles
Crew-cut
micelles
Star-like
micelles
chydrophilichydrophobi
chydrophobi
chydrophobi
mm
m
f
+
=
fhydrophobic ~0.55 ~0.75
A B
C
~0.65
(Israelachvili)
(Discher + Eisenberg)
Figure 1.2: A) Definition of packing parameter p on geometric parameters.62
B) Definition of
hydrophobic mass ratio, fhydrophobic of block copolymer. 63
C) Typical aggregate structures
and their dependence on p62
and fhydrophobic.63
The delicate balance between these two opposing forces determines the block
copolymer morphology in water. Typical morphologies formed at low copolymer
concentrations are shown in Figure 1.2C. Methods of predicting the morphology
from the block copolymer composition has been proposed by Israelachvili62,64
and
also by Discher and Eisenberg.63
The approach by Israelachvili defines a
dimensionless packing parameter, p, as the ratio between the volume occupied by
the hydrophobic chain (V) divided by the optimum interface area of the

18
hydrophilic block (A) and the maximum length of the hydrophobe (L), (see
Figure 1.2A):62
Equation 1.12
LA
V
p
⋅
=
The type of hydrophobic polymer as well as its degree of polymerization will
govern its volume and its maximum length, whereas the type of hydrophilic
polymer and its degree of polymerization will govern its optimum interface area.
Although the exact numbers are not necessarily known, the implications of
Equation 1.12 are that polymers with long hydrophilic blocks relative to the
hydrophobic blocks will tend to form spherical micelles. Changing this ratio in
favor of the hydrophobic blocks should favor structures like cylindrical micelles
or vesicles. This is consistent with the purely empirical rule presented by Disher
and Eisenberg.63
This states that molecules with a hydrophilic mass fraction, f
~0.35 (± 0.10) tends to form vesicles, whereas micelles are formed for f > 0.45
and inverted structures can be expected for f < 0.25 (Figure 1.2).63
Although this
approach is highly simplified it may give an indication of what kind of aggregate
a diblock copolymer may form in a selective solvent and, when designing new
copolymers, what block compositions should be targeted in order to obtain a
desired structure in a selective solvent.65
These predictions are mainly useful for predicting the morphology of aggregates
of amphiphilic AB diblock copolymers but not necessarily for more complicated
copolymer structures. There are indications that similar rules may be applied to
symmetric triblock copolymers of both type ABA and BAB, with B being the
lyophilic block, which behave somewhat like a BA1/2 block copolymer.61,66
One
major difference is that in solutions of the ABA copolymer, the B block may
bridge between individual micelles leading to network formation at relatively low
concentrations.67

19
1.4 Network formation of triblock copolymers in selective
solvents
Initial studies on PMMA-PS-PMMA copolymers were reported by Krause in
1964.68
It was found that in triethylbenzene, which is a good solvent for the
styrene block but a non-solvent for the methyl methacrylate, aggregates were
formed with molecular weights up to two orders of magnitude greater than that of
the individual copolymer chains. This was ascribed to micelle formation, but at
the time the precise structure of these micelles was not elucidated. In 1991 it was
found that micelles of P2VP-PS-P2VP triblocks were formed in toluene, which is
a selective solvent for polystyrene.69
Furthermore, it was observed that these
micelles were comparable in size to those formed by a PS80-P2VP580 diblock.
Theoretical calculations showed that the formation of loops by the central block
was thermodynamically feasible, allowing the formation of intra-micellar links
and creating so-called ‘flower’ micelles as shown in Figure 1.3A. These were
found to be more stable than micellar structures comprising one end-group in a
poorly solvated state. Static and dynamic light scattering experiments as well as
viscosity studies confirmed the formation of such micelles, except for the
P2VP220-PS110-P2VP220 triblock copolymer. Unlike the PS-P2VP diblock, these
triblocks showed evidence of a significant amount of molecularly dissolved
chains in coexistence with micelles at concentrations above the c.m.c.

20
A
B
Low concentration,
molecularly dissolved
Increased concentration,
micelle formation/aggregation
High concentration,
network formation
Figure 1.3: Two pathways to micellar network formation: A) If the end-blocks are highly
incompatible with the solvent, ‘flower micelles’ are formed at relatively low copolymer
concentration. Increasing the copolymer concentration leads eventually to overlap where
bridging is facilitated. B) If the end-blocks are more compatible with the solvent, a looser
structure is formed at intermediate concentrations as the penalty of ‘dangling ends’ is lower.
This eventually leads to a network structure on increasing the copolymer concentration.
ABA block copolymers in a solvent that is selective for the central B block have
been modeled using Monte-Carlo methods, in order to assess the critical micelle
concentration, the critical gelation point and the precipitation point.70
This method
allows for a more thorough investigation of the possible states of the blocks. The
data obtained from this simulation study was the variation of the fraction of
chains in their free, fully solvated state, in their dangling state (with one end
dangling from a micelle) in their looped state (both outer blocks within the same
micelle) and in their bridged inter-micelle state. The parameters varied were the
degree of incompatibility between the solvent and the end blocks and the mean
degree of polymerization of the middle block. The effect of changing these
parameters as a function of the copolymer volume fraction was examined.
Besides showing that dangling bonds and bridges were allowed within the
constraints of the model, it was shown that both the critical aggregation
concentration (c.a.c) and the critical gelation concentration (c.g.c) were both
mainly dependent on the incompatibility of the end blocks, with increased

21
incompatibility leading to lower critical concentrations. The effect of increasing
the length of the middle block only increased the proportion of bridged micelles
slightly and the precipitation concentration was also barely affected. The
formation of micelles can be viewed as competition between minimization of the
interfacial energy caused by having end-blocks in micelles (rather than in
solution) and the entropy loss caused by the forced looping of the middle block.
The gelation process of well-defined flower micelles is depicted in Figure 1.3A
and is described in detail in reference 67. Initially, at the critical aggregation
concentration, flower micelles form. Increasing the copolymer concentration
leads to an increase in flower micelle concentration. When this concentration
reaches a certain value, micelles begin to overlap and bridging is facilitated,
forming a network. This mechanism has been experimentally verified for various
triblock copolymers in selective solvents. For example, Xu et al.71
describes two
triblock copolymers comprising a poly(ethylene glycol) of DP = 795, end-capped
with relatively short perfluorinated alkyl groups (containing 6 and 8 carbons
respectively) characterized by NMR and rheology studies. Triblocks with longer
terminal fluoroalkyl chains formed well-defined micelles with a constant micelle
aggregation number at low copolymer concentration, whereas those with shorter
chains invariably formed larger aggregates. This was explained by reduced
solvation of the terminal groups due to their greater hydrophobicity as the
incompatibility between the end groups and the solvent is large even for a small
increase in the length of the terminal chains. The penalty of solvation thereby
becomes larger and formation of flower micelles is favored. The shorter chains
have a greater tendency to be solvated and less well-defined structures are
favored.
The formation of loose aggregates, rather than discrete micelles, has been
reported by Raspaud et al.,72
who examined a PS200-PI1500-PS200 triblock
copolymer in n-heptane, which is a selective solvent for polyisoprene. Below 0.02
g/mL this copolymer was molecularly dissolved. Above this concentration, light
scattering, neutron scattering and solution viscosity studies did not show evidence
of discrete micelles, but rather of non-uniform inter-connected structures.
Theoretical predictions were made based on the entropic gain of the middle block
on removing one end-block from the micelle compared to the increase in

22
interfacial energy arising from poor solvation. This led to the following condition
for the instability of flower micelles:
Equation 1.13
)3/(2
...
1
β
ϕ ⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
≥
cac
B
p
N
where NB is the number of statistical segments of the middle block, p is the
aggregation number, φc.a.c is the critical aggregation concentration and β is the
‘looping coefficient’ for the solvated chain, which varies between ~0.4- ~1.3. A
high β indicates a high entropic penalty for looping.72
NB is proportional to the
degree of polymerization of the middle block and the critical aggregation
concentration is dependent on the interaction between the solvent and the terminal
blocks. Although Equation 1.13 is based on several approximations, the
qualitative results are intuitive; a large soluble chain experiences a larger loss in
entropy on looping and this decreases the probability of looping. A higher
aggregation number increases the probability that both chain ends are present in
the same aggregate. A low critical aggregation concentration indicates high
incompatibility between solvent and A blocks, which lowers the probability of
dangling bonds and formation of bridges.
Comparison of these results with those of the Monte-Carlo simulation70
shows a
similar trend with respect to the formation of flower micelles compared to larger
aggregates or micellar gel networks: The fraction of dangling chains decreases on
increasing the solvent incompatibility. The aggregation number decreases on
increasing the size of the central block, while the proportion of loops is reduced
on increasing the block length of the central block. However, this effect was
larger for an increase in the degree of polymerization from 10 to 20 than for an
increase in DP from 20 to 40.
The design of efficient gelators requires copolymers with a relatively limited
tendency to form flower micelles as bridging is desired. This can be achieved
using terminal blocks that have relatively high compatibility with the liquid phase
or by having a central block that is less prone to back-folding. High compatibility
with the liquid phase can be facilitated by the ABA copolymer having short A
blocks or by preparing these from relatively compatible components, i.e. less

23
lyophobic polymers. On the other hand, if the end-blocks are too lyophilic, there
may be a high fraction of dangling bonds and the aggregation number may be
low. This will affect the mechanical properties of the network, as the dangling
bonds will not be elastically active.67,73
The tendency of the central block to back-
fold decreases as its block length increases, as discussed above. However, it may
be synthetically demanding to prepare very long blocks. Another possibility may
be to suppress back-folding by using either relatively rigid polymers or to use
polyelectrolytes (to take advantage of electrostatic repulsion between chains. It
has been shown that copolymers with a central polyelectrolyte block are very
efficient gelators.74,75
1.5 Gel structure of amphiphilic block copolymers
The gel structure may to some degree reflect the structure of the aggregates and
how these aggregates pack at sufficiently high concentration. Thus, for AB
diblock copolymers, cubic (spherical micelles), hexagonal (cylindrical micelles)
and lamellar phases (vesicles/membranes) are commonly observed.59
Similar
observations have been reported for triblock copolymers, but the relationship
between block composition and the morphological and rheological properties of
the gel phase is less well understood.59,76
The structure of the gel phase largely
governs the moduli and thereby the gel ‘strength’.59,77,78
A useful rule of thumb is
that the storage modulus obtained by shearing at low frequency and low
amplitude is indicative of the type of packing. A modulus of the order of 106
Pa
indicates a cubic phase, a modulus of around 104
Pa suggests a hexagonal phase
and a modulus around 102
Pa is indicative of a lamellar phase. Below this value, a
micellar solution is expected.79
Random gels, without a well-defined structure
may also exist, especially in the case of ABA triblock copolymers where micelles
may bridge to form a network.76,80
The moduli of these networks are normally
relatively low. Gels presented in this work generally have moduli of 101
-102
Pa
and no evidence of long-range order has been found indicating random gels.

24
1.6 Preparation of thiol-functional polymers
1.6.1 Why thiols?
Disulfide-thiol redox chemistry is commonly found in biochemistry.81-84
From a
synthetic point of view, the thiol group is attractive due its relative ease of
preparation and its ability to react with a variety of functional groups under mild
conditions in high yields. Thus, a macromolecule such as a peptide or a synthetic
polymer containing one or more thiol groups can be derivatised with
functionalities such as labels for tracking purposes,85-95
specific groups for
interaction with cell components96-100
or other macromolecules.96,98,101-114
Table 1.1 illustrates several synthetic routes utilized to prepare thiols from various
substrates.

25
Substrate Reaction Reagent
Disulfide
R S RS R SH
i
Reductants
Multiple
bond
R
R
SH
R
S
Rii
+ Free-radical initiators or
acid/base catalysis
Alcohol
R OH R SH
iii
Miscellaneous reagents
H2S or NaSH
Alkyl
halide
R X
R SH
R S Y
R S R
R S
iv
+
vi, vii
v
Miscellaneous reagents
Hydrolysis with acid
catalysis
Thiol
ester
R'
Y
S R
R'
Y
OH
SHR
R'
Y
O
SR
+vi
vii
+
Y=O,S
Hydrolysis with base
catalysis
Table 1.1: Common synthetic routes to aliphatic thiols. Typical conditions: (i) Zinc in dilute
acid,115,116
sodium boronhydride, NaBH4 in ethanol,117
triphenylphosphine and water in
methanol and dimethoxyethane,118
dithiothreitol, DTT, in various solvents119-122
or
trialkylphosphines and water in various solvents.123-126
In aqueous solutions, tris(2-
carboxyethyl)phosphine, TCEP, is frequently used due to its solubility and high efficiency.127
(ii) The addition of hydrogen sulfide to double bonds is efficient in the presence of free-
radical initiators. The reaction can also be catalyzed by proton or Lewis acids but only
nucleophilic substrates undergo base-catalyzed addition. Since the resulting thiol is capable
of adding to a second double bond, sulfides are often by-products.128
(iii) Various reagents
have been employed for this reaction.129
(iv) Alkyl halides can be reacted directly with
hydrogen sulfide or sodium hydrogen sulfide although sulfides are often by-products.130
(v)
Indirect methods include reaction of the alkyl halide with either thiourea or thiosulfate
followed by hydrolysis of the thiol esters or dithioesters under (vi) acidic or (viii) basic
conditions.131
1.6.2 Thiols from disulfides
Disulfides are easily reduced to thiols using various mild reductants. Although
thiol-containing amino acids or peptides such as cysteine or glutathione can be

26
used, their efficiency is relatively low due to the similarity of reduction potentials
of the respective disulfides. Therefore it is necessary to use a relatively large
excess of the reductant.132
However, cheaper or more efficient reactants are
normally used for synthetic purposes.115-127,133
The broad range of reduction
agents allows one to be selected that minimizes the purification. For
macromolecules, it is beneficial to choose conditions whereby residual reduction
agent and small-molecule by-products can easily be removed. In choosing these
conditions, the facile oxidation of thiols, especially in their basic thiolate form
should be considered.134
For example, macromolecules that are soluble in dilute
aqueous acid can be reduced using zinc, followed by dialysis. Water-soluble
macromolecules can also be reduced using tris(2-carboxyethyl)phosphine (TCEP)
or dithiothreitol (DTT), followed by acidification and/or dialysis or precipitation.
Macromolecules that are soluble in ethanol or methanol can be reduced using
NaBH4 or DTT, followed by acidification and/or precipitation or dialysis. If the
macromolecule is soluble in common organic solvents, then DTT or phosphines
like triphenylphosphine, triethylphosphine or tributylphosphine can be used. With
phosphines, it is necessary to add one equivalent of water per disulfide to avoid
sulfide formation.135
Thus, the optimal choice of reductant depends on the
macromolecule in question.
1.6.3 Thiols from double bonds
Addition of hydrogen sulfide to double bonds to give thiols may also lead to
formation of sulfides, since the formed thiol can also react under the same
conditions. Hence this reaction is less useful for the preparation of thiols. The
addition of thiols to double bonds to form sulfides has been widely used in
polymer chemistry for many years and has found new uses recently (see
later).136,137
1.6.4 Thiols from alcohols
The direct conversion of alcohols to thiols has been reported using a number of
reagents,129
with the Lawesson reagent (Figure 1.4) possibly being the most
efficient.138,139

27
O
CH3
P
S
S P
S
O
CH3
S
Figure 1.4: The Lawesson reagent, 4-Methoxyphenylthiophosphoric cyclic di(thioanhydride)
Although this transformation has found relatively wide use in organic chemistry,
there appears to be no examples where it has been applied to polymers, although
there is one example where reducing sugars have been converted into their
corresponding thiols using the Lawesson reagent in good yields.139
Instead, a
multi-step approach has typically been used for the conversion. For example, a
three-step conversion of the terminal alcohol of a PEO-based (co)polymer to the
corresponding thiols was achieved by tosylation of the hydroxy groups, followed
by addition of potassium thioacetate, and finally hydrolysis of the thioester.140,141
This is actually a combination of several of the approaches summarized in Table
1.1. Since PEO-based copolymers are at least as stable as reducing sugars, it is
seems plausible that conditions can be found where the terminal hydroxy groups
of the former can be converted to thiols in a single step.
1.6.5 Thiols from alkyl halides
The direct reaction between alkyl halides and hydrogen sulfide or alkali hydrogen
sulfides suffers from the same drawbacks as the equivalent addition to double
bonds, described above; the formed thiol may react with another halide to form
sulfides. Therefore this transformation is normally avoided. Instead, indirect
methods are normally used. For example, the formation of thiols from alkyl
halides has recently found use in converting the end-groups of polymers prepared
by ATRP into thiols using an indirect method based on reaction of the halide with
N,N-dimethylthioformamide, followed by methanolysis.120
The conversion of the
polymer was kept at 50 % in order to avoid de-bromination of chain ends due to
termination; it was found that the majority of the end-groups had become
thiolated.

28
1.6.6 Hydrolysis of thiol esters and related compounds
Hydrolysis of thiocarbonyls to afford thiols is commonly used to convert the
thiocarbonylthio end-groups of RAFT-synthesized polymers into thiols.142-145
This transformation has been conducted in various solvents using aliphatic
primary or secondary amines144,145
or aqueous sodium hydroxide.143
1.6.7 Thiolated macromolecules
Most reported examples of thiol-functional macromolecules have been obtained
by reduction of disulfide groups or by hydrolysis of thiocarbonylthio residues as
described above. There are several obvious reasons for this. First, the disulfide
group is common in biological macromolecules. In addition, the disulfide bond is
relatively inert during ATRP and RAFT polymerizations, allowing for disulfide-
containing monomers or initiators that can be cleaved after polymerization and
purification.120,123
Polymers prepared using RAFT polymerization are end-
functionalized with thiocarbonylthio moieties originating from the RAFT agent.16
This group is normally hydrolytically unstable and colored, which is why its
removal is normally desired for several practical purposes. If a thiol functionality
is desired, hydrolysis or aminolysis is an obvious choice, since this reaction
proceeds in high yield (see section 1.7.4). Nevertheless, as the examples in Table
1.1 show, it is possible to transform a number of functional groups on polymers
into thiols using well-known organic chemistry. It should be noted that these
examples are only representative and merely serves to illustrate that a large
variety of functional groups can be converted into thiols.146
1.7 Reactions of thiol-functional polymers
1.7.1 Direct oxidation of thiols, formation of symmetrical disulfides
Table 1.2 shows examples of common reactions of macromolecular thiols. The
reduction potential of the disulfide group is on the order of -0.2 V to -0.3 V132
for
most aliphatic disulfides.147
The relative ease of oxidation allows the reaction to
be carried out using a range of common oxidation agents.148
Aerial oxygen
oxidizes thiols to disulfides, although the reaction is relatively slow in the absence

29
of suitable catalysts such as transition metal salts.148,149
Other oxidants such as
iodine101
and dimethyl sulfoxide, DMSO141,150
have been used and the reaction
can be controlled electrochemically.151
However, further oxidation of the disulfide
may occur with certain oxidants when used in excess.148
The direct oxidation of thiols to disulfides has been exploited to prepare oxygen-
sensitive gelators.140,152
Three thiol-terminated poly(ethylene oxide-b-propylene
oxide-b-ethylene oxide), PEO-PPO-PEO copolymers of increasing length but
with the same weight fractions of PEO and PPO were prepared.140
Aerial
oxidation led to disulfide formation of the terminal thiol groups and the resulting
copolymers had significantly higher molecular weights and polydispersities.
These end-functionalised copolymers behaved differently to the native PEO-PPO-
PEO triblock copolymers because of their ability to form inter-micellar bridges.
This increases the size of aggregates and significantly extends the hydrogel
duration from hours to days. In addition, the release of a hydrophobic model drug,
paclitaxel, was highly dependent on the glutathione concentration, demonstrating
that cleavage of thiol groups led to release of encapsulated drugs. A similar
approach was used to prepare poly(ethylene oxide) (homo)polymers with central
disulfide bonds by oxidising thiol-terminated PEO precursors using DMSO.141
These polymers were found to be non-cytotoxic and were cleavable using
glutathione

30
Reaction Type Reaction Reagent / Reference
Symmetric disulfide formation R SH
R
S
R
S2 Oxidant
Example B r
O
O S H
n
B r
O
O S
n
B r
O
OS
n
FeCl3
D M F 120
Asymmetric disulfide formation
R
S
R'
SR' S
Y
R SH + Oxidant
Examples
S
S S
30
S
O
O
O16
S
S S
30
S
O
O
O16
+
CH2Cl2/H2O
I2
153
N S
S O
O
n
OO
OH
SHBSA S S pHEMABSA+
103
Hydro-alkylthio-addition
(Thiol-ene coupling)
R SH
R' R'SR
+ Free-radical initiator
Example
NN
N OO
O
SH
OH OH
Ph Ph
MeO OMe
O
S
OH OH
S
OH OH
S
OHOH
NN
N OO
O
hν
Dendrimer 154
Hydro-alkylthio-addition
(Michael-type addition)
E ESR
R SH +
Where E is an electron-withdrawing group
Base
Example
N
O
O
-
- -
-
-
-
SH
-
- -
-
-
-
N
O
O
S
+
Phosphatidylcholine vesicle
Thiolated polyelectrolyte
107
Alkylthio-de-halogenation (Halogen substitution) R SH
X
R'
S
R'
R
+ Base
Example OH OH
n
N
O
O
O
Cl
OH OH
m
N
O
O
O
OH OH
N
O
O
O
Cl
6
+ Ac-HTSTYWWLDGAPC-Am
Thiolated peptide
6 6
n-m
pH 8
Ac-HTSTYWWLDGAPC-Am
m
155
Table 1.2: Common reactions of thiols applied to macromolecules and/or
biomacromolecules.
Aliyar et al. describes the synthesis of polyacrylamide hydrogels crosslinked with
N,N’-bisacryloylcystamine.152
Reduction of such hydrogels with DTT led to
soluble polyacrylamides with multiple pendant thiol groups. These soluble
polymers formed polydisperse nanoparticles (nanogels), when air was passed
through dilute solutions, due to disulfide formation. The same polymer was used
at higher concentration as a replacement lens material in porcine eyes, as it was
found that the mechanical and optical properties of the natural lens could be
closely mimicked.156
In this case, disulfide formation was facilitated by a thiol

31
exchange reaction after filling the lens capsular bag to increase the rate, rather
than waiting for the relatively slow aerial oxidation to occur.
1.7.2 Formation of asymmetric disulfides
The direct formation of asymmetric disulfides by oxidation is normally not very
efficient, since the symmetric disulfides are generally more stable. Nevertheless,
this approach was used to prepare diblock copolymers of poly(propylene sulfide),
PPS, and PEO, with a disulfide bond placed between these two blocks.153
In order
for this approach to succeed, it was necessary to use excess thiolated PEO and to
remove the symmetric PEO-based disulfide by-product after reaction. These PPS-
PEO copolymers formed vesicles in aqueous solution and it was demonstrated
that reduction of the disulfide bond led to rupture of the vesicles. Under these
conditions, the calcein payload was released. Further, it was demonstrated that
these vesicles were internalized into cells and that the reductive cytosolic
environment caused the payload to be released in situ. In many cases it is not
convenient to use an excess of one thiol to prepare asymmetric disulfides; for
example, the symmetric disulfide by-product may not be easy to remove from the
desired product or both thiols may be expensive or only available in small
amounts. In such cases it is necessary to use another approach. The direct
formation of asymmetric disulfides has been mediated using diethyl
azodicarboxylate (DEAD), or related compounds. This reagent rapidly reacts with
thiols to give a reactive intermediate, which couples with a different thiol to give
the desired asymmetric disulfide in good yield.148
Although the reagent is tolerant
towards a number of functionalities, it reacts with alcohols and carboxylic acids,
which precludes its use for coupling peptides or hydroxy-containing reactants. In
addition, it is necessary to use aprotic solvents for the reaction. Thus, for most
biologically interesting molecules it is necessary to use an indirect method.102-
104,148
Normally a two-step synthesis is used, where the first step involves
formation of a crossed aromatic-aliphatic disulfide. This crossed disulfide is an
aliphatic thiol that is efficiently activated towards the preferential reaction with
another aliphatic thiol at relatively low pH (pH 3.5-6.5). The reaction is driven by
the lower pKa of the aromatic thiol product, rendering this species more stable in

32
solution than the aliphatic thiol under weakly acidic conditions.102
This method
has been used to join two different heme-coordinating peptides in excellent yield
to give an asymmetric dipeptide.102
The formation of a disulfide bridge using an
excess of activated thiolated hexahistidine and thiolated oligonucleotides was
found to be quantitative using this procedure.104
The crossed aromatic-aliphatic
disulfide that constitutes the activated thiol is stable under various conditions
when no free thiol is present. This approach was exploited to prepare an ATRP
initiator that was used for preparing well-defined poly(2-hydroxyethyl
methacrylate) polymers in deuterated methanol.103
The polymerization was found
to be well-controlled, giving polymers with narrow polydispersities, although the
initiator efficiency was relatively low. After purification, these polymers could be
coupled efficiently to bovine serum albumin (BSA) by incubating at room
temperature for 30 minutes at pH 8.0. Hence in this case thiol activation also
functions as a protecting group, depending on the environment.
1.7.3 Free-radical mediated coupling of thiols to double bonds
Recently, the well-known addition reaction of thiols to double bonds has gained
renewed interest as an attractive so-called ‘click’ process.137,157
This thiol-ene
coupling can proceed under a wide range of conditions as shown in Table 1.2.
One approach applicable to most double-bond containing substrates is a free-
radical mediated process. In general, yields for such reactions are high. In
addition, the reaction is orthogonal to a broad range of functional groups and may
proceed in water in the presence of oxygen. These are some of the important
properties that an ideal ‘click’ reaction should possess.158
Furthermore, the free
radicals necessary for the reaction to proceed can be generated either thermally or
photochemically. Thus, if the reactants contain light-sensitive groups, the reaction
can be conducted using a thermal free-radical initiator. On the other hand, if there
are thermally unstable functional groups it may be possible to conduct the
reaction at low temperature using a photoinitiator. Perhaps one of the most
thorough investigations of the applicability of the thiol-ene coupling to polymers
was recently published by Hawker’s group.157
Here various polymers bearing
alkene side-groups and alkene end-groups were synthesized. The copolymers with

33
alkene side-groups were based on polystyrene, poly(methyl methacrylate) and
poly(ε-caprolactone) (PCL) and in all cases there were 10-20 mol % alkene side-
groups. In all cases the polydispersities were relatively narrow, as the copolymers
were prepared using living or controlled polymerization methods. Polymers with
terminal alkene groups included near-monodisperse PS, PMMA and PEO. These
alkene groups were reacted with five different thiol-containing compounds using
either a photoinitiator or a thermal initiator. The photochemical reactions were
rapid and essentially quantitative with most of the thiols, whereas the thermal
reactions were slower and less quantitative. The orthogonality between the thiol-
ene ‘click’ reaction and the reaction most frequently used for ‘click’ chemistry,
the copper-catalyzed azide/alkyne cycloaddition was also investigated by
preparing PS with an azide-group at one end and an alkene in the other end.
Reaction of these functional groups in turn with a thiol and an alkyne gave 100 %
conversion no matter which reaction was performed first. Hawker’s group has
also used the high efficiency of the thiol-ene reaction to prepare dendrimers as
shown in Table 1.2.154
These were prepared using 2,4,6-triallyloxy-1,3,5-triazine
as the starting material and reacting this with 1-thioglycerol under UV irradiation,
using a photoinitiator. The resulting intermediate was reacted with 4-pentenoic
anhydride to give ene-functional first generation dendrimers. Both reactions were
highly efficient and the fourth-generation dendrimer was obtained with very few
defects, as confirmed by mass spectroscopy and NMR. Using this procedure, the
dendrimer chain ends could easily be functionalized since many functional thiols
are commercially available.
Schlaad’s group has reported on the reaction between 1,2-polybutadiene, PBD,
and various thiols in a series of publications (Scheme 1.5).114,159,160
In general, it
was found that the double bonds were consumed, suggesting high conversion.
However 1
H NMR indicated that the degrees of functionalization were less than
quantitative. This could be explained by the mechanism shown in Scheme 1.5;
after formation through thermally or photoinduced free-radical generation, the
thiol radical adds to a double bond to give a carbon-radical (Scheme 1.5A).

34
*
*
R SH
I *
*
C
S
R
R SH
R S
*
*
S
R
H
*
*
C
S
R
*
*
S
R
R SH
R S
*
S
R
S
R
*
x y z
+
+
-
+
-1,2-polybutadiene
Resulting 1,2-polybutadiene thiol adduct
A
B
C
D
Scheme 1.5: Possible reactions of thiol radicals and structure of the addition product
according to reference 114
This radical can abstract a hydrogen from another thiol, leading to formation of
the direct thiol-ene adduct and a new thiol radical (Scheme 1.5B). Another
reaction pathway goes through the vicinal double bond to form a six-membered
cyclic ring, followed by hydrogen abstraction (Scheme 1.5C). The final product
becomes a mixture of the direct thiol-ene adduct which only involves one double-
bond and the cyclic adduct, which involves two double bonds (Scheme 1.5).
Depending on the conversion there may be residual pendant double bonds. In
general, the conversion was found to be high, only leaving a few pendant double
bonds. The ratio between direct adducts and cyclic adduct depended on the type
of thiol used. The reaction of the polybutadiene units of a PBD85-PS351 block
copolymer with a thiolated sugar, 2,3,4,6-tetra-O-acetyl-β-D-1-thioglucopyranose
was examined using AIBN and irradiation.160
This approach gave high
conversions with an approximate 1:1 molar ratio between cyclic and non-cyclic
thioether groups. After deacetylation of the glucopyranose, the copolymer formed
vesicles as determined by light scattering and TEM when the solvent was
switched from THF to water. Related work involved block copolymers based on
polybutadiene and poly(ethylene oxide), with PEO-rich compositions.114
The
PBD block was derivatised with either an acetylated L-cysteine or a dipeptide,
(L,L)-cysteine-phenylalanine. Both the original block copolymers formed
spherical micelles in aqueous solutions and this morphology did not change on
derivatizing the PBD with the cysteine derivatives, although the diameter
decreased by 10-30 %. This decrease was explained by the relatively higher
hydrophilic nature of the cysteine derivatised polymers relative to the starting
polybutadiene. On the other hand, the addition of the dipeptide led to the
formation of giant worm-like micelles and giant vesicles. Close examination of

35
the giant worm-like micelles by fluorescence microscopy revealed that they had
helical superstructure, which was confirmed by circular dichroism spectroscopy.
This structure originated from the hydrogen-bonding and π-π interactions imposed
by the dipeptide.
The free-radical mediated thiol-ene reaction was also used to attach weak
electrolytes, zwitterions and permanently hydrophilic groups to PBD
homopolymer.159
The degree of functionalization was 0.5-0.9 and the ratio
between cyclic and direct adducts varied depending on the thiol. All the
copolymers were found to form vesicles in aqueous solutions if the attached
group was in its water-soluble form. The permanently hydrophilic polymer
formed uni-lamellar vesicles, whereas the other polymers formed multilamellar
vesicles. Thus, these systems are polymeric amphiphiles, where the hydrophobic
part consists of the polymer backbone, whereas the hydrophilic block consists of
the attached pendant groups.
1.7.4 Michael-type addition of thiols to electron-deficient double-bonds
A variant of the thiol-ene reaction is the reaction between thiols and electron-
deficient double bonds (Table 1.1) in the presence of base. This reaction is similar
to the well-known Michael addition and is commonly designated a Michael-type
reaction.161
It forms the basis of several thiol-labeling reagents where a thiol is
reacted with a fluorescent dye that is functionalized with a maleimide group.85,162-
164
If the maleimide is attached directly to the aromatic system it will act as an
intramolecular fluorescence quencher. As a thiol reacts with the double bond,
conjugation of the quencher is broken and the fluorescence increases to a value
that is close to that of the fluorescent dye without the maleimide group.162,163
The
fluorescence intensity can then be correlated with the thiol content by the creation
of an appropriate calibration curve. Hence the efficiency of this reaction is
generally close to 100 %. In addition, appropriate electron-deficient alkenes
include a wide range of acrylate esters, which are relatively easily prepared or
commercially available. Furthermore, the reaction can proceed in water under
near-neutral conditions and it is relatively tolerant towards the presence of
oxygen.

36
End-functionalization of polymers prepared by RAFT polymerization with
acrylates is of particular interest, since it is possible to remove the
thiocarbonylthio end group and so functionalize the thiol in a one-pot
reaction.144,145
This was demonstrated by Winnik’s group, who prepared a poly(N-
isopropylacrylamide) polymer with a isobutylsulfanylthiocarbonylsulfanyl moiety
at each end.144
Aminolysis of this macro-chain transfer agent with excess 1-
aminobutane in the presence of a small amount of TCEP gave the thiol. Addition
of excess acrylate gave the end-functionalized polymer, since this reaction is
catalyzed by excess 1-aminobutane. 1
H NMR and thiol analysis indicated > 99 %
efficiency for this reaction. A similar procedure was recently applied by Chan et
al.145
to prepare PNIPAM three-arm star polymers (Scheme 1.6): The linear
PNIPAM polymers were prepared by RAFT. Then the terminal dithioester was
cleaved by aminolysis using 1-aminohexane. This reaction was conducted in the
presence of dimethylphenylphosphine since this reagent was found to efficiently
catalyze the Michael-type reaction. To this reaction mixture was added
trimethylolpropane triacrylate (thiol:ene molar ratio = 1.5:1) and the three-arm
star was formed in high yield within 5 minutes, as determined by a range of
analysis methods. It is noteworthy that both 1-aminohexane and
dimethylphenylphosphine are nucleophiles and therefore in principle can
participate in Michael-type reactions.165,166
Presumable the reaction is successful
because the thiol reacts significantly faster than both the phosphine and the amine.
S
NC
S
Ph
ON
n
O N
nNC S
O N
n
NC S
O
O
O
O
OO
S
ON
n
CN
O
O
O
O
OO
1) C6H13NH2
Me2PPh3
2)
Trimethylolpropane triacrylate
PNIPAM-CTA 3-arm PNIPAM star
Scheme 1.6: Preparation of 3-arm PNIPAM star copolymer according to reference 145.

37
The Michael-type addition reaction is also an attractive pathway to functionalize
biologically-active molecules due to its relatively high tolerance towards a
number of functional groups and the mild reaction conditions that are required.
Thus thiolated heparin was attached to the termini of thermoresponsive acrylate-
terminated poly(lactic-co-glycolic acid)-poly(ethylene oxide)-poly(lactic-co-
glycolic acid), PLGA-PEO-PLGA, gelators.96
Hydrogels based on this material
slowly released heparin, primarily due to the hydrolytic degradation of the PLGA
blocks.
The sulfone-based PEGylation reagent described by Brocchini’s group is capable
of reacting with two thiol groups (Scheme 1.7).167
This allows for PEGylation of
cysteine residues without leading to denaturation due to destruction of the tertiary
structure originally stabilized by the disulfide bond(s). Some decrease in
bioactivity was found for the functionalized protein but this was attributed to the
steric shielding caused by the PEG chains.
S
R''O
O
O
R' PEG
S
R''O
O
O
R' PEG
S
SS
S
O
R' PEG
S
S S O
R' PEGS
pH 7.8
4 °C
+
Michael-type addition Sulfinic acid elimination Michael-type addition Bridged disulfide
H+
- R''SO2
-
Scheme 1.7: Mechanism for the PEGylation of protein thiols described by Brocchini and co-
workers.167
Poly(2-ethylacrylic acid) was attached to the surface of a maleimide
functionalized phosphatidylcholine vesicle (Table 1.2).107
In its acidic form, the
polyacid binds strongly to the phosphatidylcholine membrane, thus changing its
the permeability. Linking the polyacid covalently to the surface led to vesicles
that released a payload of calcein rapidly on lowering the pH from 7.0 to 6.5.
The formation of hydrogels has been facilitated by crosslinking using Michael-
type addition chemistry.106,108-113,168,169
Thus, thiol-mediated Michael-type
crosslinking of PNIPAM-based thermoresponsive hydrogels were reported by
Vernon’s group.111,168
Typically, NIPAM was copolymerized with a comonomer

38
that could be converted into either an acrylate168
or a thiol.111
These copolymers
gelled above the LCST of PNIPAM. Reaction with multifunctional thiols or
acrylates, depending on the copolymer, led to temperature-responsive covalently-
crosslinked networks that had improved physical properties relative to the purely
physical networks where the crosslinking was based on the LCST of PNIPAM
alone.111,168
Hubbell’s group reported the formation of hydrogels by the reaction
of acrylate or vinyl-sulfone with end-functionalized poly(alkylene oxide)
(co)polymers with multifunctional thiols.109,112,113
The network formation and
degradation of 4- and 8-arm PEO stars with bifunctional thiols was modeled and
compared to experimental data.109
It was found that the cross-linking density was
lower than anticipated due to the formation of structural imperfections
(intramolecular cycles) unless the precursor concentration and the ene-
functionality were sufficiently high. Degradation of these networks occurs
through hydrolysis of the thioether-ester links under physiological conditions. The
developed model was also successful in predicting the gel degradation kinetics.
Vinylsulfone-terminated PEGs were crosslinked with the cysteine groups of
proteins that had been engineered to possess a high level of cysteine groups and
water-solubility, as well as various biological functionalities that may have
implications for cell adhesion or wound-healing.112
Gel formation was found to
occur in less than an hour at physiological pH and 23 °C and the mechanical
properties of the final gels could be controlled by varying the stoichiometry
between the reagents as well as the precursor concentration. Therefore, mixtures
of these two components may find applications as injectable hydrogels.
Thermogelling 4-arm (PEO-PPO)4 star copolymers were used as the starting
material to prepare a synthetic alternative to calcium alginate for cell
encapsulation (Figure 1.5).113
The functionalized ends of these Tetronics® were
converted to either thiols or acrylates and the system was optimized to mimic that
of alginate with respect to viscosity by varying the degree of reaction between
thiolated and acrylated Tetronics®. These gels could be engineered to have
diffusion profiles that compared favorably to those found for calcium alginate. In
addition, the gels broke down over a few days due to hydrolysis of the thioether-
ester bond.

39
2) AIBN, Toluene
60-65 °C 16 h
O
O
20
H
60
O
O
20
H
60
N N
O
O
20
H
60
O
O20
H
60
N
N
O
O
O
20
60
O
O
O
20
60
O
O
20
60
O
O
O20
60
O
O
O
20
60
SH
N N
O
O
20
60
SH
O
O
20
60
SH
O
O
20
60
SH
Cl
O
Et3N, Toluene
0-20 °C, 12 h
Br
1) NaH, Toluene
0-20 °C 12 h
O
SH
3) NaOH, H2O
2-3 °C 1.5 h
+
Low pH, 30 w/v %
> 21 °C
HS
HS
SH SH
SH
SH
HS
HS
SH SH
SH
SH
pH increase
Tetronic T1107
Acrylated T1107
Thiolated T1107
Physical micellar gelChemically crosslinked gel
pH ~7
O
SO
Figure 1.5: Schematic of the Tetronics® T1107-based gels described by Cellesi et al.113
The
system was optimized to give gels with alginate-mimetic viscosity in one step from the
acrylated T1107 and a protected form of the thiolated T1107. The gels degraded due to
hydrolysis of the acrylate ester over several days, this is not shown.
Feijen’s group has investigated the crosslinking of dextran-based hydrogels.106,169
Two strategies were pursued: Firstly, the dextran was thiolated and reacted with
either acrylated Tetronics® or vinylsulfonated dextran.106
Secondly, a
vinylsulfonated dextran was reacted with a bis-thiolated PEO to form a gel.169
In
all cases, the mechanical properties of the final hydrogel could be adjusted by the
density of reactive groups on the dextran, its molecular weight, type of crosslinker
and concentration of reactants. Furthermore, the gels were found to be degradable
due to ester hydrolysis on a time-scale of weeks. In general, gels based on
vinylsulfone-containing dextran degraded faster than those based on acrylate-
based Tetronics®. It was found that if the length of the spacer between the

40
thioether and the ester group was increased, the degradation time could be
extended.169
1.7.5 Reaction between thiols and alkyl halides
The reaction between thiols and α-iodoacetamides is the basis of a range of
commercially available thiol-labeling fluorescent dyes.85,164
These are highly
reactive and may react with amines or even phenols in the absence of available
thiols. In addition, the iodine makes them highly light-sensitive. Similar
chemistries have been used to convert pendant amino or hydroxy-groups on
polymers into thiol-reactive groups through formation of the α-haloester or α-
haloamide.155,170,171
Both formation of the α-halocarboxyl-derivatised polymer
and the subsequent thiol substitution were quantitative, provided that a
sufficiently strong base was used to deprotonate the thiols.171
Thus it was
demonstrated that polymers could be derivatised with peptides155
as well as a
variety of other biologically-relevant thiolated molecules such as various
carbohydrate- and biotin derivatives.170,171
1.7.6 Reactions of thiols and disulfides with metal surfaces
The selective adsorption of thiols and disulfides onto metal surfaces is a
commonly used procedure to form monolayers.172
These monolayers may protect
the underlying substrate from the environment and can introduce convenient
chemistry on the surface. The high specificity of thiols with a large range of metal
surfaces makes for facile surface derivatization. In addition, disulfides tend to
form monolayers with similar structures to those formed by the corresponding
thiols.172
This may be convenient since disulfides are oxidatively stable compared
to thiols. In some cases, the disulfides are less soluble than the thiols, which is
why the latter may be preferred.
1.8 Phospholipids and phosphorylcholine-based polymers
Phospholipids are a component of essentially all biological membranes.173,174
These consist of a lipophilic tail and a hydrophilic head, which can be anionic or

41
zwitterionic.174
If anionic phospholipids are exposed to blood, they will lead to
clot formation, i.e. they are thrombogenic.175
On the other hand, zwitterionic
phospholipids such as phosphorylcholines, are non-thrombogenic and surfaces
coated with the latter suppress clot formation when in contact with blood.176,177
1.8.1 2-(methacryloyloxy)ethyl phosphorylcholine, MPC
The MPC monomer was originally prepared by Kadoma et al. who also used it to
prepare copolymers with methyl methacrylate.178
These materials were found to
be highly soluble in water but to exhibit haemolytic activity, i.e. red blood cells
were destroyed when exposed to the polymer. This property was later attributed to
the low purity of the MPC monomer. Indeed, when an improved synthetic method
for the monomer was developed,179
non-haemolytic copolymers in specific and
anti-fouling copolymers in general could be prepared.180,181
The anti-fouling properties and uses of (co)polymers prepared using
phosphorylcholine-based monomers such as 2-(methacryloyloxy)ethyl
phosphorylcholine (MPC) has been described in several reviews.180,181
Most work
has focused on MPC-based statistical copolymers. Various comonomers allow the
mechanical properties to be tuned or have functionalities that allow further
reactions such as cross-linking. PMPC-based copolymers are typically
amphiphilic and bear a close structural resemblance to naturally-occurring
phospholipids.180
Studies show that dipalmitoylphosphatidylcholine (DPPC), a
phospholipid that is found in human plasma, is preferentially adsorbed onto
surfaces containing a significant fraction of PMPC.180
This leads to formation of
an organized bilayer of plasma-lipid on the surface, which in turn is responsible
for the suppression of protein adhesion. However, since surfaces containing
PMPC also suppress protein adsorption in the absence of phospholipids,180
the
formation of the bilayer cannot be the sole mechanism for the suppression.
Besides being able to form a lipid bilayer, PMPC-containing surfaces are highly
hydrated. The amount of ‘free water’ in PMPC-containing copolymers (i.e. water
with an internal structure that is similar to that of pure water), has been found to
be essentially equal to the water content of the copolymer. This is in contrast to
other hydrophilic polymers used for biomedical applications as determined by

42
differential scanning calorimetry.182
These conclusions were confirmed by
Raman183
and ATR-FTIR184
measurements. Ellipsometry and neutron reflectivity
showed that significantly less protein was adsorbed onto PMPC-based surfaces
than on bare silicon-wafers.185,186
Thus, neither the bilayer mechanism or the free-
water mechanism can account fully for the suppression of protein adsorption and
it has been suggested that the high mobility of the PC headgroups may also exert
an influence.185,186
In addition to efficiently suppressing the adhesion of proteins, studies have shown
that phosphorylcholine-based surfaces, specifically those based on copolymers of
MPC, efficiently suppress the adhesion of a wide range of cells, including
platelets, fibroblasts and a variety of bacteria.180
Therefore, these surfaces may
find uses in, for example, implants that come in direct contact with blood and also
for surfaces that traditionally might be otherwise susceptible to bacterial growths
such as wound dressings and contact lenses.
PMPC-based copolymers are currently being used in commercial applications
such as coatings, coronary stents and contact lenses180
as well as in different
brands of cosmetics.187
Numerous other applications have been suggested,181
including grafting of PMPC from artificial joints to avoid bone-loss
(periprosthetic osteolysis),188
formation of more biocompatible membranes for
biological purification purposes189
and orally administered drug delivery
systems.190
In addition, numerous drug release applications based on PMPC-based
copolymer have been demonstrated191-193
and specially designed PMPC-
containing copolymers can be used for reversible cell encapsulation.194
The
salient point is that biological systems are very tolerant towards PMPC-based
copolymers and this property makes these ideal candidates for applications where
biocompatibility is an issue.
1.8.2 Hydrogels based on random copolymers of PMPC
Ishihara et al. reported that mixing aqueous solutions of random copolymers of
MPC/n-butyl methacrylate (BMA) and MPC/methacrylic acid spontaneously
formed a transparent free-standing gel.195
The mechanism of gelation was shown
to be due to formation of hydrogen bonds between the acid groups in hydrophobic

43
domains created by the BMA units (see Figure 1.6). The hydrophobic
surroundings suppress dissociation and the hydrogen-bonded dimers that are
formed act as crosslinks within the network.195,196
These gels are stable when
formed by mixing solutions of the two components dissolved in distilled water
but dissociate gradually in neutral or basic solution due to ionization of the acidic
groups.190
This makes them potential candidates for slow release applications in
drug delivery. The rate of dissolution can be controlled by changing the
copolymer concentration and molecular weight.190
C
O
O H
C
O
OH
C
O
O
H
C
O
O
H
C
O
-
O
C
O
-
O
C
O
O-
C
O
-
O
+
BMA-rich, hydrophobic domains
Water
Water
Gelled mixture
0.2 0.8
O O
O
PO O
N
O O
C
0.7 0.3
O O
H
O O
O
PO O
N
Figure 1.6: Schematic representation of the gelation mixture described in reference 195. The
two MPC-based statistical copolymers are made up as 5 % aqueous solutions. On mixing
these solutions the acid groups form dimers in the hydrophobic domains created by the
BMA groups and these serve as physical crosslinks.
1.8.3 Controlled Polymerization of MPC
The low solubility of the MPC monomer in aprotic solvents precludes
conventional living anionic polymerization for the preparation of MPC polymers

44
with low polydispersity. Therefore, polymerization methods such as ATRP and
RAFT have been employed to form well-defined MPC polymers. The first
example of controlled polymerization of MPC was described by Lobb et al. in
2001.38
In this study it was shown that PMPC homopolymers could be obtained
with polydispersities as low as 1.18 when prepared in aqueous solution and as low
as 1.12 in methanolic solution (Scheme 1.8). It was found that
homopolymerization was complete in around 10 minutes in water. However, the
semi-logarithmic plot was only linear up to around 75 % MPC conversion,
indicating that the polymer radical polymerization was no longer constant. In
addition, it was found that MPC auto-polymerized in aqueous solution in the
absence of a radical source. MPC polymerization in methanol was significantly
slower, but polydispersities were lower and the semi-logarithmic plot remained
linear up to at least 95 % conversion, indicating improved living character in this
solvent. In this study, the preparation of diblock copolymers was demonstrated as
well: If 2-(diethylamino)ethyl methacrylate (DEA) was added to the MPC
polymerization conducted in methanol at high MPC conversion the chain
continued to grow, resulting in a PMPC-PDEA diblock copolymer. As DEA is a
tertiary amine methacrylate, these copolymers exhibited pH-responsive behavior
in aqueous solution, forming PDEA-core micelles at pH 8 or above.
The polymerization of MPC by ATRP was further optimized by Ma et al.49
The
effect of solvent, ligand, degree of polymerization and temperature was examined
by kinetic plots of the polymerization and ‘self-blocking’ experiments in order to
examine the living character on adding a second batch of MPC monomer at high
conversion.
In water, the polydispersity was found to increase during the polymerization,
which indicated poor living character. The final polydispersities were around
1.20, suggesting reasonable control. However, the chain-extended copolymers
had polydispersities of 1.3-1.5, indicating poor blocking efficiency. In contrast,
when methanol was used as solvent, the polydispersity decreased throughout the
polymerization. In addition, self-blocking only led to a modest increase in
polydispersity from 1.17 to 1.22. If isopropanol was used as solvent, the
polymerization was slower than in methanol, but the final polydispersities were
very similar. Addition of 10 % water to the isopropanol led to faster

45
polymerization with retention of low polydispersity. Three different ligands were
examined and it was found that 2,2’-bipyridine gave the lowest polydispersities in
methanol. Increasing the temperature led to an increased rate of polymerization as
would be expected; at 20 °C and 40 °C, the semi-logarithmic plots in methanol
were linear, whereas slight curvature was observed at 60 °C. At all temperatures,
the polydispersities decreased throughout the polymerization with the final value
being below 1.2. It was found that, as the target degree of polymerization was
increased, the final polydispersities also increased. Thus, for a degree of
polymerization of 300, the final polydispersity was found to be 1.48, indicating
relatively poor control. Based on these results, the ATRP of MPC is normally
conducted in methanol at 20 °C unless other factors such as solubility or
reactivity197
necessitate the use of alternative solvents or different temperatures.
The use of RAFT polymerization for preparation of well-defined polymers of
MPC has been reported by Yusa et al.198
The homopolymerization was carried out
at 70 °C in water using 4-cyanopentanoic acid dithiobenzoate as a water-soluble
chain transfer agent and 4,4’-azobis(4-cyanopentanoic acid) as the thermally
activated initiator (Scheme 1.8). Under these conditions, the conversion reached
90 % in 60 minutes and more than 99 % in 240 minutes for a target degree of
polymerization of 73. The semi-logarithmic plot was linear after a short induction
period and the evolution of molecular weight increased linearly with conversion.
The polydispersity decreased throughout the polymerization to a final value of
1.26, indicating good living character of the polymerization under these
conditions. Surprisingly, no autopolymerization of MPC was observed in this
work in contrast to what was reported when aqueous ATRP was used.49
The
preparation of PMPC-PBMA block copolymers via RAFT was also reported.
After isolation, the PMPC-based macro-CTA was used to polymerize BMA in
methanol.198
A significant deviation from first-order kinetics was observed in the
semi-logarithmic plot. Nevertheless, two diblock copolymers with degrees of
polymerization of the PBMA blocks of 22 and 76 were prepared. These
copolymers formed micelles in aqueous solution. The critical micelle
concentration decreased by more than one order of magnitude as the DP of the
PBMA block was increased from 22 to 76.

46
OO
O
PO O
O
N
+
S
S
CN
O
OH
O
OH S
S
N
O
O
O
Br
O
O
O
CH2
C
CH3
OO
O
PO
O
N
+
O
n
OO
O
PO O
O
N
+
CN
O
OH
CH2
C
CH3
OO
O
PO
O
N
+
S
O
n
S
OO
O
PO O
O
N
+
S
N
O
OH
CH2
C
CH3
OO
O
PO
O
N
+
S
O
n
MeOH, 20 °C
Cu(I)Br, bpy
4-cyanopentanoic acid dithiobenzoate
4-(N,N-diethyldithiocarbamoylmethyl)
benzoic acid
+
OEGBr
n
ATRP:
H2O, 70 °C, 2h
0.12 eq. AIBN
RAFT:
1:5 THF:MeOH , 20 °C 3h
hν
Photoinduced LRP
+ n
MPC
+ n
A
B
C
Scheme 1.8: Approaches to controlled polymerization of MPC by A: ATRP 38,49
, B: RAFT195
and C: photoinduced living radical polymerization.196
MPC has also been polymerized by photoinduced living radical polymerization
using 4-(N,N-diethyldithiocarbamoylmethyl) benzoic acid, which is a
photoiniferter (Scheme 1.8 C).199
This polymerization was conducted in ethanol
with target DPs ranging from 15-150 and final polydispersities of 1.23-1.35,
indicating reasonable living character. However, no kinetic studies were
undertaken. The terminal carboxylic acid group of the polymer was subsequently
conjugated to an enzyme (papain). This conjugation led to a reduction in the
enzyme activity similar to that reported for PEO conjugation, but the long-term
activity was improved relative to that of the native enzyme.

47
1.8.4 Well-defined PMPC-based block copolymers
There are a few reports of the preparation of block copolymers of MPC using
RAFT198
and similar techniques.196,199
The PMPC-PBMA diblock copolymers
described by Yusa et al.198
were shown to form micelles in water and these
micelles increased the solubility of the hydrophobic anticancer drug, paclitaxel.
The solubility of this drug increased as the hydrophobic PBMA fraction of the
copolymer increased, as expected.
The preparation of a symmetrical PMA-PMPC-PMA triblock copolymers using
photo-iniferter chemistry was reported by Kimura et al.196
The target degree of
polymerization of PMPC in this block copolymer was 500 and in the final
copolymer the mole fraction of MPC was measured to be 0.33. However,
polydispersities ranged from 1.7 to more than 2, indicating poor control. Mixing a
solution of this copolymer with a solution of a random copolymer of MPC and
BMA produced gels and these results were compared to similar gel-forming
mixtures of an PMPC/PBMA random copolymer with an PMPA/PMA random
copolymer.195
It was found that the use of the symmetrical triblock copolymer led
to slower gel formation than when an PMPC/PMA random copolymer was used,
but the mechanical properties of the final gels, were very similar. Compared to the
random copolymers, the block copolymers need to rearrange significantly to form
networks, hence their longer gelation times.
Apart from these relatively few examples, the majority of PMPC-based block
copolymers have been prepared using ATRP. Two strategies for the preparation
of well-defined PMPC-based block copolymers have been pursued. One takes
advantage of the fact that the polymerization of MPC is very well-controlled in
methanol.38,49,200
Thus, diblock copolymers can be prepared by adding a second
methacrylic monomer to the polymerization mixture when the conversion is more
than 95 %. This approach leads to fairly well-defined diblock copolymers with
polydispersities of around 1.30 for a broad range of methacrylic monomers
(Figure 1.7).38,200

48
O O
N
O O
N
O O
N
O O
OH
O O
OH
OH
O O
OH
O O
N
+ Cl
O O
N
+
Cl
O O
N
+
SO3
O O O O
O
7
O
O
O O
N
+
-
DMA DEA DPA HEMA HPMA GMA
Me-DMA Bz-DMA CBMA SBMA MMA OEGMA
Figure 1.7: Monomers that form well-defined block copolymers with MPC. DMA: 2-
(dimethylamino)ethyl methacrylate. DEA: 2-(diethylamino)ethyl methacrylate. DPA: 2-
(diisopropylamino)ethyl methacrylate. HEMA: 2-hydroxyethyl methacrylate. HPMA: 2-
hydroxypropyl methacrylate. GMA: glycerol monomethacrylate. Me-DMA: 2-
(trimethylammonium)ethyl methacrylate hydrochloride. Bz-DMA: benzyl dimethyl 2-
(methacryloyloxy)ethyl ammonium chloride. CBMA: N-(carboxymethyl)-N-
(methacryloyloxy)ethyl-N,N-dimethylammonium betaine. SBMA: N-(3-sulfopropyl)-N-
(methacryloxyethyl)-N,N-dimethylammonium betaine. MMA: methyl methacrylate.
OEGMA: monomethoxy-capped oligo(ethylene glycol) methacrylate
The second route to diblock copolymers has focused on preparing macroinitiators
from commercially available end-functional polymers such as poly(ethylene
oxide), poly(propylene oxide) and poly(dimethylsiloxane), PDMS to prepare both
di- and triblock copolymers.200
Due to the hydrophobicity of PPO and PDMS, it
was only possible to determine the evolution of polydispersity versus conversion
by GPC in case of the PEO-PMPC block copolymer. The polydispersity decreases
slightly up to around 80 % conversion and remained below 1.20 up to 100 %
conversion, indicating good living character. On polymerizing MPC from the
PPO and PDMS macroinitiators, high conversions were obtained. Moreover, it
was not possible to extract residual macroinitiator after the polymerization

49
indicating that it was all incorporated into the diblock copolymer. This procedure
could also be used to form ABC triblock copolymers with a PMPC central block,
since the procedure of sequential monomer addition can be used for formation of
the third C block.
Both the sequential monomer addition approach and the macroinitiator approach
can be used to prepare symmetric ABA triblock copolymers using a bifunctional
ATRP (macro) initiator. One example was given by Ma et al.,200
where a
bifunctional PDMS-based macroinitiator was used to polymerize MPC, giving
PMPC-PDMS-PMPC triblock copolymers with degrees of polymerization of
PMPC of 10, 30 or 50. Armes’ group has used this approach to prepare a range
symmetrical MPC-based ABA-diblocks.201-203
The approach can also be used to
prepare MPC-containing ‘stars’ by using a tri-functional ATRP initiator.204,205
Our group has mainly focused on preparing stimuli-responsive PMPC-containing
block copolymers, where a change in pH or temperature causes a change in
solubility of one of the blocks. This change may facilitate the retention or release
of drugs.
1.8.5 PMPC-based pH-responsive block copolymers
The first example of a pH-responsive MPC-containing block copolymer was
reported by Lobb et al.38
Here 2-(diethylamino)ethyl methacrylate (DEA) was
added to the polymerizing MPC solution to give an PMPC30-PDEA100 diblock
copolymer. At pH 1.7, the 1
H NMR spectrum in D2O showed various signals
originating from both the PMPC block and the PDEA block, indicating that both
were solvated. However, on adjusting the pH to 8.0, the signals from the PDEA
block disappeared, indicating deprotonation of these chains. This led to
dehydration and micelle formation, consistent with DLS measurements, which
showed well-defined aggregates with an intensity-average diameter of 43 nm.
The preparation of well-defined diblock copolymers of MPC with three different
tertiary methacrylic amines, 2-(dimethylamino)ethyl methacrylate (DMA), 2-
(diethylamino)ethyl methacrylate (DEA) and 2-(diisopropylamino)ethyl
methacrylate (DPA) were reported by Ma et al.200
The mean DP of the PMPC
block was 30 and the DP of the tertiary amine block was varied from 30 to 100.

50
Copolymers with a fixed DP of 60 for the tertiary amine block were investigated
by 1
H NMR and fluorescence using pyrene as a hydrophobic probe. For the
PDMA-based copolymer, attenuation of the signals assigned to PDMA was
observed on increasing the temperature, which is consistent with the well-known
thermo-response of this polymer.206
However, no shift in the fluorescence was
observed on increasing the pH, indicating that no micelles were formed or that
pyrene is not taken up in these micelles because they are not sufficiently
hydrophobic. The PDMA block is highly cationic at physiological pH and this
property has been exploited for DNA complexation (Figure 1.8).207,208
MPC block DMA block
+
+
+
+
+
+
DNA
- -
- --
-- -
- --- --
-
+ +
+
++
+ 100 nm
DMA homopolymer PMPC30-PDMAPMPC30-PDMA10 PMPC30-PDMA40
Figure 1.8: A) Formation of PMPC-PDMA/DNA complexes. B) TEM images of PMPC-
PDMA / DNA complexes formed at a 2:1 DMA/nucleotide molar ratio. Scale bar is 500 nm.
207,208
1
H NMR studies of the PDPA-based copolymers were undertaken on increasing
the pH from 2.3 to 10.8; at pH 2.3, the PDPA chains are molecularly dissolved
and both blocks appear in the spectrum.200
Increasing the pH to 7.1 leads to
significant attenuation of all the signals from the PDPA blocks, and at pH 10.8
these signals have completely disappeared. Increasing the pH also led to a
significant change in the pyrene fluorescence spectrum. The fluorescence spectra
were used to estimate the pKa of the protonated amines. For the PMPC30-PDPA60
diblock copolymer this pKa was found to be 5.6 and for the PMPC30-PDEA60
copolymer, the value was 6.9. These pKa values were close to the reported values
obtained by acid titration for the two homopolymers.209
Additional fluorescence
A
B

51
studies indicated that the pyrene partition coefficients in the PDPA-based
copolymers were comparable to those of highly hydrophobic polystyrene micelle
cores.200
The aqueous solution properties of PMPC30-PDPAn were further
investigated.210
It was found that the aggregation number increased from 130 to
300 when n was increased from 30 to 60. The micelles based on the copolymer
with the longer PDPA block had a loading capacity of almost twice that of the
shorter copolymer for the cardiovascular drug, dipyridamole. In addition,
diffusion-controlled release was observed over a 5 h period, leading to 80 % of
the drug being released for the PMPC30-PDPA30 copolymer system and 60 % in
case of the PMPC30-PDPA60. After 20 h, release of the drug from the former
copolymer was almost complete, while around 20 % was retained in the latter
copolymer.
A related system of PMPCm-PDPAn diblock copolymers has also been studied.211
Here, m was 25 and n was increased to 120-160. These block copolymers formed
vesicles rather than micelles on increasing the pH above 6. When these vesicles
were loaded with a water-soluble anti-cancer drug (doxorubicin), a significantly
retarded release was found compared to the drug in the absence of the copolymer.
The loading efficiency of these vesicles was found to be 27 %. It has recently
been demonstrated that such biomimetic vesicles can deliver DNA and proteins
intracellularly and that the presence of these vesicles did not affect the cell
viability and the metabolic activity.212
These vesicles can retain the encapsulated
DNA for at least two weeks at physiological pH.213
At endocytic pH these
vesicles dissociate, leading to rapid payload release. In the case of DNA, the
protonated copolymer form complexes with the anionic phosphate backbone and
this serves to sterically-stabilize the DNA. Thus, this diblock copolymer serves a
dual role: At physiological pH, DNA can be encapsulated into vesicles and used
for ‘stealth’ delivery into cells. As the vesicle is incorporated into a cell and the
local pH drops, the DNA forms an electrostatic complex with the copolymer,
which now acts as a steric stabilizer, preventing aggregation and leading to very
high transfection efficiencies.
Symmetrical ABA triblock copolymers have been synthesized where the B block
comprises PMPC while the A blocks are either PDPA or PDEA.201
At low
concentration and high pH, these triblock copolymers form flower micelles,

52
where the ‘petals’ consist of the back-folded PMPC-blocks and the cores consist
of the hydrophobic tertiary amine blocks. At higher concentration, it is
thermodynamically feasible for some of the PMPC blocks form ‘bridges’ between
individual micelles. Thus at high enough concentrations, free-standing gels can be
obtained due to the relatively high degree of intermicellar bridging.214
For the
PDPA-based copolymers, free-standing gels obtained at 10 w/v %, depending on
the block composition. The PDEA-based copolymers required higher degrees of
polymerization for the PDEA blocks relative to the PMPC block to obtain free-
standing gels at the same copolymer concentration. If these gels were loaded with
a hydrophobic fluorescent drug, slow sustained release over several hours was
observed at physiological pH for the PDPA-based gels, and in general the higher
the copolymer concentration, the lower the rate of release. If the pH was lowered
to pH 3, drug release became very rapid due to molecular dissolution of the gel. It
was also found that drug release was significantly faster when a PDEA-based gel
was used, reflecting the relatively lower hydrophobicity of the PDEA chains
compared to PDPA.
Li et al. described the preparation of a pH-responsive ‘star’ copolymer using a
trifunctional ATRP initiator.205
In this example, MPC was polymerized first as
before and the second monomer was added at high conversion. The star
architecture promotes the formation of networks so it was anticipated that these
copolymers would be more efficient gelators. Indeed, this was found to be the
case, with free-standing gels being formed down to 5 w/v % at pH 8.2 for a
(PMPC125-PDPA100)3 copolymer. Unfortunately, preliminary studies indicated
that these copolymers were cytotoxic, presumably due to their weakly cationic
character at neutral pH.
1.9 PMPC-based temperature-responsive block copolymers
Work on PMPC-based temperature-responsive copolymers has been focused on
preparing ABA or ABC triblock gelators where the B block comprises PMPC and
the A and C blocks are different thermoresponsive polymers with LCST
behaviour.58,203
Li et al. described the preparation of ABA triblock copolymers,
where the A blocks comprise either PDMA, PHEMA or PNIPAM with a triblock
target composition of A90B250A90 using a commercially available bifunctional

53
initiator.202
Although both PDMA and PHEMA homopolymers are known to
exhibit LCST behaviour,206,215
none of the triblock copolymers based on these
two polymers formed gels on heating. On the other hand, copolymers where the
terminal blocks consisted of PNIPAM led to gelation on heating. Thus this system
was examined in more detail, although the synthesis of these latter copolymers
was more demanding. Free-standing gels could be formed at only 6.5 w/v %.
These gels were sufficiently biocompatible to allow V79 chinese hamster lung
cells to be grown in them. The critical gelation temperature was close to that of
PNIPAM, and subsequent FTIR studies were consistent with the dehydration of
the PNIPAM blocks as the driving force for the gelation, indicating that the
mechanism for network formation is similar to that depicted for the pH-
responsive gels.216
A related system was described in by Li et al.,203
whereby ABC triblock
copolymers of composition PPO33-PMPC180-PNIPAM90 and PPO43-PMPC160-
PNIPAM81 were prepared using PPO-based macroinitiators. However, copolymer
concentrations of 20 w/v % were necessary to obtain gels and then only for the
copolymer with the longest PPO block; thus these copolymers are inefficient
gelators. Nevertheless, light scattering and viscosity measurements on aqueous
solutions of the PPO43-PMPC160-PNIPAM81 copolymer confirmed its double
thermoresponse on heating; between 10 and 20 °C an increase in the light
scattering signal and the viscosity was observed, corresponding to micellization of
the PPO block. Above 30 °C another, larger increase was observed that
corresponded to aggregation of the PNIPAM blocks. Differential scanning
calorimetry confirmed these DLS results; a small exothermal peak was observed
indicating PPO micellization and a larger exothermal peak indicating PNIPAM
aggregation. Hence the two blocks behaved almost independently, showing the
characteristics of the two respective homopolymers, but the intermediate PMPC
block leads to the formation of a gel network at sufficiently high concentration,
rather than precipitation.

54
Molecularly dissolved
triblock
PPO-core micelles
from 15 to 32 oC Micellar network above 32 oC
6-10 w/v %
copolymer
Molecularly dissolved at 20 oC since
blue NIPAM blocks are hydrophilic
PMPC
PNIPAM PNIPAM
PMPC
PNIPAM PNIPAM
Molecularly dissolved at 20 oC since
blue NIPAM blocks are hydrophilicMolecularly dissolved triblock copolymer Triblock copolymer micellar gel
32 °C
PNIPAM PPO
PMPC
10 °C
20 w/v %
copolymer
32 °C
Figure 1.9: Schematic of thermoresponsive gelation of PNIPAM-PMPC based copolymers.
A) Gelation of PNIPAM-PMPC-PNIPAM copolymers202
B) Gelation of PPO-PMPC-
PNIPAM copolymer.203
ABA triblock copolymers based on 2-hydroxypropyl methacrylate (HPMA) as the
A blocks exhibit pronounced thermo-responsive behavior in aqueous solution.217
This is surprising, since the HPMA monomer has limited solubility in water and
PHPMA homopolymers are considered water-insoluble.218,219
Nevertheless, a
copolymer with a composition of PHPMA44-PMPC250-PHPMA44 formed gels on
heating in aqueous solutions down to 4.0 w/v %. The critical gelation temperature
could be varied from 40 °C for a 4.0 % solution down to 5 °C for a 10.0 %
solution. In contrast, two control polymers based on the same PMPC B block,
with either poly(methyl methacrylate) (PMMA) or PHEMA as A blocks
respectively, did not exhibit any temperature response. The PMMA-based
copolymer gave a highly opaque dispersion in water, indicating was poorly
dissolution, while the PHEMA-based copolymer gave a free-flowing liquid as
observed earlier.58
A
B

55
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Chapter 2: Preparation and Aqueous Solution Properties of New Thermo-responsive Biocompatible ABA Triblock
Copolymer Gelators
64
Chapter 2: Preparation and Aqueous Solution
Properties of New Thermo-responsive
Biocompatible ABA Triblock Copolymer
Gelators

Copolymer Gelators
65
2.1 Introduction
ABA triblock copolymers with water-soluble central B blocks and water-
insoluble outer A blocks are known to form gels in semi-dilute aqueous solution.1-
6
The copolymer chains form aggregates known as ‘flower’ micelles and the
water-soluble B block can act as a bridge between these micelles. Computer
simulations indicate that critical copolymer volume fractions of 0.05 - 0.10 are
required for gelation, depending on the overall molecular mass.3
This work, as
well as other theoretical studies,1,2
also predicts that the ability to form networks
depends mainly on the degrees of polymerization of the two blocks, as well as the
hydrophobic character of the outer A block. These findings have been confirmed
by a large number of experimental studies.1,4-6
In this chapter is reported the synthesis and aqueous gelation behavior of new
ABA copolymers in which the hydrophobic character of the A blocks is
systematically varied, while the central B block in each case is PMPC, a highly
hydrophilic polymer that confers clinically proven biocompatibility.7,8
Three
types of ABA triblock copolymers of approximately the same overall composition
were prepared in which the A block is PMMA, PHPMA or PHEMA. MMA
monomer is water-immiscible and PMMA is hydrophobic, whereas HPMA
monomer is water-miscible up to 13 % at 25o
C but PHPMA is water-insoluble.
On the other hand, HEMA monomer is water-miscible in all proportions and
PHEMA is water-soluble up to a mean degree of polymerization of approximately
45, becoming water-insoluble thereafter.9
Thus the relative hydrophobic character
of the A blocks ranks as: PMMA > PHPMA > PHEMA.
2.2 Experimental
2.2.1 Materials
2-(Methacryloyloxy)ethyl phosphorylcholine monomer (MPC, 99.9 % purity) was
kindly donated by Biocompatibles Ltd., UK (Farnham, UK). 2-Hydroxyethyl
methacrylate (HEMA) and 2-hydroxypropyl methacrylate (HPMA) were kindly
donated by Cognis Performance Chemicals (Hythe, UK). Methyl methacrylate

Copolymer Gelators
66
(MMA, 99%) was purchased from Sigma-Aldrich UK (Dorset, UK) and passed
through a silica column prior to use. Copper(I) bromide (CuBr, 99.999 %), 2,2’-
bipyridine (bpy, 99 %), tris(hydroxymethyl)aminomethane (Trizma, ≥ 99.9 %),
tris(hydroxymethyl)aminomethane hydrochloride (Trizma hydrochloride ≥ 99.0
%) and diethyl meso-2,5-dibromoadipate (DEDBA, 98 %) were purchased from
Sigma-Aldrich UK (Dorset, UK) and were used as received. Sodium nitrate
(NaNO3, ACS reagent) and lithium bromide (LiBr, 99 +%) was obtained from
Acros Organics (Geel Belgium). The silica gel 60 (0.063 – 0.200 µm) used to
remove the spent ATRP catalyst was purchased from E. Merck (Darmstadt,
Germany) and was also used as received. Acetonitrile, chloroform and methanol
were all HPLC-grade solvents obtained from Fisher Scientific (Loughborough,
UK) and used as received. Hydrochloric acid (32 %, general purpose grade) was
purchased from Fisher Scientific (Loughborough, UK) and used as received.
Near monodisperse PEO and PMMA GPC calibration standards were obtained
from Polymer Laboratories (Church Stretton, UK).
2.2.2 Triblock copolymer syntheses using the diethyl meso-2,5-
dibromoadipate initiator
These one-pot syntheses were carried out in two successive steps using sequential
monomer addition without purification of the intermediate PMPC macro-initiator.
Due to the poor solubility of MMA in methanol, the polymerization of MMA was
conducted at 50°C to aid solubility of this triblock copolymer. A typical synthesis
was as follows: MPC (7.40 g, 25.0 mmol, 250 eq.) was mixed with the diethyl
meso-2,5-dibromoadipate initiator (36.7 mg, 0.10 mmol, 1 eq) and 2,2’-bipyridine
(63.3 mg, 0.41 mmol, 4.1 equivalents) was dissolved in 10 mL methanol. This
solution was degassed using a nitrogen purge for 30 minutes to remove oxygen.
Then CuBr (30.1 mg, 2.1 mmol, 2.1 eq.) was added to commence the first-stage
polymerization. After 20 h, HPMA (1.44 g, 10.0 mmol, 100 eq.) was added to the
dark brown viscous solution by syringe and the reaction mixture was stirred for a
further 48 h. After this time period no vinyl signals were observed in the 1
H NMR
spectrum, hence the reaction mixture was diluted with either methanol or
methanol:chloroform and passed through a silica column to remove the spent
catalyst. After evaporation of the solvent, water was added and the final
copolymer was obtained as a white powder by freeze-drying overnight.

Copolymer Gelators
67
2.2.3 1
H NMR spectroscopy
PHEMA-based copolymer: 1
H NMR spectra were obtained at room temperature
in deuterated methanol using a Bruker AC250 NMR spectrometer.
PHPMA-based copolymer: 1
H NMR spectra were obtained at 45°C in deuterated
methanol at 500 MHz using a Bruker DRX-500 NMR spectrometer.
PMMA-based copolymer: 1
H NMR spectra were obtained at room temperature in
a 1:1 CDCl3/CD3OD mixture using a Bruker AC250 NMR spectrometer.
2.2.4 Molecular weight determination
Non-aqueous chromatograms were assessed using a Hewlett Packard HP1090
Liquid Chromatograph as the pumping unit and two Polymer Laboratories PL Gel
5 μm Mixed-C 7.5 x 300 mm columns in series with a guard column at 40°C
connected to a Gilson Model 131 refractive index detector. The eluent was a 3:1
v/v % chloroform/methanol mixture containing 2 mM LiBr (unless otherwise
stated) at a flow rate of 1.0 ml min-1
. A series of near-monodisperse PMMA
standards were used as calibration standards. Toluene (2 μl) was added to all
samples as a flow rate marker. Data analysis was carried out using CirrusTM
GPC Software supplied by Polymer Laboratories.
Aqueous chromatograms were assessed using the refractive index detector of a
Polymer Laboratories PL-GPC 50 Integrated GPC System. A 0.05 M Trizma
buffer with 0.2 M sodium nitrate was used as the mobile phase. The column
system consisted of a PL Aquagel-OH 40 and a PL Aquagel-OH 30 in series. The
GPC system was calibrated with a series of near-monodisperse PEO calibration
standards. Data analysis was carried out using CirrusTM GPC Software supplied
by Polymer Laboratories.

Copolymer Gelators
68
2.2.5 HPMA composition assessed by HPLC
The HPLC system consisted of an autosampler (Varian Model 410), a solvent
delivery module (Varian Module 230) and a UV detector (Varian Model 310).
The chromatographic column was a standard 150 x 4.6 mm C18-column. The
eluent system consisted of 0.10 % aqueous trifluoroacetic acid (TFA) and
acetonitrile. A gradient was applied from 15 % acetonitrile to 40 % acetonitrile in
20 minutes. The detection wavelength was set to 254 nm. Data were collected
with Star Chromatography Workstation system control version 6.20.
2.2.6 Preparation of copolymer solutions for rheology studies
Gel samples for rheology studies were prepared by weighing out the desired
amount of copolymer (60-200 mg) and adding deionized water (2.00 mL). Each
triblock copolymer solution was then stored at 4 °C for 6 to 48 h. The PHEMA-
PMPC-PHEMA and PHPMA-PMPC-PHPMA copolymers each produced
transparent solutions at 4 °C, whereas the PMMA-PMPC-PMMA copolymer
gave an opaque aqueous gel, indicating incomplete and/or non-uniform
dissolution. The latter copolymer solution was also heated to 50°C for 2 h and
then allowed to equilibrate overnight at room temperature to aid molecular
dissolution, but no improvement in transparency was achieved. In order to
efficiently remove air from the more concentrated copolymer solutions, it was
necessary to subject them to several freeze-thaw cycles.

Copolymer Gelators
69
2.3 Results and discussion
2.3.1 NMR characterization of triblock copolymers
For the characterization of the PHEMA-PMPC-PHEMA and PHPMA-PMPC-
PHEMA triblock copolymers by NMR, deuterated methanol (CD3OD) was used,
since methanol is a good solvent for both blocks. However, for the
characterization of PMMA-PMPC-PMMA copolymers this solvent is not suitable
due to the poor solubility of PMMA in methanol.10
This is illustrated in the inset
of Figure 2.1, where 1
H NMR spectra of the PMMA55-PMPC240-PMMA55
triblock copolymer are recorded in CD3OD and a mixture of 6 parts of CDCl3 and
4 parts of CD3OD. Although the copolymer appears to dissolve in CD3OD, the 1
H
NMR spectrum does not show the PMMA signal due to the pendant methoxy
group at 3.6 ppm. In contrast, both peaks are observed in the CDCl3:CD3OD
mixture. Figure 2.1 shows that there is almost no change in the molar ratio
between the integrals of the 1
H NMR signals assigned to PMPC and PMMA when
the volume fraction of CDCl3 increases from 0.2 to 0.6. A further reduction in the
CDCl3 content reduces this ratio, indicating gradually poorer solvation of PMPC
and, in the absence of CD3OD, the copolymer does not dissolve at all.

Copolymer Gelators
70
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
ApparentPMPC/PMMAblockmolarratio
V(CDCl3
)/(V(CDCl3
)+V(CD3
OD))
Figure 2.1: Apparent molar ratio between pendent methylene group of PMPC and the
pendent methoxy group of PMMA in a PMMA55-PMPC240-PMMA55 triblock copolymer as a
function of the volume fraction of CDCl3. The inset shows 250 MHz 1
H NMR spectra in pure
CD3OD and at a CDCl3:CD3OD volume fraction of 0.6.
2.3.2 Gel Permeation Chromatography (GPC) in chloroform:methanol
mixture
In order to characterize copolymers of hydrophilic MPC and hydrophobic
monomers such as HPMA or MMA by Gel Permeation Chromatography, it was
necessary to develop a novel protocol in a solvent that dissolved both blocks.
Such protocols have been described in the literature for random copolymers of
MPC and BMA11,12
or n-dodecyl methacrylate (DoMA).12
These are based on
solvent mixtures of 3-4 parts chloroform with 1-2 parts alcohol such as
methanol11
or ethanol.12
Therefore mixtures of chloroform and methanol were
tried as eluent for the non-aqueous GPC. Figure 2.2 shows chromatograms of an
OEG-PMPC150 polymer, where the OEG stands for monomethoxy-capped
oligoethyleneoxide with a mean DP of 7, using a 3:1 mixture of chloroform and
methanol as eluent with different amounts of lithium bromide (LiBr). In the
4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4
PMPC
PMMA
3:2 CDCl3
:CD3
OD
CD3
OD
δ / ppm

Copolymer Gelators
71
absence of LiBr, the polymer signal is seen as a broad signal eluting at the solvent
front, with a sharper peak superimposed on this. On decreasing the copolymer
concentration, the peak was found to gradually disappear, leaving only the
broader signal. This phenomenon is common in size-exclusion of
polyelectrolytes and can be ascribed to the surface charges on the
chromatographic column; most solids have a small surface charge and a charged
solute will be repelled or adsorbed by this surface, depending on the sign of the
charge.13
Once the surface charge is neutralized, the remaining copolymer elutes
by size-exclusion. Although the zwitterionic PMPC should be electrically neutral,
the chromatography experiments indicate a small permanent charge, since charge-
repulsion occurs. The problem is generally solved by adding an electrolyte to the
eluent. In this case, LiBr was used. LiBr is known to be soluble in methanol14
and
it was found to be adequately soluble in the 3:1 chloroform:methanol mixture
applied here.
11 12 13 14 15 16 17
2.0 mM LiBr
1.0 mM LiBr
2.5 mM LiBR
No LiBr
Elution time / minutes
Figure 2.2: Gel permeation chromatograms of a OEG-PMPC150 polymer in
chloroform:methanol 3:1 v:v with different concentrations of LiBr. Flow rate: 1.0 mL / min.
Temperature: 40 °C. Columns: Two Polymer Laboratories PL Gel 5 µm Mixed-C (7.5 x 300
mm) columns in series with a guard column.
Figure 2.2 shows that addition of LiBr leads to a significant narrowing of the
chromatograms, indicating that size-exclusion is the main separation mode. In

Copolymer Gelators
72
addition, the peak values shifts to longer retention times. This was also observed
to a lesser extent with the PMMA-standards used for calibration and is probably a
consequence of a decrease in hydrodynamic volume with an increased ionic
strength.13
Furthermore, the chromatograms exhibit some tailing towards lower
molecular weight (higher retention times), indicating adsorption to the columns.
This tailing is significantly less with 2.5 mM LiBr compared with 1.0 mM LiBr
and 2.0 mM LiBr, indicating that the adsorption is decreased and that size-
exclusion is dominant at this LiBr concentration.
In order to further examine the validity of the protocol, a series of OEG-PMPC
polymers were analyzed using protocols with 1.0 mM LiBr and 2.5 mM LiBr.
The chromatographic system was calibrated with a series of narrow-disperse
PMMA standards. These results were compared to results obtained by an aqueous
GPC-protocol which was previously used for analyzing PMPC polymers (Table
2.1).15

Chapter 2: Preparation and Aqueous Solution Properties of New Thermo-responsive Biocompatible ABA Triblock Copolymer Gelators
73
Target 1.0 mM LiBr Aqueous eluent
Entry Sample DP M theory Mn Mw/Mn Mn Mw/Mn % Deviation from aqueous Mn % Deviation from theoretical Mn
1 OEG-MPC100 100 30,019 26,000 1.32 23,000 1.24 13% -13%
2 OEG-MPC50-MPC50 100 30,019 38,000 1.38 30,000 1.22 27% 27%
3 OEG-MPC150 150 44,784 37,000 1.39 33,000 1.26 12% -17%
4 OEG-MPC200 200 59,549 41,000 1.35 46,000 1.28 -11% -31%
Target 2.5 mM LiBr Aqueous eluent % dev from aqueous results % dev from calculated results
5 OEG-MPC100 100 30,019 23,000 1.21 23,000 1.24 0% -23%
6 OEG-MPC50-MPC50 100 30,019 31,000 1.2 30,000 1.22 3% 3%
7 OEG-MPC150 150 44,784 35,000 1.15 33,000 1.26 6% -22%
8 OEG-MPC200 200 59,549 36,000 1.21 46,000 1.28 -22% -40%
Table 2.1: GPC-data for OEG-MPC polymers using two different eluents. All polymers were prepared using an oligo(ethylene glycol) initiator with DP~7. Entries
1-4 are the same polymers as entries 5-8, analyzed with different amount of LiBr in the eluent. The target DPs and calculated molecular weights of the samples are
given in column 3 and 4. In column 5 and 6, the measured number-average molecular weights and polydispersities are given. Columns 7 and 8 give the
corresponding numbers for the same polymers in an aqueous eluent at pH 7.0 The percentage deviation of the number-average molecular weight in the non-
aqueous eluent vs. the aqueous eluent and vs. the theoretical value is given in columns 9 and 10 respectively. Details of preparation of the polymers are given in
reference 15.

Copolymer Gelators
74
In general all the measured Mn values are lower than the calculated values for all
three eluents. This is ascribed mainly to the calibration, since the standards are
different from the actual polymers. This is a general phenomenon for SEC and
emphasizes that the technique is relative and not absolute when universal
calibration is used. The eluent with 1.0 mM LiBr (Table 2.1, entries 1-4) gives
relatively high values for Mn and Mw/Mn compared to the values obtained using
the aqueous eluent. In addition, entry 2 elute in the wrong order compared to
entry 3. Entry 2 was prepared by chain-extension of a PMPC50 copolymer and
although it should be identical to entry 1, the aqueous results indicate that the
degree of polymerization is between 100 and 150. However, using the protocol
with 1 mM LiBr indicates that this copolymer is slightly larger than the
copolymer with a target DP of 150.
If the LiBr concentration is increased to 2.5 mM, the Mn and Mw/Mn values
decrease when compared to the results obtained using the eluent with less LiBr. In
addition, the results obtained for the copolymers with DPs up to 150 mM
correspond reasonably well to the results obtained using the aqueous eluent and
the copolymers elute in the same order. The Mw/Mn values are in general lower
than for the aqueous analysis but still with a magnitude that is generally expected
for ATRP.
The eluent with 2.50 mM LiBr was chosen for analysis of homopolymers of MPC
and copolymers incorporating MPC, since the elution order of the test polymers
was correct and since the number average molecular weights corresponded well to
those obtained from aqueous size-exclusion chromatography on the same
copolymers over a range of target DPs.
2.3.3 Hydroxypropyl methacrylate
The hydroxypropyl methacrylate used throughout the majority of this work was
donated by Cognis Performance Chemicals (Cognis).16
This monomer is
commonly prepared via alkoxylation of methacrylic acid with propylene oxide as
shown in Scheme 2.1.17-19

Copolymer Gelators
75
O
O
OH
O
O
OH
O
OH O
*
*
75 %
2-hydroxypropyl
methacrylate
(HPMA)
25 %
2-hydroxyisopropyl
methacrylate
(HIPMA)
+
Methacrylic acid Propylene oxide
1,2-addition
1,3-addition
Base catalyst
*
Scheme 2.1: Synthetic route to the HPMA monomer. The asterisk denotes a chiral center.
In neutral or basic solution, the substrate is the free epoxide and the substitution
normally involves an SN2 mechanism.20
As the least substituted carbon undergoes
SN2 more readily, the major product is 2-hydroxypropyl methacrylate.
Nevertheless, some 2-hydroxyisopropyl methacrylate is formed during the
reaction (Figure 2.3), so polymers based on this monomer are technically
speaking random copolymers. In addition, each of the isomers has a chiral center
(denoted by an asterisk in Scheme 2.1) resulting in two enantiomeric forms of
each. As the isomers are structurally very similar, the polymers are generally
treated as homopolymers.
2.3.4 Characterization of commercially available grades of hydroxypropyl
methacrylate
Sigma-Aldrich® sells a grade of hydroxypropyl methacrylate which is depicted as
the 3-hydroxypropyl methacrylate in their catalogue and it is stated that it is a
“Mixture of hydroxypropyl and hydroxyisopropyl methacrylate”.21
The CAS
number is the same as for the COGNIS product discussed above, however,
indicating that it is in fact the same compound. The two compounds were
compared by 1
H and 13
C NMR, FTIR and RP-HPLC.

Copolymer Gelators
76
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
e
i
f
a
OH
j
c + d
h
gg
bb
e
i
a
h OH
j
c + d f
gg
bb
Aldrich Product No.: 268542
COGNIS Product No.: 678372
δ / ppm
200 180 160 140 120 100 80 60 40 20 0
H + G
M
AF
N
E
L
I
B
C
JK
D
δ / ppm
O
O
OH
O
O
OH
b
a
c
d
e
g
f
h
j
i
O
O
OH
O
O
OH
B
A
C
D
E
F
G
I
H
J
K
L
M
N
2.0 1.8 1.6 1.4 1.2 1.0
a
f
e
i
δ / ppm
A
B
Figure 2.3: (A) Assigned 1H-NMR spectra in CDCl3 at 400 MHz of HPMA from Aldrich
and Cognis respectively. The inset shows the region from 1-2 ppm enlarged. (B) Assigned
13C JMOD spectrum of the Cognis product in CDCl3 at 400 MHz (1
H-frequency). C=O, CH2
positive, CH, CH3 negative
Figure 2.3A shows the assigned 1
H-NMR spectra of the two products in CDCl3.
These are very similar, indicating that the contents are the same. The minor
differences between the two products can probably be related to the water content.
The relative integrals fit reasonably well with the composition given in previous
reports18,19
and the fact that peaks e and i are doublets rather than multiplets
(doublet of doublet, Figure 2.3, inset) indicates that the Aldrich product is a
mixture of the 2-hydroxypropyl methacrylate and 2-hydroxyisopropyl

Copolymer Gelators
77
methacrylate. This is substantiated by the 13
C JMOD spectrum in Figure 2.3B,
which only shows peaks with a negative sign corresponding to methyl (CH3) or
methine (CH) carbons in the region from 0 to 40 ppm. This is consistent with the
proposed structure. If the compound is 3-hydroxypropyl methacrylate, there
should be a methylene (CH2) carbon in this region. Since there are no positive
resonances, the presence of this compound can be excluded. Figure 2.4A shows
the ATR-FTIR spectra of the two samples. In order to distinguish the two it was
necessary to offset one relative to the other, substantiating that the two samples
are essentially identical. In addition, Figure 2.4B shows HPLC chromatograms of
the two compounds and of a one-to-one mixture of the two. Both samples show
two peaks as expected since both samples are a mixture of isomers. The retention
times are slightly different, however, leaving the possibility that both samples
have 2-hydroxyisopropyl methacrylate in common as well as their individual
components. Running the mixture of the two excludes this, since the mixture only
shows two peaks and not three. Thus the difference in the chromatograms is due
to small variances in elution conditions.
3750 3000 2250 1500 750
30
40
50
60
70
80
90
100
110
120
130
Transmittance/%
Wavenumber / cm
-1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
COGNIS + Aldrich, 1:1 V/V
Absorption/A.U.
Elution time / min
A B
Figure 2.4: (A) ATR-FTIR spectra of HPMA from Aldrich and Cognis respectively. The (B)
HPLC chromatograms of HPMA from Aldrich, Cognis and a 1:1 V/V mixture of the two.
Conditions: 15-40 % acetonitrile/0.1 % aqueous trifluoroacetic acid in 20 minutes, 1
mL/min, detection at 254 nm, Column: Alltima HP C18 HL 5µ 150 x 4.6 mm.
Table 2.2 compares the mole fractions of HIPMA in the two products, calculated
by 1
H NMR and HPLC respectively. The two methods are seen to give relatively
consistent results, although 1
H NMR generally indicates a slightly lower HIPMA
content than HPLC. The reason for this is not known, but at least two factors may
contribute: Firstly, the uncertainty on the 1
H NMR integrals is on the order of 5 %
and secondly in the HPLC calculations, it is assumed that the molar extinction

Copolymer Gelators
78
coefficient of HPMA and HIPMA is exactly the same at 254 nm. Given the
structural similarity of the two compounds, this assumption is probably valid
within a few percent. The combination of these two sources of error is believed to
account for the small difference between the two methods.
Sample HIPMA mole fraction /
1
H NMR HIPMA mole fraction / HPLC
Aldrich Product No. 268542 0.228 ± 0.004 0.251
Cognis Product No. 678372 0.228 ± 0.006 0.255
Table 2.2: Mole fractions of 2-hydroxyisopropyl methacrylate (HIPMA) measured by 1
H
NMR (400 MHz in CDCl3) and HPLC (15-40 % CH3CN in 0.1 % aqueous TFA, 254 nm,
Column: GraceSmart R.P.18 5 m 150 mm x 4.6mm). The mole fractions from the 1
H NMR
measurements were obtained by calculating the ratio between well-separated peaks assigned
to on isomer (peaks h, c+d and j in Figure 2.3A respectively) and peaks assigned to both
isomers (peaks b+g, a+f and e+i in Figure 2.3A). These were averaged and the error is the
standard error. The mole fractions from HPLC were obtained by calculating the ratio
between the area of the minor peak at 10-11 min in the chromatograms (Figure 2.4B) and
the sum of the areas of both peaks.
Given the discussion above, HPMA is a well-defined mixture rather than a pure
substance, no matter the source, as the product from Aldrich is almost identical to
the product from Cognis Performance Chemicals and the depiction in the Aldrich
Catalogue is misleading. Thus, if a pure compound is desired, it is necessary to
separate the isomers, for example by chromatographic means. Similarly, if single
enantiomers are desired it would be necessary to separated these for example by
chromatography on chiral columns. Such separations have not been attempted in
this work but may be relevant in future applications. Preparation of polymers
from the structural isomers may give polymers with different properties to those
obtained from a mixture, i.e. the temperature-induced transitions may be sharper.
Preparation of polymers from a single enantiomer will probably not have any
significant effect on the observed thermal transitions but may be relevant if
copolymers are used for the release of optically active drugs; the interaction with
one isomer may be higher than with the other. Often one enantiomer of a drug is
more potent or beneficial than the other and polymers enriched in one active form
may be useful in selecting the more attractive enantiomer from a mixture.22

Copolymer Gelators
79
2.3.5 Copolymer synthesis
Each of these three ABA triblock copolymers was synthesized by ATRP in
methanol using sequential monomer addition23
as shown in Scheme 1 for
PHPMA-PMPC-PHPMA. Diethyl meso-2,5-dibromoadipate (DEDBA, ex.
Aldrich) was used as a bifunctional initiator. In the case of the PMMA55-
PMPC240-PMMA55 triblock synthesis, the reaction solution was heated to 50 °C
prior to addition of the MMA monomer due to the marginal solubility of PMMA
in methanol at ambient temperature.10
Scheme 2.2: ATRP synthesis of the PHPMA-PMPC-PHPMA triblock copolymer
The three copolymers were characterized by GPC and 1
H NMR (see Table 2.3).
The PHPMA44-PMPC250-PHPMA44 and PHEMA55-PMPC250-PHEMA55 triblocks
were both soluble in CD3OD. Solubilization of the PMMA55-PMPC240-PMMA55
copolymer required the use of a CDCl3/CD3OD mixture for a reliable 1
H NMR
spectrum.

Chapter 2: Preparation and Aqueous Solution Properties of New Thermo-responsive Biocompatible ABA Triblock Copolymer Gelators
80
Target block composition
a 1
H NMR block composition
a
Mol % MPC
Conversion
first block
b)
Conversion
second block
b)
Mn
c)
Mw/Mn
c)
PHPMA50-PMPC250-PHPMA50 PHPMA44-PMPC250-PHPMA44 74 > 98 % > 98 % 84,700 1.39
PMMA49-PMPC240-PMMA49 PMMA55-PMPC240-PMMA55 69 > 98 % > 98 % 89,000 1.72
PHEMA51-PMPC250-PHEMA51 PHEMA55-PMPC250-PHEMA55 73 > 98 % > 98 % 91,900 1.62
Table 2.3: Summary of the 1
H NMR and GPC data for the three ABA triblock copolymers examined in this chapter. a)
Subscripts indicate the mean degrees of
polymerization (DP) of each block. b)
As determined by 1
H NMR. c)
As determined by GPC conducted in a 3:1 chloroform/methanol mixed eluent using poly(methyl
methacrylate) calibration standards.

Copolymer Gelators
81
Similarly, GPC characterization was carried out using a 3:1 CHCl3/CH3OH mixed
eluent for all copolymers (Figure 2.5). The copolymer compositions estimated
from 1
H NMR spectroscopy are close to those targeted (Table 2.3). It is worth
emphasizing that the PMPC precursor block has a relatively low polydispersity
(around 1.30) in each case, so these triblock copolymers have reasonably well-
defined molecular architectures. However, the polydispersities of the overall
triblock copolymers are somewhat higher (1.39 to 1.72), which simply reflects the
fact that rather high degrees of polymerization are being targeted in these one-pot
syntheses, leading to some loss of control over the living character of the ATRP
chemistry. Nevertheless, our GPC data are generally comparable to those
previously reported for related pH-responsive triblock copolymers synthesized by
sequential monomer addition by both ATRP23
and also by group transfer
polymerization (GTP).6
10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0
PHPMA44
-PMPC250
-PHPMA44
Mn
= 84,700
Mw
/Mn
= 1.39
PHEMA55
-PMPC250
-PHEMA55
Mn
= 91,900
Mw
/Mn
= 1.62
Elution time / min
PMMA55
-PMPC240
-PMMA55
Mn
= 89,000
Mw
/Mn
= 1.72
RI-signal
Figure 2.5: GPC traces obtained for the various PMPC-based triblock copolymers in 3:1
CHCl3:CH3OH with 2 mM LiBr
2.3.6 Aqueous solution behavior
The temperature-dependent viscosity behavior of 10 w/v % aqueous solutions of
each copolymer is shown in Figure 2.6. PMMA55-PMPC240-PMMA55 produced a

Copolymer Gelators
82
highly opaque gel that flowed slowly on tube inversion and exhibited a relatively
high, almost temperature-independent viscosity. The highly hydrophobic nature
of the PMMA blocks prevents molecular dissolution and leads to long micelle
residence times (i.e. ‘frozen’ micelles).3,4
In contrast, a 10 w/v % aqueous
solution of PHEMA55-PMPC250-PHEMA55 was highly transparent and had a low
viscosity (i.e. was free-flowing) over the entire temperature range. Again, the
viscosity of this copolymer does not exhibit any significant temperature
dependence, although gelation was achieved at 60 °C for more concentrated
copolymer solutions (> 20 w/v %). Thus, the thermo-responsive behavior of the
PHEMA chains is suppressed significantly when they are attached to the much
more hydrophilic PMPC block.
Figure 2.6: Temperature dependence of the solution viscosity for 10 w/v % aqueous solutions
of the three PMPC-based triblock copolymers shown in Table 2.3
The viscosity of a 10 w/v % aqueous solution of the PHPMA44-PMPC250-
PHPMA44 copolymer unexpectedly increased by almost two orders of magnitude
on heating from 0°C (free-flowing liquid) to 30°C (transparent free-standing gel,

Copolymer Gelators
83
Figure 2.7). As this behavior was unforeseen, this copolymer was investigated in
more detail.
Low
[copolymer]
High
[copolymer]
MPC
HPMA HPMA
Heat Heat
‘Molecularly’ dissolved
at low temperature
Micellar gel network at
elevated temperature
Flower micelles at
elevated temperature
Figure 2.7: Top: From left to right: (A) a free-flowing 10 % PHPMA55-PMPC250-PHPMA55
solution at 50 °C; (B) an opaque 10 % PMMA55-PMPC240-PMMA55 gel at 50 °C; (C) 7.5 %
PHPMA44-PMPC250-PHPMA44 at 4°C (free-flowing solution) and (D) the same copolymer
solution at 50 °C (now a transparent, free-standing gel). Bottom: Consequence of heating a
PHPMA44-PMPC250-PHPMA44 solution: At low temperature, the copolymer is molecularly
dissolved. Increasing the temperature leads to formation of ‘flower-micelles’. At sufficiently
high concentration, bridges between individual micelles may form, leading to a micellar gel
network.
In Figure 2.8A the temperature-dependent storage and loss moduli of 5 % and 10
% aqueous solutions of this copolymer are shown. In both cases the storage
modulus increases by two orders of magnitude and becomes larger than the loss
modulus, confirming gel formation.
A
B
C
D

Copolymer Gelators
84
0 10 20 30 40 50
0.01
0.1
1
10
A
G'
G''
10 %
5 %
Modulus/Pa
T / °C
4 5 6 7 8 9 10
0
10
20
30
40 B
Tgel
/°C
Copolymer concn. / %
Figure 2.8: (A) Storage (G’) and loss (G’’) moduli obtained for 5 and 10 % aqueous solutions
of the PHPMA44-PMPC250-PHPMA44 triblock copolymer, respectively. (B) The G’ – G’’
cross-over temperature as a function of concentration for the same PHPMA44-PMPC250-
PHPMA44 copolymer
The critical gelation temperature (Tgel) was determined from the cross-over of the
storage and loss modulus curves and decreases monotonically from 40°C to 5°C
as the copolymer concentration is increased from 4.0 to 10.0 w/v % (see Figure
2.8B). This is similar to the behavior of Pluronic-type triblock copolymers, where
the hydration of the poly(propylene oxide) block is both concentration- and
temperature-dependent.24,25

Copolymer Gelators
85
0 5 10 15 20 25 30 35 40 45 50 55
500
1000
1500
2000
2500
3000
3500
4000
4500
d~60 nm
Countrate/kcps
Temperature / °C
d~25 nm
Figure 2.9: Temperature dependence of the scattered light intensity count rate obtained for
a 0.10 w/v % aqueous solution of PHPMA44-PMPC250-PHPMA44. Note the upturn at around
10 °C due to micellar self-assembly. The diameters are the calculated hydrodynamic
diameter from the correlation functions.
In contrast, the aqueous solution behavior of classical thermo-responsive poly(N-
isopropylacrylamide)-based copolymers is largely concentration-independent.26
Our dynamic light scattering experiments indicate the presence of weakly
interacting ‘flower’ micelles even for highly dilute solutions at 5 °C, with
increased light scattering being observed at elevated temperatures (see Figure
2.9). Close examination of variable temperature 1
H NMR spectra recorded in D2O
(see Figure 2.10) suggests only partial solvation of the PHPMA blocks at low
temperature. The signal at 0.95 ppm due to the pendent methyl groups on the
PHPMA blocks is visible at low temperature but is attenuated at higher
temperatures relative to the PMPC signals at 2.95 ppm, 3.42 ppm and 3.70-4.00
ppm and also the methacrylic backbone signals at 0.50-0.90 ppm and 1.65 ppm
which increase in intensity and become sharper along with a downfield shift due
to temperature. Similarly, FTIR studies have shown that the intensity of the
absorption band assigned to the pendent methyl groups in poly(propylene oxide)
is strongly correlated with the dehydration of this polymer at higher
temperatures.27,28

Copolymer Gelators
86
1.50 1.25 1.00 0.75 0.50 0.25 0.00
46 °C
δ / ppm
5 °C
H NMR spectra recorded for a 3.7 % w/V PHPMA44-PMPC250-
PHPMA44 triblock copolymer solution in D2O at 5°C and 46°C. The pendent methyl groups
and part of the backbone signals are assigned. Spectral shifts are due to differences in
temperature
2.4 Summary and conclusions
In summary, although PHPMA homopolymer is not normally considered to be a
water-soluble polymer, it can be rendered water-soluble (or at least water-
dispersible) by covalent attachment to a much more hydrophilic block, in this case
PMPC. Thus, the unexpected thermo-responsive behavior of the PHPMA44-
PMPC250-PHPMA44 triblock copolymer is most likely due to the weakly
hydrophilic nature of the PHPMA blocks, which are clearly capable of intra-chain
and inter-chain hydrogen bonding via C=O…
HO type interactions. In contrast, the
analogous PHEMA-based triblock copolymer is simply too hydrophilic to
undergo efficient gelation (at least in semi-dilute aqueous solution), while the
PMMA-based triblock copolymers cannot be molecularly dissolved/dispersed in
water under any conditions, leading to opaque viscous solutions with no thermo-
responsive behavior. Unlike NIPAM, HPMA monomer is cheap and has
relatively low toxicity. Moreover, the marked concentration dependence observed
a
a
b+c
b+c
CH2
CH3
O O
CH3
50
OH
CH2
CH3
OO
O
P
O
O O
N
CH3
CH3
CH3
250
CH2
CH3
OO
CH3
50
OH
bb
c c
+
b
c
a
a

Copolymer Gelators
87
for the critical gelation temperature suggests that de-gelation may be easily
achieved simply by dilution, which may be useful in certain biomedical
applications.
2.5 References
1
Balsara, N. P., Tirrelli, M., Lodge, T. P. Macromolecules 1991, 24, 1975-
1986
2
Semenov, A. N., Joanny, J.- F., Khokhlov, A. R. Macromolecules 1995, 28,
1066-1075
3
4
Zhang, K., Macdonald, P. M., Menchen, S. Langmuir 1997, 13, 2447-2456
5
Raspaud, E., Lairez, D., Adam, M., Carton, J.- P. Macromolecules 1994, 27,
2956-2964
6
Gotzamanis, G.T., Tsitsilianis, C., Hadjiyannakou, S.C., Patrickios, C.S.,
Lupitskyy, R., Minko, S. Macromolecules 2006, 39, 678-683
7
8
9
10
Fox, T.G., Kinsinger, J.B., Mason, H.F., Schuele, E.M. Polymer 1962, 3, 71-
95
11
Iwasaki, Y., Tabata, E., Kurita, K., Akiyoshi, K. Bioconj. Chem. 2005, 16,
567-575
12
Ishihara, K., Nomura, H., Mihara, T., Kurita, K., Iwasaki, Y., Nakabayashi,
N. J. Biomed. Mat. Res. A 1998, 39, 323-330
13
Stenlund, B., Adv. Chrom. 1976, 14, 37-74
14
Weast, R. C., Astle, M. J., Beyer, W. H. (Editors): CRC Handbook of
Chemistry and Physics, CRC Press, Boca Raton, United States of America,
1985-1986, 66th
edition
15
Ma, I.Y., Lobb, E.J., Billingham, N.C., Armes, S.P., Lewis, A. L., Lloyd,
A.W., Salvage, J. Macromolecules 2002, 35, 9306-9314
16
Cognis Bisomer HPMA, Product Number 678,372
17
Hayes, R. A., Boutsicaris, S. P. U.S. Patent 2,929,835, 1960
18
Weaver, J.V.M. PhD Thesis University of Sussex, Sussex, United Kingdom
2003
19
2002, 35, 1152-1159
20
Edition
21
Aldrich Hydroxypropyl methacrylate, Product number 268,542
22
Nguyen, L.A., He, H., Pham-Huy, C. Int. J. Biomed. Sci. 2006, 2, 85-100
23
Ma, Y., Tang, Y., Billingham, N. C., Armes, S. P. Biomacromol. 2003, 4,
864-868
24
Hvidt, S., Jørgensen, E.B., Brown, W., Schillén, K. J. Phys. Chem. 1994, 98,
12320-12328

Copolymer Gelators
88
25
Mortensen, K., Brown, W., Jørgensen, E. Macromolecules 1994, 27, 5654-
5666
26
Wu, C., Wang, X. Phys. Rev. Lett. 1998, 80, 4092-4094
27
Cabana, A., Aїt-Kadi, A., Juhász, J. J. Coll. Interface Sci. 1997, 190, 307-312
28
Su, Y., Wang, J., Liu, H. Langmuir 2002, 18, 5370-5374

Chapter 3: New Biocompatible Wound Dressings based on Chemically Degradable Triblock Copolymer Hydrogels
89
Chapter 3: New Biocompatible Wound Dressings
based on Chemically Degradable
Triblock Copolymer Hydrogels

90
3.1 Introduction
It is well known in the literature that ABA triblock copolymers with water-soluble
central B blocks and water-insoluble outer A blocks can form either flower-
micelles, or free-standing gels in aqueous solution, depending on the copolymer
concentration and copolymer composition1-8
The gels are of particular interest and
comprise a three-dimensional network of inter-connected micelles, with the
water-soluble B blocks acting as bridges between adjacent micelles. Computer
simulations indicate that critical copolymer volume fractions of 0.05 - 0.10 are
required for gelation, depending on the overall molecular mass.3
This work and
other theoretical studies1,2
also predicts that network formation depends mainly on
the molecular weights of both blocks, as well as the hydrophobic character of the
outer A block. These findings have been confirmed by a number of experimental
studies.1,6
Recently our group reported an example of a pH-responsive triblock
gelator.9
Here the A blocks were based on PDPA and the central B block
comprised PMPC, a highly hydrophilic polymer with excellent
biocompatibility.10,11
The PDPA chains become protonated below their pKa of
around 6.2, which allows molecular dissolution of the triblock copolymer chains
in acidic solution. Neutralization of this solution leads to free-standing gels.
However, precise control over the in situ pH adjustment is somewhat problematic
and biological actives such as cells, DNA or proteins may not survive the initial
acidic conditions. Given these disadvantages, a second-generation thermo-
responsive triblock gelator was designed12
in which the PDPA blocks were
replaced with PNIPAM, a water-soluble polymer that is well-known for its
inverse temperature solubility behavior.13,14
This modification allowed the
formation of transparent free-standing gels at 37 °C, which were sufficiently
biocompatible to allow in situ cell proliferation. Moreover, using a disulfide-
based initiator allowed the synthesis of a third-generation PNIPAM-PMPC-
PNIPAM triblock containing a single S-S bond within the backbone of the central
PMPC block. Selective cleavage of this S-S bond using a naturally-occurring
tripeptide such as glutathione will convert this triblock copolymer into the
corresponding diblock copolymer of approximately half the original triblock
molecular weight. Since the resulting PMPC-PNIPAM diblock copolymer cannot

91
form free-standing gels, this leads to the concept of a biochemically-responsive
gel based on a disulfide ‘keystone’. Unfortunately, NIPAM is not an ideal
building block for the design of thermo-responsive copolymers for biomedical
applications. This monomer is relatively expensive, requires purification prior to
use and is a potent neurotoxin.15
It is also not trivial to copolymerize NIPAM with
methacrylic monomers such as MPC using ATRP,12,16
which is our preferred
synthetic methodology. In view of this, the use several hydrophilic methacrylic
monomers in place of NIPAM was recently explored. It was already shown that
PHEMA exhibited thermo-responsive behavior in aqueous solution,17
but
unfortunately PHEMA-PMPC-PHEMA triblocks proved insufficiently
hydrophobic to form free-standing gels. On the other hand, an analogous triblock
copolymer based on HPMA instead of HEMA gave very encouraging preliminary
results (see chapter 2).18
Thus a PHPMA50-PMPC250-PHPMA50 triblock
copolymer could be dissolved in cold water, but formed free-standing gels at
higher temperatures. Moreover, the critical gelation temperature was relatively
sensitive to the copolymer concentration, which is not the case for PNIPAM-
based copolymers. In the present chapter, the studies of a series of PHPMA-
PMPC-PHPMA triblock copolymers of varying block composition and molecular
weight are reported. Their aqueous gelation behavior is examined using gel
rheology, light scattering, variable temperature 1
H NMR spectroscopy and
transmission electron microscopy and the biocompatibility of selected gels was
assessed for potential wound dressing applications.
3.2 Experimental Section
3.2.1 Materials
donated by Biocompatibles Ltd., UK. 2-Hydroxypropyl methacrylate (HPMA)
was donated by Cognis Performance Chemicals (Hythe, UK). Bis(2-
hydroxyethyl)disulfide (98 %), 2-bromoisobutyryl bromide (98 %), basic alumina
(Brockmann I, standard grade, ~150 mesh, 58 Å), dithiothreitol (DTT, 99 %),
glutathione (99 %), L-Glutathione (reduced form ≥ 95 %), anhydrous methanol
(MeOH 99.8 %), copper(I) bromide (CuBr, 99.999 %), 2,2’-bipyridine (bpy, 99
%) 4-(dimethylamino)pyridine (99 %) (DMAP), trifluoroacetic acid (TFA, 99+

92
%) and diethyl meso-2,5-dibromoadipate (DEDBA, 98 %) were purchased from
Sigma-Aldrich UK and were used as received. Lithium bromide (LiBr, 99 +%)
was from Acros Organics (Geel, Belgium) and used as received. The silica gel 60
(0.063 – 0.200 µm) used to remove the spent ATRP catalyst was purchased from
E. Merck (Darmstadt, Germany) and was also used as received. Magnesium
sulfate (MgSO4), sodium hydrogen carbonate (NaHCO3), anhydrous sodium
carbonate (Na2CO3) sodium chloride (NaCl) and triethylamine (Et3N) were
laboratory reagent grade from Fisher Scientific (Loughborough, UK) and used as
received. Acetonitrile, tetrahydrofuran, dichloromethane, chloroform and
methanol were all HPLC-grade solvents obtained from Fisher Scientific
(Loughborough, UK). Near monodisperse PMMA GPC calibration standards
were obtained from Polymer Laboratories (Church Stretton, UK). Near
monodisperse PNaStS GPC calibration standards were obtained from Polymer
Standard Service (Mainz, Germany).
3.2.2 Synthesis of the disulfide-based bifunctional ATRP initiator, bis[2-(2-
bromoisobutyryloxy)ethyl] disulfide, (BiBOE)2S2
The disulfide-based bifunctional ATRP initiator (BiBOE)2S2 was synthesized
according to a literature protocol.19,20
Bis(2-hydroxyethyl) disulfide (15.4 g, 0.1
mol) was dissolved in 200 ml dry THF, excess triethylamine (42.0 ml, 0.30 mol)
was added under a nitrogen atmosphere and this solution was cooled in an ice
bath. 2-Bromoisobutyryl bromide (59.8 g, 0.26 mol) was added dropwise from a
dropping funnel over a 1 h period so as to minimize the reaction exotherm and the
reaction solution slowly turned reddish brown. The solution was allowed to warm
up to ambient temperature and stirred for 24 h. The insoluble triethylammonium
bromide salt was removed by filtration and the resulting colorless solution was
concentrated under vacuum. The concentrated solution was stirred with 0.10 M
aqueous sodium carbonate to hydrolyze any residual 2-bromoisobutyryl bromide.
The crude product was then extracted three times with dichloromethane using a
separating funnel. The combined dichloromethane extracts were first dried with
anhydrous magnesium sulfate and then concentrated to afford a reddish brown oil
(31.2 g; yield = 69 %), which was stored in a refrigerator prior to use. The crude
product was purified by dissolution in dichloromethane, followed by passage

93
through a basic alumina column to yield a pale yellow liquid that crystallized in
the freezer (-25 °C).
1
H NMR (CDCl3) δ 4.45 (t, 2H, J=6.6 ,-CH2OOC-), 2.96 (t, 2H, J=6.6 ,-CH2S-),
and 1.95 (s, 6H, (CH3)2C-) ppm
13
C NMR (CDCl3) δ 171.4 (C=O), 63.5 (-CH2OOC-), 55.5 (Br-C), 36.7 (S-CH2),
30.7 (CH3) ppm
Elemental microanalyses gave C = 31.94 % (theory 31.87 %), H = 4.68 % (theory
4.46 %), Br = 35.54 % (theory 35.34 %), and S = 14.44 % (theory 14.18 %),
suggesting that the initiator purity exceeded 98 % based on S.
3.2.3 Synthesis of the propanediol-based bifunctional ATRP initiator, 1,3-
bis (2-bromoisobutyryloxy) propane (BiB)2P
Propan-1,3-diol (2.014 g, 0.026 mol) was dispersed in 10 mL dichloromethane.
To this was added triethylamine (8.3 mL, 6.0 g, 0.06 mol) and 4-
(dimethylamino)pyridine (0.7355 g, 0.0060 mol). The resulting solution was
placed under nitrogen and cooled on ice. 2-Bromoisobutyryl bromide (7.45 mL,
13.9 g, 0.060 mol) in 25 mL dichloromethane was added dropwise over 20
minutes. The reaction mixture was left for 72 h after which 150 mL
dichloromethane was added. The organic phase was washed with water (2 x 50
mL), saturated sodium hydrogen carbonate (2 x 50 mL) and water (2 x 50 mL).
After drying over anhydrous magnesium sulfate, the drying agent was filtered off
and the solvent was removed at 50 °C to give a yellow oil. This was dissolved in
dichloromethane and passed through a silica column (eluent: dichloromethane).
Isolated yield: 4.66 g corresponding to 47 %.
1
H NMR (CDCl3) δ 4.29 (t, 4 H, J=6.3 Hz, -CH2OOC-), 2.09 (m, 2H, -C-CH2-C-
), 1.93 (s, 12 H, (CH3)2C-) ppm
13
C NMR (CDCl3) δ 171.5 (C=O), 62.1 (-CH2OOC-), 55.6 (Br-C), 30.7 (CH3),
27.5 (CH2) ppm
MS (ES+), m/z (%) 397 (M + Na+
, 15)
4.85 %), Br = 42.63 % (theory 42.72 %) suggesting that the initiator purity
exceeded 99 % (based on C and Br).

94
3.2.4 Copolymer Synthesis and Purification
One-pot copolymer syntheses were conducted using sequential monomer addition
without purification of the intermediate PMPC macro-initiator. A typical
synthesis was carried out as follows: MPC (10.002 g, 33.9 mmol, 250 eq.) was
placed under nitrogen. (BiBOE)2S2 (61.2 mg, 0.135 mmol, 1 eq) was dissolved in
12 mL anhydrous methanol and added to the MPC through a cannula. The
reaction mixture was purged with nitrogen for 30 min. 2,2’-Bipyridine (83.8 mg,
0.537 mmol, 4.0 equivalents) and CuBr (38.6 mg, 0.269 mmol, 2.0 eq.) was
added to commence the first-stage polymerization. After 6 h, HPMA (1.9533 g,
13.5 mmol, 100 eq.) was added to the dark brown viscous solution by cannula and
the reaction mixture was stirred for a further 70-100 h until no vinyl signals were
observed in the 1
H NMR spectrum. After this time period, the reaction mixture
was diluted with methanol and passed through a silica column to remove the spent
catalyst. The solution was partly evaporated and precipitated into excess THF
(500 mL) to remove residual monomer and traces of 2,2’-bipyridine. After
filtration, residual THF was removed by co-evaporation with three 50 mL
portions of methanol. To the solid residue was added 200 mL water and this was
stirred until a uniform mixture was obtained. The water was evaporated at 50-60
°C under reduced pressure to obtain a solution volume of approximately 50 mL
prior to addition of 150 mL water. Approximately 150 mL water was again
removed under vacuum and the resulting solution was freeze-dried overnight.
Finally, the copolymer was dried at 80 °C at high vacuum for 48 h, then for 5-6 h
at 90 °C. These additional co-evaporation steps were essential for the cell studies,
since it was found that traces of cytotoxic methanol were very difficult to remove
by simply drying the copolymer in a vacuum oven. In contrast, repeated co-
evaporation of residual methanol with water under reduced pressure proved to be
a reliable means of ensuring sufficient purification to achieve biocompatibility.
This protocol produced 9-10 g of purified triblock copolymer (75-83 % yield).
3.2.5 Bipyridine content assessed by HPLC
The HPLC system consisted of an autosampler (Varian Model 410), a solvent
delivery module (Varian Module 230) and a UV detector (Varian Model 310).
The chromatographic column was a standard 150 x 4.6 mm C18-column,

95
commonly a GraceSmart R.P. 18, 5 µm, 150 mm x 4.6 mm. The eluent system
consisted of 0.10 % aqueous trifluoroacetic acid and acetonitrile. A gradient was
applied from 5 % acetonitrile to 100 % acetonitrile in 20 minutes. The detection
wavelength was set to 300 nm. Data were collected with Star Chromatography
Workstation system control version 6.20.
Solutions of around 10 mg copolymer (mass determined to three significant
digits) in 1.000 mL 0.10 % v/v trifluoroacetic acid in methanol were analyzed by
HPLC. A stock solution of 0.010 M bipyridine in 0.10 % v/v trifluoroacetic acid
in methanol was diluted with 0.10 % v/v trifluoroacetic acid in methanol to create
a calibration curve (Figure 3.1). A linear fit through data points forced through
(0,0) gave a calibration factor of 4.94·10-7
µg x detector count with a coefficient
of determination of 0.999.
0.0 3.0x10
6
6.0x10
6
9.0x10
6
1.2x10
7
1.5x10
7
1.8x10
7
0
1
2
3
4
5
6
7
8
9
10
2,2'-bipyridinemass/µg
Detector Count
Figure 3.1: Calibration curve of mass of 2,2’-bipyridine versus detector count at λ=300 nm.
Conditions: 1 mL/ min, 5-100 % acetonitrile in 0.1 % aqueous TFA over 20 minutes.
Column: GraceSmart R.P.18, 5µm. 150 mm x 4.6 mm. A linear fit through (0,0) gave a
straight line with equation: m(bpy) [µg] =4.94·10-7
µg x Detector Count, R2
=0.999.
Mixtures of bpy and copolymer were prepared in order to examine any potential
interference effects of the copolymer content on the measured amounts of 2,2’-
bipyridine. In principle, some bipyridine may elute with the copolymer and this

96
may lead to lower recovered amounts. Figure 3.2 shows the result of adding
various known amounts of 2,2’-bipyridine to a solution of a copolymer. The
measured detector count rate is seen to be very close to the count rate calculated
using the fit from Figure 3.1, as indicated by the correlation coefficient of the
straight line being very close to 1. This shows that essentially all 2,2’-bipyridine
in the sample is recovered.
0.0 2.0x10
6
4.0x10
6
6.0x10
6
8.0x10
6
1.0x10
7
0.0
2.0x10
6
4.0x10
6
6.0x10
6
8.0x10
6
1.0x10
7
Measured Count Rate = 0.9989 x Calculated Count Rate
R
2
= 0.9997
MeasuredCountRate
Calculated Count Rate
Figure 3.2: Measured Count Rate versus Calculated Count Rate of a PHPMA90-PMPC200-S-
S-PMPC200-PHPMA90 triblock copolymer solution spiked with known concentrations of 2,2’-
bipyridine using the calibration constant derived from Figure 3.1. Conditions: 1 mL/ min, 5-
100 % acetonitrile in 0.1 % aqueous TFA over 20 minutes. Column: GraceSmart R.P.18,
5µm. 150 mm x 4.6 mm.
3.2.6 1
H NMR Spectroscopy
1
H NMR spectra were recorded in either D2O or CD3OD using either a 400 MHz
Bruker AV1-400 or a 500 MHz Bruker DRX-500 spectrometer. For the variable
temperature studies, the integrated peak intensity due to the pendent methyl
groups in the PHPMA chains at 1.3 ppm was compared to that due to the pendent
methylene groups of the PMPC chains at 3.7 ppm. This numerical value was

97
normalized with respect to the actual block composition of the copolymer, as
determined by 1
H NMR in CD3OD, which is a good solvent for both PHPMA and
PMPC. Thus the apparent block composition could be estimated at any given
temperature.
3.2.7 Molecular Weight Determination
Chromatograms were assessed using a Hewlett Packard HP1090 Liquid
Chromatograph as the pumping unit and two Polymer Laboratories PL Gel 5µm
Mixed-C (7.5 x 300 mm) columns in series with a guard column at 40°C
connected to a Gilson Model 131 refractive index detector. The eluent was a 3:1
v/v % chloroform/methanol mixture containing 2 mM LiBr at a flow rate of 1.0
ml min-1
. A series of near-monodisperse PMMA samples were used as calibration
standards. Toluene (2 µL) was added to all samples as a flow rate marker. Data
analyses were conducted using CirrusTM GPC Software supplied by Polymer
Laboratories.
For the disulfide cleavage experiments with glutathione, chromatograms were
assessed using a Polymer Laboratories LC1120 HPLC pump, as the pumping unit
and two Polymer Laboratories Aquagel-OH 8 mm columns (Type 40 first,
followed by Type 30) in series with a guard column at 25 °C connected to a
Polymer Laboratories ERC-7515A refractive index detector. The eluent was 70 %
aqueous 0.2 M NaNO3, 0.01M NaH2PO4, adjusted to pH 7.0 with 30 % methanol
was used. A series of near-monodisperse PNaStS samples were used as
calibration standards.
3.2.8 Dynamic Light Scattering
Copolymer solutions for light scattering studies were prepared as 1.00 wt. %
aqueous solutions in PBS. These stock solutions were diluted to the desired
concentration and filtered through a 0.43 µm Nylon filter prior to use. Dynamic
light scattering experiments were performed with a Zetasizer Nano-ZS instrument
(Malvern Instruments, UK) at a scattering angle of 173 °. Dispersion Technology
Software version 4.20 supplied by the manufacturer was used for cumulants
analysis according to ISO 13321:1996.

98
3.2.9 Transmission Electron Microscopy
Samples were mounted on pre-coated carbon-coated copper grids. These grids
were submerged for 1 minute into a 0.40 % aqueous copolymer solutions at 25 °C
and then placed in an aqueous uranyl acetate solution (1 % w/w) for 20 seconds.
Imaging was performed on a FEI Tecnai Spirit TEM operating at 120 kV
equipped with a Gatan 1K MS600CW CCD camera. Hannah Lomas is
acknowledged for the TEM pictures.
3.2.10 Gel Rheology Studies
Copolymer (30.0-300.0 mg) was dissolved in aqueous PBS solution (1.00 mL) for
rheology studies. These solutions were left to stand in a refrigerator at 4 °C
overnight. For more concentrated copolymer solutions (10-30 %, depending on
the copolymer composition and its molecular weight), the solutions were
subjected to several freeze-thaw cycles in order to remove trapped air. A
Rheometric Scientific SR-5000 rheometer equipped with cone-plate geometry
(40.0 mm, 0.05 radians) was used for the oscillatory temperature sweeps,
employing a frequency of 1 rad/s, a stress of 0.5 Pa and a heating rate of 3
°C/min. This instrument was fitted with a Peltier element for temperature control
and a thermostatted water-bath was used as a heat sink.
3.2.11 Disulfide Gel Cleavage Experiments with Dithiothreitol (DTT)
Aqueous triblock copolymer solutions were prepared in PBS buffer that had been
purged with nitrogen for several hours prior to use in order to exclude oxygen.
Sample preparation was otherwise identical to that used for the temperature-
sweep experiments. DTT concentrations were calculated assuming that the Mn of
the PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 was 150,000. Addition of the
DTT reductant was achieved by placing 1.0 mL of a 11.0 % copolymer solution
and 0.10 mL of the reductant solution in two separate syringes. These syringes
were connected by a three-way valve and thermostatted in a water-bath at the
desired temperature for 1.5 minutes. These solutions were then mixed for 10

99
seconds by pushing the plungers forward and back. The valve was opened and the
resulting aqueous mixture of copolymer and reductant was placed in the
thermostatted rheometer. Measurements commenced approximately 35-40
seconds after mixing using the cone-and-plate geometry. The applied stress was
0.06 Pa and the frequency was 1 rad per second.
3.2.12 Disulfide Cleavage Experiments with Glutathione
Addition of glutathione to the copolymer gels did not have any significant effect
on the mechanical properties. Instead, a solution of 30.4 mg PHPMA88-PMPC200-
S-S-PMPC200-PHPMA88 was dissolved in 2.000 mL PBS buffer that had been
purged with nitrogen for several hours prior to use in order to exclude oxygen.
This solution contains approximately 0.1 mM disulfide. It was stored at 4 °C
overnight to ensure complete dissolution and then placed at 37 °C in an incubator.
Glutathione (11.6 mg, 37.7 µmol) was dissolved in 2.000 mL nitrogen-purged
PBS (pH 7.3) to make up a 18.9 mM solution which was placed in an incubator at
37 °C.
In order to initiate the reaction, 0.100 mL glutathione solution (1.89 µmol) was
added to the copolymer solution. This corresponds to a final concentration of
0.900 mM glutathione or approximately 9 times the molar amount of disulfide
bonds.
The solution was rapidly divided into separate vials with 0.35 mL each and
replaced at 37 °C. At regular time intervals, a vial was removed and placed in the
freezer at -25 °C to quench the disulfide cleavage reaction.
Immediately prior to analysis, the vials were allowed to thaw and analyzed
directly using an appropriate GPC eluent (70 % aqueous 0.2 M NaNO3, 0.01 M
NaH2PO4, adjusted to pH 7.0 with NaOH, 30 % v/v methanol).
3.3 Results and Discussion
3.3.1 Synthesis of bifunctional initiators with and without disulfide
The disulfide initiator, bis[2-(2-bromooisobutyryloxy)ethyl] disulfide
(BiBOE2S2), was prepared according to the published procedure.19,20
In addition,

100
a slightly modified version of the procedure was used for the initiators (Scheme
3.1).
Br
O
S
O
S
O
O
BrOH
S
S
OHBr
Br
O
Br
O
OO
O
BrBr
Br
O
OHOH
+2
DMAP/Et3N
CH2Cl2
0 °C-20 °C
+2
DMAP/Et3N
CH2Cl2
0 °C-20 °C
a)
b)
Scheme 3.1: a) Preparation of bis[2-(2-bromooisobutyryloxy)ethyl] disulfide, BiBOE2S2 b)
Preparation of 1,3-bis (2-bromoisobutyryloxy) propane BiB2P
Dichloromethane was used as a solvent instead of THF.19,20
As the former is a
poor solvent for the dihydroxy precursors but a good solvent for the diester
products, a turbid mixture formed. To this two-phase system, triethylamine and
DMAP was added and the mixture was cooled using an ice-bath. 2-
Bromoisobutyryl bromide was slowly added as a solution in dichloromethane. On
standing overnight, the mixture gradually cleared. Washing with water and
aqueous sodium hydrogen carbonate removed triethylammonium bromide,
unreacted alcohol and residual acid. The resulting oils were passed through a
basic alumina column with dichloromethane efficiently removed any residual 2-
bromoisobutyric bromide and 2-bromoisobutyric acid to give colorless products.
These crystallized on standing at -25 °C, indicating high purity. The purity was
confirmed by 1
H NMR and the compounds were characterized by 13
C NMR, mass
and elemental analysis.

101
3.3.2 Copolymer Synthesis
O
O
O Br-PMPCn-S-S-PMPCn-Br
HPMA
P
O
N
O O
O
O
HO
MPC
S S
O O
O
Br
O
Br
Cu(I)Br, bpy
methanol, 20°C
PHPMAm-PMPCn-S-S-PMPCn-PHPMAm
BiBOE2S2
20°C 2 m
2 n
Scheme 3.2: Synthesis of PHPMA-PMPC-S-S-PMPC-PHPMA triblock copolymers via
ATRP
The PHPMA-PMPC-S-S-PMPC-PHPMA triblock copolymers were synthesized
by ATRP in a one-pot synthesis according to Scheme 3.2 using sequential
monomer addition, as reported previously.18
Copolymer characterization data are
summarized in Table 3.1. In general the polydispersities are reasonably narrow,
especially when the high target degrees of polymerization are taken into
consideration. The MPC conversion was 98-100 % for all syntheses, whereas the
HPMA conversion was lower in most cases (82-96 %). The polymerizing mixture
becomes very viscous after the first-stage MPC polymerization. This is believed
to contribute to the relatively slow second-stage HPMA block copolymerization,
since HPMA homopolymerization under these conditions is significantly faster.21
Entry 1 corresponds to the triblock copolymer gelator previously reported in
Chapter 2 (see also ref. 18). This particular copolymer was prepared using a
commercially available ATRP initiator, diethyl meso-2,5-dibromoadipate
(DEDBA); the alternative bis[2-(2-bromoisobutyryloxy)ethyl] disulfide
(BiBOE2S2) initiator used for the majority of the copolymer syntheses was
prepared according to a literature procedure.19,20
For the disulfide-containing
copolymers it was found that disulfide-based copolymers generally had lower
overall molecular weights when the same degrees of polymerization as originally
used for the DEDBA-based copolymers were targeted. This point is illustrated by
comparing Entries 1 and 5 in Table 3.1. In this case, the number-average

102
molecular weight of entry 5 should be approximately 3,000 higher than that of
Entry 1, but the actual measured value is 23,600 lower. The most likely
explanation is that the DEDBA initiator is less efficient due to its acrylate-like
secondary radicals: methacrylate-like, tertiary radicals are usually considered to
be preferred for the ATRP of methacrylic monomers.22
By comparing Entry 6 and
Entry 1 in Table 3.1 it is clear that, in order to obtain similar copolymer molecular
weights using the disulfide initiator, it is necessary to target a higher degree of
polymerization. Thus, the actual triblock composition shown for Entry 1 assumes
that the BiBOE2S2 initiator is 100 % efficient and ignores the small difference
between the PHPMA block lengths for Entries 1 and 6. In order to investigate
whether radical transfer to the disulfide additionally lowered the efficiency as
previously suggested,19
a triblock copolymer was prepared using the BiB2P
initiator, which is structurally similar to the BiBOE2S2 but does not contain a
disulfide group (Scheme 3.1b). Entry 7a in Table 3.1 shows the data for the first
block of this copolymer. The number-average molecular weight is slightly lower
than for the corresponding copolymer obtained using the BiBOE2S2 initiator, see
Entry 6a. However, the difference between these molecular weights is relatively
small, and does not suggest any significant radical transfer. This is consistent with
related work on disulfide-based branched copolymers from the Armes group.23
The disulfide-containing copolymers can be classified according to the target DP
of the middle block; Entries 2-4 all have a central block with a DP of 250 as their
PHPMA content is increased from 10 to 20 wt %. Entry 5 has a longer central
block but a similar PHPMA content to Entries 1 and 3, whereas Entry 6 has the
longest central block and slightly higher PHPMA content than entries 1-3 and 5.

103
Entry No. Target Triblock Composition Triblock Composition by
1
H-NMR Mn Mw/Mn PHPMA content / wt. %
1 PHPMA50-PMPC250-PHPMA50 PHPMA70-PMPC390-PHPMA70 84,700 1.39 15
2 PHPMA30-PMPC127-S-S-PMPC127-PHPMA30 PHPMA30-PMPC127-S-S-PMPC127-PHPMA30 56,300 1.64 10
6a PMPC200-S-S-PMPC200 PMPC200-S-S-PMPC200 54,200 1.44 -
7 PHPMA80-PMPC400-PHPMA80 PHPMA71-PMPC400-PHPMA71 71,400 1.43 15
7a PMPC400 PMPC400 51,600 1.44 -
H NMR and GPC studies of the triblock copolymers. All
copolymers were prepared using the disulfide initiator, except for the first entry, which was prepared using the commercially available DEDBA
initiator. 1
H NMR were recorded at 400 MHz. GPC data were obtained using a 3:1 v/v chloroform/methanol eluent and a series of PMMA calibration
standards

104
3.3.3 Purification of copolymers
The rather tedious purification protocol described in the experimental section was
empirically found to be required, if the copolymers were to be used in biological
applications. If these steps were not performed, the cytotoxicity of the copolymer
gels was too high to be useful, i.e. cell viabilities were less than 80 % after 24 h.
The purification steps are summarized in Table 3.2.
Purification
step no.
Description Purpose
1 Passage through a silica column Removal of Cu
2 Precipitation with THF Removal of 2,2'-bipyridine ligand
3 Addition and evaporation of water Removal of methanol
4 Freeze-drying Removal of water
5 Vacuum oven at 80-90 C
Removal of residual water and remaining volatiles
(methanol, THF)
Table 3.2: Steps used in purification of copolymers for cytotoxicity studies
The purification steps can be rationalized as follows:
The first step removes the copper catalyst as described previously.24
The residual
copper levels are typically found to be on the order of 1 ppm by inductively
coupled plasma atomic emission analysis25
and the observed cytotoxicity could
not be correlated to the measured copper concentration.
The second step, precipitation into excess tetrahydrofuran, removes the 2,2’-
bipyridine ligand from the catalyst system. Although a significant fraction was
removed by the silica column, sufficient ligand remained to be detected by 1
H
NMR, although the concentration was too low for reliable quantification. After
precipitating the copolymer into excess tetrahydrofuran once, no ligand could be
detected by 1
H NMR.
The addition and evaporation of water to remove residual volatiles such as
methanol was found to be a necessary intermediate step before the final drying. If
the copolymer was washed or precipitated with another solvent such as diethyl
ether or dichloromethane, the final drying step gave an insoluble polymer. The
nature of this crosslinking was not investigated in detail, however.
Freeze-drying was applied for removal of water and the final drying step at high
temperature removed traces of water and volatiles.

105
After these drying steps, the cell viability for cells exposed to a 20.0 % w/v
copolymer gel over 72 h was in general more than 80 %. However, the rate of cell
proliferation was typically slower compared to cells not exposed to gels. As stated
above, this viability could not be correlated to the Cu content. In addition, the
methanol content was measured to be around 20-30 ppm of the copolymer by
Headspace Gas Chromatography.26
At these concentrations, methanol did not
affect cell viability.27
In order to examine the 2,2’-bipyridine ligand content of the copolymers, an
HPLC method was developed. 2,2’-bipyridine has a maximum absorption at 302
nm in 0.0125 M HCl, with an extinction coefficient of 1.47×104
M-1
·cm-1
.28
This
relatively high extinction coefficient allows for ligand detection down into the
micromolar range using a calibration. It was found that injected amounts of
5.00×10-11
mol, corresponding to 7.80×10-9
g could be measured quantitatively
and the ligand could be detected at even lower concentrations.
0 2 4 6 8 10 12 14 16 18 20
0.000
0.002
0.004
0.006
0.008
0.010
0.012
JMASh469 x 3
JMASh469 x 1
Polymer
bpy
Absorption(300nm),A.U.
Elution time / min
Figure 3.3: HPLC traces of a PHPMA90-PMPC200-S-S-PMPC200-PHPMA90 copolymer batch
(JMASh469) precipitated with tetrahydrofuran once (JMASh469 x 1) and thrice
(JMASh469 x 3). Conditions: 1 mL/ min, 5-100 % acetonitrile in 0.1 % aqueous TFA over 20
minutes. Column: GraceSmart R.P.18, 5µm. 150 mm x 4.6 mm

106
This is illustrated in Figure 3.3, where chromatograms of the same copolymer
batch are shown after a single precipitation into tetrahydrofuran (JMASh469 x 1)
and after three precipitations (JMASh469 x 3). The shift of the peak assigned to
the bipyridine ligand was commonly observed and may be due to either
concentration differences, temperature fluctuations or changes of the column
surfaces. The identity of the peak was confirmed by adding a bpy standard to the
sample (spiking). This led to an increase in the sample area, and not occurrence of
a second peak. The effect of repeated precipitations with tetrahydrofuran is
clearly seen as a substantial reduction in the area of the ligand peak. The results
for several copolymer batches, all with the same target composition are seen in
Table 3.3. The measured 2D cell viabilities of a 10.0 % copolymer gel are also
given for comparison.
Cell Viability
Entry Batch Name
µg bpy / g
polymer 24 h 48 h 72 h Average
1 JMASh469x3 20 4.3
b
106.9 92.3 94.7 98.0
2 JMASh352-2 20 16.3 ± 1.6 98.3 78.6 69.3 82.1
3 JMASh381-2 20 78.1 ± 2.8 85.0 85.1 72.5 80.9
4 JMASh234-2 20 83.5 ± 5.2 70.7 76.4 78.1 75.1
5 JMASh303-2 20 77.4 ± 1.9 104.7 83.3 74.3 87.5
6 JMASh268-2-2 20 107.6 ± 8.5 74.2 96.8 86.3 85.8
7 JMASh559 44.5 ± 0.2 N/A N/A N/A N/A
8
a
JMASh469x1 20 183.8 ± 5.7 N/A N/A N/A N/A
Table 3.3: 2,2’-Bipyridine content and measured 2D viability for a 10.0 % gel of a series of
copolymer batches with composition PHPMA~90PMPC200-S-S-PMPC200-PHPMA~90. Primary
human dermal fibroblast viability was assessed using a MTT assay and ThinCert inserts.
Cell viability studies were performed by K. Bertal and details of the assay can be found in
reference 29. a
This sample was only precipitated once into THF. The result of two further
precipitations is shown in entry 1. b
This measurement was only repeated once.
Entry 1 shows the copolymer that had been precipitated three times with
tetrahydrofuran. Its bpy content is around 4 µg bpy/g copolymer, the lowest of all
the copolymers, and in addition the viability is close to 100 % even after 72 h.
The remaining copolymers were all precipitated only once, and in most batches
there is between 40 and 110 µg bpy / g copolymer. The viabilities for most of the
copolymers are comparable but significantly higher than for the copolymer

107
containing ~4 µg bpy/g copolymer. There are, however, quite large deviations in
the viability data, which is expected since only a single repeat was performed and
there are significant uncertainties in cell counts, as well as between different
strains of cells. Nevertheless, the bpy content does seem to have an influence on
the viability. As 2,2’-bipyridine isomers are known to be toxic,30
this
contamination is believed to be a major contribution to the residual toxicity of the
gels.
10 11 12 13 14 15 16 17
Heat treated
Mn = 51,300
Mw/Mn = 1.46
No heat treatment
Mn = 52,300
Mw/Mn = 1.36
Elution time / min
Figure 3.4: Gel Permeation Chromatograms recorded for a PHPMA90-PMPC200-S-S-
PMPC200-PHPMA90 before and after being subjected to the heating protocol described in the
experimental section (80 °C for 48 h, followed by 90 °C for 5 h)
The relatively high temperatures employed in the purification protocol might
conceivably lead to elimination of the disulfide bonds. Unfortunately, the sulfur
content of these copolymers was below the microanalytical detection limit ( < 0.1
%). However, gelation can only occur with ABA triblock copolymers: the
corresponding disulfide-cleaved AB diblock copolymer does not form micellar
gels since there are no chains that can bridge between adjacent micelles. In
addition, GPC analyses did not show any significant copolymer degradation after
thermal treatment (Figure 3.4). Thus there is no experimental evidence to suggest

108
that disulfide cleavage / degradation occurs to any extent, either during the ATRP
synthesis of these copolymers or during their long-term storage.
3.3.4 Aqueous Solution Properties
10
-3
10
-2
10
-1
10
15
20
25
30
G',G''/Pa
Strain
G'
G''
Figure 3.5: Storage and loss modulus as a function of strain at 1 Hz for a 10.0 % w/v
aqueous gel of PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 at 37 °C. The graph shows three
consecutive measurements obtained for the same solution recorded directly after one
another.
Figure 3.5 shows the storage and loss moduli of a 10.0 % aqueous solution of
PHPMA88-PMPC200-S-S- PMPC200-PHPMA88 as a function of the strain at 37 °C.
At low strains (below approximately 0.005) the data are scattered due to the low
sensitivity of the instrument. From 0.005 to the highest measured value at 0.20,
the moduli are almost strain-independent, indicating linear viscoelasticity. The
figure shows three consecutive data sets obtained on the same solution recorded
directly after one another. As these are essentially identical, the solution has
completely relaxed between the measurements, demonstrating that increasing the
strain does not lead to any permanent changes in the copolymer gel.

109
Figure 3.6 shows the effect of increasing the temperature on the storage and loss
modulus of a 15.0 % PHPMA43-PMPC125-S-S-PMPC125-PHPMA43 copolymer
solution.
5 10 15 20 25 30 35 40 45 50
10
-4
10
-3
10
-2
10
-1
10
0
10
1
G',G''/Pa
T / °C
G' (0.5 °C/min concentric cylinder)
G'' (0.5 °C/min concentric cylinder)
G' (3 °C/min cone and plate)
G'' (3 °C/min cone and plate)
Figure 3.6: Comparison of rheometer geometry and heat rate for 15.0 % PHPMA43-
PMPC125-S-S-PMPC125-PHPMA43 copolymer solution in PBS (pH 7.2) at 1 rad/s, 0.5 Pa. The
measurement with the concentric cylinder was covered with a layer of paraffin oil to
suppress water evaporation.
Two different experimental set-ups were used; a cone-and-plate measuring cell at
a relatively fast heating rate of 3 °C/min and a concentric cylinder at a scan rate of
0.5 °C/min where evaporation was suppressed by a layer of paraffin oil. Since
evaporation was not suppressed using the cone-and-plate geometry, the validity of
this protocol was examined for one copolymer solution performing the same
measurement using a concentric cylinder set-up with the solution covered with
paraffin oil. It was found that these two types of measurements gave almost
identical results over the 0-50 °C range, indicating that effects due to evaporation
losses for the cone-and-plate geometry were negligible. In addition, the internal
dynamics of the system are sufficiently fast to allow the faster heating rate, which
significantly shortens measurement times.

110
Figure 3.7 shows the moduli as a function of heating and cooling of a 10.0 %
PHPMA88-PMPC200-S-S- PMPC200-PHPMA88 copolymer solution. Gelation is
reversible and the system exhibits very little hysteresis.
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
1
10
100
G' , heat
G'', cool
G', cool
G'' , heat
G',G''/Pa
Temperature / °C
Figure 3.7: Temperature-corrected heating and cooling scans of a 10.0 % aqueous solution
of PHPMA88-PMPC200-S-S- PMPC200-PHPMA88 copolymer. Conditions: 0.5 °C/min, 1.0 Hz,
0.5 Pa, concentric cylinders. The solution was covered with a layer of paraffin oil to suppress
water evaporation.
Figure 3.8 shows the effect of increasing the temperature on the storage and loss
moduli. For all the triblock copolymers, the same general trends are observed. At
low temperature, both the loss and the storage moduli are low. The loss modulus
is always higher than the storage modulus, which is typical for a free-flowing
liquid. On increasing the temperature, both moduli increase by one to two orders
of magnitude to reach an almost constant high level. This increase occurs
gradually over a temperature range of 20 °C to 30 °C. The exact values of the
moduli, as well as the onset temperature, vary according to the copolymer
composition. The storage moduli for copolymers with a PMPC block DP of ~250
are around 0.002 Pa at low temperature. On increasing the solution temperature,

111
the storage moduli for PHPMA43-PMPC125-S-S-PMPC125-PHPMA43 (Table 3.1,
Entry 3) and PHPMA66-PMPC126-S-S-PMPC126-PHPMA66 (Table 3.1, Entry 4)
begin to increase at around 20 °C.
0 5 10 15 20 25 30 35 40 45 50 55 60
10
-3
10
-2
10
-1
10
0
10
1
PHPMA66
-PMPC126
-S-S-PMPC126
-PHPMA66
PHPMA43
-PMPC125
-S-S-PMPC125
-PHPMA43
PHPMA88
-PMPC200
-S-S-PMPC200
-PHPMA88
PHPMA70
-PMPC390
-PHPMA70
T / °C
G'/Pa
PHPMA30
-PMPC127
-S-S-PMPC127
-PHPMA30
0 5 10 15 20 25 30 35 40 45 50 55 60
10
-3
10
-2
10
-1
10
0
10
1
PHPMA66
-PMPC126
-S-S-PMPC126
-PHPMA66
PHPMA30
-PMPC127
-S-S-PMPC127
-PHPMA30
PHPMA43
-PMPC125
-S-S-PMPC125
-PHPMA43
PHPMA70
-PMPC390
-PHPMA70
PHPMA88
-PMPC200
-S-S-PMPC200
-PHPMA88
T / °C
G''/Pa
Figure 3.8: Temperature dependence of: (A) storage and (B) loss moduli of various 10.0 w/v
% PHPMA-PMPC-PHPMA copolymer solutions in PBS buffer (pH 7.2). Conditions: 1
rad/s, 3 °C/min, 0.5 Pa.
A
B

112
This increase continues until a modulus of 4 Pa is reached at around 50 °C, and
the two curves are almost indistinguishable. On the other hand, the storage
modulus for PHPMA30-PMPC127-S-S-PMPC127-PHPMA30 (Table 3.1, Entry 2)
does not increase until a temperature of 45 °C is reached (Figure 3.8A). The final
value of 0.01 Pa is attained at a temperature of 55 °C. Thus, this increase
commences at significantly higher temperature and the relative increase is more
than two orders of magnitude lower than for the two other copolymers.
The development of the loss modulus with temperature shows greater variation
(Figure 3.8B). The copolymers with an PMPC DP of 250 have a loss modulus of
0.01 Pa at 4 °C, which is five times that of the storage modulus. The loss modulus
of PHPMA43-PMPC125-S-S-PMPC125-PHPMA43 and PHPMA66-PMPC126-S-S-
PMPC126-PHPMA66 starts to increase immediately, with the latter copolymer
solution increasing more rapidly. The final value of the latter copolymer reaches
approximately 10 Pa at 50 °C, which is three times that of the former. The loss
modulus of PHPMA30-PMPC127-S-S-PMPC127-PHPMA30 is approximately
constant up to 40 °C, where an order of magnitude increase is observed over a 15
°C range. In all cases, the loss modulus is larger than the storage modulus for 10
% copolymer solutions with PMPC DPs of 250. Copolymers with a DP of ~400
for the central PMPC block also exhibit an increase in moduli with temperature,
but there are significant differences with increasing molecular weight. A 10.0 w/v
% aqueous solution of PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 solution has
a storage modulus of 0.003 Pa at 0 °C. On heating, this modulus increases rapidly
up to 20 Pa at 30 °C. The loss modulus (G’’) follows a similar trend but starts at
0.08 Pa, which is significantly higher than the corresponding storage modulus.
G’’ increases with temperature to reach a final value of 10 Pa at 40 °C. Thus, if a
gel is defined as a substance that has a larger storage modulus than its loss
modulus, then this aqueous copolymer solution exhibits thermally-induced
gelation.31,32
A 10.0 w/v % aqueous solution of PHPMA70-PMPC390-PHPMA70
has a storage modulus of 0.12 Pa at 0 °C, which is significantly higher than that
for PHPMA88-PMPC200-S-S-PMPC200-PHPMA88. The loss modulus at this
temperature is only slightly higher at 0.2 Pa. Increasing the temperature leads to
an increase in both moduli, but the increase in G’ is more rapid than that of G’’,
so this aqueous solution also undergoes thermally-induced gelation.

113
The temperature at which G’ equals G’’ is designated the critical gelation
temperature, Tgel. Below Tgel, the copolymer solution behaves like a liquid, while
above Tgel it behaves as an elastic solid. It was found empirically that Tgel
generally increased at lower applied frequencies (data not shown). This behavior
is typical for thermo-responsive ABA block copolymer gels in which the physical
cross-links have a relatively short residence time (in the present case, this is
because the PHPMA blocks are only weakly hydrophobic, rather than strongly
hydrophobic).31
A frequency of 1 rad s-1
was chosen because this is equivalent to
a mean residence time of 1 s, which corresponds to the approximate time scale
that characterizes simple tube-inversion experiments.32

114
0 5 10 15 20 25 30
5
10
15
20
25
30
35
40
45
50
PHPMA70
-PMPC390
-PHPMA70
PHPMA88
-PMPC198
-S-S-PMPC198
-PHPMA88
TGel
/°C
PHPMA43
-PMPC125
-S-S-PMPC125
-PHPMA43
Copolymer concentration / w/v %
0 5 10 15 20 25 30
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
PHPMA70
-PMPC390
-PHPMA70
G'( ),G''( )
PHPMA88
-PMPC198
-S-S-PMPC198
-PHPMA88
G'( ),G''( )
PHPMA43
-PMPC125
-S-S-PMPC125
-PHPMA43
G'( ),G''( )
G',G''/Pa
Copolymer concentration / w/v %
Figure 3.9: (A) Critical gelation temperature (Tgel) as a function of copolymer concentration
for three PHPMA-PMPC-PHPMA triblock copolymers; (B) storage and loss moduli
determined at 37 °C as a function of copolymer concentration for the same three
copolymers. Vertical arrows indicate the critical copolymer concentration required for
gelation in each case.
Figure 3.9A shows Tgel plotted against the copolymer concentration where a
cross-over point was obtained at concentrations up to 30 w/v %. For the
A
B

115
remaining copolymers, G’ was always lower than G’’ at all temperatures and
concentrations measured, indicating no gelation. Tgel decreases on increasing the
copolymer concentration. PHPMA70-PMPC390-PHPMA70 and PHPMA88-
PMPC200-S-S-PMPC200-PHPMA88 behave differently at lower concentration, with
the former copolymer forming a free-standing gel event at 4.0 w/v %. The latter
does not form a gel below 6.0 w/v %, but at 7.5 w/v % (or higher) their gelation
behavior is remarkably similar. PHPMA43-PMPC125-S-S-PMPC125-PHPMA43 also
exhibited thermally-induced gelation, but only above 19.0 w/v %. The
corresponding moduli are shown in Figure 3.9b. G’ and G’’ both increase with
concentration, as expected. This is because these parameters are related to the
number of elastically active chains,33
which are expected to increase at higher
copolymer concentration due to the larger probability of overlap between
neighboring aggregates.

116
A
B
Low concentration
(<0.1 %) molecularly
Increased concentration
(0.1-5 %) micelle formation/aggregation
High concentration
(>5 %) network formation
Figure 3.10: Two pathways to formation of physical networks: (A) If the end-blocks are
highly incompatible with the solvent, ‘flower micelles’ are formed at relatively low
concentration. Increasing the concentration leads eventually to overlap where bridging is
facilitated and this leads to a micellar gel network. (B) If the end-blocks are more compatible
with the solvent, a looser structure is formed at intermediate concentrations as the penalty of
‘dangling ends’ is lower. This eventually leads to a network structure on increasing the
concentration, however, the constituents of this network are less well-defined than in the
case of the micellar gel. If the solvent compatibility changes with temperature, this may
cause formation of a well-defined micellar network gel.
The gelation of ABA-type triblock copolymers with solvent-incompatible outer A
chains has been considered in a number of theoretical studies.1,2,5,34
Generally
speaking, the storage modulus is proportional to the number of elastically active
chains and the absolute temperature.31,35
The number of elastically active chains
can be related to the aggregation number if the copolymer concentration is
constant. In general, a higher aggregation number provides more elastically active
chains due to the larger amount of network junctions.2,31
If the incompatibility of
the end-blocks with the solvent is high, so-called ‘flower’ micelles are formed.
These consist of a core of the solvent-incompatible groups with relatively high
aggregation numbers, with the ‘petals’ made up of the lyophilic central block,
and the end-blocks typically being located within the same micelle Figure
3.10A).3,4,31
The central block is entropically constrained, so if the incompatibility

117
of the end-groups is low, the aggregation of end-blocks in larger, looser structures
in co-existence with molecularly dissolved unimers is favoured.5,7
These looser
aggregates tend to have much lower aggregation numbers than ‘flower’ micelles
(Figure 3.10B).5
The rheological behavior may therefore be due to an increase in the aggregation
number at higher temperature. Figure 3.11 shows the raw light scattering
intensity for selected copolymers at 0.1 w/v %. This low copolymer concentration
was selected to suppress the formation of large aggregates due to inter-micellar
bridging. Thus, these studies probe the early onset of gelation (i.e. micellar self-
assembly), rather than the free-standing gels themselves. Copolymers with an
PMPC block of DP 250 (Table 3.1, Entries 2-4), exhibit similar scattering
intensities at low temperatures. Increased scattering is observed at higher
temperatures, suggesting a higher degree of aggregation.

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
3x10
2
5x10
2
7x10
2
8x10
2
1x10
3
2x10
3
3x10
3
5x10
3
PHPMA43
-PMPC125
-S-S-PMPC125
-PHPMA43
PHPMA49
-PMPC150
-S-S-PMPC150
-PHPMA49
PHPMA88
-PMPC200
-S-S-PMPC200
-PHPMA88
PHPMA70
-PMPC390
-PHPMA70
Lightscatteringintensityat173°/kcps
T / °C
PHPMA66
-PMPC126
-S-S-PMPC126
-PHPMA66
PHPMA30
-PMPC127
-S-S-PMPC127
-PHPMA30
Figure 3.11: Temperature dependence of the light scattering intensity at 173 ° for 0.10 %
aqueous solutions of six triblock copolymers in PBS buffer (pH 7.2).
Each copolymer was examined by temperature-dependent light scattering in PBS.
The scattering intensity for PHPMA30-PMPC127-S-S-PMPC127-PHPMA30 and
PHPMA66-PMPC126-S-S-PMPC126-PHPMA66 increases less rapidly with

118
temperature than that for PHPMA43-PMPC125-S-S-PMPC125-PHPMA43. The
greater scattering intensity obtained on increasing the PHPMA chain length from
30 to 43 is also believed to be mainly due to a higher aggregation number. This
behavior is observed for functionally similar triblock copolymers36
and is related
to the lower aqueous solubility of the longer PHPMA chains. However, the
reduced scattering observed on further increasing the PHPMA DP to 66 is
somewhat unexpected.36
One possibility is that triblock copolymers with longer
PHPMA blocks are more prone to phase separation. Such precipitation would
remove scatterers from the solution. Indeed, the rheology data obtained for this
copolymer are not in disagreement of this hypothesis (Figure 3.8). However,
visual inspection of this copolymer solution after heating did not indicate any
precipitation. Another possibility is that the aggregation number attains a
maximum value and then decreases at higher DP. Such behavior has been
observed in certain Pluronic copolymer solutions when the
hydrophobic/hydrophilic block ratio is increased.37
An alternative explanation for
the rheological data is that the higher aggregation number from the longer
hydrophobic blocks leads to a lower physical crosslink density, since more
copolymer chains are associated.38
This would inevitably lead to weaker gels. The
light scattering data presented here does not allow us to distinguish between these
scenarios. The scattering from copolymers with an PMPC central block of
DP~400 (Table 3.1, Entries 1 and 7) is approximately ten times higher at low
temperature than for copolymers with an PMPC DP of 250 or 300. The increased
scattering intensity with temperature is very similar for these two copolymers.
This correlates well with the GPC and rheology data. The higher scattering
intensity indicates larger aggregates than those obtained with shorter copolymers.
This is consistent with the behavior observed in Figure 3.9, where gelation occurs
at lower concentrations. For all copolymers, there is an increase in the light
scattering intensity between 10 °C and 20 °C.

119
0.1 1 10 100 1000
Time (µs)
1 10 100 1000
Time (µs)
1 10 100 1000
Time (µs)
1.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.1 1 10 100 1000
Time (µs)
37 °C
19 °C
4 °C
1 10 100 1000
Time (µs)
1 10 100 1000
Time (µs)
C(τ)
1.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
37 °C
19 °C
4 °C
37 °C
19 °C
4 °C
37 °C
19 °C
4 °C
37 °C
19 °C
4 °C
37 °C
19 °C
4 °C
C(τ)
Figure 3.12: Autocorrelation functions obtained from dynamic light scattering studies of six triblock copolymers (0.10 w/v % aqueous solutions in PBS
buffer, pH 7.2 at 4 °C, 19 °C and 37 °C. Scattering angle = 173 ° in each case.
PHPMA66-PMPC126-S-S-PMPC126- PHPMA66 PHPMA43-PMPC125-S-S-PMPC125-PHPMA43 PHPMA30-PMPC127-S-S-PMPC127-PHPMA30
PHPMA49-PMPC150-S-S- PMPC150-PHPMA49 PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 PHPMA70-PMPC390-PHPMA70

120
Correlation functions obtained for 0.1 w/v % copolymer solutions are shown in
Figure 3.12 at 4 °C, 19 °C and 37 °C. Analysis of these correlation functions
using cumulants analysis was attempted so as to obtain intensity distributions of
the decay rates (and thereby the diffusion coefficients). Unfortunately, the data
fits were quite poor, especially below 20 °C. This is most likely due to the ill-
defined, highly polydisperse nature of the aggregates. Nevertheless, each
correlation function clearly shows an increase in the diffusion coefficient on
increasing the temperature, indicating a concomitant reduction in the
hydrodynamic radius. This behavior is consistent with a transition from a mixture
of loose aggregates and unimers at low temperature to a more compact micellar
structure at higher temperature. The diffusion coefficient of the aggregate/unimer
mixture should be low, since the scattering signal is dominated by the spatially
large, low density aggregates. On the other hand, the flower micelles have a
higher aggregation number but are smaller and denser, leading to greater
diffusional mobility at higher temperatures. Cumulants analyses of 0.1 w/v %
aqueous solutions above 20 °C indicated hydrodynamic radii of around 30 nm for
copolymer micelles with PMPC DPs of 250-300 and 80 nm for copolymer
micelles with an PMPC DP of 400; these micelle dimensions do not vary very
much from 20 °C to 60 °C. TEM images of a 0.4 w/v % PHPMA88-PMPC200-S-S-
PMPC200-PHPMA88 aqueous solution dried at 25 °C (Figure 3.13) indicate the
presence of spherical aggregates with radii of 20-30 nm, which is significantly
less than the 80 nm radius indicated by DLS for this particular copolymer.

121
Figure 3.13: Transmission electron microscopy images of dried ‘flower-like’ micelles
obtained by drying a 0.40 w/v % aqueous solution of PHPMA88-PMPC200-S-S-PMPC200-
PHPMA88, followed by staining with uranyl acetate. H. Lomas is acknowledged for the
image.
However, DLS is known to over-size relative to TEM, particularly for
polydisperse systems. Moreover, the negative staining protocol used in the TEM
specimen preparation primarily emphasizes the hydrophobic micelle cores,
whereas DLS ‘sees’ the highly hydrated micelle coronas as well. Also significant
shrinkage may occur on drying these micelles prior to TEM examination. In
addition, since these triblock copolymer micelles are strongly associative,
micellar aggregates may be present even at low copolymer concentration. Such
aggregates would dominate the light scattering measurements due to their much
greater scattering intensity. Nevertheless, the image shown in
Figure 3.13 confirm that spherical aggregates are formed at room temperature,
which is consistent with the formation of ‘flower’ micelles.
Micellar self-assembly was also examined by variable temperature 1
H NMR
studies of PHPMA30-PMPC127-S-S-PMPC127-PHPMA30 and PHPMA88-PMPC200-
S-S-PMPC200-PHPMA88 in D2O at 7.0 w/v %. The integrated peak intensities of

122
the pendent methyl group of the PHPMA block and the ammonio-methylene
group due to the PMPC block were calculated and compared to that observed in
CD3OD, which is a good solvent for both blocks. The results are shown in Figure
3.14.
0 5 10 15 20 25 30 35 40
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
PHPMA88
-PMPC200
-S-S- PMPC200
-PHPMA88
PHPMA66
-PMPC126
-S-S- PMPC126
-PHPMA66
PHPMA43
-PMPC125
-S-S- PMPC125
-PHPMA43
PHPMA30
-PMPC127
-S-S- PMPC127
-PHPMA30
ApparentPHPMAcontentof
triblockcopolymer(normalizedwith
respecttofullysolvatedcopolymer)
T / °C
Figure 3.14: Temperature dependence of the apparent PHPMA contents of 7.0 w/v %
solutions of four triblock copolymers in D2O normalized with respect to their corresponding
block compositions determined in CD3OD. The apparent reduction in PHPMA content that
occurs on increasing the temperature indicates poorer solvation and/or lower mobility.
Spectra recorded at 21 °C in D2O and CD3OD were obtained using a 400 MHz spectrometer,
the remaining spectra were recorded at a 500 MHz spectrometer.
For both copolymers, the peak intensity ratio is less than unity, even at
temperatures well below 20 °C. This indicates that the PHPMA block is not fully
solvated under these conditions. As expected, the relative degree of hydration of
the PHPMA blocks is higher for the PHPMA30-PMPC127-S-S-PMPC127-
PHPMA30 copolymer, which supports the hypothesis that the shorter PHPMA
blocks are more easily solvated. This is reasonable, since the HPMA monomer is
water-soluble. Heating above 20 °C leads to a 33 % reduction in the normalized
PHPMA signal, which indicates that the PHPMA chains become less mobile, as
expected. This degree of attenuation is less than that observed for the
corresponding PNIPAM-based triblock copolymers.12,16
Thus, a significant
proportion of the PHPMA chains remain solvated, either as molecularly dissolved

123
unimers or as ‘dangling ends’. The transition around 20 °C for the semi-
concentrated 7.0 w/v % copolymer solutions corresponds quite well with the
onset of the increase in the light scattering signal observed in more dilute solution
(Figure 3.11). Moreover, preliminary tensiometry studies do not show any
difference in surface activity below and above 20 °C (not shown).
In summary, the combined rheology, DLS, TEM and 1
H NMR data are consistent
with a gradual transition from loose, ill-defined aggregates and unimers at low
temperatures to relatively well-defined, interacting flower micelles at higher
temperatures. The former state is predicted by Monte Carlo simulations,
particularly for weak segregation,8
and experimental evidence for such structures
has been reported.5
Stronger segregation favors flower micelle formation: in the
present case, this is achieved by increasing the solution temperature, which
increases the hydrophobic character of the PHPMA chains. This is schematically
shown in Figure 3.10. The precise molecular event(s) involved in this transition
has not been identified, but given the literature data for related thermo-responsive
copolymers such as PNIPAM39
and PPO,40,41
as well as the 1
H NMR studies
presented in Figure 3.14, it seems reasonable to suggest that dehydration of the
pendent methyl groups of the PHPMA chains may well be a driving force.
3.3.5 Cleavage of disulfide bonds in disulfide-based triblock copolymer gels
with dithiothreitol (DTT)
It is well known that disulfide bonds can be cleaved by mild reducing agents such
as dithiothreitol (DTT).19
If the central disulfide bond is cleaved, the triblock
chains are converted into PHPMA–PMPC-SH diblock chains that are
approximately half of the original copolymer molecular weight. This is illustrated
in Scheme 3.3 and Figure 3.15A. More importantly, the inter-micellar bridges in
the 3D gel network are destroyed, which should lead to rapid gel dissolution.
Hence the central disulfide bond acts as a ‘keystone’ for the gel, as recently
demonstrated for the analogous thermo-responsive PNIPAM-PMPC-S-S-PMPC-
PNIPAM copolymers.42
Cleavage of the disulfide bonds in free-standing gels
formed from 10.0 % w/v PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 was
conducted with varying DTT / disulfide molar ratios, see Figure 3.15B.

124
DTT
Free-standing micellar gel at 37 oC Free-flowing micelles at 37 oC
pH 7.4, 37 oC
S
S
S
S
S S
S S
S
S
S S
S
S
SS
Scheme 3.3: Chemical degradation of the free-standing aqueous micellar gel formed by the
PHPMA–PMPC-S-S-PMPC–PHPMA triblock copolymer after cleavage of the disulfide
bonds by using dithiothreitol (DTT).
If no DTT is added to a 10.0 % copolymer solution, no change is observed in the
complex viscosity over time, provided that water evaporation is suppressed. This
is ensured by using a concentric cylinder set-up, whereby a thin layer of paraffin
oil on top of the aqueous copolymer gel minimizes evaporation losses, at least on
the time scale of the rheological measurements. A measurable reduction in
viscosity is observed when using a DTT/disulfide molar ratio of 1.0. However, the
final viscosity is still around 0.5 Pa·s, which corresponds to a viscous solution. On
further ageing of this degraded copolymer solution in the presence of air at 37 °C,
the viscosity increases. This is due to (i) combination of re-oxidized thiol end-
groups and/or (ii) evaporation of water, leading to a higher copolymer
concentration. In contrast, when 2-10 equivalents of DTT are added to the free-
standing gel, its complex viscosity is reduced to below the limit of detection of
the rheometer (i.e. approximately 3×10-2
Pa·s) within approximately 11 minutes.
The precise time required for complete gel dissolution clearly depends on the
amount of reductant added. As expected, higher DTT/disulfide molar ratios lead
to greater rates of chemical degradation and hence gel dissolution.

125
10 11 12 13 14 15 16
Retentiontime / min
0 1 2 3 4 5 6 7 8 9 10 11
0.01
0.1
1
DTT:S-S = 10.0
DTT:S-S = 5.0
DTT:S-S = 2.0
DTT:S-S = 1.0
Complexviscosity/Pa.s
t / min
DTT:S-S = 0
Figure 3.15: (A) Gel permeation chromatograms recorded for a PHPMA88-PMPC200-S-S-
PMPC200-PHPMA88 triblock copolymer before and after exposure to DTT. Conditions:
DTT/S-S molar ratio = 10, methanol, 25 °C, 12 h. (B) Kinetics of gel dissolution caused by
cleavage of the disulfide bonds in a 10.0 w/v % gel comprising a PHPMA88-PMPC200-S-S-
PMPC200-PHPMA88 copolymer in PBS buffer (pH 7.2) at 37 °C using DTT/disulfide molar
ratios of 10.0, 5.0, 2.0, 1.0 and zero.
3.3.6 Cleavage of disulfide bonds in disulfide-based triblock copolymer gels
with glutathione
Although DTT is not biologically relevant, similar thiol-disulfide redox chemistry
occurs in biological systems. For example, glutathione is present in millimolar
concentrations within mammalian cells.43
This thiol-containing tripeptide has
already been shown to lead to reductive dissolution of PNIPAM-PMPC-S-S-
PMPC-PNIPAM copolymer gels.42
Preliminary studies indicate that the
PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 gels can be cleaved with
glutathione as well, although the time-scale is on the order of days for a 1.50 %
copolymer solution (Figure 3.16). Under these conditions, avoiding evaporation
losses and aerial oxidation becomes important. This is in contrast to the data
presented in Figure 3.15B, where the cleavage is on the order of minutes using
DTT. The GPC data for the copolymers in Figure 3.16 are very different from the
GPC data presented earlier on (Table 3.1) which serves to illustrate the relative
nature of the size-exclusion chromatography method; GPC measures the
hydrodynamic radius of a polymer, rather than its molecular weight, provided
there is no interaction with the column. Thus the numbers obtained are related to
the hydrodynamic radius of the polymer relative to the hydrodynamic radii of the
(PHPMA88-PMPC200S)2
Mn = 89,500
Mw/Mn = 1.35
PHPMA88-PMPC200SH
Mn = 45,200
Mw/Mn = 1.47
A B

126
calibration standards. Since the two GPC protocols apply different columns,
solvent mixtures and sets of calibration standards are used the observed
differences are not too surprising. In addition, the measured molecular weights are
in general smaller than the values calculated for the 3:1 chloroform:methanol
protocol but too large for the 7:3 water:methanol protocol. This may indicate that
there is a contribution to the retention time from column adsorption in the former
case and possibly aggregation in the latter case. Nevertheless, the observed
decrease in the molecular weight is significant and can only be due to disulfide
cleavage, since no other parameters have changed.
11 12 13 14 15 16 17 18
t = 23.2 h
Mn = 221,700
Mw/Mn = 1.57
t = 2.4 h
t = 1 h
t = 0
Mn = 327,500
Mw/Mn = 1.51
RISignal
Elution time / min
Figure 3.16: Cleavage of a 1.0 % solution of a 1.5 % w/v PHPMA88-PMPC200-S-S-PMPC200-
PHPMA88 copolymer solution, 9 eq. glutathione, N2-purged PBS pH 7.2, 37 °C. GPC
conditions: 70 % 0.2 M NaNO3, 0.01 M NaH2PO4, adjusted to pH 7.0; 30 % methanol.
Calibrated with near-monodisperse poly(sodium 4-styrenesulfonate) standards.
In addition, several redox reactions are known to occur within human skin.44
The
assessment of the effect of these reactions on the gel dissolution is expected to be
challenging and is outside the scope of the current study.

127
3.3.7 Properties of thiol-terminated diblock copolymers
Detailed characterization of the thiol-functionalized PMPC-PHPMA micelles is
of interest but has not been examined in depth in the current work due to time
constraints. Briefly, an increase in count rate is observed on increasing the
temperature of a dilute aqueous solution of such diblock copolymer micelles,
similar to that observed for the corresponding triblock copolymers at the same
copolymer concentration. The overall count rate of the reduced copolymer is
always lower at all temperatures, suggesting a reduced degree of aggregation (see
Figure 3.17).
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
1x10
3
1x10
3
2x10
3
3x10
3
4x10
3
5x10
3
PHPMA88
-PMPC200
-SH
PHPMA88
-PMPC200
-S-S-PMPC200
-PHPMA88
Lightscatteringintensityat173°C/kcps
T / °C
Figure 3.17: Temperature dependence of the light scattering intensity at 173 ° for 0.10 %
aqueous solutions of PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 and PHPMA88-PMPC200-
SH in PBS buffer (pH 7.2). PHPMA88-PMPC200-SH was prepared by adding 2000 equivalent
of DTT to the 0.1 % PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 solution, leaving this for
10 minutes at 25 °C followed by filtering through a 0.22 µm nylon filter immediately before
starting the measurement.
3.4 Conclusions
In summary, a series of thermo-responsive ABA triblock copolymer gelators
based on PMPC and PHPMA was synthesized. Depending on their copolymer

128
molecular weights and relative block compositions, these copolymers can form
transparent free-standing gels in aqueous solution. More specifically, gels are
obtained for copolymers with PHPMA contents of between 14 and 19 wt. %. The
critical gelation temperature and gel strength are strongly dependent on the
copolymer concentration, thus judicious selection of a particular triblock
copolymer and its solution concentration allows the formation of gels with desired
physical properties. Dynamic light scattering, TEM and 1
H NMR studies indicate
that gelation is due to the self-assembly of individual copolymer chains to form a
micellar gel network, with bridging chains between adjacent micelles. The
introduction of a central disulfide bond within these bridges allows rapid de-
gelation to be achieved under mild conditions using reductants such as DTT. In
addition, K. Bertal has shown that these copolymer gels had no significant
adverse effects when placed directly on tissue-engineered skin under conditions
that mimic those found for human skin in a related work.29
Thus these copolymers
appear to offer some potential as wound dressings.
3.5 References
1
Balsara, N. P., Tirrell, M., Lodge, T. P. Macromolecules 1991, 24, 1975-1986
2
Semenov, A. N., Joanny, J.-F., Khokhlov, A. R. Macromolecules 1995, 28,
1066-1075
3
4
Zhang, K., Macdonald, P. M., Menchen, S. Langmuir 1997, 13, 2447-2456
5
Raspaud, E., Lairez, D., Adam, M., Carton, J.-P. Macromolecules 1994, 27,
2956-2964
6
Gotzamanis, G. T. , Tsitsilianis, C. , Hadjiyannakou, S. C. , Patrickios, C. S. ,
Lupitskyy, R. , Minko, S. Macromolecules 2006, 39, 678-683
7
Alami, E., Almgren, M., Brown, W., Francois, J. Macromolecules 1996, 29,
2229-2243
8
Wang, Y., Mattice, W. L., Napper, D. H. Macromolecules 1992, 25, 4073-
4077
9
Ma, Y., Tang, Y., Billingham, N. C., Armes, S. P. Biomacromol. 2003, 4,
864-868
10
11
12
Li, C., Tang, Y. , Armes, S. P., Morris, C. J., Rose, S. F., Lloyd, A. W.,
13
Heskins, M., Guillet, J. J. Macromol. Sci. Chem. 1968, A2, 1441-1455
14
Wu, C., Wang, X. Phys. Rev. Lett. 1998, 80, 4092-4094
15
Hayashi, M., Tanii, H., Horiguchi, M., Hashimoto, K. Arch. Toxicol. 1989,
63, 308-313

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16
Li, C., Buurma, N. J., Haq, I., Turner, C., Armes, S. P., Castelletto, V.,
Hamley, I. W., Lewis, A. L. Langmuir 2005, 21, 11026-11033
17
18
Madsen, J., S. P. Armes, S. P, Lewis, A. L. Macromolecules 2006, 39, 7455-
7457
19
20
21
2002, 35, 1152-1159
22
Matyjaszewski, K., Xia, J. Chem. Rev. 2001, 101, 2921-2990
23
Li, Y., Armes, S.P. Macromolecules 2005, 38, 8155-8162
24
Ma, I.Y., Lobb, E.J., Billingham, N.C., Armes, S.P., Lewis, A. L., Lloyd,
A.W., Salvage, J. Macromolecules 2002, 35, 9306-9314
25
These analyses were performed by the Centre for Chemical Instrumental
Analysis and Services (CCIAS), Department of Chemistry, University of
Sheffield, UK.
26
These analyses were performed by Biocompatibles UK Ltd.
27
Unpublished data by K. Bertal, Department of Engineering Materials,
University of Sheffield, UK. Fibroblast activity was essentially unaffected by
aqueous methanol containing up to 0.10 % v/v methanol.
28
Westheimer, F. H., Benfey, O. T. J. Am. Chem. Soc. 1956, 78, 5309-5311
29
Madsen, J., Armes, S. P., Bertal, K., Lomas, H., MacNeil, S., Lewis, A. L.
Biomacromol. 2008, 9, 2265–2275
30
Li, S., Crooks, P.A., Wei, X., de Leon, J. Cr. Rev. Tox. 2004, 34, 447–460
31
Fairclough, J. P. A., Norman, A. I. Ann. Rep. Sect. "C" (Phys. Chem.) 2003,
99, 243-276
32
Almdal, K., Dyre, J., Hvidt, S., Kramer, O. Pol. Gels Netw. 1993, 1, 5-17
33
Tanaka, F. Pol. J. 2002, 34, 479-509
34
Pham, Q. T., Russel, W. B., Thibeault, J. C., Lau, W. Macromolecules 1999,
32, 5139-5146
35
Green, M. S., Tobolsky, A. V. J. Chem. Phys. 1946, 14, 80-92
36
Booth, C., Attwood, D., Price, C. Phys. Chem. Chem. Phys. 2006, 8, 3612-
3622
37
Linse, P. Macromolecules 1993, 26, 4437-4449
38
Vermonden, T., Besseling, N. A. M., van Steenbergen, M. J. , Hennink, W. E.
Langmuir 2006, 22, 10180-10184
39
Maeda, Y., Higuchi, T., Ikeda, I. Langmuir 2000, 16, 7503-7509
40
Cabana, A., Aїt-Kadi, A., Juhász, J. J. Coll. Int. Sci. 1997, 190, 307-312
41
42
Li, C., Madsen, J., Armes, S. P., Lewis, A. L. Angew. Chem. Int. Ed. 2006,
45, 3510-3513
43
Carelli, S., Ceriotti, A., Cabibbo, A., Fassina, G., Ruvo, M., Sitia, R. Science
1997, 277, 1681-1684
44
Moseley, R., Hilton, J. R., Waddington, R. J., Harding, K. G., Stephens, P.,
Thomas, D. W. Wound Rep. Reg. 2004, 12, 419-429

Chapter 4: Preparation and Aqueous Solution Properties of Thermoresponsive Biocompatible AB Diblock Copolymers
130
Chapter 4: Preparation and Aqueous Solution
Properties of Thermoresponsive
Biocompatible AB Diblock Copolymers

131
4.1 Introduction
The aggregation behavior of amphiphilic AB diblock copolymers in solvents that
are selective for one of the blocks has been of considerable interest for several
decades.1-5
A wide range of aggregate morphologies have been identified,
including spherical micelles, cylindrical micelles and vesicles.6,7
If the selective
solvent is water, such self-assembled nano-structures have potential biomedical
and pharmaceutical applications for controlled drug release.3,8,9
Probably the most
extensively investigated system comprises copolymers of water-soluble PEO, and
thermo-responsive PPO.1
Many other examples of PEO-based diblock
copolymers, where the second block comprises either a permanently hydrophobic
or a stimulus-responsive block, have been reported over the last decade or so.1,3,4
One alternative to PEO is PMPC, which can be readily prepared via ATRP.10,11
The MPC repeat units are biomimetic, thus MPC-based copolymers confer
clinically proven biocompatibility on a range of surfaces, including coronary
stents, ear grommets, soft contact lenses and artificial hip joints.12-14
Examples of
pH-responsive PMPC-based diblock copolymers were recently reported, where
the second block comprises PDPA.15,16
PDPA is highly hydrophobic in its neutral
form at physiological pH, thus these PMPC-PDPA diblock copolymers form
micellar or vesicular aggregates, depending on the relative block lengths. Below
pH 6.3, the PDPA blocks become protonated, causing molecular dissolution of
the copolymer chains. These vesicular aggregates can be used to deliver DNA
efficiently to cell nuclei, with high transfection efficiencies being achieved.8,9
In
addition, a series of ABA triblock gelators, where the B blocks comprise PMPC
and the A blocks comprise various stimulus-responsive blocks such as PDPA,17
PNIPAM18,19
or PHPMA20,21
was recently reported. The PDPA-based triblocks
proved to be efficient pH-responsive gelators.17
On the other hand, both the
PNIPAM- and PHPMA-based triblocks were found to be thermo-responsive
gelators.18-21
Copolymer gelators based on PNIPAM exhibited a critical gelation
temperature close to that reported for PNIPAM homopolymer,18
whereas the
gelation properties of the PHPMA-PMPC-PHPMA triblock copolymers were
highly dependent on the copolymer composition and concentration.20,21
In

132
addition, these latter copolymer gels did not exhibit any cytotoxicity, making
them potential candidates for biomedical application such as wound dressings.
The thermoresponsive behavior of the PHPMA-PMPC-PHPMA triblock
copolymers was initially unexpected, since PHPMA homopolymer is known to be
water-insoluble.22,23
In the present chapter the synthesis of a series of analogous
PMPC-PHPMA diblock copolymers is reported. In contrast to the earlier triblock
copolymers, these diblocks do not form inter-connected gel networks. This
fundamental difference simplifies their aqueous solution behavior, which was
studied by means of variable temperature dynamic light scattering and variable
temperature 1
H NMR spectroscopy. In particular, our aim was to examine the
effect of varying the mean degree of polymerization of the PHPMA block at a
fixed PMPC block length.
4.2.1 Materials
donated by Biocompatibles Ltd., UK. 2-Hydroxypropyl methacrylate (HPMA)
was donated by Cognis Performance Chemicals (Hythe, UK). Basic alumina
(Brockmann I, standard grade, ~150 mesh, 58 Å), anhydrous methanol (MeOH
99.8 %), copper(I)bromide (CuBr, 99.999 %), 4-(dimethylamino)pyridine
(DMAP, 99%) and 2,2’-bipyridine (bpy, 99 %) were all purchased from Sigma-
Aldrich UK and used as received. The silica gel 60 (0.063 – 0.200 µm) used to
remove the spent ATRP catalyst was purchased from E. Merck (Darmstadt,
Germany) and was also used as received. 2-Phenoxyethanol (99 %) and lithium
bromide (LiBr, 99+ %) was from Acros Organics and used as received.
Magnesium sulfate (MgSO4), sodium hydrogen carbonate (NaHCO3), sodium
chloride (NaCl) and triethylamine (Et3N) were laboratory reagent grade from
Fisher Scientific (Loughborough, UK) and used as received. Dichloromethane,
chloroform and methanol were all HPLC-grade solvents obtained from Fisher
Scientific (Loughborough, UK) and used as received. Phosphate-buffered saline
(PBS) was prepared from tablets obtained from Oxoid (Basingstoke, UK).
Regenerated cellulose dialysis membrane (1,000 MWCO) was purchased from

133
Spectra/Por. Disposable UV-grade cuvettes were obtained from Fisher Scientific
(Loughborough, UK).
4.2.2 Synthesis of the 2-phenoxyethyl 2-bromoisobutyrate initiator, PhOBr
2-Phenoxyethanol (5.013 g, 0.0363 mol) was dissolved in dichloromethane (20
mL). DMAP (0.3299 g, 0.0027 mol) and triethylamine (3.63 g, 5.0 mL, 0.0359
mol) were added and the resulting solution was cooled on ice and kept under a
nitrogen atmosphere. 2-Bromoisobutyryl bromide (11.5 g, 6.2 mL, 0.050 mol)
was dissolved in dichloromethane (20 mL) and added dropwise over 40 minutes
to this solution, which was then stirred overnight at room temperature (~20 °C).
The reaction mixture was filtered and the precipitate was washed with additional
dichloromethane (50 mL). The combined organic fractions were washed with
water (2 x 20 mL), saturated NaHCO3 (3 x 30 mL), water (3 x 30 mL) and
saturated NaCl (50 mL). The organic phase was dried over MgSO4, filtered and
passed through basic alumina using dichloromethane as eluent and evaporated at
50 °C followed by thorough drying under reduced pressure. Yield: 4.84 g (46 %).
5.27 %), Br = 27.46 % (theory 27.83 %), which suggested that the initiator purity
exceeded 98 % (based on Br).
1
H NMR (400 MHz, CDCl3) δ 7.19 (m, 2H, Ar), 6.83 (m, 3H, Ar), 4.41 (t, 2H, J =
4.9 Hz, -CH2-O-C=O), 4.09 (t, 2H, J = 4.9 Hz, -CH2-O-Ar), 1.83 (s, 6H,
(CH3)2C) ppm
13
C NMR δ (400 MHz, CDCl3) δ 171.6 (CH2-O-C=O), 158.5 (Ar), 129.6 (Ar),
121.3 (Ar), 114.8 (Ar), 65.6 (CH2-O-C=O), 64.3 (CH2-O-Ar), 55.7 (Br-C-) 30.8
(Br-C-(CH3)2) ppm
EI-MS, m/z 286 (M+
), 288 (M+
)
4.2.3 Copolymer Synthesis and Purification
One-pot ATRP syntheses of the PMPC-PHPMA diblock copolymers were
conducted using sequential monomer addition without purification of the
intermediate PMPC macro-initiator, as reported earlier.21
A typical synthesis was
conducted as follows: to MPC (5.0027 g, 16.94 mmol, 25 eq.) under nitrogen was

134
added a solution of PhOBr (0.1945 g, 0.6773 mmol, 1 eq.) in anhydrous methanol
(3.0 mL) via cannula. The flask was washed with anhydrous methanol (3.0 mL),
which was added to the MPC solution. After purging this solution with nitrogen
for 25 min, a mixture of CuBr (97.1 mg, 0.677 mmol, 1 eq.) and bpy (212.0 mg,
1.357 mmol, 2 eq.) was added. After 30 min, an aliquot was analyzed by 1
H NMR
and GPC to determine the monomer conversion and molecular weight of the
PMPC block. HPMA monomer (8.7880 g, 60.96 mmol, 90 eq.), which had been
purged with nitrogen for 3.5 h prior to its addition, was added via cannula
immediately after removing this aliquot. After 44 h, 1
H NMR confirmed the
disappearance of the vinyl signals, and the reaction solution was diluted with
methanol and exposed to aerial oxygen to quench the polymerization. The
resulting green copolymer solution was passed through a silica column to remove
the spent copper catalyst and the residual solution was dialyzed first against
methanol for three days, and then against a 3:1 chloroform: methanol mixture for
three days, with daily changes of solvent. Solvent was removed under reduced
pressure, 50 mL water was added and this aqueous solution was also evaporated
under reduced pressure at 50 °C. Water (50 mL) was again added and removed
under reduced pressure at 50 °C. Finally water (50 mL) was added for a third time
and the aqueous solution was freeze-dried overnight. The dry copolymer was
placed in a vacuum oven at 80 °C for 48 h and then subjected to further heating a
90 °C for 3 h. Overall yield: 10.0 g (73 %). This somewhat time-consuming
purification protocol was previously found to be necessary to removes traces of
methanol from PHPMA-PMPC-PHPMA triblock copolymers in order to ensure
excellent biocompatibility with various cell types.21
4.2.4 1
H NMR Spectroscopy
1
H NMR spectra were recorded in CD3OD to determine block compositions and
mean degrees of polymerization. Copolymer spectra were also recorded in D2O
using either a 400 MHz Bruker AV1-400 or a 500 MHz Bruker DRX-500
spectrometer. For the variable temperature studies in D2O, the integrated peak
intensity due to the pendent methyl groups in the PHPMA chains at 1.3 ppm was
compared to that due to the pendent methylene groups of the PMPC chains at 3.7

135
ppm. This numerical value was normalized with respect to the actual diblock
copolymer composition, as determined by 1
H NMR in CD3OD, which is a good
solvent for both the PHPMA and the PMPC blocks. Thus the apparent relative
PHPMA content of each diblock copolymer in D2O could be estimated at any
given temperature.
4.2.5 Molecular Weight Determination
Chromatograph pump unit and two Polymer Laboratories PL Gel 5µm Mixed-C
(7.5 x 300 mm) columns in series with a guard column at 40°C connected to a
Gilson Model 131 refractive index detector. The eluent was a 3:1 v/v %
chloroform/methanol mixture containing 2 mM LiBr at a flow rate of 1.0 ml min-
1
. A series of near-monodisperse poly(methyl methacrylate) [PMMA] samples
were used as calibration standards. Toluene (2 µL) was added to all samples as a
flow rate marker. Data analyzes were conducted using CirrusTM GPC Software
supplied by Polymer Laboratories.
4.2.6 Dynamic Light Scattering
Copolymer solutions for light scattering studies were prepared as either 1.0 or 5.0
w/v % stock solutions in PBS at pH 7.2. The initial mixtures were then
equilibrated for 24 h at 4 °C to ensure complete homogeneity. These stock
solutions were diluted to the desired concentration and filtered through a 0.43 µm
Nylon filter immediately before the measurements. Dynamic light scattering
experiments were performed using a Zetasizer Nano-ZS instrument (Malvern
Instruments, UK) at a scattering angle of 173°. Dispersion Technology Software
version 4.20 supplied by the manufacturer was used for cumulants analysis
according to ISO 13321:1996.

136
4.3 Results and Discussion
4.3.1 Initiators
The PhOBr initiator was prepared according to a previously reported protocol
using the commercially available 2-phenoxyethanol instead of bis(2-
hydroxyethyl)disulfide.21 1
H and 13
C NMR spectroscopy, mass spectroscopy and
elemental microanalyses were consistent with the target compound being isolated
in high purity (> 98 %). This aromatic initiator was selected to aid determination
of mean degrees of polymerization from 1
H NMR spectra. In addition, its initiator
efficiency was close to 100 % and the aromatic ester group appeared to be
hydrolytically stable during work-up.
4.3.2 Copolymer Synthesis
O
O
O PhO-PMPCm-Br
HPMA
P
O
N
O O
O
O
HO
MPC
Cu(I)Br, bpy
methanol, 20°C
PhO-PMPCm-PHPMAn
20°C
O
O
O
Br
PhOBr
Scheme 4.1: Synthesis of PMPCm-PHPMAn diblock copolymers via ATRP using sequential
monomer addition (MPC monomer polymerized first).
The PMPC-PHPMA diblock copolymers were synthesized by ATRP in a one-pot
protocol according to Scheme 4.1 using sequential monomer addition following a

137
previously published protocol.21
In our previous study, PMPC-rich triblock
copolymers were easily purified by precipitation into excess THF.21
However, the
PHPMA blocks are highly soluble in THF (and most other common organic
solvents), thus purification of these PHPMA-rich diblock copolymers required
non-aqueous dialysis to remove residual catalyst and unreacted monomer.
Characterization data for the purified PMPC-PHPMA diblock copolymers are
summarized in Table 4.1. The block compositions determined by 1
H NMR
utilized the aromatic end-group signals originating from the PhOBr initiator. This
approach yielded copolymer compositions that corresponded well with the target
compositions, indicating high initiator efficiencies.
Entry Target Composition
1
H NMR Composition
Mn
1
H NMR
Mn
GPC
Mw/Mn Wt % HPMA
1 PMPC25-PHPMA25 PMPC23-PHPMA24 10,500 18,300 1.18 34
H
NMR and GPC studies of the diblock copolymers. 1
H NMR spectra were recorded at 400
MHz. GPC data were obtained using a 3:1 v/v chloroform/methanol eluent and a series of
PMMA calibration standards.
All GPC traces proved to be unimodal and polydispersities for these diblock
copolymers were generally below 1.30, indicating well-controlled
polymerizations (Figure 4.1). PMPC25-PHPMA120 has a small high molecular
weight shoulder. This is possibly due to a very low degree of branching, since
HPMA monomer contains a small amount of dimethacrylate impurity due to its
slow transesterification during storage.23
Another plausible reason for this high
molecular weight shoulder may be radical recombination of the active chain ends.
This alternative explanation is perhaps less likely, since a similar shoulder was
not observed for copolymers with shorter PHPMA blocks, although the overall
comonomer conversions were equally high (>99 %) in all cases, as judged by 1
H

138
NMR (data not shown). Nevertheless, the polydispersity of this copolymer is 1.29
(Table 4.1), indicating that the polymerization is reasonably well controlled.
12 13 14 15 16 17
PMPC25
-PHPMA120
PMPC25
-PHPMA90
PMPC25
-PHPMA58
PMPC25
-PHPMA39
PMPC23
-PHPMA24
NormalizedRIsignal
Elution time / min
12 13 14 15 16 17
PMPC49
-PHPMA67
PMPC49
-PHPMA49
PMPC49
-PHPMA26
NormalizedRIsignal
Elution time / min
Figure 4.1: Gel permeation chromatograms of the PMPC-PHPMA diblock copolymers
obtained using a 3:1 chloroform: methanol eluent and a series of near-monodisperse
poly(methyl methacrylate) calibration standards.

139
4.3.3 Temperature-dependent dynamic light scattering studies
The temperature dependence of the count rate and hydrodynamic diameter
observed for 1.0 w/v % aqueous solutions of the PMPC-PHPMA diblock
copolymers are shown in Figure 4.2. The observed behavior is highly dependent
on the copolymer composition. For example, neither the scattering intensity nor
the hydrodynamic radius of PMPC23-PHPMA24 is significantly affected by the
temperature. Its hydrodynamic radius is approximately 4 nm, which is consistent
with a molecularly dissolved copolymer. In contrast, the scattered light intensity
obtained for PMPC25-PHPMA39 increases by almost two orders of magnitude
between 4 °C and 15 °C. The corresponding hydrodynamic radii increase from 70
nm to 130 nm between 4 °C and 7 °C, followed by a reduction to 110 nm between
10 °C and 15 °C.
0 5 10 15 20 25 30 35 40 45 50
10
2
10
3
10
4
10
5
10
6
PMPC25
-PHPMA39
PMPC25
-PHPMA58
PMPC25
-PHPMA120
PMPC23
-PHPMA24
PMPC25
-PHPMA90
Scatteringintensity/kcps
Temperature / °C
0 5 10 15 20 25 30 35 40 45 50
10
2
10
3
10
4
10
5
10
6
PMPC49
-PHPMA67
PMPC49
-PHPMA26
PMPC49
-PHPMA49
Scatteringintensity/kcps
Temperature / °C
0 5 10 15 20 25 30 35 40 45 50
11
5
10
50
100
PMPC25
-PHPMA39
RH
/nm
Temperature / °C
PMPC23
-PHPMA24
PMPC25
-PHPMA90
PMPC25
-PHPMA58
PMPC25
-PHPMA120
0 5 10 15 20 25 30 35 40 45 50
11
5
10
50
100
PMPC49
-PHPMA67
PMPC49
-PHPMA26
PMPC49
-PHPMA49
RH
/nm
Temperature / °C
Figure 4.2: (A,B) Scattering intensity vs. temperature plots for 1.0 w/v % PMPC-PHPMA
diblock copolymers in PBS (pH 7.2). (C,D) Hydrodynamic radius vs. temperature plots for
the same aqueous diblock copolymer solutions.
A B
C D

140
These relatively large aggregates suggest that at least some fraction of this
diblock copolymer may not be molecularly dissolved even at low temperature.
Visual inspection of this aqueous copolymer solution revealed that it had
significant turbidity at all temperatures. In addition, cumulants analyses indicated
that some degree of aggregation occurred even at the lowest temperature
examined (see Figure 4.3).
4 °C
37 °C
22 °C
PMPC25-PHPMA90
4 °C
37 °C
22 °C
PMPC25-PHPMA90
PMPC23-PHPMA24
1 10 100 1000
RH / nm
4 °C
22 °C
37 °C
4 °C
22 °C
37 °C
4 °C
22 °C
37 °C
PMPC25-PHPMA120
4 °C
22 °C
37 °C
4 °C
22 °C
37 °C
1 10 100 1000
RH / nm
PMPC25-PHPMA58
4 °C
22 °C
37 °C
PMPC25-PHPMA58
4 °C
22 °C
37 °C
PMPC25-PHPMA39
4 °C 37 °C
22 °C
PMPC25-PHPMA39
4 °C 37 °C
22 °C
PMPC49-PHPMA67
37 °C
22 °C
4 °C
PMPC49-PHPMA67
37 °C
22 °C
4 °C
PMPC49-PHPMA49
4 °C
37 °C
22 °C
PMPC49-PHPMA49
4 °C
37 °C
22 °C
PMPC49-PHPMA26
4 °C 22 °C
37 °C
PMPC49-PHPMA26
4 °C 22 °C
37 °C
Figure 4.3: Temperature dependence of hydrodynamic radii determined from cumulants
analyses of 1.0 w/v % aqueous PMPC-PHPMA diblock copolymer solutions in PBS at pH
7.2.

141
The scattered light intensity for PMPC25-PHPMA58 increased by approximately a
factor of two from 4 °C to 12 °C, with no further change at higher temperatures.
However, the corresponding hydrodynamic radii of 60 nm are almost constant
over the entire temperature range.
The scattered light intensities obtained for PMPC25-PHPMA90 and PMPC25-
PHPMA120 diblock copolymers both increase between 4 °C and 10 °C, with no
further changes occurring up to 50 °C. The hydrodynamic radii are almost
constant for these two copolymer solutions over the entire temperature range,
with the PMPC25-PHPMA90 forming slightly smaller aggregates. At first sight, it
may seem surprising that the hydrodynamic radii are not affected by the
temperature for all these copolymer solutions, since the greater scattering
intensity suggests either a larger aggregation number or a higher concentration of
copolymer aggregates. However, similar behavior has been reported for thermo-
responsive diblock copolymers based on poly(ethylene oxide) (PEO) and
poly(propylene oxide) (PPO).24
In this case, the reduced solvation of the PEO
block on raising the solution temperature is compensated by a higher aggregation
number. A similar mechanism may well operate for the current copolymer
system. Raising the temperature causes progressive dehydration of the PHPMA
chains, which leads to gradual contraction of the aggregates. However, for
copolymers where micelles and individual copolymer chains (unimers) co-exist,
the greater hydrophobic character at higher temperature may lead to additional
aggregation of unimers. This would lead to a larger hydrodynamic diameter,
which offsets the effect of dehydrating the PHPMA chains. The net effect is that
there is very little change in the hydrodynamic diameter.
Although the PMPC23-PHPMA24 diblock copolymer did not undergo detectable
aggregation over the entire temperature range studied (4 °C to 50 °C), the four
copolymers with longer PHPMA blocks formed aggregates at all temperatures.
Somewhat surprisingly, the hydrodynamic radii of these aggregates at 22 °C
follow the order: PMPC25-PHPMA39 (106 nm) > PMPC25-PHPMA58 (58 nm)>
PMPC25-PHPMA120 (35 nm) > PMPC25-PHPMA90 (27 nm). Thus the copolymer
with the shortest hydrophobic PHPMA block forms the largest aggregates.
Moreover, extending this hydrophobic block leads to a reduction in the
hydrodynamic radius, which is in contrast to the majority of the literature data
reported for amphiphilic block copolymers. In the vast majority of cases, the

142
hydrodynamic size increases as the mean degree of polymerization of the
hydrophobic block is increased which is interpreted in terms of a higher
aggregation number.24,25
The behavior of the series of diblock copolymers with a fixed PMPC DP of 49
and a variable PHPMA DP (see Figure 4.2b and Figure 4.2d) is slightly different
to the series comprising a shorter fixed PMPC block (DP ~ 25). At 4 °C, 1.0 w/v
% solutions of PMPC49-PHPMA26 and PMPC49-PHPMA49 have hydrodynamic
radii below 10 nm, indicating molecular dissolution. Increasing the temperature
leads to thermo-responsive behavior, with large increases in both scattering
intensity and hydrodynamic radius. For PMPC49-PHPMA49, enhanced scattering
begins at 12 °C. The hydrodynamic radius increases up to 100 nm at 25 °C,
followed by a reduction to around 80 nm at 50 °C. A 1.0 w/v % solution of
PMPC49-PHPMA26 behaves similarly, although additional scattering ensues at 20
°C and a hydrodynamic radius of more than 200 nm is attained at 34 °C. PMPC49-
PHPMA67 does not exhibit any significant thermo-responsive behavior: its
hydrodynamic radius is reduced from ~ 90 nm at 4 °C to ~ 70 nm at 50 °C. It is
also noteworthy that the high temperature behavior of these three copolymers
follows the same anomalous behavior observed for the diblock copolymers
containing shorter PMPC blocks, i.e. the copolymer with the shortest PHPMA
block forms the largest aggregates.
One possible explanation for this anomalous behavior is that the degree of
hydration of the PHPMA block is strongly dependent on its degree of
polymerization. Cloud point data obtained for poly(propylene oxide),26
poly(2-
(dimethylamino)ethyl methacrylate),27
poly(2-(N-morpholino)ethyl
methacrylate),28
poly(2-hydroxyethyl methacrylate)29
also shows this trend and is
in accordance with both classical Flory-Huggins theory30
and also the observation
that HPMA monomer is water-miscible up to 13 %. In contrast, cloud point data
obtained for PNIPAM31,32
indicates somewhat weaker dependence on the mean
degree of polymerization, while the LCST values of statistical copolymers of 2-
(2-methoxyethyoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate
recently reported by Lutz et al. exhibit little or no dependence on copolymer
molecular weight.31
It is noteworthy that, if shorter PHPMA blocks aggregate but
have some affinity for water, the formation of relatively large, hydrated colloidal
aggregates is likely. There are a few literature examples of other diblock

143
copolymers that form similarly large aggregates.22,33,34
Diblock copolymers
comprising poly(methyl methacrylate) and poly(sulfonated glycidyl
methacrylate), [PMMA-b-PSGMA] reported by Jerome et al.25
form large
colloidal aggregates in water, apparently due to slow dissolution kinetics caused
by the glassy PMMA cores. There are several literature reports describing
systems in which the hydrophobic cores are partially hydrated.16,26
For example,
Save and co-workers state that diblock copolymers comprising PPO and PGMA
form spherical aggregates of 150-200 nm diameter in aqueous solution as judged
by DLS. Similarly, Ikerni et al.34
studied an ABA triblock copolymer where A =
PHEMA, (DP ~ 13) and B = PEO (DP ~ 165) by fluorescence spectroscopy, static
light scattering and small angle x-ray spectroscopy. These copolymer aggregates
had hydrophobic PHEMA cores and PEO coronas. However, the hydrodynamic
radius was almost 100 nm and there was apparently a distinct boundary between
the hydrophobic and hydrophilic domains. It is perhaps noteworthy that, based on
the PHEMA DP alone, these copolymers should be fully water-soluble.29
It is
likely that hydrogen bonding between the PHEMA and the PEO chains plays a
significant role in this particular case.
4.3.4 Concentration-dependent dynamic light scattering
Our Malvern DLS instrument detects scattered light at 173o
, which allows
meaningful measurements to be made on significantly more concentrated
solutions than those used in conventional DLS experiments. Figure 4.4 shows the
hydrodynamic radius as a function of copolymer concentration at both 4 °C and
37 °C. The hydrodynamic radius of PMPC23-PHPMA24 at 4 °C is less than 5 nm
regardless of the copolymer concentration, suggesting that this copolymer is
molecularly dissolved. However, raising the temperature to 37 °C leads to the
formation of large aggregates for copolymer concentrations exceeding 1.0 w/v %
(see Figure 4.4B). The temperature dependence of the hydrodynamic radius for
three PMPC23-PHPMA24 concentrations is shown in Figure 4.4C. As already
shown in Figure 4.2, this copolymer remains molecularly dissolved as a 1.0 w/v
% solution at all temperatures. Large colloidal aggregates are formed above 30 °C
in a 2.0 w/v % solution.

144
0 1 2 3 4 5 6
1
5
10
50
100
500
1000
4 °C
PMPC25
-PHPMA120
PMPC25
-PHPMA39
PMPC25
-PHPMA90
PMPC25
-PHPMA58
PMPC23
-PHPMA24
RH
/nm
Concentration / % w/v
0 1 2 3 4 5 6
1
5
10
50
100
500
1000
37 °C
PMPC25
-PHPMA120
PMPC25
-PHPMA39
PMPC25
-PHPMA90
PMPC25
-PHPMA58
PMPC23
-PHPMA24
RH
/nm
0 1 2 3 4 5 6
1
5
10
50
100
500
1000
4 °C
PMPC49
-PHPMA26
PMPC49
-PHPMA49
PMPC49
-PHPMA67
RH
/nm
0 1 2 3 4 5 6
1
5
10
50
100
500
1000
37 °CPMPC49
-PHPMA26
PMPC49
-PHPMA49
PMPC49
-PHPMA67
RH
/nm
0 5 10 15 20 25 30 35 40 45 50 55
1
5
10
50
100
500
1000 PMPC23
-PHPMA24
1.0 wt/v %
2.0 wt/v %
5.0 wt/v %
RH
/nm
Temperature / °C
0 5 10 15 20 25 30 35 40 45 50 55
1
5
10
50
100
500
1000 PMPC49
-PHPMA49
1.0 wt/v %
2.0 wt/v %5.0 wt/v %
RH
/nm
Temperature / °C
A B
C D
E F
Figure 4.4: Concentration dependence of the apparent hydrodynamic radius of solutions of
(A) PMPC~25-PHPMAn diblock copolymers in PBS, pH 7.2 at 4 °C; (B) PMPC~25-PHPMAn
diblock copolymers in PBS, pH 7.2 at 37 °C; (C) PMPC49-PHPMAn diblock copolymers in
PBS, pH 7.2 at 4 °C; (D) PMPC49-PHPMAn diblock copolymers in PBS, pH 7.2 at 37 °C. (E)
Hydrodynamic radius as a function of temperature for 1.0 w/v %, 2.0 w/v % and 5.0 w/v %
solutions of PMPC23-PHPMA24. Dotted lines indicate aggregation/precipitation. (F)
Hydrodynamic radius as a function of temperature for 1.0 w/v %, 2.0 w/v % and 5.0 w/v %
solutions of PMPC49-PHPMA49. Dotted lines indicate aggregation/precipitation.
More concentrated copolymer solutions causes aggregation to occur at around 15
°C. However, the size of these aggregates is 500-900 nm, which is clearly far too
large to be simple ‘core-shell’ micelles. Visual inspection confirmed gradual

145
precipitation of the 5.0 w/v % copolymer solution on standing at 22 °C. This
precipitate redissolved on cooling to 4 °C. These observations suggest that the
colloidal aggregates formed in more concentrated aqueous solution are at best
metastable structures. This is in contrast to the temperature-responsive PHPMA-
PMPC-PHPMA triblock copolymer gelators described earlier, which form
transparent solutions or gels with no signs of precipitation up to 30 w/v %.21
However, it is noteworthy that these triblocks are PMPC-rich, with PHPMA
contents of only 10-20 wt. %. In contrast, the diblock copolymers described in the
present work have PHPMA contents ranging from 20 to 70 wt. %.
The hydrodynamic radii of the four PMPC-PHPMA diblock copolymers with a
fixed PMPC DP of 25 all increase at higher copolymer concentrations. Similar
observations have been made for other copolymers where aggregates and unimers
co-exist in equilibrium and the phenomenon is attributed to enhanced
incorporation of unimers within aggregates.24
In the present study, this appears to
be strongly dependent on the DP of the PHPMA block. At 4 °C, the mean
hydrodynamic radius of the PMPC25-PHPMA39 aggregates increases from 10 nm
at 0.1 w/v % to 500 nm at 5.0 w/v % (Figure 4.4A). Clearly, the copolymer is
only weakly aggregated in dilute solution at this temperature since its radius is
close to that expected for individual copolymer chains (~ 5 nm). At higher
copolymer concentrations, inter-chain interactions such as hydrogen bonding may
well contribute to the formation of very large aggregates. Copolymer aggregates
with a mean radius of ~ 35 nm in 0.1 w/v % solution are formed at 37 °C, with
this radius increasing up to 400 nm for copolymer concentrations of 5.0 w/v %.
Thus, the copolymer’s propensity towards aggregation is greater at 37 °C than at
4 °C in dilute solution, probably due to progressive dehydration of the PHPMA
chains. At higher copolymer concentrations, smaller aggregates are formed at 37
°C than at 4 °C due greater dehydration at the higher temperature, leading to more
compact aggregates. This implies that, at some critical copolymer concentration
where the two curves shown in Figure 4.4A and Figure 4.4B for PMPC25-
PHPMA39 cross over, the dehydration-driven contraction eventually outweighs
the formation of larger aggregates due to unimer incorporation. This cross-over
concentration seems to lie between 1.0 and 2.0 w/v % for PMPC25-PHPMA39.
The hydrodynamic radii of copolymers with longer PHPMA blocks also increase
with copolymer concentration. However, the relative increase is much smaller

146
than for PMPC25-PHPMA39 (see Figure 4.4A and Figure 4.4B). For example, in
the case of PMPC25-PHPMA58 at 4 °C, the aggregate radius increases from
approximately 40 nm at 0.1 w/v % up to 90 nm at 5.0 w/v %. At 37 °C, the radii
are typically 5-10 nm smaller than at 4 °C (except at 0.10 w/v %, where the radius
is larger by almost 20 nm). This behavior is reminiscent of that observed for the
PMPC25-PHPMA39 copolymer, albeit with the critical copolymer concentration
shifted to a much lower value. This is to be expected, since the hydrophobicity of
the PHPMA block should be greater for higher degrees of polymerization.
Unfortunately, the excess scattering intensity at such dilutions is very low, which
adds significant uncertainty to the data. Therefore the exact position of this cross-
over concentration is rather hard to determine reliably. Increasing the
concentration of either PMPC25-PHPMA90 or PMPC25-PHPMA120 from 0.1 w/v
% to 5.0 w/v % leads to an increase of a few nm in aggregate size, indicating a
shift in the unimer-aggregate equilibrium. The concentration dependence of the
hydrodynamic radius of the series of diblock copolymers with a fixed PMPC DP
of 50 is more complex (see Figure 4.4C and Figure 4.4D).
At 4 °C, PMPC49-PHPMA49 has a hydrodynamic radius of approximately 5 nm
up to a concentration of 3.0 w/v %, indicating molecular dissolution. A further
increase in the copolymer concentration leads to the abrupt formation of
aggregates with a hydrodynamic radius of approximately 100 nm at 5.0 w/v %. In
contrast, the hydrodynamic radius of PMPC49-PHPMA26 aggregates increases
almost exponentially from 6 nm at 0.5 w/v % up to 200 nm at 5.0 w/v % (Figure
4.4C). This is unexpected, since a more hydrated, shorter PHPMA block should
favor molecular dissolution. This behavior may be due to the higher
polydispersity of this copolymer. Indeed, cumulants analyses of the light
scattering data indicate at least two populations for this copolymer at 4 °C, which
correspond to molecularly dissolved chains and colloidal aggregates (Figure 4.5).

147
PMPC23-PHPMA24
1 10 100 1000 10000
0.1 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 % 5.0 %
0.1 %
2.0 % 5.0 %
0.1 %
1 10 100
0.1 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
PMPC25-PHPMA90
4 °C
22 °C
37 °C
10 100 1000
0.1 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
0.1 %
PMPC25-PHPMA58
5.0 %
2.0 %
5.0 %
2.0 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
0.1 %
RH / nm RH / nm RH / nm
0.1 %
2.0 %
5.0 %
0.1 %
2.0 %
5.0 %
10 100 1000
0.1 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
0.1 %
PMPC25-PHPMA120
RH / nm
Intensity
2.0 %
5.0 %0.1 %0.1 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
0.1 %
2.0 %
0.1 %
10 100 10001 10000
PMPC25-PHPMA39
RH / nm
2.0 %
5.0 %
0.1 %
5.0 %
2.0 %
5.0 %
0.1 %
5.0 %
0.1 %
2.0 %
0.1 %
2.0 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
0.1 %
10 100 1000110 100 10001
2.0 %
0.1 %
5.0 %2.0 %
0.1 %
5.0 %
0.1 %
5.0 %
2.0 %
0.1 %
5.0 %
2.0 %
5.0 %0.1 %
2.0 %
5.0 %0.1 %
2.0 %0.1 %
10 100 10001
5.0 %
2.0 %0.1 %
5.0 %
2.0 %
PMPC49-PHPMA26 PMPC49-PHPMA49 PMPC49-PHPMA67
4 °C
22 °C
37 °C
Intensity
0.1 % 5.0 %
2.0 %
0.1 % 5.0 %
2.0 %
Figure 4.5: (A) Hydrodynamic radii from cumulants analyses of 0.1 w/v % , 2.0 w/v % and
5.0 w/v % solutions of the PMPC25-PHPMAn diblock copolymers at 4 °C, 22 °C and 37 °C.
(B) Hydrodynamic radii from cumulants analyses of 0.1 w/v % , 2.0 w/v % and 5.0 w/v %
solutions of the PMPC49-PHPMAn diblock copolymers at 4 °C, 22 °C and 37 °C.
This aggregation from a small but measurable fraction of the copolymer leads to a
larger calculated hydrodynamic radius. The hydrodynamic radius of PMPC49-
PHPMA67 increases between 0.1 w/v % and 1.0 w/v %, after which an almost
constant value of approximately 200 nm is attained. This indicates that the
equilibrium is shifted towards micelles, which is consistent with the cumulants
analysis (see Figure 4.5B). Increasing the temperature also leads to aggregation of
PMPC49-PHPMA26 and PMPC49-PHPMA49 between 0.5 and 1.0 w/v %. In both
cases, increasing the copolymer concentration apparently leads to a modest
A
B

148
reduction in the hydrodynamic radius, which may be due to either formation of
more compact aggregates or a lower aggregation number.35
In general, the DLS
data indicates the presence of large non-micellar aggregates, whose size is largely
independent of the copolymer concentration above 1.0 w/v %. However, PMPC49-
PHPMA67 exhibits qualitatively different behavior, since its apparent
hydrodynamic radius increases monotonically with increasing copolymer
concentration. This is somewhat surprising, since the longer PHPMA block
should favor aggregate formation, in accordance with the behavior observed for
the copolymer series with a fixed PMPC DP of 25. Inspection of the cumulants
analyses for this copolymer (Figure 4.5B) indicates that it forms two types of
aggregate: one with a hydrodynamic radius of around 20 nm and the other with a
radius in excess of 200 nm. Increasing the copolymer concentration shifts the
equilibrium towards the larger species, as expected.
Figure 4.4F shows how the hydrodynamic radius varies with temperature for three
PMPC49-PHPMA49 concentrations. Below 15 °C, the hydrodynamic radius
observed for 1.0 w/v % and 2.0 w/v % solutions is less than 5 nm, which indicates
molecular dissolution. Increasing the temperature leads to formation of aggregates
of 100 nm radius at both concentrations. At 5.0 w/v %, the same copolymer has a
hydrodynamic radius of around 10 nm below 12 °C. This is around twice as large
as the molecularly dissolved unimers observed at lower concentrations, indicating
that the 5.0 w/v % copolymer is weakly aggregated even at low temperature,
although the effect of a change in refractive index cannot be excluded (a
refractive index for pure water of 1.330 was assumed for all measurements, which
is a valid approximation to within 0.5 % over the temperature interval studied36
).
However, the change in solution refractive index due to dissolved solids may have
an influence especially at the relatively high concentrations used. This effect was
not investigated. Above 12 °C, aggregates are formed with radii of 150-200 nm.
Thus the aqueous solution behavior of PMPC49-PHPMA49 is somewhat different
from that of PMPC23-PHPMA24, even though the PHPMA content is almost
identical. Essentially, the critical aggregation temperature is much less
concentration-dependent for the larger copolymer. In addition, the aggregate size
is much smaller for the larger copolymer and macroscopic precipitation is not
observed. Hence it appears that longer PHPMA blocks leads to the formation of

149
more compact, smaller aggregates. In addition, the longer PMPC block is more
efficient in forming colloidally stable aggregates in solution.

150
4.3.5 Temperature-dependent 1
H NMR studies
The degree of solvation of these PMPC-PHPMA copolymers was examined by
temperature-dependent 1
H NMR spectroscopy in D2O. Figure 4.6 shows 1
H NMR
spectra recorded for 1.0 w/v % solutions of PMPC25-PHPMA39 in (i) CD3OD at
22 °C and (ii) in D2O at three different temperatures. Since CD3OD is a good
solvent for both blocks, this solvent was used to determine the true block
composition. D2O is a good solvent for the PMPC blocks at all temperatures and
therefore the PMPC signals were used as internal standards. The 1
H NMR
spectrum of the molecularly dissolved copolymer in CD3OD is quite complex,
with several overlapping peaks. Moreover, the HPMA repeat unit is actually a
75:25 mixture of two isomers.34
This is why the signals labeled h and f comprise
more than one peak. In order to assess the block composition in CD3OD, the
integral of signal g (1H; assigned to the major HPMA isomer). Multiplying this
integral by 4/3 and comparing it to an appropriate PMPC signal (a, 2H) allows
calculation of the block composition. This approach produced results that were
consistent with the target block compositions (see Table 4.1). Alternatively, if the
peak integrals for a (2H) and f (3H) were compared, similar compositions were
obtained (within a few percent) despite the overlap between f and the backbone
signals. Increasing the temperature of the diblock copolymer solutions in D2O
leads to gradual attenuation of the PHPMA signals (Figure 4.6). This attenuation
is due to the reduced mobility of these chains and/or a reduction in the number of
molecularly dissolved polymer chains due to their incorporation into aggregates.38
Inspecting the 1
H NMR spectra recorded in D2O (Figure 4.6), only the signal from
the side-chain methyl groups (f) of the PHPMA chains is readily detectable in
aqueous solution, with signals g and h being either almost completely attenuated
or obscured by overlapping PMPC signals. Therefore, the integrals of signals a
and f were compared to calculate apparent block compositions for each of the
diblock copolymers at a given temperature. These compositions were then
normalized with respect to the actual block composition obtained from the
spectrum recorded in CD3OD to produce an ‘apparent’ PHPMA content.

151
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
4.6 °C
25 °C
37 °C
CD3
OD
e
h g
h
Backbone
Backbone
f
g
f
e
b,c,d
d
c
b
a
a
O
O
O
O
O
PO
O
O
N
+
25
O
O
OH
39
O
δ / ppm
H NMR spectra of PMPC25-PHPMA39 recorded in CD3OD at 21 °C
and in D2O at 4.6 °C, 25 °C and 37 °C. All spectra are normalized relative to peak ‘a’. The
arrows indicate those PHPMA signals that are significantly attenuated at elevated
temperature.
The results are shown in Figure 4.7. The apparent PHPMA content of a 1.0 w/v %
solution of PMPC23-PHPMA24 in D2O at 5 °C is identical to the true content
within experimental error, indicating that this copolymer is molecularly dissolved.
Increasing the temperature leads to an apparent reduction in the PHPMA content,
but even at 37 °C this block has a degree of solvation of more than 85 %. Thus
these NMR results are consistent with the DLS data obtained for this copolymer,
which indicated molecular dissolution up to 50 °C (Figure 4.2).
Increasing the DP of the PHPMA block leads to a reduction in the apparent
PHPMA content regardless of the temperature. This is because longer PHPMA
blocks are more hydrophobic and hence more prone to aggregation. For both
PMPC25-PHPMA39 and PMPC25-PHPMA58 the apparent PHPMA content is
around 70 % of the true value at 5 °C. This value is progressively reduced to 20 %
on heating to 37 °C. For these two copolymers, the light scattering intensity

152
increased significantly with temperature, whereas their hydrodynamic radii were
relatively large but did not change significantly (Figure 4.2). In addition,
concentration-dependent light scattering indicated that unimers and micelles co-
exist in such solutions (Figure 4.4). Thus attenuation of the NMR signals due to
PHPMA at higher temperature is consistent with the incorporation of unimers into
aggregates, although there may also be some contribution due to a reduction in
segmental motion within the aggregates.

153
0 5 10 15 20 25 30 35 40
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
PMPC25
-PHPMA39
PMPC25
-PHPMA120
PMPC25
-PHPMA90
PMPC25
-PHPMA58
PMPC23
-PHPMA24
ApparentPHPMAcontent
Temperature / °C
0 5 10 15 20 25 30 35 40
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
PMPC49
-PHPMA67
PMPC49
-PHPMA26
PMPC49
-PHPMA49
Temperature / °C
Figure 4.7: Temperature dependence of the apparent PHPMA content of 1.0 w/v % aqueous
solutions of various PMPC-PHPMA diblock copolymers in D2O normalized with respect to
their actual block compositions (as determined in CD3OD). The monotonic reduction in
apparent PHPMA content on increasing the temperature indicates progressively poorer
solvation and/or lower mobility for this block; this is consistent with the onset of micellar
self-assembly. (A) Data set obtained for PMPC-PHPMA diblock copolymers with a fixed
PMPC DP of ~ 25; (B) data set obtained for PMPC-PHPMA diblock copolymers with a fixed
PMPC DP of ~ 49. Lines are guides for the eye, rather than fits to the data.
A
B

154
PHPMA blocks with higher DP values are significantly less solvated: above 15
°C, the apparent PHPMA content is less than 10 % and remains relatively
constant for both PMPC25-PHPMA90 and PMPC25-PHPMA120. At 5 °C, the
apparent PHPMA content is 20-40 %, with PMPC25-PHPMA90 being more
solvated as expected. For these two copolymers, slightly more intense light
scattering was observed between 4 °C and 15 °C, whereas the hydrodynamic
radius did not change significantly. In addition, there was almost no increase in
the hydrodynamic radius with concentration, suggesting that few, if any,
copolymer chains are present as unimers (Figure 4.4). Thus, the PHPMA chains
are highly dehydrated for these two copolymers and the additional attenuation
observed on raising the temperature from 5 °C to 15 °C is probably mainly due to
further dehydration of the aggregates.
Figure 4.7B shows the apparent PHPMA content as a function of temperature for
three copolymers with a fixed mean PMPC DP of 49. Their behavior is similar to
that observed for the series of copolymers comprising shorter PMPC chains.
Increasing the temperature leads to reduced PHPMA signal intensities and this
attenuation is highly dependent on the mean DP of the PHPMA chains. It is
noteworthy that, for all copolymers, these variable temperature 1
H NMR
experiments do not indicate a well-defined critical aggregation temperature but
rather continuous dehydration and a progressive shift in the unimer/aggregate
equilibrium. This is in contrast to the behavior observed for other thermo-
responsive polymers such as PPO38
or PNIPAM,18,40
where a critical aggregation
temperature or LCST could be inferred from the attenuated 1
H NMR signals.

155
20 40 60 80 100 120 140
0.01
0.05
0.1
0.5
1
5 °C
37 °C
Mean DP of PHPMA block
20 40 60 80 100 120 140
1
10
100
1000
37 °C
4 °C
PMPC~25
-PHPMAn
PMPC~50
-PHPMAn
RH
/nm
Mean DP of PHPMA block
Figure 4.8: (A) Apparent PHPMA content measured by 1
H NMR spectroscopy in 1.0 w/v %
solutions in D2O for PMPC25-PHPMAn (triangles) and PMPC50-PHPMAn (circles) diblock
copolymers as a function of the actual degree of polymerization of the PHPMA block at 5 °C
(open symbols) and 37 °C (closed symbols). (B) DLS hydrodynamic radius obtained for 1.0
w/v % solutions in PBS at pH 7.2 containing PMPC25-PHPMAn (triangles) and PMPC50-
PHPMAn (circles) diblock copolymers as a function of the actual degree of polymerization of
the PHPMA block at 4 °C (open symbols) and 37 °C (closed symbols). Lines are guides to the
eye, rather than data fits.
B
A

156
Figure 4.8A shows the variation in the apparent PHPMA content with the DP of
this block at 5 °C and 37 °C for both series of PMPC-PHPMA diblock
copolymers. At 5 °C, the apparent PHPMA content decreases continuously from
100 % to 17 % on increasing the DP of the PHPMA block from 24 to 120. At 37
°C, the apparent PHPMA content decreases from 100 % to 4 % on increasing the
DP of the PHPMA block from 24 to 120. Intermediate apparent block
compositions were observed at 22 °C (data not shown). Hence the degree of
solvation of the PHPMA chains is very sensitive to their mean degree of
polymerization, whereas the influence of the PMPC block is relatively weak. In
addition, the reduced apparent PHPMA content at elevated temperature is also
highly dependent on the DP of this block. Figure 4.8B shows the effect of varying
the PHPMA DP on the hydrodynamic radius at 4 °C and 37 °C. Copolymers
containing relatively short PHPMA blocks may be either molecularly dissolved or
thermo-responsive. Increasing the PHPMA DP leads to aggregation, with no
discernable difference in aggregate dimensions observed at 4 °C and 37 °C. For
those copolymers that do aggregate, the hydrodynamic radius is reduced as the
PHPMA DP is increased up to 90. In view of our 1
H NMR data, this indicates that
the aggregation behavior is highly dependent on the hydration of the PHPMA
blocks. Preliminary variable angle light scattering studies only indicate a weak
dependence of the diffusion coefficient on the scattering angle in most cases,
indicating approximately spherical aggregates (Figure 4.9).

157
2.0x10
10
4.0x10
10
6.0x10
10
8.0x10
10
1.0x10
11
2.0x10
-8
4.0x10
-8
6.0x10
-8
8.0x10
-8
1.0x10
-7
1.2x10
-7
1.4x10
-7
1.6x10
-7
1.8x10
-7
2.0x10
-7
PMPC25
-PHPMA90
4 °C
38 °C
Γ/q
2
/cm
2
/s
q
2
/ cm
-2
2.0x10
10
4.0x10
10
6.0x10
10
8.0x10
10
1.0x10
11
2.0x10
-8
4.0x10
-8
6.0x10
-8
8.0x10
-8
1.0x10
-7
1.2x10
-7
1.4x10
-7
1.6x10
-7
PMPC25
-PHPMA39
38 °C
4 °C
Γ/q
2
/cm
2
/s
q
2
/ cm
-2
Figure 4.9: Angular dependence of the diffusion coefficient for two 1.00 w/v % copolymer
solutions in PBS at 4 °C and 38 °C
4.3.6 Aggregation mechanism
In general, the thermo-responsive behavior of non-ionic water-soluble polymers is
due to a significant reduction in hydrogen bonding interactions between the
polymer chains and water molecules occurring at a particular temperature. For
example, temperature-dependent IR studies of PPO in water confirmed weaker
hydrogen bonding between water and the ether oxygens on the polymer

158
backbone,40
as well as partial dehydration of the pendent methyl groups.41
Similar
studies of aqueous solutions of PNIPAM provided spectroscopic evidence for
hydrogen bonding between the N-H protons and the carbonyl oxygens in the
collapsed state above the LCST, but not for the soluble chains below the LCST.42
PHPMA mean degree of polymerisation
Temperature
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
Unimers
Unimers
Unimers
Large hydrated aggregates /
unimers
Large hydrated aggregates /
unimers
Micelles /
unimers
Micelles / unimers
Micelles
Figure 4.10: Schematic representation of the effect of raising the solution temperature and
increasing the mean degree of polymerization of the PHPMA block on the colloidal
aggregates produced by self-assembly.
The aggregation of PMPC-PHPMA diblock copolymers in aqueous solution
strongly depends on both the DP of the PHPMA block and the solution
temperature. This behavior is summarized in Figure 4.10. Copolymers with
sufficiently short PHPMA blocks remain molecularly dissolved at all
temperatures in dilute solution. Increasing the DP of the PHPMA block or the
copolymer concentration eventually leads to aggregation, although a sufficiently
long PMPC block may suppress this significantly. The aggregates formed by
diblock copolymers with relatively short PHPMA blocks are very large,
presumably due to their highly hydrated nature. These aggregates exist in
equilibrium with molecularly dissolved copolymer chains at low concentrations.
Increasing the solution temperature causes progressive dehydration of the
PHPMA blocks, which leads to incorporation of unimers into aggregates as well

159
as to more compact aggregates (presumably due to expulsion of water).
Increasing the PHPMA DP has a similar effect; the equilibrium between unimers
and aggregates shifts towards aggregates and these aggregates are significantly
less hydrated and therefore smaller. At a PHPMA DP of 90 or above, there are
essentially no unimers present.
There are two main differences between these PHPMA-based thermo-responsive
diblock copolymers and those based on other thermo-responsive blocks: First,
these block copolymers form relatively large, water-rich aggregates if the
PHPMA block has a relatively low DP. Perhaps counter-intuitively, relatively
short copolymers form larger aggregates than those with longer PHPMA blocks.
Second, whether a given diblock copolymer forms large aggregates or becomes
molecularly dissolved seems to depend on the DP of the water-soluble PMPC
block, with longer ‘buoy’ blocks favoring unimers. For those copolymers that do
exhibit thermo-responsive behavior, this transition is relatively ill-defined,
typically occurring over a temperature range of 10-15 °C.
Similar broad transitions have been observed for thermo-responsive polymers
based on PPO24
and PHEMA,29
whereas thermo-responsive polymers based on
PNIPAM30,31
and poly(2-(2-methoxyethoxy)ethyl methacrylate43
typically exhibit
sharper transitions. It is conceivable that the isomeric nature of the PHPMA block
may be important in dictating its aqueous phase behavior. Another possibly may
be a polydispersity effect; the diblock copolymers described in the present work
typically have polydispersities ranging from 1.2 to 1.3 (Table 4.1). If the aqueous
solubility of the PHPMA block at a given temperature is sensitive to its degree of
polymerization, such polydispersities may well ‘smear out’ any thermal transition.
In this context, Sugihara32
recently studied thermo-responsive diblock copolymers
based on poly(2-(2-ethoxy)ethoxyethyl vinyl ether) [PEOEOVE] and poly(2-
methoxyethyl vinyl ether) [PMOVE], where phase separation of PEOEOVE200-
PMOVE400 with a polydispersity of 1.10 occurred over less than 5 °C. In contrast,
an ad-mixture comprising three near-monodisperse diblock copolymers
(PEOEOVE100-PMOVE200, PEOEOVE200-PMOVE400 and PEOEOVE300-
PMOVE600) had an overall polydispersity of 1.81 and only underwent partial
phase-separation over a temperature range of approximately 20 °C.44
Apart from
this earlier work, there seem to be few, if any, detailed studies on the influence of

160
the polydispersity on the aqueous phase behavior of stimulus-responsive diblock
copolymers.
4.4 Conclusions
A novel class of amphiphilic PMPC-PHPMA diblock copolymers has been
synthesized via ATRP. In general, these syntheses were well-controlled, affording
copolymers with polydispersities between 1.20 and 1.30 and actual compositions
that were close to the targeted compositions. These copolymers were readily
dissolved or dispersed in cold aqueous solution and exhibited a range of phase
behavior depending on the degree of polymerization of the PMPC and PHPMA
blocks, the copolymer concentration and the solution temperature. Thus, a
PMPC24-PHPMA23 diblock copolymer is molecularly dissolved at 4 °C in
concentrations up to 5.0 w/v %. Increasing the temperature led to the formation of
very large aggregates with dimensions of several hundred nanometers at
concentrations above 2.0 w/v %, with a critical aggregation temperature that
ranged from 30 °C at 2.0 w/v % to 15 °C at 5.0 w/v %. Increasing the PHPMA
block length for a fixed PMPC block length of ~ 25 led to concentration-
dependent aggregation at all temperatures. PMPC49-PHPMA49 diblock copolymer
is molecularly dissolved at 4 °C up to 3.0 w/v %, with large colloidal aggregates
being formed at 5.0 % w/v. In this particular case, aggregation occurred at a
temperature of approximately 10-12 °C almost independent of copolymer
concentration. The solution behavior of PMPC49-PHPMA26 or PMPC49-PHPMA67
was more complicated due to the co-existence of several colloidal species, as
revealed by cumulants analyzes. In addition, 1
H NMR analyses indicated that the
PHPMA chains within these aggregates remained at least partially solvated,
suggesting coexistence with unimers. Furthermore, longer PHPMA blocks led to
the formation of smaller, less hydrated aggregates whose size was only weakly
concentration-dependent.
Since most of these copolymers exhibit relatively high critical aggregation
concentrations, they may offer some potential for intracellular drug delivery.
After delivering the drug, gradual dilution should lead to dissolution of the
aggregates, allowing their excretion in the form of molecularly dissolved chains.

161
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Ikerni, M., Odagiri, N., Tanaka, S., Shinohara, I., Chiba, A. Macromolecules
1981, 14, 34-39
35
Yusa, S.-i., Shimada, Y., Mitsukami, Y., Yamamoto, T., Morishima, Y.
36
Mitra, S. K., Dass, N., Varshneya, N. C. J. Chem. Phys. 1972, 57, 1798-1799
37
Candau, F., Heatley, F., Price, C., Stubbersfield, R. B. Eur. Polym. J. 1984,
20, 685-690
38
Ma, J.-h., Guo, C., Tang, Y.-l., Liu, H.-z. Langmuir 2007, 23, 9596-9605
39
Tokuhiro, T., Amiya, T., Mamada, A., Tanaka, T. Macromolecules 1991, 24,
2936-2943
40
Cabana, A., Aїt-Kadi, A., Juhász, J. J. Coll. Int. Sci. 1997, 190, 307-312
41
42
Maeda, Y., Higuchi, T., Ikeda, I. Langmuir 2000, 16, 7503-7509
43
Lutz, J.F., Hoth, A., Macromolecules 2006, 39, 893-896
44
Sugihara, S., Ph.D. Thesis: Stimuli-Responsive Block Copolymers by Living
Cationic Polymerization: Precision Synthesis and Self-Association with High
Sensitivity, Department of Macromolecular Science, Graduate School of
Science, Osaka University, Osaka, Japan 2003, Chapter 3

Chapter 5: Derivatization of Rhodamine 6G and Preparation of Fluorescent PMPC-based (co)polymers
163
Chapter 5: Derivatization of Rhodamine 6G and
Preparation of Fluorescent PMPC-based
(co)polymers

164
5.1 Introduction
The use of synthetic polymers for the intracellular delivery of drugs requires a
detailed knowledge of the final fate of the macromolecular vector. One solution to
this problem is to fluorescently label the polymer chains. This allows its diffusion
within tissue and/or live cells to be monitored using established techniques such
as fluorimetry and confocal fluorescent microscopy.1-4
Ideally, the dye label
should emit in a part of the spectrum where there is little auto-fluorescence from
either the cell constituents or the body fluids under investigation. In addition, high
quantum yields are clearly advantageous since this minimizes the degree of
labeling that is required. For relatively expensive dyes this allows significant cost
savings and it may also minimize any possible toxic or physical effects caused by
the dye label. Finally, dyes with high photostabilities are preferred. One class of
dyes that fulfill all of the above requirements is rhodamines, which are
characterized by high quantum yield, emit in the red part of the spectrum, are
cost-effective and offer good photostability.5
In general, there are several methods for the covalent attachment of a dye label
onto a polymer chain: specific labeling of a reactive site, copolymerization with a
monomeric dye or using dye-labelled initiators. Functionalized rhodamines are
widely available for coupling via various chemistries.6
These dyes are commonly
used for labeling specific sites in biological macromolecules. However, these
compounds are significantly more expensive than non-reactive dyes.7
In addition,
there are several literature examples of polymerizable vinylic rhodamine
derivatives8-11
with at least one commercially available rhodamine-labelled
monomer.12
Usually such polymerizable dyes are copolymerized with
conventional vinyl monomers to give statistical copolymers with relatively low
dye contents. In contrast, using monofunctional fluorescent initiators allows the
chromophore to be placed precisely at the polymer chain-end.
There appears to be no reports on rhodamine-based initiators for ATRP, although
a number of other fluorescent dye initiators have been used to prepare labelled
copolymers.13-18
For example, a 2-bromoisobutyrate ester of fluorescein allowed

165
good control to be obtained in the polymerization of NIPAM.13
However, the
relatively poor photostability of fluorescein5
combined with the hydrolytic
instability of aromatic esters19
suggests that polymers derived from this initiator
may not be ideal for biomedical applications where monitoring is required over
extended periods of time (days to weeks) in aqueous solution. According to
Zhang and co-workers, an ATRP initiator based on phenyl oxazole14
has proved
to be efficient. However, this chromophore has an emission maximum at 370 nm
and at this wavelength autofluorescence of cellular constituents may be
problematic. Similarly, the anthracene-based initiator reported by Klumperman’s
group15
should have an emission maximum at around 400 nm (i.e. similar to that
of native anthracene) which may also lead to autofluorescence problems.
Initiators based on substituted naphthalimides exhibit maximum emissions at
around 500 nm,15,18
which is close to that of fluorescein. Thus the former may be
useful alternative labels to the latter commonly used dye. On the other hand,
rhodamine dyes are generally more photostable than fluorescein5
and are also
relatively water-soluble.20,21
In principle, the facile modification of rhodamine 6G
should allow functional dyes to be incorporated into polymers either as ATRP
initiators or monomers. This should complement the existing labelled initiators
and vinyl monomers and also allow more efficient tracking of synthetic
macromolecules in living systems. The use of such rhodamine-labelled
copolymers in the context of monitoring the intracellular delivery of various
drugs22,23
and also for monitoring diffusion into tissue-engineered human oral
mucosa24
was recently reported. Here, the facile synthetic protocols for modifying
rhodamine 6G so as to obtain both pH-dependent and pH-independent fluorescent
labels that can be subsequently converted into the corresponding monofunctional
ATRP initiators and also a vinyl monomer based on a pH-independent label are
described.
Rhodamines exist in a fluorescent hydroquinone form at neutral and acidic pH
and a non-fluorescent spirolactone form at basic pH (Scheme 5.1).25

166
ON
O
N
+
OH
HH
ON
O
N
O
H H
2'
H+
OH- 2'
Hydroquinone form Spirolactone form
Scheme 5.1: Base-induced conversion of hydroquinone to spirolactone for 2’-substituted
rhodamine 6G
Amide formation in the 2’ position of rhodamine esters has been reported in
several recent papers and patents.7,25-29
Primary amines react directly with the
cyclic ester to form secondary amides under mild conditions. For these
compounds, conversion to the cyclic spirolactam occurs at lower pH than for the
rhodamine ester starting material and no significant fluorescence was observed
above pH ~ 6.30
A pH value below 6 is rarely encountered in biological systems
and therefore these compounds most likely are of limited use for fluorescent
probing of living tissue. However, they may offer applications as fluorescent pH
indicators. A synthetically elegant but somewhat laborious solution to this
problem is to couple the rhodamine-based secondary amide to a fluorescein dye.30
This fluorophore emits light over a wide pH range at a wavelength that depends
on the solution pH.
If a tertiary amide derivative of the rhodamine dye is used instead of a secondary
amide, then internal amide formation at high pH is prevented. Thus conjugation is
retained and no loss of fluorescence is observed in alkaline solution.7
Formation
of the tertiary amide does not occur under mild conditions, but it has been
reported using either benzotriazole coupling agents28,29
or highly reactive Lewis
acids;7
in contrast, other commonly used amidation reagents such as
carbodiimides afforded only low yields.7
In addition, the synthesis of rhodamine-
based acid halides has been described in the patent literature. These highly
reactive compounds have been used to prepare a range of tertiary amide
derivatives.26,27
However this approach precludes the use of functional amines

167
such as γ-aminoalcohols, unless protecting groups are employed. Therefore, an
additional synthetic step is required to prepare hydroxy-functional rhodamine
dyes that exhibit pH-independent fluorescence.7
On the other hand, the direct
reaction between cyclic lactones and secondary amines has been described to
proceed in high yields under relatively mild conditions, particularly if a large
excess (up to 20 equivalents) of amine is used.31
This approach, where the amine
is used as solvent and reactant has not been reported for the preparation of
rhodamine-based tertiary amides.
In this chapter, a convenient one-step synthesis of a range of hydroxy-functional
rhodamine 6G-based dyes with tertiary amides is reported. In addition, protocols
for the esterification of both hydroxy-functional secondary amides and tertiary
amides to produce a series of 2-bromoisobutyryl esters are described. These
compounds are useful as fluorescently-labeled ATRP initiators. In addition, the
synthesis of a methacrylic ester has also been conducted. In principle, this
monomer can be statistically copolymerized to produce fluorescently-labeled
copolymer chains.
2-Bromoisobutyryl esters are usually considered to be particularly effective
ATRP initiators.32
Thus, the rhodamine 6G esters prepared herein have been used
for the ATRP synthesis of a range of well-defined fluorescently-labeled
biocompatible homopolymers based on the biomimetic monomer, 2-
(methacryloyloxy)ethyl phosphorylcholine (MPC). MPC-based stimulus-
responsive block copolymers comprising either 2-(diisopropylamino)ethyl
methacrylate (DPA) or 2-hydroxypropyl methacrylate (HPMA) have also been
prepared by sequential monomer addition.
However, when preparing the above copolymers it was found that their number-
average molecular weights measured by end-group analysis were in general
higher than the target molecular weight. Thus the effect of the ATRP catalyst on
the chemical stability of these new ATRP initiators was examined. To act as an
ATRP initiator, a 2-bromoisobutyryl ester must react with a copper(I) catalyst to
form a radical species according to Scheme 5.2a.33
In the presence of monomer,
ATRP should be the prevailing reaction (Scheme 5.2b). In the absence of
monomer, the initial radical may react with other radicals (Scheme 5.2c) or with

168
solvent (Scheme 5.2d).34,35
Since the concentration of the ester initiator is of the
order of 10-2
M and the equilibrium is shifted towards the halide form, the radical
concentration is very low. Therefore, the probability of radical recombination is
also very low (Scheme 5.2c), whereas transfer to solvent is much more likely
(Scheme 5.2d).34,35
O
O
Br
R
O
O
R
O
O
R
M
O
O
CH3
CH3
R
H
O
O
R
O
O
R
O
O
Br
CH3
R OH
+ Cu+-(bpy)2
-Br-
+.
M
ka
'
kda
'
Cu2+-(bpy)2-(Br-)2
.
ATRP
MeOH
x
x x'
a)
b)
c)
Recombination
Transfer to solvent
d)
MeOH
MeOH
+
Transesterification
e)
Scheme 5.2: a) Reaction of 2-bromoisobutyric esters with a Cu(II)(bpy)2 complex to form a
radical species.33
b) ATRP with a monomer according to Matyjaszewski.33
c) Radical
recombination.34,35
d) Transfer to solvent.34,35
e) Transesterification with methanol.36
In addition, transesterification with methanol may also occur (Scheme 5.2e). This
reaction is formally an equilibrium, which should adjust on mixing the reactants
according to the equilibrium constant and Le Chatelier’s principle.36
However, in
the absence of a catalyst the reaction is often very slow. The rate of
transesterification is significantly accelerated by either acids or bases, but several
other compounds have also been used.36
In particular, Yamamoto et al. reported
that alkoxy-triphenylphosphine-copper(I) complexes are very efficient
transesterification catalysts.37
In addition, complexes between copper(II) and
terpyridines are known to be efficient transesterification catalysts for
phosphodiesters.38,39
Thus it is reasonable to assume that copper/bipyridine
complexes may well act as transesterification catalysts, in addition to their
primary role as ATRP catalysts. If the aim is to prepare end-functionalized
polymers via functionalized ester initiators, knowledge of the rate and degree of

169
transesterification becomes essential, since the transesterified initiators and
polymers will not have the desired chain-end functionality.
5.2.1 Materials
Rhodamine 6G (99 %), N-(2-hydroxyethyl)piperazine (98.50 %) and lithium
bromide (LiBr, 99 +%) were obtained from Acros Organics (Geel, Belgium) and
were used as received. 3-Aminopropan-1-ol (99 %), 2-(methylamino)ethanol (99
%), sodium hydrogen carbonate (99.7 +%, A.C.S. grade), 2-bromoisobutyryl
bromide (98 %), anhydrous methanol (MeOH, 99.8 %), CuBr (99.999 %), 2,2’-
bipyridine (bpy, 99 %) morpholine (> 99%, ReagentPlus®), methacrylic acid (99
%), methacrylic anhydride (94 %), 2,6-di-tert-butyl-4-methylphenol (BHT, ≥ 99
%), ethyl 2-bromoisobutyrate (EtOBr, 98 %), deuterated methanol (CD3OD,
99.96 atom %), trifluoroacetic acid (TFA, 99+ %), triethylamine (Et3N, ≥ 99 %),
and 2-(butylamino)ethanol (98+ %) were all purchased from Sigma Aldrich UK
(Dorset, UK) and were used as received. The silica gel 60 (0.063 – 0.200 µm)
used to remove the spent ATRP catalyst was purchased from E. Merck
(Darmstadt, Germany) and was used as received. 2-Bromoisobutyric acid (>98 %)
was obtained from Fluka (Dorset, UK) and was used as received. HPLC grade
acetonitrile, diethyl ether, dichloromethane, methanol, tetrahydrofuran,
isopropanol and n-heptane were obtained from Fisher Scientific (Loughborough,
UK) and were used as received. Magnesium sulfate (MgSO4), sodium chloride
(NaCl), triethylamine (Et3N) and sodium sulfate (Na2SO4) were laboratory
reagent grade from Fisher Scientific (Loughborough, UK) and were used as
received. Sodium bromide (NaBr, 99 + %) and hydrochloric acid (HCl, 32 %,
general purpose grade) were purchased from Fisher Scientific (Loughborough,
UK) and were used as received.
donated by Biocompatibles UK Ltd. (Farnham, UK) and was used as received. 2-
Hydroxypropyl methacrylate (HPMA) was donated by Cognis Performance
Chemicals (Hythe, UK) and used as received. 2-(Diisopropylamino)ethyl
methacrylate (DPA, >98 %) was purchased from Scientific Polymer Products
(Ontario, US) and passed through a DHR-4 column (provided by the

170
manufacturer) to remove inhibitor prior to polymerization. Phosphate-buffered
saline (PBS) was prepared from tablets obtained from Oxoid (Basingstoke, UK).
Regenerated cellulose dialysis membrane (1,000 MWCO) was purchased from
Spectra/Por. Disposable UV-grade cuvettes were obtained from Fisher Scientific
(Loughborough, UK).
2-phenoxyethyl 2-bromoisobutyrate initiator, PhOBr was prepared according to
chapter 4 of this thesis.
5.2.2 Preparation of 2-bromoisobutyric anhydride
2-Bromoisobutyric anhydride was prepared according to a previously published
procedure.40
2-Bromoisobutyric acid (10.0133 g, 60.0 mmol) was dissolved in
dichloromethane (75 mL) and N,N’-dicyclohexylcarbodiimide (6.8106 g, 33.0
mmol) was added to this solution. The resulting opaque mixture was stirred at
25°C overnight. The precipitate was filtered off, and the filtrate was concentrated
through rotary evaporation and precipitated into cold, dry n-heptane. The residue
was filtered, washed with cold n-heptane, and dried under reduced pressure to
give 4.18 g (44 %) of a white solid.
1
H NMR (CDCl3): δ 1.99 (s, 12H, CH3) ppm.
13
C NMR (CDCl3): δ 165.71, 54.90, 30.12 ppm.
5.2.3 Reaction between rhodamine 6G and 3-aminopropan-1-ol to give
rhodamine 6G N-(3-hydroxypropyl)amide, 1
This reaction was conducted according to a previously published procedure,
except that DMF was substituted for acetonitrile.25,30
Rhodamine 6G (10.011 g,
20.7 mmol) was dissolved in 200 mL acetonitrile. To this solution was added 3-
aminopropan-1-ol (4.8 mL, 63 mmol). The reaction mixture became gradually
heterogeneous and lost color. After 20 h, most of the solvent was evaporated to
obtain a concentrated solution (approximately a third of its original volume) and
the mixture was filtered. The solid was washed thoroughly with water and dried
under vacuum till constant weight to give 8.71 g (89 %) of an off-white product,
1.

171
1
H NMR (400 MHz, 3:1 CDCl3: CD3OD) δ 8.09 (m, 1H), 7.71 (m, 2H), 7.27 (m,
1H), 6.55 (s, 1H), 6.33 (s, 1H), 3.52 (t, 2H, J = 5.5 Hz), 3.41 (m, 6H), 2.09 (s,
6H), 1.52 (t, 6H, J = 7.09 Hz), 1.34 (m, 2H) ppm
13
C NMR (400 MHz, 3:1 CDCl3: CD3OD) δ 172.02, 156.26, 154.54, 150.44,
135.65, 133.26, 131.07, 130.67, 126.63, 125.24, 120.99, 107.75, 99.23, 68.75,
61.39, 40.86, 38.92, 33.19, 19.07, 16.87 ppm
ESI-MS, m/z (M+H)+
472
5.2.4 Esterification of 1 with 2-bromoisobutyryl bromide to give rhodamine
6G N-(3-(2-bromoisobutyryl)propyl)amide, 2
To 1 (3.0 g, 6.3 mmol) was added acetonitrile (200 mL) and 32 % hydrochloric
acid (1.0 mL, 10 mmol). On refluxing this mixture, a dark red solution was
formed within 45 minutes. Then 2-bromoisobutyryl bromide (1.0 mL, 7.9 mmol)
was added to the refluxing solution. After a further 2.5 h, an additional charge of
2-bromoisobutyryl bromide was added (0.5 mL, 4.0 mmol). After a total reaction
time of 5 h, the reaction mixture was evaporated to afford a viscous oil. Addition
of ether (100 mL) led to precipitation overnight at -25 °C. The precipitate was
dispersed in water and an excess of sodium hydrogen carbonate was added. After
stirring for 3 h, the aqueous dispersion was extracted with dichloromethane (three
50 mL portions).
The combined organic extracts were dried over dry magnesium sulfate and
filtered, washing the filtrate with dichloromethane. Evaporation and drying in
vacuum afforded the desired ATRP initiator 2 (3.70 g, 94 %) in its neutral form.
1
H NMR (400 MHz, CDCl3) δ 7.91 (m, 1H), 7.46 (m, 2H), 7.04 (m, 1H), 6.33 (s,
2H), 6.21 (s, 2H), 3.94 (t, 2H, J = 6.0 Hz), 3.21 (m, 6H), 1.90 (s, 6H), 1.87 (s,
6H), 1.51 (m, 2H), 1.32 (t, 6H, J = 7.21 Hz) ppm
13
C NMR (400 MHz, CDCl3) δ 171.37, 168.11, 153.51, 151.73, 147.41, 132.42,
131.33, 128.49, 128.04, 123.80, 122.74, 117.97, 106.07, 96.54, 65.00, 63.79,
56.01, 38.38, 37.10, 30.70, 27.27, 16.70, 14.74 ppm
ESI-MS, m/z (M+H)+
622

172
5.2.5 General reaction between rhodamine 6G and a secondary amine
In a round-bottomed flask, rhodamine 6G (10.0 g, 0.021 mol) was dissolved in the
secondary amine (10.0 g). The flask was fitted with a reflux condenser, placed
under nitrogen and heated to 90 °C for approximately 24 h. After cooling, the
solution was dissolved in the minimum amount of methanol and poured into 500
mL water. After filtering, the aqueous solution was saturated with sodium
chloride and extracted with 50 mL aliquots of a 2:1 isopropanol:dichloromethane
mixture until only a faint color remained in the aqueous phase. The combined
organic phases were dried over anhydrous sodium sulfate, filtered and evaporated.
The resulting solid was recrystallized from methanol. The hydrochloride salt or
hydrobromide salt were prepared by dissolving this solid in water, adding 1.1
molar equivalents of the corresponding acid and freeze-drying the aqueous
solution overnight.
5.2.6 Reaction between rhodamine 6G and 2-(methylamino)ethanol to give
rhodamine 6G N-(2-hydroxyethyl)-N-methyl amide, 3
Yield: 52 % (as HBr salt)
1
2H), 6.50 (m, 2H), 3.23 (q, 4H, J = 7.21 Hz), 3.03 (m, 4H), 2.71 (s, 1.7H), 2.44 (s,
1.3H), 1.91 (s, 6H), 1.13 (t, 6H, J = 7.21 Hz) ppm
13
140.02, 134.76, 134.15, 133.93, 133.67, 133.38, 131.53, 129.17, 117.53, 97.75,
63.11, 53.63, 42.33, 36.07, 21.02, 17.31 ppm
ESI-MS, m/z (M+H)+
472
Accurate Mass (Calculated), (M): 471.251960 (471.252192) corresponding to an
elemental composition of C29H33N3O3 (C29H33N3O3)
5.2.7 Reaction between rhodamine 6G and diethanolamine to give
rhodamine 6G N-(bis(2-hydroxyethyl))amide, 4
Yield: 75 % (as HCl salt)

173
1
1H), 6.80 (s, 2H), 6.56 (s, 2H), 3.47 (t, 2H, J = 5.50 Hz), 3.33 (q, 4H, J = 7.21
Hz), 3.15 (m, 8H), 1.99 (s, 6H), 1.20 (m, 6H, J = 7.21 Hz) ppm
13
139.07, 133.84, 132.90, 132.56, 131.48, 130.33, 128.15, 116.71, 96.81, 62.26,
61.84, 55.46, 50.36, 41.43, 20.01, 16.44
ESI-MS, m/z (M+H)+
502
Accurate Mass (Calculated), (M+H)+
: 502.2685 (502.2706) corresponding to an
5.2.8 Reaction between rhodamine 6G and N-(2-hydroxyethyl)piperazine to
give rhodamine 6G N-(4-(2-hydroxyethyl)piperazine) amide, 5
Yield: 65 % (in neutral form)
1
1H), 6.73 (s, 2H), 6.55 (s, 2H), 3.46 (t, 1H, J = 5.50 Hz), 3.40 (t, 2H, J = 5.62 Hz),
3.29 (q, 7.27 Hz), 3.16 (br m, 2H + MeOH), 2.94 (br t, 2H, J~5.1 Hz), 2.54 (br t,
2H, J~5.0 Hz), 2.40 (t, 1H, J = 5.38 Hz), 2.24 (t, 2H, J = 5.62 Hz), 1.97 (s, 6H),
1.17 (t, 6H, J = 7.21 Hz) ppm
13
138.00, 136.75, 133.94, 133.05, 132.82, 132.27, 130.42, 128.20, 116.52, 96.66,
61.20, 55.50, 52.98, 46.41, 41.25, 20.00, 16.23 ppm
ESI-MS, m/z (M+H)+
527
5.2.9 Reaction between rhodamine 6G and 2-(butylamino)ethanol to give
rhodamine 6G N-(4-hydroxy butyl)-N-methyl amide, 6
Yield: 56 % (in HCl salt form)
Recrystallized from chloroform
1
H NMR (400 MHz, 3:1 CDCl3: CD3OD) δ 7.50 (m, 3H), 7.19 (m, 1H), 6.83 (ss,
2H), 6.58 (ss, 2H), 3.41 (br t, 1H, J = 5.75 Hz), 3.33 (q, 4H, J = 7.21 Hz), 3.08 (br
t, 1H, J = 5.75 Hz), 2.97 (br m, 2H), 2.90 (br t, 1H, J = 5.38 Hz), 2.81 (m, 1H),

174
2.00 (ss, 6H), 1.20 (t, 6H, J = 7.21 Hz), 0.95 (br q, 1H, J = 7.46 Hz), 0.61 (br m,
4H), 0.46 (br t, 2H, J = 6.11 Hz) ppm
13
C NMR (400 MHz, 3:1 CDCl3: CD3OD) δ 168.9, 159.0, 157.3, 156.1, 136.7,
131.0, 129.8, 129.2, 128.0, 126.9, 125.6, 123.5, 114.0, 94.0, 60.4, 57.8, 51.1,
48.3, 38.6, 30.6, 27.8, 19.9, 18.1, 13.9 ppm
ESI-MS, m/z (M+H)+
514
5.2.10 Reaction between rhodamine 6G and morpholine to give rhodamine
6G N-morpholinamide, 11
Yield: 10 % (in neutral form)
Recrystallized from dichloromethane
1
H NMR (400 MHz, 3:1 CDCl3: CD3OD) δ7.54 (m, 2H), 7.39 (m, 1H), 7.18 (m,
1H), 6.73 (s, 2H), 6.57 (s, 2H), 3.30 (q, 4H, J = 7.21 Hz), 3.27-3.10 (m, br, 8H),
1.98 (s, 6H), 1.19 (t, 6H, J = 7.21 Hz) ppm
13
138.72, 135.07, 134.22, 134.07, 134.04, 133.32, 131.57, 129.30, 117.64, 97.86,
70.36, 46.11, 42.42, 21.04, 17.37 ppm
ESI-MS, m/z (M+H)+
484
5.2.11 Reaction between hydroxy-functional rhodamine derivatives and 2-
bromoisobutyric anhydride to give a monofunctional ATRP initiator
using 2-bromoisobutyric acid as solvent.
In a round-bottomed flask was placed hydroxy-functional rhodamine derivative (3
or 5) (neutral form, 500 mg, ~ 1 mmol) and 5.0 g 2-bromoisobutyric acid (30
mmol). The mixture was placed under nitrogen and heated to the stated
temperature (see below). Once a homogeneous solution had formed, 2-
bromoisobutyric anhydride (635 mg, 2.0 mmol) was added. After 24 to 48 h, no
further reaction occurred and the reaction mixture was cooled to room
temperature and diethyl ether (100 mL) was added. After filtration and washing
with diethyl ether, the solid residue was partitioned between dichloromethane

175
(100 mL) and water (50 mL). Sodium hydrogen carbonate was added until gas
evolution ceased and the aqueous phase was washed with aliquots of
dichloromethane (3 x 50 mL). The combined organics were washed with water
(five 50 mL portions) and finally with a saturated sodium bromide solution (50
mL). The organic phase was dried over anhydrous sodium sulfate, filtered and
evaporated. The crude product was recrystallized from THF.
5.2.12 Reaction between 3 and 2-bromoisobutyric anhydride to give a
monofunctional initiator, rhodamine 6G N-(2-(2-bromoisobutyryl)-
ethyl)-N-methyl amide, 7
Temperature: 70 °C. Yield 0.327 g (53 %) of neutral product after
recrystallization.
1
1H), 7.00 (m, 2H), 6.80 (m, 2H), 3.90 (t, 2H, J = 5.26 Hz), 3.49 (m, 5 H), 2.98 (s,
3H), 2.16 (s, 6H), 1.81 (s, 6H), 1.36 (t, 6H, J = 7.34) ppm
13
154.58, 135.64, 130.84, 130.23, 129.97, 129.83, 129.30, 127.57, 125.42, 113.57,
93.90, 67.89, 63.41, 49.84, 46.529, 38.42, 30.50, 17.64, 13.64 ppm
ESI-MS, m/z (M+H)+
620
elemental composition of C33H39BrN3O4 (C33H39BrN3O4)
5.2.13 Reaction between 5 and 2-bromoisobutyric anhydride to give a
monofunctional initiator, rhodamine 6G N-(4-(2-(2-
bromoisobutyryloxy)ethyl))piperazine amide, 8
Temperature: 50 °C. Yield: 0.420 g (66 %) of neutral product after
recrystallization.
1
1H), 7.18 (s, 2H), 6.99 (s, 2H), 4.48 (t, 2H, J = 5.50 Hz), 3.75 (q, 4H, J = 7.21
Hz), 3.62 (br m, 4H), 2.87 (t, 2H, J = 5.50 Hz), 2.59 (br m, 4H), 2.43 (s, 6H), 2.13
(s, 6H), 1.60 (t, 6H, J = 7.21 Hz) ppm

176
13
156.17, 136.58, 132.40, 131.67, 131.47, 131.41, 130.88, 129.02, 126.73, 115.06,
95.27, 69.26, 64.38, 57.09, 43.15, 39.84, 31.79, 26.80, 18.51, 14.78 ppm
ESI-MS, m/z (M+H)+
675
elemental composition of C36H44BrN4O4 (C36H44BrN4O4)
5.2.14 Reaction between 5 and methacrylic anhydride to give a
monofunctional monomer, rhodamine 6G N-(4-(2-
(methacryloyloxy)ethyl))piperazine amide, 9
In a round-bottomed flask was placed 5 (neutral form, 1.0051 g, 1.9 mmol) and
methacrylic acid (20.0 mL, 20.3 g, 0.236 mol). To this mixture was added 50 mL
chloroform and 12.6 mg BHT. Once a homogeneous solution had formed,
methacrylic anhydride was added (2 mL, 2.07 g, 13.4 mmol) was added. After 40
h, 2 mL methanol was added to quench residual methacrylic anhydride and the
reaction mixture was left for further 2 h. Chloroform was evaporated at 30 °C
under reduced pressure and the residue was poured into 200 mL diethyl ether.
After filtration and washing with diethyl ether, the solid residue was partitioned
between dichloromethane (100 mL) and water (50 mL). Sodium hydrogen
carbonate was added until gas evolution ceased and the aqueous phase was
washed with aliquots of dichloromethane (3 x 50 mL). The combined organics
were washed with water (five 50 mL portions) and finally with a saturated sodium
bromide solution (50 mL). The organic phase was dried over anhydrous sodium
sulfate, filtered and evaporated. This product was found to be ≥ 95 % pure by 1
H
NMR. If necessary, the product could be recrystallized from THF.
Yield: 0.859 g (76 %) of neutral product after precipitation from diethyl ether.
1
H NMR (400 MHz, 3:1 CDCl3: CD3OD) δ 7.78 (m, 2H), 7.65 (m, 1H), 7.46
(m,1H), 7.00 (s, 2H), 6.85 (s, 2H), 6.09 (s, 1H), 5.63 (s, 1H), 4.23 (t, 2H, J = 5.62
Hz), 3.55 (q, 4H, J = 7.15 Hz), 3.41 (br m, 4H), 2.65 (t, 2H, J = 5.75 Hz), 2.37 (br
m, 2H), 2.32 (br m, 2H), 2.21 (s, 6H), 1.93 (br s, 3H), 1.40 (t, 6H, J = 7.21 Hz)
ppm

177
13
153.43, 136.05, 131.51, 130.28, 129.69, 128.97, 127.51, 126.05, 119.66, 113.49,
93.64, 61.84, 56.32, 53.47, 52.69, 47.53, 41.79, 38.44, 19.80, 18.52, 13.76 ppm
ESI-MS, m/z (M+H)+
595
5.2.15 Reaction between 4 and 2-bromoisobutyric anhydride to give a bi-
functional initiator, rhodamine 6G N-(bis((2-
bromoisobutyryloxy)ethyl))amide, 10 using phase-transfer conditions.
Rhodamine 6G N-(bis(2-hydroxyethyl))amide (Cl-salt, 1.030 g, 1.914 mmol) was
dissolved in 50 mL water. 2-Bromoisobutyric anhydride (1.0439 g, 3.304 mmol)
was dissolved in dichloromethane (9 mL) and transferred to the reaction mixture
with dichloromethane (11 mL). Further, dichloromethane (10 mL) was then
added. After 47 h, the reaction mixture was transferred to a separating funnel with
saturated sodium hydrogen carbonate. The aqueous phase was extracted with
dichloromethane (4 x 50 mL). The combined organic phases were washed with
water (3 x 50 mL), then with brine (50 mL). After drying over sodium sulfate and
filtering, the solution was concentrated at 50 °C, cooled to room temperature and
precipitated with diethyl ether. After filtration, the solid was redissolved in
dichloromethane and precipitated with diethyl ether and this procedure was
repeated until no more acid could be detected by NMR. The resulting dark red
solid was dried in vacuum overnight to give 0.22 g (14 %) of neutral product.
1
H NMR (CD3OD) δ 7.64 (m, 3H), 7.29 (m, 1H), 6.82 (s, 2H), 6.56 (s, 2H), 4.09
(m, 2H), 3.65 (m, 2H), 3.45 (m, 8H), 2.19 (s, 6H), 1.81 (s, 6H), 1.71 (s, 6H), 1.28
(s, 6H) ppm
13
C NMR (CDCl3) δ 171.14, 169.40, 157.02, 156.04, 155.95, 153.10, 135.18,
130.82, 130.34, 128.93, 128.00, 127.75, 126.13, 113.23, 93.86, 63.49, 48.00,
43.57, 38.37, 30.60, 18.93, 13.86 ppm
ESI-MS (M+H)+
800

178
5.2.16 Preparation of PMPC homopolymers using a rhodamine-based
initiator
In a typical experiment, 7 (0.17 mmol, 1.0 equivalent) and MPC (1.00 g, 3.38
mmol, 20 equivalents) was dissolved in 1.5 mL anhydrous methanol. After
purging the solution for 20 minutes, CuBr (24.3 mg, 0.17 mmol, 1.0 equivalent)
bpy (52.9 mg, 0.34 mmol, 2.0 equivalents) was added. After 1.5 h, methacrylic
protons were no longer detected by 1
H NMR and the reaction mixture was diluted
with methanol and exposed to air. The homogeneous solution was then diluted
with methanol and passed through a silica column to remove the spent copper
catalyst. The dark red solution was evaporated and washed thoroughly with THF
to remove residual bpy followed by acetonitrile to remove any unreacted initiator.
Finally the polymer was redissolved in water and freeze-dried overnight, followed
by drying in a vacuum oven at 80 °C for two days. Yield: ~75 %.
These polymers were also efficiently purified by dialysis against methanol,
typically using dialysis membranes with a MWCO of 1,000 Da.

179
5.2.17 Preparation of pH-responsive PMPC-PDPA diblock copolymers using
a rhodamine-based ATRP initiator
In a typical experiment, MPC (1.00 g, 3.34 mmol, 25 equivalents) under nitrogen
was dissolved in anhydrous methanol (1.5 mL) containing 7 (0.135 mmol, 1
equivalent) and purged with nitrogen for 20 minutes. Then bpy (42.7 mg, 0.273
mmol, 2 equivalents) and CuBr (19.7 mg, 0.137 mmol, 1 equivalent) were mixed
as solids and added. After 37 minutes a nitrogen-purged solution of DPA (2.60 g,
12.2 mmol, 90 eq.) in anhydrous methanol (4 mL) was added to the polymerizing
solution via cannula. After 63 h, the reaction mixture was exposed to air and
diluted with isopropanol. The homogeneous solution was then passed through a
silica column to remove the spent catalyst and evaporated. The dark red residue
was washed thoroughly with acetonitrile. Then the solid was dispersed in water
followed by careful evaporation of the water under reduced pressure at 50 °C.
This procedure was repeated twice. Finally the solid was dispersed in water and
freeze-dried overnight, followed by drying in a vacuum oven at 80 °C for two
days.
5.2.18 Preparation of a temperature responsive PMPC-PHPMA diblock
copolymer using a rhodamine-based ATRP initiator
In a typical experiment, MPC (1.0039 g, 3.40 mmol, 25 equivalents) was placed
under nitrogen. 7 (143.0 mg, 0.231 mmol) was dissolved in anhydrous methanol
(2.5 mL). 1.5 mL of this solution (85.8 mg, 0.138 mmol, 1 equivalent) was added
to the MPC. After purging the solution with nitrogen for 30 minutes, bpy (44.0
mg, 0.282 mmol, 2 equivalents) and CuBr (20.3 mg, 0.142 mmol, 1 equivalent)
was mixed and added. After 30 minutes, a sample was removed for 1
H NMR
analysis and nitrogen-purged HPMA (1.1728 g, 8.134 mmol, 60 equivalents) was
added.
After 24 h, the reaction mixture was exposed to air and diluted with methanol.
The homogenous solution was passed through a silica column to remove spent
catalyst and evaporated. The polymer was then re-dissolved in methanol and
precipitated using a 3:1 v/v mixture of tetrahydrofuran and 40-60 petroleum ether.
After stirring overnight and cooling to -25 °C, the mixture was filtered. The solid
was redissolved in 50 mL methanol and evaporated. Then 50 mL water was

180
added, the mixture was stirred until homogenous and the water was evaporated
partially at 60 °C. Addition of further water, homogenization and evaporation was
repeated twice in order to thoroughly remove residual methanol. Then 50 mL
water was added, and the solution was frozen and freeze-dried. Finally the solid
polymer was placed in a vacuum oven at 80 °C for 48 h.
5.2.19 Preparation of temperature responsive PHPMA-PMPC-10-PMPC-
PHPMA triblock copolymer gelators using a bifunctional rhodamine-
based initiator
MPC (4.9877 g, 16.89 mmol, 250 equivalent) and 10 (54.2 mg, 0.0678 mmol, 1
equivalent) were dissolved in anhydrous methanol (6.0 mL) under nitrogen. The
solution was purged with nitrogen for 40 minutes. Then bpy (43.8 mg, 0.280
mmol, 4 equivalents) and CuBr (20.7 mg, 0.1446 mmol, 2 equivalents) was mixed
as solids and added. After 5 h, nitrogen-purged HPMA (0.9764 g, 6.77 mmol, 100
equivalents) was added through cannula. After 5 days, the reaction mixture was
exposed to air and diluted with methanol. The homogenous solution was then
passed through silica with methanol to remove the spent catalyst. The solvent was
evaporated and the polymer was precipitated with tetrahydrofuran. After
filtration, the solid was redissolved in methanol, which was evaporated. This was
repeated twice in order to remove tetrahydrofuran. Then the residue was dispersed
in water followed by careful evaporation at 50 °C and this was repeated twice.
Then, water was added and the solution was frozen and freeze-dried. Finally the
polymer was dried in a vacuum oven at 80 °C for 2 days.
5.2.20 Synthesis of deuterated methyl 2-bromoisobutyrate
To 1 mL deuterated methanol in an NMR tube was added 2 drops of 2-
bromoisobutyryl bromide. The solution was left for 1 h and used directly for 1
H
NMR and HPLC measurements.

181
5.2.21 General protocol for examining transesterification of 2-
bromoisobutyryl esters in methanol in the presence of the CuBr/2 bpy
ATRP catalyst.
In a typical protocol, 2-bromoisobutyryl ester (approximately 10-4
mol determined
to three significant figures) was dissolved in 2.0 mL anhydrous methanol or
deuterated methanol. The reaction mixture was purged with nitrogen for 5
minutes. Then CuBr (1 equivalent) and bpy (2 equivalent) was mixed and added.
Aliquots were taken out, diluted with aerated CD3OD and analyzed by 1
H NMR
and HPLC at regular intervals. In addition, selected samples were analyzed by
electrospray mass spectroscopy (ESI-MS).
A similar procedure was followed for the control experiments; CuBr was either
replaced with one equivalent of CuBr2, or omitted altogether, whereas two
equivalents of bpy ligand were used in all cases.
5.2.22 Calculation of the fraction of remaining 2-bromoisobutyryl ester
initiator in the presence of the ATRP catalyst
The fraction of remaining initiator was defined as the initiator concentration
divided by the sum of the initiator concentration and the cleaved alcohol.
Alternatively, the fraction of remaining initiator is given by the initiator
concentration divided by the sum of the initiator concentration and the methyl
ester by-product concentration. This fraction was calculated using either 1
H NMR
or HPLC. In the latter case, it was assumed that the initiator and the methyl ester
by-product or the cleaved alcohol had the same UV extinction coefficient.
5.2.23 Gel permeation chromatography
Chromatograph and two Polymer Laboratories PL Gel 5µm Mixed-C 7.5 x 300
mm columns in series with a guard column at 40°C connected to a Gilson Model
131 refractive index detector. The eluent was a 3:1 v/v % chloroform/methanol
mixture containing 2 mM LiBr at a flow rate of 1.0 ml min-1
. A series of near-
monodisperse PMMA samples were used as calibration standards. Toluene (2 µl)

182
was added to all samples as a flow rate marker. Data analysis was carried out
using CirrusTM GPC Software supplied by Polymer Laboratories.
5.2.24 Reverse-phase high performance liquid chromatography
HPLC chromatograms were acquired using a Varian ProStar HPLC system
consisting of an autosampler (Varian Model 410), a solvent delivery module
(Varian Module 230) and a UV-detector (Varian Model 310). The column was
either a 150 x 4.6 mm ProGemini 5µ C18 110 Å or a 100 x 4.6 mm Thermo
Hypersil Keystone 3µ Betabasic-18. Chromatographic conditions: 95 % 0.1 %
aqueous TFA:acetonitrile to 100 % acetonitrile in 20 minutes followed by
equilibration for 10 minutes at the original conditions prior to injection of a new
sample. Sample: Approximately 0.5 % solution in methanol, 0.2 µl injected.
Detection: UV at 254 nm. Data were collected with Star Chromatography
Workstation system control version 6.20.
5.2.25 Molar absorption coefficient determination
Solutions for measuring the molar absorption coefficient of the rhodamine
derivatives were prepared by weighing approximately 20.0 mg of dye in a 25 mL
or a 100 mL volumetric flask using a microbalance and filling to the mark with
either 0.1 M aqueous HCl or methanol or methanol containing 0.1 % v/v
trifluoroacetic acid. Serial dilution of these stock solutions using pipettes and
volumetric flasks gave solutions with absorbances ranging between 1.0 and 1.5.
Further sequential dilutions allowed evaluation of the molar absorption
coefficient, which is expressed as an average of either two or three values. Stock
solutions for determining the polymer molecular weight were obtained in a
similar fashion; polymers were weighed into a 100 mL volumetric flask and
serially diluted to give solutions with absorbances ranging between 1.0 and 1.5.
This ‘maximum absorbance’ solution was then further diluted and the Beer-
Lambert law (A = ε·c·l) was used to calculate the apparent initiator concentration
for each solution, using the molar absorption coefficient, ε, at maximum
wavelength (λ = 538 nm to 541 nm depending on the polymer and initiator as
given in Table 5.2). The stated value of ε is an average of three measurements. A

183
PC-controlled Perkin-Elmer Lambda 25 uv/visible absorption spectrophotometer
was used to record spectra from 300 nm to 700 nm at a scan rate of 240 nm min-1
with a slit width of 1 nm. All measurements were performed using disposable
UV-grade cuvettes.
5.2.26 pH-dependent absorption and emission of 1 and 3
1 and 3 (2-3 mg, 50 µmol) in a 25 mL measuring flask was dissolved in 0.1 M
HCl (25 mL). This solution was further diluted with 0.1 M HCl to give a final
solution with an absorbance between 0.1 and 2.0 (approximately 10-4
to 10-5
M) A
2 mL aliquot was removed after measuring the solution pH using a calibrated pH
meter (Hanna Instruments). The pH was then slowly increased using NaOH
concentrations of 0.50 M, 0.05 M and 0.001 M. A 2 mL aliquot was removed at
approximately every pH unit. Each aliquot was analyzed by uv/visible absorption
spectroscopy, fluorescence spectroscopy and dynamic light scattering.
A PC-controlled Perkin-Elmer Lambda 25 uv/visible absorption
spectrophotometer was used for recording spectra from 300 nm to 700 nm at a
scan rate of 240 nm min-1
with a slit width of 1 nm. A PC-controlled Fluoromax-3
fluorimeter was used for obtaining fluorescence spectra under the following
conditions: excitation wavelength = 530 nm, emission scans from 540-700 nm at
240 nm min-1
, an excitation slit width of 5 nm and an emission slit width of 2.5
nm.
5.2.27 pH-dependent absorption, emission and dynamic light scattering of
PMPC-PDPA diblock copolymers
A literature protocol was followed:41
In a typical procedure, diblock copolymer 7-
PMPC22-PDPA84 (50.0 mg) was dissolved in 0.1 M HCl (25.0 mL). A 2 mL
aliquot was removed after measuring the pH using a calibrated pH meter (Hanna
Instruments). The pH was then slowly increased using NaOH concentrations of
0.50 M, 0.05 M and 0.001 M. A 2 mL aliquot was removed at approximately
every pH unit. Each aliquot was analyzed by uv/visible absorption spectroscopy,
fluorescence spectroscopy and dynamic light scattering. A PC-controlled Perkin-
Elmer Lambda 25 spectrophotometer was used for recording spectra from 300 nm

184
to 700 nm at a scan rate of 240 nm min-1
with a slit width of 1 nm. A PC-
controlled Fluoromax-3 fluorimeter was used for obtaining fluorescence spectra
under the following conditions: excitation wavelength = 530 nm, emission scans
from 540 to 700 nm at 240 nm/min, an excitation slit width of 5 nm and an
emission slit width of 2.5 nm (Unless otherwise stated). Dynamic light scattering
experiments were performed with a Zetasizer Nano-ZS (Malvern Instruments,
UK) at a scattering angle of 173°. Dispersion Technology Software version 4.20
from Malvern Instruments was used for the data analyses.
5.2.28 Temperature-dependent absorption and fluorescence emission of 7-
PMPC30-PHPMA60
7-PMPC30-PHPMA60 (7.7 mg) was dissolved in water (7.7 mL) to give a 0.1 w/v
% solution. A PC-controlled Perkin-Elmer Lambda 25 spectrophotometer was
used for recording spectra from 300 nm to 700 nm at a scan rate of 240 nm min-1
with a slit width of 1 nm. The temperature was controlled using a PTP-1 Peltier
system in conjunction with a PCP 150 Peltier system. A PC-controlled
Fluoromax-3 fluorimeter was used for obtaining fluorescence spectra under the
following conditions: excitation wavelength = 530 nm, emission scans from 540
to 700 nm at 240 nm/min, an excitation slit width of 5 nm and an emission slit
width of 2.5 nm. The temperature was controlled using a water bath (nüve BS302)
5.2.29 Thermogravimetric analysis
Analyses were conducted using a Perkin-Elmer Pyris 1 TGA instrument. Samples
dried at 80 °C were heated in air to 800 °C at a heating rate of 10 °C min-1
. J.
Balmer is acknowledged for the TGA analysis.
5.2.30 Gel Rheology Studies
Copolymer (100.0-200.0 mg) was dissolved in demineralised water (1.00 mL) for
rheology studies. These solutions were subjected to several freeze-thaw cycles in
order to remove trapped air and left to stand in a refrigerator at 4 °C overnight. A
Rheometric Scientific SR-5000 rheometer equipped with cone-plate geometry

185
(40.0 mm, 0.05 radians) was used for the oscillatory temperature sweeps,
employing a frequency of 1 rad/s, a stress of 0.5 Pa and a heating rate of 3
°C/min. This instrument was fitted with a Peltier element for temperature control
and a thermostatted water-bath was used as a heat sink.
5.2.31 Evaluation of the extent of hydrolysis of the initiator end-groups
PMPC homopolymer (25.0 mg) was dissolved in PBS at pH 7.2 (2.500 mL) to
give a 1.00 % aqueous solution. This was divided into eight sealed vials (0.300
mL in each vial). One vial was analyzed by gel permeation chromatography
immediately, while the remaining two vials were placed in an incubator at 37 °C.
After the specified times, a vial was removed from the oven and immediately
frozen at - 25 °C (approximate freezing time ~ 5-10 min). At the end of the
experiment, these vials were thawed and filtered through a 0.45 µm nylon filter
immediately prior to GPC analysis. The extent of end-group hydrolysis was
assessed by aqueous gel permeation chromatography (GPC) at 20°C using two
Polymer Laboratories Aquagel-OH 8 mm columns (Type 40 first, followed by
Type 30) in series with a Polymer Laboratories LC1200 UV/visible detector at
530 nm followed by a Polymer Laboratories ERC-7515A refractive index
detector. The aqueous mobile phase was a mixture of 0.2 M NaNO3 and 0.01 M
NaH2PO4 adjusted to pH 7 using aqueous NaOH.
5.3 Results and discussion
5.3.1 Reaction between rhodamine 6G and 3-aminopropan-1-ol
The literature reaction25,30
between rhodamine 6G and primary amines to give is
shown in Scheme 5.3 a). This reaction was reported to proceed spontaneously at
room temperature in DMF, with spirolactam yields ranging from 54 % to 92 %
depending on the primary amine used. Replacing DMF with acetonitrile, CH3CN,
gave 89 % isolated yield when 3-aminopropan-1-ol was used as the primary
amine. Since CH3CN is significantly easier to remove than DMF, the former
solvent was preferred for such reactions. As both reactants are water-soluble,
purification is easily achieved by washing the water-insoluble product with excess

186
water. 1
H and 13
C NMR spectra and ESI-MS analysis were all consistent with the
target structure.
5.3.2 Direct reaction between secondary amines and rhodamine 6G
The direct reaction between rhodamine 6G and a secondary amine is shown in
Scheme 5.3 b). The secondary amine was used as a reactive solvent, typically
using 1.0 g rhodamine 6G dye per gram of amine. Maintaining the reaction
mixture at 90°C for 17-23 h gave the desired tertiary amide in 10-75 % yield with
the main by-product being the cyclic lactone, as determined by electrospray
ionization mass spectroscopy (ESI-MS). Various secondary amines were
evaluated as shown in Scheme 5.3. Yields for the reactions of rhodamine 6G with
alcoholic secondary amines were in excess of 50 %, whereas the reaction with
morpholine only afforded 10 % yield. This difference is believed to be due to the
relatively poor solubility of rhodamine 6G in morpholine.42
ON
O
N
+
O
Cl
N
R1
R2H
ON
O
N
N
R2
R1
ON
O
N
NR1
N
R1
HH
O
N
OH
90 °C
17-23 h
1: R1 = -CH2CH2CH2OH (89 %)
4: R1 = R2 = -CH2CH2OH (75 %)
6: R1 = -CH2CH2OH, R2 = -CH2CH2CH2CH3 (56 %)
3: R1 = -CH2CH2OH, R2 = -CH3 (52 %)
25 °C
24 h
CH3CN
pH-dependent rhodamine derivative, 1
pH-independent rhodamine derivative, 3-6
a)
b)
11: R (= R1 = R2) = (10 %)
5: R (= R1 = R2) = (65 %)
Scheme 5.3: General reaction of rhodamine 6G with various secondary amines to form the
corresponding substituted amides. Numbers in parentheses are yields of isolated purified
compounds.
In this context, it is perhaps noteworthy that the synthesis of a similar hydroxy-
terminated rhodamine B derivative has been reported with an overall yield of 50

187
% in two steps.7
Thus the one-step protocol produces similar or better yields
without requiring protecting group chemistry. All products were highly water-
soluble, whereas the cyclic lactone by-product is water-insoluble. Aqueous
solubility was also been reported for the similar rhodamine B-based compound.7
The reaction shown in Scheme 5.3 was also attempted using rhodamine B instead
of rhodamine 6G. However, no detectable amount of the target tertiary amide was
formed using this dye. The main difference between the two dyes is that
rhodamine 6G is an ethyl ester, whereas rhodamine B is in its free carboxylic acid
form. Thus, the difference in reactivity may be explained as follows: In case of
the ethyl ester rhodamine 6G, the reaction probably proceeds by direct
displacement of ethanol by the amine. The acidic rhodamine B, on the other hand,
becomes de-protonated by the amine and this leads to formation of the cyclic
lactone,7
which is unreactive towards secondary amines.

188
5.3.3 Esterification of hydroxy-functional rhodamine derivatives
ON
O
N
N
NXO
ON
O
N
N
O
O
Br
N
ON
O
N
O
O
Br
O
O
Br
N
ON
O
N
O
O
Br
ON
O
N
N
OH
N
ON
O
N
OH
ON
O
N
N
NOH
OH
N
ON
O
N
OH
5
b or c
8: X=2-bromoisobutyryl 9: X=methacryloyl
1
a
ATRP initiator, 2
ATRP initiator 73
b
ATRP initiator 104
d
Scheme 5.4: Esterification of three hydroxyfunctional rhodamine derivatives to produce
various fluorescently-labelled ATRP initiators and a fluorescently-labelled methacrylic
monomer. Reaction conditions: a) (i) CH3CN, 32 % HCl, reflux. (ii) 2-bromoisobutyryl
bromide, 3h, reflux (iii) Aqueous NaHCO3:CH2Cl2. Yield: 89 % ; b) (i) 2-bromoisobutyric
acid, 70 °C, (ii) 2-bromoisobutyric anhydride, 70 °C. (iii) Aqueous NaHCO3:CH2Cl2. Yield:
66 %; c) (i) methacrylic acid, CHCl3, 25 °C, (ii) methacrylic anhydride, 25 °C, (iii) Aqueous
NaHCO3:CH2Cl2. Yield: 76 % d) (i) 2-bromoisobutyric anhydride in water:dichloromethane
5:3, 47 h, 25 °C (ii) Aqueous NaHCO3:CH2Cl2. Yield: 14 %
The synthetic routes to the various ATRP ester initiators are shown in Scheme
5.4. The secondary amide, 1, was isolated in its non-protonated spirolactam form
(Scheme 5.3a). Addition of a small excess of 32 % aqueous HCl to a suspension
of this compound in acetonitrile gave a deep red solution, indicating protonation
of the amine groups and formation of the conjugated hydroquinone form. Heating

189
to reflux afforded better solubility and addition of 2-bromoisobutyryl bromide
gave the target product in 94 % yield within 3 h (Scheme 5.4). The resulting
ATRP initiator, 2, was isolated in sufficient purity (≥ 95 % by 1
H NMR and
analytical HPLC) to be used directly for polymer syntheses. Further purification
(> 99 %) was achieved by recrystallization from methanol:water mixtures or by
preparative reverse phase HPLC.25,30
Applying the same protocol to the tertiary amide, 3, gave the desired product but
in a much lower yield. ESI-MS analysis indicated that amide hydrolysis was
significant giving the acid as the main by-product. This illustrates that the tertiary
amide is significantly more prone to acidic hydrolysis than the secondary amide.
In addition, it was found that if the amine hydrochloride salt form of the dye was
used, a significant amount of the 2-chloroisobutyryl ester was obtained due to a
halogen exchange side-reaction. In general, the yield of the final ester was only
around 10 % using this protocol, thus alternative approaches were examined. The
classic solution is to add a weak base to remove the hydrobromic acid formed in
situ. However, in this particular case, this will lead to deprotonation of the
aromatic amines thereby rendering them prone to substitution. Thus, when the
reaction was conducted in the presence of pyridine, ESI-MS analysis revealed the
formation of aromatic 2-bromoisobutyryl amides in addition to the desired ester.
Use of 2-bromoisobutyric anhydride instead of the acid bromide gives 2-
bromoisobutyric acid as a by-product instead of HBr. 2-Bromoisobutyric acid is a
weaker acid and it was found that virtually no amide hydrolysis occurred when
the anhydride was used. Unfortunately, the reaction was very slow in common
solvents such as acetonitrile and DMF, with only 10-20 % conversion being
achieved over 4-5 days even at 80-90 °C; this may be related to the low solubility
of the tertiary amide, 3, in non-protic solvents. Using 2-bromoisobutyric acid as
the solvent was found to significantly improve the yield. This acid melts at around
47 °C, which is why it is necessary to work above this temperature. This approach
is illustrated for compound 5 in Scheme 5.4. The reaction could be monitored by
HPLC by directly comparing the intensity of the peaks assigned to starting
material and product as the reaction produced very few by-products. Two
different temperatures were compared (Figure 5.1) and it was found that at 70 °C,
a conversion of around 70 % was obtained after 24 h for the reaction with 3, while
the reaction was significantly slower at 50 °C. Further increasing the temperature

190
did not lead to a significantly higher conversion but more by-products were
formed. Therefore the reaction was generally carried out at 70 °C.
0 10 20 30 40 50 60 70 80
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
70 °C
50 °C
Initiatorfraction
Time / h
Figure 5.1: Kinetics of formation of rhodamine 6G-based initiator 7 versus time as
determined by reverse phase HPLC.
Since the product ester was highly soluble in dichloromethane after neutralization,
purification was relatively straightforward given the low solubility of the starting
material in this solvent; after neutralization with sodium hydrogen carbonate, the
product was extracted selectively into dichloromethane. The same procedure was
found to be applicable for conversions of the piperazine-based adduct 5 into the
ester initiator 8. The deprotonated form of 8 proved to be less soluble in water
than 7. This is believed to be the main reason for the higher isolated yields of 8.
Both esters could be purified by recrystallization from THF, whereas attempts to
purify these products by column chromatography (silica) were not successful
(both column adsorption and a significant degree of hydrolysis were observed).
A similar approach was used for the preparation of a rhodamine-based
methacrylic monomer to make compound 9 (Scheme 5.4). This reaction was
conducted at 20 °C, which is above the m.p. of methacrylic acid (16o
C) but
sufficiently low to avoid thermal polymerization. As compound 5 has relatively

191
low solubility in pure methacrylic acid at this temperature, it was necessary to add
chloroform as a co-solvent. This gave the reported yield of 76 %.
The bi-functional ATRP initiator, 10, was prepared using phase-transfer
conditions; compound 4 is highly soluble in water but has a relatively low
solubility in dichloromethane. The 2-bromoisobutyric anhydride on the other
hand is poorly soluble in water but highly soluble in dichloromethane. Rapid
mixing of the two-phase system led to formation of the initiator 10, probably
because compound 4 has a certain affinity for dichloromethane and therefore acts
as a phase transfer agent. The isolated yield of this compound was only 14 % in
48 h. This indicates that the reaction is relatively inefficient. In addition, a
significant of monosubstituted product was formed even when a large excess of
anhydride was used. Although this may reflect a different reactivity of the two
hydroxy-groups, it may also be related to the phase-transfer conditions. However,
the 1
H NMR spectra indicates that the 2-bromoisobutyryl groups are in different
chemical environments (Figure 5.2).

192
O
Br
O
O
O
Br
ON
O
N
N
HH
1.02.03.04.05.06.07.08.0 1.02.03.04.05.06.07.08.0
δ / ppm
a
b
c
df
h
i
j
k
g
m
m
a
b
k
i
j
eg
h’
m’
m’
k
6 H
m, m’
12 H
j
6H
h, h’
4 H
g, i
8 H
CH2Cl2
a, b, c, d, e, f
8 H
Figure 5.2: Assigned 1
H-NMR spectrum of the pH-independent bifunctional rhodamine-
based ATRP initiator 10
A similar difference between the nitrogen substituents could be distinguished in
the starting compound 4 and also for most of the products of rhodamine 6G and
secondary amines. This phenomenon is often observed for substituted amides and
is due to conformational isomerism arising from hindered rotation around the
amide bond.43
This isomerism probably accounts for the two chemically
distinguishable hydroxyl groups in 4. In addition, the different environments for
the two bromine atoms may lead to a difference in reactivity when 10 is used as
an ATRP initiator. This could potentially lead to the formation of polymer chains
of unequal length.

193
5.3.4 Elemental analyses of rhodamine 6G derivatives
Selected rhodamine 6G derivatives were analyzed by elemental microanalysis in
order to assess their purity. It was found that the halogen content was typically
20-30 % higher than the theoretical values. In contrast, values for carbon, nitrogen
and hydrogen were less than 10 % lower than the theoretical values. However,
microanalysis of the commercially available rhodamine 6G (hydrochloride salt)
used in all experiments gave a chlorine content that was 18 % higher than the
theoretical value, whereas the results for carbon, nitrogen and hydrogen were
around 5 % lower than their theoretical values. As the purity of this dye precursor
was stated to be 99 %, it appears that elemental analysis is not a particularly
reliable method for determining the halogen content of these compounds.
Therefore these microanalytical results are omitted from the characterization data.
5.3.5 Absorption maxima and molar absorption coefficients obtained for
various rhodamine derivatives
Table 5.1 shows the absorbance maxima and molar absorption coefficients
obtained for the various rhodamine 6G derivatives. For the two dyes containing a
secondary amide group (1 and 2) the maximum absorption wavelength is
essentially the same as for rhodamine 6G, both in water and acidic methanol. The
molar absorption coefficient of 1 in methanol with 0.1 % added trifluoroacetic
acid is similar to that of rhodamine 6G in methanol, whereas it is significantly
lower for 2. The molar absorption coefficients observed in 0.1 M HCl are
significantly smaller than those reported for compounds prepared using diamines,
which are typically of the order of 60,000.30
On the other hand, a more complex
coupling product of normetanephrine and rhodamine 6G has a reported molar
absorption coefficient of 41,400,25
which is significantly closer to the value of
34,000 obtained for 2. Both of these dyes were isolated in the spirolactam form
(Scheme 5.1), which is not directly soluble in water. These dyes only dissolved
very slowly in 0.1 M HCl, even with heating and ultrasonic treatment. Dissolution
was rapid in 1.0 mL of 32 % HCl, which could be diluted with water without
precipitation. However, the magnitude of extinction coefficients using this

194
procedure was of the order of 5,000 M-1
·cm-1
which is significantly lower than for
rhodamine 6G and for the derivatives with tertiary amides. Therefore, solutions
were prepared by dissolving the dyes in methanol with 0.1 % v/v TFA and
diluting this stock solution with 0.1 M aqueous HCl. This indicates that the
conversion of each dye to its fluorescent hydroquinone form is relatively slow and
may not go to completion in aqueous acid. Moreover, it is known that poorer
solvent quality tends to lower the extinction coefficient.44
Such a solvent effect is
also observed in the present work: when dissolved in methanol, 3 and 4 both have
molar absorption coefficients close to that of rhodamine 6G, whereas the
corresponding values in 0.1 M aqueous HCl are generally significantly lower.
Compound
λmax
nm
(MeOH)
λmax
nm
(0.10 M HCl)
εmax
cm
-1
·M
-1
(MeOH)
10
-4
x εmax
cm
-1
·M
-1
(0.10 M HCl)
Rhodamine 6G 529 N/M 111,000 ± 900 -
1 529
a)
530
b)
10,900 ± 1,000
a)
52,000 ± 800
b)
2 529
a)
530
b)
88,000 ± 3,000
a)
34,000 ± 2,000
b)
3 533 532 114,000 ± 600 87,000 ± 2,500
4 533 533 116,000 ± 250 87,000 ± 3,900
5 533 534 100,000 ± 2,300 84,000 ± 2,500
6 534 533 100,000 ± 200 85,000 ± 5,600
7 534 534 84,000 ± 1,900 67,000 ± 1,200
8 534 535 87,000 ± 850 89,000 ± 2,100
9 534 534 93,000 ± 1,100 91,000 ± 2,900
10 536 - 94,000 ± 1,200 -
11 533 532 87,000 ± 9,000 90,000 ± 500
Table 5.1: Maximum wavelength and corresponding molar absorption coefficients in MeOH
and 0.10 M HCl for various rhodamine 6G derivatives. a)
These measurements were
performed in methanol containing 0.1 % v/v trifluoroacetic acid. b)
The dye was dissolved in
25.0 mL methanol containing 0.1 % v/v trifluoroacetic acid and diluted with 0.1 M aqueous
HCl. The error is the standard error for the three measurements. Each uncertainty is the
standard error of three measurements at three different concentrations. ‘N/M’ simply means
not measured.
The molar absorption coefficients of 5 and 6 in methanol are around 10 % lower
than that of rhodamine 6G. This effect is larger for compounds 7-11, which each
have molar absorption coefficients of around 80 % of that of rhodamine 6G in
methanol. The molar absorption coefficient of dyes 3-7 in 0.1 M HCl is

195
significantly lower than in methanol, which is probably related to the reduced
solvation between these two solvents. This is illustrated in Figure 5.3, where
absorption spectra recorded for 7 dissolved in both 0.1 M HCl and methanol are
shown. Despite the differing concentrations, the maximum values are almost
identical. The main difference is in the relative intensity of the shoulder at
approximately 508 nm, which is significantly weaker in methanol than in 0.1 M
HCl. This feature is directly related to the aggregation of the dye molecules.21,45
As the dimer is not fluorescent, the solvent quality will affect the emission output.
400 425 450 475 500 525 550 575 600 625 650
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.26x10
-5
M in MeOH
1.63x10
-5
M in 0.1 M HCl
Absorbance
Wavelength / nm
Figure 5.3: Absorption spectra obtained for 7 in methanol and 0.1 M HCl. Scan speed: 240
nm/min.
In contrast, the ATRP initiator and monomer based on the piperazine derivative (8
and 9) exhibit very similar molar absorption coefficients in 0.1 M HCl and
methanol. These compounds have additional amine functionality due to the
piperazine ring. This extra amine group becomes protonated at low pH, which
improves aqueous solubility relative to the other derivatives. The morpholine
derivative, 11, exhibits a similar behavior and this cannot be attributed to
protonation. This compound does not have a hydrophobic 2-bromoisobutyryl or
methacrylate group why it would be expected that this compound is more water-
soluble.

196
5.3.6 pH-dependence of absorption and emission behavior
Figure 5.4 shows typical normalized absorption and emission fluorescence spectra
obtained for the derivatized rhodamine dyes in acidic aqueous solution. These are
similar to those reported for rhodamine 6G.21,45
400 425 450 475 500 525 550 575 600 625 650 675 700
EmissionAbsorption
NormalisedAbsorption/Emission/A.U.
Wavelength / nm
Figure 5.4: Normalized absorption and emission spectra of 3 in aqueous HCl at pH 2.0. The
emission spectrum was recorded with an excitation wavelength of 530 nm.
The effect of increasing the pH of a 10-5
M solution of 1 on the emission and
absorption spectra is shown in Figure 5.5A and Figure 5.6A. Both the maximum
emission and the absorption at 530 nm increase monotonically from pH 1 to pH 4.
This is because pH adjustment involved addition of aqueous base and this dilution
changes the dye unimer/dimer ratio in favor of the unimers.21,45
Since dimers act
as fluorescence quenchers,45
increasing the relative unimer concentration can lead
to an increase in absorption at 530 nm and emission, provided that the former
effect is larger than the dilution factor. This is apparent from inspection of the
absorption spectra up to pH 2 (Figure 5.5A). Raising the pH leads to precipitation,
which leads to an increase of the background scattering in the absorption spectra.
This precipitation is due to formation of the water-insoluble non-fluorescent
spirolactam form of the dye (Scheme 5.1). Above pH 4, this becomes the
dominant factor in the attenuation in the absorption and emission spectra. This
effect is also evident in digital photographs of the aqueous solutions/suspensions

197
(Figure 5.6A). Figure 5.6B shows how the relative emission and the relative
A530/A508 absorbance ratio vary as a function of pH for a 10-5
M solution of 3. The
peak assigned to dimer formation is observed as a shoulder at approximately 508
nm. This feature is at a longer wavelength than the reported peak of 496 nm for
the rhodamine 6G dimer in aqueous solutions.45
400 425 450 475 500 525 550 575 600
pH 9.2pH 7.0
pH 6.1
pH 4.0
pH 2.0
pH 1.0
Absorption/A.U.
Wavelength / nm
400 425 450 475 500 525 550 575 600
pH 9.0
pH 7.2
pH 6.0
pH 4.0
pH 2.0
Absorption/A.U.
Wavelength / nm
A B
Figure 5.5: (A) Absorption spectra of 1 versus pH. (B) Absorption spectra of 3 versus pH
This spectral shift may be due to the slightly different molecular structures, but
overlapping peaks make precise location of such features rather problematic.
Nevertheless, the change in the absorbance ratio gives an approximate
unimer/dimer concentration ratio. Increasing the pH from 1.5 to 10 more than
doubles the emission intensity, despite dilution of the solution. As the A530/A508
absorbance ratio increases simultaneously, this increase in emission is believed to
be related to a shift in the unimer-dimer equilibrium.

198
0 1 2 3 4 5 6 7 8 9 10
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Imax
/Imax,pH1
/A.U.
pH
Abs530nm
1 2 3 4 5 6 7 8 9 10
0.0
0.4
0.8
1.2
1.6
2.0
2.4
1.6
1.7
1.8
1.9
2.0
Imax
/Imax,pH1.5
/A.U.
pH
Abs530nm
/Abs508nm
/A.U.
pH:
0.5 1.0 2.0 3.0 4.0 5.0 6.1 7.0 8.0 9.2
pH:
1.5 2.0 3.0 4.0 5.0 6.0 7.0 7.2 8.0 9.0 10.0
A B
C D
Figure 5.6: (A) Effect of increasing the solution pH on the maximum emission normalized
with respect to pH 1.0 and absorbance at 530 nm for a solution initially containing 5•10-5
M
1; (B) Effect of increasing the pH on the maximum emission and the relative absorbance at
530 nm and 508 nm respectively for a solution initially containing 1•10-5
M 3. (C) Digital
image of 5•10-5
M 1 at different pH (D) Digital image of 1•10-5
M 3 at different pH.
5.3.7 Use of rhodamine-based ATRP initiators to prepare PMPC
homopolymers
ATRP initiators, 7,8 and 10 were used to prepare PMPC homopolymers using a
previously reported ATRP protocol as shown in Scheme 5.5.46,47

199
O
O
O
P
O
N
O O
MPC
Cu(I)Br, bpy
methanol, 20°C
7, 8 or 10
7-PMPCn
8-PMPCn
PMPCn-10-PMPCn
or
or
Scheme 5.5: Synthesis of PMPC homopolymers by ATRP using the rhodamine 6G-based
initiators
Table 5.2 summarizes the characterization data obtained for the various
homopolymers. The maximum absorption wavelength was red-shifted by 5-8 nm,
relative to that of the corresponding initiators for all molecularly-dissolved
copolymers (Table 5.1). This shift indicates a change in the molecular
surroundings44
and must be due to the polymer.
The homopolymer based on initiator 7 with a target DP of 20 (Table 5.2, entry 1),
has a number average molecular weight determined by 1
H NMR that is a little
higher than the targeted. However, it was not possible to obtain reliable degrees of
polymerization for the higher molecular weight copolymers based on this
initiator; the aromatic rhodamine signals could be detected but the uncertainties in
the NMR integrals was relatively high due to poor signal-to-noise ratios. The
number average molecular weight determined by 1
H NMR for the 7-PMPC25
homopolymer was significantly lower than that indicated by absorption
spectroscopy using the molar absorption coefficient of the initiator (Table 5.1).
For 7-PMPC50, 7-PMPC100 and 7-PMPC200 the Mn values determined by
absorption spectroscopy were also significantly higher than targeted. TGA
analyses (Figure 5.7) indicated that these copolymers contained around 15 %
water, even after extensive drying under vacuum at 90 °C. This is not unexpected,
since it is known that water binds tenaciously to PMPC.48
However, even

200
allowing for such water contents cannot account for the high Mn values. The Mn
was also determined in methanol for all samples with essentially identical results
to those obtained in 0.1 M HCl (data not shown).
100 200 300 400 500 600 700 800
10
20
30
40
50
60
70
80
90
100
7-PMPC100
7-PMPC25
Weight%
Temperature / °C
Figure 5.7: Weight loss as a function of heating in air of 7-PMPC20 and 7-PMPC100. J.
Balmer is acknowledged for the TGA experiments.

201
Entry
Target
Composition
1
H NMR
Composition
Mn
Target
Mn
NMR
a)
Mn
GPC
b)
Mw/Mn
GPC
b)
Mn
Amax
(λmax)
c)
1 7-PMPC20 7-PMPC25 6,500 8,000 15,000 1.21
16,000
± 200
(539)
2 7-PMPC50 N/A
d)
15,400 N/A 25,000 1.22
54,000
± 1,600
(539)
3 7-PMPC100 N/A
d)
30,200 N/A 30,000 1.25
66,000
± 40
(539)
4 7-PMPC200 N/A
d)
59,700 N/A 57,000 1.47
98,000
± 20,200
(540)
5 8-PMPC20 8-PMPC26 6,600 8,300 15,000 1.18
11,000
± 2,400
(542)
6
PMPC10-10-
PMPC10
PMPC13-10-
PMPC13
6,700 8,500 16,000 1.23
16,000
± 200
(538)
H NMR, GPC and absorption data for homopolymers prepared
using two rhodamine-based ATRP initiators, 7 and 8. a) 1
H NMR spectra recorded in
CD3OD. b)
GPC in 3:1 CHCl3:CH3OH using poly(methyl methacrylate) calibration
standards. c)
Mn Calculated from the εmax value of the initiator (see Table 5.1) in methanol.
The uncertainty values are the standard error of three measurements at three different
concentrations. d)
Signals from the rhodamine monomer could not be integrated due to their
low intensity.
The homopolymer of MPC with a target DP of 20, prepared using initiator 8
(Table 5.2, Entry 5) has the same Mn by GPC as that based on 7 (Table 5.2, Entry
1). This indicates similar initiator efficiencies. 8-PMPC20 was purified by dialysis
in methanol using a dialysis membrane with a molecular weight cut-off of 1,000.
GPC analysis of the dialysate confirmed removal of oligomers, as expected (data
not shown). Therefore, the molecular weight of the dialyzed homopolymer is
expected to be higher than targeted and this is supported by the 1
H NMR results
that indicates that the composition is 8-PMPC26. However, the number average
molecular weight of 11,000 measured by visible absorption spectroscopy is

202
significantly closer to the number obtained by 1
H NMR of 8,300. Assuming 85 %
water content, the two numbers are 12 % apart which is a reasonable number
considering the combined uncertainties on the absorption data (Table 5.2) and 1
H
NMR data (~10-20 %). This indicates that the longer distance between the dye
and the initiating group leads to a smaller change of the molar absorption
coefficient of the dye.
The number-average molecular weight of the copolymer prepared using
bifunctional initiator 10 for a target degree of polymerization of 20 (Table 5.2,
Entry 6) was determined by 1
H NMR and visible absorption spectroscopy,
respectively. These values are similar to that of a PMPC homopolymer prepared
with the same target degree of polymerization using monofunctional initiator 7
(Table 5.2, Entry 1). This indicates that initiators 10 and 7 have similar
efficiencies.
5.3.8 Ethyl 2-bromoisobutyrate (EtOBr) under ATRP conditions
EtOBr is commercially available and has previously been used as an ATRP
initiator using methanol as the solvent.49,50
In addition, the 1
H NMR spectrum is
relatively simple and most signals are well-separated and easily integrable (Figure
5.8A).
Initially, EtOBr was subjected to ATRP conditions using deuterated methanol as
the solvent. According to Scheme 5.2, transesterification should lead to formation
of ethanol and the deuterated d3-methyl ester. The 1
H NMR spectra in Figure
5.8A confirms the formation of new signals that can be assigned to ethanol (peaks
d, e). However, no new signals due to the d3-methyl ester can be observed. In
addition, the two methyl groups next to the bromine atom for the d3-methyl ester
are indistinguishable from those of the starting ethyl ester. This is not surprising
given the close structural similarities between these two compounds. The kinetic
samples were also analyzed by HPLC with UV detection at 254 nm.
Representative chromatograms are shown in Figure 5.8B. Due to its lack of a UV
chromophore, ethanol cannot be detected. However, the normalized
chromatograms clearly show an enhanced signal intensity for the peak assigned to
the deuterated methyl ester at 9.1 minutes relative to the peak assigned to EtOBr

203
at 10.4 minutes with increasing reaction times. Thus, these measurements
complement the 1
H NMR measurements, which indicated ethanol formation but
could not confirm the presence of the d3-methyl ester.
The 1
H NMR spectra also show a new signal at 1.3 ppm (x,x’) with increasing
reaction time. This signal corresponds well with methyl isobutyrate according to a
manufacturer of this compound.51
This non-halogenated ester would be formed if
there is significant radical transfer to the solvent during the reaction, which would
gradually remove bromine from the 2-bromisobutyrate ester (Scheme 5.2d). This
may also account for the origin of the unassigned signal at 7.1 min in the HPLC
chromatograms (Figure 5.8B), which also increases with time. However, since no
standard was available, the identity of this compound could not be confirmed due
to time constraints.

204
6 5 4 3 2 1 0
1380 min
30 min
1 min
δ / ppm
5 6 7 8 9 10 11 12
180 min
30 min
5 min
2 min
1 min
Normalisedabsorptionat254nm/A.U.
Retention time
O
O
CH3
CH3
Br
D
D
D
a'
a'
C
H2
OCH3
O
CH3
CH3
Br
ab
c
a
a,a’
a,a’
a,a’
a
c
c
c
b
b
b
b
O
O
Br
O
O
Br
D
D
D
OH
O
Br
a’
CH2
OHCH3
d e
d
c d
d
d
e
x,x’
x,x’
x,x’
A
B
e
Figure 5.8: Kinetics of the reaction of ethyl 2-bromoisobutyrate:CuBr:bpy at a relative
molar ratio of 1:1:2 in CD3OD in the absence of any added monomer. (A) 400 MHz 1
H NMR
spectra recorded for ethyl 2-bromoisobutyrate, kinetic samples, deuterated methyl 2-
bromoisobutyrate and ethanol. (B) HPLC chromatograms recorded for ethyl 2-
bromoisobutyrate, kinetic samples, deuterated methyl 2-bromoisobutyrate and 2-
bromoisobutyric acid. HPLC column: Thermo Hypersil Keystone 100 x 4.6 mm, 3µ
Betabasic-18 Detection: UV at 254 nm.
The initiator fraction could be calculated from the integrals assigned to EtOBr and
ethanol in the 1
H NMR spectra (Figure 5.8A). Similarly, the relative
concentrations of EtOBr and deuterated methyl 2-bromoisobutyrate were

205
estimated by the relative areas under the peaks in the HPLC chromatograms.
(Figure 5.8B). The rate of decomposition of the initiator as a function of time is
shown in Figure 5.13. The two analytical methods give essentially identical
results, which suggests that the assumption of identical absorption coefficients for
the esters is a good approximation. The initiator concentration decreases rapidly
in the first 30 minutes. After 3 h, the concentration of this species is 70 % of its
original value and after 23 h there is only around 50 % of the initiator left. As
Figure 5.8A suggests, side-products cannot be ignored at these long reaction
times, which is why the actual EtOBr concentration is even lower.
The ability of the CuBr2:bpy ATRP catalyst to catalyze transesterification was
also investigated. This is important, since the kinetic samples analyzed in Figure
5.8 were quenched by aerial oxidation of the copper(I) species to the copper(II)
species. Thus if the latter species is also a transesterification catalyst then the
‘quenching’ protocol is ineffective, thereby invalidating the results shown in
Figure 5.13. However, as Figure 5.9 confirms, no further transesterification
occurred over a 48 h period.
6 5 4 3 2 1 0
+
CuBr2
:bpy 1:2
48 h
δ / ppm
O
O
Br
O
O
Br
A
B
Figure 5.9: 1
H NMR spectra recorded for: (A) EtOBr:CuBr2:bpy 1:1:2 reaction mixture in
CH3OH after 48 h; (B) EtOBr in CD3OD

206
5.3.9 Chemical stability of the 2-phenoxyethyl 2-bromoisobutyrate (PhOBr)
initiator under ATRP conditions
The PhOBr initiator was also subjected to ATRP conditions in (non-deuterated)
methanol. The 1
H NMR spectra shown in Figure 5.10A indicate the disappearance
of signals assigned to b and c due to the methylene groups of PhOBr.
Unfortunately, the signals assigned to b’ and c’ for phenoxyethanol overlap with
the large methanol signal, rendering quantification rather unreliable. However, the
HPLC chromatograms in Figure 5.10B clearly show that PhOBr disappears,
whereas peaks assigned to phenoxyethanol and deuterated methyl 2-
bromisobutyrate both appear gradually under ATRP conditions. The extent of
initiator decomposition calculated from the HPLC data (assuming identical
extinction coefficients for PhOBr and phenoxyethanol) is shown in Figure 5.13.
Within 1 minute, less than 50 % of the original initiator is left. Almost no initiator
could be detected in a reaction mixture that had been left overnight.

207
8 7 6 5 4 3 2 1 0
1380 min
30 min
1 min
δ / ppm
5.0 7.5 10.0 12.5 15.0
1380 min
180 min
30 min
5 min
2 min
1 min
Normalisedabsorptionat254nm/A.U.
Retention time / min
O OH
HH
HH
HH
H
H H b'
c'
d'
d'
e'
e'
f'
O O
O
CH3
CH3
Br
HH
H
H H
HH
HH
a
b
e
c
de
a
d
f
a,a’
a,a’
a,a’
a
x
x
x
d,e,f
d,d’,e,e’,f,f’
d’,e’,f’
c’
b’
c
c
c
b
b
b
O O
O
Br
O OH
O
O
Br
D
D
D
A
B
Figure 5.10: Kinetics of the reaction of PhOBr: CuBr: bpy at a relative molar ratio of 1:1:2
in CH3OH. (A) 400 MHz 1
H NMR spectra recorded for phenoxyethanol, kinetic samples and
PhOBr. (B) HPLC chromatograms obtained for deuterated methyl 2-bromoisobutyrate,
phenoxyethanol, kinetic samples and PhOBr. Column: Thermo Hypersil Keystone 100 x 4.6
mm, 3µ Betabasic-18 Detection: UV at 254 nm.

208
0.0 2.5 5.0 7.5 10.0 12.5 15.0
Normalisedabsorption(254nm)/A.U.
8 7 6 5 4 3 2 1 0
+
CuBr2
:bpy 1:2
48 h
δ / ppm
O OH
O O
O
Br
N N
O O
O
Br
O O
O
Br
A
B
Figure 5.11: (A) HPLC chromatograms recorded for a 1:2 PhOBr:bpy mixture after 120
min in methanol at 22 °C. Column: Thermo Hypersil Keystone 100 x 4.6 mm, 3µ Betabasic-
18 Detection: UV at 254 nm. (B) 400 MHz 1
H NMR of a PhOBr: CuBr2: bpy mixture at a
relative molar ratio of 1:1:2 after 48 h in CH3OH compared to PhOBr.
The influence of bpy ligand and the CuBr2/bpy complex was also investigated for
this system. Figure 5.11A shows a HPLC chromatogram obtained for PhOBr in
methanol in the presence of two molar equivalents of bpy after 120 min. During
this time, no phenoxyethanol is formed, demonstrating that the bpy ligand alone
does not promote transesterification. Figure 5.11B shows an 1
H NMR spectrum of
PhOBr in the presence of one equivalent of CuBr2 and two equivalents of bpy
recorded after 48 h. This is indistinguishable from that of PhOBr before addition

209
of the complex, thus demonstrating that the copper(II) species does not promote
transesterification of PhOBr.
5.3.10 Chemical stability of rhodamine 6G-based initiators under ATRP
conditions
The rhodamine 6G-based initiators 7, 8 and 10 were also examined under ATRP
conditions. As shown in Figure 5.12, transesterification occurred for all the
initiators. However, transesterification occurred much more slowly for initiator 8
(the adduct between rhodamine 6G and N-(2-hydroxyethyl)piperazine) than for
the other two initiators (Figure 5.12A). This is illustrated in Figure 5.13, which
shows that after 2 minutes, the fraction of 8 remaining attains an almost constant
value of 0.55, whereas the corresponding amounts of 7 and 10 are essentially zero
within 1 minute. Thus initiator 8 is significantly more stable towards
transesterification than PhOBr. The latter initiator was previously demonstrated to
be highly efficient, since the number-average molecular weight of PMPC
homopolymer determined by 1
H NMR corresponded well to the target molecular
weight.52
Based on these results, initiator 8 should be ideal for preparing
fluorescently-labelled polymers by ATRP in methanol, whereas the use of 7 and
10 may be problematic due to rapid transesterification. This hypothesis agrees
well with the data presented in Table 5.2, where the molecular weight determined
by visible absorption spectroscopy studies of an PMPC homopolymer synthesized
using initiator 8 lies within the experimental uncertainty of the value determined
by 1
H NMR spectroscopy. In contrast, the molecular weights obtained for the
PMPC homopolymers prepared using initiators 7 and 10 are significantly higher.

210
5 6 7 8 9 10 11 12 13 14 15
5
30 min
5 min
2 min
1 min
8 (No catalyst)
400 450 500 550 600 650 700
1380 min
5 min
Initiator
5
8
%
m/z
5 6 7 8 9 10 11 12 13 14 15
3
1380 min
180 min
30 min
5 min
2 min
1 min
7 (No catalyst)
400 450 500 550 600 650 700
1380 min
5 min
Initiator
7
3
%
m/z
5.0 7.5 10.0 12.5 15.0
4
180 min
1357 min
30 min
5 min
2 min
1 min
10 (No catalyst)
400 450 500 550 600 650 700 750 800 850 900
1357 min
5 min
10
Initiator
%
m/z
Figure 5.12: Analysis of the chemical degradation of rhodamine 6G-based initiators under
ATRP conditions. (A) HPLC chromatograms obtained for compound 5, kinetic samples of 8
with CuBr and bpy (8: CuBr: bpy = 1:1:2) and compound 8. (B) ESI-MS of selected kinetic
samples of 8 with CuBr and bpy (8: CuBr: bpy = 1:1:2) and initiator 8. (C) HPLC
chromatograms obtained for compound 3, kinetic samples of 7 with CuBr and bpy (7: CuBr:
bpy = 1:1:2) and compound 7. (D) ESI-MS of selected kinetic samples of 7 with CuBr and
bpy (7: CuBr: bpy = 1:1:2) and initiator 7 under ATRP conditions. (E) HPLC
chromatograms obtained for compound 4, kinetic samples of 10 with CuBr and bpy (10:
CuBr: bpy = 1:2:4) and compound 10. (F) ESI-MS of selected kinetic samples of 10 with
CuBr and bpy (10: CuBr: bpy = 1:2:4) and initiator 10 under ATRP conditions.
A B
C D
E
OHNRH
O
O
BrNRH
N NRH O
O
Br
N NRH OH
O
O
Br
O
O
Br
NRH
OH
OH
NRH
O
O
Br
OH
NRH
F

211
0 1 2 3 4 5 100 1000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
10, HPLC @ 254 nm
7, HPLC @ 254 nm
8, HPLC @ 254 nm
PhOBr, HPLC @ 254 nm
EtOBr,
1
H NMR
EtOBr, HPLC @ 254 nm
[Init]/([Init]+[R-OH])
Time / min
Figure 5.13: Fraction of EtOBr, PhOBr and rhodamine initiators present as a function of
time. For EtOBr, the fraction was calculated by both 1
H NMR and HPLC. For the
remaining compounds, only the HPLC data were used. These calculations assumed no side-
reactions and identical absorption coefficients for both the initiator and its by-product.
5.3.11 Copper(I)bromide:2,2’-bipyridine as a transesterification catalyst
The results in the previous sections clearly show that the CuBr:bpy catalyst
system can act as an effective transesterification catalyst for several 2-
bromoisobutyryl esters in methanolic solution. Although this may have
consequences for the end-group functionality, this is not necessarily the case for
several reasons: Firstly, essentially all the monomers used in this work are
methacrylate esters and these might also be subject to transesterification, i.e. this
reaction may compete with transesterification of the initiator. Secondly,
polymerization also competes with the transesterification and the polymer repeat
units may be less prone to transesterification. Nevertheless, it is quite possible
that the end-group functionalities undergo transesterification, especially under
monomer-starved conditions. In fact, transesterification of the initiator end-group
would explain why the absorbance-derived molecular weights of the PMPC
homopolymers prepared using 7 (Table 5.2, Entry 2-3) differ significantly from
the target molecular weights than that of the homopolymer with a target DP of 20

212
(Table 5.2, Entry 1). This is particularly likely in view of the lower initiator
concentrations and longer reaction times for the former two homopolymers.
Based on the relative initiator stabilities (Figure 5.13), this phenomenon should be
less pronounced for the more stable initiator 8 since the stability of this is higher
than that of PhOBr which has been successfully used to prepare well-defined
diblock copolymers52
but this has not been investigated in depth due to time
restraints.
5.3.12 Use of rhodamine-based ATRP initiators to prepare pH-responsive
PMPC-PDPA diblock copolymers and PMPC-PHPMA di- and
triblock copolymers
Initiators 2 and 7 were also used to prepare diblock copolymers of MPC and DPA
or HPMA. In addition, ABA triblock copolymers of MPC and HPMA with PMPC
‘B’-blocks were prepared using initiator 10. Similar polymerization conditions
were used for all copolymers (Scheme 5.6). The PMPC-PDPA diblock
copolymers exhibit pH-responsive behavior, since the tertiary amine groups on
the PDPA chains are protonated (and hence hydrophilic) at low pH but become
deprotonated (and hence hydrophobic) above approximately pH 6.3 (the pKa for
PDPA). Therefore, these copolymers are molecularly dissolved at low pH, but
form colloidal aggregates (micelles or vesicles) above pH 6.41,53,54
Copolymers of
PMPC and PHPMA exhibit thermo-responsive aggregation.52,55-57
In addition,
PHPMA-PMPC-PHPMA triblock copolymers may exhibit thermo-responsive
gelation.55,56

213
O
O
O
DPA
P
O
N
O O
O
O
N
MPC
Cu(I)Br, bpy
methanol, 20°C
20°C
2 or 7
PMPCn
PMPCn-PDPAm
O
O
HO
HPMA
PMPCn-PHPMAm
or
or
Scheme 5.6: Synthesis of PMPCn-PDPAm and PMPCn-PHPMAm diblock copolymers by
ATRP.
Table 5.2 summarizes the characterization data obtained for the various block
copolymers prepared using rhodamine-labelled initiators. As with the
homopolymers, the maximum absorption wavelength was red-shifted by 1-5 nm
relative to that of the corresponding initiators for all molecularly-dissolved
copolymers, indicating a slight change in the local environment of the
chromophore.44
The PMPC-PDPA diblock copolymers prepared using the pH-dependent initiator,
2, have relatively narrow polydispersities. The number-average molecular weight
(Mn) estimated by 1
H NMR and calculated from visible absorption spectroscopy
corresponds closely to the target molecular weight for 2-PMPC28-PDPA56, while
the calculated value is too high for 2-PMPC24-PDPA115. 2-PMPC28-PDPA56 was
purified by dialysis against a 3:1 chloroform/methanol mixture, while 2-PMPC24-
PDPA115 was purified by centrifugation of a methanolic solution. Neither of these
purification methods is expected to affect the block composition much. However,
the mean degree of polymerization is determined by comparing the initiator NMR
signal to the PMPC NMR signals in the homopolymer precursor and by
comparing NMR signals for each block in the final diblock copolymer. If low
molecular weight material has been removed, this molecular weight will be

214
higher. This may be the reason for the discrepancy between the two number-
average molecular weights. Nevertheless, the relatively narrow polydispersity
indicates reasonably good living character for the polymerization, suggesting
minimal homopolymer contamination.
The pH-independent initiator 7 also gives well-defined PMPC-PDPA and PMPC-
PHPMA diblock copolymers with relatively low polydispersities. In general, the
1
H NMR data indicate relatively high initiator efficiencies, with experimental
degrees of polymerizations being close to those targeted. The difference between
the 1
H NMR results and those obtained from visible absorption spectroscopy is
not unexpected expected, given the results observed for the PMPC homopolymers
(Table 5.2).
The bifunctional initiator 10 was used to prepare three examples of PHPMA-
PMPC-PHPMA triblock copolymers. Copolymers with target compositions of
PHPMA50-PMPC125-10-PMPC125-PHPMA50 had relatively low polydispersities of
around 1.30. These data are comparable to those reported for similar copolymers
prepared using non-fluorescent bifunctional initiators.55,56
Compared to similar
copolymers based on a disulfide-based initiator,56
the number-average molecular
weight of PHPMA50-PMPC125-10-PMPC125-PHPMA50 measured by GPC is
closer to PHPMA88-PMPC200-S-S- PMPC200-PHPMA88 (Mn = 89,500) than to
PHPMA43-PMPC125-S-S- PMPC125-PHPMA43 (Mn = 57,200). This suggests
relatively poor initiator efficiency for 10. Thus PHPMA90-PMPC200-10-
PHPMA90-PMPC200, which was designed to be an ‘efficient’ gelator,56
in fact has
a much higher molecular weight than that targeted, which may be probably
related to its relatively high polydispersity. The number-average molecular weight
determined on the basis of visible absorbance spectroscopy increases with the
copolymer molecular weight as measured by GPC. However, these molecular
weights are significantly larger than expected, which suggests a significant
amount of initiator degradation (or inactivity). This is not unexpected, since
bifunctional initiator 10 appears to be particularly prone to transesterification
under ATRP conditions.

215
Target
Composition
1
H NMR
Composition
Mn
Target
Mn
NMR
a)
Mn
GPC
b)
Mw/Mn
GPC
b)
Mn
Amax
(λmax / nm)
c)
2-PMPC30-PDPA60 2-PMPC28-PDPA56 22,000 21,000 26,000 1.27 21,400 ± 800 (533)
7-PMPC25-PDPA70 7-PMPC25-PDPA69 22,900 22,600 31,000 1.27 34,000 ± 3,400 (538)
7-PMPC25-PHPMA60 7-PMPC30-PHPMA60 16,000 18,000 33,000 1.22 38,000 ± 2,800 (535)
PHPMA50-PMPC125-10-PMPC125-PHPMA50 (PHPMA30-PMPC125)2-10 89,000 N/A 75,000 1.34 171,000 ± 5,900 (538)
PHPMA50-PMPC125-10-PMPC125-PHPMA50 PHPMA50-PMPC125)2-10 89,000 N/A 80,000 1.30 319,000 ± 800 (537)
PHPMA100-PMPC200-10-PMPC200-PHPMA100 (PHPMA90-PMPC200)2-10 147,800 N/A 131,000 1.71 2,112,000 ± 276,000 (538)
H NMR, GPC and absorption data for PMPC-based block copolymers prepared using three rhodamine-based ATRP initiators, 2, 7 and
10. a) 1
H NMR spectra recorded in CD3OD for PMPC homopolymers and in a 3:1 CDCl3:CD3OD mixture for PMPC-PDPA diblock copolymers. The initiator end-
group could be used for assessing the degree of polymerization for the PMPC homopolymers, which in turn allowed assessment of the block composition (N.B. the
initiator end-groups were not quantifiable in the block copolymer spectra. b)
GPC data obtained for a 3:1 CHCl3:CH3OH eluent using poly(methyl methacrylate)
calibration standards. c)
Mn calculated from the εmax value of the initiator (see Table 5.1) in methanol for PMPC homopolymers, PMPC-PHPMA diblock
copolymers and PHPMA-PMPC-PHPMA triblock copolymers and in 0.10 M HCl for PMPC-PDPA diblock copolymers. The uncertainties are the standard error
of three measurements recorded at three different concentrations.

216
5.3.13 pH-dependent self-assembly behavior of rhodamine-labelled PMPC-
PDA diblock copolymers
Figure 5.14A shows the effect of varying the solution pH on the hydrodynamic
radius, RH, for 0.20 % aqueous solutions of 2-PMPC28-PDPA56 and 2-PMPC24-
PDPA115.
Figure 5.14B shows the same data for an aqueous solution of 7-PMPC25-PDPA90.
2 3 4 5 6 7 8 9
10
100
2-PMPC28
-PDPA56
2-PMPC24
-PDPA115
RH
/nm
pH
2 3 4 5 6 7 8 9 10
10
100 7-PMPC25
-PDPA90
RH
/nm
pH
A B
Figure 5.14: Variation of hydrodynamic diameter with solution pH obtained by dynamic
light scattering at 25o
C for 0.20 % aqueous solutions of pH-responsive diblock copolymers:
(A) 2-PMPC28-PDPA56 and 2-PMPC24-PDPA115 and (B) 7-PMPC25-PDPA90.
For the diblock copolymers based on the pH-responsive initiator, 2, a
hydrodynamic radius of 6-7 nm (and relatively weak scattering) is obtained up to
pH 5, indicating molecularly dissolved unimers. Between pH 5 and pH 6, a ten-
fold increase in RH is observed (and much more intense scattering), indicating
vesicle formation. This is consistent with previous work on unlabelled PMPC-
PDPA diblock copolymers of similar compositions.41,53
The diblock copolymer
based on the pH-independent initiator 7 exhibit a similar behavior although in this
case, the vesicle formation occurs at slightly higher pH between pH 6 and 7.
The absorption and emission spectra for 2-PMPC28-PDPA56 at pH 3.0 and pH 8.0
are shown in Figure 5.15A. At pH 3.0, both absorption and emission is essentially
similar to those obtained for the low molecular weight pH-dependent rhodamine
derivatives at low pH (Figure 5.3 and Figure 5.5). Increasing the pH to 8.0 leads

217
to complete disappearance of the 530 nm band and almost complete attenuation of
the emission. This indicates formation of the spirolactam form, as expected.
Figure 5.15B shows the maximum emission as a function of pH, normalized to
the emission at pH 3.0. Up to pH 4.0, the emission is almost constant, which is
consistent with the behavior of the corresponding low molecular weight
compound 1 (Figure 5.6A). Between pH 4.0 and 6.0, the emission decreases by
approximately three orders of magnitude due to formation of the non-fluorescent
cyclic spirolactam. Although the transition for the low molecular weight
compound was quite sharp between pH 4 and 5, the transition for the copolymer
appears to be significantly broader. Hence the emission starts to decrease at pH
4.0 but does not reach a stable level until pH 6-7. This is possibly due to slower
kinetics and/or less favorable thermodynamics in forming the spirolactam when a
polymer chain is attached.
300 350 400 450 500 550 600 650 700
pH 8.0
pH 3.0
Absorption/A.U.
Wavelength / nm
2-PMPC28
-PDPA56
Fluorescenceemission/A.U.
2 3 4 5 6 7 8 9 10
10
-3
10
-2
10
-1
10
0
2-PMPC24
-PDPA115
2-PMPC28
-PDPA56
MaxFl.Emission/MaxFl.Emission(pH3)
pH
A B
PMPC28-PDPA56 at pH 3.0 and pH 8.0. Note the logarithmic scale on the emission spectra.
(B) Fluorescence intensity versus pH normalized to pH 3.0. The initial concentration was
0.20 % in 0.1 M HCl. Excitation wavelength = 530 nm, emission slit = 5 nm.
Figure 5.16A shows absorption and emission spectra for 7-PMPC22-PDPA84 at pH
3.0 and pH 8.0. At low pH absorption and emission is similar to the
corresponding spectra of the low molecular weight starting compounds (Figure
5.3 and Figure 5.5) and of 2-PMPC28-PDPA56 (Figure 5.15). Increasing the pH
leads to a more prominent shoulder at 508 nm, eventually giving an absorption
spectrum with two distinct peaks at pH 7.0. This spectrum is characteristic of
rhodamine dimer formation.45
In addition, the baseline of the spectra increases at

218
pH 8, suggesting scattering due to colloidal aggregates; digital photographs of the
solutions recorded at different pH also indicate subtle systematic changes in
visual appearance (Figure 5.16C).
450 500 550 600 650 700
508 nm
530 nm
pH 8
pH 3
Absorption/Emission/A.U.
Wavelength / nm
0 1 2 3 4 5 6 7 8 9 10
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
0.2
0.4
0.6
0.8
1.0
1.2
1.4
7-PMPC25
-PDPA90
Abs(530nm)/Abs(508nm)
pH
MaxEmission/MaxEmission(pH3)
PMPC25-PDPA90 at pH 3.0 and pH 8.0. The initial concentration was 0.20 % in 0.1 M HCl.
Excitation wavelength = 530 nm (B) Ratio between the magnitude of the 530 nm and 508 nm
bands compared to the maximum normalized fluorescence intensity versus pH. (C) Digital
photographs of a 0.20 % w/v solution of 7-PMPC22-PDPA84 at increasing pH. Notice the
color shift due to dimer formation above pH 6.5.
Figure 5.16B shows the ratio between the absorption at 530 nm and 508 nm as
well as the maximum fluorescence intensity normalized at pH 3.0 as a function of
pH. The absorption ratio does not change between pH 2.0 and pH 6.0, while the
emission decreases slightly. According to Figure 5.14B, this copolymer is
molecularly dissolved in this pH range and the absorption spectra are
characteristic of rhodamine unimers. The decrease in the fluorescence intensity is
mainly due to dilution upon addition of base. Above pH 6.0, the PDPA blocks
A B
C

219
become hydrophobic and vesicles are formed. This leads to an increase in the
local concentration of rhodamine, which results in dimer formation.58
5.3.14 Temperature-dependent self-assembly of rhodamine-labelled PMPC-
PHPMA block copolymers.
The relative fluorescence intensity and the hydrodynamic radius versus
temperature of 7-PMPC30-PHPMA60 copolymer solutions are shown in Figure
5.17A. The apparent fluorescence intensity increases from 5 °C to 7.5 °C and then
decreases continuously up to 37 °C. The temperature of maximum fluorescence
correlates well with the corresponding aggregation temperature. The origin of the
fluorescence increase between 5 °C and 7.5 °C is not known, but may be related
to the reduced formation of rhodamine dimer as the water becomes a better
solvent at higher temperatures. The reduction in emission above 7.5 °C is
probably related to the aggregation leading to a higher local concentration of
rhodamine labels. Similar observations were made for the pH-responsive PMPC-
PDPA diblock copolymers on increasing the solution pH (Figure 5.16).
0 5 10 15 20 25 30 35 40
5.0
7.5
10.0
12.5
15.0
17.5
20.0
0.85
0.90
0.95
1.00
7-PMPC30
-PHPMA60
RH
/nm
Temperature / °C
MaxEmission/MaxEmission(7.5°C)
460 480 500 520 540 560 580 600
0.0
0.5
1.0
1.5
2.0
5 °C
37 °C
Absorption
Wavelength /nm
Figure 5.17: (A) Relative fluorescence intensity and hydrodynamic radius of the rhodamine-
based diblock copolymer, 7-PMPC30-PHPMA60. A 0.10 w/v % aqueous solution with
excitation at 530 nm was used for the fluorescence studies. Light scattering studies were
conducted using a 1.00 w/v % aqueous solution filtered through a 0.22 µm Nylon filter prior
to measurements. The average of three consecutive light scattering measurements is shown.
(B) Absorption spectra recorded at 5 °C (blue), 20 °C (black) and 37 °C (red) for a 0.10 w/v
% solution of 7-PMPC30-PHPMA60. The arrows designate increasing temperature.
A B

220
The reduced emission of 7-PMPC30-PHPMA60 with aggregation is of the order of
10 % from 7.5 °C to 37 °C (Figure 5.17A). This should be compared to a
reduction of more than 50 % observed for aggregation of the corresponding pH-
responsive copolymer, 7-PMPC25-PDPA90, on increasing the pH. This is
emphasized by the absorption spectra acquired at 5 °C, 20 °C and 37 °C shown in
Figure 5.17B; as expected, increasing the temperature leads to a decrease in the
maximum absorbance at around 540 nm. However, the relative reduction is very
small and, in addition, the shoulder at 508 nm assigned to dimer formation does
not become very prominent. This is in contrast to that observed for aggregation of
the pH-responsive diblock copolymer (Figure 5.16A). This behavior is probably
related to the influence of the temperature on the absorption and emission
properties of aqueous rhodamine solutions. From 0 °C to 10 °C, the absorption is
reported to increase with temperature due to a decrease in dye aggregation.59
This
should lead to an increase in the fluorescence intensity, which is probably what is
observed between 5 °C and 7.5 °C in Figure 5.17a. This effect is countered by the
onset of aggregation, which increases the local concentration of rhodamine dyes.
At higher temperatures (typically above 20 °C), reduced fluorescence has been
reported for a number of rhodamine-dyes, apparently due to a decrease in the
fluorescent quantum yield.60
This would lead to the observed continuous decrease
in emission at elevated temperature.
5.3.15 Temperature-dependent gelation of thermo-responsive triblock
copolymers
Despite low initiator efficiency and concomitant initiator degradation of the
fluorescent bifunctional initiator, it was still possible to prepare fluorescent
PHPMA-PMPC-PHPMA triblock copolymer gelators similar to those reported
earlier using non-fluorescent bifunctional initiators.55-57
Figure 5.18 shows the
storage and loss moduli observed for a 10.0 w/v % and a 20.0 w/v % aqueous
solution of PHPMA50-PMPC125-10-PMPC125-PHPMA50 versus temperature. At
low temperatures, the storage moduli are approximately one-tenth of the loss
moduli, indicating a free-flowing liquid. Increasing the temperature leads to an
increase in both moduli. Since the storage modulus increases faster with
temperature than the loss modulus, a free-standing gel is obtained. The critical

221
gelation temperature is highly dependent on the copolymer concentration, which
was also observed for the non-fluorescent triblock copolymers.55-57
The moduli of
a 10.0 w/v % solution are in general lower than for the 10.0 w/v % PHPMA88-
PMPC200-S-S-PMPC200-PHPMA88 solution described earlier and, in addition, the
critical gelation temperatures are higher. This is not too surprising, since the
overall molecular weight of PHPMA50-PMPC125-10-PMPC125-PHPMA50 is lower
and, for copolymers with similar PHPMA fractions, larger copolymers always
proved to be more efficient gelators.56
In addition, the chemical difference
between the two bromoisobutyryl groups in 10 may lead to different initiator
efficiencies, which would give asymmetric ABA’ triblock copolymers. However,
such asymmetry is very difficult to verify experimentally.
0 5 10 15 20 25 30 35 40 45 50
10
-3
10
-2
10
-1
10
0
10
1
10
2
G' = G''
10.0 % w/v G'' 10.0 % w/v G'
20.0 % w/v G'
20.0 % w/v G''
G',G''/Pa
T / °C
Figure 5.18: Temperature dependence of the loss and storage modulus for 10.0 w/v % and
20.0 w/v % PHPMA50-PMPC125-10-PMPC125-PHPMA50 aqueous solutions. Experimental
parameters: 1 rad/s, 0.5 Pa, 3 °C/min. Insert shows a digital picture of a 10.0 w/v % solution
of PHPMA50-PMPC125-10-PMPC125-PHPMA50 in water.
Despite some loss of fluorescent chromophore during polymerization, the
solutions remained highly colored, see inset in Figure 5.18. In general, these

222
fluorescent triblock copolymers are best employed as additives to non-labelled
triblock copolymers. This allows the diffusion of individual copolymers into
biological tissue and/or cells to be tracked using various fluorescence
techniques.22-24
For example, Bertal et al. has recently used PHPMA50-PMPC125-
10-PMPC125-PHPMA50 to investigate the mechanism of the unexpected anti-
bacterial action of such thermo-responsive copolymer gelators.61
5.3.16 Stability of initiator group in aqueous solution.
The pH-responsive PMPC-PDPA diblock polymers are being evaluated as
fluorescently-labelled vehicles for drug delivery applications.22-24
In this context,
it is important that no cleavage of the bond linking the rhodamine label to the
copolymer chain occurs under physiological conditions over long time scales.
Thus the hydrolytic stability of the initiator end-group on a 7-PMPC25
homopolymer at 37°C in PBS at pH 7.2 was investigated by dual detection
aqueous GPC.
12 13 14 15 16 17 18 19
7-PMPC25
t=7 days
t=0 days
Refractive index
Absorption at 530 nm
Abs530nm
andRIsignal/A.U.
Retention time / minutes
Figure 5.19: Visible absorption (λ = 530 nm) and refractive index detector GPC traces for
1.0 % 7-PMPC25 at zero time and after 7 days storage in PBS buffer at pH 7.2 and 37o
C.
Eluent: 0.2 M NaNO3 and 0.01 M NaH2PO4 adjusted to pH 7; flow rate = 1.0 mL min-1
.

223
Unlabelled PMPC homopolymer does not absorb at 530 nm, so the observed
signal at this wavelength is solely due to the rhodamine end-groups, whereas the
refractive index signal is proportional to the polymer concentration. The good
agreement between the chromatograms obtained using the uv-visible detector and
the refractive index detector indicates that every polymer chain is labelled, as
expected. Figure 5.19 shows the chromatograms recorded for two 7-PMPC25
copolymer solutions; one was freshly made up, while the other was stored in PBS
buffer at pH 7.2 for 7 days at 37 °C. The two traces are essentially identical,
indicating that little or no degradation of the rhodamine end-groups occurs on this
time scale. The integrated GPC signals normalized with respect to zero time for 7-
PMPC25 and 7-PMPC100 are compared in Figure 5.20. Again there is little change
over one week, showing that the covalent bond between polymer and
chromophore remains intact within this time period. Of course, in vitro and in
vivo conditions will differ from those used in this model study and further work is
certainly required to confirm the fidelity of the rhodamine label. Nevertheless,
these preliminary results suggest that the fate of dye-labelled copolymer chains
can be monitored in live cells using techniques such as confocal microscopy over
time scales of at least a few days. In principle, this should be sufficient to assess
the polymer distribution within body tissue and hence to assess the extent of its
renal excretion.

224
0.0
0.2
0.4
0.6
0.8
1.0
0 1 47 168
Time / hours
Norm(Area(Abs530nm)/Area(RI),t=0)
Figure 5.20: Evolution of the rhodamine end-group functionality of 1.0 % aqueous solutions
of 7-PMPC25 and 7-PMPC100 in PBS buffer (pH 7.2) stored at 37 °C determined by
comparing the integrated absorbance signal at 530 nm with the integrated refractive index
signal and normalizing the ratio to the ratio at t=0.01 days (15 min).
5.4 Conclusions
A series of hydroxy-functional rhodamine 6G derivatives were prepared. A
literature protocol was followed for the preparation of derivatives that were
fluorescent at low pH, while a novel one-step procedure was developed for the
preparation of derivatives that exhibited pH-independent fluorescence. Protocols
for esterification of the protonated hydroxy-functional precursors using either 2-
bromoisobutyryl bromide or 2-bromoisobutyric anhydride were developed so as
to afford both monofunctional and bifunctional rhodamine-based ATRP initiators.
One of these initiators exhibited fluorescence below pH 4 but was non-fluorescent
at higher pH, while the remaining initiators proved to be highly fluorescent over a
wide pH range (from pH 1 to pH 10). A new permanently fluorescent rhodamine-
based methacrylic monomer was also synthesized by esterification with
methacrylic anhydride, using a mixture of methacrylic acid and chloroform as a
solvent. The ATRP initiators were used to prepare well-defined, rhodamine-
labelled PMPC homopolymers as well as pH-responsive PMPC-PDPA diblock
copolymers and thermo-responsive AB diblock and ABA triblock copolymers
with PMPC and PHPMA blocks.
RH-MeEt-PMPC25 RH-MeEt-PMPC100

225
The number-average molecular weights for the homopolymers calculated from
visible absorption spectroscopy analysis of the fluorescent end-groups were in
general larger than those estimated by 1
H NMR spectroscopy, with the latter
technique lying closer to the targeted values. There may be several explanations
for this observation. For example, the separation distance between the
chromophore and the initiator group appears to be important; the homopolymer
prepared using initiator 8, which has a larger separation distance between the 2-
bromoisobutyryl group and the aromatic ring than initiator 7, has much better
agreement between the molecular weight calculated from 1
H NMR and that
obtained from its absorption spectrum. In addition, the water content of the PMPC
homopolymers was of the order of 10-15 w/w %. This is important when
preparing stock solutions for visible absorption spectroscopy but does not lead to
experimental error for the 1
H NMR measurements.
The agreement between the theoretical molecular weight and the molecular
weight measured by visible absorption spectroscopy became poorer as the target
molecular weight was increased. For these copolymers, the relative end-group
content was too small to be quantified by 1
H NMR. Studies of several isobutyryl
esters under ATRP conditions in methanol revealed that the CuBr/bpy ATRP
catalyst can also act as an effective transesterification catalyst. Such a side
reaction would lead to (partial) loss of the chain end-groups, which would lead to
significantly lower apparent molecular weights. Since the degree of
transesterification is dependent on the reaction time, this may be the reason that
longer polymers apparently have fewer end-groups than targeted. However, more
experiments are needed to support this hypothesis.
It was found that the rate of transesterification was highly dependent on the
initiator structure. Thus, initiator 8 reacted much slower with methanol than
initiator 7. Initiator 8 was more stable than the previously used PhOBr initiator
which is known to give copolymers with excellent agreement between target
molecular weights and 1
H NMR molecular weights. These degradation studies
indicate that this initiator should be preferred in terms of stability and preliminary
polymerization results suggest that this is indeed the case.
The Mn values determined for the diblock copolymers were in general higher than
the target values, again indicating the likelihood of in situ transesterification.

226
The PMPC-PDPA diblock copolymers were pH-responsive and formed vesicles
between pH 5 and pH 7. For the PMPC-PDPA diblock copolymers based on the
pH-responsive initiator, complete disappearance of fluorescence was observed on
increasing the pH, which is consistent with formation of the non-fluorescent
spirolactam form of the dye. The transition was shifted by approximately one pH
unit relative to the small molecule precursor and occurred over almost the same
range as the copolymer aggregation. Aggregation also had an influence on the
fluorescence of the PMPC-PDPA diblock copolymers based on the pH-
independent initiators. However, in this case the disappearance of the
fluorescence was due to the formation of non-fluorescent dimers due to the larger
density of chromophores in the aggregates.
Although the PMPC-PHPMA diblock copolymers formed aggregates upon
heating, the changes in the visible absorption spectra and the fluorescence
emission intensity was much less pronounced than those found for the pH-
responsive copolymers. This is probably due to the enhanced water-solubility of
the dye at elevated temperature, which offsets the tendency to form non-
fluorescent dimers.
Like their non-fluorescent counterparts, PHPMA-PMPC-PHPMA triblock
copolymers based on the rhodamine-based bifunctional initiator proved to be
thermo-responsive gelators in aqueous solution. The rhodamine label proved to
be hydrolytically stable over at least one week at pH 7 at 37 °C, suggesting that
the distribution of these rhodamine-labelled copolymer chains within living cells
or tissue can be monitored over time scales of at least a few days.
5.5 References
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MacNeil, S. manuscript in preparation

Chapter 6: Conclusions and Future Work
229

230
Chapter 2: Preparation and Aqueous Solution Properties of New
Thermo-responsive Biocompatible ABA Triblock
Copolymer Gelators
Chapter 2 presented the use of mixtures of chloroform and methanol as a suitable
solvent for analyzing PMPC homopolymers and block copolymers of PMPC and
various hydrophobic blocks by NMR and GPC. The rather complex nature of the
‘HPMA’ monomer was then discussed and the chapter went on to describe the
synthesis of three ABA block copolymers by ATRP using a commercially
available bifunctional initiator. In all the examples, the B block consisted of
PMPC whereas the A blocks were either PHEMA, PHPMA or PMMA. In
addition the aqueous solution properties were investigated: A 10 w/v % aqueous
solution of PHEMA-PMPC-PHEMA was free-flowing at all temperatures. A 10
w/v % aqueous solution of PMMA-PMPC-PMMA was opaque and highly
viscous independent of the temperature, which was taken to indicate poor
dissolution. In contrast, 10 w/v % PHPMA-PMPC-PHPMA was transparent and
the solution viscosity in water increased two orders of magnitude on increasing
the temperature from 5 °C to 30 °C. Thus, it was demonstrated that although
PHPMA is normally considered water-insoluble, it can be rendered water-soluble
when attached to a hydrophilic block. Gelation was found down to concentrations
of 4 w/v % and the critical gelation temperature was found to be highly dependent
on the copolymer concentration.
Chapter 3: New Biocompatible Wound Dressings based on
Chemically Degradable Triblock Copolymer
Hydrogels
In this chapter the synthesis of a series of PHPMA-PMPC-PHPMA triblock
copolymers with different composition and molecular weights was described.
These copolymers could form transparent free-standing gels in aqueous solution
with mechanical properties that were dependent on the copolymer molecular
weights and relative block compositions. Thus, gels were obtained for copolymers
with PHPMA contents of between 14 and 19 w/v %. The critical gelation

231
temperature and gel strength was also found to be strongly dependent on the
copolymer concentration. Dynamic light scattering, TEM and 1
H NMR studies
indicated that gelation is due to the self-assembly of individual copolymer chains
to form a micellar gel network, with bridging chains between adjacent micelles.
When these copolymers were based on a disulfide-containing ATRP initiator,
reductive cleavage under mild conditions led to rapid degelation.
This chapter also described the essential purification that was necessary to obtain
biocompatible polymeric gels. In addition, it was found that residual 2,2’-
bipyridine ligand from the ATRP catalyst in concentrations of the order of 10-100
ppm is the most likely reason for cytotoxicity and that purification should aim at
removing this.
Preliminary results show that these non-cytotoxic copolymer gels give controlled
release of for example anti-psoreasis drugs. In addition the copolymers have been
found to be capable of intracellular delivery and to exhibit inherent antimicrobial
activity. Thus, the triblock copolymers have several beneficial properties which
may be useful for biomedical applications such as wound dressings.
The possibility of derivatization of the thiol group formed by reductive cleavage
of the disulfide group has not been examined in depth in this work. However,
since thiols can be derivatized quantitatively, this is a possible route to end-
functional diblock copolymers.
Chapter 4: Preparation and Aqueous Solution Properties of
Thermoresponsive Biocompatible AB Diblock
Copolymers
In chapters 2 and 3 it was found that amphiphilic PHPMA-containing triblock
copolymers exhibited temperature-dependent behavior in aqueous solutions. In
chapter 4, the preparation of a series of amphiphilic PMPC-PHPMA diblock
copolymers was reported. The diblock copolymers do not form inter-micellar
bridges, why the study of their solution properties should be relatively simpler.
These copolymers could be dissolved or dispersed in cold aqueous solution and
exhibited a rich phase behavior depending on the degree of polymerization of the

232
PMPC and PHPMA blocks, the copolymer concentration and the solution
temperature. In particular it was found that copolymers with short PHPMA blocks
formed large aggregates, whereas extending the PHPMA block led to relatively
smaller aggregates. This was explained by a decrease in hydration on increasing
the PHPMA block length. Both critical aggregation concentrations and critical
aggregation temperatures were found to be dependent on the degree of
polymerization of both blocks as well as on the relative block composition in a
complex way.
The results presented in this chapter led to some insight into the thermoresponsive
behavior of PHPMA. From a fundamental point of view, future work might
include the preparation of diblock copolymers with PMPC and PHPMA contents
that are comparable to half of the triblock copolymers prepared in chapters 2 and
3, i.e. with a higher PMPC content. The solution behavior of these should give a
fuller picture of the influence of the PMPC block. In addition, preparation of
similar diblock copolymers with other water-soluble block such as for instance
PEO might give a more complete picture of the influence of the water-soluble
block on the aggregation behavior of PHPMA. Variable temperature IR studies
might lead to insight into the dehydration mechanism in line with similar work on
other polymers with critical aggregation behavior.
Most of these copolymers were found to exhibit relatively high critical
aggregation concentrations, why they may offer some potential for intracellular
drug delivery. After delivering the drug, gradual dilution should lead to
dissolution of the aggregates, allowing their excretion from the cells in the form
of molecularly dissolved chains.
Chapter 5: Derivatization of Rhodamine 6G and Preparation of
Fluorescent PMPC-based (co)polymers
Chapter 5 described the modification of the fluorescent dye rhodamine 6G to
prepare monofunctional ATRP initiators with and without pH dependent
fluorescence. In addition a bifunctional pH-independent ATRP initiator and a
monofunctional pH-independent methacrylic monomer were prepared.

233
Initially, these initiators were used to prepare PMPC homopolymers. In addition,
pH-responsive diblock copolymers of PMPC and PDPA as well as
thermoresponsive di- and triblock copolymers of PMPC and PHPMA were
prepared. During this work, it was found that 2-bromoisobutyryl ester-based
ATRP initiators in general were prone to transesterification with methanol in
presence of the CuBr:bpy ATRP catalyst. This may be part of the explanation for
the observed poor correlation between target molecular weights and experimental
molecular weights. A rhodamine-based initiator with an larger distance between
the chromophore and the 2-bromoisobutyryl ester were prepared. This compound
was found to have a reduced rate of transesterification. Preliminary results
indicated that the use of this novel initiator led to improved agreement between
target and experimental molecular weights. The chemical structure of the initiator
was also found to have some significance; in general the absorption coefficient of
a polymer-bound chromophore was different from the absorption coefficient of
the initiator precursor. Again, preliminary results indicated that this difference
was less significant for the compound with a larger distance between the
chromophore and the 2-bromoisobutyryl ester.
Despite the observed loss of fluorescent groups due to transesterification with the
solvent, these initiators gave highly fluorescent polymers. The fluorophore in
these copolymers was found to be hydrolytically stable on a time-scale of at least
one week, which makes them promising candidates for tracking copolymers in
living tissue.
The promising preliminary results obtained when using the more stable ATRP
initiator should be confirmed by preparing more PMPC homopolymers with
different target degrees of polymerization. This should lead to better correlation
between the target and experimental values. In addition, the degree of
transesterification of the polymer end-group should be investigated and possibly a
solvent system should be identified that reduces transesterification. This could for
instance be based on a mixture of water and propan-2-ol.
For polymer tracking purposes, the use of the rhodamine monomer might be
preferred instead of the initiator. Although this method offers less control over the
exact amount fluorophores in the chain and the position, there may be advantages
of having it embedded inside the chain. Thus, the more shielded chromophore

234
may lead to reduced specific interaction with cell constituents as well as
decreased transesterification.

Temperature-responsive Biocompatible Block Copolymers PhD JMadsen

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