shuko suzuki thesis (pdf 6mb)
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THE QUEENSLAND UNIVERSITY OF TECHNOLOGY
IN VITRO MINERALISATION OF WELL-DEFINED POLYMERS AND SURFACES
A thesis presented in fulfilment of the requirements for the degree of
Doctor of Philosophy
Shuko Suzuki M. App. Sc.
Tissue Repair and Regeneration Program
Institute of Health and Biomedical Innovation School of Physical and Chemical Sciences
November 2007
ABSTRACT
Currently, many polymeric biomaterials do not possess the most desirable surface
properties for direct bone bonding due to the lack of suitable surface functionalities.
The incorporation of negatively charged groups has been shown to enhance calcium
phosphate formation in vitro and bone bonding ability in vivo. However, there are
some conflicting literature reports that highlight the complicated nature of the
mineralisation process as well as the sometimes apparent contradictory effect of the
negatively charged groups. Surface modification using well-defined polymers offer a
more precise control of the chain structures. The aims of this study were to
synthesise well-defined polymers containing phosphate and carboxylic acid groups,
and perform various surface modification techniques. The influence of the polymer
structure on mineralisation was examined using a series of specially synthesised
phosphate-containing polymers. The mineralisation ability of the fabricated surfaces
was also tested.
Soluble poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-
(methacryloyloxy)ethyl phosphate) (PMOEP) were synthesised using reversible
addition fragmentation chain transfer (RAFT)-mediated polymerisation. The
polymerisation conversions were monitored by in situ Raman spectroscopy.
Subsequently 31P NMR investigation revealed the presence of large amounts of diene
impurities as well as free orthophosphoric acids in both the MAEP and MOEP
monomers. Elemental analyses of the polymers showed loss of phosphate groups due
to hydrolysis during the polymerisation. Both gel and soluble PMAEP polymers
were found to contain large amounts of carboxyl groups indicating hydrolysis at the
C-O-C ester linkages. Block copolymers consisting of PMAEP or PMOEP and
poly(2-(acetoacetoxy) ethyl methacrylate) PAAEMA were successfully prepared for
the purpose of immobilisation of these polymers onto aminated slides.
Well-defined fluorinated polymers, (poly(pentafluorostyrene) (PFS),
poly(tetrafluoropropyl acrylate) (TFPA) and poly(tetrafluoropropyl methacrylate)
(TFPMA)) were synthesised by RAFT-mediated polymerisation. It was found that
the Mn values of PFS at higher conversions were significantly lower than those
i
calculated from the theory, although the PDI’s were low (<1.1). One possible
explanation for this is that it may be a result of the self-initiation of FS which created
more chains than the added RAFT agents. Both TFPA and TFPMA showed well-
controlled RAFT polymerisations. Chain extension of the fluorinated polymers with
tert-butyl acrylate (tBA) followed by hydrolysis of the tBA groups produced the
amphiphilic block copolymers containing carboxylic acid groups. Block copolymers
consisting of PAAEMA segments were further reacted with glycine and L-
phenylalanyl glycine.
Three types of surface modifications were carried out: Layer-by-Layer (LbL)
assemblies of the soluble phosphate- and carboxylic acid-containing homopolymers,
coupling reactions of block copolymers consisting of phosphate and keto groups onto
aminated slides, and adsorption of fluorinated homo and block copolymers
containing carboxylic acid groups onto PTFE. For LbL assemblies XPS analyses
revealed that the thickness of the poly(acrylic acid) (PAA) layer was found to be
strongly dependent on the pH at deposition. AFM images showed that the PMAEP
LbL had a patchy morphology which was due to the carboxylate groups that were not
deprotonated at low pH. Successful coupling of the block copolymers consisting of
phosphate and keto groups onto aminated slides was evident in the XPS results. The
conformation of attached P(MOEP-b-AAEMA) was investigated by ToF-SIMS.
Adsorption of the fluorinated polymers onto the PTFE film was examined using
different solvents. PFS showed the best adsorption onto PTFE. The block
copolymers consisting of PFS and PtBA or PAA were successfully adsorbed onto
PTFE. Contact angle measurements showed that the adsorbed block copolymers
reorganised quickly to form a hydrophilic surface during the investigation.
In vitro mineralisation of various phosphate-containing polymers and the fabricated
surfaces were studied using the simulated body fluid (SBF) technique. The
SEM/EDX investigation showed that either brushite or monetite, with a tile-like
morphology, was formed on both soluble and gel PMAEP polymers after seven days
in SBF. The PMOEP gel formed a similar layer as well as a secondary growth of
hydroxyapatite (HAP) that exhibited a typical globular morphology. Fourier
transform infrared (FTIR) spectroscopy of the PMOEP film prepared from soluble
PMOEP showed large amounts of carbonated HAP formation after seven days in
SBF. Carbonated HAP is the phase that most closely resembles that found in
ii
biological systems. Both the LbL surfaces and the block copolymer-attached
aminated slides showed only patchy mineralisation even after 14 days in SBF. This
indicates that ionic interactions of the negatively charged phosphates or carboxylates
and protonated amines prevented chelation of calcium ions, which is believed to be
the first step in mineralisation. The P(FS-b-AA) adsorbed PTFE film also showed
only small amounts of mineral formation after 14 days in SBF. These results
highlight the many features controlling the mineralisation outcomes.
iii
LIST OF PUBLICATIONS
Papers: • S. Suzuki, L. Rintoul, M. J. Monteiro, E. Wentrup-Byrne, L. Grøndahl (2007),
“In vitro mineralization of phosphate-containing polymer ad-layers”, Polymer Preprints, 48 (1), 430-431
• S. Suzuki, M. R. Whittaker, L. Grøndahl, M. J. Monteiro, E. Wentrup-Byrne (2006). “Synthesis of Soluble Phosphate Polymers by RAFT and their In Vitro Mineralization”, Biomacromolecules, 7, 3178-3187
• S. Suzuki, M. Jasieniak, M. J. Monteiro, E. Wentrup-Byrne, H. Griesser, L. Grøndahl, “Probing the Orientation of a Bi-functional Di-block Copolymer Ad-layer by XPS and Static ToF-SIMS” (in preparation)
• S. Suzuki, M. R. Whittaker, L. Grøndahl, E. Wentrup-Byrne, M. J. Monteiro, “Synthesis of Well-Defined Fluorine-Containing Polymers by RAFT” (in preparation)
• E. Wentrup-Byrne, A. Chander-Temple, S. Suzuki, J.J. Suwanasilp, L. Grøndahl “Structural Characterisation of Phosphate Polymers by FTIR and XPS” (in preparation)
• E. Wentrup-Byrne, L. Grøndahl, S. Suzuki (2005). “Methacryloxyethyl phosphate-grafted expanded polytetrafluoroethylene membranes for biomedical applications”, Polymer International, 54, 1581-1588
• S. Suzuki, L. Grøndahl, D. Leavesley and E. Wentrup-Byrne (2005). “In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications” Biomaterials, 26 (26), 5303-5312
Oral Presentations: • International Symposium on Polymeric Materials for Regenerative Medicine
(PMRM 2007), April, 2007, Montreal Canada
• 28th Australian Polymer Symposium (APS) and 16th Australian Society for Biomaterials (ASB), February 2006, Rotorua NZ
Poster Presentations: • 233rd American Chemical Society (ACS) National Meeting, March 2007,
Chicago USA
• 27th Australian Polymer Symposium (APS), February 2005, Adelaide AU
iv
DECLARATION OF ORIGINAL AUTHORSHIP
The work submitted in this thesis has not been previously submitted for a degree or
diploma at this or any other educational institution. To the best of my knowledge and
belief, the information contained in this thesis contains no material previously
published or written by any other person except where due reference is made.
Signed ………………………..
Dated ………………………..
v
ACKNOWLEDGEMENTS This document is the result of the contributions of many people – those that have
contributed direct information that is described within, and those that have shaped
me to be able to produce this document. My work has spanned across several
Universities, with the majority of work being performed at the Queensland
University of Technology (QUT) and the University of Queensland (UQ), and thus
have collaborated with many people that have provided positive support for the
research that I was undertaking. I appreciate their assistance and thank them all.
Dr. Edeline Wentrup-Byrne, who not only took on the large role of being my
principle supervisor, but also for providing constant help and guidance through the
project and in particular for poring over my thesis for many hours. Moreover, I am
grateful for her encouraging and challenging me to get out of the comfort zone and
explore the international nature of research.
Dr. Lisbeth Grøndahl, my co-supervisor, for her guidance and dedication in ensuring
I was doing things right. Dr. Grøndahl also accommodated me with a laboratory and
office at the University of Queensland for the majority of my time performing the
research. In addition, I appreciate her spending her own time to assist with the
research and papers written.
A/Prof. Michael Monteiro, my co-supervisor, for initiating the new direction of the
research project and with guidance with RAFT polymerisation, and without whom it
would have not been possible to perform the large amount of synthesis.
Dr. Michael Whittaker, for training me with synthesis skills and his great help and
encouragements through the project, as well as running DLS samples.
The specialists, their experienced knowledge of the mechanics and intricacies of the
equipment were critical to the research process, in particular:
• Prof. Hans Griesser and Mr. Marek Jasieniak at the Ian Wark Research
Institute, University of South Australia, for performing ToF-SIMS and with
its interpretation.
• Dr. Barry Wood for his entertainment and expert assistance with the XPS
vi
• Dr. Llew Rintoul for sharing his knowledge and technical expertise in
spectroscopies
• Dr. Tri Le for his patient and sharing knowledge of NMR and in particular
helping me to understand the phosphorous-NMR
• Dr. John Bartley for assistance with the proton-NMR analysis
• Dr. Thor Bostrom, Dr. Deb Stenzel, Mr. Loc Dong and Mr. Lambert Bekessy
for their technical support with SEM and EDX characterisations
• Dr. Ihwa Tan and Mr. Yoosup Park for the technical support of the water
phase GPC
• Mr. George Blazak for performing microanalysis
Prof. Graeme George for sharing his experienced knowledge and providing kind
support.
Prof. David Hill for assistance with solving, from my perspective, difficult problems.
The members of the QUT and UQ polymer groups who afforded me a friendly and
very supportive environment.
The Queensland University of Technology for financial support. In addition, the
QUT Grant-in-Aid scheme and IHBI travel fund made possible the international
visits and presentations.
I also received travel awards from the Royal Australian Chemical Institute (RACI)
and RACI QLD polymer group which I greatly appreciate.
In addition, my colleagues, who provided enormous entertainment and motivation.
And finally, I thank my family and my friends who all have been very supportive
over the course of the research.
vii
TABLE OF CONTENTS ABSTRACT……………………………………………………………………. i
LIST OF PUBLICATIONS …………………………………………………... iv
DECLARATION OF ORIGINAL AUTHORSHIP…………………………. v
ACKNOWLEDGEMENTS…………………………………………………… vi
TABLE OF CONTENTS……………………………………………………… viii
LIST OF FIGURES ………………………………………………………….... xiv
LIST OF TABLES……………………………………………………………... xxii
LIST OF SCHEMES ………………………………………………………….. xxiv
ABBREVIATIONS…………………………………………………………….. xxvi
Chapter 1: Introduction
1.1 Biomaterials and Biocompatibility……………………………………... 1
1.2 Bone………………………………………………………………………. 2
1.3 Host Response to Polymeric Biomaterials……………………………... 5
1.3.1 Wound healing……………………………………………………. 5
1.3.2 Tissue response to implants………………………………………. 6
1.4 Material/Bone Interface………………………………………………… 8
1.5 Surface Modification: Improving the Material/Bone Interface………. 10
1.5.1 Physicochemical Surface Modification…………………………… 10
1.5.2 Morphological surface modification……………………………… 11
1.5.3 Incorporation of biological molecules…………………………….. 12
1.6 Expanded PTFE (ePTFE) in Medicine………………………………… 14
1.7 Surface Modification of PTFE and Other Fluoropolymers…………... 14
1.8 Project Outline…………………………………………………………… 18
1.9 References………………………………………………………………... 20
Chapter 2: Polymer Synthesis
2.1 Introduction……………………………………………………………… 25
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2.1.1 Living Radical Polymerisation (LRP)…………………………….. 25
2.1.2 Reversible Addition Fragmentation Chain Transfer (RAFT) Method …………………………………………………………...
28
2.1.3 Living Radical Polymerisation of Phosphorous-Containing Monomers………………………………………………………….
33
2.1.4 Living Radical Polymerisation of Fluorinated Monomers………... 34
2.2 Experimental……………………………………………………………... 37
2.2.1 Materials…………………………………………………………... 37
2.2.2 Methods…………………………………………………………… 38
2.2.2.1 MOEP/MAEP Homopolymer Synthesis………………….. 38
2.2.2.2 MOEP/MAEP Block Copolymer Synthesis………………. 38
2.2.2.3 Hydrolysis of PMOEP and PMAEP Polymers…………… 38
2.2.2.4 Fluorinated Homopolymer Synthesis……………………... 39
2.2.2.5 Fluorinated Block Copolymer Synthesis………………….. 40
2.2.2.6 Hydrolysis of tBA Segments……………………………… 41
2.2.2.7 Model Biomolecule Modification of Functional Fluorinated Block Polymers………………………………
42
2.2.2.8 Stability of the PFS RAFT end-groups…………………… 43
2.2.3 Analytical Techniques…………………………………………….. 43
2.2.3.1 FT-Raman spectroscopy…………………………………... 43
2.2.3.2 Gel permeation chromatography (GPC)………………….. 44
2.2.3.3 Elemental Analyses……………………………………….. 45
2.2.3.4 Nuclear Magnetic Resonance (NMR)…………………….. 45
2.2.3.5 UV/VIS Spectroscopy…………………………………….. 45
2.2.3.6 Fourier Transform Near Infrared (FT-NIR)……………..... 45
2.3 Results…………………………………………………………………….. 46
2.3.1 RAFT-Mediated Polymerisation of Phosphate-Containing Monomers………………………………………………………….
46
2.3.1.1 Monoacryloxyethyl phosphate (MAEP) polymerisation…. 46
2.3.1.2 Methacryloyloxyethyl phosphate (MOEP) polymerisation 51
2.3.1.3 NMR of monomers and polymers………………………… 54
2.3.1.4 Elemental Analyses of Monomers and Polymers………..... 61
ix
2.3.1.5 Hydrolysis of gel polymers……………………………….. 61
2.3.1.6 Synthesis of MAEP and MOEP block copolymers with AAEMA…………………………………………………...
62
2.3.2 RAFT-Mediated Polymerisations of Fluorine Containing Monomers………………………………………………………….
65
2.3.2.1 Pentafluorostyrene (FS) polymerisation…………………... 65
2.3.2.2 Tetrafluoropropyl acrylate (TFPA) polymerisation………. 72
2.3.2.3 Tetrafluoropropyl methacrylate (TFPMA) polymerisation. 74
2.3.2.4 Block copolymerisation of macrofluorinated polymers with pentafluorostyrene (FS), tert-butyl acrylate (tBA), acetoacetoxyethyl methacrylate (AAEMA) and acetoacetoxyethyl acrylate (AAEA)………………………
77
2.3.3 Biomolecule Attachment………………………………………….. 83
2.4 Discussion……………………………………………………………….... 87
2.4.1 RAFT-Mediated Polymerisation of Phosphate-Containing Monomers …………………………………………………………………
87
2.4.1.1 Polymerization with PEPDTA…………………………….. 87
2.4.1.2 Polymerization with CDB………………………………..... 87
2.4.1.3 Loss of phosphate groups………………………………….. 88
2.4.1.4 Mechanism of gel formation……………………………..... 89
2.4.1.5 Block Copolymerisation with AAEMA………………….... 91
2.4.2 RAFT-Mediated Polymerisation of Fluorine-Containing Monomers………………………………………………………….
92
2.4.2.1 FS Polymerisation…………………………………………. 92
2.4.2.2 Tetrafluoropropyl Acrylate (TFPA) and Tetrafluoropropyl Methacrylate (TFPMA) Polymerisations………………….
95
2.4.2.3 Chain Extension of Fluorinated Macro-RAFT……………. 95
2.4.3 Biomolecule Attachment…………………………………………. 95
2.5 Conclusion……………………………………………………………….. 97
2.6 References……………………………………………………………….. 98
Chapter 3: Surface Fabrication
3.1 Introduction…………………………………………………………….... 103
3.1.1 LbL Assembly…………………………………………………….. 103
x
3.1.2 Attachment of Block Copolymers onto Aminated Slides………… 107
3.1.3 Adsorption of Fluorinated Polymers…………………………….... 109
3.1.4 Surface Characterisation Techniques……………………………... 113
3.1.4.1 X-ray photoelectron spectroscopy (XPS)………………….. 113
3.1.4.2 Static time-of-flight secondary ion mass spectroscopy (ToF-SIMS)……………………………………………….
114
3.1.4.3 Infrared reflection-adsorption spectroscopy (IRRAS)…….. 115
3.1.4.4 Atomic force microscopy (AFM)………………………….. 116
3.1.4.5 Contact angle measurement……………………………....... 116
3.2 Experimental…………………………………………………………….. 118
3.2.1 Materials…………………………………………………………... 118
3.2.2 Methods…………………………………………………………… 118
3.2.2.1 Layer by Layer (LbL) assembly: PEI-PAA, PEI-PMAEP, and PEI-PMOEP…………………………………………..
118
3.2.2.2 Coupling of block copolymers to aminated slide………….. 119
3.2.2.3 Fluorinated homo and copolymer adsorption onto PTFE…. 119
3.2.3 Instrumentation……………………………………………………. 120
3.2.3.1 X-ray photoelectron spectroscopy (XPS)………………….. 120
3.2.3.2 Infrared reflection-adsorption spectroscopy (IRRAS)…….. 120
3.2.3.3 Atomic force microscopy (AFM)………………………….. 120
3.2.3.4 Static time of flight secondary ion mass spectrometry (static ToF-SIMS)…………………………………………
121
3.2.3.5 Sessile drop contact angle measurements…………………. 121
3.2.3.6 Dynamic light scattering (DLS)……………………...……. 122
3.3 Results……………………………………………………………..……… 123
3.3.1 Layer-by-Layer (LbL) Assembly………………………….……… 123
3.3.1.1 LbL of PAA…………………………………...…………… 123
3.3.1.2 LbL of phosphate-containing polymers…...………………. 127
3.3.2 Block Copolymers Coupled onto Aminated Slides…...…………... 135
3.3.2.1 Quantitative XPS investigation of attached polymers...…… 136
3.3.2.2 ToF-SIMS investigation of the conformation of attached block copolymers………………………………………….
140
xi
3.3.3 Adsorption of Fluorinated Polymers onto PTFE……………...…... 148
3.3.3.1 Effect of monomer structure and solvents for adsorption..... 148
3.3.3.2. Adsorption of PFS with varying Mn’s…………………….. 152
3.3.3.3. Adsorption of P(FS-b-tBA) and P(FS-b-AA) block copolymers onto PTFE………………………....................
153
3.4 Discussion………………………………………………………………… 159
3.4.1 Layer-by-Layer (LbL) Assembly………………………………..... 159
3.4.1.1 LbL assembly of PAA…………………………………….. 159
3.4.1.2 LbL assembly of PMAEP and PMOEP…………………… 160
3.4.2 Block Copolymer Attachment onto Aminated Slides…………….. 162
3.4.2.1 Qualitative analysis of attached polymers…………………. 162
3.4.2.2 Conformation of attached block copolymers……………… 163
3.4.3 Adsorption of Fluorinated Polymers onto PTFE………………….. 165
3.4.3.1 Homopolymer adsorption………………………………...... 165
3.4.3.2. Block copolymer adsorption onto PTFE………………….. 167
3.5 Conclusions………………………………………………………………. 170
3.6 References………………………………………………………………... 172
Chapter 4: In Vitro Mineralisation
4.1 Introduction…………………………………………………………….... 177
4.1.1 Mineralisation……………………………………………………... 177
4.1.2 Simulated Body Fluid (SBF)……………………………………… 178
4.1.3 Negatively Charged Groups………………………………………. 180
4.2 Experimental……………………………………………………………... 184
4.2.1 Materials…………………………………………………………... 184
4.2.2 Methods………………………………………………………….... 184
4.2.2.1 Synthesis of cross-linked PAA gels……………………….. 184
4.2.2.2 Simulated body fluid (SBF) experiments……………….... 185
4.2.2.3 Scanning electron microscopy with energy dispersive x-ray analysis……………………………………………………..
185
4.2.2.4 Fourier transform infrared spectroscopy – attenuated total reflectance .………………………………………………...
185
xii
4.2.2.5 XPS………………………………………………………… 186
4.3 Results…………………………………………………………………….. 187
4.3.1 SBF studies of PMAEP and PMOEP……………………………... 187
4.3.2 SBF studies of Layer-by-Layer (LbL) films……………………… 195
4.3.3 SBF studies of block copolymers coupled to aminated slides……. 199
4.3.4 SBF studies of fluorinated amphiphilic block copolymers attached onto PTFE Films…………………………………………………………..
202
4.4 Discussion…………………………………………………………………. 206
4.4.1 SBF studies of PMAEP and PMOEP……………………………… 206
4.4.2 SBF Studies of Layer-by-Layer (LbL) Films……………………... 208
4.4.3 SBF Studies of Block Copolymers Coupled to Aminated Slides…. 209
4.4.4 SBF Studies of Fluorinated Amphiphilic Block Copolymers Adsorbed onto PTFE Films………………………………………………..
210
4.5 Conclusion………………………………………………………………… 212
4.6 References………………………………………………………………… 213
Chapter 5: Overall Conclusions and Future Work
5.1 Chapter 2………………………………………………………………..... 215
5.2 Chapter 3………………………………………………………………..... 216
5.3 Chapter 4………………………………………………………………..... 217
5.4 General Discussion……………………………………………………….. 217
5.5 Future work……………………………………………………………..... 218
xiii
LIST OF FIGURES
Chapter 1
Figure 1.1: Hierarchical structure of bone(Reproduced from ref.5)............. 3
Figure 1.2: Cellular activities at wound repair (Reproduced from ref.11)…. 5
Figure 1.3: Schematic illustration of the successive events following implantation of a material (Reproduced from ref.15)…………..
9
Figure 1.4: Covalent immobilisation of RGD peptides with carboxyl groups on a modified polymer surface…………………………
12
Figure 1.5: Entrapment of biomolecules into a polymer substrate………… 13
Figure 1.6: Adsorption of functional polymers at the FEP/water interface... 17
Chapter 2
Figure 2.1: Heating block setup for in situ Raman polymerisation………... 44
Figure 2.2: Raman spectra of MAEP polymerisation solution ([PEPDTA] = 1 × 10-2 M (expt.2)) (a) initial and (b) after 7.25 h in the region of (A) 3800-360 cm-1 and (B) 1800-900 cm-1…………..
46
Figure 2.3: Conversion versus time of a MAEP polymerisation in methanol using the RAFT agent PEPDTA or CDB, and AIBN as initiator: (a) no RAFT (expt.1), (b) [PEPDTA] = 1 × 10-2 M (expt.2), and (c) [PEPDTA] = 2 × 10-2 M (expt.3) and (d) [CDB] = 1 × 10-2 M (expt.5)…………………………………...
48
Figure 2.4: 1H NMR (400 MHz) spectra of (A) linear PMAEP (expt. 2, Table 2.1) in methanol-d4 and (B) hydrolyzed PMAEP in D2O……………………………………………………………..
49
Figure 2.5: GPC traces of hydrolysed PMAEP with different conversions from expt.2, Table 2.1 (A) and expt.3, Table 2.1 (B)………….
50
Figure 2.6: Mn and PDI of PMAEP polymerized with PEPDTA after hydrolysis (a) [PEPDTA] = 1 × 10-2 M (expt.2, Table 2.1) and (b) 2 × 10-2 M (expt.3, Table 2.1). The lines show the theoretical evolution of Mn with conversion for PAA…………………………………………………………….
56
Figure 2.7: Raman spectra of MOEP polymerisation solution ([CDB] = 1 × 10-2 M (expt.9, Table 2.1)) (a) initial and (b) after 19.5 h…...
52
Figure 2.8: Conversion versus time of a MAEP polymerisation in methanol using the RAFT agent PEPDTA or CDB, and AIBN as initiator: (a) no RAFT (expt.6, Table 2.1), (b) [PEPDTA] = 1 × 10-2 M (expt.7, Table 2.1), and (c) [PEPDTA] = 2 × 10-2 M (expt.8, Table 2.1), and (d) [CDB] = 1 × 10-2 M (expt.9, Table
xiv
2.1) and (e) [CDB] = 2 × 10-2 M (expt.10, Table 2.1)…………. 52
Figure 2.9: GPC traces of hydrolysed PMOEP with different conversions from expt.9, Table 2.1 (A) and expt.10, Table 2.1 (B)………...
54
Figure 2.10: Mn and PDI of PMOEP polymerized with CDB after hydrolysis (a) [CDB] = 1 × 10-2 M (expt.9, Table 2.1) and (b) 2 × 10-2 M (expt.10, Table 2.1)…………………………………..
55
Figure 2.11: 1H NMR spectrum of MAEP in methanol-d4………………….. 55
Figure 2.12: 31P-NMR of MAEP monomer in methanol-d4 A) H-decoupled and B) H-coupled………………………………………………
56
Figure 2.13: 1H NMR spectrum of MOEP in methanol-d4………………….. 55
Figure 2.14: 31P NMR of MOEP monomer in methanol-d4 A) H-decoupled and B) H-coupled..……………………………….….................
57
Figure 2.15: 1H NMR spectrum of PMAEP (expt.4, Table 2.1) in methanol-d4………………………………………………………………..
58
Figure 2.16: 31P NMR spectrum of PMAEP (expt.4, Table 2.1) in methanol-d4………………………………………………………………..
58
Figure 2.17: 1H NMR of PMOEP (expt.10, Table 2.1) in methanol-d4………………………………………………………………..
60
Figure 2.18: 31P-NMR of PMOEP (expt.10, Table 2.1) in methanol-d4………………………………………………………………..
60
Figure 2.19: Raman spectra of PFS polymerisation solution ([PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM) (a) initial and (b) after 25 h..
66
Figure 2.20: Conversion based on the normalised 1620 cm-1 bands form Raman spectra versus time of a bulk PFS polymerisation using the RAFT agent PEPDTA, and Vazo-88 or AIBN as initiator: Curve a: [PEPDTA] = 29 mM and [AIBN] = 2.9 mM, 60 ºC (expt.1, Table 2.3), b: [PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM, 80 ºC (expt.3, Table 2.3), c: [PEPDTA] = 56 mM, and [Vazo-88] = 5.6 mM, 80 ºC(expt.4, Table 2.3)……………
67
Figure 2.21: Mn and PDI of PFS polymerized with PEPDTA (a) [PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM (extp.3, Table 2.3), b: [PEPDTA] = 56 mM, and [Vazo-88] = 5.6 mM (extp.4, Table 2.3). The lines show the theoretical evolution of Mn with conversion……………………………………………………...
68
Figure 2.22: FT-NIR spectra of FS polymerisation solution ([PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM) (a) initial and (b) after 25 h..
69
Figure 2.23: Conversion versus time of bulk FS polymerisation ([PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM, 80 ºC): Curve a, from in situ FT-NIR polymerisation, and b, from in situ Raman polymerisation…………….…………….…………….………..
70
xv
Figure 2.24: 1H NMR spectrum of PFS polymerisation mixture after 67h for calculation of the conversion (CDB) (this corresponds to 81% conversion). ………………………………………………
71
Figure 2.25: Raman spectra of TFPA polymerisation solution ([PEPDTA] = 26 mM and [AIBN] = 2.6 mM) (a) initial and (b) after 3 h……
72
Figure 2.26: Conversion versus time of bulk TFPA polymerisation using the RAFT agent PEPDTA, and AIBN as initiator at 60 ºC: Curve a: [PEPDTA] = 28 mM and [AIBN] = 2.8 mM (expt.11, Table 2.3), and b: [PEPDTA] = 56 mM, and [AIBN] = 5.6 mM (expt.12, Table 2.3). ………………..…………….……………
73
Figure 2.27: Mn and PDI of TFPA polymerized with PEPDTA (a) [PEPDTA] = 28 mM and [AIBN] = 2.8 mM (expt.11, Table 2.3), b: [PEPDTA] = 56 mM, and [AIBN] = 5.6 mM (expt.12, Table 2.3). The lines show the theoretical evolution of Mn with conversion. …………………………..………………………...
73
Figure 2.28: Raman spectra of TFPMA polymerisation solution ([CDB] = 25 mM and [AIBN] = 2.5 mM) (a) initial and (b) after 23 h …………………..…………….…………….………………..
74
Figure 2.29: Conversion versus time of bulk TFPMA polymerisation using the RAFT agent CDB, and AIBN as initiator at 60 ºC: Curve a: [CDB] = 25 mM and [AIBN] = 2.5 mM (expt.13, Table 2.3), and b: [CDB] = 50 mM and [AIBN] = 5.0 mM (expt.14, Table 2.3). ……………………...…………….…………….…………
75
Figure 2.30: Mn and PDI of TFPMA polymerized with CDB (a) [CDB] = 25 mM and [AIBN] = 2.5 mM (expt.13, Table 2.3), b: [CDB] = 50 mM, and [AIBN] = 5.0 mM (expt.14, Table 2.3). The lines show the theoretical evolution of Mn with conversion…...
75
Figure 2.31: Effect of CDB purity on the rate of polymerisation of TFPMA (in bulk) in the presence of 25 mM CDB and 2.5 mM AIBN at 60 oC. Using hexane as eluent, CDB was passed through: a neutral activity aluminium oxide column followed by a silica column: Curve a: once and twice, respectively (expt.13, Table 2.3), and b: twice each (expt.15, Table 2.3). …………………..
76
Figure 2.32: GPC chromatograms of the chain extension of PFS with tBA carried out in (A) tetrahydrofuran (expt.2, Table 2.4), and (B) ethyl acetate (expt.3, Table 2.4), using PDA detection at 262 nm (full line, styrenic side groups) and at 310 nm (dashed line, RAFT end group)…………........................................................
80
Figure 2.33: Degradation of the RAFT end-groups of PFS (expt.7, Table 2.3) stored in tetrahydrofuran (curve a) and ethyl acetate (curve b) monitored by UV-vis absorbance spectroscopy at 310 nm. …………………………..…………….………………
81
Figure 2.34: 1H NMR spectra of P(TFPA-b-tBA) from expt.6, Table 2.4: (A) neat polymer (in acetone-d6) and, (B) after hydrolysis with
xvi
TFA (in DMSO-d6) (* = solvent) ……………………………... 82
Figure 2.35: 1H NMR spectrum of; (A) P(TFPMA-b-AAEMA) from expt.9, Table 2.4, in DMSO-d6, (B) glycine in 1:1 = D2O:DMSO-d6 and (C) glycine modified P(TFPMA-block-AAEMA) in DMSO-d6 (* = solvent)…….. …………….…………….……..
83
Figure 2.36: FTIR-ATR of (a) P(TFPMA-b-AAEMA) and (b) after glycine attachment in the ranges of (A) 3600-547 cm-1 and (B) 1670-1340 cm-1…………….…………….…………….……………..
84
Figure 2.37: 1H NMR spectra of (A) P(TFPMA-b-AAEMA) from expt.9, Table 2.4, (B) L-phenylalanyl glycine and (C) L-phenylalanyl glycine modified P(TFPMA-b-AAEMA), all in DMSO-d6 (* = solvent) ………………..…………….…………….…………...
86
Chapter 3
Figure 3.1: Illustration of attached polymer layers………………………… 104
Figure 3.2: Chain entanglement of polymeric chains by binary-hooking structure....…………….…………….…………….……………
112
Figure 3.3: Beam geometry and polarisation of IR radiation at the interface…………….…………….…………….………………
115
Figure 3.4: Idealised electrostatically driven LbL assembly deposition…... 123
Figure 3.5: XPS survey scans of (A) blank glass slide (sample 1A), (B) PEI film (sample 1B1) and (C) PEI-PAA LbL (sample 1B2)…..
124
Figure 3.6: C 1s narrow scans of (A) PEI film (sample 1B1) and (B) PEI-PAA LbL (sample 1B2) ………………………………………..
125
Figure 3.7: N1s narrow scans of (A) PEI film (sample 1B1) and (B) PEI-PAA LbL (sample 1B2). ……………………………………….
126
Figure 3.8: XPS survey scans of (A) blank silicon wafer (sample 2A), (B) PEI film (sample 2B), (C) PEI-PMAEP LbL (sample 2C) and (D) PEI-PMOEP LbL (sample 2D) ……………………………
128
Figure 3.9: C1s narrow scans of (A) PEI film (sample 2B), (B) PEI-PMAEP LbL (sample 2C) and (C) PEI-PMOEP LbL (sample 2D). ……………………………….…………….……………...
129
Figure 3.10: N1s narrow scans of (A) PEI film (sample 2B), (B) PEI-PMAEP LbL (sample 2C) and (C) PEI-PMOEP LbL (sample 2D) ……………………………….…………….………………
130
Figure 3.11: 2D and 3D AFM images of A) silicon wafer, B) PEI film (sample 2B), C) PEI-PMAEP LbL (sample 2C) and D) PEI-PMOEP LbL (sample 2D) (analysed area 1.0×1.0 μm) ……………………………………….……………………
132
xvii
Figure 3.12: 2D and 3D AFM images of PMAEP (analysed area 10×10 μm)……… …………….…………….…………….…………..
133
Figure 3.13: FTIR spectra of (A) soluble PMOEP (ATR) and (B) PEI-PMOEP LbL (sample 2D) (IRRAS)…………………………...
134
Figure 3.14: Idealised coupling reaction of block copolymers with aminated slide……………….…………….…………….………………..
135
Figure 3.15: XPS spectra of aminated slides (A) as received (Sample 3A), (B) PAAEMA functionalized (Sample 3B), (C) PMOEP functionalized (Sample 3C) and (D) P(MOEP-b-AAEMA) functionalized (Sample 3D). …………………………………..
136
Figure 3.16: C1s narrow scans of aminated slides (A) as received (sample 3A), (B) PAAEMA functionalized (sample 3B), (C) PMOEP functionalized (sample 3C) and (D) P(MOEP-b-AAEMA) functionalized (sample 3D). …………………………………...
138
Figure 3.17: N 1s narrow scans of aminated slides (A) as received (sample 3A), (B) PAAEMA functionalized (sample 3B), (C) PMOEP functionalized (sample 3C) and (D) PMOEP-b-PAAEMA functionalized (sample 3D). …………………………………...
138
Figure 3.18: Possible conformations of block copolymers reacted with aminated slide………………………………………………….
140
Figure 3.19: Positive and negative SSIMS spectra for APS-treated amianted glass slide. Note: sodium peak (23 amu) was scaled to 25% of its original intensity; peaks above 25 amu in negative mass spectrum were magnified by 10 folds………………………….
141
Figure 3.20: Positive SSIMS spectra for: (a) Sample 3A (aminated slide), (b) Sample 3B (PAAEMA attached), (c) Sample 3C (PMOEP attached), and (d) Sample 3D (P(MOEP-b-AAEMA) attached) ………………….…………….……………………...
142
Figure 3.21: Negative SSIMS spectra for: (a) Sample 3A (aminated slide), (b) Sample 3B (PAAEMA attached), (c) Sample 3C (PMOEP attached), and (d) Sample 3D (P(MOEP-b-AAEMA) attached).
142
Figure 3.22: Score plots on PC1 and PC2 for aminated glass slide and its modifications. (a) scores derived from the positive fragments; (b) scores derived from the negative fragments………………..
144
Figure 3.23: Loadings of selected positive (a) and negative (b) fragments on PC1s.. …………….…………….…………….………………..
145
Figure 3.24: Normalised intensities of Si+ and PO3- for APS-treated
aminated glass slide and its modifications. (a) Si+ intensity reflects the APS coverage with polymers; (b) PO3
- intensity is a sign of the surface density of the PMOEP segment………….
146
Figure 3.25: Negative mass spectrum of P(MOEP-b-AAEMA) attached aminated slide and the lateral distribution of the terminating
xviii
phosphate groups (PO3-) across the sample (analysed area →
100x100 μm) …………….…………….……………………… 147
Figure 3.26: XPS Survey spectra of PTFE films (A) untreated, (B) PFS attached (sample 3B3), (C) PTFPMA attached (sample 3C1), (D) PTFPA attached (sample 3D1) ……………………………
149
Figure 3.27: C1s narrow scans of PTFE films (A) untreated (sample 4A) and (B) PFS adsorbed (sample 4B3) …………………………...
151
Figure 3.28: C1s narrow scans of PTFE films after fluorinated homopolymer adsorption using different solvents. ……………
151
Figure 3.29: Relationship between molecular weight of PFS and adsorbed amounts of PFS onto PTFE films………………………………
153
Figure 3.30: C1s narrow scans of block copolymer attached PTFE films (A) P(FS101-b-tBA237) (sample 4F), (B) P(FS101-b-tBA141) (sample 4G), (C) P(FS101-b-AA237) (sample 4H), (D) P(FS101-b-AA141) (sample 4I1) and (E) P(FS101-b-AA141) (sample 4I2) …………..
154
Figure 3.31: Water droplet (5 μL) profiles on the surfaces of untreated PTFE (sample 4A) (A) advancing and (B) receding, P(FS101-b-tBA141) adsorbed PTFE (sample 4G) (C) advancing and (D) receding, and P(FS101-b-AA141) adsorbed PTFE (sample 4I1) (E) advancing and (F) receding ……………………………….
156
Figure 3.32: Schematic representation of PAA deposition onto PEI deposited surface at different pH ……………………………...
160
Figure 3.33: Schematic representation of PEI-PAA LbL showing free-functional groups ………………………………………………
161
Figure 3.34: Possible conformations of block copolymers reacted with aminated slide…………………………………………………..
163
Figure 3.35: Schematic representation of fluoropolymer adsorption onto PTFE by (A) fluorine adsorption and (B) chain entanglement...
166
Figure 3.36: Possible adsorption behaviour of PFS onto PTFE in different solvents.. …………….…………….…………….……………..
167
Figure 3.37: Aggregate adsorption onto PTFE surfaces in different solvents. 169
xix
Chapter 4
Figure 4.1: SEM images of sample 1A (PMAEP gel) (A) before treatment, and after SBF immersion for 7 days with (B) carbon coating and (C) gold coating, (D) sample 1B (soluble PMAEP film after 7 days in 1.5 SBF), (E) sample 1G (PAA gel) after 7 days immersion in SBF, and (F) EDX spectrum of the mineral on sample 1G………………………………………………………
188
Figure 4.2: SEM images of sample 1C (PMOEP gel) (A) before treatment, and after SBF immersion for 7 days with (B) carbon coating and (C) gold coating, sample 1D (PMOEP gel) (D) before treatment and (E) after SBF immersion for 7 days and (F) minerals dislodged from sample 1D. …………………………..
189
Figure 4.3: SEM images of sample 1E (PMOEP gel) (A) before, and (B) after SBF immersion for 7 days, (C) sample 1F (soluble PMOEP cast on glass) after SBF immersion for 7 days, and (D) EDX spectrum of the mineral on sample 1E. ……………..
190
Figure 4.4: ATR-FTIR spectra of polymer samples after 7 days immersion in SBF (solid line) and initial, untreated polymers (dotted line). (A) sample 1A (PMAEP gel); (B) sample 1B (PMAEP film) (Experiment done in 1.5×SBF.); (C) sample 1C (PMOEP gel); (D) sample 1D (PMOEP gel); (E) sample 1E (PMOEP gel); (F) sample 1F (PMOEP film on a glass surface), and (G) sample 1G (PAA gel). y-axis is absorbance…...……………….
193
Figure 4.5: ATR-FTIR spectra of Sample E (PMOEP gel) reacted with Ca(OH)2 (solid line) and untreated (dotted line). y-axis is absorbance…………..…………….…………….……………...
195
Figure 4.6: SEM images of minerals formed on the LbL surfaces after SBF immersion for 7 days: (A) sample 2A (PEI-PMAEP), (B) sample 2B (PEI-PMOEP), (C) sample 2C (PEI only), and (D) sample 2D (silicon wafer), and 14 days: (E) sample 2A, (F) sample 2B, (G) sample 2C, and (H) sample 2D………………..
197
Figure 4.7: SEM images of minerals formed on the block copolymer functionalized aminated slides after SBF immersion for 7 days: (A) sample 3A, (B) sample 3B, (C) sample 3C and (D) sample 3D, and 14 days: (E) sample 3A, (F) sample 3B, (G) sample 3C and (H) sample 3D. …………………………….…………..
200
Figure 4.8: SEM images of minerals formed on the PAAEMA functionalized and untreated aminated slides after SBF immersion for 7 days: (A) sample 3E (PAAEMA) and (B) sample 3F (untreated aminated slide), and 14 days: (C) sample 3E (PAAEMA) and (D) sample 3F. ………………...................
201
Figure 4.9: EDX spectra of the mineral (A) and the non-mineral area (B) on sample 3B (see Figure 4.7F). ………………………………
201
xx
Figure 4.10: SEM images of minerals formed on P(FSm-b-AAn) attached and untreated PTFE films after SBF immersion for 7 days: (A) sample 4A, (B) sample 4B, (C) sample 4C and (D) sample 4D (untreated PTFE), and 14 days: (E) sample 4A, (F) sample 4B, (G) sample 4C and (H) sample 4D……………………………..
204
Figure 4.11: Proposed structures of a P(AAEMA-b-MOEP) block copolymer on an aminated slide (A) in vacuum and (B) in SBF solution. ……………….…………….…………….…………...
210
xxi
LIST OF TABLES
Chapter 1
Table 1.1: Composition of human trabecular and cortical bone (vol%)4…… 2
Chapter 2
Table 2.1: Experimental conditions of MAEP and MOEP polymerisation reactions and characteristics of the polymers obtained………….
47
Table 2.2: Experimental conditions of chain extension reactions and molecular weight of the polymers obtained after hydrolysis……
64
Table 2.3: Experimental results for the RAFT polymerisation of fluorinated macromers……………………………………………………......
65
Table 2.4: Experimental Results from the Chain Extension of Fluorinated Macromers via RAFT……………………………………………
79
Table 2.5: Expected IR bands from glycine attachments64…......................... 85
Chapter 3
Table 3.1: Comparison of energies associated with intermolecular forces… 111
Table 3.2: Literature values of Ne of PTFE………………………………… 112
Table 3.3: Properties of PEI and PAA……………………………………… 124
Table 3.4: Atomic % of O, C, N and Si…………………………………….. 125
Table 3.5: Normalized atomic % of nitrogen species from the curve fitting of the N1s peak…………………………………………………..
127
Table 3.6: Properties of PMAEP/PMOEP used for this study……………… 128
Table 3.7: Atomic % of C, N, P and Si from XPS survey scans…………… 129
Table 3.8: Normalized atomic % of nitrogen species from curve fitting of the N1s peak……………………………………………………..
130
Table 3.9: Mean roughness (nm) of LbL films from the AFM images in Figure 3.7.………………………………………………………..
131
Table 3.10: Block-copolymers attached to aminated slides for SBF………… 135
Table 3.11: XPS data: Atomic % of elements concentrations from XPS survey scans……………………………………………………..
136
Table 3.12: Normalized atomic % of nitrogen species from curve fitting of the N1s peak……………………………………………………..
139
xxii
Table 3.13: Positive and negative fragments used in PCA………………….. 139
Table 3.14: Polymer characteristics………………………………………….. 148
Table 3.15: Atomic % of elements from XPS survey scans and atomic C% of different C elements from the high resolution C1s scans………..
150
Table 3.16: Properties of PFS and atomic % from C 1s scans of PFS adsorbed PTFE films……………………………………………..
152
Table 3.17: Properties of PFS block copolymers…………………………….. 153
Table 3.18: Atomic % of C-F2 and C-others…………………………………. 155
Table 3.19: Advancing and receding water contact angles of polymer-adsorbed PTFE films. ……………………………………………
157
Table 3.20: Advancing and receding contact angles of P(FS-b-AA) adsorbed surfaces after soaking in MilliQ water for 2 days……………......
158
Table 3.21: DH of P(FS-b-AA) in DMF and MEK a………………….……… 158
Chapter 4
Table 4.1: Different calcium phosphate structures8………………….……... 178
Table 4.2: Ion concentrations of human blood plasma, different SBF solutions,14 and SPF16………………….………………………....
179
Table 4.3: Properties of PMAEP and PMOEP polymers subjected to SBF studies………………….………………….……………………...
187
Table 4.4: LbL samples for SBF and their atomic % of Ca and P after 7 days in SBF (obtained from XPS survey scans) ………………...
196
Table 4.5: Block-copolymer-attached aminated slides for SBF……………. 199
Table 4.6: Properties of fluorinated block copolymer absorbed PTFE films.. 203
Table 4.7: Atomic % of Ca and P from XPS survey scans and the Ca/P ratios………………………………………………………….......
205
xxiii
LIST OF SCHEMES
Chapter 1
Scheme 1.1: Synthetic route to poly(vinylamine) with dextran lactone and N-(perfluoroundecanoyloxy)succinimide……………………...
17
Chapter 2
Scheme 2.1: Schematic representation of nitroxide-mediated polymerisation of styrene with TEMPO, monomer (e.g. styrene) and initiator (e.g BPO = benzyl peroxide). Pn• and Pm• are propagating polymer radicals……………………...…………………….......
26
Scheme 2.2: Schematic representation of the ATRP polymerisation process, Pn = polymer, X = halide, L = ligand, and M = monomer……..
27
Scheme 2.3: General structure of a RAFT agent, Z = activation group (e.g. aryl and alkyl) and R = good leaving group (e.g. cumyl and cyanoisopropyl) ……………………...…………………….......
28
Scheme 2.4: Schematic representation of proposed RAFT polymerisation process. 29
Scheme 2.5: Structures of RAFT agents……………………...……………... 32
Scheme 2.6: Structures of phosphate-containing monomers………………... 33
Scheme 2.7: Structures of fluorinated monomers……………………............ 36
Scheme 2.8: Hydrolysis of polymeric side-chains and dithiocarbonate moiety of PMAEP/PMOEP using NaOH solution; R1 = H (PMAEP) or CH3 (MOEP), R2 = H (PEPDTA) or CH3 (CDB) and R3 = CH2 (PEPDTA) or none (CDB) ……………………..
49
Scheme 2.9: Chain extension of (A) PAAEMA with MAEP/MOEP and (B) PMOEP with AAEMA, R = H: MAEP, CH3: MOEP…………………………………………………………...
63
Scheme 2.10: Schematic representations of chain extensions of PFS, PTFPA and PTFPMA, and further reactions (× = reaction did not proceed) ……………………...……………………...................
78
Scheme 2.11: Reaction scheme of PAAEMA block copolymer with glycine... 83
Scheme 2.12: Possible hydrolysis sites on the side-chain of the polymer and structure of resulting polymer (R = H: MAEP, R = CH3: MOEP) ……………………...……………………....................
89
Scheme 2.13: Structure of polymer from monomer-diene mixture(R = H: MAEP, R = CH3: MOEP) ……………………...……………...
90
Scheme 2.14: Mechanism of Diels-Alder dimer and 1,4-diradical formations of styrene. (Reproduced from Ref:86) M = styrene, AH = Diels-Alder dimer, •M2• = 1,4-diradical and DCB = 1,2
xxiv
diphenylcyclobutane…………………………………………. 94
Chapter 3
Scheme 3.1: Various reaction schemes of PAAEMA…………………… 107
Scheme 3.2: Structures of fluorinated homopolymers…………………….... 148
Chapter 4
Scheme 4.1: Structure of carboxylated polyphosphazene…………………... 181
Scheme 4.2: Structures of DEAEMA and VP……………………................. 183
Scheme 4.3: Possible hydrolysis sites on the side-chain of the polymer and structure of resulting polymer (R = H: MAEP, R = CH3: MOEP) ……………………...……………………...………….
207
xxv
ABBREVIATIONS POLYMERS
ePTFE Expanded Polytetrafluoroethylene
FEP Tetrafluoroethylene-hexafluoropropylene copolymer
PAA Poly(acrylic acid)
PAAEA Poly(2-(acetoacetoxy)ethyl acrylate)
PAAEMA Poly(2-(acetoacetoxy)ethyl methacrylate)
PAH Poly(allylamine hydrochloride)
PBT Poly(buthylene terephthalate)
PCL Poly(ε-carprolactone)
PDMS Poly(dimethyl siloxane)
PEG Poly(ethylene glycol)
PEI Polyethyleneimine
PEO Poly(ethylene oxide)
PET Poly(ethylene terephthalate)
PFS Poly(pentafluorostyrene)
PGA Poly(L-glutamic acid)
PHBV Poly(3-hydroxybutyrate-co-3-hydroxyvalerate
PHEMA Poly(2-hydroxyethyl methacrylate)
PLA Poly(lactic acid)
PMA Poly(methacylic acid)
PMAEP Poly(monoacryloxyethyl phosphate)
PMOEP Poly(methacryloyloxyethyl phosphate)
PNVF Poly(N-vinylformamide)
PS Polystyrene
PTFE Polytetrafluoroethylene
PTFPA Poly(tetrafluoropropyl acrylate)
PTFPMA Poly(tetrafluoropropyl methacrylate)
PU Polyurethane
PVAm Poly(vinylamine)
PVDF Poly(vinylidene fluoride)
PVP Poly(2-vinylpyridine)
xxvi
MONOMERS
AA Acrylic acid
AAEA 2-(acryloyloxy)ethyl acetoacetate
AAEMA 2-(methacryloyloxy)ethyl acetoacetate
AAm Acrylamide
DEAEMA (Diethylamino)ethyl methacrylate
DMAA N,N-dimethylacrylaminde
DMAEMA (N,N-dimethylamino)ethyl methacrylate
FS 2,3,4,5,6-pentafluorostyrene
HEMA 2-Hydroxyethyl methacrylate
MAA Methacrylic acid
MAEP Monoacryloxyethyl phosphate
MOEP Methacryloyloxyethyl phosphate
NaSS Sodium styrenesulfonate
tBA tert-butyl acrylate
TFPA 2,2,3,3-tetrafluoropropyl acrylate
TFPMA 2,2,3,3-tetrafluoropropyl methacrylate
VBC 4-vinylbenzyl chloride
VP 1-vinyl-2-pyrrolidinone
SOLVENTS AND OTHER CHEMICALS
APS 3-Aminopropyltrimethoxysilane
AIBN 2,2-Azobis(isobutyronitrile)
BMPs Bone morphogenic proteins
CDB Cumyl dithiobenzoate
CPDA Cumyl phenyldithioacetate
DCB 1,2-Diphenylcyclobutane
DCM Dichloromethane
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
EA Ethyl acetate
EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
FB Fluorobenzene
xxvii
hTM Human Thrombomodulin
MEK Methyl ethyl ketone
PBS Phosphate-buffered saline
PEPDTA 1-Phenylethyl phenyl dithioacetate
PPDB 2-Phenyl-prop-2-yl dithiobenzoate
RGD Arginine-Glycine-Aspartate
SBF Simulated body fluid
SPF Simulated physiological fluid
TEMPO 2,2,6,6-Tetramethylpiperidinyloxy
TFA Trifluoroacetic acid
THF Tetrahydrofuran
Vazo 88 1,1’-Azobis(cyclohexanecarbonitrile)
INSTRUMENTS AND TECHNIQUES
AFM Atomic Force Microscope
ATR Attenuated Total Reflection
CVD Chemical vapour deposition
DLS Dynamic light scattering
EDX Electron Dispersive X-ray
ESCA Electron Spectroscopy for Chemical Analysis
FTIR Fourier Transform Infrared Spectroscopy
FT-NIR Fourier Transform Near-Infrared Spectroscopy
GPC Gel Permeation Chromatography
IRRAS Infrared Reflection-Adsorption Spectroscopy
NMR Nuclear Magnetic Resonance
SEM Scanning Electron Microscope
SSIMS Static Secondary Ion Mass Spectroscopy
ToF Time-of-Flight
XPS X-ray Photoelectron Spectroscopy
xxviii
OTHERS
ATRP Atom transfer radical polymerisation
CSIRO Commonwealth Scientific and Industrial Research Organization
DH Number-average hydrodynamic diameter
GBR Guided bone regeneration
ITP Iodine transfer polymerisation
LbL Layer-by-Layer
Mn Molecular weight
NMP Nitroxide-mediated polymerisation
PDI Polydispersity index
RAFT Reversible addition fragmentation chain transfer
SAMs Self Assembled Monolayers
TCPS Tissue Culture Polystyrene
xxix
Chapter 1: Introduction
Chapter 1: Introduction
1.1 Biomaterials and Biocompatibility
A biomaterial is defined as a material used to replace part of a living system or used
in close contact with a living system. Metals, ceramics, polymers, glasses, and
composites have all been widely investigated and used as biomaterials.1 Biomaterials
science is a multidisciplinary field, which encompasses not only the synthesis and
development of suitable materials but also the evaluation of their degradation,
mechanical, physical and chemical properties. The study of the complex host
response to the introduced bulk material as a whole, as well as the complex cascade
of events occurring at the material’s surface are essential parts of biomaterial-science.
Biocompatibility is the ability of a biomaterial to perform with the appropriate
response in a specific application.2 Biocompatibility, therefore, cannot be defined in
terms of the properties of a material alone; instead, it is a combination of its specific
properties and the function for which it is intended. Biocompatibility assessment is
an essential factor for determining the success of devices or materials intended for
use in contact with the body.2
Polymeric biomaterials have proven their use in a range of applications in many
areas of medicine due to their diverse chemical compositions, chemical and
mechanical properties as well as their ability to be manufactured in a wide range of
structural forms.3 There are many polymeric biomaterials on the market that meet
their bulk biocompatibility requirements. However, not many of these polymers
possess ideal surface properties. Since the first interaction of an implant with the
body is through its surface, the initial acceptance of the material in the body is highly
dependent on its surface properties.
Polymer surfaces are not rigid, which makes them very complicated as the surface
composition depends on the environment (e.g. air vs liquid). Consideration of many
parameters is required, including topography, chemical composition, surface energy,
wettability, crystallinity, surface mobility and heterogeneity. It is not yet fully
understood which parameters are the most important when considering biological
response to polymer surfaces. However, analysing as many parameters as possible
1
Chapter 1: Introduction
gives a more complete understanding of the surface and its interface reactions with
the body.
The material/tissue interface is a very complex system and is still not fully
understood. Agreement as to what constitutes, optimal surface property is also a
matter of some contention and, of course, depend on the specific application. For
example, calcification and protein adhesion onto contact lenses surfaces is not ideal
and there has been significant research focusing on how to prevent these processes
from occurring. On the other hand, bone implants require bioactive surfaces that can
support cell growth and mineralisation for direct bone bonding. Since the focus of
this PhD thesis is the creation of more reactive surfaces in clinically important
polymers in order to facilitate better bone bonding, it is necessary to consider the
nature of bone and bone fracture healing as well as the polymer/bone interface.
1.2 Bone
Bone is a unique composite material, in the sense that it consists of both an inorganic
mineral phase and organic macromolecules. It is often referred to as a “living
mineral” because it undergoes continual growth: both dissolution and remodelling
occur in response to either internal signals or external force fields, such as gravity.
The chemical composition of the two types of human bone is shown in Table 1.1.
Table 1.1: Composition of human trabecular and cortical bone (vol%).4
The inorganic mineral component of human bone is carbonated hydroxyapatite. The
organic matrix is composed of an insoluble framework of collagen fibrils (~90%),
and water soluble non-collageneous proteins (~10%) as well as trace amounts of
many other types of proteins. This special combination of inorganic and organic
2
Chapter 1: Introduction
components leads to a biological organic-inorganic composite material, with unique
mechanical properties.
The two principal types of bone are identifiable by their macroscopic structures:
cortical (or compact) bone and trabecular (or cancellous) bone. Cortical bone is a
dense tissue with a porosity of about 10%, and is primarily found in the shaft of long
bones and the outer shell around cancellous bone at the end of joints. Trabecular
bone is highly porous (50-90% porosity), and is found in the end of long bones and
in flat bones like the pelvis. Many of the unique properties of bone are a result of its
hierarchically-organized structure, which is shown in Figure 1.1.
.
Figure 1.1: Hierarchical structure of bone. (Reproduced from ref.5)
At the nanostructure level, the three main components of fibrils: mineral apatite
crystals, collagens, and non-collageneous proteins are observed. Collagen molecules
together with non-collagenous proteins constitute the organic matrix that performs
important functions including the following:
• Mechanical design control – strength and elasticity of bone. The combination
of organic and inorganic materials produces a strength greater than either the
components alone.4
3
Chapter 1: Introduction
• Mineral stabilisation – surface stabilisation of minerals from dissolution or
phase transition.6,7
• Mineral nucleation – controls the location, as well as organization of the
nucleation sites. This in turn leads to control of the structure and orientation
of the inorganic phase.6
Although the collagen matrix, itself, does not possess either nucleation sites
or the template for mineral deposition, the non-collagenous proteins, such as
osteonectin and some phosphoproteins (which have high proportion of
anionic groups that have high affinities for calcium ions) absorbed onto the
collagen network are believed to be the biomineralisation nucleators.6,8
Four different types of bone cells have been identified: osteoblasts (bone-forming
cells), osteocytes (mature cells embedded in the bone matrix), bone-lining cells
(living in the surface of bone), and osteoclasts (bone-resorbing cells). Osteoblasts are
derived from the mesenchymal stem cells of the bone marrow stroma. They
synthesise, and lay down, the precursors of type I collagen. Osteoblasts produce
osteocalcin, as well as proteoglycans, and are rich in alkaline phosphatase (an
organic phosphate-splitting enzyme). Hence, these cells are know as bone-forming
cells and are important for biomineralisation.
Osteocytes are developed from osteoblasts and live in the bone. It has been suggested
that osteocytes can sense the mechanical load in bone from the fluid flow stresses
occurring inside the bone.8 These cells then send signals which activate the
osteoclasts and osteoblasts. Osteocytes may also participate in bone resorption to
increase the level of calcium ions whenever it is demanded.9
Bone lining cells are thought to be either inactive osteoblasts which may be activated
or a cell type of their own. They are elongated, thin cells which cover the bone
surfaces that are not under remodeling.
Osteoclasts, typically multinucleated, are derived from monocytes which are
originally derived from hematopoietic stem cells. Osteoclasts are the only cell type
that can resorb bone. They may be recruited and activated through the signals from
osteoblasts which are initially activated by osteocytes.10 It has been shown that
4
Chapter 1: Introduction
osteoclastic bone resorption does not occur in the absence of osteoblasts or added
stimuli.
1.3 Host Response to Polymeric Biomaterials
The host tissue reaction induced by an implant material is an important factor that
determines the biocompatibility of any material. Another factor is the degradation
behaviour of the material in the body. These two factors are often correlated, hence a
separate interpretation may prove difficult. Moreover, the host response to the
implant is dependent not only on material properties, such as its physical structure
and chemistry, but also the nature of the implantation site.
1.3.1 Wound healing
There are many changes in both the types and numbers of cells present at a site of
soft-tissue repair, as shown in Figure 1.2.11 Varying concentrations of chemicals and
electrolytes in body fluid are also observed during soft-tissue repair. The three
processes that occur following injury are: hemostasis, inflammation, and wound
healing.
Figure 1.2: Cellular activities at wound repair. (Reproduced from ref.11)
5
Chapter 1: Introduction
The first stage (hemostasis) happens within a few minutes. Blood platelets fill the
injury site forming a temporary shield from the environment. Two important types of
leukocytes (white blood cells) that participate in the inflammation stage are
neutrophils and macrophages. These cells migrate to the wound as a result of a
number of chemicals released during injury.11 Neutrophils kill bacteria, while
macrophages remove cellular and foreign debris from the wound site. Macrophages
stay at the injury site and participate in the next stage of wound healing.
Macrophages break up the fibrous clot formed by the blood platelets so that new
blood vessels can penetrate the wound site. Capillaries bud and grow from the blood
vessels into nearby tissue, and proliferate into the injury zone and form an
interconnecting network. This network supplies oxygen and nutrients required by
other cells present at the site. Fibroblasts then migrate and synthesise extracellular
substances, such as collagen. The synthesised collagen binds into fibres, which
randomly fill the wound, forming scar tissue.
The difference between the healing mechanism of hard tissue from that of soft tissue
is the unique ability of hard tissue to regenerate without scarring.12 Where bone is
concerned, in general, there are two types of fractures: closed and open fractures.
Closed fractures have no contact with the external environment; therefore, the
healing process is only affected by local factors. Since open fractures have contact
with the outside environment, they must heal with concomitant ongoing soft tissue
regeneration, which may involve additional signals and cell types at the bone
surfaces. When a biomaterial is implanted, it is thus more complex healing scenario
that is required.
It is important to note the terms “bone bonding”, “osteointegration” and
“osseointegration” all have the same meaning of “direct bone contact of a material
with bone without interposition of non-bone tissues such as fibrous capsules”. This
provides a direct structural and functional connection between living bone and the
materials surface.
6
Chapter 1: Introduction
1.3.2 Tissue response to implants
The surgical procedures required in the introduction of any biomaterial device create
a wound and hence triggers a “wound-healing response”. Inflammation, wound
healing and foreign body response, as well as fibrous encapsulation, are generally
considered as the typical host/tissue responses to an implant.13 Inflammation is an
essential reaction occurrence at the implantation site before healing can begin. The
normal reaction of biological systems to introduced materials is to biodegrade them
or attempt digestion. Failing this, the alternative is encapsulation of the introduced
material within a fibrous collagen capsule.13 This foreign body reaction involves
foreign body giant cells and other cells such as macrophages and fibroblasts that are
already present at the wound site.
Once a fibrous capsule is formed, the material is isolated from the body.
Vascularisation cannot occur inside the capsule; therefore, no cell growth is possible.
If bacterial infections occur inside this fibrous capsule, the infection may be
prolonged as macrophages are unable to access the interior of the capsule and do the
job for which they are designed. Excessive fibrous growth can result in tissue
damage and ultimately tissue death.
As mentioned earlier, when a biomaterial is inserted into bone, a situation similar to
that of an open fracture is created. The resulting activation of the macrophages can
result in the formation of a fibrous layer or even fibrous encapsulation. This, in turn,
can lead to serious consequences such as micromotion which leads to implant
loosening and even bone resorption. In other words, good bone bonding is critical for
the successful functioning of the biomaterial implant or device.
Consequently, many implants including materials for cranio-maxillo-facial
applications require direct bone bonding without the interposition of fibrous capsules.
It is generally believed that bone bonding will take place when a layer of apatite,
similar to bone in crystallinity and composition, forms spontaneously on their surface
in contact with blood plasma.14 The mineralisation process can occur through cell-
and material-directed pathways. This depends on the material surface properties
which will be discussed in the following section.
7
Chapter 1: Introduction
1.4 Material/Bone Interface
When a material is implanted, a cascade of events occurs at the surface (Figure
1.3).15 More specifically, water molecules, which constitute 70 wt% or more of our
body, are the first to come in to contact with the implant material surface and form a
water layer (Figure 1.3 A-B) that is followed by hydrated ions (C). The behaviour of
water near the surface has been recognised to play an important role for the
following events.15,16 The various biomolecules, in particular proteins, come and
interact with the hydrated surface either through or by displacing a water layer (D-E).
When the cells arrive at the surface, they encounter the adsorbed protein layers (F). If
the proteins are denatured, the cells will not attach or may even activate a
macrophage response if they recognise the material as a foreign substance. This can
lead to fibrous encapsulation. Therefore cell and tissue interaction with material
surfaces is mediated by the adsorbed proteins.17,18
Figure 1.3: Schematic illustration of the successive events following implantation of a material. (Reproduced from ref.15)
8
Chapter 1: Introduction
The proteins involved in osteoblast adhesion onto material surfaces include
extracellular matrix proteins, cytoskeletal proteins, integrins and cadherins.19 The
types, amounts, and conformations of adsorbed proteins on material surfaces
determine the osteoblast cell behaviour. Eventually cell-directed mineralisation can
occur if the adsorbed proteins signal osteoblast adhesion, spreading and
differentiation.6
Functional groups play an important role in cell behaviour via protein attachment as
well as for mineralization. The effect of functional groups on protein adsorption and
endothelial cell growth have been investigated using self-assembled monolayers
(SAMs) of alkanethiolates on gold.20 It was found that cell growth increased in the
following order -CH2OH < -C(O)OCH3 < -CH3 « -C(O)OH. However, tissue culture
polystyrene (TCPS) showed better cell growth than SAMs. XPS analysis of TCPS
revealed a surface composition of over 12% oxygen present as various polar groups
(i.e. hydroxyl, carbonyl, carboxylic acid, and esters). As well as oxygen, small
amounts of nitrogen (0.56%) were also detected by XPS analysis.21 The results of
such cell growth studies seem to indicate that multiple functionalities provide a
pronounced synergistic effect.20
The incorporation of functional groups to increase the hydrophilicity of polymer
surfaces is a common technique for improving cell adhesion on hydrophobic polymer
surfaces.22 Hydrophobic surfaces are known to induce strong irreversible protein
adsorption. This denatures their conformation and hence causes a loss in bioactivity.
Very hydrophilic surfaces, on the other hand, inhibit protein adhesion. Grafting of
water soluble poly(ethylene oxide) (PEO) has been shown to decrease the interfacial
free energy and the steric repulsion forces between the PEO chains and proteins in
polymers used for vascular grafts applications.23 It is now accepted that moderately
hydrophilic surfaces induce a positive cell response because these surfaces are able
to adsorb proteins without causing denaturing.
In addition to introducing functional groups to change the wettability of a material’s
surface, mineralisation induced by different functional groups is also important.
Apatite formation was found to be significantly enhanced on negatively charged
SAMs, compared to positively charged, or neutral surfaces.24,25 The mineral growth
9
Chapter 1: Introduction
rate decreased in the order: -PO4H2 > -COOH » -CONH2 ≃ -OH > -NH2 » -CH3 ≃
0.24
1.5 Surface Modification: Improving the Material/Bone Interface
1.5.1 Physicochemical Surface Modification
The physicochemical surface modification of polymers in order to incorporate new
functional groups has long been studied for use in a wide range of applications.
Many techniques are available, including chemical, plasma, and grafting methods.
The introduction of new functional groups gives rise to different surface properties
such as: surface energy, water-retaining capacity, and surface mobility. All of which
are important for tissue response.26
Chemical treatments, including acid or base etching that produce hydroxyl and
carboxyl groups, have been used for both non-degradable21 and biodegradable
polymeric biomaterials.27,28 Aminolysis techniques have been used for modifying
ester-containing polymers such as poly(3-hydroxybutyrate-co-3-
hydroxyvalerate)(PHBV), poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA) and
polyurethane (PU).29-32 In such treatments, the polymers are treated with solutions
containing diamino compounds (e.g. diaminohexane). Despite their effectiveness,
chemical methods have several disadvantages. Degradation of the bulk properties by
the harsh chemical reagents often occurs. For biodegradable polymers, decreases in
molecular weight have been observed. It is also difficult to monitor and control the
modification depth profile. In the case of poly (lactic-co-glycolic acid) (PLGA),
chemical treatments have been optimised for minimal degradation.30
Plasma modification using glow discharges from gases such as air, O2, H2O vapour,
CO2, NH3, N2, and SO2 are frequently used to introduce functional groups such as
hydroxyl, carboxyl, amino and sulphate groups on polymeric surfaces.33 Plasma
treatment is also used for the deposition or immobilization of molecules.34 An O2-
plasma-treated PCL surface, which was subsequently alternatively dipped into
calcium and phosphate solutions, showed HAP formation after 24 hours in SBF.35 A
poly(ethylene glycol)/poly(butylene terephthalate) (PEG/PBT) segmented block
10
Chapter 1: Introduction
copolymer, which was treated with an O2 plasma exhibited improved bone marrow
stromal cell attachment and growth, which was equivalent to the result obtained for
an O2-plasma-treated polymer/HAP composite.36 However, a serious drawback of
these plasma techniques is the aging effect of the treated surface in air, which is
dependent on the environment and temperature. Although its technical operation is
simple, it is very difficult to control the actual amount of a particular functional
group formed on a surface.
Graft copolymerisation offers stable functional groups by covalently immobilising
new polymer brushes.37 Active sites (radicals or peroxides) are produced randomly
along the surface polymer chains by an electron beam, ozone oxidation, X-rays, γ-
rays or Ar plasma treatments. These sites initiate graft copolymerisation. Grafting is
applicable to many monomer/polymer combinations. The grafting of phosphate-
containing monomers has been found to induce calcium-phosphate growth38-40 with
grafted materials showing direct bone bonding in vivo.41 The incorporation of
negatively-charged functional groups onto polymeric surfaces is discussed further in
Chapter 4.
1.5.2 Morphological surface modification
Surface micro- and nano-scale patterning has shown significant influence on cell
behaviour such as: cell shape, migration, and protein synthesis.42,43 Osteoblast
proliferation has been found to be sensitive to surface topography.44 Surfaces with
grooves have been shown to induce “contact guidance”, which influences cell
spreading, alignment and migration.
It has been reported that bone cells aligned themselves parallel to the direction of the
grooves on a polystyrene (PS) surface with 5μm-deep grooves, but they did not
respond to the surface with 0.5 μm-deep grooves (both grooves were 5 μm wide).45
On the other hand, poly(L-lactic acid) (PLA) and PS surfaces with microgrooves of
0.5, 1.0, and 1.5 μm depth and 1, 2, 5, and 10 μm width showed enhanced
differentiation of osteoblast-like cells and mineral formation.46 Another study
showed that a polydimethylsiloxane (PDMS) surface with microtextures of 6 μm
11
Chapter 1: Introduction
high and 5-40 μm in diameter promoted spreading and adhesion of human bone-
marrow-derived cells.47
In general, smooth surfaces exhibit less cell adhesion than rough surfaces. In the case
of biodegradable scaffolds, surface topography and roughness have been shown to be
important in allowing the migration of cells on the scaffold surface.48 Osteoblast-like
cells seeded onto a rough PLLA surface, which had an island pattern, preferred to
elongate and showed uniform growth compared to those on a smooth PLLA
surface.49
1.5.3 Incorporation of biological molecules
The two techniques discussed in the previous sections cannot induce specific cell
behaviour due to non-specific protein adhesion. Immobilization of biomolecules onto
the surface, on the other hand, provides a way to control specific cell and tissue
responses by directly delivering molecules with a defined function to the tissue-
implant interface. Many different biologically active molecules have been either
covalently or physically immobilised onto surfaces in order to enhance biological
interaction. Some examples are proteins, peptides, polysaccharides, lipids, drugs,
ligands, and nucleic acids.50
Covalent immobilisation requires the presence of reactive groups such as –OH, –SH,
–NH2, or –COOH. Because many polymeric biomaterials do not possess these
reactive groups, surface modification, such as graft copolymerisation, is necessary in
order to introduce them. Figure 1.4 shows a schematic representation of the
introduction of peptides onto a pre-treated polymer substrate.
OO
Figure 1.4: Covalent immobilisation of RGD peptides with carboxyl groups on a modified polymer surface.
OH
O
OH
O
NOH
OO
NO
O
O
NO
O
O
NH
O
RGD
NH
O
RGDEDC
H2N–RGD
e.g poly(acrylic acid) grafting
COOH incorporation
12
Chapter 1: Introduction
RGD peptides (R: arginine, G: glycine, D: aspartic acid) have been found to promote
cell adhesion and many RGD-functionalized polymeric biomaterials have been
widely studied.51 RGD-functionalized poly(ethylene glycol-b-D,L-lactic acid),52 and
a RGD-functionalized poly(lactic acid) scaffold53 have shown significant
improvements in osteoblast attachment and spreading compared to the unmodified
materials.
Growth factors, such as bone morphogenic proteins (BMPs), are also known to
stimulate local bone regeneration. Biodegradable polymers are often used as carriers
for BMPs.54-57 BMPs have also been covalently immobilised on an allyl amine
plasma polymerised surface, where it induced significant osteoblastic activity in
vitro.58
Some examples of physical immobilisation are coating of protein layers, electrostatic
interaction, entrapment, and Layer-by-Layer techniques using negatively and
positively charged biomolecules. The entrapment technique involves immobilising
biomolecules onto the swollen polymer surface, as shown in Figure 1.5.22
Figure 1.5: Entrapment of biomolecules into a polymer substrate.
Klee et al.59 investigated human osteoblast behaviour on surfaces with both
physically and covalently attached fibronectin. Poly(vinylydenefluoride) (PVDF)
was first pre-treated by either the plasma graft copolymerisation of acrylic acid, or
the chemical vapour deposition (CVD) of an amine-containing monomer, followed
by immobilisation of fibronectin using different techniques. Although both
physically adsorbed and covalently attached fibronectin surfaces showed enhanced
cell attachment, proliferation was only enhanced on the surfaces with the covalently
Surface swelling Entrapment
e.g. PLA In a solvent and
non-solvent mixture with biomolecules
In pure non-solvent
biomolecules
13
Chapter 1: Introduction
bound protein. This was interpreted as supporting the view that the long term
presence of biomolecules is necessary for cell adhesion and proliferation.59
Despite showing many promising results, there are also some concerns with covalent
immobilization. It has been shown that the covalent immobilization of enzymes and
proteins resulted in a substantial loss of activity due to the loss of active sites by
bonding or reorientation of the biomolecules after bonding.60 Sterilization becomes
more difficult, fouling by other biomolecules occurs, and adverse biological
responses of enzyme-supported surfaces have all been reported.50
1.6 Expanded PTFE (ePTFE) in Medicine
In 1969, expanded PTFE (ePTFE) was developed by W.L. Gore and associates.61
ePTFE has a highly fibrillated structure with an improved resistance to creep.
Although ePTFE is soft, and very flexible, it is also very strong and extremely
resistant to stretching or tearing. Because of its bioinertness, ePTFE has found many
applications in medicine, including vascular grafts,62-65 cranio-maxillo-facial
surgery,62 and guided bone regeneration (GBR).66-68 It is also used for catheter
coatings, sutures, aneurism clips, and oxygenation membranes. In general, ePTFE
does not support soft-tissue growth and ingrowth in vivo, which is a benefit for use in
vascular grafts and GBR. However, for cranio-maxillo-facial applications, where
implants are in contact with both soft and hard tissues, a more bioactive surface is
desirable.
1.7 Surface Modification of PTFE and Other Fluoropolymers
Fluoropolymers have a wide range of applications in industry due to their
outstanding properties such as high-temperature stability, excellent chemical
resistance, low water sorption, and low dielectric constant. Depending on the
application, the low surface energy of fluoropolymers, which results in poor adhesion
to other materials, is not desirable. Surface modification of these materials has been
widely studied, including modification for medical applications.69,70
Chemical modification of fluoropolymers has been successfully used for
defluorination and refunctionalisation.69 However, as mentioned before harsh
14
Chapter 1: Introduction
chemical treatments alter PTFE bulk properties. The chemical treatment of ePTFE
has been reported to cause destruction of the structural integrity even under relatively
mild conditions.71 Although fluoropolymers are sensitive to irradiation treatment,
radiation treatments, such as: X-ray, γ-ray, UV, laser, electron beam and ion beam
methods have all been investigated for the surface modification of
fluoropolymers.69,72 Most of the irradiation treatments result in the formation of
alkyl-type radicals and lead to the formation of carbon-carbon double bonds. The
surface modification of ePTFE (Gore-Tex) by N2+, Ar+, and Ca+ ion implantation has
also been reported.73
Plasma treatments of fluoropolymers have been carried out with glow discharges
generated from various gases including: H2,74 CH4,74 He,74,75 Ne,75 O2,76 H2,77 N2,76,78
Ar,74,76,78,79 a H2O/Ar mixture,80 SO281, NH3
74,76,82-84, and an NH3/O2 mixture85. The
aging process of O2, Ar, N2, and NH3 plasma-treated PTFE surfaces in air, and
phosphate-buffered saline (PBS), have been investigated for up to 1 month.86,87
Lappan et al.88 reported the surface modification of PTFE using either O2 or NH3
plasma-treatment followed by adsorption of polyelectrolytes.
Radiation-induced grafting onto fluoropolymers is a well-established method89 and
wide varieties of monomers have been grafted onto PTFE (and ePTFE) this way.
Such monomers include: styrene,90-94 MMA,94-98 2-hydroxyethyl methacrylate
(HEMA),94,97 acrylic acid (AA),99-102 methacrylic acid,97,98 acrylamide (AAm),103
N,N-dimethylacrylamide (DMAA)104-106, PEO107,108, MAEP102 and MOEP109.
Another common grafting technique of PTFE is plasma activation. Kang and
coworkers have reported UV-induced graft copolymerisation of hydrophilic
monomers onto Ar-plasma-pretreated PTFE.110 The hydrophilic monomers used in
Kang’s study included: AAm, AA, the Na salt of styrenesulfonic acid (NaSS),
DMAA, and (N,N-dimethylamino)ethyl methacrylate (DMAEMA).110 Tu et al.111
used ozone treatment on H2-plasma-pre-treated PTFE to create hydroperoxides, and
peroxides, which were then used to graft AAm and NaSS. AA grafting onto plasma-
pre-treated PTFE has also been extensively studied.112,113
More recently, the surface-initiated living radical polymerisation of fluoropolymers
has been reported using atom transfer radical polymerisation (ATRP),114 and
reversible addition-fragmentation chain transfer (RAFT)115 polymerisation. The
15
Chapter 1: Introduction
PTFE surfaces were pre-treated with plasma, in combination with ozone114, to create
active sites. In the case of ATRP, the initiators were immobilised on the plasma-
treated PTFE surface. For the RAFT techniques, graft copolymerisation of HEMA
onto plasma-treated tetrafluoroethylene-hexafluoropropylene copolymer (FEP) was
conducted in the presence of a RAFT agent and N,N-dimethylaniline.115 The addition
of N,N-dimethylaniline was to accelerate the decomposition of the peroxides formed
during the surface pre-treatment. Yu et al.116 grafted comb-copolymer brushes onto
PTFE using both RAFT, and ATRP techniques. Glycidyl methacrylate was first
grafted onto an Ar-plasma-treated PTFE surface using a RAFT technique. ATRP
initiators were then attached onto the epoxide side-chains of the grafted polymer.
Hydrophilic monomers were finally grafted from the ATRP initiator sites to form
comb structures.
Localised grafting onto a PTFE surface has also been reported. The grafting of 2-
dimethylaminoethyl methacrylate (DMAEMA) through a redox catalysis process
onto n-doped PTFE surfaces which had been obtained by local scanning
electrochemical microscopy has been achieved.117 This concept developed from
work on electro-grafting of polymers onto conducting, and semi-conducting
electrodes. The electro-grafting process is made possible by the electro-reduction of
vinylic monomers.
Grafted PTFE surfaces have also been used to immobilise biomolecules. Covalent
immobilisation of fibronectin with the carboxylic acid groups of poly(methacrylic
acid)-grafted PTFE has shown improved healing for vascular grafts.118,119 Human
thrombomodulin (hTM) has been immobilised onto poly(acrylic acid)-grafted PTFE.
The modified surface exhibited the expected improved anticoagulation activity.120,121
Fluorosurfactants can be used for the surface modification of fluoropolymers. This
modification method involves noncovalent interactions between the surfactant and
the polymer. Marchant and coworkers investigated the surface modification of PTFE
by the physisorption of fluorosurfactant polymers.122 Poly(vinylamine) (PVAm),
which was obtained from the hydrolysis of poly(N-vinylformamide) (PNVF), was
reacted with dextran lactone, followed by reaction with perfluorocarbon succinimide
(Scheme 1.1).
16
Chapter 1: Introduction
NH
Scheme 1.1: Synthetic route to poly(vinylamine) with dextran lactone and N-(perfluoroundecanoyloxy)succinimide.
XPS was used to characterise the fluorosurfactant-adsorbed PTFE surfaces. It was
found that adsorbed polymer, which contained 45 mol% fluorocarbon chains (y =
366 in Scheme 1.3), was stable under shear stress (1-20 dyn/cm2), whereas some
delamination of the polymers containing 15 and 21 mol% fluorocarbon chains was
observed. Marchant also created fluorosurfactants containing an RGD peptide, or
endothelial cell (EC)-selective peptide, for ePTFE surface modification.123,124 These
surfactants were used as PTFE and ePTFE surface modifiers for vascular graft
applications. The resulting surfaces facilitated endothelial cell attachment, growth,
and function.
Although PTFE is thought to be “non-stick”, due to its low surface energy,
spontaneous adsorption of biopolymers125,126 as well as poly(L-lysine) (PLL)127 from
aqueous solution, due to hydrophobic interactions, have been reported. Coupe and
coworkers have investigated the adsorption of functional polymers onto
poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) from aqueous solution.128
Since the interfacial energy of FEP/water is high, the polymers are thought to adsorb
with the functional groups extending to water (see Figure 1.6).
Figure 1.6: Adsorption of functional polymers at the FEP/water interface.
CH
O
** n
NH2
** n
NH* x
NH2
*m
Dextran
NH* x
NHy
Dextran(CF2)9
O
CF3
NH2
*n
Dextran Lactone NaOH
DMSO DCC, NHSH2O
C10F21COOH
x = 179 y = 122-366 n = 269-512 Total = 814
X X
X X
X X X
X = COOH or NH2
H2O Polymer Adsorption
FEP
17
Chapter 1: Introduction
Poly(acrylic acid) (PAA), poly (allylamine hydrochloride) (PAH), polyethyleneimine
(PEI) and PLL all showed adhesion to FEP. The highest adsorption of these
polymers to FEP was observed at pH values where the polymers were not charged.
This is due to the low solubility of these polymers in this state. Salt addition
increased adsorption of these polymers to FEP due to screening of the repulsive
interactions between charged groups.
1.8 Project Outline
It is widely accepted that polymeric materials used in orthopaedic applications
generally require some kind of surface modification to improve their bioactivity.
Surface modification has been shown to enhance osseointegration and bone ingrowth.
In previous studies, it has been shown that it is possible to graft phosphate-containing
monomers onto expanded PTFE (ePTFE) using a radiation-induced grafting
technique.102,109 The modified surfaces showed enhanced mineralisation in vitro.38,39
However, it was difficult to control the structure of the grafted polymers since the
phosphate-containing monomers used in the early studies are known to form
insoluble gels by conventional polymerisation.129,130
The approach in this study represents a new direction in the surface modification of
biomaterials in particular PTFE. It involves synthesis of well-defined phosphate- and
carboxylate-containing polymers by RAFT-mediated polymerisation, followed by
the surface fabrication using these synthesised polymers. The fluorinated segment of
the block copolymers allows adsorption onto PTFE surfaces, while not requiring pre-
treatment of PTFE. This technique allows control of the molecular weights of
polymers, as well as control over the absorbed mass, and spatial distribution of
adsorbed polymers.
There are three equally important experimental sections within this thesis: synthesis
(Chapter 2), surface fabrication (Chapter 3) and in vitro mineralisation (Chapter 4).
These are followed by the overall conclusions and proposed future work (Chapter 5).
The outlines for chapters 2-4 are as follows:
18
Chapter 1: Introduction
Chapter 2: Synthesis
1. Synthesis of well-defined phosphate-containing polymers from
monoacryloxyethyl phosphate (MAEP) and methacryloyloxyethyl phosphate
(MOEP) using RAFT-mediated polymerisation
2. Chain extension of phosphate polymers with an 2-(methacryloyloxy)ethyl
acetoacetate (AAEMA) keto group
3. Synthesis of well-defined fluorine-containing polymers from 2,3,4,5,6-
pentafluorostyrene (FS), 2,2,3,3-tetrafluoropropyl acrylate (TFPA), and
2,2,3,3-tetrafluoropropyl methacrylate (TFPMA) using RAFT-mediated
polymerisation
4. Chain extension of fluorinated polymers with FS, tert-butyl acrylate tBA, and
AAEMA/2-(acryloyloxy)ethyl acetoacetate (AAEA)
5. Amino acid attachment onto PAAEMA block copolymers
Chapter 3: Surface Fabrication
1. Layer-by-Layer (LbL) assembly of phosphate- and carboxylate-containing
homopolymers
2. Coupling of PAAEMA block copolymers onto aminated slides
3. Adsorption of fluorinated homo- and copolymers onto PTFE films
1 and 2 are model systems and the substrates used were silicon wafer/glass slides and
aminated glass slides, respectively. These functional groups can be introduced onto
PTFE surfaces, or other biomaterials’ surfaces, by pre-treatments such as plasma, as
discussed in section 1.3. Adsorption of fluorinated polymers onto PTFE does not
require any pre-treatment.
Chapter 4: In vitro mineralisation
Mineralisation of phosphate-containing homopolymers, and fabricated surfaces was
studied using SBF for 1-2 weeks. Samples used for this study are as follows:
1. Phosphate-containing gel and soluble polymers
2. Phosphate- and carboxylate-containing LbL surfaces
3. Phosphate-containing block copolymers coupled to aminated slides
4. Carboxylic acid-containing fluorinated copolymers adsorbed onto PTFE films
19
Chapter 1: Introduction
1.9 References
(1) Ratner, B. D. In Biomaterials science : an introduction to materials in medicine; Ratner, B.D., Hoffman, A.S., Schoen, F.S., and Lemons, J.E., Ed.; Academic Press: San Diego, 1996.
(2) Jones, A. J., and Denning, N.T. Polymeric Biomaterials (Bio- and Eco-compatible polymers): A perspective for Australia; Dept. of Industry, Technology and Commerce: Canberra, 1988.
(3) Visser, S. A., Hergenrother, R.W., and Cooper, S.L. In Biomaterials science : an introduction to materials in medicine; Ratner, B.D., Hoffman, A.S., Schoen, F.S., and Lemons, J.E., Ed.; Academic Press: San Diego, 1996.
(4) Ontanon, M., Aparicio, C., Ginebra, M.P., and Planell, J.A. In Structural biological materials; Elices, M., Ed.; Pergamon: UK, 2000.
(5) Rho, J. Y., Kuhn-Soearing, L., and Zioupos, P. Medical Engineering & Physics 1998, 20, 92-102.
(6) Mann, S. Biomineralization : principles and concepts in bioinorganic materials chemistry; Oxford: N.Y., 2001.
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24
Chapter 2: Polymer Synthesis
Chapter 2: Polymer Synthesis
2.1 Introduction
The aim of this synthesis section of the work was to create well-defined polymers for
the purpose of surface modification of polymers to be used in biomedical
applications. Living radical polymerisation (LRP) was the technique of choice since
it provides good control over both polymer structure and composition, and due to the
fact that it requires moderately simple reaction conditions.
2.1.1 Living Radical Polymerisation (LRP)
LRP enables us to engineer well-defined polymers with tuneable structures,
compositions and properties.1 In an ideal LRP the molecular weight (Mn) increases
linearly with conversion and narrow molecular weight distributions can be obtained
(Mw/Mn<1.1). The three main techniques that have found widespread applications are:
nitroxide-mediated polymerisation (NMP), atom transfer radical polymerisation
(ATRP) and reversible addition fragmentation chain transfer (RAFT). Advantages of
these methods over anionic/cationic living polymerisation are that they can be carried
out under moderate reaction conditions and in the presence of small amounts of
impurities that are difficult to eliminate.
In NMP, stable nitroxide free radicals (e.g. 2,2,6,6-tetramethylpiperidinyloxy;
TEMPO) act as reversible terminating agents.2 NMP-mediated polymerisations is
carried out by two methods: one involves the thermal decomposition of an
alkoxyamine into a reactive radical and a stable radical, and the other uses a mixture
of a conventional radical initiator and the nitroxide radical. Scheme 2.1 represents
the mechanism of styrene polymerisation using TEMPO.
25
Chapter 2: Polymer Synthesis
O
Pn + Pm Pn+mTermination
Pn + Pm= H
(Combination)
(Disproportionation)
Scheme 2.1: Schematic representation of nitroxide-mediated polymerisation of styrene with TEMPO, monomer (e.g. styrene) and initiator (e.g BPO = benzyl
peroxide). Pn• and Pm• are propagating polymer radicals.
NMP process involves an equilibrium between dormant species (i.e. reversibly
terminated with the stable free radical) and active chains (i.e. propagating polymer
radicals). Hence, the amount of free nitroxide in the polymerization solution is a key
because an excess of free nitroxide shifts the equilibrium to the dormant species
which results in very little monomer addition.3
NMP requires elevated temperatures (>120oC) and long reaction times (1-3 days) to
activate highly stable TEMPO-capped dormant chains. It has been successfully
applied to limited numbers of monomers such as acrylate and styrenic systems. It has
been traditionally difficult to obtain low polydispersity indexes (PDI’s) for other
monomers.4 However, Hawker and coworkers developed alkoxyamine-based
initiators which can be applied to a wide range of monomers resulting in good
molecular weight control and lower PDI’s (1.05-1.15).3,5
ATRP reactions involve an organic halide that undergoes a reversible redox process
catalyzed by a transition metal compound such as cuprous halide.6 The mechanism
by which ATRP proceeds is described below:
NNO
NO
NO
NO
Monomer
BPO
TEMPO
26
Chapter 2: Polymer Synthesis
Pn – X + Cu(I)/L2 Pn• + Cu(II)X/L2
M
Pn+m / Pn= + Pm
HTermination:
Scheme 2.2: Schematic representation of the ATRP polymerisation process, Pn = polymer, X = halide, L = ligand, and M = monomer.
The copper (I) complex accepts a halogen atom (X; usually Cl, Br) and undergoes a
one-electron oxidation to form a Cu(II) complex. At the same time, an organic
radical is generated that either reacts with a monomer, or it can reabstract the halogen
atom from the copper (II) complex. This technique has been successfully applied to
obtain well-defined polymers with various structures (e.g. linear, block, star and
comb) from a variety of monomers.6,7 However, there are some disadvantages:8
• Specialised initiators are required, although they are currently commercially
available
• Stringent purification of the resulting polymers is required to remove the
metal complex (This is particularly important for biomedical applications
where there is concern over the release of even trace amounts of metals when
polymer materials or devices are implanted.)
• Reactions are sensitive to traces of oxygen since Cu(I) will oxidise to form
Cu(II) deactivator
• Generally acidic monomers need to be protected
In comparison to NMP and ATRP reactions which control chain growth by
reversible termination, the RAFT process involves reversible chain transfer reactions.
This is a more versatile technique since a much wider range of monomers can be
successfully polymerised, including many functional monomers. RAFT
27
Chapter 2: Polymer Synthesis
polymerisations have been carried out in both aqueous and organic solvents and
generally use a lower polymerisation temperature than either ATRP or NMP
systems.9
2.1.2 Reversible Addition Fragmentation Chain Transfer (RAFT) Method
The RAFT process was invented by Rizzardo and coworkers at the Commonwealth
Scientific and Industrial Research Organization (CSIRO) (Australia) in 1998.9 It is a
radical polymerisation which uses a thiocarbonylthio compound added which acts as
a highly efficient RAFT agent (Scheme 2.3). This transfer of the S=C(Z)S- moiety
between the active and dormant chains maintains the living characteristics of the
polymerisation.
S S
z
R
Scheme 2.3: General structure of a RAFT agent, Z = activation group (e.g. aryl and alkyl) and R = good leaving group (e.g. cumyl and cyanoisopropyl).
There are four groups of RAFT agents categorised according to the nature of the Z
group: (1) dithioesters (Z = aryl or alkyl), (2) trithiocarbonates (Z = substituted
sulfur), (3) dithiocarbonates (xanthates: Z = substituted oxygen) and (4)
dithiocarbamates (Z = substituted nitrogen).10 A wide range of RAFT agents have
been synthesized and utilized for different types of monomers.11
The choice of RAFT agent is important since different monomers may require
different RAFT agents depending on the reactivity of the propagating polymer
radical.12 For example, methacrylate monomers form radicals that are very good
leaving groups. To obtain an ideal living polymerisation of this monomer, the RAFT
agent has to have an equally good or superior leaving group, such as a cumyl or
cyanoisopropyl group. Several reviews have summarized RAFT agents and the
polymerisations in which they have been applied.11,13,14
28
Chapter 2: Polymer Synthesis
S
z
S R S
z
RS S
z
S
S
z
SS
z
S S
z
S
Pm
Initiator 2I
I Pm
+ Pm Pm + R
Monomer
R PnMonomer
Pn + Pm + Pm
Pm Pn Pn
Pn + Pm Pn+m
(I) Initiation
(II) Chain Transfer
(III) Reinitiation
(IV) Addition/Fragmentation
(V) Termination
Pn + Pm= H
(Combination)
(Disproportionation)
kad, 1
kad, 2
kad
kad
kβ, 1
kβ, 2
kβ
kβ
Pre-equilibrium
Main equilibrium
Scheme 2.4: Schematic representation of proposed RAFT polymerisation process.
The mechanism of the RAFT process is illustrated in Scheme 2.4. In the early stage
of the polymerisation, the propagating polymeric radical (Pm•, mainly oligomeric
nature) reacts with the initial RAFT agent, forming the intermediate radical, which
then fragments into a polymeric thiocarbonylthio compound and a new radical (R•)
(pre-equilibrium, II). The reaction of the radical with a monomer forms a new
propagating radical Pn• (reinitiation, III). Subsequent addition fragmentation steps
form an equilibrium between the propagation polymeric radicals (Pn• and Pm•) and
the dormant polymeric RAFT agents (main equilibrium, IV). This step results in an
equal probability for all chains to grow, which results in the formation of polymers
with low PDI’s.11 Polymerisation is unavoidably terminated by either combination or
disproportionation (V), which is dictated by the amount of initiator that is
decomposed. The most of the polymer chains contain thiocarbonylthio groups at one
end and re-initiating groups (R) at the other.
Kinetically, the individual reactions of pre-equilibrium (II) and main equilibrium (II)
are called “addition rate coefficients” (kad) and “fragmentation rate coefficients”
(kβ).15 It is important to note that, in pre-equilibrium, the reactions are asymmetrical
since different radical species are attacking and leaving. Hence as mentioned before,
29
Chapter 2: Polymer Synthesis
the choice of R group is crucial. Contrarily, these reactions are symmetrical in the
main equilibrium.
The rate of polymerisation (Rp) in RAFT polymerisations using dithiobenzoates has
been shown to be retarded, and decreases with increasing the initial RAFT agent
concentrations. There are two rate retardation effects: “induction period” and “rate
retardation”.15 An induction period is at the beginning of the polymerisation where
there is no polymerisation occurring. Rate retardation is when the polymerisation rate
is slower than that of conventional polymerisation without a RAFT agent. There are
two models which have been proposed to describe this behaviour: intermediate
radical termination or slow fragmentation.15-18 The former model explains the severe
retardation through the termination of the intermediate radicals with other radicals in
the system that results in the formation of three- and four-arm stars.17,18 The latter
model involves the slow fragmentation of the stable intermediate radicals.16 These
models are currently being widely debated in the literature. It has also been found
that impurities such as dithiobenzoic acid, which is the starting reagent for CDB,
induces inhibition in the polymerisations of 2-hydroxyethyl methacrylate (HEMA),
styrene and methyl acrylate.19 Despite this, the polymerisation can proceed in a
controlled manner.
The molecular weight increases linearly with conversion in the RAFT process, and
the equation (1) has been traditionally used to predict the molecular weight:
Mn = [M]0 x
[RAFT]0 Mw(M) + Mw(RAFT)×
where [M]0 is the initial monomer concentration, x is the fractional monomer
conversion to the polymer, [RAFT]0 is the initial RAFT agent concentration, and
Mw(M) and Mw(RAFT) are the molar masses of the monomer unit and RAFT agent,
respectively. More precisely, taking into account the initiator-derived chains, as well
as the chain transfer constant (Ctr) of RAFT agent, the equation becomes as
follows:18
(1)
Mn = [M]0 x
([RAFT]0 – [RAFT]x) + af([I]0 – [I]x) Mw(M) + Mw(RAFT) (2) ×
30
Chapter 2: Polymer Synthesis
where [RAFT]x is the RAFT agent concentration at x, a is the mode of termination (a
equals 1 for termination by combination and 2 for disproportionation), f is the
initiator efficiency, [I]0 is the initial initiator concentration and [I]x is the initiator
concentration at x. The concentration of the RAFT agent at x can be calculated from
the equation (3).
Many types of monomers have been successfully polymerized by the RAFT process.
These include functional (meth)acrylates, (meth)acrylamide, styrenic, and vinyl
monomers. However, there are certain types of monomers where RAFT-mediated
polymerisation can not be used. For example, olefins such as ethylene and propylene
are difficult to homopolymerise by the RAFT process.11 Other examples are
monomers containing primary or secondary amines that are known to undergo facile
reaction with the thiocarbonylthio compounds.
Depending on the intended application such as in a medical use, the residual RAFT
end-group can sometimes be problematic. Stenzel et al.20 investigated the
cytotoxicity of two RAFT agents: benzyl dithiobenzoate and 3-
benzylsulfanylthiocarbonylsulfanyl propanoic acid. The assay they used was based
on the Australian Standards Protocol for Biological Evaluation of Medical Devices:
test for in vitro cytoxicity AS/ISO10993.5-2002. The leached liquid from the RAFT
agent (supernatant of 0.125g of RAFT agent in 2.5mL cell culture medium) was
diluted 1:6 in the medium. The 0.8mL of this solution was placed onto the cultured
mouse fibroblast L929 cell line, and after 48 hours cell numbers were counted and
compared to a control culture without RAFT agent. Benzyl dithiobenzoate, a
commonly used RAFT agent inhibited cell growth by 72% when compared to the
controls, whereas only 9.7% inhibition for 3-benzylsulfanylthiocarbonylsulfanyl
propinoic acid was observed.20 Hence less toxic RAFT agents should be
preferentially used for polymers used in medical applications, or alternatively RAFT
end-group removal is required.
The thiocarbonylthio groups can be cleaved by several methods which includes
radical induced reduction (to provide a hydrocarbon end-group), thermal elimination
(to provide an unsaturated end-group) and reaction with a nucleophile e.g. amine,
[RAFT]x = [RAFT]0(1-x)Ctr (3)
31
Chapter 2: Polymer Synthesis
hydroxide, borohydride (to provide a thiol end-group).21 When forming thiol groups,
it is important to eliminate air completely from the reaction, since thiols undergo
oxidative coupling to give the disulfide and lead to a doubling of the molecular
weight of the polymers.22
In this study, RAFT-mediated polymerisation was chosen since acidic monomers
were being used. Both ATRP and NMP are not suitable for acidic monomers.
Initially the intention was to synthesise phosphate- and fluorine-containing block
copolymers using RAFT but this approach proved unfeasible because of the fact that
the solvents for these monomer-polymer combinations proved to be non-common.
Two RAFT agents, 1-phenylethyl phenyldithioacetate (PEPDTA) and cumyl
dithiobenzoate (CDB) were synthesised and used in this research (Scheme 2.5).
SS
SS
1-Phenylethyl phenyldithioacetate (PEPDTA) Cumyl dithiobenzoate (CDB)
Scheme 2.5: Structures of RAFT agents.
PEPDTA has shown good control of acrylate and styrene polymerisations, as well as
faster rates of polymerisation compared to that of CDB.23-25 CDB is one of the most
versatile RAFT agent, and used for methacrylate monomers. In the literature, 2-
cyanoprop-2-yl dithiomethacrylate (CPDB) has been shown to induce less rate
retardation of methyl methacrylate (MMA) polymerisation than CDB while still
providing good control.26-28 However, the synthesis of this RAFT agent requires
large amounts of AIBN currently not commercially available in Australia. Due to
the limited supply of AIBN and the necessity of avoiding the costly and time-
consuming AIBN synthesis experiments, this RAFT agent was not used.
32
Chapter 2: Polymer Synthesis
2.1.3 Living Radical Polymerisation of Phosphorous-Containing Monomers
Phosphorous-containing polymers have attracted significant attention over several
decades due to their wide range of applications including biomedical use.
Incorporation of these groups in polymers provides properties such as enhanced
mineralisation, imprinting of biological molecules and improved blood and other
biocompatibilities.29-34
Several studies have reported the successful LRP of phosphorous-containing
monomers. ATRP of phosphate-containing monomers in their non-acidic forms such
as dimethyl(1-ethoxycarbonyl)vinyl phosphate and deprotonated 2-
methacryloyloxyethyl phosphate (MOEP) have been successfully carried out.35,36
However, limited monomer conversion was observed suggesting that complexation
between the copper ions and the phosphoryl oxygen of the phosphonate groups was
occurring.35,36
2-Methacryloyloxyethyl phosphorylcholine was polymerised using ATRP in both
aqueous or alcoholic solutions37 as well as RAFT20,38 processes. Incorporation of
phosphorylcholine groups onto biomaterial surfaces has shown extremely high
biocompatibility and antithrombogenicity due to the reduction of protein
adsorption.33,34
Phosphonated methacrylates have been successfully copolymerised with vinyldene
chloride and methyl acrylate using the RAFT technique to obtain statistical, gradient
and diblock terpolymers.39
Two commercially available phosphate-containing monomers monoacryloxyethyl
phosphate (MAEP) and MOEP were polymerised using the RAFT technique in this
study (Scheme 2.6). These monomers were chosen since previous studies have
shown that their graft copolymers are capable of inducing calcium phosphate
nucleation.29,31,40
OO
PO
OHOH
O
OO
PO
OHOH
O Monoacryloxyethyl phosphate (MAEP) Methacryloyloxyethyl phosphate (MOEP)
Scheme 2.6: Structures of phosphate-containing monomers.
33
Chapter 2: Polymer Synthesis
2.1.4 Living Radical Polymerisation of Fluorinated Monomers
Fluorine-containing polymers are of industrial interest due to their excellent physical
and chemical properties.41,42 However, there are only a few fluoropolymers that
possess functional groups. Since LRP offers the possibility of introducing
functionalisation and unique architectures in polymers, there have been several
studies which have investigated its potential in the synthesis of fluoropolymers.43
2,3,4,5,6-Pentafluorostyrene (FS) has been polymerised mainly by the ATRP process,
possibly due to the fast polymerisation rate (e.g. 90% conversion in 90 min). PFS and
its block copolymers with styrene have been prepared by ATRP and extensively
characterized.44 Borkar et al.45 investigated the synthesis of highly fluorinated
styrene monomers by nucleophilic substitution of FS with 1H,1H-
pentafluoropropane-1-ol and 1H,1H-pentadecafluorooctane-1-ol. These monomers
were subsequently homo and copolymerised with fluorinated styrene and styrene by
copper-mediated ATRP.45 Triblock copolymers based on central poly(ethylene
glycol) (PEG) or poly(ethylene glycol-co-propylene glycol) (PEGPG) blocks with FS
outer blocks have also been prepared by ATRP and showed low PDI’s (1.2-1.3).46
These triblock copolymers were further functionalized by complexing lithium
bis(trifluoromethylsulfonyl)imide salt and liquid PEGPG precursor to form ion
conducting polymer electrolytes.
Fu et al.47 synthesised well-defined block copolymers of PFS and poly(tert-butyl
acrylate) (PtBA) via ATRP. Amphiphilic block copolymers of PFS and poly(acrylic
acid) (PAA) were prepared by the hydrolysis of the tBA segments of the
corresponding P(FS-b-tBA) copolymers. These block copolymers formed
membranes with well-defined pores in sizes in the micrometer range due to inverse
micelle formation in aqueous media.
Fluorinated methacrylate, 2-[(perfluorononenyl)oxy]ethyl methacrylate, has been
used to prepare a series of di- and triblock copolymers by ATRP.48,49 Perrier et al.
have successfully synthesised poly(methacrylate)s and polystyrenes incorporating
fluorinated moieties as either the initiator or monomer by copper-mediated ATRP
with pyridine imine ligands.50,51 Shemper and Mathias52 reported the synthesis of a
fluorinated macroinitiator from a fluorinated surfactant for ATRP polymerisations.
34
Chapter 2: Polymer Synthesis
Statistical and block copolymers with linear and star-like architectures were prepared
using this initiator.
Only a few studies of fluorinated polymer syntheses have been carried out using the
RAFT technique. Pai et al.53 modified poly(dimethyl siloxane) (PDMS) to form a
di(trithiocarbonate) functional molecule to use in the RAFT process. A-B-A triblock
copolymers were synthesized from this macro-RAFT and polymerized with N,N-
dimethyl acrylamide (DMA) and 2-(N-butyl perfluorooctanefluoro-sulfonamido)
ethyl acrylate (BFA). PDI’s were under 1.25. An A-B block copolymer of
poly(ethylene oxide)-b-poly(1,1,2,2-tetrahydroperfluorodecyl acrylate) was
synthesized via RAFT polymerisation, iodine transfer polymerisation (ITP), and
ATRP, in the presence of either degenerative transfer agents or a macroinitiator
based on poly(ethylene oxide).54 Both RAFT and ATRP gave better control of the
expected copolymer preparation than ITP.
Yu et al.55 have investigated the surface-initiated RAFT graft polymerisation of 4-
vinylbenzyl chloride (VBC) hydrogen-terminated silicon surfaces using cumyl
dithobenzoate (CDB) or cumyl phenyldithioacetate (CPDA) as the RAFT agent.
Tethered poly(4-vinylbenzyl chloride) (PVBC) brushes were further copolymerized
with FS to prove the livingness of the chain ends. The formation of block copolymer
brushes was confirmed by ellipsometry and XPS. Surface-grafted styrene brush-
based homopolymers and diblock copolymers bearing semifluorinated alkyl side
groups of different length, i.e., -(CH2)2(-CF2)n (n=6 or 8), have been synthesized by
NMP on silicon oxide surfaces.56
Three types of commercially available fluorine-containing monomers have been bulk
polymerised by the RAFT technique in this study (Scheme 2.7). In the literature,
only one study showing the chain extension of surface-initiated polymers with RAFT
end-groups using FS.55 As far as I can assertain, there are no reports on RAFT
polymerisation of 2,2,3,3-tetrafluoropropyl acrylate (TFPA) and 2,2,3,3-
tetrafluoropropyl methacrylate (TFPMA).
35
Chapter 2: Polymer Synthesis
O
O
HF F
F F
2,2,3,3-tetrafluoropropyl
acrylate (TFPA)
O
O
HF F
F F
2,2,3,3-tetrafluoropropyl methacrylate (TFPMA)
F
FF
F
F
2,3,4,5,6-pentafluorostyrene
(FS)
Scheme 2.7: Structures of fluorinated monomers.
36
Chapter 2: Polymer Synthesis
2.2 Experimental
2.2.1 Materials
MOEP was purchased from Aldrich (USA, no stated-purity) and the inhibitor
removed by toluene extraction.57 Inhibitor-free MAEP (Polysciences, USA, stated
purity of 97%) was used as received. 2-(methacryloyloxy)ethyl acetoacetate
(AAEMA, Aldrich, 95%) was distilled under vacuum to remove the inhibitor and
polymer. 2,3,4,5,6-pentafluorostyrene (PFS, Aldrich, USA, 99%), 2,2,3,3-
tetrafluoropropyl methacrylate (TFPMA, Aldrich, 99%), 2,2,3,3-tetrafluoropropyl
acrylate (TFPA, Matrix Scientific, USA, 97%), 2-(acryloyloxy)ethyl acetoacetate
(AAEA, Aldrich, 95%), tert-butyl acrylate (tBA, Aldrich, 98%) were all passed
through a column of basic alumina to remove the inhibitor. 2,2-azobis
(isobutyronitrile) (AIBN, Fluka, 98%) and 1,1’-Azobis(cyclohexanecarbonitrile)
(Vazo 88, DuPont, 98%) were recrystallised twice from methanol prior to use. AR
grade reagents ethyl acetate (99.5%), methanol (99.8%), n-hexane (95%), DMSO
(99.9%), and the ACS reagent acetone (99.5%) were used without further
purification. HPLC grade tetrahydrofuran (THF, 99.8%) was used in most cases,
except for the polymerisation when inhibitor-free anhydrous THF (Aldrich, 99.9%)
was used. Trifluoroacetic acid (TFA, 98%, Aldrich) and sodium cyanoborohydride
(95%, Aldrich), glycine (Ajax Finechem, AU) and L-phenylalanyl glycine (Aldrich)
were used as received. Benzoylated dialysis tubing (Mw cut off of 1200, Aldrich)
was used for the purification of PMOEP and PMAEP. Snake skin dialysis tubing
(Mw cut off of 3000, Pierce) was used for other polymers.
The RAFT agents, 1-phenylethyl phenyl dithioacetate (PEPDTA) and cumyl
dithiobenzoate (CDB) were synthesized according to the literature.58,59 PEPDTA was
purified by passing through a neutral aluminium oxide column using petroleum spirit
(40-60 ºC) as an eluent. The solid product was further purified by recrystallisation
from methanol. CDB was purified by passing through silica gel and neutral
aluminium oxide columns using n-hexane as an eluent.
37
Chapter 2: Polymer Synthesis
2.2.2 Methods
2.2.2.1 MOEP/MAEP Homopolymer Synthesis
Solutions containing monomer, RAFT agent and AIBN in methanol were prepared in
the concentrations given in Table 2.1. A typical homopolymerisation included the
following; 2 g of MAEP (1.01 M), 27.2 mg of PEPDTA (1 × 10-2 M), and 3.3 mg of
AIBN (2 × 10-3 M) were dissolved in 6.7 g of methanol. Aliquots of 1.5 mL were
transferred to five individual ampoules which were degassed by four freeze-
evacuate-thaw cycles and sealed. These samples were placed in an oil bath at 60 ºC
and removed after the required time such that five different time points were reached
for each experiment. Conversion was measured by Raman spectroscopy. The
reaction was stopped by cooling the solution in liquid N2. Soluble polymer samples
were purified by dialysing against methanol for 3 days. Gels were extensively
washed with methanol followed by acetone. The samples were dried in a vacuum
oven at 40 ºC for 3 days.
PAAEMA homopolymer was prepared with CDB as the RAFT agent (EXPT 10 in
Table 2.2, p.63). Solutions containing monomer (2.92 M), CDB (0.125 M), and
AIBN (0.017 M) in ethyl acetate were prepared. Polymerisation was carried out as
described previously. Polymerisation was stopped after 16.7 h. The polymer was
precipitated twice in methanol and dried under vacuum at 25 ºC for 1 day.
2.2.2.2 MOEP/MAEP Block Copolymer Synthesis
The conditions of block copolymer synthesis (macro-RAFT agent, monomer, and
concentrations) are given in Table 2.2 (in p.63). The same procedure as that in the
polymerisation of the homopolymer was used except ethyl acetate was used as the
solvent. The polymer samples were purified by dialysing against methanol for 3
days.
2.2.2.3 Hydrolysis of PMOEP and PMAEP Polymers
The soluble PMOEP and PMAEP polymers were hydrolysed to poly(acrylic acid)
(PAA) and poly(methacrylic acid) (PMA) respectively, by stirring in excess 5M
38
Chapter 2: Polymer Synthesis
NaOH at 80 ºC for 24 hours (see Scheme 2.6). The resulting polymers were purified
by dialysing against water in benzoylated dialysis tubing. Gel samples with high
conversions were hydrolysed with 5 or 10M NaOH at 80 ºC for 7 days. Full
conversion to the acid analogues was confirmed by 1H NMR.
2.2.2.4 Fluorinated Homopolymer Synthesis
The concentrations of monomers, RAFT agents and AIBN are given in Table 2.3 (p.
64). A typical polymerisation procedure is given below.
Typical RAFT Polymerisation of FS
21.8 mg of PEPDTA (8.00×10-5 mol, 2.81×10-2 M), and 1.95 mg of Vazo 88
(8.00×10-6 mol, 2.81×10-3 M) were dissolved in 4.00 g of FS (2.06×10-2 mol, 7.25
M). Aliquots of 0.5 mL were transferred to six individual ampoules which were
degassed by four freeze-evacuate-thaw cycles and sealed. These samples were placed
in an oil bath at 80 ºC and removed after the required time such that six different
time points (i.e. conversions) were obtained for each experiment. Conversion was
measured by Raman spectroscopy. The polymerisation was stopped by quenching
with liquid nitrogen, exposure to air and dilution with ethyl acetate. The polymer
(PFS) was purified by precipitating into methanol, vacuum filtered and dried
exhaustively under vacuum at room temperature.
Typical RAFT Polymerisation of TFPMA
19.6 mg of CDB (7.20×10-5 mol, 2.50×10-2 M), and 1.18 mg of AIBN (7.20×10-6 mol,
2.50×10-3 M) were dissolved in 3.60 g of TFPMA (1.80×10-2 mol, 6.25 M). Aliquots
of 0.5 mL were transferred to six individual ampoules which were degassed by four
freeze-evacuate-thaw cycles and sealed. These samples were placed in an oil bath at
60 ºC and removed after the required time such that six different time points were
reached for each experiment. Conversion was measured by Raman spectroscopy. The
polymerisation was stopped by quenching with liquid nitrogen, exposure to air and
dilution with ethyl acetate. The polymer (PTFPMA) was purified by precipitating
into n-hexane, vacuum filtered and dried exhaustively under vacuum at room
temperature.
39
Chapter 2: Polymer Synthesis
Typical RAFT Polymerisation of TFPA
21.8 mg of PEPDTA (8.00×10-5 mol, 2.63×10-2 M), and 1.31 mg of AIBN (8.00×10-6
mol, 2.63×10-3 M) were dissolved in 4.00 g of TFPA (2.15×10-2 mol, 7.08 M).
Aliquots of 0.5 mL were transferred to six individual ampoules that were degassed
by four freeze-evacuate-thaw cycles and then sealed. These samples were placed in
an oil bath at 60 ºC and removed after the required time such that six different
conversions were reached for each experiment. Conversion was measured by Raman
spectroscopy. The polymerisation was stopped by quenching with liquid nitrogen,
exposure to air and dilution with ethyl acetate. The polymer (PTFPA) was purified
by precipitating into n-hexane, and dried exhaustively under vacuum at room
temperature.
2.2.2.5 Fluorinated Block Copolymer Synthesis
The conditions of block copolymer synthesis (macro-RAFT agent, monomer, solvent
and concentrations) are summarised in Table 2.4. Examples of each macro-RAFT
agent are given below:
Typical Chain Extension of PFS: Synthesis of Poly(FS-b-tBA),
expts.1 and 2, Table 2.4:
0.18 g of PFS (Exp. 7, Table 2.3:, Mn = 29209, PDI = 1.06, 6.03×10-6 mol, 6.86×10-3
M) was dissolved into a solution of tBA (0.406g, 3.17×10-3 mol, 3.45M), AIBN (0.29
mg, 1.77 ×10-6 mol, 1.92×10-3 M) and THF (0.403 g, 5.59×10-3 mol, 6.09M). The
mixture was placed in a glass ampoule, deoxygenated by five freeze-thaw-pump
cycles and sealed. The sample was polymerized at 60 ºC for 1.7 hr (expt.1, Table 2.4)
and 2.8 hr (expt.2, Table 2.4) and conversion was monitored by Raman spectroscopy.
The polymerisation was stopped by quenching with liquid nitrogen, exposure to air
and dilution with THF. The polymer was purified by precipitating into n-hexane,
vacuum filtered and dried exhaustively under vacuum at room temperature. In the
case of P(FS-b-AAEA), polymer was purified by precipitating into methanol.
40
Chapter 2: Polymer Synthesis
Typical Chain Extension of PTFPA: Synthesis of Poly(TFPA-b-AAEA),
expt.5, Table 2.4:
0.16 g of PTFPA (expt.11, Table 2.3: Mn = 39959, PDI = 1.06, 3.91×10-6 mol,
9.57×10-3 M) was dissolved into a solution of AAEA (0.20 g, 9.95×10-4 mol, 2.44 M),
AIBN (0.07 mg, 4.26×10-7 mol, 1.04×10-3 M) and ethyl acetate (0.20 g, 2.27×10-3
mol, 5.56 M). The mixture was placed in a glass ampoule, deoxygenated by five
freeze-thaw-pump cycles and sealed. The sample was polymerized at 60 ºC and
conversion was monitored by Raman spectroscopy. The polymerisation was stopped
by quenching with liquid nitrogen, exposure to air and dilution with ethyl acetate.
The gel did not dissolve in either ethyl acetate or THF. The extract obtained from the
gel by ethyl acetate was precipitated in n-hexane, centrifuged and dried exhaustively
under vacuum at room temperature for GPC. In the case of P(TFPA-b-tBA), the
polymer was purified by precipitating into methanol/water (1:1), and dried
exhaustively under vacuum at room temperature.
Typical Chain Extension of PTFPMA: Synthesis of Poly(TFPMA-b-tBA),
expt.8, Table 2.4:
0.19 g of PTFPMA (expt.15, Table 2.3: Mn = 38025, PDI = 1.11, 5.01×10-6 mol,
8.46×10-3 M) was dissolved into a solution of tBA (0.26 g, 2.03×10-3 mol, 3.43 M),
AIBN (0.16 mg, 9.74×10-7 mol, 1.65×10-3 M) and THF (0.26 g, 3.63×10-3 mol, 6.13
M). The mixture was placed in a glass ampoule, deoxygenated by five freeze-thaw-
pump cycles and sealed. The sample was polymerized at 60 ºC and conversion was
monitored by Raman spectroscopy. The polymerisation was stopped by quenching
with liquid nitrogen, exposure to air and dilution with THF. The polymer was
purified by precipitating into n-hexane, vacuum filtered and dried exhaustively under
vacuum at room temperature.
2.2.2.6 Hydrolysis of tBA Segments
The hydrolysis of tBA side groups on the block copolymers to acrylic acid was
carried out according to the literature procedure.60
41
Chapter 2: Polymer Synthesis
Synthesis of Poly(TFPMA-b-AA):
A 5-fold molar excess of TFA (18.1 mg, 1.58×10-4 mol, 3.00×10-1 M) with respect to
the tBA groups of the block copolymer was added dropwise to a solution of 20 mg
P(TFPMA-b-tBA) (expt.8, Table 2.4) dissolved in 0.5 mL of DCM. The reaction was
carried out at room temperature with stirring for 16 hours. DCM was evaporated by
blowing N2 on the surface of the solution, and the polymer was dried exhaustively at
room temperature under high vacuum overnight.
2.2.2.7 Model Biomolecule Modification of Functional Fluorinated Block
Polymers
Glycine attachment
A 2.5-fold molar excess of glycine (14.0 mg, 1.86×10-4 mol) with respect to the
AAEMA groups was added to the solution of P(TFPMA-b-AAEMA) (expt.9, Table
2.4, 50mg, 7.3×10-5 mol AAEMA units) in acetone (5 mL). The mixture was stirred
at room temperature for 7 days in order to react the amines with the ketones to form
an imine which was then stabilized by mild reduction with addition of NaBH3CN (10
mg, 1.59×10-4 mol, 3.18×10-8 M) to form a stable secondary amine bond by stirring
for a further 2 days. After evaporating the acetone to half the volume, the polymer
was precipitated into n-hexane, vacuum filtered and washed extensively with MilliQ
water. It was dried exhaustively under vacuum at 40 ºC overnight. The modified
polymer was characterized by 1H NMR.
L-Phenylalanyl glycine attachment
A 2-fold molar excess of L-phenylalanyl glycine (19.5 mg, 8.76×10-5 mol) with
respect to the AAEMA groups and equal mole of triethylamine (TEA, 8.9 mg,
8.76×10-5 mol, 2.50×10-8 M) were added to the solution of P(TFPMA-b-AAEMA),
(expt.9, Table 2.4, 30.0 mg, 4.38×10-5 mol AAEMA units) in anhydrous
dimethylformamide (DMF) (3.5 mL). The mixture was stirred at 60 ºC for 1 day to
react the amines with the ketones to form an imine which was then stabilized by mild
reduction with addition of NaBH3CN (29.0 mg, 4.16×10-4 mol, 1.32×10-7 M) to form
a stable secondary amine bond by stirring further 2 days at 60 ºC. The DMF was
evaporated to half the volume and the polymer was precipitated into MilliQ water,
42
Chapter 2: Polymer Synthesis
and washed extensively with water. It was dried exhaustively under vacuum for 2
days at 40 ºC. The modified polymer was characterized by 1H NMR.
2.2.2.8 Stability of the PFS RAFT end-groups
A 1 mg/mL solution of fluorinated polystyrene prepared from the RAFT
polymerisation using PEPDTA (expt.7, Table 2.3) in THF or ethyl acetate was left at
room temperature without stirring. Changes in the UV-vis absorption at 310nm,
indicating hydrolysis of the dithioester RAFT end-groups were monitored over an 18
day period.
2.2.3 Analytical Techniques
2.2.3.1 FT-Raman spectroscopy
To obtain the degree of conversion, samples were prepared in ampoules and FT-
Raman spectra (PE Spectrum 2000 NIR FTIR, 64 scans, 8 cm-1 resolution, wave
number range 4000 – 360 cm-1) were recorded at various time points. Spectral
information was extracted by means of spectral analysis software (GRAMS/32,
Galactic Inductries Corp., Salem, NH). The area under the C=C double bond
stretching band at 1640 cm-1 (1620 cm-1 for PFS) was normalised to the non-
changing signal (for methanol at 1000 cm-1) and used for the conversion calculation.
Although Raman intensity is linear to the concentration of the species, normalisation
using an internal signal was performed to compensate any changes due to the
instrumentation (e.g. laser power and instrument arraignments). In addition, a control
reaction involving one sample for each reaction condition was polymerised in situ in
the FT-Raman spectrometer (8 cm-1 resolution, wavenumber range 4000 – 360 cm-1)
at 60 ºC to obtain a conversion/time curve. Figure 2.1 shows the set up of heating
block inside the FT-Raman spectrometer.
43
Chapter 2: Polymer Synthesis
Heating Block
Figure 2.1: Heating block setup for in situ Raman polymerisation.
Spectra were collected as follows: every 3 minutes, 16 scans for MOEP/MAEP
polymerisations, every 30 minutes, 64 scans for FS polymerisations, and every 10
minutes, 32 scans for TFPMA and TFPA polymerisations.
2.2.3.2 Gel permeation chromatography (GPC)
Average molar mass and molar mass distributions of the hydrolysed MOEP and
MAEP homo and block copolymers were measured by GPC using a Waters system
(Alliance GPCV 2000) equipped with three Ultrahydrogel columns (7.8 × 300 mm),
a UV detector, and a refractive index (RI) detector. The sample was prepared by
diluting the polymer solution with 5 mM NaOH to obtain a 10 mg/mL solution. The
analyses were carried out in a 5 mM NaOH solution at 60 ºC, with a flow rate of 0.5
mL/min. Calibration was relative to 10 PAA standards (Mn range 830 – 888,900 )
(Polymer Standards Service, Mainz, Germany).
GPC measurements for fluorinated polymers were performed using a Waters
Alliance 2690 Separations Module equipped with three 7.8 × 300mm Waters
Styragel GPC columns (2 linear Ultrastyragel and one Styragel HR3 columns), an
autosampler, column heater, differential refractive index detector and a Photo Diode
Array (PDA) connected in series. HPLC grade tetrahydrofuran was used as eluent at
a flow rate of 1 mL min-1. Polystyrene standards Mn ranging from 517 – 2000000
44
Chapter 2: Polymer Synthesis
were used for calibration. Molecular weights of all polymers are reported relative to
polystyrene standards.
2.2.3.3 Elemental Analyses
Elemental carbon, hydrogen and sulphur were determined using a Carlo Erba
Elemental analyzer model 1106. Elemental phosphorous was determined using an
ICPAES Spectro spectroflame P instrument using a forward power of 1200W, a flow
rate of 1.0 mL/min and a Meinhard concentric nebuliser. Soluble polymers,
monomers, and standards were prepared in methanol. The insoluble gels were acid
digested prior to characterization.
2.2.3.4 Nuclear Magnetic Resonance (NMR)
1H-NMR spectra were recorded using either a 300, 400, or 500 MHz spectrometer
(Avance Bruker). Software used was TOPSPIN 1.3. Chemical shifts are given in
ppm relative to the residual solvent peak.
31P-NMR spectra were recorded using a 400 MHz spectrometer (Avance Bruker).
Orthophosphoric acid, H3PO4, in D2O was used as an external reference. The
polymers were analysed in methanol-d4.
2.2.3.5 UV/VIS Spectroscopy
UV spectra were recorded on a Hitachi U-3000 UV-VIS spectrometer in the
wavelength range from 190 to 700 nm.
2.2.3.6 Fourier Transform Near Infrared (FT-NIR)
The in situ FT-NIR measurements of FS polymerisation were performed using a
Nicolet 5700 FTIR spectrometer with a heating block set to 80 °C. Each spectrum in
the NIR region ranging from 7000-5000 cm-1 was recorded every 30 min, 32 scans,
with a resolution of 8 cm-1. The decrease in area of the vinyl C-H stretching overtone
band of the monomer was calculated to give percent conversion.
45
Inte
nsity
(a.u
.)
Chapter 2: Polymer Synthesis
46
(b)
2.3 Results
2.3.1 RAFT-Mediated Polymerisation of Phosphate-Containing Monomers
Two molecular weights of 10 and 20k were the targeted for both MAEP and MOEP
polymerisations involving the changing RAFT concentrations only. Therefore the
concentrations of initiator, monomer and solvent were kept constant (e.g. for 10k,
[RAFT]:[Initiator] = 10:1, and for 20k, [RAFT]:[Initiator] = 5:1) (See Table 2.1 for
polymerisation conditions). All polymerisation reactions were monitored by Raman
spectroscopy.
2.3.1.1 Monoacryloxyethyl phosphate (MAEP) polymerisation
The homopolymerisation of MAEP was carried out in the presence of either
PEPDTA or CDB with AIBN as initiator in methanol at 60ºC. The experimental
conditions and characteristics of the polymer structure (i.e. either cross-linked or
soluble in methanol) are given in Table 2.1. Figure 2.2 shows the Raman spectra of a
typical MAEP polymerisation mixture both before and after polymerisation.
Conversion was calculated from the C=C band at 1638 cm-1 which was normalized
against the methanol solvent band at 1032 cm-1. The local linear baselines used for
these peaks were 1653–1607 cm-1 and 1079–961 cm-1, respectively.
A B
1638
1032 (a) (a)
Figure 2.2: Raman spectra of MAEP polymerisation solution ([PEPDTA] = 1 × 10-2 M (expt.2)) (a) initial and (b) after 7.25 h in the region of (A) 3800-360 cm-1 and (B) 1800-900 cm-1.
Raman Shift (cm-1)
(b)
Raman Shift (cm-1)
Chapter 2: Polymer Synthesis
47
Table 2.1: Experimental conditions of MAEP 1 and MOEP 2 polymerisation reactions 3 and characteristics of the polymers obtained.
1: [MAEP] = 1.01 mol/L
Monomer Experimental 5 Elemental Analyses ICP Expt.
RAFT [RAFT] (mol/L)
Time (h)
Conv (%)
Characteristics
Theoretical Mn
4
Mn PDI %C %H %S %P 1 MAEP ─ ─ 3 90 Gel 125000 6 2.98 6 35.6 5.5 0 9.5 2 MAEP PEPDTA 1 × 10-2 7 75 Soluble 7200 9000 1.18 3 MAEP PEPDTA 2 × 10-2 10 84 Soluble 4113 5500 1.23 4 MAEP PEPDTA 1 × 10-2 9 83 Soluble 7325 5500 1.46 39.3 5.9 0.2 7.0 5 MAEP CDB 1 × 10-2 40 35 Soluble 3532 1300 1.22 6 MOEP ─ ─ 3 96 Gel 215000 6 2.07 6 37.7 5.9 0 11.2 7 MOEP PEPDTA 1 × 10-2 0.7
2 7
23 44 81
Gel Gel Gel
2499 4545 8502
263000 100000
1.82 3.63
37.6
5.9
0
11.5 8 MOEP PEPDTA 2 × 10-2 7 95 Gel 9 MOEP CDB 1 × 10-2 20 74 Soluble 7780 10500 1.94 38.3 6.0 0.1 9.9
10 MOEP CDB 2 × 10-2 36 44 Soluble 2438 2800 1.84 MAEP 29.3 4.9 15.9
MOEP 34.5 5.5 14.7 Theoretical MAEP 7 30.6 4.6 15.8 Theoretical MAEP based on the MAEP-Diene-H3PO4 mixture 8 30.1 4.6 16.1 Theoretical PMAEP based on the MAEP-Diene mixture 9 34.7 4.8 13.7 Theoretical MOEP 7 34.3 5.3 14.8 Theoretical MOEP based on the MOEP-Diene-H3PO4 mixture 8 34.7 5.2 14.8 Theoretical PMOEP based on the MOEP-Diene mixture 9 38.9 5.6 12.5
2: [MOEP] = 0.96 mol/L 3: Solvent = methanol, [AIBN] = 2 × 10-3, Reaction temperature = 60 ºC 4: Theoretical Mn was calculated as conversion × a × [Monomer]/[RAFT] + b. a = 94 or 108 (molecular weight of sodium acrylate for MAEP or sodium methacrylate for MOEP), and b = 106 or 174 (molecular weight of polymer end-groups after hydrolysis for PEPDTA or CDB) 5: Polymers were hydrolysed for GPC analysis with 5M NaOH at 80 ºC for 24 hours. 6: After hydrolyses with 10M NaOH 7: Theoretical values calculated from the monomer structure. 8: Theoretical values calculated from the monomer-diene-H3PO4 mixture: ratios obtained from NMR. Monomer: diene: H3PO4 = 50.9: 22.8: 26.3(%, MAEP) and 50.8: 24.9: 24.2 (%, MOEP). 9: Theoretical values calculated from the monomer-diene mixture: ratios obtained from NMR. Monomer: diene = 69.0: 31.0 (%, PMAEP), and 65.8: 34.2 (%, PMOEP).
Chapter 2: Polymer Synthesis
Figure 2.3 shows conversion versus time plots of MAEP polymerisations. A control
MAEP polymerisation was carried out under the same experimental conditions as above
but in the absence of a RAFT agent. This experiment resulted in rapid polymerisation,
reaching 100% conversion in under 1 h (curve a, Figure 2.3), and resulted in the
formation of an insoluble cross-linked gel. Polymerisation of MAEP in the presence of
PEPDTA at two different concentrations gave polymers that were soluble in methanol
with no evidence of gel formation. The rate of polymerisation was retarded in the
PEPDTA-mediated polymerisation and there is a marked increase in the inhibition time
with increased PEPDTA (curves b and c, Figure 2.3) from approximately 50 min (at
[PEPDTA]= 1 × 10-2 M) to 120 min (at [PEPDTA]= 2 × 10-2 M). When MAEP was
polymerized with CDB, the rate of polymerisation was much slower than that of
PEPDTA but with no inhibition period (curve d, Figure 2.3).
Time (min)0 500 1000 1500 2000
Con
vers
ion
(%)
0
20
40
60
80
100(a)
(b)
(d)
(c)
Figure 2.3: Conversion versus time of a MAEP polymerisation in methanol using the RAFT agent PEPDTA or CDB, and AIBN as initiator: (a) no RAFT (expt.1), (b) [PEPDTA] = 1 × 10-2 M (expt.2), and (c) [PEPDTA] = 2 × 10-2 M (expt.3) and (d) [CDB] = 1 × 10-2 M (expt.5).
Although PMAEP was soluble in water, the eluent used in the GPC analysis, it was
hydrolyzed with 5 M NaOH to give poly(acrylic acid) (PAA), and its molecular weight
48
Chapter 2: Polymer Synthesis
distribution was determined with high accuracy using PAA calibration standards. The
proposed hydrolysis reaction is shown in Scheme 2.8.
O
OP O
OHOH
OS n
S
R
RR3 2
1
+ excess NaOH 80 oC
ONaO
NaS n
R
R
1
2
ONaS
R3 NaOONa + Na3PO4+ +
Scheme 2.8: Hydrolysis of polymeric side-chains and dithiocarbonate moiety of PMAEP/PMOEP using NaOH solution; R1 = H (PMAEP) or CH3 (MOEP), R2 = H (PEPDTA) or CH3 (CDB) and R3 = CH2 (PEPDTA) or none (CDB).
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 ppm 8.5 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 ppm
a
b
water CH2
CH
* *
OHO
n
b
a
Water
-(CH2)2-
methanol
b
a
CH2
CH
* *
OCH2
CH2
O
OPO OHOH
n
b
a
A B
Figure 2.4: 1H NMR (400 MHz) spectra of (A) linear PMAEP (expt. 2, Table 2.1) in methanol-d4 and (B) hydrolyzed PMAEP in D2O.
49
Chapter 2: Polymer Synthesis
This method was used in the analysis of all the PMAEP and PMOEP as well as their
block copolymer samples. The disappearance of the methylene resonances (δ = 3.6-4.6)
from the side-chain in the 1H NMR spectra (Figure 2.4) showed that the polymer was
quantitatively converted to the acid form.
Figure 2.5 shows GPC chromatograms of hydrolysed PMAEP with different conversions.
Evolution of Mn is observed with increasing conversion. The values of Mn and PDI
obtained from GPC are shown in Figure 2.6. The Mn increased linearly with conversion
for the two PEPDTA concentrations, and the PDI’s decreased from 1.3 to approximately
1.2 over the conversion range. Although the Mn profile for the PEPDTA at 1 x 10-2 M
was close to theory (solid line, Figure 2.6), at the higher concentration of PEPDTA the
experimental Mns were greater than theory by a factor of approximately two (dotted line,
Figure 2.6) and converged towards the theoretical line above conversions of 70% due to
the increased amount of dead polymer arising from initiator-derived radicals. The
experimental Mn of hydrolysed polymer obtained from the CDB-mediated
polymerisation was lower (1300, expt.5 in Table 2.1) than the theoretical value (3400)
and the PDI was close to 1.22.
75% 63% 38%
16% 37% 69% A B
20% 84%
Figure 2.5: GPC traces of hydrolysed PMAEP with different conversions from expt.2, Table 2.1 (A) and expt.3, Table 2.1 (B).
50
Chapter 2: Polymer Synthesis
Conversion (%)0 20 40 60 80
Mn
0
2000
4000
6000
8000
PDI
1.01.11.21.3
(a)
(b)
Figure 2.6: Mn and PDI of PMAEP polymerized with PEPDTA after hydrolysis (a) [PEPDTA] = 1 × 10-2 M (expt.2, Table 2.1 ) and (b) 2 × 10-2 M (expt.3, Table 2.1 ). The lines show the theoretical evolution of Mn with conversion for PAA. Theoretical Mn was calculated as: conversion × 94 × [MAEP]/[PEPDTA] + 106. The molecular weight of sodium acrylate is 94 and 106 is the molecular weight of polymer end-groups after hydrolysis.
2.3.1.2 Methacryloyloxyethyl phosphate (MOEP) polymerisation
The MOEP homopolymerisation was carried out in the presence of either PEPDTA or
CDB with AIBN as initiator in methanol at 60ºC. The experimental conditions and
characteristics of the polymer structure are given in Table 2.1. The polymerisations were
monitored by Raman spectroscopy and the conversion was calculated from the C=C
band at 1638 cm-1 which was normalized against the methanol solvent band at 1032 cm-1
(Figure 2.7). The local linear baselines used for these peaks were 1656–1620 cm-1 and
1069–979 cm-1, respectively. The conversion profiles are shown in Figure 2.8.
51
Chapter 2: Polymer Synthesis
A B
(a) (a)
1032
1638
Inte
nsity
(a.u
.)
(b) (b)
Raman Shift (cm-1) Raman Shift (cm-1)
Figure 2.7: Raman spectra of MOEP polymerisation solution ([CDB] = 1 × 10-2 M (expt.9, Table 2.1)) (a) initial and (b) after 19.5 h in the region of (A) 3800-360 cm-1 and (B) 1800-900 cm-1.
Time (min)0 500 1000 1500 2000
Con
vers
ion
(%)
0
20
40
60
80
100 (c) (a) (b)
(d)
(e)
Figure 2.8: Conversion versus time of a MOEP polymerisation in methanol using the RAFT agent PEPDTA or CDB, and AIBN as initiator: (a) no RAFT (expt.6, Table 2.1), (b) [PEPDTA] = 1 × 10-2 M (expt.7, Table 2.1), and (c) [PEPDTA] = 2 × 10-2 M (expt.8, Table 2.1), and (d) [CDB] = 1 × 10-2 M (expt.9, Table 2.1) and (e) [CDB] = 2 × 10-2 M (expt.10, Table 2.1).
52
Chapter 2: Polymer Synthesis
In the absence of a RAFT agent, the polymerisation was fast and reached high
conversion (over 85%) after 200 min (curve a, Figure 2.8). This resulted in the formation
of an insoluble gel. Polymerisation in the presence of PEPDTA showed only a slight
decrease in rate with increased RAFT agent concentration (curves b and c, Figure 2.8),
suggesting that re-initiation is not the rate determining step. The polymers formed for
the two PEPDTA concentrations were also cross-linked gels, even at low (23%)
conversion (Table 2.1). However, when CDB is used as the RAFT agent at the same
concentrations both severe retardation and inhibition of the polymerisation rates (curves
d and e, Figure 2.8) were observed. The resulting polymer was found to be soluble.
Figure 2.9 shows GPC chromatograms of hydrolysed PMOEP obtained from the CDB
mediated polymerisations (expts. 9 and 10 in Table 2.1) with different conversions. The
Mn and PDI values are plotted in Figure 2.10.
58% 43%
25%
30%
27% 20%
A B
71% 44%
Figure 2.9: GPC traces of hydrolysed PMOEP with different conversions from expt.9, Table 2.1 (A) and expt.10, Table 2.1 (B).
53
Chapter 2: Polymer Synthesis
Conversion (%)0 20 40 60 80
Mn
0
2000
4000
6000
8000
10000PD
I1.01.21.41.61.82.02.2
(a)
Figure 2.10: Mn and PDI of PMOEP polymerized with CDB after hydrolysis (a) [CDB] = 1 × 10-2 M (expt.9, Table 2.1 ) and (b) 2 × 10-2 M (expt.10, Table 2.1 ). The solid lines show the theoretical evolution of Mn with conversion calculated as: conversion × 108 × [MOEP]/[CDB] + 174. The molecular weight of sodium acrylate is 108 and 174 is the molecular weight of polymer end-groups after hydrolysis.
(b)
The Mn values increased linearly with conversion and were close to theory for both CDB
concentrations (Figure 2.10), suggesting that the RAFT agent had been consumed early
in the polymerisation. However, the PDI profiles deviate from ideal ‘living’ behaviour.
The PDI profiles (Figure 2.10) are similar for the two CDB concentrations, and increase
from 1.6 at low conversion to over 2 at high conversion.
2.3.1.3 NMR of monomers and polymers
When intended for medical application, it is important to characterise the polymer
thoroughly as in this case, factors such as the amount and distribution of phosphate
groups as well as their states are predicted to affect mineralisation in SBF (details in
Chapter 4).
54
Chapter 2: Polymer Synthesis
7 6 5 4 3 2 1 ppm
0.
68
9
0.
68
5
2.
00
41
.9
65
0.
96
5
1.
00
0
0.
94
5
CH2 C
H
C
O
O CH2
CH2
O P
O
OH
OHa,a’
b
c d a
a’
b c
d
I I
Methanol Water
I = Impurity
I
Figure 2.11: 1H NMR spectrum of MAEP in methanol-d4.
OHPO
OHOH
22.8%
26.3% 50.9%
A
OO
PO
OHOH
O
OO
PO
OHO
OO
O
B
Figure 2.12: 31P-NMR of MAEP monomer in methanol-d4 A) H-decoupled and B) H-coupled.
55
Chapter 2: Polymer Synthesis
The 1H-NMR of MAEP monomer as received is shown in Figure 2.11. Characteristic
signals for MAEP were observed with the correct integrations. However, some
impurities were also evident at around 2.5-2.8, 3.6-3.8 and 3.9-4.1 ppm.
The 31P-NMR spectrum of MAEP monomer is shown in Figure 2.12. The proton
decoupled 31P-NMR spectrum (Figure 2.12 A) exhibits three peaks at 1.09, 0.13 and -
0.87 ppm. When coupled with protons, the patterns of these peaks become singlet, triplet
and pentet, respectively. Thus, they were assigned to free-orthophosphate (i.e.
orthophosphoric acid), MAEP monomer and diacrylate (or diene), respectively. It is
important to note that the phosphorus external standard (i.e. orthophosphoric acid) was
run in D2O, whereas all samples were in methanol-d4, which caused small shifts. The
integration of these peaks gives the following molar ratio: free-phosphate, 26.3%;
MAEP, 50.9%; diene, 22.8%.
7 6 5 4 3 2 1 ppm
3.
04
8
0.
48
1
2.
07
12
.1
44
1.
12
3
1.
00
0
0.
44
8
a,a’
b
c d
a
a’
b
c
d
I
Water
Methanol
I = Impurity Benzene
CH2 C
CH3
C
O
O CH2
CH2
O P
O
OH
OH
Figure 2.13: 1H NMR spectrum of MOEP in methanol-d4.
56
Chapter 2: Polymer Synthesis
Figure 2.14: 31P NMR of MOEP monomer in methanol-d4 A) H-decoupled and B) H-coupled.
The 1H NMR of MOEP as received is shown in Figure 2.13. Besides the characteristic
signals for MOEP with the correct integrations, the presence of less impurities (at 3.7-
3.8, 4.0-4.1 ppm) compared to that of MAEP was observed. A benzene resonance at 7.3
ppm was also found to be present and is possibly due to residual benzene used for the
purification of the monomer.
The 31P NMR of MOEP also showed three peaks at 0.91, 0.07, -0.74 ppm, with the same
H-coupling pattern as MAEP (Figure 2.14). The molar ratios of these peaks are: free-
phosphate, 24.2%; MOEP, 50.8%; diene, 24.9%.
The 31P NMR spectra above unexpectedly revealed that large amounts of diene are
present in both the MAEP and MOEP monomers. However, this was discovered late in
the project and therefore, purification of the monomers was not performed. It is
generally accepted that diene is formed during the purification of monomer by
distillation due to the esterification process, hence further distillation will probably not
remove this completely. Other purification techniques such as solvent extraction need to
be established.
OO
PO
OHOH
O
O
50.8%O
OPO
OHO
OO
O24.9%
OHP
OHOH
24.2%
A
B
57
Chapter 2: Polymer Synthesis
8 7 6 5 4 3 2 1 ppm
19
7.
16
6
12
2.
84
1
11
1.
62
4
20
4.
65
6
10
.0
00
Water
Methanol
b a
OCH2
CH2O
P OOH
OH
O
CH2
Sn
HS
c
d
d
d
a
b
c
Figure 2.15: 1H NMR spectrum of PMAEP (expt.4, Table 2.1) in methanol-d4.
-3-2-13 2 1 0 ppm
6.
66
9
31
.4
84
61
.8
47
PMAEP
Polydiene I
I
I = Impurity
Figure 2.16: 31P NMR spectrum of PMAEP (expt.4, Table 2.1) in methanol-d4.
58
Chapter 2: Polymer Synthesis
Figure 2.15 shows the 1H NMR of PMAEP (expt.4, Table 2.1). The methylene
resonance of the side-chains shows a broad peak at 4.0 – 4.5 ppm. There is another peak
at 3.5 – 3.8 ppm which is unidentified. Since after hydrolysis this peak also disappeared,
it is thought to be another methylene with an ester linkage. The peaks around 7.1-7.4
ppm are from the phenyl protons of the RAFT end-groups. Using these peaks, the
number of repeating units on PMAEP was calculated to be 107, which is higher than that
obtained from GPC (n=76).
The 31P NMR spectrum of this polymer is shown in Figure 2.16. Broadening of peaks is
often observed for polymers. The peaks at 0.1 and -0.5– -1.15 ppm are assigned to the
phosphorus from MAEP and diene in PMAEP. The broad peak at -1.9 ppm is
unassigned. There are also unidentified sharp peaks at 0.5 and -0.2 ppm. The
orthophosphate peak at 1.09 ppm is no longer present, since these polymers were
dialysed after polymerisation and thus the orthophosphates have been removed.
Figure 2.17 shows the 1H NMR spectrum of PMOEP. There is a broad peak at 4.0 – 4.7
ppm assigned to the methylene groups in the side-chain. The unidentified peak at 3.5 –
3.9 ppm is much smaller compared to that of PMAEP. The number of repeating unit in
PMOEP calculated using the RAFT end-group peaks was 26, which is close to that
obtained from GPC (n=24). The 31P-NMR spectrum of PMOEP exhibited two broad
peaks of monomer and diene units of PMOEP at 0.0 and -0.7 – -1.43 ppm, respectively
(Figure 2.18). Again, the orthophosphate peak at 0.9 ppm was no longer present.
59
Chapter 2: Polymer Synthesis
OCH2
CH2O
P OOH
OH
O
CH2
Sn
CH3S
8 7 6 5 4 3 2 1 ppm
68
.8
49
34
.2
34
11
.0
15
73
.6
76
10
.0
00
Water Methanol
b a
c
d
d
b
a
c
d
Figure 2.17: 1H NMR of PMOEP (expt.10, Table 2.1) in methanol-d4.
-3-2-13 2 1 0 ppm
29
.7
57
70
.2
43
PMOEP
Polydiene
Figure 2.18: 31P-NMR of PMOEP (expt.10, Table 2.1) in methanol-d4.
60
Chapter 2: Polymer Synthesis
2.3.1.4 Elemental Analyses of Monomers and Polymers
Elemental analyses were used to establish the total amounts of C, H, and P in the
polymer. Table 2.1 shows the theoretical C, H, and P% and experimental values of
monomers and polymers. Two monomer theoretical values from the monomer structures
and the monomer-diene-H3PO4 mixture with the concentrations obtained from 31P NMR
are listed. Their values are very close. Expected theoretical values for polymers are
calculated from the monomer-diene mixture from 31P NMR. Both experimental values
of MAEP and MOEP monomers showed good agreements of C, H, and P% with the
theoretical value calculated from the monomer structures.
Free-orthophosphates are removed during the purification of the polymers by dialysis as
is evident from the lack of a resonance ~1.0 ppm in the 31P NMR spectra. Therefore, the
expected polymer elemental composition can be estimated from the monomer-diene
mixture, assuming that the reactivity of monomer and diene are similar. The soluble
(expt.2) and gel (expt.1) PMAEP showed phosphorous contents of 7.0 and 9.5%,
respectively, which are much smaller than the expected value for the MAEP-diene
mixture (13.7%). Their carbon% (35.6 and 39.3% for gel and soluble PMAEP,
respectively) was also higher than the expected value (34.7%).
The PMOEP gels (expts. 6 and 7) showed P% that is slightly lower (~11%) than the
expected value of the MOEP-diene mixture (12.5%). In contrast, the soluble PMOEP
(expt.9) displayed a much lower phosphorous content (9.9%). However, the carbon% for
this sample is 38.3%, which is close to the theoretical value of the MOEP-diene mixture
(38.9%). Diene impurities and hydrolysis of phosphates are discussed in Section 2.4.1.
2.3.1.5 Hydrolysis of gel polymers
In order to gain some insight into the mechanism of gel formation, the gel polymers
obtained from the non-living systems were hydrolysed with excess NaOH for 7 days at
80ºC. This quite harsh treatment of the polymer should result in the conversion of all
ester groups in the polymer side-chains to carboxylic acid groups. Therefore, if as a
result of this treatment the polymers become soluble in good solvents, we can
61
Chapter 2: Polymer Synthesis
confidently infer that the crosslinks are formed through the polymer side chains and not
the backbone carbons.
PMOEP gels from the PEPDTA-mediated polymerisation (expt.7) were hydrolysed with
5 M NaOH for 7 days. The two samples, 23 and 44% conversions were rendered soluble
and successfully characterised by GPC. In contrast the high conversion sample remained
a gel. The results showed very high molecular weights and high PDI’s. For the 23%
conversion, the value of Mn obtained was 263,000 (theoretical Mn =1,800) and the PDI
was 1.82. The experimental Mn of the PMA from the 44% PMOEP conversion was
100,000 (theoretical Mn = 3,500) and the PDI was 3.63. These results strongly indicate
that the polymerisation must have occurred in a non-controlled way.
Hydrolysis with 10 M NaOH solution was necessary to dissolve the PMAEP (expt.1)
and PMOEP (expt.6) synthesised without RAFT agent since no reaction was observed
even after 7 days using 5M NaOH. As expected for conventional polymerisation these
polymers showed very high Mn’s and high PDI’s (expt.1 and expt.6 in Table 2.1). These
results suggest that most if not all cross-linking of PMOEP and PMAEP gels is through
the side-chain and not through the polymer backbone. (See scheme 2.13 in the
discussion)
2.3.1.6 Synthesis of MAEP and MOEP block copolymers with AAEMA
The aim of this series of reactions was to synthesise block copolymers where one block
contained functional groups enabling immobilisation onto a surface for the
mineralization study. AAEMA has a ketone side group that can react under mild
conditions with amines to yield an imine, which can then be reduced to the more stable
secondary amines.61 In this study, a selection of block copolymers containing PAAEMA
were synthesised in order to immobilise the soluble PMOEP and PMAEP on to aminated
surfaces through the AAEMA units.
The homopolymerisation of AAEMA with CDB (expt.10, Table 2.2) gave a Mn of 5000
which is double that of the theoretical value (i.e. 2200). It had a low PDI (1.13). One
explanation could be that the efficiency of CDB for AAEMA polymerisations is only
62
Chapter 2: Polymer Synthesis
~50%. It is also possible that the use of PS standards is inappropriate in this system. It
was noted earlier that CDB is an effective RAFT agent for MOEP polymerisation since
the theoretical and the experimental Mn values agreed. In that case, hydrolysis before
GPC and the use of PAA standards produced an appropriate calibration set. Krasia et al.
found similar results when AAEMA was polymerised with either 2-cyano- or 2-phenyl-
prop-2-yl dithiobenzoate (CPDB or PPDB, respectively) as the RAFT agent.62 Their
results showed a Mn value ~70% higher than the theoretical value.
OO
OO
O
n SS
OO
PO
OHOH
O
R
OO
OO
O
O
OP O
OHOH
Om n
SSR
O
OP O
OHOH
O
Sn S
OO
OO
O
m
O
OP O
OHOH
On
SSO
OO
O O
(A)
(B)
Scheme 2.9: Chain extension of (A) PAAEMA with MAEP/MOEP and (B) PMOEP with AAEMA, R = H: MAEP, CH3: MOEP.
PAAEMA was further chain extended with either MAEP (expts.11 and 12, Table 2.2)
and MOEP (expt.14, Table 2.2) (See Scheme 2.9A). The block copolymers were
hydrolysed before GPC measurements. The Mn’s for these polymerisations were double
that of theory and the PDI’s close to 1.38 which are satisfactory for our purpose since
the majority of chains contain both MOEP/MAEP and AAEMA units. It was also
necessary to couple a P(AAEMA) block of much greater than 22 units. Therefore,
PMOEP made with CDB (expt.13, Table 2.2) was chain extended with AAEMA (See
63
Chapter 2: Polymer Synthesis
64
Scheme 2.9B) to give a Mn of 22500 (109 units of AAEMA) and a PDI of 1.38, again an
acceptable PDI for the purpose of this study.
Chapter 2: Polymer Synthesis
65
Table 2.2: Experimental conditions of chain extension reactions and molecular weight of the polymers obtained after hydrolysis.
GPC Units Expt. Macro RAFT Mn PDI Monomer [M]
(mol/L)
[RAFT]
(mol/L)
[AIBN]
(mol/L)
Polym
Time
(min)
Conv
(%)
Theoretical
Mn Mn PDI PAAEMA PMOEP/
PMAEP
10 CDB AAEMA 3.4 0.15 0.019 980 44 2200 5000 2 1.13 22
11 PAAEMA-X 1 5000 2 1.13 MAEP 0.53 0.005 0.001 270 40 6711 12000 1.38 22 99
12 PAAEMA-X 1 5000 2 1.13 MAEP 0.53 0.005 0.001 545 66 9302 17500 1.38 22 159
13 PMOEP-X 1 10500 1.94 AAEMA 0.22 0.005 0.0006 160 20 6664 22500 1.38 109 97
14 PAAEMA-X 1 5000 2 1.13 MOEP 0.49 0.005 0.001 180 39 6857 19500 1.41 22 155
1: X ≡ SC(Ph)=S 2: Mn obtained without hydrolysis using THF GPC
Chapter 2: Polymer Synthesis
2.3.2 RAFT-Mediated Polymerisations of Fluorine Containing Monomers
In this series of experiments, two targeted molecular weights of 25 and 50k were set for
all monomers. The RAFT to the initiator concentrations were set to 10:1 in all cases.
Table 2.3: Experimental results for the RAFT polymerisation of fluorinated macromers. expt. Monomer [RAFT] [I] Temp Time Conv Theory GPC
(mmol L-1) (mmol L-1) (oC) (h) (%) 1 Mn Mn PDI
1 FS 29 (PEPDTA) 2.9 (AIBN) 60 60.7 27 12000 13000 1.09
2 FS 28 (PEPDTA) ― 100 36 2
3 FS 28 (PEPDTA) 2.8 (V- 88) 80 25 92 46500 29000 1.05
4 FS 56 (PEPDTA) 5.6 (V- 88) 80 18.5 96 24000 16500 1.06
5 FS 28 (PEPDTA) 2.8 (V- 88) 80 22.5 53 27000 20000 1.06
6 FS 56 (PEPDTA) 5.6 (V- 88) 80 21.5 95 23000 14000 1.03
7 FS 28(PEPDTA) 2.8 (V-88) 80 14 87 44000 29000 1.06
8 FS 28(PEPDTA) 2.8 (V-88) 80 25 96 2 48500 31500 1.37
9 FS 28(PEPDTA) 2.8 (V-88) 80 24.5 72 78 3
36000 39000 4
28500 1.08
10 FS 28(CDB 5) 2.8 (V-88) 80 67 73 81 3
36500 40500 4
29000 1.08
11 TFPA 26 (PEPDTA) 2.6 (AIBN) 60 3 90 44500 40000 1.06
12 TFPA 53 (PEPDTA) 5.3 (AIBN) 60 2.6 88 22000 18500 1.07
13 TFPMA 25 (CDB 5) 2.5 (AIBN) 60 23 88 41000 39000 1.13
14 TFPMA 50 (CDB 5) 5.0 (AIBN) 60 18 91 21500 22500 1.14
15 TFPMA 25 (CDB 6) 2.5 (AIBN) 60 15 76 36500 38000 1.11
1: conversion obtained from FT-Raman, 2: conversion obtained from FT-NIR, 3: conversion obtained from 1H NMR, 4: Theoretical Mn calculated based on the conversion from 1H NMR, 5 and 6: CDB was purified by passing it through a neutral activity aluminium oxide column followed by a silica column in one of two protocols: (5) once and twice, respectively, or (6) twice each, using hexane as eluent.
2.3.2.1 Pentafluorostyrene (FS) polymerisation
The bulk hompolymerisation of pentafluorostyrene (FS) was carried out in the presence
of 1-phenylethyl phenyldithioactetate (PEPDTA) and 1,1’-
azobis(cyclohexanecarbonitrile) (Vazo 88) or AIBN at 80 or 60 oC, respectively. Raman
spectra of a PFS polymerisation solution are shown in Figure 2.19. The olefinic band at
1620 cm-1 was normalized against the 385 cm-1 band for conversion calculations. The
66
Chapter 2: Polymer Synthesis
local linear baselines used for these peaks were 1629–1603 cm-1 (see Figure 2.19C) and
416–360 cm-1, respectively. The overlapping peak at 1645 cm-1 is assigned to the C=C
ring band. This band comes at 1600 cm-1 for styrene. With fluorine substitution, the band
shifts to a higher wavenumber.
Raman Shift (cm-1)
(b)
B A (a)
Figure 2.19: Raman spectra of PFS polymerisation solution ([PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM) (a) initial and (b) after 25 h, in the region of (A) 3800-200 cm-1, (B) 1800-260 cm-1 and (C) 1700-1560 cm-1, showing the local linear baseline for the area analysis under the 1620 cm-1 peak.
(b)
Inte
nsity
(a.u
.)
Raman Shift (cm-1)
(a) 1620
Raman Shift (cm-1)
C
385
(a)
67
Chapter 2: Polymer Synthesis
0
20
40
60
80
100
Figure 2.20: Conversion based on the normalised 1620 cm-1 bands from Raman spectra versus time of a bulk PFS polymerisation using the RAFT agent PEPDTA, and Vazo-88 or AIBN as initiator: Curve a: [PEPDTA] = 29 mM and [AIBN] = 2.9 mM, 60 ºC (expt.1, Table 2.3), b: [PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM, 80 ºC (expt.3, Table 2.3), c: [PEPDTA] = 56 mM, and [Vazo-88] = 5.6 mM, 80 ºC(expt.4, Table 2.3).
Initially, AIBN was used as the initiator at a polymerisation temperature of 60 ºC
(expt.1, Table 2.3). However the rate of polymerisation was very slow (Figure 2.20,
curve a), and it took 60.7 h to obtain a conversion of only 26.8%. Since styrene and
substituted styrenes are known to thermally initiate, PFS and PEPDTA without an
initiator in a degassed ampule were heated at 100 ºC for 36 hours. However, only a 2%
conversion was achieved (expt.2, Table 2.3). Hence a higher temperature initiator (i.e.
1,1’-Azobis(cyclohexanecarbonitrile) (Vazo-88)) was ultimately selected for this
experiment.
The conversion profiles for two concentrations of PEPDTA (where [PEPDTA]/[Vazo 88]
= 10), targeting 25 and 50 K polymers at full conversion, are shown in Figure 2.20,
curves b and c, respectively. The rates of polymerisation for both reactions were
relatively linear until approximately 50% conversion, after which a gel effect is observed
resulting in a significantly increased rate. High conversions (> 90%) were obtained for
the 25 and 50 K polymerisations after 1500 and 1000 min, respectively.
Time (min)
(b) (c)
(a)
Con
vers
ion
(%)
0 1000 2000 3000
68
Chapter 2: Polymer Synthesis
Conversion (%) 0 20 40 60 80 100
Mn
0
10000
20000
30000
40000
PDI
1.001.021.041.061.081.10
(a)
(b)
Figure 2.21: Mn and PDI of PFS polymerized with PEPDTA (a) [PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM (extp.3, Table 2.3 ), b: [PEPDTA] = 56 mM, and [Vazo-88] = 5.6 mM (extp.4, Table 2.3 ). The lines show the theoretical evolution of Mn with conversion.
Figure 2.21 shows the Mn and PDI of PFS obtained from the GPC. All the GPC
chromatograms of PFS showed narrow and unimodal distributions. There was no
evidence of termination by recombination since no high molecular weight shoulders
were observed. As can be see in Figure 2.21, the Mn for both polymerisations increased
linearly with conversion, and was in good agreement with theory up to 50% conversion.
At higher conversions, the Mn values deviated significantly from the theory. The PDI
values started at below 1.1 and at full conversion were below 1.06, suggesting that the
polymer chains were of near uniform length.
Conversion measurements using FT-NIR and NMR
In order to confirm the accuracy of the conversion obtained from FT-Raman, FT-NIR
and 1H NMR were used. In the case of FT-NIR, in situ polymerisation was performed.
69
Chapter 2: Polymer Synthesis
The PFS polymerisation solution was placed in the heating block in the FT-NIR
instrument and conversion was monitored using the change in the absolute area under
the vinyl C-H stretching overtone at 6200 cm-1 (Figure 2.22).
6200 cm-1
(a)
(b)
6600 6200 5800 5400Wavenumber
Figure 2.22: FT-NIR spectra of FS polymerisation solution ([PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM) (a) initial and (b) after 25 h.
The conversion plot is shown in Figure 2.23(a). Compared to that of the FT-Raman
(Figure 2.23(b), 92% conversion in 25h), slightly higher rate of polymerisation was
observed (93% conversion in 20h). This can be attributed to the different heating block
setups. The FS polymerisation temperature of 80 ºC is higher than the boiling point of
PFS under high pressure (62-63 ºC at 50mmHg), and the experiments showed that
depending on whether a different heating block or oil bath was used, some deviation in
the rate of polymerisation was observed. This was confirmed by measuring the
conversion by the same technique (i.e. FT-Raman). Nevertheless, the polymer obtained
with 96% conversion from FT-NIR showed a lower Mn than the theoretical (expt.8,
Table 2.3). The high PDI (1.37) of this sample can be explained by the fact that extended
polymerisation after reaching high conversion (as shown in Figure 2.23a) and some
termination by combination has occurred.
(cm-1)
Abs
orba
nce
(a.u
.)
70
Chapter 2: Polymer Synthesis
0
20
40
60
80
100
0 200 400 600 800 1000 1200 1400Time (min)
Con
vers
ion
(%)
(a)
(b)
Figure 2.23: Conversion versus time of bulk FS polymerisation ([PEPDTA] = 28 mM and [Vazo-88] = 2.8 mM, 80 ºC): Curve a, from in situ FT-NIR polymerisation, and b, from in situ Raman polymerisation.
The 1H-NMR was also used to calculate the conversion rates. After the polymerisation
was performed by in situ Raman, the ampoules were opened to take samples for NMR
analysis for direct comparison of conversions from these two techniques. Figure 2.24
shows the 1H NRM of the PFS polymerisation mixture in CDCl3. The normalised area
for the olefinic –CH=CH2 (6.65 ppm) and –CH=CH2 (5.85 and 6.19 ppm) protons for
FS compared to the sum of the aliphatic –CH-CH2 protons (1.7-3.0 ppm) for PFS were
used. Table 2.3 shows the conversions of polymerisation samples obtained from Raman
and 1H NMR (expts.9 and 10). In order to establish whether or not the nature of the
RAFT agent would influence the reaction outcome, CDB was used in a control
experiment (expt.10).
71
Chapter 2: Polymer Synthesis
H HH
Figure 2.24: 1H NMR spectrum of PFS polymerisation mixture after 67h for calculation of the conversion (CDB) (this corresponds to 81% conversion).
The conversions from 1H NMR and FT-Raman were found to be in good agreement,
although the 1H NMR gave a slightly higher conversion than that from the Raman. It is
possible that during the sample preparation for 1H NMR, some monomer evaporated.
Also the polymerisation solution at this conversion is very viscous and since the sample
for NMR was taken from the top, this may have let to some discrepancy. There may also
be some variations in conversion in the polymerisation solution due to the gel effect.
PFS obtained from CDB-mediated polymerisation also showed a lower Mn compared to
the theoretical value. Both FT-NIR and 1H NMR measurements agree with the
conversion obtained from Raman.
F
FF
F
F
HH
F
FF
F
F
n
Hp b
c a
CHCl3
Water
Hc
HaHb
3 Hp
p p
72
Chapter 2: Polymer Synthesis
2.3.2.2 Tetrafluoropropyl acrylate (TFPA) polymerisation
The bulk homopolymerisation of the fluorinated acrylate monomer (TFPA) was carried
out in the presence of PEPDTA and 2,2-azobis(isobutyronitrile) (AIBN) at 60 oC. Figure
2.25 shows the Raman spectra of a bulk TFPA polymerisation. The C=C band at 1636
cm-1 was normalized against the 360 cm-1 band in order to measure the conversion. The
local linear baselines used for these peaks were 1672–1588 cm-1 and 382–332 cm-1,
respectively.
(a)
(b)
A
(a)
1636
B
(b)
360
Raman Shift (cm-1) Raman Shift (cm-1)
Inte
nsity
(a.u
.)
Figure 2.25: Raman spectra of TFPA polymerisation solution ([PEPDTA] = 26 mM and [AIBN] = 2.6 mM) (a) initial and (b) after 3 h, in the region of (A) 3800-200 cm-1 and (B) 1900-200 cm-1.
The conversion profiles at two concentrations of PEPDTA, targeting 25 and 50 K at full
conversion, are shown in Figure 2.26 (a) and (b), respectively. With increased PEPDTA
concentration the inhibition time increased from 20 to 40 min despite the increased
initiator concentration. After this initial inhibition period the rates were similar and rapid,
reaching high conversions (> 80%) in under 160 min.
73
Chapter 2: Polymer Synthesis
0
20
40
60
80
100
Figure 2.26: Conversion versus time of bulk TFPA polymerisation using the RAFT agent PEPDTA, and AIBN as initiator at 60 ºC: Curve a: [PEPDTA] = 28 mM and [AIBN] = 2.8 mM (expt.11, Table 2.3), and b: [PEPDTA] = 56 mM, and [AIBN] = 5.6 mM (expt.12, Table 2.3).
Conversion (%)0 20 40 60 80 100
Mn
0
10000
20000
30000
40000
PDI
1.001.041.081.121.16
Figure 2.27: Mn and PDI of TFPA polymerized with PEPDTA (a) [PEPDTA] = 28 mM and [AIBN] = 2.8 mM (expt.11, Table 2.3 ), b: [PEPDTA] = 56 mM, and [AIBN] = 5.6 mM (expt.12, Table 2.3 ). The lines show the theoretical evolution of Mn with conversion.
Time (min)
Con
vers
ion
(%)
(a)
(b)
0 50 100 150 200
(a)
(b)
74
Chapter 2: Polymer Synthesis
The Mn and PDI obtained from GPC are shown in Figure 2.27. The Mn profiles were in
excellent agreement with the theoretical values for both PEPDTA concentrations and the
PDI’s for all conversions were low (between 1.05 and 1.14).
2.3.2.3 Tetrafluoropropyl methacrylate (TFPMA) polymerisation
CDB was the RAFT agent of choice to mediate the fluorinated methacrylate monomer,
as PEPDTA has been shown to be a poor agent for most methacrylates including MOEP.
The bulk homopolymerisation of TFPMA was carried out in the presence of CDB and
AIBN at 60 oC. Raman spectra of TFPMA polymerisation solutions are shown in Figure
2.28 The C=C peak at 1630 cm-1 was normalized against the 602 cm-1 peak to measure
the conversion. The local linear baselines used for these peaks were 1675–1601 cm-1 and
629–568 cm-1, respectively.
A B
1630 (a) (a)
(b) (b)
602
Inte
nsity
(a.u
.)
Raman Shift (cm-1) Raman Shift (cm-1)
Figure 2.28: Raman spectra of TFPMA polymerisation solution ([CDB] = 25 mM and [AIBN] = 2.5 mM) (a) initial and (b) after 23 h, in the region of (A) 3800-200 cm-1 and (B) 1820-550 cm-1.
75
Chapter 2: Polymer Synthesis
Figure 2.29 shows the conversion profiles for the TFPMA polymerisation. High
conversions (~90%) were obtained after 1380 and 1080 min for the target 25 and 50 K
polymers, respectively.
Time (min)0 200 400 600 800 1000 1200 1400
Con
vers
ion
(%)
0
20
40
60
80
100
(b) (a)
Figure 2.29: Conversion versus time of bulk TFPMA polymerisation using the RAFT agent CDB, and AIBN as initiator at 60 ºC: Curve a: [CDB] = 25 mM and [AIBN] = 2.5 mM (expt.13, Table 2.3), and b: [CDB] = 50 mM and [AIBN] = 5.0 mM (expt.14, Table 2.3).
Conversion (%)0 20 40 60 80 100
Mn
0
10000
20000
30000
40000
PDI
1.001.101.201.301.40
(a)
Figure 2.30: Mn and PDI of TFPMA polymerized with CDB (a) [CDB] = 25 mM and [AIBN] = 2.5 mM (expt.13, Table 2.3 ), b: [CDB] = 50 mM, and [AIBN] = 5.0 mM (expt.14, Table 2.3 ). The lines show the theoretical evolution of Mn with conversion.
(b)
76
Chapter 2: Polymer Synthesis
Figure 2.30 shows the Mn and the PDI values obtained from GPC. The Mn profiles
showed excellent agreement between experiment and theory, although the PDI’s were
slightly higher than that observed for the other two monomers, decreasing from 1.3 at
low conversion to 1.13 at high conversion.
Figure 2.31 shows the conversion profiles for the TFPMA polymerisations in the
presence of CDB with different purities. In the reaction shown as curve (a), the CDB had
been purified by passing through an aluminium oxide column once and then twice
through a silica column. The rate was significantly increased (curve (b)) when the CDB
was more intensively purified by passing it first through an aluminium oxide column
twice and followed by a further twice through a silica column. This significant change in
the rate of polymerisation by increased purification of the RAFT agent highlights both
the sensitivity of this type of reaction and the importance of reagent purity. However, the
rate of TFPMA polymerisation did not affect the control over Mn or PDI.
Time (min)0 200 400 600 800 1000 1200 1400
Con
vers
ion
(%)
0
20
40
60
80
100
(b)
(a)
Figure 2.31: Effect of CDB purity on the rate of polymerisation of TFPMA (in bulk) in the presence of 25 mM CDB and 2.5 mM AIBN at 60 oC. Using hexane as eluent, CDB was passed through: a neutral activity aluminium oxide column followed by a silica column: Curve a: once and twice, respectively (expt.13, Table 2.3), and b: twice each (expt.15, Table 2.3).
77
Chapter 2: Polymer Synthesis
2.3.2.4 Block copolymerisation of macrofluorinated polymers with
pentafluorostyrene (FS), tert-butyl acrylate (tBA), acetoacetoxyethyl methacrylate
(AAEMA) and acetoacetoxyethyl acrylate (AAEA)
Scheme 2.10 summarises the chain extension reaction of fluorinated homopolymers
(PFS, PTFPA and PTFPMA) with with FS, tBA, AAEMA or acetoacetoxyethyl acrylate
(AAEA). Amphiphilic block copolymers can be prepared from the blocks consisting of
tert-butyl groups (from PtBA) through hydrolysis to form carboxylic acid groups
(Scheme 2.10D). AAEMA and AAEA have a reactive keto functionality which allows
the coupling of glycine or L-phenylalanyl glycine (Scheme 2.10H). Blocks consisting of
methacrylate and acrylate fluorinated polymers with FS were also synthesised. The
molecular weight data for all these polymerisations are given in Table 2.4.
78
Chapter 2: Polymer Synthesis
79
Scheme 2.10: Schematic representations of chain extensions of PFS, PTFPA and PTFPMA, and further reactions (× = reaction did not proceed).
O
O
O
OO
O O
F
FF
F
F
n S S
F
FF
F
Fn
O
Om S S
F
FF
F
Fn
OO
m
OO
O
S S
(A)
(B)AAEA
tBA
TFA
(C)
(D)
(E)
OO
n
F
F
FF
H
S S O
O
O
OO
O O
O
n
F
F
FF
H
OO
m S S
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F
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F
F
O
n
F
F
FF
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m S S
O
F
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F
F
O
O
n
F
F
FF
H
OHO
m S S
O
O
n
F
F
FF
H
OO
m
OO
O
S S
(F)
(G)
(H)AAEMA
OO
n
F
F
FF
H
S S O
O
O
OO
O O
O
n
F
F
FF
H
OO
m
OO
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S SO
NH2 OH
O
F
FF
F
F
O
n
F
F
FF
H
m S S
O
F
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F
F
OO
n
F
F
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m S S
O
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n
F
F
FF
H
OO
m
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S S
NH
OH
O
1.
NaBH3CN2.
Chapter 2: Polymer Synthesis
80
Table 2.4: Experimental Results from the Chain Extension of Fluorinated Macromers via RAFT.
Expt Macro-RAFT Solvent [Monomer] [MacroRAFT] [AIBN] Polym time Conv. Theory GPC
Polymer Mn PDI (mol L-1) (mol L-1) (mol L-1) (mol L-1) (h) (%) Mn Mn PDI
1 PFS 29000 1.06 6.1 (THF) tBA (3.4) 0.00686 0.00192 1.7 39.92 54500 50000 1 1.23
2 PFS 29000 1.06 6.1 (THF) tBA (3.4) 0.00686 0.00192 2.8 71.86 74000 80000 1 1.31
3 PFS 20000 1.06 4.9 (EA) tBA (3.4) 0.00860 0.00092 1.8 46.24 43500 62500 1.15
4 PFS 14000 1.03 7.7 (EA) AAEA (1.1) 0.00456 0.00049 5.0 23.10 25000 19500 1.09
5 PTFPA 40000 1.06 5.6 (EA) AAEA (2.4) 0.00957 0.00104 3.0 50.47 65500 37500 1.10
6 PTFPA 40000 1.06 4.9 (EA) tBA (3.4) 0.00845 0.00093 1.9 59.09 70000 81500 1.11
7 PTFPA 40000 1.06 6.1 (EA) FS (2.8) 0.01038 0.00114 15.0 19.49 49500 56000 1.09
8 PTFPMA 38000 1.11 6.1 (THF) tBA (3.4) 0.00846 0.00165 20.1 56.71 64000 69000 1.18
9 PTFPMA 38000 1.11 6.5 (THF) AAEMA (2.5) 0.00903 0.00165 9.0 52.84 67000 55500 1.22
10 PTFPMA 38000 1.11 4.7 (EA) FS (2.3) 0.00840 0.00089 16.8 19.95 48000 56500 1.24
1: GPC chromatogram was bimodal
Chapter 2: Polymer Synthesis
Initially chain extension of PFS with tBA was carried in tetrahydrofuran (THF).
However, the GPC chromatogram of this polymer showed a bimodal distribution
(Figure 2.32A, solid line, using photodiode array (PDA) detection at 262 nm
(styrenic side groups)) (expts.1 and 2, Table 2.4). When PDA detection at 310 nm
was used to measure the RAFT end-group (Figure 2.32A, dashed line), it showed
that the polymer in the low Mn peak had lost this group. As a consequence, the
polymerisation solvent was then changed to ethyl acetate. GPC chromatograms of
P(PFS-b-tBA) obtained from polymers synthesised in ethyl acetate (expt.3, Table 2.4)
showed a unimodal distribution as well as the presence of RAFT end-groups over the
whole PFS distribution (Figure 2.32B). The chain extension of PFS with AAEA was
also successful using ethyl acetate (expt.4, Table 2.4).
Retension Time (min)20 22 24 26
UV
Res
pons
e
0.00
0.02
0.04
0.06
Retension Time (min)20 22 24 26
UV
Res
pons
e
0.00
0.02
0.04
0.06
0.08
(A) (B)
Retention Time (min) Retention Time (min)
Figure 2.32: GPC chromatograms of the chain extension of PFS with tBA carried out in (A) tetrahydrofuran (expt.2, Table 2.4), and (B) ethyl acetate (expt.3, Table 2.4), using PDA detection at 262 nm (full line, styrenic side groups) and at 310 nm (dashed line, RAFT end group).
The stability of the RAFT end-group of PFS in THF and ethyl acetate at room
temperature was investigated over an 18 day period. UV-vis spectroscopy was used
to measure the loss of dithioester absorbance at 310 nm and a plot of the absorbance
of this peak versus time is shown in Figure 2.33.
81
Chapter 2: Polymer Synthesis
0.08
0.12
0.16
0.20
0.24
0.28
0.32
0.36
0 2 4 6 8 10 12 14 16 18 20Day
Abs
(a)
(b)
Figure 2.33: Degradation of the RAFT end-groups of PFS (expt.7, Table 2.3) stored in tetrahydrofuran (curve a) and ethyl acetate (curve b) monitored by UV-vis absorbance spectroscopy at 310 nm.
These results clearly show that PFS stored in THF resulted in the significant loss of
RAFT end-groups, presumably due to oxidation of these end-groups with residual
amounts of peroxides in THF, whereas there is little or no loss when stored in ethyl
acetate.63 Electrospray ionization mass spectrometry (ESI-MS) has been used for
end-functional structural characterization.63 In future work, analysis of polymers
before and after storage in THF using this technique would make it possible to
confirm the UV-vis spectroscopy results as well as to identify the nature of the end
groups.
Chain extension of PTFPA with either tBA or FS was successfully carried out in
ethyl acetate solution. The Mn and PDI’s of the block copolymers are shown in Table
2.4 (expts.6 and 7). GPC traces of these polymers show a unimodal distribution
indicating the involvement of essentially all the macro-RAFT chains in the
polymerisation. In both monomers, Mn was well-controlled and the PDI was low. In
the case of chain extension with AAEA in ethyl acetate, some formation of gel was
observed already in the early stages of polymerisation (within 10 min). The gel
obtained was insoluble in both excess ethyl acetate and THF. The polymer that could
be extracted with THF was purified and analysed by GPC (expt.5, Table 2.4). The
Mn was close to that of the macro-RAFT, indicating no chain extension had occurred.
82
Chapter 2: Polymer Synthesis
The result was the same when the solvent was changed to dimethyl sulfoxide
(DMSO).
PTFPMA was successfully chain extended with tBA, AAEMA or PFS and the Mn
and PDI’s of the block copolymers are shown in Table 2.4 (expts.8-10). In all cases,
the PDI was below 1.25. For chain extension with tBA or AAEMA, THF was used
as a solvent (expts.8 and 9). This prevented the loss of RAFT end-groups, although
oxidation was observed for the PFS-PEPDTA macro-RAFT. This indicates that
methacrylate-dithiobenzoate is much more stable to peroxides compared to PFS-
phenylditioacetate.
Amphiphilic block copolymers consisting of carboxylic acid groups were prepared
by the hydrolysis of the t-butyl groups by stirring with TFA (5-fold molar excess) in
DCM solution at room temperature overnight.60 Figure 2.34A shows the 1H NMR
spectrum of P(TFPA-b-tBA) (expt.6, Table 2.4) before hydrolysis. The chemical
shifts between 1.5-2.6 ppm are attributed to the protons of the aliphatic –CHCH2– of
both PTFPA and PtBA polymer backbones (peaks 1, 2, 5, and 6). The triplets at 4.6
and 6.4 ppm (peaks 3 and 4) correspond to the protons of –CH2-CF2– and –CHF2 of
PTFPA block, respectively. The large peak at 1.4 ppm (peak 7) is attributed to the
protons of –C(CH3)3 of the PtBA block. After hydrolysis, this large peak at 1.4 ppm
disappeared which is consistent with the hydrolysed structure (Figure 2.34B). The
broad peak at 3.4 ppm is from the water present in the system.
H2O
Figure 2.34: 1H NMR spectra of P(TFPA-b-tBA) from expt.6, Table 2.4: (A) neat polymer (in acetone-d6) and, (B) after hydrolysis with TFA (in DMSO-d6) (* = solvent).
83
Chapter 2: Polymer Synthesis
2.3.3 Biomolecule Attachment
Glycine and L-phenylalanyl glycine were coupled to P(TFPMA-b-AAEMA) (expt.9,
Table 2.4) by reacting the primary amine groups with the keto group to form imines,
followed by reductive amination to form secondary amines (Scheme 2.11).
Scheme 2.11: Reaction scheme of PAAEMA block copolymer with glycine.
Figure 2.35: 1H NMR spectrum of; (A) P(TFPMA-b-AAEMA) from expt.9, Table 2.4, in DMSO-d6, (B) glycine in 1:1 = D2O:DMSO-d6 and (C) glycine modified P(TFPMA-block-AAEMA) in DMSO-d6 (* = solvent).
O
n
F
F
FF
H
OO
m
OO
O
S S
O
n
F
F
FF
H
OO
m
OO
S S
N OH
O
NH2 OH
O
O
n
F
F
FF
H
OO
m
OO
S S
NH
OH
O
NaBH3CN
Acetone
+ -
2
84
Chapter 2: Polymer Synthesis
Glycine was attached to P(TFPMA-b-AAEMA) by stirring the reagents in acetone
for 7 days at room temperature, followed by mild reduction using NaBH3CN and
further stirring for 2 days. Glycine is sparingly soluble in acetone and the reaction
mixture was slightly cloudy. The resulting polymer was extensively washed with
MilliQ water. Figure 2.35 shows the 1H NMR spectra of P(TFPMA-b-AAEMA) (A),
glycine (B) and polymer after coupling with glycine (C).
The glycine peak at 3.2 ppm (peak 12) is overlapped with the water peak in Figure
2.35C. There is a peak at 8.8 ppm (peak 11), possibly from the protonated secondary
amine. Since proton exchange between amines and residual water can occur,
quantification of attached amino acid using this peak integration will not be accurate.
The shape of the peak 9 has also changed after attachment, as well as peak 9’
disappeared.
Figure 2.36 shows FTIR-ATR of P(TFPMA-b-AAEMA) (a) before and (b) after
glycine attachment in the ranges of (A) 3600-547 cm-1 and (B) 1670-1340 cm-1.
(b)
(a)
(b)
(a)
1579
1394
(A) (B)
Figure 2.36: FTIR-ATR of (a) P(TFPMA-b-AAEMA) and (b) after glycine attachment in the ranges of (A) 3600-547 cm-1 and (B) 1670-1340 cm-1.
85
Chapter 2: Polymer Synthesis
Table 2.5: Expected IR bands from glycine attachments64.
Table 2.5 shows positions of expected IR bands from glycine attachments. Peaks at
1579 and 1394 cm-1 (Figure 2.36 B) indicate the presence of carboxylic acid salts
(antisymmetric and symmetric CO2 stretching bands at 1650-1540 cm-1 and 1450-
1360 cm-1, respectively). Carboxylic acid groups of glycine were possibly
deprotonated by the presence of trace amounts of cations in MilliQ water (confirmed
by ICP analysis). N-H stretching from secondary amine at 3320-3280 cm-1 is weak
and overlapped with OH stretching of bound water. There was also no evidence of
protonated amines which can be identified from the Fermi resonance at 2700-2300
cm-1.
The evidence of glycine attachment is not strong probably because the solubility of
the glycine in acetone limited the process, so it was then changed to L-phenylalanyl
glycine which is soluble in DMF or DMSO at elevated temperatures. This dipeptide
has the additional advantage that it has a phenyl group which is easy to detect by 1H
NMR.
86
Chapter 2: Polymer Synthesis
Figure 2.37: 1H NMR spectra of (A) P(TFPMA-b-AAEMA) from expt.9, Table 2.4, (B) L-phenylalanyl glycine and (C) L-phenylalanyl glycine modified P(TFPMA-b-AAEMA), all in DMSO-d6 (* = solvent).
The DMF solution containing L-phenylalanyl glycine, P(TFPMA-b-AAEMA) and
triethylamine was stirred at 60 ºC for 1 day followed by the addition of NaBH3CN
and then further stirred for 2 days. The polymer was extensively purified by washing
with MilliQ water before NMR. Figure 2.37 shows the 1H NMR spectra of
P(TFPMA-b-AAEMA) (A), L-phenylalanyl glycine (B) and polymer after coupling
with L-phenylalanyl glycine (C). From Figure 2.37(C), peaks from the dipeptide,
especially the phenyl group (peak 13), are clearly observed indicating successful
attachment. From the integrations of peaks 13 and 10 (from PAAEMA), 1 out of 8
AAEMA units was found to contain the dipeptide.
87
Chapter 2: Polymer Synthesis
2.4 Discussion
2.4.1 RAFT-Mediated Polymerisation of Phosphate-Containing Monomers
In this study, the homo and block copolymerisation of MAEP and MOEP by RAFT
techniques using two different RAFT agents; PEPDTA and CDB were investigated.
The polymerisations without a RAFT agent were fast (high conversions at 100 and
200 min for MAEP and MOEP, respectively), but gave insoluble cross-linked gels.
However, judicious choice of RAFT agent gave soluble PMAEP and PMOEP.
2.4.1.1 Polymerization with PEPDTA
MAEP polymerisation with PEPDTA as the RAFT agent gave soluble polymers with
low PDI’s. Although there was inhibition as well as retardation, the PEPDTA-
mediated polymerisation proceeded faster compared to that using CDB. It has been
proposed that the intermediate radical becomes less stable when the Z activation
group is a benzyl group rather than a phenyl group, resulting in faster fragmentation
and hence reducing retardation.58,65 The inhibition periods increased with the
increase in the concentration of the RAFT agent, from approximately 50 min (at
[PEPDTA]= 1 x 10-2 M) to 120 min (at [PEPDTA]= 2 x 10-2 M). This suggests that
the leaving radical R (Ph(CH3)CH.) is slow to re-initiate polymerisation.12,66,67 (see
Scheme 1 (III) Reinitiation step)
The rate of MOEP polymerisation with PEPDTA was slightly retarded but no
inhibition period was observed. However, the resulting polymer was an insoluble gel
even at low conversion (23%). For methacrylate monomers, the phenylethyl group
(R group) of PEPDTA is a poor leaving group with respect to the methacrylyl
propagating radical, therefore, polymerisation is not controlled.68
2.4.1.2 Polymerization with CDB
Although the PMAEP obtained from the CDB-mediated polymerisation was soluble,
the rate of polymerisation was extremely slow and conversion only reached 40%
88
Chapter 2: Polymer Synthesis
after 40 hours. Significant retardation in the polymerization of acrylates, as well as
other monomers such as styrenes and methacrylates in the presence of CDB is well
known, and has been discussed in the introduction (section 2.1.2).
The molecular weight of PMAEP-CDB obtained from GPC was 2.6 times smaller
than that of the theoretical value. Loiseau et al.69 found that the polymerization of
acrylic acid (AA) with trithiocarbonic acid dibenzyl ester as the RAFT agent resulted
in a lower Mn than the theoretical value when the [AA]/[RAFT] ratio was more than
100. This was ascribed to chain transfer to the solvent. This was observed even in
methanol which is known for its low chain transfer constant. It is possible that a
similar chain transfer reaction is occurring in the MAEP polymerisation with CDB in
methanol, although this was not tested in this study. Another possibility is that
although acrylates do not undergo self-initiation, the other initiation sources such as
impurities in the monomer and the long polymerisation time caused creation of more
chains than the RAFT agents and therefore shorter chains were obtained.
MOEP polymerisation was found to be well controlled with CDB, although the rate
of polymerisation was retarded to a lesser degree than that in the MAEP
polymerisation. However, the PDI was quite high (1.6–2.1) which increased with
conversion followed by a slight decrease at the high conversion. The reason for this
is possibly due to the high diene content and this will be further discussed in Section
2.4.1.4.
2.4.1.3 Loss of phosphate groups
From the elemental analysis of the polymers, it becomes apparent that phosphorous
groups were being lost in all systems, and that this phenomenon was most
pronounced for the soluble polymers produced by RAFT-mediated synthesis. This
loss could be attributed to hydrolysis during the polymerisation reaction by the
orthophosphoric acid present in the monomer at elevated temperatures as well as the
additional presence of acidic protons since the solvents used may contain small
amounts of water that would catalyse the reaction.
Polymerisation conducted in the presence of a RAFT agent generally took longer
(~20h) compared to without (~2h), hence hydrolysis could be expected to occur to a
89
Chapter 2: Polymer Synthesis
larger extent. In addition, it was noted that the PMAEP systems were more prone to
such hydrolysis than the PMOEP systems. This is also in agreement with the general
observation that methacrylate polymers are much more stable than acrylate polymers
towards spontaneous hydrolysis in aqueous media.38
There are two possible cleavage sites on the side chains: the C-O-P phosphate ester
bond (1, Scheme 2.12) and the C-O-C ester bond (2). Cleavage 1 forms a hydroxyl
group whereas cleavage 2 leads to a carboxylic acid group.
O
OP O
OHOH
O*
R
n
O
OH
O
R
m
OHO
*
R
l
O
OP O
OHOH
O**
R
n
1
2
Scheme 2.12: Possible hydrolysis sites on the side-chain of the polymer and structure of resulting polymer (R = H: MAEP, R = CH3: MOEP).
The most likely cleavage site is the C-O-P bond (1) which is known to be unstable.
From the FTIR investigation (Section 4.3.1 in Chapter 4), it was evident that both
soluble and gel PMAEP contained a high proportion of carboxylic acid groups and
thus must have undergone hydrolysis at the C-O-C ester bond (2). This was not
observed in the FTIR spectra of PMOEP polymers.
2.4.1.4 Mechanism of gel formation
It has been proposed that phosphate-containing monomers and other monomers (e.g.
HEMA70) crosslink in free-radical polymerisations due to the presence of either
small amounts of residual diacrylate or dimethacrylate contaminants or as a result of
chain transfer to monomer or polymer.71,72 From the 31P NMR spectra, it was evident
that both MAEP and MOEP contain unexpectedly large amounts of dienes (22.8 and
24.9%, respectively), although suppliers do not mention this. Although this is
90
Chapter 2: Polymer Synthesis
expected to cause extensive cross-linking, even with these high diene concentrations
with the right choice of RAFT agent, it was possible to obtain non-crosslinked
soluble polymers.
O
O
O
R
OP OOH
O
OP O
OHOH
O*
R
n
OO
R
*m
Scheme 2.13: Structure of polymer from monomer-diene mixture(R = H: MAEP, R = CH3: MOEP).
Scheme 2.13 shows the expected structure of polymer obtained from what we now
know to be more of a monomer-diene starting reagent mixture. In a system where all
the double bonds (i.e. monomer and both bonds in diene) have the same reactivity,
the critical extent of reaction at the gel point pc is given by:
[Diene ] Xw
[Monomer] + [Diene] = pc (4)
where Xw is the weight-average degree of polymerisation that would be observed in
the polymerisation of monomer in the absence of diene.73 In other words, cross-
linking in this case is inversely dependent upon the chain length of the polymer: the
greater the chain length the greater the conditional probability for crosslinking.74,75
Based on the Mn values obtained from the hydrolysis of the gels and RAFT-mediated
polymerisation (Table 2.1), it was clear that cross-linked polymer forms when high
91
Chapter 2: Polymer Synthesis
molecular weight polymer is obtained and soluble polymer when molecular weights
below 20 K are produced. Therefore, by targeting low molecular weights (in this case
20K) using the RAFT technique, non-cross-linked polymers were obtained.
Another possible cross-linking mechanism is hydrogen abstraction from anywhere on
the polymer chain: more likely from the methylene groups on the side chain. This has
been observed for PMOEP synthesised by radiation.76 However, chain transfer
reactions to polymer (or even monomer) is conversion dependent (or more precisely
dependent upon the weight fraction of polymer in the reaction mixture) and not
molecular weight dependent. Therefore we can confidently exclude this mechanism
from this system.
The fact that monomers contain large amounts of dienes means that polymers formed
from these monomers by the RAFT technique are most probably highly branched
instead of having a liner structure. This would explain the high PDI’s obtained from
the CDB-mediated MOEP polymerisation (1.6–2.1). In addition, PMOEP had less
hydrolysis compared to that of PMAEP. The PDI’s of PMAEP were low (1.2–1.3),
and PMAEP contained high amounts of carboxylic acids. This indicates that since
PMAEP (and MAEP) are prone to hydrolysis, early in the polymerisation, the
hydrolysis took place resulting in less dienes present in the system.
Sherrington et al.77 have shown that a soluble branched polymer can be formed when
a monomer is polymerised with a bifunctional monomer (i.e. diene) in the presence
of a chain transfer agent. This concept can be applied to living radical polymerisation.
ATRP78-80 and RAFT81 have both been used to create branched polymers. In general,
PDI’s of branched polymers are very high. Wang78 and Li79 have used dienes that
contain cleavable linkages. The GPC data of their hydrolysed polymers showed
living character as well as narrow PDI’s. In this study the concentration of diene was
found to be ~30% and the hydrolysed PMOEPs still showed high PDI’s, whereas in
some literature reports, they are as low as 1-2%.78,79
2.4.1.5 Block Copolymerisation with AAEMA
The block copolymers containing PMAEP or PMOEP and PAAEMA were prepared
in order to immobilise the linear PMOEP and PMAEP on a surface suitable for SBF
92
Chapter 2: Polymer Synthesis
studies. RAFT polymerization is known as one of the most versatile methods for the
synthesis of block copolymers since the homopolymers obtained by the RAFT
technique have RAFT end-groups which can be further chain extended with suitable
monomers. The rule of block copolymer synthesis in RAFT is that the propagating
radical for the first formed block must be a good homolytic leaving group with
respect to that of the second block.11
In the case of PMAEP blocks, since the acrylate propagating radical is not a good
leaving group with respect to the methacrylate propagating radical, PAAEMA was
synthesised first and then used as a macro-RAFT to chain extend using MAEP. The
living nature of the soluble PMOEP prepared with CDB was observed when it was
used as a macro-RAFT agent to chain extend with AAEMA to form the block
copolymer. Chain extension of PAAEMA with MOEP was also successful. In all
cases, the experimental Mns after hydrolysis of the side chains were much higher
than the corresponding theoretically calculated values. However, the PDI was
reasonable and in the range from 1.38-1.41.
2.4.2 RAFT-Mediated Polymerisation of Fluorine-Containing Monomers
2.4.2.1 FS Polymerisation
The bulk hompolymerisation of FS was carried out in the presence of PEPDTA and
Vazo 88 at 80 oC. High conversions (>90%) were obtained after 1500 and 1000 min
for polymerisations targeting 25 and 50 K at full conversions, respectively. In
comparison, the ATRP polymerisation of FS in the bulk at 110 ºC was very rapid.
When a [M]0:[Initiator]0 ratio of 55:1 was employed, around 90% conversion was
obtained in 90 min.44 In this study, the [M]0:[Initiator]0 ratios are 2579:1 and 1290:1
for targeting 25 and 50 K at full conversion, respectively.
The Mn for both polymerisation conditions showed good agreement with theoretical
values up to 50% conversion, whereas large deviations were observed at higher
conversion. Since the PDI values decreased from 1.1 to below 1.06 at full conversion,
as well as the fact that the polymerisation rate at the high conversion was not
93
Chapter 2: Polymer Synthesis
retarded, this indicates that the RAFT process was dominating. Three possible
explanations were considered for the observed lower Mn compared to the theory:
• The conversion obtained from FT-Raman was incorrect
• Mn obtained from GPC was inaccurate
• The creation of more chains during the polymerisation
The conversion of the FS polymerisation was also monitored by FT-NIR and 1H
NMR which are often the techniques of choice in the literature.25,44 Although, there
were slight variations, possibly due to the experimental errors, the results correlated
well with the initial FT-Raman analysis.
For highly fluorinated polymers, it was found that a deviation occurred between the
calculated Mn and the Mn obtained from GPC. This was explained by the smaller
hydrodynamic volume of the fluorinated polymers compared to the polystyrene
standards. This is also commonly observed for other types of monomers when
standard and sample are different. However in the case of PFS synthesised by the
ATRP technique, this was not observed.44 Good agreement was found between the
calculated Mn and the experimental data obtained from the GPC with the same GPC
conditions used here (i.e. PS standards, THF as an eluent, room temperature).
Therefore it can be concluded that GPC values in this study should be reasonably
accurate. However, in future work, these polymers should be characterised with more
advanced GPC, such as with multiple angle laser light scattering (MALLS) detectors,
to obtain the absolute molecular weights. Although 1H NMR has been used to
calculate the molecular weights in the literature, the chains derived from the other
initiation sources do not contain the RAFT end groups. Therefore the Mn vs
conversion based on 1H NMR is not an accurate measure of the RAFT efficiency.
According to the RAFT mechanism, the lower Mn than the theoretical value may be
an indication of the existence of other sources of radicals than the RAFT-derived
ones, such as initiator-derived chains. This should not be the case, since the initiator
concentration was kept small to minimise initiator-derived chains (i.e. [RAFT]:[I] =
10:1).18
94
Chapter 2: Polymer Synthesis
Styrene and substituted styrenes are known to undergo thermal polymerisation by
two proposed mechanisms. Mayo proposed the formation of a Diels-Alder dimer
(AH) followed by transfer of a hydrogen atom from the dimer to a monomer
(mechanism shown in Scheme 2.14).73,82,83 Flory’s mechanism involves the
formation of 1,4-diradicals (•M2•, in Scheme 2.14).84 This diradical can transfer a
radical to a monomer and then start monoradial polymerisation, or start diradical
polymerisation. It has also been proposed that a diradical transform to a Diels-Alder
dimer or 1,2-diphenylcyclobutane (DCB) that is inactive in polymerisation.84 In the
case of FS, it is suggested that 1,4-diradicals form which then initiate the
polymerisation.85 This study also showed thermal initiation of FS, although it was
very slow (2% conversion after 36 hours).
Scheme 2.14: Mechanism of Diels-Alder dimer and 1,4-diradical formations of styrene. (Reproduced from Ref: 86) M = styrene, AH = Diels-Alder dimer, •M2• =
1,4-diradical and DCB = 1,2-diphenylcyclobutane.
ATRP polymerisations of FS did not show a deviation of GPC determined Mn
compared to theory, possibly due to the fast polymerisation time (~90 min). For
RAFT-mediated FS polymerisations with PEPDTA, thermal initiation effects are
possibly more pronounced due to the long polymerisation times (1000 and 1500 min).
95
Chapter 2: Polymer Synthesis
The use of another RAFT agent, CDB, which showed even longer polymerisation
time also showed a lower Mn than the theoretical value.
2.4.2.2 Tetrafluoropropyl Acrylate (TFPA) and Tetrafluoropropyl Methacrylate
(TFPMA) Polymerisations
The PTFPAs obtained from PEPDA-mediated polymerisation showed the excellent
agreement of Mn and the theory as well as the low PDI (1.05-1.14), indicating that
this is a well controlled RAFT polymerisation. TFPMA polymerisation was carried
out in the presence of CDB, and the Mn obtained from the GPC correlated well with
the theoretical values. The PDI was in the range 1.13-1.3, which is slightly higher
than those of the other two monomers. It was also observed that the purity of CDB
was an important consideration in controlling the rates of polymerisation for TFPMA,
in agreement with previous findings.19
2.4.2.3 Chain Extension of Fluorinated Macro-RAFT
The fluorinated homopolymers were chain extended with FS, tBA, AAEMA or
AAEA. The residual peroxides in THF were found to cleave the RAFT end-groups
of PFS-PEPDTA by oxidation. Whereas, PTFPMA-CDB was found to be stable in
THF resulting in successful chain extension in this solvent. Chain extension of
PTFPA-PEPDTA with AAEA did not proceed successfully. This suggests that
although TFPA and AAEA are both acrylates, the PTFPA macro-RAFT is less stable
and sterically hindered the propagating radical compared to the AAEA radical. One
way to overcome this may be by polymerizing AAEA first and then chain extending
with TFPA. Amphiphilic block copolymers were successfully prepared from the
blocks consisting of t-butyl groups (from t-butyl acrylate) through hydrolysis to form
acids.
2.4.3 Biomolecule Attachment
Attachment of biomolecules to functional polymers has been widely studied due to
many applications in medicine such as drug delivery, gene therapy and improving the
96
Chapter 2: Polymer Synthesis
biocompatibility. For example, functionalisation of polymer surfaces with peptides
containing the cell recognition motif RGD (R: arginine, G: glycine, D: aspartic acid)
is one approach to instigating cell-material interaction.87-90 The functional groups of
polymers most often used for coupling are: carboxylic acid, amine, and hydroxyl
groups. These groups are first preactivated (e.g. carboxyl group with N-
hydroxysuccinimide (NHS) to form the active ester), followed by the coupling with
the amines of biomolecules.
Polymers containing reactive AAEMA or AAEA allow attachment of biomolecles,
by simple coupling of the keto group with the amines. Attachment of glycine and L-
phenylalanyl glycine, onto P(TFPMA-b-AAEMA) was investigated in this study.
Glycine attachment onto P(TFPMA-b-AAEMA) was not clearly observed from 1H
NMR. This is possibly due to the low solubility of glycine in acetone, which was the
reaction solvent. L-phenylalanyl glycine is soluble in DMF at elevated temperatures,
and the coupling reaction onto P(TFPMA-b-AAEMA) was successful in this solvent.
In literature, amine compounds have been successfully coupled onto PAAEMA in
the common solvent.91 (This will be further discussed in Chapter 3, Section 3.2.1) To
overcome solubility problem, protected amino acids can be used.87 However, this
strategy also has a drawback of the need of harsh conditions to remove the protecting
groups after attachment.
97
Chapter 2: Polymer Synthesis
2.5 Conclusion
To the best of my knowledge, this is the first successful report of soluble PMAEP
and PMOEP synthesised by RAFT-mediated polymerization. It is also the first time
that such large amounts of diene impurities were identified from the 31P NMR of
both MAEP and MOEP. Such dienes are known to cause cross-linking, and this
study was also able to show that the cross-linking is molecular weight dependent.
Therefore, by limiting the molecular weight below 20K using the RAFT technique, it
was possible to prevent cross-linking. Block copolymers consisting of PMAEP or
PMOEP with PAAEMA were successfully synthesised for the purpose of
immobilising them onto aminated slides.
Well-defined fluorinated homopolymers were synthesised using the RAFT
technique. PFS homopolymers showed much lower Mns compared to the theoretical
values. This is proposed to be due to the thermal initiation of PFS and the longer
polymerisation times compared to the ATRP technique. The purity of the RAFT
agent, CDB, has also been shown to be a factor important in maintaining fast rates of
polymerization, in agreement with literature. It was also found that storing PFS in
THF resulted in the loss of the RAFT end-groups, presumably due to oxidation by
peroxides. Block copolymers consisting of fluorinated polymers as the first block
and either the protecting (t-butyl) or reactive groups (ketones) as the second were
successfully synthesized. It was found that the formation of amphiphilic block
copolymers from the hydrolysis of the t-butyl groups was facile, and the attachment
of amino acids (glycine or L-phenylalanyl glycine) to the ketones of PAAEMA
provided a simple methodology for the attachment of important model biomolecules.
The resulting well-defined fluorinated homo and copolymers are highly suitable for
investigation of surface modification of fluoropolymers by simple adsorption.
98
Chapter 2: Polymer Synthesis
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426.
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Chapter 3: Surface Fabrication
Chapter 3: Surface Fabrication
3.1 Introduction A wide range of surface modification techniques has been used to introduce new
surface properties onto biomaterials. However, in many cases it has been difficult to
modify only the outermost layer of the materials using conventional techniques
without affecting more than the desired depth.
The greatest challenge is to alter the surface of materials in a controlled way. The
attachment of well-defined homo and copolymers produced by living radical
polymerisation is one approach to modifying the outermost surface of a polymeric
material with the ability to restrict the penetration of the change. Three relatively
simple yet robust techniques that fall into this category of surface fabrication are
listed below:
1. Layer-by-Layer (LbL) assemblies of soluble phosphate- and carboxylate-
containing homopolymers
2. Coupling reactions of keto-containing block copolymers onto aminated
surfaces
3. Adsorption of fluorinated homo and block copolymers containing carboxylic
acid groups onto PTFE
3.1.1 LbL Assembly
The Layer-by-Layer (LbL) ultrathin film fabrication was pioneered by Decher and
coworkers in 1991.1-4 This technique employs the alternating immersion of a solid
material into oppositely charged polyelectrolyte solutions to produce a
polyelectrolyte multilayer as an insoluble polymeric complex film on the material
surface. The surface charge is reversed upon each exposure. LbL films can be
prepared with up to a hundred layers.
There are many advantages of this innovative technique including the following:
103
Chapter 3: Surface Fabrication
• A much greater thermal and mechanical stability compared to the Langmuir-
Blodgett film technique
• It can be applied to materials of any shape, including colloids5 and porous
scaffolds6
• A large choice of polyelectrolytes can be employed
• The incorporation of other molecules, such as inorganic particles,5,7,8 DNA,9-
11 and proteins12-16 is possible
When polymers are attached onto substrate surfaces, this is conventionally illustrated
as involving conformational orientation of molecular “loops”, “tails” and “trains” as
shown in Figure 3.1.
Loop
Tail
Train
Figure 3.1: Illustration of attached polymer layers.
In LbL assemblies, it is possible to control the amounts of trains and loops through
altering the physicochemical conditions of the dipping solutions, such as the ionic
strength, and controlling the multilayer structure. The nature of the polyelectrolytes
and the concentration of the polyelectrolyte solution as well as charge density are
also important factors for tuning surface functionality and thickness.17,18 It is also
known that the pH of the solution strongly affects the deposition of weak
polyelectrolytes such as poly(acrylic acid) (PAA). Small changes in pH can cause
dramatic differences in film properties such as film thickness19,20 and morphology,21
hence this aspect has been highly studied.
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Chapter 3: Surface Fabrication
Shiratori et al. 20 showed that the pH of the deposition solution has a large effect on
the thickness of poly(allylamine hydrochloride) (PAH)/PAA films. The PAH and
PAA bilayer thickness can be altered from 5 to 80 Å by controlling the pH. Kim and
Bruening19 investigated the deposition of hyperbranced poly(amidoamine)
dendrimers/PAA films in the pH range 2.0-8.0. The film thickness increased with
decreasing the deposition pH for PAA, or by increasing the deposition pH for the
dendrimers. Thicker films are formed because the lower charge density of the
polyelectrolytes requires more adsorption to compensate for the surface charge.
Although most of the polyelectrolytes used in LbL studies are not well defined,
Morgan et al.22 used well-defined strong and weak homo and block
copolyelectrolytes, synthesised using the RAFT technique, to investigate the pH
response on LbL film production. Their weak polyanion/strong polycation film
showed exponential film thickness growth when deposited in a pH 5.5 solution
without salt. This behaviour is indicative of strong hydrogen bonds between multiple
partially ionized weak polyanions via carboxylates. The addition of an 0.1 M NaCl
solution disrupted this hydrogen bonding and the layer thickness displayed linear
growth. Multilayer adsorption caused or enhanced by hydrogen bonding has also
been reported by other groups.23-25 The LbL films fabricated from polyanion
copolymers, either block or random structure, showed dramatic differences in film
dimensions and morphology even though these copolymers possessed equivalent
degrees of ionization, Mn’s, PDI’s, and compositions.22
Due to the ease of assembly and the high structural versatility, the LbL technique has
found a wide range of applications in many fields of science and technology.
Examples include sensors, optical materials, filtration membranes, corrosion
prevention, conducting polymers, electrochromics, and light emitting systems.
Another area where it has found interesting use is in biomedical applications. Caruso
et al.26,27 used the LbL technique to form hollow polymeric microspheres (or
capsules) by removing template particles (e.g. silica) chemically after LbL
assemblies. This technique can generate hollow polyelectrolyte spheres of a
controlled diameter, wall thickness and composition. The same technique has been
used to create hollow capsules consisting solely of DNA multilayers by hydrogen
bonding of the base pairs,28 as well as nanoporous PAH/PAA films.29 Caruso and
coworkers also were able to create PAA-only capsules using click chemistry of PAA
105
Chapter 3: Surface Fabrication
copolymers with alkyne or azide alternatively assembled onto a silica template.27
These capsules showed a pH response as well as further functionalisation. Some of
the applications for these capsules are in drug delivery, sensing and biocatalysis.
Michel et al.30 developed a technique to load a high quantity of an active substance
into LbL films in one step using large unilamellar liposome vesicles. In one study,
they loaded calcium ions, spermin and alkaline phosphatase into liposomes and
coated them with a layer of poly(D-lysine). This was then embedded into poly(L-
glutamic acid)/poly(allylamine hydrochloride) (PGA/PAH) LbL films.12 The films
were then put in contact with a solution of paranitrophenyl phosphate (PNP) which
diffused through the layer as well as liposomes and the hydrolysed phosphate esters
of alkaline phosphatase to free phosphates. After 18 hours of reaction, calcium
phosphate (CaP) mineralisation had occurred inside the closed space. This was
characterised using ATR-FTIR and AFM.
Although not within the scope of this particular project, the stability of LbL films can
be further increased by post modification to form covalent bonds. Cross-linking of
these films has been found to greatly improve film stability. For example, layers
constructed from a photoreactive diazo resin as the polycation with polyanions
containing sulphonate or carboxylic groups were post UV treated to change ionic
interactions into covalent bonds.31 Another example is that of PAH/PAA multilayer
films which were further treated by heating32 or EDC coupling29 to form amide
bonds. Lawrie et al.33 used gamma radiation to cross-link PEI layers on silica
particles.
In this study, PEI/PAA LbL films were prepared from polymer solutions with
different pHs. The thickness of the PAA film was found to be strongly dependent on
the deposition pH. Soluble PMAEP and PMOEP synthesised by RAFT
polymerisation were also used to create phosphate-containing surfaces by the LbL
technique using PEI. Although these polymers are strong polyelectrolytes due to the
phosphate groups, since PMAEP was also shown to contain large amounts of
carboxylic acid groups (discussed in Chapter 4, Section 4.3.1) this ultimately
affected the film morphology.
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Chapter 3: Surface Fabrication
3.1.2 Attachment of Block Copolymers onto Aminated Slides
Functional polymers have attracted much attention over the years.34 According to the
IUPAC Compendium of Chemical Terminology, a functional polymer is defined as:
• A polymer that bears specific chemical groups; or
• A polymer that has specified physical, chemical, biological, pharmacological,
or other uses which depend on specific chemical groups
Functional polymers are ubiquitous even in daily life. Some examples which are
widely studied are: polymeric catalysts, polymers that are photoresponsive,
conductive, magnetic and oxygen carrying-polymers, polymers used in drug delivery,
biomaterials and as biosensors.34
2-(acetoacetoxy)ethyl methacrylate (AAEMA) is a functional monomer containing
β-ketoester moieties. The potential of this monomer has been increasingly explored
over the last few decades mainly due to two types of reactions it undergoes. Some
example reactions are shown in Scheme 3.1. The most important of these involve the
methylene and ketone carbonyl groups of these monomers participating in numerous
crosslinking reactions such as enamine formation, Michael addition, reductive
amination, as well as conventional methods involving melamines and isocyanates.35
AAEMA has attracted a wide range of applications in thermoset coatings, adhesives
and paints.
Enamine formation
Reductive amination
Michael addition
Metal Chelation
*O
OO
O O
*
n
NHR1
R2
NH2 R1
R1
*O
OO
O
*
n O
R1
*O
O O
*
n
O
O
*O
OO
O
*
n NR1 R2
NR1
*O
OO
O
*
n *O
OO
O
*
n NHR1
*
O
O
O
*n O
OM
M
Imine
Scheme 3.1: Various reaction schemes of PAAEMA.
107
Chapter 3: Surface Fabrication
A secondary, but important reaction is metal chelation.36-39 Acetoacetoxy groups act
as strong bidentate ligands capable of coordinating a wide range of metal ions with
different geometries.
The aim of this study is to immobilise block copolymers consisting of PAAEMA and
PMAEP or PMOEP onto aminated slides by reaction of the keto groups of PAAEMA
with the primary amines. The reaction of β-keto groups and amines is well
established.35 Moszner et al.40 synthesised monomers containing enamines by
reacting AAEMA with various aliphatic mono- and diamines in THF. These
monomers were successfully polymerised with AIBN as an initiator at 60 °C. They
were also able to modify PAAEMA with n-butyl amines at room temperature. Park
et al.41 synthesised composite latexes based on polystyrene and poly(n-butyl
acrylate-co-AAEMA) followed by post-crosslinking using 1,6-hexanediamine at
room temperature. The resulting film showed a significant increase in tensile strength.
Yu and coworkers42 reported that AAEMA-based resins can selectively remove
primary amines in the presence of secondary amines.
AAEMA has been homo and copolymerised with various monomers using
conventional polymerisation techniques.43 A hyperbranched AAEMA polymer has
also been produced by the Michael addition of this monomer.44 More recently,
Schlaad and coworkers synthesised well-defined homo and copolymers based on
AAEMA via RAFT-mediated polymerisation.38,45 The resulting polymers showed
strong coordination to metals and metal ions. Interestingly, it was found that
PAAEMA homopolymers self-assembled into hollow, double-stranded hierarchical
superstructures driven by the hydrogen-bridging interactions between adjacent
acetoacetoxy groups and compensation of dipole moments.45 The researchers also
produced block copolymers containing acetoacetoxy groups from
transacetoacetoxylation of PHEMA copolymer and the Claisen acylation of esters of
poly(2-hydroxyethyl ethylene) copolymer with sodium acetate.39
In this study the coupling reactions of block copolymers consisting of PAAEMA and
either PMAEP or PMOEP onto aminated glass slides was successfully accomplished.
The unstable imines formed through reaction of the amines and ketones were then
reduced to form stable secondary amines by the addition of NaCNBH3. XPS was
used to characterise the successful immobilisation. Since the PMOEP homopolymer
108
Chapter 3: Surface Fabrication
was found to be capable of attaching onto the aminated slides through electrostatic
interactions, the conformation of attached block copolymers was also investigated
using ToF-SIMS.
3.1.3 Adsorption of Fluorinated Polymers
Surfactants are known for their ability to adsorb at an interface (i.e. air/liquid,
liquid/liquid and solid/liquid) in order to reduce surface tension. The term surfactant
is a blend of the phrase “surface active agent”. They contain hydrophobic (tail) and
hydrophilic (head) groups which make them soluble in both organic solvents and
water.
Fluorinated surfactants are an important class of surfactants with a wide range of
applications, such as in coatings, textiles, moldings, fire fighting foams and
lubrication.46 Fluorocarbons are substantially greater than hydrocarbons in terms of
thermal, chemical and biological inertness, surface activity, gas dissolving capacity,
hydrophobicity and lipophobicity.47,48
Block copolymers are known to exhibit similar behaviour as surfactants. The
adsorption of block copolymers onto substrates, including polymers, has been widely
investigated.49 The driving force for adsorption is dependent on the nature of the
system. Surface micellisation and micelle adsorption of neutral block copolymers
onto hydrophilic and hydrophobic surfaces have been well studied.46 Greater rates of
adsorption for micellar solutions over non-micellar solution have been observed. For
block copolymers containing polyelectrolyte segments, adsorption is strongly
influenced by the charge on the copolymer relative to the surface.
Segregation of A-B block copolymers can be used to enhance the interfacial strength
between homopolymers A and B.46 According to Leibler’s theory, if the degree of
polymerization of the A block of the copolymer is greater than the degree of
polymerization of the A homopolymer, the homopolymer chains will penetrate the
copolymer brush.50,51
Several important applications of the adsorption of block copolymers are in the
detergency industry, in oil recovery and lubrication uses.49 However, the surface
109
Chapter 3: Surface Fabrication
modification of polymers by this technique is not well suited for industrial
application due to limitations in the thin film fabrication technology.52
Koberstein reported that if the block copolymer consist of functional groups with low
surface tension, the surface segregation of these groups occurs.53 However, this is not
applicable if the block copolymer contains high-energy reactive functional groups.
Adsorption of poly(styrene-b-tBA) (P(S-b-tBA)) onto a polystyrene substrate has
been carried out by spin coating followed by annealing.54 The block copolymers
were segregated on the surface of the polystyrene substrate due to the low surface
tension of the polyacrylate blocks. The t-butyl ester groups were subsequently
hydrolysed to form surface carboxylic acid groups.
In another study, Koberstein and coworkers formed poly(styrene-b-dimethylsiloxane)
films onto polystyrene by adsorption of these block copolymers in super critical
carbon dioxide which swelled the polystyrene.52 The driving force for adsorption in
this case is the reduction of interfacial tension between the supercritical fluid and the
polymeric substrate. The polystyrene segment of the block copolymer provides a
mechanical anchor through the formation of entanglements with the polymer chains
of the substrate.
As mentioned in the Chapter 1 (Section1.8), Marchant et al.55-57 used fluoropolymers
for the surface modification of PTFE films and expanded PTFE (ePTFE). In their
studies, random copolymers containing side-chains of perfluroundecanoyloxy groups
were adsorbed onto PTFE substrates from water. These modified polymers were
found to be stable under shear stress and supported endothelial cell attachment,
growth and function.
Some of the relevant intermolecular forces are shown in Table 3.1. Weak-electron
sharing bonds (or Lewis acid-Lewis base bonds) occur between electron donor (e.g.
I2) and electron deficient (e.g. aromatic compound) groups.58 Hydrophobic
interactions occur between non-polar groups in an aqueous solution when the
hydrogen-bonding structure of water is altered.
110
Chapter 3: Surface Fabrication
Table 3.1: Comparison of energies associated with intermolecular forces.
Intermolecular force Energy (kJ/mol)
Hydrogen bond58,59 10-50
Weak-electron sharing bond58 50
Dipole-dipole59 5-25
London dispersion59 0.05-40
Dipole-induced-dipole59 2-10
Hydrophobic60 4-21
In the case of fluorinated polymers, fluorine-fluorine (F-F) interaction, which is one
type of van der Waals force, can be expected between the fluorinated substrate and
the polymers. Halogen-halogen interactions have long been known in X-ray
crystallographic structures and are well studied. Alkorta and Elguero61 studied F-F
interaction using NMR and Atoms in Molecules (AIM) analysis. Boyd and
coworkers62 investigated F-F bonding in aromatic compounds based on electron
density. They found that the presence of F-F bonds can impart as much as 14
kcal/mol (or 59 kJ/mol) of local stabilisation in the molecules.62
Poly(pentafluorostyrene) (PFS) has previously been used for the surface modification
of PTFE, however, not by adsorption. Minko and coworkers63 fabricated amine-
functionalised PTFE surfaces and reacted this with carboxyl terminated PFS (PFS-
COOH) by coating substrates with this polymer and heating for 6 h at 150 °C. The
same procedure was used to immobilise poly(2-vinylpyridine) (PVP-COOH) onto
the PFS-functionalised surface. These grafted polymers were reversibly tuneable
from highly hydrophobic to highly hydrophilic surfaces by soaking in a solvent
selective to one of the components of the grafted brush.
One characteristic feature of polymer is known as “chain entanglement”.64 The
entanglement structure (or junction) involves binary-hooking geometry as shown in
Figure 3.2.
111
Chapter 3: Surface Fabrication
Figure 3.2: Chain entanglement of polymeric chains by binary-hooking structure.
Chain entanglement creates physical cross-linking and is a major factor controlling
the melt rheological, solid mechanical and adhesive properties of polymers.65 The
molecular weight between entanglements (Me) is often determined from the flow
properties of polymer melts. The literature values of the number of chain backbone
atoms between entanglements (Ne) of PTFE are listed in Table 3.2.
Table 3.2: Literature values of Ne of PTFE.
Ne Published Year Reference
93 1988 66
110 1988 67
112 1989 65
119 1983 68
132 1963 69
If the chain length of fluorinated segments of block copolymers is longer than this
value, the entanglement of these segments and the PTFE chain may be possible as
long as the right solvent is used to swell PTFE outer layer. This will further improve
the adhesion forces.
This study showed the adsorption of three types of fluorinated homopolymers (PFS,
PTFPMA, and PTFPA) as well as block copolymers consisting of PFS and PtBA or
PAA onto the PTFE substrate was performed. Of particular interest is the question
whether or not the polymer chains can entangle with the swollen PTFE outermost
chains. The effect of the chain length of PFS was also investigated.
112
Chapter 3: Surface Fabrication
3.1.4 Surface Characterisation Techniques
Surface analysis techniques have become vital since the recognition of the
importance of the surface properties of materials, and surface modification has
become commonplace in such a wide variety of applications. Since each technique
has inherent advantages and disadvantages, typically a multi-technique approach is
used. The choice of which depends on the information required. An important point
to note is that polymer surfaces or even polymer films that are attached are not in fact
rigid and reorientation of the chains can occur depending on the environment. This
makes it difficult to fully characterise polymer surfaces under one set of conditions
(e.g. wet or dry). In this study, the polymer on the surface could consist of even a
monolayer in thickness, meaning that the possibility that the techniques used covered
a wide range and included XPS, ToF-SIMS, AFM, IRRAS and contact angle
measurements.
3.1.4.1 X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron spectroscopy (XPS) is also known as electron spectroscopy for
chemical analysis (ESCA). XPS is a sensitive technique used to determine the
chemical composition of a surface. Typically it measures the first 5-10 nm for all
elements except hydrogen. A detection limit of about 0.1% atomic concentration can
be measured.70 Although most polymers can be analysed by this technique,
specimens must be vacuum compatible, and the degradation of some radiation-
sensitive polymer samples may occur due to the extended measurement times
required. However, an advantage is that liquid samples can also be analysed in
conjunction with cryogenic sampling.
Although X-rays penetrate deeply into the sample, the photoelectrons only eject from
the surface region. The equation expressing the sampling depth (d) of XPS is given
by:
d = 3λ sin θ (1)
λ is the attenuation length of the photoelectron and θ is the angle between the sample
surface and the analyser. λ for PTFE at 1000 eV is reported to be 2.4 nm.71
113
Chapter 3: Surface Fabrication
3.1.4.2 Static time-of-flight secondary ion mass spectroscopy (ToF-SIMS)
The underlying principle of static secondary ion mass spectroscopy (SIMS)
resembles that of XPS, but the source of bombardment is an accelerated ion beam.72
The atomic and molecular fragments emitted from the material surface on
bombardment of the high-energy ion beam are subsequently detected. Static time-of-
flight (ToF) SIMS has greatly enhanced both the reliability and surface sensitivity
(sampling depth of 1-2 nm). Trace amounts (pico or even femto mole) of organic
molecules can be detected and identified.72
ToF-SIMS is widely used for polymer surface characterization. Polymer
identification is possible from the finger print spectrum (m/z ≤ 200). The polymer
surface tacticities of both poly(methyl methacrylate) (PMMA) and polystyrene (PS)
have been studied using ToF-SIMS.73
Over the past decade, ToF-SIMS has become a widely used technique for the
characterisation of adsorbed protein films. Since the sampling depth is less than the
typical dimension of most proteins (4-10 nm), this technique has proven invaluable
for studying not only the amount and conformation of the adsorbed proteins but also
their orientation.74-76 ToF-SIMS has also been used to identify the molecular
orientation of phospholipids on alkane thiol SAMs on gold.77
ToF-SIMS data are generally generating complex spectra which contain hundreds of
peaks. Therefore, multivariate analysis technique (MVA), such as principal
component analysis (PCA), have increasingly been used to aid in the interpretation of
ToF-SIMS spectra.78,79 Before PCA, data sets are commonly pre-treated with, for
example, normalization to the total sum of intensities of the selected peaks and
mean-centering. The input to PCA is a matrix where the rows are samples (i.e.
spectra) and the columns are variables (i.e. peak intensities). Mathematically, PCA
consists of the singular value decomposition of the variance-covariance matrixes.
Thus the new variables (PC1, PC2, etc) formed by this transform are linear
combinations of the original variables (the ToF-SIMS peak intensities). The process
of data decomposition using PCA results in the formation of two new matrices: a
score matrix and a loading matrix. The scores show the relationship between the
samples in the new coordinate (PC defined) space, whilst the loadings illustrate the
relationship between the original variables and the principal components.
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Chapter 3: Surface Fabrication
3.1.4.3 Infrared reflection-adsorption spectroscopy (IRRAS)
IRRAS is a well-established technique used for the analysis of thin films, monolayers
and molecules adsorbed on flat surfaces.80 The substrate of choice is often a metal for
several reasons including its 100% reflectivity regardless of the incident angle and
polarisation. The technique utilizes a polarized IR beam: either p- or s-. That means
it either parallel or perpendicular polarized radiation with respect to the plane of
incidence, respectively (Figure 3.3).
p
s Reflected
Transmitted
Incident Beam θ θ1 1
θ2
Figure 3.3: Beam geometry and polarisation of IR radiation at the interface.
The signal-to-noise ratio (S/N) is calculated from the following equation80:
S/N = = ( – )R R
where R and R are the reflectivities of the clean and film-covered substrates,
respectively, at the frequency of the absorption maximum, R is the intrinsic noise
level of the single-beam sample spectrum, and ρ =1/R . Hence, the S/N is
proportional to the reflectivity difference between sample and reference (R –R ). For
dielectric substrates, the R –R strongly depends on polarisation, incidence angle and
the optical properties of the particular substrate. For silicon, optimum incidence
angles (θ ) are 86º and 0-50º for p- and s-, respectively.
0 F
N
N
0 F
0 F
opt80 When a beam is nearly
parallel to the surface, it is called grazing angle. Grazing angle IR has been widely
used to characterise LbL films,81 self-assembled monolayers82-84 and adsorbed
proteins.85
ρ(R0 – RF)0 F
(2) RN
115
Chapter 3: Surface Fabrication
3.1.4.4 Atomic force microscopy (AFM)
Atomic force microscopy (AFM) investigates the surface morphology on a
nanometer scale. Most types of samples can be examined and they are not required to
be conductive. AFM measures the forces of interaction between a probe and a
surface.
While a cantilever probes the sample surface, the motion of the cantilever, which is
deflected depending on the surface features, is accurately measured by a laser beam
which is reflected from the top of the cantilever. Subsequently three-dimensional
images are reconstructed. In addition to the morphology, the electrical charge and
hydrophilicity can also be assessed using AFM, leading to its increasing popularity
as a tool for characterising biomaterial surfaces as well as interfaces. Soft biological
samples such as cell surfaces and proteins on biomaterials have been successfully
characterised by non-contact AFM techniques.86,87
3.1.4.5 Contact angle measurement
Contact angle measurement techniques are one of the most sensitive measurements
for obtaining true surface information from the outermost few Angstroms of solid
surfaces.88 Although widely used for the investigation of the surface free energy of
many materials, the data can be difficult to interpret since artefacts often occur.
According to Young’s equation, the surface free energy of the solid can be derived
from the cosine of the contact angle of the liquid to the surface and the surface
tension of this liquid.86
LV
SLSV
γγγ
θ−
=cos (3)
where θ = contact angle, γ = interfacial tensions of SV (solid –vapour), SL (solid–
liquid), and LV (solid–vapour).
The assumptions which need to be valid for the straightforward interpretation of
contact angles are: be thermodynamically equilibrated, smooth, homogeneous, rigid,
immobile, non-deformable surfaces which do not swell or dissolve in the test
liquid.88 Many polymer surfaces do not satisfy all of these conditions. However, in
116
Chapter 3: Surface Fabrication
spite of this limitation, raw data from wetting experiments often contain useful
information such as the degree of hydrophilicity and molecular reorientation.
The hysteresis of contact angles can be measured by increasing (advancing) and
decreasing (receding) the test droplet volume. There are two types of hysteresis; true
or thermodynamic hysteresis and kinetic hysteresis.88 True or thermodynamic
hysteresis is generally based on the microscopic domains of non-equilibrium
transitions. Slow curve changes with time or frequency are due to the kinetic
hysteresis. In many polymer surface systems, reorientation and mobility of the
functional groups and macromolecules depending on the environment can be
observed due to thermodynamic hysteresis.
117
Chapter 3: Surface Fabrication
3.2 Experimental
3.2.1 Materials
Silicon wafers (100) and microscope glass cover slips were used as the substrates for
the LbL fabrications. Branched polyethyleneimine (PEI, Mw = 70k, 30 % aqueous
solution) was obtained from Polysciences (USA), poly(acrylic acid) (PAA, Mw = 2k)
from Aldrich. The syntheses of soluble PMAEP and PMOEP are described in
Chapter 2. HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, 99.9%)
and MES were used as buffers to make 0.1M buffer solutions with pH 7 and pH 5.5,
respectively. 1M NaOH and 1M HCl were used to adjust the pH.
3-Aminopropyltrimethoxysilane (APS) treated glass slides (or aminated slides) were
kindly donated by Asper Biotech (ES). The syntheses of PAAEMA homopolymer
and block copolymers are described in Chapter 2. Anhydrous dimethylformamide
(DMF) and NaCNBH3 were bought from Aldrich and used as received.
Polytetrafluoroethylene (PTFE) virgin tape (1.5 mm thickness) was obtained from E-
Plas, Victoria, Australia. The thickness was measured to be 1.54-1.58 mm. Syntheses
of fluorinated homopolymers and block copolymers are described in Chapter 2.
Dichloromethane (DCM), fluorobenzene (FB), methyl ethyl ketone (MEK) and DMF
were analytical grade and used as received.
3.2.2 Methods
3.2.2.1 Layer by Layer (LbL) assembly: PEI-PAA, PEI-PMAEP, and PEI-
PMOEP
Glass cover slips (1.0 × 1.0 cm2) and silicon wafers (approx. 1.0 × 0.5 cm2) were
acid-piranha treated (3:1 (v:v) conc H2SO4/H2O2) for at least 6 hours at 80 ºC to
ensure clean and uniformly oxidised surfaces. They were then washed extensively
with MilliQ water and dried under a gentle stream of N2. The clean substrates were
used immediately. LbL film formation was carried out by immersing the substrate in
an aqueous solution of branched polyethyleneimine (PEI) (2 mg/mL) for 20 min
followed by washing with MilliQ water. Then they were immersed in an aqueous
polyanion (either PAA, PMAEP or PMOEP) solution (2mg/mL) for 20 min and
118
Chapter 3: Surface Fabrication
washed again before drying under stream of N2. Only one layer of each polycation
and polyanion was deposited on all samples.
In the case of the PEI-PAA films, for deposition solutions with pH 5.5 and 7.0, 0.1M
MES and HEPES buffers were prepared, and after polyelectrolyte addition (2mg/mL),
the pH was adjusted using 1M HCl and NaOH.
3.2.2.2 Coupling of block copolymers to aminated slide
The aminated slide (approx. 0.5 × 0.5 cm2) was immersed in either 0.5 mL of the
PAAEMA homopolymer or block copolymer solution in dry DMF (2 mg/mL). After
reacting these individually in a glass tube with a septa seal for around 16 hours to
react the primary amine on the glass surface with the ketone group on the PAAEMA
to form the unstable imine. This linkage was then stabilized by mild reduction to
form the stable secondary amine bonds by the addition of excess NaCNBH3 (~1 mg).
The solution was left to react for over 4 hours. The PMOEP homopolymer was
reacted with the aminated slide following the same procedure except for the
reduction step. The slides were then washed thoroughly with DMF, rinsed with
acetone, and dried under vacuum.
3.2.2.3 Fluorinated homo and copolymer adsorption onto PTFE
PTFE films (approx. 1.0 × 0.5 cm2) were washed in a series of solvents (2h each in
chloroform and n-hexane and overnight in methanol with stirring at room
temperature) and dried before use. The film was then soaked in the relevant polymer
solution (1 mg/mL) in different solvents (e.g. DCM, FB, MEK or DMF) in a glass
tube overnight. The sample was then washed more than three times (~10 seconds
each) with the soaking solvent and then dried.
119
Chapter 3: Surface Fabrication
3.2.3 Instrumentation
3.2.3.1 X-ray photoelectron spectroscopy (XPS)
XPS spectra were recorded using a Kratos Axis Ultra X-ray photoelectron
spectrometer with monochromated Al Kα X-ray source (1486.6 eV) running at 150
W (15 kV, 10 mA emission current). The survey scans were collected at 1200-0 eV
with 1.0 eV steps at a pass energy of 160 eV; the narrow scans at 0.1 eV steps at a
pass energy of 20 eV. Vision 2 software was used for data acquisition and processing.
The binding energies were charge-corrected using a saturated hydrocarbon C1s peak
(285.0 eV) for LbL and aminated slide samples.89 For adsorption of fluoropolymers
onto PTFE, the binding energies were charge corrected to 292.5 eV for the C-F2 of
PTFE.90
High resolution spectra were resolved into individual Gaussian-Lorentzian peaks
using a least squares fitting program (CasaXPS, Casa Software Ltd.). Component
energies, number of peaks and peak widths (FWHM of 1.0 and 1.3 for all Cs and Ns,
respectively) were fixed initially and refinement was done only for peak heights. In a
final refinement cycle, component energies and peak widths were also refined and
these changed by less than 1.0 %. Peak fit results were imported into Excel for final
illustrations.
3.2.3.2 Infrared reflection-adsorption spectroscopy (IRRAS)
IRRAS was carried out using a 19650 Series grazing angle accessory (SPECAC) in a
Nicolet FTIR spectrometer, under continuous purging with CO2 free dry air. The s-
polarized light was incident at 40º relative to the substrate surface and an MCT
detector was used. To increase the signal-to-noise ratio, four spectra recorded at 8
cm-1 resolution and 512 scans were averaged.
3.2.3.3 Atomic force microscopy (AFM)
A NanoScope IVa Mutimode AFM instrument (Veeco, USA) was used in tapping
mode to obtain information on surface roughness. Measurements were performed
using a phosphorous-doped silicon cantilever (Veeco, USA) with a nominal tip
120
Chapter 3: Surface Fabrication
radius of less than 10 nm and a scan speed of approximately 3.05 Hz. The cantilever
had a spring constant of 20-80 N/m. A nominal area of either 1.0 μm × 1.0 μm or
10.0 μm × 10.0 μm was scanned. The Ra value is obtained from analyzing the total
area of 1.0 μm × 1.0 μm of the AFM image (Figure 3.7).
3.2.3.4 Static time of flight secondary ion mass spectrometry (static ToF-SIMS)
The ToF-SIMS analyses were performed using a PHI TRIFT II (model 2100)
spectrometer (PHI Electronics Ltd, USA) equipped with a 69Ga liquid metal ion gun
(LMIG). A 15 keV pulsed primary ion beam was used to desorb and ionise species
from the sample surface. Pulsed, low energy electrons were used for charge
compensation. Stainless steel grids were additionally used to minimise charging
effects. Mass axis calibration was done with CH3+, C2H5
+ and C3H7+
in positive mode
and with CH-, C2H- and Cl- in negative mode of operation. A mass resolution m/Δm
of ~ 4500 at nominal m/z = 27 amu (C2H3+) was typically achieved. Each sample was
characterised by 8 positive and 5 negative spectra: 49 positive and 49 negative peaks
were selected for sample characterisation. Principal Component Analysis was
accomplished using PLS_Toolbox Version 3.0 (Eigenvector Research, Inc., Manson,
WA) working in the MATLAB Platform (MATLAB Version 6.5, the MathWorks
Inc., Natic, MA).
3.2.3.5 Sessile drop contact angle measurements
A custom build apparatus fitted with a Kodak Digital Science DC120 camera linked
to a Kodak Digital Science Picture Postcard Software imaging program was used to
measure the contact angles. The measurements were performed manually at room
temperature.
For advancing contact angle measurements, a drop of MilliQ water (5 μL) was
placed into contact with the flat surface using a microsyringe. An image was
recorded immediately. The syringe tip was then placed in contact with the drop and
another 5 μL was added to advance the drop edge slowly. This addition was repeated
twice to create a total of 20 μL, with images recorded each time. The images of 5, 10
121
Chapter 3: Surface Fabrication
and 15 μL drops were used for determining the advancing angle measurements.
Receding contact angles were measured following the same procedure after
withdrawing water from the drop. Only the image of the last 5μL drop was used.
The following equation was used to calculate the average contact angle (θ) using
height (h) and distance (d):58
The reported values are the average values of measurements for two samples from
three different locations for each sample. The errors in the angle values are the
standard deviations.
3.2.3.6 Dynamic light scattering (DLS)
Dynamic light scattering measurements were performed using a Malvern Zetasizer
Nano Series instrument running DTS software and operating a 4 mW He-Ne laser at
633 nm. Analysis was performed at an angle of 90° and a constant temperature of 25
°C. All samples were run 10 times. The number average particle size is reported.
h θd
(3) h = d/2 tan(× θ / 2)
122
Chapter 3: Surface Fabrication
3.3 Results 3.3.1 Layer-by-Layer (LbL) Assembly
In this study, LbL assembly of PEI and PAA, PMAEP or PMOEP was carried out to
create carboxylate- or phosphate-containing surfaces (Figure 3.4). Only one layer of
each of the polycation and polyanions is adsorbed on either glass or silicon surfaces.
XPS was used to analyse the adsorbed layers. The topographies of PMAEP and
PMOEP LbLs were imaged by AFM. IRRAS was also used to investigate the
polyelectrolytes complex.
pHopt =
Figure 3.4: Idealised electrostatically driven LbL assembly deposition.
3.3.1.1 LbL of PAA
Table 3.3 shows properties of polymers used for this study. Since PAA is a weak
polyanion, its deposition is expected to be strongly affected by the pH of the solution.
The literature values of pKa of PEI (Mw = 25k) and PAA are 8.591and 5.5,
respectively. The optimum pH (pHopt) for deposition is calculated using the
following equation:
For PEI and PAA, the pHopt is 7. In this study, two pH values 7 and 5.5 as well as a
control, unadjusted MilliQ water were used. The pHs of the PEI and PAA solutions
(2 mg/mL) in unadjusted water were found to be 9.9 and 3.3, respectively (Table 3.3).
pKa(polyanion) + pKa(polycation)
2(4)
OH
OH
OH
OH
OH
OH
O-
O-
O-
O-
O-
O-
+ +
+ +
+ +
+ +
+
O-
O-
O-
+
O-
O-
O-
+ +
+ +
+ +
+ +
_ _
_ _
_
Piranha Solution Polycation Polyanion
(PEI) (PAA/ PMAEP/ PMOEP)
_
Carbon contaminants
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Chapter 3: Surface Fabrication
Table 3.3: Properties of PEI and PAA.
Mw pKa pH of 2mg/mL solution
PEI 70,000 ~8.5 9.9
PAA 2,000 5.5 3.3
020040060080010001200Binding Energy (eV)
(C) (B) (A)
C1sSi2s Si2p N1s
O1s
O KLL
O2s
Figure 3.5: XPS survey scans of (A) blank glass slide (sample 1A), (B) PEI film (sample 1B1) and (C) PEI-PAA LbL (sample 1B2).
After preparation of the PEI-PAA LbL, the XPS survey spectrum was compared to
those of the PEI and the untreated slides as shown in Figure 3.5. The peak labelled
KLL arises from the Auger process. Glass slides contain O, C, Si, as well as trace
amounts of Na (0.2%), Ti (0.4%), N (1.2%), K (0.2%), B (2.15%) and S (0.8%)
(Figure 3.5A). PEI deposition was clearly observed from the increase in the N 1s and
C 1s peaks (Figure 3.5B). The PAA film also showed an increase in C 1s (Figure
3.5C). Table 3.4 summarises the atomic % of O, C, N and Si obtained from the XPS
survey scans of all samples.
The C/N ratio is also shown in Table 3.4. After PEI deposition, the ratios of C/N
were in the range of 3.3-4.0, which was slightly higher than the expected value from
the PEI structure (C/N=2). This is most probably due to the co-contribution of
hydrocarbons from the contaminants on the glass slide. After PAA deposition, the
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Chapter 3: Surface Fabrication
C/N ratios increased to 5.9-9.6, indicating successful film formation at all pH values
studied.
Table 3.4: Atomic % of O, C, N and Si.
Sample Solution Polymer O C N Si C(=O)O C/N
1A a ― ― 65.0 10.3 1.2 19.8 ― 8.6
1B1 Water PEI 58.8 16.3 4.9 20.0 ― 3.3
1B2 Water PEI-PAA 46.3 35.0 4.1 14.6 7.1 8.5
1C1 pH 5.5 PEI 58.3 15.0 4.5 22.3 ― 3.3
1C2 pH 5.5 PEI-PAA 55.0 22.0 3.7 19.3 2.5 5.9
1D1 pH 7 PEI 61.2 12.3 3.1 23.3 ― 4.0
1D2 pH 7 PEI-PAA 58.5 15.3 1.6 24.6 4.0 9.6 a: Untreated glass slide
The deposition of PEI was not dramatically affected by pH, since the atomic C% was
found to lie in a reasonably close range (12.3-16.3%). On the other hand the atomic
C % of the PAA deposited surface showed large differences depending on the pH. In
pH 7, the PAA deposition only increased its atomic C% from 12 (PEI deposited
surface) to 15%, whereas at pH 3.3 (unadjusted water), it increased from 16 to 35%.
This indicates that the PAA film fabricated at pH 3.3 was much thicker than that
obtained from the pH 7 solution.
282285288291294
Binding Energy (eV)
282285288291294
Binding Energy (eV)(A) (B)
K1s 1 2
3
4
1
2
3 5
4 5
6
Figure 3.6: C 1s narrow scans of (A) PEI film (sample 1B1) and (B) PEI-PAA LbL (sample 1B2).
125
Chapter 3: Surface Fabrication
Figure 3.6 shows the XPS C1s high resolution spectra of PEI and PAA deposited
surfaces. The C1s peak from the PEI deposited surface (Figure 3.6A) required five
peaks to fit, and these peaks were assigned to C*-C (peak 1, 285.0 eV), C*-C-O
(peak 2, 285.8 eV), C*-N (amino, peak 3, 286.1 eV), C-O, (peak 4, 286.9 eV) and N-
C*=O (amide, peak 5, 288.1 eV).89,92,93 Peak 1, 2 and 4 possibly arise from the
carbon contaminants. The small amide peak indicates some possible oxidation of
amines. The C1s spectrum of the PAA deposited surface (Figure 3.6B) showed one
additional peak at 289.1 eV (peak 6) due to C*(=O)-O. The atomic % of this
carboxylic acid carbon against the total elements is also shown in Table 3.3. Peak 2
at 285.5 eV is also assigned to C*-C(=O)O from PAA.
396398400402404406Binding Energy (eV)
396398400402404406
Binding Energy (eV) (B) (A)
1
2
3 1
2
3
Figure 3.7: N1s narrow scans of (A) PEI film (sample 1B1) and (B) PEI-PAA LbL (sample 1B2).
Figure 3.7 shows the N1s high resolution spectra of the PEI film and PEI-PAA LbL
on glass surface. Three peaks were required for fitting and assigned to the amine
(399.5±0.2 eV),33,94 the amide (400.7±0.2 eV),33 and the protonated amine
(401.9±0.2 eV).17,33,94-98 The presence of amide peaks correlates well with C1s peak
(peak 5 in Figure 3.6). The XPS spectrum of the thicker PEI film obtained from
drying the original PEI solution did not show evidence of any amide peak. This result
is in agreement with the fact that the oxidation of amines is more likely to occur
when it is very thin or monolayer film.33,99 Figure 3.7B shows a large increase in the
protonated amine peak when PAA was deposited (sample 1B2). Table 3.5
summarises the relative ratios of the fitted N1s peaks.
126
Chapter 3: Surface Fabrication
Table 3.5: Normalized atomic % of nitrogen species from the curve fitting of the N1s peak.
Sample Solution Polymer N1 a N2 b N3 c
1B1 Water PEI 59.1 10.6 30.4
1B2 Water PEI-PAA 33.9 8.8 57.3
1C1 pH 5.5 PEI 36.8 15.8 47.4
1C2 pH 5.5 PEI-PAA 40.0 14.3 45.7
1D1 pH 7 PEI 43.3 16.7 40.0
1D2 pH 7 PEI-PAA 31.8 27.3 40.9
a: amine (399.5±0.2eV), b: amide (400.7±0.2eV) and c: protonated amine (401.9±0.2 eV)
Although the atomic C% values are similar for all the PEI films at different pHs, the
high resolution N1s peaks show different patterns. For samples 1D1 and 1C1 (pH 7
and 5.5), there were more protonated amines (40 and 47%, respectively) compared to
the 30% found for sample 1B1 deposited in unadjusted water (pH 9.9). At pH 7 and
5.5, the ionization of PEI is 100% and thus very probably leads to stronger
interaction with the glass slides. This also indicated that fewer amine groups are
available for interaction with PAA and therefore less PAA chains were attached to
these surfaces compared to PEI deposited in unadjusted water (pH 9.9).
When PAA was attached in unadjusted water (sample 1B2), a large increase in the
protonated amine peak was observed. This indicates successful PAA deposition. At
pHs 5.5 and 7, only slight increases in the protonated amine peaks were observed,
which correlates well with small increase in the total C% that was observed in the
survey scans (samples 1C2 and 1D2).
3.3.1.2 LbL of phosphate-containing polymers
The LbL deposition of PEI and PMAEP or PMOEP onto silicon wafers was carried
out in unadjusted water. The pH of the PMAEP and PMOEP solutions (2 mg/mL)
were found to be 2.7 and 2.8, respectively (Table 3.6). The same branched PEI was
used.
127
Chapter 3: Surface Fabrication
Table 3.6: Properties of PMAEP/PMOEP used for this study.
n Mn a PDI pH of 2mg/mL solution
PMAEP 57 11500 1.46 2.7
PMOEP 97 20500 1.94 2.8 a: calculated from Mn of hydrolysed polymers obtained from GPC, assuming no hydrolysis of side-chains. The actual Mn is expected to be lower than stated here due to hydrolysis of some side-chains.
020040060080010001200Binding Energy (eV)
(C)
(B)
(A)
(D)
O1s
N1s
C1s
Si2p
Si2s P2p O KLL
Plasmon
Figure 3.8: XPS survey scans of (A) blank silicon wafer (sample 2A), (B) PEI film (sample 2B), (C) PEI-PMAEP LbL (sample 2C) and (D) PEI-PMOEP LbL (sample 2D).
XPS survey scans (Figure 3.8) were used to analyse LbL formation. Table 3.7
summarises the elemental compositions of the surfaces. After acid Piranha treatment,
clean silicon wafers showed only 5.5% carbon, which corresponds to a clean
surface.100 As layers of polymers were deposited, increases in the C% and decreases
in the Si% were observed, indicating both successful film and LbL formations. The
appearance of the P peak gave further evidence of successful PMAEP and PMOEP
film formations. The lower P% found for sample 2C (PMAEP layer) compared to
that of sample 2D (PMOEP layer) can be explained by the lower phosphorous
content of the PMAEP polymer as characterised and discussed in Chapter 2.
128
Chapter 3: Surface Fabrication
Table 3.7: Atomic % of C, N, P and Si from XPS survey scans.
Sample Polymer O C N P Si C(=O)O C/N
2A a ― 46.1 5.5 ― ― 48.4 ― ―
2B PEI 31.9 21.4 5.9 ― 40.8 ― 3.6
2C PEI-PMAEP 33.7 44.1 2.8 2.2 17.3 7.2 15.8
2D PEI-PMOEP 34.9 48.5 4.2 3.5 9.0 6.5 11.5
a: Untreated silicon wafer
Table 3.7 also shows the C/N ratios. The PEI deposited surface showed a C/N ratio
of 3.6, which is similar to that obtained for the PEI layer on the glass surface (sample
1B1, Table 3.4). LbL results for the phosphate polymers showed higher atomic C%
and C/N ratios than for the PAA samples.
282284286288290292294
Binding Energy (eV)
282284286288290292294
Binding Energy (eV)282284286288290292294
Binding Energy (eV)
(A)
(B) (C)
1 2 3
4
5
1 2
3 4 4’ 6
1
2 4 4’ 6
3
Figure 3.9: C1s narrow scans of (A) PEI film (sample 2B), (B) PEI-PMAEP LbL (sample 2C) and (C) PEI-PMOEP LbL (sample 2D).
The high-resolution C1s spectra of these samples are shown in Figure 3.9. The
spectrum of the PEI film (sample 2B) was fitted with five peaks: C*-C (peak 1, 285.0
129
Chapter 3: Surface Fabrication
eV), C*-C-O (peak 2, 285.6 eV), C*-N (amino, peak 3, 286.1 eV), C-O, (peak 4,
286.7 eV), and N-C*=O (amide, peak 5, 288.2 eV). Peaks 1, 2 and 4 are from the
carbon contaminants.
For the C1s spectra of the PMAEP and PMOEP LbL’s, peaks 2, 4 and 4’ were
assigned to C*-C=O (285.6 eV) and C*-O-C (286.1 eV), C*-O-P (286.5 eV),
respectively. Appearance of the C(=O)-O peak (peak 6) was observed. Atomic % are
shown in Table 3.7. No amide peaks were observed in these spectra.
396398400402404406408
Binding Energy (eV)396398400402404406408
Binding Energy (eV)
396398400402404406408
Binding Energy (eV)
(C)
(A)
(B)
1
2 3
1
3 3 1
2 2
Figure 3.10: N1s narrow scans of (A) PEI film (sample 2B), (B) PEI-PMAEP LbL (sample 2C) and (C) PEI-PMOEP LbL (sample 2D).
Table 3.8: Normalized atomic % of nitrogen species from curve fitting of the N1s peak.
Sample Polymer N1 a N2 b N3 c
2B PEI 68.1 19.3 24.5
2C PEI-PMAEP 29.3 2.9 67.8
2D PEI-PMOEP 31.2 2.2 66.6 a: amine (399.5±0.1eV), b: amide (400.9eV) and c: protonated amine (401.9eV)
130
Chapter 3: Surface Fabrication
Curve fitting of the N1s peak spectra required three peaks: the same as for LbL on
glass slides (Figure 3.10). The normalised atomic % of these peaks are shown in
Table 3.8. Sample 2B (PEI film) showed amine, amide and protonated amine
concentrations of 68, 19 and 25%, respectively. In the case of samples 2C and 2D
(PMAEP and PMOEP LbL’s), only small amide peaks were observed (2.9 and 2.2%,
respectively). This could be attributed to the low resolution of the spectra. As
expected, large increases in the protonated amine peaks were observed for these
samples (~67%).
To investigate the topography of these samples, tapping mode AFM was employed
(Figure 3.11). The mean roughness (Ra) from these images is shown in Table 3.9.
The clean silicon surface showed smooth topography (Figure 3.11A). The PEI film
(sample 2B) did not show increased roughness compared to the silicon wafer (Figure
3.11B). When PMAEP was deposited (sample 2C), the surface became much
rougher compared to the PEI film surface (Ra = 0.69 nm). Some “aggregates” (or
“patches”) of polymer molecules were clearly visible (Figure 3.11C). The PEI-
PMOEP LbL surface (sample 2D, Figure 3.11D) was much smoother than that of the
PEI-PMAEP surface, with the Ra value of 0.27 nm.
Table 3.9: Mean roughness (nm) of LbL films from the AFM images in Figure 3.7.
Sample Attached Polymer Ra (nm)
2A ― 0.10
2B PEI 0.10
2C PEI-PMAEP 0.69
2D PEI-PMOEP 0.27
131
Chapter 3: Surface Fabrication
Data Scale = 5.0 μm
Data Scale = 5.0 μm
Data Scale = 15.0 μm
Data Scale = 10.0 μm
A
B
C
D
Figure 3.11: 2D and 3D AFM images of A) silicon wafer, B) PEI film (sample 2B), C) PEI-PMAEP LbL (sample 2C) and D) PEI-PMOEP LbL (sample 2D) (analysed area 1.0×1.0 μm).
132
Chapter 3: Surface Fabrication
Large area scans (10×10 μm, Figure 3.12) were also carried out because surface
roughness and layer homogeneity could be expected to play an important role in
subsequent mineralisation studies. Although patchy adsorption was observed, the
PMAEP was found to uniformly cover the surface especially at the scale of SEM
images (area of 25×25 μm and 50×50 μm) which were subsequently used in the SBF
study (Chapter 4, section 4.3.2).
Data Scale = 50.0 μm Figure 3.12: 2D and 3D AFM images of PMAEP (analysed area 10×10 μm).
FTIR is a useful technique commonly used to characterise functional groups. In more
specific applications, IRRAS has been used to analyse thin films including
monolayers on silicon wafers and flat metal surfaces. Attenuated total reflectance
FTIR (FTIR-ATR) has been used throughout this study for other samples, since it
does not require any special sampling (experimental in Chapter 4, Section 4.2.2). The
FTIR-ATR spectrum of soluble PMOEP showed a large band at 974 cm-1,
corresponding to the P-O(H) stretching (Figure 3.13A). The IRRAS spectrum of the
LbL surface showed the two large bands at 1065 and 967 cm-1 corresponding to the
out-of-phase and in-phase P-O stretches of phosphate salts, respectively (Figure
3.13B).6 This indicates that large amounts of phosphate groups are interacting with
protonated amines.
133
Chapter 3: Surface Fabrication
Wavenumber (cm-1)
967 1065
(B)
974 (A)
Figure 3.13: FTIR spectra of (A) soluble PMOEP (ATR) and (B) PEI-PMOEP LbL (sample 2D) (IRRAS).
134
Chapter 3: Surface Fabrication
3.3.2 Block Copolymers Coupled onto Aminated Slides
Coupling of the block copolymers to the aminated slides was carried out in dry DMF
at room temperature followed by mild reduction with NaCNBH3 to convert the
unstable imines to the more stable secondary amines (Figure 3.14).
Figure 3.14: Idealised coupling reaction of block copolymers with aminated slide.
Table 3.10: Block-copolymers attached to aminated slides for SBF.
Coupled Polymer Sample
m a n b PDI
3B PAAEMAm 22 ― 1.13
3C PMOEPn ― 19 1.98
3D P(MOEPn-b-AAEMAm) 109 97 1.38
3E P(AAEMAm-b-MOEPn) 22 132 1.41
3F P(AAEMAm-b-MAEPn) 22 99 1.38
3G P(AAEMAm-b-MAEPn) 22 160 1.38 a: m = unit of PAAEMA segment, b: n = unit of PMOEP/PMAEP segment
Table 3.10 summarizes the properties of the polymers used for this study. PAAEMA
and PMOPE homopolymers were also reacted with aminated slides as controls. The
successful immobilization of the polymers onto the aminated slides was verified by
analysing the XPS spectral changes. ToF-SIMS was also used to identify the
conformation of one of the coupled block copolymers (sample 3D).
NH2
NH2
NH2
NH2
N
N
N
N
NH
NH
NH
NH
PA
AE
MA
PMAEP/PMOEP
Reduction
NaCNBH3
Coupling
Polymer in dry DMF
135
Chapter 3: Surface Fabrication
3.3.2.1 Quantitative XPS investigation of attached polymers
Figure 3.15 shows the XPS survey spectra of the control slides (untreated aminated,
PAAEMA and PMOEP), as well as the P(MOEP-b-AAEMA) functionalised slides.
Table 3.11 summarises the elemental compositions of the polymer attached surfaces
obtained from the XPS survey spectra.
020040060080010001200Binding Energy (eV)
(A) (B)
(C) (D)
O1s
N1s C1s
Si2p Si2s
P2p Na1s
Na KLL Ca2pK1sP2s
O KLL
Figure 3.15: XPS spectra of aminated slides (A) as received (Sample 3A), (B) PAAEMA functionalized (Sample 3B), (C) PMOEP functionalized (Sample 3C) and (D) P(MOEP-b-AAEMA) functionalized (Sample 3D).
Table 3.11: XPS data: Atomic % of elements concentrations from XPS survey scans.
Sample Polymer Attached Na O N Ca C P Si C(=O)O
3A – 4.0 54.4 2.6 0.9 22.3 – 15.9 0.9
3B PAAEMA 1.6 49.7 1.8 1.2 26.3 – 19.4 2.0
3C PMOEP 1.8 49.9 1.7 1.0 26.1 1.3 18.1 2.2
3D P(MOEP-b-AAEMA) 3.5 44.3 1.5 0.5 36.0 1.5 12.8 4.3
3E P(AAEMA-b-MOEP) 3.3 38.6 1.5 0.3 46.1 1.8 8.5 6.6
3F P(AAEMA-b-MAEP) 2.3 47.5 1.5 0.8 30.7 1.0 16.2 4.3
3G P(AAEMA-b-MAEP) 1.9 46.5 1.5 0.8 32.2 0.9 16.1 4.4
136
Chapter 3: Surface Fabrication
The XPS spectrum of the 3-aminopropyltrimethoxysilane (APS) treated glass slide
(aminated slide), showed the presence of N and C from the APS, as well as O, Si and
small amounts of Na and Ca from the glass slide.
Immobilisation of the homopolymers PAAEMA (sample 3B) and PMOEP (sample
3C) onto aminated slides resulted in a reduction in the amount of N and an increase
in the amount of C (to 26 atom% in both systems) indicating successful attachment
of the homopolymers. In addition, the PMOEP-modified slide showed the
appearance of P as expected (1.3%).
The amounts of C increased to 30.7-46.1% when block copolymers were attached.
This suggests that a higher amount was attached for the copolymer than for the
homopolymers. For both PMOEP and PMAEP copolymers, those with the longer
PMOEP or PMAEP segments (samples 3E and 3G) showed higher attachments
compared to those with shorter chains (samples 3D and 3F). In all cases, the amount
of N was reduced to 1.5% and presence of P was observed (0.9-1.8%).
A decrease in Si% was only observed for samples 3D and 3E (PMOEP copolymers)
compared to the aminated slide. Others showed an increase in Si% after polymer
attachment. Most probably this can be attributed to the carbon contaminants on the
aminated slides which were either partially replaced after polymer adsorption or
removed by the washing steps.
Figure 3.16 shows the high resolution C 1s peaks for samples 3A-3D. The untreated
aminated slide (Figure 3.11A) shows peaks related to APS (C*-C (peak 1, 285.0 eV)
and C*-N (amino, peak 3, 286.1 eV)) as well as from carbon contaminants (C*-C-O
(peak 2, 285.5 eV), C-O, (peak 4, 286.9 eV), and C*(=O)-O (peak 6, 288.8 eV)).
Amide peaks (N-C*=O (peak 5, 287.8 eV)) was also observed. When polymers were
attached, changes in the peak shapes were observed. The increase in the ester peak
(peak 6) after polymer attachment was clearly observed and the atomic % of this
peak is shown in Table 3.11.
137
Chapter 3: Surface Fabrication
282284286288290292294
Binding Energy (eV)
282284286288290292294
Binding Energy (eV)
282284286288290292294
Binding Energy (eV)
282284286288290292294
Binding Energy (eV)
(A) (B)
(C) (D)
1
2 3 4
5 6
1
2
4 4’ 6
5 3
1
2
4 4’ 6
5 3
1
2
4 4’ 6
5 3
Figure 3.16: C1s narrow scans of aminated slides (A) as received (sample 3A), (B) PAAEMA functionalized (sample 3B), (C) PMOEP functionalized (sample 3C) and (D) P(MOEP-b-AAEMA) functionalized (sample 3D).
395397399401403405
Binding Energy (eV)
395397399401403405
Binding Energy (eV)
395397399401403405
Binding Energy (eV)
395397399401403405
Binding Energy (eV)
(A)
(C) (D)
(B)
Figure 3.17: N 1s narrow scans of aminated slides (A) as received (sample 3A), (B) PAAEMA functionalized (sample 3B), (C) PMOEP functionalized (sample 3C) and (D) PMOEP-b-PAAEMA functionalized (sample 3D).
138
Chapter 3: Surface Fabrication
The high-resolution spectra of the N1s peak for all the samples (Figure 3.17)
required three peaks for the fit. These peaks were assigned to amine (399.6±0.2 eV),
amide (400.7±0.2 eV) and the protonated amine (401.8±0.2 eV), as for the LbL films.
No oxidised nitrogen species (i.e. NO3-) were detected for any of the samples by XPS.
The shape of the N1s peak changed depending on which polymer was attached to the
slide. The relative ratios of the fitted nitrogen peaks are given in Table 3.12.
Table 3.12: Normalized atomic % of nitrogen species from curve fitting of the N1s peak.
Sample Polymer Attached N1 a N2 b N3 c
3A – 46.2 27.3 26.4
3B PAAEMA 55.2 22.7 22.1
3C PMOEP 30.6 26.9 42.6
3D P(MOEP-b-AAEMA) 41.7 26.7 31.7
3E P(AAEMA-b-MOEP) 38.2 26.8 35.0
3F P(AAEMA-b-MAEP) 40.5 26.2 33.3
3G P(AAEMA-b-MAEP) 37.2 27.2 35.6 a: amine (399.6±0.1eV), b: amide (400.7±0.2eV) and c: protonated amine (401.8±0.2eV)
The APS-treated aminated slide (sample 3A) showed 46.2% amines and 26.4%
protonated amines. The presence of protonated amines on this sample indicate that
some APS amines have reacted with the hydroxyl groups on the glass surface during
silanation possibly by as a result of residual water or proton exchange.95 Polymer
attachment did not alter the amount of the amide peak (in the range of 22.7–27.3 %),
suggesting that no further oxidation occurred as a result of the procedures used to
attach the polymers to the slides. When PAAEMA was attached (sample 3B), there
was an increase in the amine peak (55.2%) and a decrease in the protonated amine
(22.1%) peak. Attachment of the PMOEP homopolymer caused an increase in
protonated amine (42.6%) and a decrease in amine (30.6%) peaks (sample 3C).
Compared to the aminated slide, the block copolymer attachment resulted in a slight
decrease in amine (37.2-41.7%) and an increase in protonated amine peaks (31.7-
35.6%). These values are in fact between those of the PAAEMA and PMOEP-
139
Chapter 3: Surface Fabrication
functionalised slides. For both PMOEP and PMAEP copolymers, there is a trend that
those with longer PMOEP/PMAEP segments (samples 3E and 3G) showed larger
protonated amine peaks compared to the shorter ones (samples 3D and 3F). However,
differences between them are only ~3%, and this may not be significant.
3.3.2.2 ToF-SIMS investigation of the conformation of attached block
copolymers
ToF-SIMS was used to assess the conformation of one of the attached block
copolymers on the aminated surfaces as well as control samples: samples 3A
(aminated slide), 3B (PAAEMA-attached), 3C (PMOEP-attached), and 3D
(P(MOEP-b-AAEMA)-attached). Figure 3.18 represents the possible conformations
of the block copolymers through different chemical interactions: (A) coupling
reaction of keto groups of PAAEMA segment and amines, (B) ionic interaction of
PMAEP/PMOEP segment of phosphates and protonated amines, and (C)
combination of both types of interaction.
Figure 3.18: Possible conformations of block copolymers reacted with aminated slide.
Since XPS showed some variations of APS-treatments between the slides, it was
determined to use the same slide for the samples and the untreated one for ToF-SIMS.
Figure 3.19 shows the positive and negative SSIMS spectra of the APS-treated
aminated slide surface (sample 3A). The positive spectrum showed distinct
hydrocarbon peaks such as C+, CH3+, C2H3
+, C3H3+ and C3H5
+. The signal at m/z 28
contains 2 peaks corresponding to Si+ and C2H4+. The spectrum also reveals the
presence of sodium confirming its identification by XPS. The negative spectrum is
O
NH
O
PAAEMA
PMAEP/PMOEP
(A) (B) (C)
O
OP O
O-O
NH3+
140
Chapter 3: Surface Fabrication
dominated by signals at m/z = 16 (O-) and at m/z = 17 (OH-). Signals at m/z = 46 and
62 amu indicate oxidized nitrogen species (NO2- and NO3
- respectively). XPS
investigation did not reveal the presence of these species. Hence it appears that these
groups are only present on the outer surface in very low concentration and thus
appear over-represented in the negative mass spectrum.
0 20 40 60 80
Inte
nsity
20050610A.TDC + Ions 100µm 1496964 cts
11 67 53 15 71 69 39 55
57 43 41
28 23
27 29
x0.25
m/z
+ SIMS
0
20050615A.TDC - Ions 100µm 1372525 cts
25
13
16
26
62 46
x10
0 40 60
Inte
nsity
m/z
- SIMS
80 20
35
Figure 3.19: Positive and negative SSIMS spectra for APS-treated amianted glass slide (samples 3A). Note: sodium peak (23 amu) was scaled to 25% of its original intensity; peaks above 25 amu in negative mass spectrum were magnified by 10 folds.
141
Chapter 3: Surface Fabrication
30 40 50 60 70 80 90In
tens
ity
20050610A.TDC + Ions 100µm 14969
53 67 716939 575543 27 41 29
28 + SIMS (a)
m/z
20050625A.TDC + Ions 100µm 12791
53 716939 5755
43 27 41
29 28 + SIMS (b)
30 40 50 60 70 80 90
Inte
nsity
m/z
20050640A.TDC + Ions 100µm 13868
53 67 71 69
39 57 55
43 27 41 29
28 + SIMS (c)
30 40 50 60 70 80 90
Inte
nsity
m/z
20050655A.TDC + Ions 100µm 123955
53 67 71 69
39 57 55
43 27 41 29 28
+ SIMS (d)
30 40 50 60 70 80 90
Inte
nsity
m/z
Figure 3.20: Positive SSIMS spectra for: (a) sample 3A (aminated slide), (b) sample 3B (PAAEMA attached), (c) sample 3C (PMOEP attached), and (d) sample 3D (P(MOEP-b-AAEMA) attached).
30 40 50 70 80 90
20050615A.TDC - Ions 100µm 137252
37 42 32 29 46 35 62
26
Inte
nsity
m/z
(a) - SIMS
20050630A.TDC - Ions 100µm 171669
30 40 50 70 80 90
37 41 32
29 46 35 62
26
Inte
nsity
m/z59
(b) - SIMS
20050645A.TDC - Ions 100µm 166643
30 40 50 70 80 90
37 41
32
29
46
35 62
26
Inte
nsity
m/z59
63
79
(c) - SIMS
20050660A.TDC - Ions 100µm 104055
30 40 50 70 80 90
37 41
32
29
46 35
62
26
Inte
nsity
m/z
59
63
79
- SIMS (d)
Figure 3.21: Negative SSIMS spectra for: (a) sample 3A (aminated slide), (b) sample 3B (PAAEMA attached), (c) sample 3C (PMOEP attached), and (d) sample 3D (P(MOEP-b-AAEMA) attached).
142
Chapter 3: Surface Fabrication
Figure 3.20 shows the positive SSIMS spectra for samples 3A-3D. Although the
qualitative characteristics show similarities, the intensity patterns differ between the
samples. The block copolymer attached slide (Figure 3.20d) shows substantial
reduction in the yield of the Si+ ion (m/z = 28).
The negative mass SSIMS spectra for samples 3A-3D are shown in Figure 3.21.
When PMOEP and P(MOEP-b-AAEMA) (samples 3C and D, respectively) were
attached, the spectra clearly showed the appearance of peaks at m/z = 63 and m/z =
79, which correspond to the PO2- and PO3
- fragments. This clearly confirms the
presence of phosphorous-containing moieties on the surface.
As described in detail in Section 3.1.4.2, the complex ToF-SIMS data sets were
subjected to both PCA and Analysis of Means (graphical procedure).101,102 Each
sample was characterised by 8 positive and 5 negative spectra: 49 positive and 49
negative peaks were selected for sample characterisation using MVA (Table 3.13).
Table 3.13: Positive and negative fragments used in PCA.
Positive Fragments Negative Fragments
C+, CH+, CH2+, CH3
+, C2H2+, C2H3
+, C2H4
+, C2H5+, C2H6
+, C3H2+, C3H3
+, C3H4
+, C3H5+, C3H6
+, C3H7+, C3H8
+, C4H3
+, C4H4+, C4H5
+, C4H6+, C4H7
+, C4H8
+, C4H9+, C4H10
+, C5H5+, C5H6
+, C5H7
+, C5H8+, C5H9
+, C5H510+, C5H11
+, Al+, Si+, SiH+, CH2Si+, CH3Si+, SiOH+, CHO+, CH3O+, C2H2O+, C2H3O+, C2H4O+, COOH+, C2H5O+, C4H5O+, CH2NO+, CH3NO+,CH4N+, C2H4N+.
C-, CH-, CH2-, CH3
-, C2-, C2H-, C2H3
-, C3-
, C3H-, C3H2-, C3H3
-, C3H4-, C4
-, C4H-, C4H2
-, C4H3-, C5H-, O-, OH-, Si-, SiH-,
CHSi-, SiO-, SiOH-, SiO2-, SiO2H-, SiO3
-, SiO3H-, P-, CP-, PO-, POH-, PO2
-, PO3-,
NO-, NO2-, NO3
-, CN-, CHN-, C2HN-, CNO-, CHNO-, CH3O-, C3O-, C2HO-, C2H3O-, COOH-, C3H3O-, C3H4O-.
Figure 3.22 shows the scores for both the positive and negative ion mass spectra. In
both cases, there are 4 distinct clusters corresponding to the 4 investigated samples.
Each group represents multiple spectra taken from different areas of the sample. The
first two PCs captured 98.89 and 97.24% of the data variance present in the positive
and the negative mass spectra respectively, indicating most of the original spectral
information has been retained. Since samples are different, the differences were most
143
Chapter 3: Surface Fabrication
pronounced between the untreated aminated slide (sample 3A) and the block
copolymer attached slide (sample 3D).
+ SIMS (a)
- SIMS (b)
Figure 3.22: Score plots on PC1 and PC2 for aminated glass slide and its modifications. (a) scores derived from the positive fragments; (b) scores derived from the negative fragments.
Block attached PMOEP attached PAAEMA attached Aminated slide
Block attached PMOEP attached PAAEMA attached Aminated slide
The relationship between the original variables and the principal components is
illustrated by the loading plots, which are given in Figure 3.23. The score and the
relevant loading plots should be evaluated together following the principal: peaks
with positive loadings are relatively more intense in spectra with positive scores and
relatively less intense in spectra with negative scores (and vice versa).75 Only the
loadings of the first PCs are given here. Both the positive and the negative
hydrocarbon fragments loaded negatively on the relevant first PCs. This in fact
indicates the opposite physical meaning. The positive hydrocarbon ions are
characteristic of the P(MOEP-b-AAEMA) modification, whilst the negative
144
Chapter 3: Surface Fabrication
hydrocarbon lines are associated with the APS treated glass slide. These plots are not
shown here due to the large number of the CxHy+/- ions selected. Considering the
peak intensities and their specificities, the probing of the orientation was performed
using 2 lines: Si+ was selected as the best mark of APS, whilst PO3- was selected as
the best marker for the PMOEP segment.
+ SIMS
APS marker
(a)
- SIMS
PMOEP marker
(b) x 0.1
Figure 3.23: Loadings of selected positive (a) and negative (b) fragments on PC1s.
Figure 3.24 shows the statistically evaluated values of the Si+ and PO3- intensities for
samples 3A-D. Figure 3.24a indicates that the block copolymer was the most
efficient in masking the substrate signal. Figure 3.24b shows that the surface
exposure of the PO3- line was higher for the block copolymer compared to that for
the PMOEP polymer when attached alone. This observation is in good agreement
145
Chapter 3: Surface Fabrication
with the quantitative XPS data. These results clearly indicate that the PMOEP
segment at the ASP surface was more exposed when associated with the PAAEMA
block. The combination of data from these two important characterisation techniques
suggest that the attachment of the P(MOEP-b-AAEMA) block copolymer onto the
aminated glass slide resulted in a conformation of the PMOEP moiety extending
from the surface surrounded by the PAAEMA fragments. The PAAEMA blocks in
the attached copolymer are lying along the substrate surface thus exposing the
phosphate groups more to the vacuum (See Figure 3.18B).
(a)
(b)
Figure 3.24: Normalised intensities of Si+ and PO3- for APS-treated aminated glass
slide and its modifications. (a) Si+ intensity reflects the APS coverage with polymers; (b) PO3
- intensity is a sign of the surface density of the PMOEP segment.
146
Chapter 3: Surface Fabrication
Figure 3.25 shows the negative mass spectrum of sample 3D (P(MOEP-b-AAEMA)
attached aminated slide) and the corresponding distribution of the PO3- fragment
(peak at 79 m/z) across the examined area (100×100 μm). The image reveals that the
lateral distribution of phosphate groups was uniform across the surface.
10 μm
Total (-)
PO3
-
Figure 3.25: Negative mass spectrum of P(MOEP-b-AAEMA) attached aminated slide (sample 3D) and the lateral distribution of the terminating phosphate groups (PO3
-) across the sample (analysed area → 100x100 μm).
147
Chapter 3: Surface Fabrication
3.3.3 Adsorption of Fluorinated Polymers onto PTFE
The adsorption of fluorinated homo and copolymers onto PTFE was carried out by
soaking the PTFE films in 1 mg/mL polymer solution in the solvent of choice
(dichloromethane (DCM), fluorobenzene (FB), methylethyl ketone (MEK) or
dimethylformamide (DMF)) for about 16 hours then washing three times (~10
seconds each) with the same solvent used for soaking and then dried. The films were
analysed by XPS and contact angle measurements. Dynamic light scattering (DLS)
was also used to investigate whether the amphiphilic block copolymers are forming
aggregates in the deposition solvents.
3.3.3.1 Effect of monomer structure and solvents for adsorption
Adsorption of three types of fluorinated homopolymers was investigated: PFS,
PTFPMA and PTFPA onto PTFE using different solvents (DCM, FB and MEK). All
polymers easily dissolved in these solvents. The structure and properties of the
polymers used are shown in Scheme 3.2 and Table 3.14, respectively.
Scheme 3.2: Structures of fluorinated homopolymers.
Table 3.14: Polymer characteristics.
Sample Polymer Mn n PDI No. F a
4B PFS 27417 140 1.06 700
4C PTFPMA 38951 193 1.13 773
4D PTFPA 39959 213 1.06 853 a: Number of fluorines on the polymer chain was calculated from the Mn and structure of repeating units
OO
F
F
FF
H
** *
n
F
FF
F
F* *
n
OO
F
F
FF
H
* n
PFS PTFPMA PTFPA PTFE
*
FF
*F
Fn
148
Chapter 3: Surface Fabrication
Figure 3.26 shows the XPS survey scans of untreated (sample 4A), PFS-adsorbed
(sample 4B3), PTFPMA-adsorbed (sample 4C2), and PTFPA-adsorbed (sample 4D2)
PTFE films.
020040060080010001200Binding Energy (eV)
270280290300500520540560
(A) (B) (C) (D)
C1s O1s
F1s
F2s
FKLL
Figure 3.26: XPS Survey spectra of PTFE films (A) untreated, (B) PFS-adsorbed (sample 4B3), (C) PTFPMA-adsorbed (sample 4C1), (D) PTFPA-adsorbed (sample 4D1).
The untreated PTFE (sample 4A) and PFS-adsorbed (sample 4B3) films showed only
F and C, whereas a small amount of O was observed on the PTFPMA- and PTFPA-
adsorbed PTFE films (samples 4C1 and 4D1, respectively). This is in agreement with
the chemical structures. The atomic concentrations of all samples are summarized in
Table 3.15.
The theoretical values of the F/C ratios of PTFE, PFS, PTFPMA and PTFPA are 2,
0.63, 0.57 and 0.67, respectively. Therefore adsorption of these polymers should
decrease the F/C ratios. The untreated PTFE showed an F/C ratio of 2.2 which is
slightly higher than the theoretical.
149
Chapter 3: Surface Fabrication
Table 3.15: Atomic % of elements from XPS survey scans and atomic C% of different C elements from the high resolution C1s scans.
Sample Polymer Solvent Atomic % from Survey
XPS
Atomic % from
C1s Narrow Scan
F O C F/C C-F2 C-others
4Aa ― ― 68.8 ― 31.2 2.2 99.3 0.7
4B1 DCM 59.2 ― 49.8 1.5 78.2 21.8
4B2 FB 53.7 ― 46.4 1.2 73.3 26.7
4B3
PFS
MEK 57.3 ― 42.7 1.3 67.9 32.1
4C1 DCM 55.5 1.3 43.2 1.3 85.4 14.6
4C2 FB 59.9 0.8 39.3 1.5 97.5 2.5
4C3
PTFPMA
MEK 62.5 0.0 37.5 1.7 99.2 0.8
4D1 DCM 54.7 1.1 44.3 1.2 87.1 12.9
4D2 FB 81.3 0.7 39.1 1.6 98.3 1.8
4D3
PTFPA
MEK 63.9 0.0 36.0 1.8 99.6 0.4 a: Untreated PTFE
Adsorption of PFS reduced the F/C ratios to 1.2-1.5, depending on the solvent used.
In the case of PTFPMA and PTFPA, the presence of O can also be used to identify
polymer adsorption. Both polymers showed the appearance of O (1.1-1.3%) when
adsorbed in DCM with a concomitant reduction in F/C ratios to 1.2-1.3. When FB
was used, there was also some O (0.7-0.8%) seen as well as a reduction in the F/C
ratios (1.5-1.6). But these changes were smaller than those obtained in DCM. In the
case of MEK, there was no O, and the F/C ratios were high (1.7-1.8).
The high resolution C1s XPS spectra were found to be useful for identifying the
polymer adsorption. Figure 3.27 shows the high resolution C1s XPS spectra of
untreated and PFS adsorbed PTFE films. The peak at 292.5 eV corresponds to the C-
F2 from PTFE. Untreated PTFE showed trace amounts of aliphatic carbon (0.7%) at
285.3 eV, indicating the presence of a small hydrocarbon impurity. The C1s
spectrum of the PFS adsorbed PTFE surface can be curve-fitted using five peak
components with binding energies at about 286.1, 286.7, 286.8, 288.9, and 292.5 eV.
These are attributed to C*-H (peak 1), C*-C6H5 (peak 2), C*-CF (aromatic, peak 3),
C*-F (aromatic, peak 4) and C*-F2 (PTFE, peak 5) species, respectively.103
150
Chapter 3: Surface Fabrication
282284286288290292294Binding Energy (eV)
F
FF
F
F
** n
(A)
282284286288290292294Binding Energy (eV)
(B)
1 2 3
4
5
3
4
2 1
**
F F
F Fn 5
5
5
Figure 3.27: C1s narrow scans of PTFE films (A) untreated (sample 4A) and (B) PFS adsorbed (sample 4B3).
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
PFS
PTFPMA
PTFPA
DCM FB MEK
Sample 4B1 Sample 4B2 Sample 4B3
Sample 4C1 Sample 4C2
Sample 4D1 Sample 4D2
Sample 4C3
Sample 4D3
282284286288290292294Binding Energy (eV)
Figure 3.28: C1s narrow scans of PTFE films after fluorinated homopolymer adsorption using different solvents.
151
Chapter 3: Surface Fabrication
Figure 3.28 shows the C1s spectra of all samples studied. The atomic % of C-F2 and
carbons from polymers (C-others) are shown in Table 3.15. PFS adsorption onto
PTFE occurred in all solvents and it was the highest in MEK (32% C-others). The
best adsorptions of PTFPA and PTFPMA were observed when DCM was used as the
solvent. Only a very small adsorption of these polymers was observed in FB, and
none in MEK.
The contact angle measurements of sample 4C1 (PTFPMA adsorbed PTFE in DCM)
was carried out. The advancing and receding angles were found to be 97 ± 3° and 91
± 4°, respectively that were slightly lower than those of PTFE (~110°) (shown in
Table 3.19).
3.3.3.2. Adsorption of PFS with varying Mn’s
The effect of PFS Mn on adsorption onto PTFE was examined using three different
molecular weights (Table 3.16). MEK was the adsorption solvent of choice, since it
showed the best PFS adsorption. The atomic % of C-F2 and C-others from the high
resolution C1s peaks are shown in Table 3.16. The results are the average of four
samples.
Table 3.16: Properties of PFS and atomic % from C 1s scans of PFS adsorbed PTFE films.
Sample Adsorbed PFS Atomic % from C 1s scans
Mn n PDI no. F C-F2 STD C-others STD
4D 6514 32 1.09 161 88.4 2.2 11.6 2.2
4E 12093 61 1.05 305 83.9 2.4 16.1 2.4
4B 27417 140 1.06 700 67.9 3.1 32.1 3.1
Figure 3.29 shows the plot of units of PFS against atomic % of C-others. The
adsorption of PFS onto PTFE was proportional to the molecular weight in the range
investigated.
152
Chapter 3: Surface Fabrication
0
5
10
15
20
25
30
35
40
0 20 40 60 80 100 120 140 160
Unit of PFS C
oth
ers
XP
S
Figure 3.29: Relationship between molecular weight of PFS and adsorbed amounts of PFS onto PTFE films.
3.3.3.3. Adsorption of P(FS-b-tBA) and P(FS-b-AA) block copolymers onto
PTFE
Table 3.17 shows the properties of the P(FS-b-tBA) and P(FS-b-AA) block
copolymers used in this study. Two different PtBA block segments were used:
sample 4F has a 1.7 times longer PtBA segment than that of sample 4G. Samples 4H
and 4I were obtained from the hydrolysis of samples 4F and 4G, respectively. All
copolymers were synthesised from the same PFS macro-RAFT (i.e. the same Mn and
PDI for the first block).
Table 3.17: Properties of PFS block copolymers.
Polymer PFS
1st block
PtBA or PAA
2nd block
Mn N no. F PDI Mn n
PDI of
full block
4F P(FS-b-tBA) 19869 101 505 1.04 30377 237 1.11
4G P(FS-b-tBA) 19869 101 505 1.04 18099 141 1.07
4H P(FS-b-AA) 19869 101 505 1.04 17078 1 237
4I P(FS-b-AA) 19869 101 505 1.04 10160 1 141 1: calculated by assuming 100% cleavage of tBA side-chains
The attachment of P(FS-b-tBA) and PtBA was carried out in MEK. Since sample 4H
(P(FS101-b-AA237)) with a long PAA chain was only soluble in DMF, the attachment
153
Chapter 3: Surface Fabrication
was carried out in this solvent. Sample 4I was soluble in DMF and MEK hence both
solvents were examined. Figure 3.30 shows the high resolution C1s XPS spectra of
these samples and the atomic % of C-F2 (from PTFE) and C-others (from polymer)
are shown in Table 3.18.
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
282284286288290292294Binding Energy (eV)
(A) (B)
(C) (D)
(E)
1 2 3 4 5
6
7
1 2 3 4 5
6
7
Figure 3.30: C1s narrow scans of block copolymer adsorbed PTFE films (A) P(FS101-b-tBA237) (sample 4F), (B) P(FS101-b-tBA141) (sample 4G), (C) P(FS101-b-AA237) (sample 4H), (D) P(FS101-b-AA141) (sample 4I1) and (E) P(FS101-b-AA141) (sample 4I2).
154
Chapter 3: Surface Fabrication
Table 3.18: Atomic % of C-F2 and C-others.
Sample Polymer Solvent C-F2 C-others
4F P(FS101-b-tBA237) MEK 78.7 21.3
4G P(FS101-b-tBA141) MEK 78.4 21.6
4H P(FS101-b-AA237) DMF 44.1 55.9
4I1 P(FS101-b-AA141) DMF 49.2 50.8
4I2 P(FS101-b-AA141) MEK 63.3 36.7
The C1s high-resolution spectra of both P(FS-b-tBA) and P(FS-b-AA) adsorbed to
the PTFE surface can be curve-fitted into seven peak components. Peaks 1-5 with
binding energies at about 285.0 285.6, 286.1, 286.6, and 286.8 eV are attributed to
C*-H (PtBA or PAA, peak 1), C*-C(=O)O (PtBA or PAA, peak 2), C*-H (PFS, peak
3), C*-C6H5 (PFS, peak 4), and C*-CF (aromatic of PFS, peak 5) species. Peak 6 at
288.8 eV is attributed to both C*-F (aromatic of PFS) and C(=O)OH/C(=O)O- (from
PtBA/PAA) species. Peak 7 at 292.5 eV is from C*-F2 (PTFE).
Both P(FS-b-tBA) block copolymers (samples 4F and 4G) had similar by adsorbed
amounts (C-others: ~21%) regardless of the PtBA chain length. P(FS-b-AA) block
copolymers (samples 4H, 4I1, and 4I2) showed a higher adsorption onto PTFE
compared to those of the P(FS-b-tBA) block copolymers. Samples 4H and 4I1 (DMF)
showed similar adsorption amounts (C-others: ~50%). Sample 4I2 (MEK) showed
less adsorption (C-others: 37%) compared to sample 4I1 (DMF).
The effect of block copolymer adsorption on the surface wettability of the modified
PTFE substrates was also characterized using water contact angle measurements.
Figure 3.31 shows photographic images of advancing and receding water droplets
(both 5 μL) on untreated PTFE, P(FS101-b-tBA141)-adsorbed (sample 4G), and
P(FS101-b-AA141)-adsorbed (sample 4I1) films. The advancing and receding angles
are shown in Table 3.19 that also contains literature values of the advancing angles
of PFS, PtBA, and PAA.
155
Chapter 3: Surface Fabrication
Advancing Receding
(A)
(F) (E)
(C) (D)
(B)
Figure 3.31: Water droplet (5 μL) profiles on the surfaces of untreated PTFE (sample 4A) (A) advancing and (B) receding, P(FS101-b-tBA141) adsorbed PTFE (sample 4G) (C) advancing and (D) receding, and P(FS101-b-AA141) adsorbed PTFE (sample 4I1) (E) advancing and (F) receding.
156
Chapter 3: Surface Fabrication
Table 3.19: Advancing and receding water contact angles of polymer-adsorbed PTFE films.
Sample Adsorbed
Polymers
Deposition
Solvent
Advancing
Angle (°)
Receding
Angle (°)
Hysteresis
(°)
4A a ― ― 109 ± 6 111 ± 6
4C1 PTFPMA DCM 97 ± 3 91 ± 4 6 ± 5
4F P(FS101-b-tBA237) MEK 88 ± 2 86 ± 5 2 ± 5
4G P(FS101-b-tBA141) MEK 98 ± 3 90 ± 6 8 ± 7
4H P(FS101-b-AA237) DMF 91 ± 6 31 ± 3 60 ± 7
4I1 P(FS101-b-AA141) DMF 88 ± 5 28 ± 3 60 ± 6
4I2 P(FS101-b-AA141) MEK 101 ± 4 49 ± 3 52 ± 5
PFSb 99104,105
PtBAc 88 ± 3106
PAAd 1554, 48107 a: Untreated PTFE film, b: PFS homopolymer (Ref: 104) and PFS grafted surface (layer thickness = 89 nm) (Ref: 105), c: PtBA homopolymer, d: spin coated PAA thin film (Ref: 54) and PAA grafted polyethylene (Ref: 107)
The advancing contact angle of water on a PTFE surface (sample 4A) was
determined to be 109 ± 6°. No hysteresis was observed, indicating a homogeneous
and stable surface. When P(FS101-b-tBA237) with the long tBA segment was adsorbed
(sample 4F), the advancing angle decreased to 88°. This is the same as the literature
value for the PtBA contact angle. For the P(FS101-b-tBA141) adsorbed surface
(sample 4G), the advancing angle was 98° which is the same as that of PFS (99°).
However, it showed a slightly larger although still small hysteresis (8°).
Both P(PFS-b-AA) polymers adsorbed to PTFE surfaces using DMF as the
deposition solvent (samples 4H and 4I1) showed advancing angles of ~90°. Receding
angles were 31° and 28°, respectively, indicating reorganization of the block
copolymer chains during the contact angle measurment. The difference in chain
length of the PAA segments did not show any significant effect on the contact angles.
Sample 4I2 (MEK) showed advancing and receding angles of 101 ± 4° and 49 ± 3°,
respectively: these are higher than those of sample 4I1(DMF).
157
Chapter 3: Surface Fabrication
To further investigate the chain reorganisation phenomenon, modified films were
soaked in MilliQ water at room temperature for 2 days and blot dried before contact
angle measurements (Table 3.20). Samples 4H and 4I1 (DMF) showed ~17° lower
advancing angles (73 ± 3°) compared to the dry samples. Receding angles also went
down slightly to 21 ± 2°. Again, regardless of the AA segment length, the results
were the same. The advancing angle of sample 4I2 (MEK) was 92 ± 4°. This was a 9°
decrease compared to the dry sample. However, it is still highly hydrophobic. The
receding angle was 50 ± 6°, which did not change from that of the dry sample.
Table 3.20: Advancing and receding contact angles of P(FS-b-AA) adsorbed surfaces after soaking in MilliQ water for 2 days.
Sample Adsorbed
Polymer
Deposition
Solvent
Advancing
Angle (°)
Receding
Angle (°)
Hysterisis
(°)
4H P(FS101-b-AA237) DMF 73 ± 3 21 ± 2 52 ± 4
4I1 P(FS101-b-AA141) DMF 73 ± 4 21 ± 2 52 ± 4
4I2 P(FS101-b-AA141) MEK 92 ± 4 50 ± 6 42 ± 7
The characterisation of block copolymers formed in DMF and MEK were performed
using dynamic light scattering (DLS). The number-average hydrodynamic diameter
(DH) is shown in Table 3.21. In DMF, sample 4H with the long PAA segment
showed a larger DH (30 nm) compared to sample 4I1 (24 nm). Sample 4I2 (MEK)
showed a slightly smaller DH (21 nm) compared to the DMF sample.
Table 3.21: DH of P(FS-b-AA) in DMF and MEK.a
Sample Polymer Solvent DH (nm)
4H P(FS101-b-AA237) DMF 30
4I1 P(FS101-b-AA141) DMF 24
4I2 P(FS101-b-AA141) MEK 21 a: All samples were run 10 times and the STD = ± 1 nm
158
Chapter 3: Surface Fabrication
3.4 Discussion
3.4.1 Layer-by-Layer (LbL) Assembly
LbL assembly is a well-recognised and robust surface modification technique. There
is no limitation in the substrate shapes and sizes, and indeed it has found a wide
range of applications including in biomaterials. In this study, carboxylate- and
phosphate-containing surfaces were prepared using PEI as the polycation and PAA,
PMAEP or PMOEP as the polyanion.
3.4.1.1 LbL assembly of PAA
The LbL assemblies of PEI and PAA were produced under three different pH
conditions: pH 5.5, 7 and water without adjustment. As expected for a weak
polyanion, the pH of the solution strongly affected the deposition of the PAA film by
altering the degree of ionisation of the PAA.
At low pH (unadjusted water, pH 3.3) where PAA is less than half ionised, the
polymer chains are in a coiled conformation which contains a high numbers of loops.
Hence more chains are required to form layers on the surface and thus results in a
thick film. Choi and Rubner suggested that when the degree of ionization falls below
a critical value, dramatic increases in bilayer thickness occur, driven by the less
densely charged polymer being in contact with the high charge density polymer of
opposite charge.108 Moreover, at low pH, the unionized carboxylic acid groups can
form strong hydrogen bonding between the PAA chains and that makes for an even
thicker layer (shown in Figure 3.32A).
At pH 7, the degree of ionisation is 100 %. The chains can be more elongated (and
have fewer loops) and hence they interact strongly with the PEI film resulting in
thinner PAA films (Figure 3.32B). There should be no hydrogen bond formation at
this pH.
159
Chapter 3: Surface Fabrication
+ +
+ + + +
+ +
+ + +
+ +
+ +
+ +
+ +
+ +
-
-
-
-
- -
- -
- - -
- -
- - -
- -
- -
- -
- - COOH COO- COOH COO-
COOH
-
- -
- -
COOH
-
- -
- -
COOH
+ +
+ + + +
+ +
+ + +
+ +
+ +
+ +
+ +
+ - - -
- -
- -
-
-
- - - - -
- - - -
- - -
- - -
- -
-
A B
pH 7 pH 3.3
- -
-
-
- -
Figure 3.32: Schematic representation of PAA deposition onto PEI deposited surface at different pH.
Hydrogen bonding plays an important role in LbL formation in many systems. There
are cases that use pure hydrogen bonding instead of electrostatic interactions to form
polyelectrolytes layers.109-112
Kim et al.19 showed that the thickest dendrimer/PAA film was obtained by the
deposition of dendrimers at pH 8 and PAA at pH 4. This study showed similar results
in that the thickest film was from PEI deposition at pH 9.9 and PAA at pH 3.3.
3.4.1.2 LbL assembly of PMAEP and PMOEP
The XPS investigation of the LbL assemblies of both PEI-PMAEP and PEI-PMOEP
prepared in unadjusted water showed a large increase in atomic C%, indicating thick
film formation. The pHs of the PMAEP and PMOEP solutions were 2.7 and 2.8,
respectively. Since the pKa values of dihydrogen phosphates are 1.7 and 7.01 (values
for hydroxymethyanephosphonic acid113), at least one proton on the phosphate
groups of PMAEP and PMOEP are deprotonated. The ionic interaction of the
protonated amines and the phosphates was evident from the XPS high resolution N1s
spectra, as well as from the IRRAS data.
AFM images revealed a smooth topography for the PMOEP LbL film surface,
whereas PMAEP layer was patchy, showing aggregates of polymers. The PMAEP
chains contain a high degree of carboxylic acid groups, most likely randomly
distributed. The degree of ionization of PAA at pH 2.5 was measured to be 20-30
160
Chapter 3: Surface Fabrication
%.20 In comparison to PMOEP, the PMAEP chains would be less ionised. Therefore
the conformation of the polymer chains should be more coiled and hydrogen bonding
between the chains would be also possible. This explains the topography observed by
AFM.
Since the aim of this experiment was only to create a carboxylate- or phosphate-
containing surface, only one layer of each-polyanion and polycation-was deposited.
However, in the literature it has been shown that a consistent surface charge
distribution (i.e. uniform polymer layer coverage) does not fully develop until after
the adsorption of a few bilayers.19,114,115 Therefore, it is not impossible that the
surfaces created here may also show PEI properties as well as some Si-OH.
Moreover, the PEI-PMAEP LbL film would contain large amounts of free-carboxylic
acid groups (see Figure 3.33). Nevertheless, the XPS investigation revealed that
these LbL systems produced surfaces that contain high amounts of carboxylate or
phosphate groups.
Figure 3.33: Schematic representation of PEI-PAA LbL showing free-functional groups.
In summary, the LbL assembly approach successfully immobilised carboxylate- and
phosphate-containing polymers on the substrate surface. Since strong ionic
interaction of polyelectrolytes is stable in solution, the mineralisation study of these
water-soluble polymers is made possible in SBF by this technique. Although these
LbL surfaces may show small amounts of hydroxyl and amine groups from either the
substrate or PEI, respectively, it is expected to have dominant carboxylate and
phosphate groups.
OH OH NH2NH2NH2 NH2
PO4H2
COOH COOH HOOC COOH PO4H2PO4H2
PO4H2
161
Chapter 3: Surface Fabrication
3.4.2 Block Copolymer Attachment onto Aminated Slides
3.4.2.1 Qualitative analysis of attached polymers
The XPS investigation revealed successful immobilization of the polymers onto the
aminated slides. From the survey scans, the increase in C% and the concomitant
decrease in N% were evident when polymers were attached.
The attached amounts of PAAEMA and PMOEP homopolymers were similar, based
on the increase in C% (samples 3B and 3C, respectively). The high resolution spectra
of the N1s peak of the PAAEMA attached slide showed an increase in the amine
peak, indicating PAAEMA was attached through reductive amination of the keto
group. The PMOEP attached slide showed an increase in the protonated amine peak,
which indicated PMOEP attachment was through ionic interactions. Although the
attachment reaction was carried out in the aprotic solvent DMF and hence is not
expected to cause deprotonation of the phosphate groups of the PMOEP moieties, it
is possible that proton transfer between the phosphate groups of PMOEP and the
amine groups of APS can occur.
From the atomic C% from the survey scans, the degree of functionalisation was
higher for all block copolymers compared to homopolymers, possibly due to the
much higher Mn’s of the block copolymers. Since both the PAAEMA and PMOEP
homopolymers are capable of attaching to the aminated slides, in the case of the
block copolymers, it was envisaged that a competition between reductive amination
(leading to attachment of the PAAEMA block) and proton transfer (leading to
attachment of the PMOEP block), might exist.
One interesting finding is the fact that the amounts of attached PMOEP copolymers
(samples 4D and 4E) were higher than those for the PMAEP copolymers (samples 4F
and 4G). Since PMAEP contains high amounts of carboxylic acid groups, the
differences observed between the PMOEP and PMAEP copolymer behaviour may
indicate better proton transfer of phosphates to amines compared to carboxylic acid
groups.
The ionic interaction of these block copolymers was also evident from the high
resolution N1s spectra. The atomic % of amines and protonated amines of block
copolymer-functionalised slides were somewhere between those of the PAAEMA
162
Chapter 3: Surface Fabrication
and PMOEP-functionalised surfaces. This indicates that both reactions are occurring
for these samples.
3.4.2.2 Conformation of attached block copolymers
In this study where the block copolymers contain phosphate groups, ToF-SIMS is the
“gold-standard” surface characterization technique because these groups have been
found to be ideal markers for the conformational analysis of phospholipids.77 ToF-
SIMS provides a higher surface specificity (1~2 nm) and sensitivity (107 – 1011
atoms/cm2) than XPS. Together with PCA, it makes a powerful tool for the
elucidation of both the conformation and orientation of adsorbed molecules such as
proteins and lipids on the surface.74,75,79,116
XPS revealed that both PAAEMA and PMOEP/PMAEP segments are capable of
interacting with amines through reductive amination and proton transfer, respectively.
Therefore, the conformation of the adsorbed block copolymers depends ultimately on
the relative reactivity of these two functional groups. The three types of
conformational models are shown again in Figure 3.34:
Figure 3.34: Possible conformations of block copolymers reacted with aminated slide.
where block copolymers were attached through (A) covalent interaction of
PAAEMA block, (B) ionic interaction of PMOEP block and (C) combined
interactions.
The positive SSIMS spectra of these samples showed differences in intensity of the
Si+ ion (m/z = 28). The block copolymers showed substantial reduction in this peak,
O
NH
O
PAAEMA
PMAEP/PMOEP
(A) (B) (C)
O
OP O
O-O
NH3+
163
Chapter 3: Surface Fabrication
which indicates higher polymer coverage. The negative SSIMS spectra of PMOEP
and block copolymer functionalised surfaces clearly showed evolution of the PO2-
(m/z = 63) and PO3- (m/z = 79) peaks.
Both score plots for positive and negative ion mass spectra showed distinctive
differences between the samples investigated and the most pronounced differences
are found between the untreated and block copolymer-attached aminated slides.
From the loadings plots, Si+ and PO3- are selected as the best markers for APS (i.e.
aminated slide) and the PMOEP segment, respectively.
From the statistically evaluated values of the Si+ intensities, the block copolymer was
found to be the most efficient in attenuating the substrate signal. The PO3- intensity
for the block copolymer was higher than that for PMOEP homopolymer attachment
alone. This observation is in agreement with the quantitative XPS data (1.3 and 1.5
atom % of P for PMOEP and block copolymer immobilized slides, respectively).
These data strongly support the view that the PMOEP segment on the aminated slide
more exposed when associated with the PAAEMA block. When the conclusions
from the data generated by the different techniques are combined, together they
suggest that the immobilization of the P(MOEP-b-AAEMA) block copolymer onto
the aminated slide resulted in a conformation where the PMOEP moieties are coiled
away from the surface and surrounded by the PAAEMA fragments situated much
closed to the surface (Figure 3.34A).
The surfaces created from this technique provide phosphate-containing polymer
chains and hence are suitable for mineralisation study of soluble PMAEP and
PMOEP. It is in contrast to the LbL films that employ deprotonated phosphate
groups for film formation by ionic interactions.
164
Chapter 3: Surface Fabrication
3.4.3 Adsorption of Fluorinated Polymers onto PTFE
The adsorption of fluorinated homo and block copolymers onto PTFE was carried
out by soaking the PTFE in the relevant polymer solution. This technique has the
great advantage that it does not require any special instrument, is robust, and can be
applied to any shapes such as microporous expanded PTFE (ePTFE). This is highly
relavant in the event of its finding a commercial viable application.
3.4.3.1 Homopolymer adsorption
PTFE is well known for its low surface tension (of 18.6 dynes/cm117), which
provides its non-stick property. In this study, three types of fluorinated
homopolymers were tested for adsorption onto PTFE in different solvents (DCM, FB
and MEK). PFS showed the greatest adsorption onto PTFE in all solvents. From XPS,
the highest PFS adsorption was found when MEK was used as the solvent. For
PTFPMA and PTFPA, some adsorption was observed only when DCM was used.
Since these polymers had higher amounts of fluorine atoms in each polymer chain
(773 and 853, respectively) compared to PFS (700), this would appear to indicate
that the structure of the repeating unit affects the adsorption.
There are two factors that need to be considered regarding the solvent: swelling of
PTFE and solubility of the polymer (which affects both adsorption and desorption).
Since the same solvent was used for adsorption and washing, a good solvent for
polymer enhances the desorption during washing. A good swelling solvent for PTFE
allows the fluropolymers to entangle and anchor well. However, it is also possible
that this solvent will remove adsorbed polymers in the washing step.
Nasef investigated the swelling of PTFE film in different solvents.118 The sorbed
liquid was found to be 0.35 wt% for DCM, 0.15% for methanol, and 0.23% for
benzene. Thus DCM was the best solvent. From this trend, it can be predicted that
PTFE swelling is higher in DCM than in MEK. It is also possible that FB is a better
swelling agent than benzene.
In this study, the adsorption of PTFPMA and PTFPA was only observed in DCM.
Although both DCM and fluorobenzene swell PTFE, the results may indicate that
interaction between the fluorinated solvent (i.e. FB) and the polymers (PTFPMA and
165
Chapter 3: Surface Fabrication
PTFPA) are so strong, they prevent adsorption onto the PTFE. There was no
adsorption of these polymers when MEK was used as the solvent, this is in
agreement with the prediction that MEK is a low-swelling solvent for PTFE.
The contact angle of PTFPMA-adsorbed PTFE was higher than that of PtBA. If only
the fluorine atoms of the PTFPMA were adsorbed into the PTFE chains (Figure
3.35A), the contact angle of this surface would be expected to be close to that of
PtBA (88°), assuming uniform polymer coverage since XPS showed that the total
atomic C% from the adsorbed polymer is over 10%. The fact that the contact angle
obtained was higher (θadv = 97°), could indicate that the whole PTFPMA chain is in
fact entangled (Figure 3.35B).
Figure 3.35: Schematic representation of fluoropolymer adsorption onto PTFE by (A) fluorine adsorption and (B) chain entanglement.
In the case of PFS, the highest adsorption was observed when MEK was used.
Jankova et al. investigated the solubility of linear PFS synthesised by ATRP and
found that FB, MEK, and DCM dissolve 0.417, 0.143, and 0.009g of PFS per gram
at room temperature, respectively.119 This supports the suggestion that in FB, PFS is
well solvated and in a stretched conformation, whereas in DCM, the chains are more
coiled. In MEK, although the chains are possibly only slightly coiled, the swelling of
the PTFE film is less, therefore more chains are packed on the surface, compared to
those adsorbed from DCM (Figure 3.36).
F F F F F F F F F F F F F F
F F F
F
F
F F
F F
F
F F
F F F F
(A) (B)
PTFE
166
Chapter 3: Surface Fabrication
Figure 3.36: Possible adsorption behaviour of PFS onto PTFE from different solvents.
The effect of PFS chain length on adsorption onto PTFE was investigated using three
different molecular weights of polymers. The chain entanglement Ne of PTFE is
~110.67 The polymers below this length which were studied showed much less
adsorption compared to the highest one. However, more measurements using
polymers with molecular weights closer to the Ne are required to confirm if the
adsorption is in fact by entanglement. Since the MEK used for this study is a low-
swelling solvent for PTFE, the entanglements may be minimal. The solubility of
polymers in MEK may also play a role: the higher the molecular weight, the less
soluble, hence the higher the adsorption.
The most important interactions for fluoropolymer adsorptions are hydrophobic and
fluorine-fluorine interactions. PFS was the most hydrophobic polymer studied and it
showed the best adsorption. It is also possible that in PFS, fluorine atoms are easier
to polarise due to the presence of the aromatic ring when in close contact with the
PTFE. Therefore, the fluorine-fluorine interaction between PFS and PTFE becomes
stronger than for the other polymers. Since PTFPMA and PTFPA contain carbonyl
groups (i.e. they are more hydrophilic), some repulsion between these polymers and
highly hydrophobic PTFE may occur. This results in competition with the fluorine-
fluorine interactions.
3.4.3.2. Block copolymer adsorption onto PTFE
XPS analysis showed that the amounts of adsorbed P(FS-b-tBA) from MEK with
different PtBA chain lengths were similar. However, the contact angle measurements
of these samples were different. The advancing angle of the copolymer-adsorbed
DCM FB MEK
(A) (B) (C)
PTFE
Swollen Region
167
Chapter 3: Surface Fabrication
PTFE with the longer PtBA segment (n = 237, sample 4F) was close to that of PtBA,
whereas the shorter PtBA (n = 141, sample 4G) was closer to that of PFS. The
homopolymer of PtBA did not adsorb to PTFE (data not shown). The experimental
results indicate that PFS segments adsorb onto the PTFE and PtBA segments are
extending from the surface. The block copolymer with the long PtBA segments
covers the surface well, whereas the short tBA segments do not fully cover the
surface, leaving some PFS regions. Since both PFS and PtBA are soluble in MEK,
there should be no polymer-aggregates in this solvent.
A higher adsorption of P(FS-b-AA) copolymers was observed compared to that of
P(FS-b-tBA), which suggests possible aggregation of P(FS-b-AA). As mentioned in
the Introduction (Section 3.1), micelles are known to adsorb readily onto surfaces.
Whittaker et al.120 investigated the micellisation of P(S-b-AA) in DMF, which is a
good solvent for both chains, and toluene, which is only good for PAA. In their study,
the number-average hydrodynamic diameters (DH) of P(S153-b-AA175) and P(S153-b-
AA234) in DMF were 6.2 and 6.3 nm, respectively, whereas in toluene, they were
42.5 and 44.5 nm. In this study, DLS analyses showed that the DH of the block
copolymers in DMF and MEK were between 21-30 nm, indicating formation of
some kind of aggregates. DMF is a good solvent only for the PAA segments,
whereas MEK is good only for the PFS segments. Therefore, it could be predicted
that inverse-structures of aggregates in DMF and MEK occur with the outer layer
PAA segments in DMF, and PFS segments in MEK.
Both P(FS-b-AA)-attached films prepared in DMF showed advancing angles of~ 90°.
This suggests that the aggregates with PAA segments as the outer layer possibly
reorganized when adsorbed and dried onto PTFE exposing the PFS segments on the
surface in order to reduce surface tension (shown in Figure 3.37). Another possibility
is that when these aggregates were adsorbed, they may have lost their aggregate
structures.
168
Chapter 3: Surface Fabrication
DMF
MEK
Drying
Drying
PAA
PFS
Figure 3.37: Aggregate adsorption onto PTFE surfaces in different solvents.
Receding angles of these surfaces were ~30°, indicating some reorganization of the
polymer chains during the contact angle measurement. After soaking these samples
in water, the advancing angles reduced to 73° (~17° decrease). This also suggests the
existence of flexible polymer chains on the PTFE surfaces.
P(FS101-b-AA141) adsorbed onto PTFE in MEK showed the highest advancing angle
(101°). In MEK, aggregates with the PFS segments in the outer layer possibly
adsorbed onto PTFE without structural change. The receding angle was 50°, and this
did not change after soaking in water, which indicates that full rearrangement occurs
during the contact angle measurement.
Hydrophilicity/hydrophobicity of the PFS containing amphiphilic block copolymers
adsorbed PTFE is found to be reversibly tuneable depending on the environment (e.g.
water vs air). Since the carboxylic acid groups on these chains are free, it is suited for
mineralisation study of these groups.
169
Chapter 3: Surface Fabrication
3.5 Conclusions
Three techniques for the fabrication of modified surfaces using well-defined
functional polymers were presented in this study:
• Layer-by-Layer (LbL) assembly of soluble phosphate- and carboxylate-
containing homopolymers
• Coupling reactions of keto-containing block copolymers onto aminated slides
• Adsorption of fluorinated homo and block copolymers containing carboxylic
acid groups onto PTFE
LbL assembly was monitored by XPS. The thickness of PAA film was found to be
strongly dependent on the pH during deposition. This is in agreement with the
literature reports for weak polyanions. Phosphate-containing LbL films were
successfully prepared using soluble PMAEP and PMOEP with PEI. AFM
investigations showed the formation of a patchy PMAEP film, whereas the PMOEP
film was smooth. Since PMAEP contains large amounts of carboxylic acid groups,
these groups are not deprotonated at a low deposition pH and hence this affected the
film morphology.
Block copolymers consisting of PAAEMA and either PMAEP or PMOEP were
attached onto aminated glass slides, by the coupling reaction of the keto and amine
groups to form imines which were then reduced to form stable secondary amines.
Thus free-phosphate polymers immobilised on a surface was successfully
accomplished. XPS investigations revealed successful attachments of block
copolymers, as well as both PAAEMA and PMOEP homopolymers. Since the
PMOEP homopolymer was found to be capable of attaching onto aminated slides
through electrostatic interactions, it is concluded that the conformation of the
attached block copolymer depends on the relative reactivity of the two functional
groups. ToF-SIMS investigations of the P(MOEP-b-AAEMA)-attached aminated
slide showed that the PMOEP segment was more extended to the vacuum and
PAAEMA segment more attached to the aminated slide surface.
Adsorption of three types of fluorinated homopolymers (PFS, PTFPMA, and PTFPA)
was investigated and it was found that PFS showed the best adsorption behaviour
170
Chapter 3: Surface Fabrication
onto PTFE. The attractive interactions governing the adsorption are thought to be
hydrophobic and fluorine-fluorine interactions. Because PFS is the most hydrophobic,
and the fluorine atoms in the aromatic rings are possibly easier to polarise, this
creates stronger fluorine-fluorine interactions. Both PTFPMA and PTFPA contain
ester groups which may create some repulsive force with the PTFE. These polymers
were only found to adsorb in DCM, possibly by entanglement. P(FS-b-AA) block
copolymers were also successfully adsorbed onto PTFE. Contact angle
measurements showed large hysteresis indicating a fast reorganisation of the
adsorbed block copolymer chains.
These techniques successfully created surfaces consisting of carboxylate and
phosphate groups. The resulting surfaces are suitable for the in vitro assessments
such as for mineralisation. The surface modification approach using well-defined
polymers by these techniques have shown to be robust and hence will find wider
applications.
171
Chapter 3: Surface Fabrication
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Chapter 4: In Vitro Mineralisation
Chapter 4: In Vitro Mineralisation
4.1 Introduction
4.1.1 Mineralisation
An ideal implant surface for hard tissue applications should provide optimal
conditions for osseointegration. One feature, which has been demonstrated to be
important, is rapid calcium phosphate layer deposition. Small changes in surface
properties have been shown to improve calcium phosphate (CaP) nucleation and
growth, and the presence of anionic functional groups on the materials surface
especially phosphates (one of the components of hydroxyapatite; HAP) has been
shown to enhance mineral growth.1-4
The first step in mineralisation, before growth proceeds, is nucleation.
Heterogeneous nucleation occurs on a substrate surface under normal body fluid
conditions. The interaction between the surface and the ions lowers the interfacial
energy; therefore, nucleation can proceed at a lower ionic concentration than that of
supersaturation.5 This process has important consequences for biomaterials as well as
in biomineralisation. In the case of bone formation through the behaviour of the
biomineralisation process, matrix proteins such as osteonectin and phosphoproteins
attach to the collagen matrix and act as nucleators.5,6 Synthetic materials, such as
polymeric biomaterials, generally do not possess nucleation sites. However, it has
been suggested that the presence of polar functional groups on the surface is a key
factor in nucleation. In such cases, nucleation is thought to be triggered by
electrostatic interactions.7 Once the apatitic nuclei are formed, the apatite crystals
grow by uptake of calcium and phosphate ions from the solution.
There are many different inorganic phases formed from calcium and phosphate
(usually referred to as the calcium phosphates, CaP). Some are listed in Table 4.1,
together with the atomic calcium to phosphate ratios (Ca/P ratio). Substitution of
other ions within the apatite lattice commonly occurs, for example, biological apatite
is usually substituted with other ions.8 Bone-like apatite is, in fact, carbonated HAP.
Carbonate ions can be substituted into the apatite lattice in two different positions:
substitution of either the OH, or phosphate groups.9 In simulated body fluid (SBF)
studies, partial substitution of Ca2+ with Mg+ ions is often observed.10 These
177
Chapter 4: In Vitro Mineralisation
substitutions have been found to reduce both the crystallinity of the apatitic crystal11,
and inhibit the crystal growth.12
Table 4.1: Different calcium phosphate structures.8
4.1.2 Simulated Body Fluid (SBF)
The need to predict how a material will behave in vivo has led to the use of the so-
called simulated body fluid techniques (SBF) as the most common method for testing
CaP mineralisation in vitro.13 In such studies, the test material is immersed in a
solution containing the inorganic ions found in blood plasma, controlled at
physiological pH, temperature and concentration. Calcium phosphate growth on the
surface of the material is subsequently investigated. Currently, several different SBF
techniques have been developed (Table 4.2). Corrected SBF (c-SBF) has been
proposed to the International Organization for Standardization as the most suitable
solution for in vitro measurement of the apatite-forming ability of materials.14
Revised SBF (r-SBF), which has equal ion concentrations to that of human blood
plasma, was chosen for this study to mimic in vivo conditions as well as to maintain
consistency with the previous study.15
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Chapter 4: In Vitro Mineralisation
Table 4.2: Ion concentrations of human blood plasma, different SBF solutions,14 and SPF.16
Ion Concentration (mM)
Na+ K+ Mg2+ Ca2+ Cl- HCO3- HPO4
2- SO42-
Human Blood Plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
Original SBF 142.0 5.0 1.5 2.5 148.8 4.2 1.0 0
Corrected SBF (c-SBF) 142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5
Revised SBF (r-SBF) 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
Newly improved SBF (n-SBF) 142.0 5.0 1.5 2.5 103.0 4.2 1.0 0.5
Simulated physiological fluid
(SPF)
152.0 5.0 1.5 2.5 136.0 27.0 2.5 0.5
Although this test only studies inorganic apatite growth, without any interaction of
biomolecules such as proteins, the formation of calcium-phosphate (CaP) minerals or
a bone-like apatitic layer in SBF is accepted as an indication of the bone bonding
ability of the material in vivo.17 Kokubo and Takadama14 has recently reviewed the
quantitative correlation of apatite formation in SBF with in vivo bone bioactivity.
Studies in this article included the evaluation of: bioglasses and other ceramic
materials such as sintered HAP, HAP/β-tricalcium phosphate and calcium sulfate.
For all of these materials, apatite formation on their surfaces in SBF is correlated
well with their in vivo bone bonding ability.
This type of correlation is also observed for polymeric materials. MOEP-grafted
polyethylene (PE) and poly(ethylene terephthalate) (PET) both form HAP in SBF
and have been shown to have bone bonding ability in vivo.1,18,19 Copolymers of
MOEP and 1-vinyl-2-pyrrolidinone,20,21 MOEP or MAEP and 2-hydroxyethyl
methacrylate (HEMA)22 failed to induce CaP nucleation in SBF. These copolymers
were subsequently reported as having performed poorly in in vivo tests. These
observations also appear to confirm the correlation between apatite formation in SBF
with in vivo behaviour.
In some cases, dynamic SBF is used. This is thought to create a more similar
environment to the living body than that of static SBF tests.23-26 The topology of the
material becomes more relevant than that of the chemistry in this system. For
example, an alkaline-treated micropatterned surface of a titanium alloy showed HAP
growth over all of the surface in static SBF, but was more pronounced in the bottom
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Chapter 4: In Vitro Mineralisation
of the microholes than the flat surfaces in dynamic SBF.27 This micropatterned
surface showed the best bone bonding ability in vivo compared to those without
patterned surfaces.
CaP phases other than HAP have also been found to form in SBF, especially in the
early stage of mineralisation. Brushite and octacalcium phosphate (OCP) are thought
to be precursors of HAP.8,28,29 OCP is known to promote osteoblast differentiation
and proliferation, and it has been reported previously that this phase has a higher
probability of precipitation from r-SBF at a pH of 7.4.30 Amorphous CaP phases have
also often been found to form initially in SBF.31 Using 31P solid state NMR
spectroscopy, Lin et al.32 identified an amorphous phase with substantial hydration as
the initial phase formed on a bioglass surface in SBF. It has been shown that
amorphous CaP is then transformed into HAP which is the most thermodynamically
stable phase.
4.1.3 Negatively Charged Groups
Many researchers have shown that negatively charged groups on a material’s
surfaces are better heterogeneous nucleators of apatite-like minerals in SBF than
positive or neutral surfaces. Hence, it can be interpreted that the accumulation of
Ca2+ ions near the negatively charged surfaces, due to chelation, increases the
likelihood of supersaturation, and as a result, initial nucleation is preferentially
triggered at such sites.33 Among these negatively charged groups, phosphates are
found to have the largest effects.
Self assembled monolayers (SAMs) have been used to study the effect of different
functional groups on the nucleation of calcium phosphate in SBF. Tanahashi and
Matsuda 3 showed significant apatite formation on negatively charged SAMs, but not
on positively charged, or neutral surfaces. The growth rate determined using quartz
crystal microbalance (QCM) measurements decreased in the order: -PO4H2 > -
COOH » -CONH2 ≃ -OH > -NH2 » -CH3 ≃ 0.3 Interestingly, -COOH terminated
surfaces exhibited an induction period of almost 10 days before apatite growth, with
a linear growth rate, whereas, -PO4H2 surfaces did not exhibit this induction period.
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Another study of calcium phosphate growth induced by SAMs with different
functional groups on titanium showed similar results.4 Nucleation occurred on the
surfaces with -PO4H2 and -COOH groups, but not on the surfaces with -OH or -
CH=CH2. Again, -PO4H2 was found to exhibit stronger propensity for nucleation
than the -COOH.
Incorporation of negatively charged functional groups such as carboxylate and
phosphate ions on the surface of biomaterials or within a polymeric framework is a
popular strategy for attempting to enhance the deposition of calcium-phosphate
minerals. The effect of these groups on the calcification of polymeric materials has
recently been the subject of an excellent review by Chirila.34 Although the
incorporation of phosphorous-containing moieties has been shown to lead, in vitro, to
the enhanced calcification of naturally occurring materials, such as: cotton,35,36
bamboo37,38 and chitin,39 the calcification results for synthetic polymers are less well-
studied and somewhat controversial.40 As Chirila pointed out in his review “the
effect of the phosphate group may be more complicated than initially thought”.
Polymers containing carboxylic acid groups have shown induction periods for apatite
nucleation, no mineralisation or even inhibitory effects.41-44 However, apatite
formation can generally be achieved, even without an induction period, if the
polymer is pre-treated with Ca(OH)2 or CaCl2 before SBF immersion.45 It has been
shown that bound Ca2+ ions get released when the polymer is soaked in SBF. The
high concentration of Ca2+ ions enhances chelation with the carboxylic acid groups
and hence accelerates apatite formation.41 In some cases, such as NaOH42 or O2
plasma46 treated poly(ε-caprolactone) (PCL), alternative dipping of samples into Ca2+
and PO42- solutions was used before the SBF study.
In the case of carboxylated polyphosphazenes (structure shown in Scheme 4.1),
which contains two carboxylic acid groups per repeating unit, nucleation of CaP has
been shown within 24 hours in SBF, without Ca2+ treatment.47
Scheme 4.1: Structure of carboxylated polyphosphazene
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Chapter 4: In Vitro Mineralisation
* N P *
O
O
COOH
COOH
n
After 5 days, the crystalline composition of this mineral was similar to that of
octacalcium phosphate, then changed to either calcium deficient apatite, or tricalcium
phosphate after 7 days. This indicates that the composition of ionic groups and the
structure of the polymer are also important factors in nucleation.
In a series of elegant experiments, Ikada et al.1,18 demonstrated that MOEP-grafted
PE and PET exhibit significantly improved mineralisation in SBF. Expanded PTFE
(ePTFE) membranes have been surface modified with MAEP,48 or MOEP49 by
radiation induced grafting. MAEP grafted ePTFE with an external surface coverage
of 44 % or more has shown secondary growth of apatite-like mineral formation in
SBF, although unmodified ePTFE did not mineralise.2 MOEP-grafted samples also
showed the formation of carbonated HAP and other types of CaP.15,49 These MAEP-
or MOEP-grafted materials all showed mineralisation in SBF without Ca2+
pretreatment.
When incorporated into hydrogels, the effect of phosphate groups on mineralisation
is not straight forward. Chirila et al.22 showed that after 9 weeks in SBF, CaP
deposition on copolymers of PHEMA and 10 mol % MAEP, or MOEP was four
times lower than that found on the PHEMA homopolymer. Subsequent animal
studies revealed no CaP deposition on PHEMA copolymers whereas PHEMA
homopolymer was extensively mineralised. This clearly demonstrates an inhibitory
effect of phosphate groups in hydrogels on mineralisation.
Stancu et al.20 copolymerized MOEP with (diethylamino)ethyl methacrylate
(DEAEMA) and 1-vinyl-2-pyrrolidinone (VP) (Structures shown in Scheme 4.2).
Globular apatite formation was only observed on P(MOEP-co-DEAEMA) with less
than 60 mol % MOEP, and on PDEAEMA after 15 days in SBF. In P(MOEP-co-
DEAEMA), MOEP alternates with DEAEMA showing uniform distribution,
whereas PMOEP forms “islands” in P(MOEP-co-VP) due to the different reactivity
ratios of these monomers. It was suggested that in PMOEP rich areas, Ca2+ ions are
coordinated by two phosphate groups from adjacent MOEP units. These strongly
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bound Ca2+ ions are no longer capable of attracting phosphate ions from SBF, thus
inhibiting further nucleation. However, this explanation cannot be applied to the
mineralisation of MAEP- and MOEP-grafted materials, and phosphate terminated
SAMs.
Scheme 4.2: Structures of DEAEMA and VP.
ON
O DEAEMA
N
O
VP
In this study, the effects of polymer structure on mineralisation were investigated in
SBF using a series of phosphate containing polymers (soluble and cross-linked
PMAEP and PMOEP). The mineralisation of fabricated surfaces (LbL films
containing phosphate or carboxylate groups, block copolymers attached to the
aminated slide, and fluorinated block copolymers attached to a PTFE substrate) were
also studied. Since PMAEPs have been found to contain some carboxylic acid groups,
the effect of this group on mineralisation was also investigated using poly(acrylic
acid) (PAA) gels.
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4.2 Experimental
4.2.1 Materials
Acrylic acid (99%), stabilised with 200 ppm hydroquinone monomethyl ether
(MEHQ), was purchased from Aldrich and used as received. The purities of the
chemicals used to prepare the SBF solution were as follows: NaCl (99.9%), NaHCO3
(99.0%), Na2CO3 (99.9%), KCl (99.0%), K2HPO4 (99.0%), MgCl2·6H2O (99.0%),
CaCl2·2H2O (99.5%), Na2SO4 (99.0%), and HEPES (2-[4-(2-Hydroxyethyl)-1-
piperazinyl]ethanesulfonic acid) (99.9%).
Samples subjected to mineralisation studies are as follows: The synthesis of gels and
soluble PMAEP and PMOEP are described in Chapter 2. The material properties of
samples investigated are listed in Table 4.3. Fabrication of LbL films and block
copolymers coupled onto aminated slides are discussed in Chapter 3, and their
properties are listed in Tables 4.4 and 4.5, respectively.
4.2.2 Methods
4.2.2.1 Synthesis of cross-linked PAA gels
Acrylates are known to cross-link when polymerised under radiation, due to
hydrogen abstraction. To obtain cross-linked PAA gels for mineralisation study,
gamma radiation-induced polymerisation of AA was carried out. A solution of 10
w/v % acrylic acid in MilliQ water was prepared in a glass tube and sealed with a
Suba cap. Dissolved oxygen in the monomer solution was removed by bubbling
nitrogen gas through for 20 minutes in an ice-bath. The sample was then subjected to
a 60Co gamma radiation source to obtain a total dose of 16 kGy (dose rate of 2.8
kGy/h) using a 220 Nordian Gamma-cell (Canada) at room temperature. The
obtained gel was soaked in MilliQ water for 3 days with regular water exchanges to
remove any residual monomer. The sample was then dried in a vacuum oven to
constant weight.
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4.2.2.2 Simulated body fluid (SBF) experiments
The simulated body fluid was prepared according to the method described by Kim et
al. 13. Chemicals were dissolved in MilliQ water (that had been boiled for one hour
prior to preparation) and buffered with HEPES (2-[4-(2-Hydroxyethyl)-1-
piperazinyl]ethanesulfonic acid) and 1M NaOH at pH = 7.4 at 36.5 ºC. The insoluble
gels were used as prepared. The soluble PMOEP was cast from methanol solution
onto the glass substrate where it was left for the mineralisation study. The soluble
PMAEP was cast onto PTFE and was used without this substrate. Approximately 10
mL of SBF solution were added to a 15 mL polystyrene tube containing a polymer
sample. The tubes were immersed in a water bath at 36.5 + 0.2 ºC for a period of
seven days. The SBF solution was changed every 2 days. After 7 days, the materials
were washed by soaking in MilliQ water for 10 minutes, this was repeated three
times. The materials were subsequently dried in a vacuum oven at 40 ºC to constant
weight.
4.2.2.3 Scanning electron microscopy with energy dispersive x-ray analysis
SEM/EDX (FEI Quanta 200 Environmental SEM equipped with an Evarhart
Thomley secondary electron detector) was performed at 10 kV to examine the
morphology of the calcium phosphate deposit and to obtain the elemental
composition of the CaP minerals on the surface. Before SEM analysis, the surface of
the sample was coated with a very thin layer of carbon using Cressington Carbon
Coater to reduce sample charging. In some cases, where it is stated, the sample was
coated with gold using a Biorad Gold Sputter Coater.
4.2.2.4 Fourier transform infrared spectroscopy – attenuated total reflectance
A Nicolet Fourier Transform Infrared Spectrometer equipped with a diamond ATR
(refractive index of 2.41 at 1000 nm and an average angle of incidence of 50 º) was
used to analyse the mineral deposits. Spectra, 64 scans at 4 cm-1 resolution, were
collected over a range of 4000 – 525 cm-1. The depth of penetration was calculated to
be between 2.85 (525 cm-1) and 0.37 μm (4000 cm-1), for an estimated refractive
index of the polymer/CaP phase of 1.5.
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4.2.2.5 XPS
XPS spectra were recorded using a Kratos Axis Ultra X-ray photoelectron
spectrometer with a monochromated Al Kα X-ray source (1486.6 eV) running at 150
W (15 kV, 10 mA emission current). The survey scans were collected at 1200-0 eV
with 1.0 eV steps at a pass energy of 160 eV; narrow scans were taken at 0.1 eV
steps and a pass energy of 20 eV. Vision 2 software was used for data acquisition and
processing. The binding energies were charge-corrected using the C1s hydrocarbon
peak (285.0 eV).
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4.3 Results
4.3.1 SBF studies of PMAEP and PMOEP
Mineralisation of a series of phosphate-containing polymers was investigated using
SBF in order to evaluate the effects of cross-linking and phosphate content. Sample
characteristics are shown in Table 4.3.
Table 4.3: Properties of PMAEP and PMOEP polymers subjected to SBF studies.
Samp
le
Expt a
Polymer [RAFT] (mol/L) Conv b
(%)
Characteristic Total
% P cSurface
% P d
1A 1 PMAEP ― 90 Gel 9.5 4.39
1B 2 PMAEP 1 × 10-2 (PEPDA) 83 Soluble 7.0 2.99
1C 7 PMOEP 2 × 10-2 (PEPDA) 95 Gel e e
1D 6 PMOEP 1 × 10-2 (PEPDA) 81 Gel 11.5 e
1E 5 PMOEP ― 96 Gel 11.2 4.33
1F 8 PMOEP 1 × 10-2 (CDB) 74 Soluble 9.9 5.33
1G PAA ― ― Gel ― ―
a: Expt no. from Table 2.1, Chapter 2, b: conv = conversion obtained from Raman spectroscopy, c: determined by ICP, w/w %, d: determined by XPS, atomic %, e: did not determined
Cross-linked gel polymers and soluble PMOEP cast on a glass surface were
immersed in SBF for seven days with regular solution changes. The films obtained
from the soluble PMAEP dissolved in SBF, hence a modified SBF solution 1.5 times
the concentration of normal SBF, was used. After seven days, the polymers were
washed and dried to constant weight. CaP depositions were characterised by
SEM/EDX and ATR-FTIR.
For identification of the mineral phases, XRD is generally used in conjunction with
SEM/EDX. However, Grøndahl et al.2 has demonstrated that minerals formed in
SBF over a period of 7-14 days are often amorphous which do not show XRD
patterns. Instead, this study used FTIR since the previous studies had already
demonstrated it to be an useful technique for mineral identification.2,15
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Chapter 4: In Vitro Mineralisation
Ca
O
C
NaMg P
Ca
A
B
C
D
E Ca/P = 0.88 (Ca+Mg/P) = 1.37
Ca/P = 1.14 (Ca+Mg)/P = 1.34
Au-Coated
Untreated
Ca/P = 3.95 (Ca+Mg)/P = 4.54
F
Figure 4.1: SEM images of sample 1A (PMAEP gel) (A) before treatment, and after SBF immersion for 7 days with (B) carbon coating and (C) gold coating, (D) sample 1B (the film obtained from soluble PMAEP after 7 days in 1.5 SBF), (E) sample 1G (PAA gel) after 7 days immersion in SBF, and (F) EDX spectrum of the mineral on sample 1G.
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D A Untreated Untreated
B E Ca/P = 0.73 (Ca+Mg)/P = 0.98
Ca/P = 0.94 (Ca+Mg)/P = 1.16
Ca/P = 1.41 Ca+Mg/P = 1.51
Ca/P = 1.51 Ca+Mg/P = 1.55
C F Carbon Tape
Ca/P = 1.49 Ca+Mg/P = 1.54
Ca/P = 1.48 Ca+Mg/P = 1.52
Au-Coated
Figure 4.2: SEM images of sample 1C (PMOEP gel) (A) before treatment, and after SBF immersion for 7 days with (B) carbon coating and (C) gold coating, sample 1D (PMOEP gel) (D) before treatment and (E) after SBF immersion for 7 days and (F) minerals dislodged from sample 1D.
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Chapter 4: In Vitro Mineralisation
O
Na Mg
P
Ca Ca
D
C
Ca/P = 0.73 (Ca+Mg)/P = 0.99
Ca/P = 1.44 (Ca+Mg)/P = 1.55
Untreated Ca/P = 1.72 (Ca+Mg)/P = 1.77
C
B
A
Figure 4.3: SEM images of sample 1E (PMOEP gel) (A) before, and (B) after SBF immersion for 7 days, (C) sample 1F (soluble PMOEP cast on glass) after SBF immersion for 7 days, and (D) EDX spectrum of the mineral on sample 1E.
Figures 4.1-4.3 show SEM images of the samples before and after SBF immersion.
EDX elemental analysis of the minerals revealed the presence of C, O, P, Ca and
small amounts of Mg and Na in all homopolymer samples after SBF immersion. The
EDX spectra of minerals on sample 1E and 1G are shown in Figure 4.3D and 4.1F,
respectively. The presence of carbon is due mainly to the sample coating used for
SEM/EDX analysis but may also be from the polymer and/or carbonate ions. In
mineralisation studies, Mg2+ substitution for Ca2+ within a CaP lattice is often
observed10 and it is therefore necessary to evaluate both the Ca/P and (Ca+Mg)/P
ratios. These ratios are shown as inserts in the following SEM images.
SEM images of sample 1A (PMAEP gel) before and after SBF immersion for 7 days
are shown in Figure 4.1 A and B, respectively. The untreated polymer surface was
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Chapter 4: In Vitro Mineralisation
smooth, whereas after soaking in SBF, the surface was covered with thin layers of
CaP mineral, which had multiple cracks due to sample preparation. EDX analysis of
this mineral yielded Ca/P and (Ca+Mg)/P ratios of 0.88 and 1.37, respectively
(Inserts in Figure 4.1B). Sample 1B (the film obtained from soluble PMAEP) also
showed the same tile-like morphology with Ca/P and (Ca+Mg)/P ratios of 1.14 and
1.34, respectively (Figure 4.1D). These values indicate that this mineral phase is
possibly either brushite (CaHPO4·H2O), monetite (CaHPO4), or octacalcium
phosphate (Ca8H2(PO4)6·5H2O). However, it is important to note that since these
mineral layers were thin, the ratios obtained from EDX are associated with large
errors due to the large sampling depth of EDX (~ 5 μm). It is also important to note
that when the SBF-treated PMAEP gel was coated with gold on top of a carbon
coating so as to obtain a clear image, this tile-like structure was completely covered
and the surface morphology was very smooth (Figure 4.1C). The thickness of gold
coatings is typically 25 nm or more, whereas carbon coatings are generally 10-15 nm,
and less dense. This suggests that the tile-like mineral layer is very thin.
The effect of carboxylic acid (-COOH) groups on mineralisation was tested using a
poly(acrylic acid) (PAA) gel (sample 1G). Figure 4.1E shows the SEM image of
PAA after 7 days soaking in SBF. Again a tile-like morphology was observed. The
Ca/P and (Ca+Mg)/P ratios were 3.95 and 4.54, respectively.
All the PMOEP gels (sample 1C, 1D and 1E) had smooth surface morphologies
before SBF (Figure 4.2 and 4.3). After 7 days in SBF, the surfaces were covered with
a tile-like mineral layer similar to those observed on the PMAEP samples. The Ca/P
and (Ca+Mg)/P ratios of this layer were 0.73-0.94 and 0.98-1.16, respectively. In
addition, a secondary growth of round mineral nodules of various sizes (ø ~ 2-5 μm)
including some large clusters, was observed. These round mineral nodules had Ca/P
and (Ca+Mg)/P ratios of 1.41-1.51 and 1.51-1.55, respectively. Figure 4.2F shows
minerals dislodged onto carbon tape during SEM sample preparation. Here, two sizes
of spherical mineral clusters were observed, one of which had particles of much
smaller diameter. Nevertheless, both of them had similar Ca/P and (Ca+Mg)/P ratios
(1.48-1.49 and 1.52-1.54, respectively). The tile-like morphology was masked when
coated with gold (Figure 4.2C), also shown on the PMAEP gel.
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Chapter 4: In Vitro Mineralisation
After 7 days, the SBF-treated soluble PMOEP cast on a glass surface (sample 1F)
was also covered with a large amount of spherical mineral clusters (Figure 4.3C). Its
clusters were much smaller in diameter than those formed on the PMOEP gels: they
were, however, similar to one of the mineral types dislodged from the PMOEP gel
(sample 1D) shown in Figure 4.2F. EDX analysis revealed Ca/P and (Ca+Mg)/P
ratios of 1.72 and 1.77, respectively; slightly higher than the theoretical value for
HAP (i.e., 1.67).
To summarise SEM results, PMAEP gel and the film obtained from soluble PMAEP
and PAA gel showed tile-like mineral morphology. PMOEP gels also showed similar
tile-like morphology covering the polymer surfaces as well as secondary growth of
globular HAP. The best mineralisation was observed on soluble PMOEP cast on
glass that was covered with high amounts of HAP like globular minerals.
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Chapter 4: In Vitro Mineralisation
1069
1723
1721
995
1075
99
3
B A
1565
1567
Wavenumber (cm-1) Wavenumber (cm-1)
17
10
1069
1645
991
959 C D
1715
1070
991
959
1652
Wavenumber (cm-1) Wavenumber (cm-1)
1482
14
18
1021
872
1710
E F
1647
1067
990
959
Wavenumber (cm-1) Wavenumber (cm-1)
1541
G
1406
14
51
1043
Wavenumber (cm-1)
Figure 4.4: ATR-FTIR spectra of polymer samples after 7 days immersion in SBF (solid line) and initial, untreated polymers (dotted line). (A) sample 1A (PMAEP gel); (B) sample 1B (PMAEP film) (Experiment done in 1.5×SBF.); (C) sample 1C (PMOEP gel); (D) sample 1D (PMOEP gel); (E) sample 1E (PMOEP gel); (F) sample 1F (PMOEP film on a glass surface), and (G) sample 1G (PAA gel). y-axis is absorbance.
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Chapter 4: In Vitro Mineralisation
Figure 4.4A and B shows the ATR-FTIR spectra of cross-linked gel and the film
obtained from soluble PMAEP (sample 1A and 1B), respectively, before (dotted line)
and after SBF immersion (solid line). After SBF treatment, both spectra showed the
presence of a large peak at 1069-1075 cm-1 corresponding to a phosphate vibration
band. The peak at 993-995 cm-1 which is also in the phosphate region, together with a
shoulder on the higher wavenumber side of the band ~1070 cm-1, indicate possible
brushite formation.50 Another important feature of FTIR spectra of the SBF-treated
PMAEP samples is the new band at 1565-1567 cm-1. This corresponds to the
carbonyl vibration of a carboxylate group, indicating that large amounts of the
PMAEP side chains were hydrolysed at the C-O-C ester bond. In the case of the
soluble PMAEP sample (Figure 4.4B), the band at 1069 cm-1 has a shoulder at 1036
cm-1. However, it is not possible to identify a mineral phase based on one band. The
bands around 1650 cm-1 observed in these samples were assigned to water bound to
either the CaP mineral or the polymers in their deprotonated state.51
ATR-FTIR spectra of all PMOEP gels showed a new band around 1070 cm-1 after
SBF-treatment (Figure 4.4C-E). This is similar to that observed for the PMAEP
samples. This band also showed a shoulder at higher wavenumbers, and the band at
990-991 cm-1 may indicate the presence of brushite; again similar to the PMAEP
samples.
According to the literature, to the best of my knowledge, the phosphate band around
1070 cm-1 does not correspond to any previously identified CaP phase.50 To
investigate whether this band can be ascribed to the phosphate groups of the
polymers with calcium ions, PMOEP gel was soaked in Ca(OH)2 solution in order to
chelate the Ca2+ ions. The resulting ATR-FTIR spectrum of this sample shows that
such an interaction results in the large broad acidic phosphate vibration around 975
cm-1 disappearing, and giving rise to two strong bands at 1060 and 962 cm-1,
corresponding to the out-of-phase stretching and in-phase stretching vibrations,52
respectively (Figure 4.5). Therefore it can be concluded that the band around 1070
cm-1 which appears after SBF-treatment does not correspond to this particular
interaction.
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Chapter 4: In Vitro Mineralisation
1163
974
1060
Wavenumber (cm-1)
961
Figure 4.5: ATR-FTIR spectra of Sample E (PMOEP gel) reacted with Ca(OH)2 (solid line) and untreated (dotted line). y-axis is absorbance.
Figure 4.4F shows the ATR-FTIR spectra of soluble PMOEP sample (sample 1F)
before and after SBF treatment. After SBF, the spectra showed a dramatic decrease
of the polymer bands, indicating a large amount of mineral formation on this sample.
There was a broad band at 1021 cm-1, which can be assigned to the P-O stretching of
the phosphates in HAP. This sample also showed bands at 1482, 1418 and 872 cm-1
corresponding to the carbonate vibrations of carbonated HAP.
The ATR-FTIR spectrum of sample 1G (PAA gel) shows a large peak at 1541 cm-1
corresponding to carboxylate-bound calcium (Figure 4.4G). This spectrum shows a
broad phosphate vibration band around 1018-1080 cm-1, possibly due to a mixture of
CaP phases.
4.3.2 SBF studies of Layer-by-Layer (LbL) films
Mineralisation of the layer-by-layer ultrathin films fabricated from soluble PMAEP
or PMOEP and PEI on silicon wafers was investigated in r-SBF for 7 and 14 days.
An LbL film with PEI, only, and the silicon wafer substrate were also used as
controls. After the periods of immersion, sample surfaces were characterised using
SEM/EDX and XPS. Mineralisation of PAA and PEI LbL films on glass slides was
also investigated by 7 days immersion in SBF, and characterised by XPS. Table 4.4
summarizes results for the LbL samples in this study.
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Chapter 4: In Vitro Mineralisation
Table 4.4: LbL samples for SBF and their atomic % of Ca and P after 7 days in SBF (obtained from XPS survey scans).
XPS XPS After 7 days in SBF Sample Substrate Polymer
P % C(=O)O % Ca % P % Ca/P
2A Si PEI-PMAEP 2.28 6.89 1.12 1.52 0.74
2B Si PEI-PMOEP 2.62 6.69 1.39 2.62 0.53
2C Si PEI ― ― 0.60 ― ―
2D Si ― ― ― 0.87 ― ―
2E Glass PEI-PAA ― 3.55 0.89 ― ―
2F Glass PEI ― ― 0.64 ― ―
2G Glass ― ― ― 0.38 ― ―
Figure 4.6A-D shows SEM images of the LbL films, and the silicon wafer, after 7
days in SBF. Sparse and patchy CaP mineral formation was observed on both
samples 2A (PMAEP) and 2B (PMOEP) LbL films (Figure 4.6A and B,
respectively). EDX analyses of these minerals showed the Ca/P ratios of 1.77 and
1.65 for samples 2A and 2B, respectively, indicating possible HAP formation
(Inserts in Figure 4.6). No Mg was detected in these minerals. The control sample 2C
(PEI) and sample 2D (silicon substrate) surfaces showed sparse and patchy calcium
mineral formation, possibly CaCO3 after 7 days (Figure 4.6C and D, respectively).
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Chapter 4: In Vitro Mineralisation
A E
B
Ca/P = 1.77
F
Ca/P = 1.65
Ca/P = 2.06
Ca/P = 1.96
GC
D H
Figure 4.6: SEM images of minerals formed on the LbL surfaces after SBF immersion for 7 days: (A) sample 2A (PEI-PMAEP), (B) sample 2B (PEI-PMOEP), (C) sample 2C (PEI only), and (D) sample 2D (silicon wafer), and 14 days: (E) sample 2A, (F) sample 2B, (G) sample 2C, and (H) sample 2D.
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Chapter 4: In Vitro Mineralisation
Table 4.4 shows atomic % of Ca and P from XPS survey scans of samples after 7
days immersion in SBF. None of the samples contained calcium before the SBF
studies according to XPS analysis. The stability of the LbL films in SBF was also
confirmed from the carbon and silicon % by comparison to those before the SBF
study. The presence of Ca was observed in all samples. P was only observed in
sample 2A (PMAEP) or 2B (PMOEP) LbL films. Due to the sampling area of XPS
(0.7 × 0.3 mm2) both the mineral-containing and the non-mineral-containing areas
are analysed by this technique. The rationale for this statement is taken from the
spatial distribution of mineral deposits observed in the SEM images.
XPS analysis of samples 2A and 2B showed that the atomic concentration of P is
higher than that of Ca. This P comes both from the mineral and the polymers. It is
proposed that not all the phosphate groups of the polymers are involved in the
chelation of Ca2+ due to strong interactions with the protonated amines in the LbL
films.
In the case of sample 2C, the PEI surface, initial attraction is through the chelation of
phosphate ions by the protonated amines. XPS did not detect any P on this surface,
although there was a small amount of Ca (0.60 %) possibly from the minerals
identified from the SEM/EDX.
The SBF-treated sample 2D, the silicon wafer surface, had slightly higher amounts of
Ca (0.87 %) compared to that of the PEI surface. There was no detectable P on this
surface; this is in agreement with the EDX analysis of the minerals. Here, the initial
interaction is the chelation of Ca2+ ions by –OH groups.
Figure 4.6E-H presents SEM images of the LbL films and the silicon wafer surface
after 14 days in SBF. No significant mineral growth was observed on the surfaces of
sample 2A (PMAEP) and 2B (PMOEP) films (Figure 4.6E and F, respectively). In
the case of samples 2C (PEI) and 2D (silicon surface), sparse and patchy CaP
mineral formation was observed after 14 days (Figure 4.6G and H, respectively), but
not after 7 days (Figure 4.6C and D) indicating there was more than a 7 day
induction period for CaP formation on these samples. Although amine and -OH
groups are not as efficient at inducing CaP growth as phosphate and carboxylate
groups, CaP nucleation has previously been observed on amine and -OH terminated
self assembled monolayers.3,16
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Chapter 4: In Vitro Mineralisation
In the case of PEI-PAA LbL film fabricated on a glass surface (sample 2E), XPS
revealed slightly higher amounts of Ca (0.9 %) than the controls (0.64 and 0.38 % for
a samples 2F (PEI) and 2G (a bare glass slide), respectively) after 7 days in SBF.
This indicates that chelation of Ca2+ ions with carboxylic acid groups is stronger than
with the Si-OH (a glass surface), and protonated amines (PEI). XPS analysis showed
no P on all of these slides.
4.3.3 SBF studies of block copolymers coupled to aminated slides
Mineralisation of block copolymers consisting of PMAEP or PMOEP and PAAEMA
fixed on aminated slides (Table 4.5) were investigated in SBF for up to two weeks. A
slide with PAAEMA attached and a slide without any polymer were also used as
controls. The samples were characterised using SEM/EDX.
Table 4.5: Block-copolymer-attached aminated slides for SBF.
Sample Coupled Block Copolymer
m a n b PDI
XPS Atomic
P % c
3A P(MOEPn-b-AAEMAm) 109 97 1.38 1.53
3B P(AAEMAm-b-MOEPn) 22 132 1.41 0.66
3C P(AAEMAm-b-MAEPn) 22 99 1.38 0.22
3D P(AAEMAm-b-MAEPn) 22 160 1.38 0.27
3E PAAEMAm 22 ― 1.13 ―
3F d ― ― ― ― ― a: unit of PAAEMA segment, b: unit of PMOEP/PMAEP segment, c: obtained from XPS survey scans of samples before SBF treatment, d: untreated aminated slide
Figures 4.7 and 4.8 show, respectively, SEM images of the samples, and the controls,
after SBF treatment.
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Chapter 4: In Vitro Mineralisation
A E Mg = 1.57 Ca = 2.85 P = 0.68
Mg = 1.62 Ca = 5.29 P = 2.04
B F Mg = 1.51 Ca = 2.85 P = 0.95
Mg = 1.86 Ca = 1.83 P = 0
C G
Mg = 1.66 Ca = 2.14 P = 0.42
Mg = 1.57 Ca = 1.75 P = 0
H Mg = 1.63 Ca = 1.79 P = 0
D
Mg = 1.25 Ca = 5.69 P = 2.69
Figure 4.7: SEM images of minerals formed on the block copolymer functionalized aminated slides after SBF immersion for 7 days: (A) sample 3A, (B) sample 3B, (C) sample 3C and (D) sample 3D, and 14 days: (E) sample 3A, (F) sample 3B, (G) sample 3C and (H) sample 3D.
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Chapter 4: In Vitro Mineralisation
A C Mg = 1.89 Ca = 2.07 P = 0
NaCl
B D Mg = 0.97 Ca = 6.96 P = 3.40
Figure 4.8: SEM images of minerals formed on the PAAEMA functionalized and untreated aminated slides after SBF immersion for 7 days: (A) sample 3E (PAAEMA) and (B) sample 3F (untreated aminated slide), and 14 days: (C) sample 3E (PAAEMA) and (D) sample 3F.
EDX spectra of the mineral on sample B (P(AAEMA-b-MOEP)) and the non-
mineral area are shown in Figure 4.9A and B, respectively.
Figure 4.9: EDX spectra of the mineral (A) and the non-mineral area (B) on sample 3B (see Figure 4.7F).
The EDX analysis of the aminated slide indicated that it contains high amounts of Ca
(1.83 %), Mg (1.86 %) and Na (8.07 %), as well as C (16.47 %), O (54.08 %), Si
C
B
O Na Mg
Si
P Cl Ca C
O Na Mg
Si
Ca
A
201
Chapter 4: In Vitro Mineralisation
(17.29 %), and K (0.47 %) (Figure 4.9A). There was no detectable P in the non-
mineral area of the slide. In contrast, the EDX spectrum of the mineral showed P
(0.95 %), Ca (2.85 %), Mg (1.51 %) and Na (5.81 %), as well as Cl (0.98 %) (Figure
4.9B). The presence of Cl may be from NaCl. The other elements detected were C
(29.23 %), O (44.42 %) and Si (14.28 %). Due to the large sampling depth of EDX, it
was not possible to quantitatively evaluate the Ca/P ratios of the minerals. However,
all the minerals showed an increase in Ca compared to that of the slides, and the
atomic concentrations of Ca, Mg, and P are shown as inserts in SEM images (Figure
4.7 and 4.8)).
The SEM images of block copolymer-functionalized aminated slides showed sparse,
patchy CaP mineral formation after 7 days in SBF (Figure 4.7A-D). After two weeks,
the mineral formation was slightly increased but still patchy (Figure 4.7E-H). Only
some areas of sample D (P(AAEMA-b-PMAEP) attached slide) showed some large
clusters of CaP minerals (Figure 4.7H).
CaP mineral formation was not observed on the control PAAEMA even after two
weeks of SBF treatment (Figure 4.8A and C). However, the aminated slide induced
CaP formation after two weeks (Figure 4.8D). Interestingly, this mineral phase
showed a distinctive morphology not seen in any of the phosphate-containing
samples. Such non-spherical mineral formations have also been observed on amine-
terminated SAMs have in simulated physiological fluid (SPF).16
4.3.4 SBF studies of fluorinated amphiphilic block copolymers attached onto
PTFE Films
The mineralisation of fluorinated amphiphilic block copolymers (P(FSm-b-AAn))
attached to PTFE was investigated in SBF for up to two weeks. Untreated PTFE film
was used as a control. The samples were characterised using SEM/EDX and XPS.
Table 4.6 shows properties of P(FSm-b-AAn) attached PTFE films.
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Chapter 4: In Vitro Mineralisation
Table 4.6: Properties of fluorinated block copolymer absorbed PTFE films.
Sample P(FSm-b-AAn) Absorption solvent Contact Angle (°)
m n PDI b Advancing Receding
4A 101 237 1.11 MEK 91 ± 6 31 ± 3
4B 101 141 1.07 MEK 88 ± 5 28 ± 3
4C 101 141 1.07 DMF 101 ± 4 49 ± 3
4D a ― ― ― ― 109 ± 6 111 ± 6
a: Untreated PTFE film, b: PDI of corresponding P(FSm-b-tBAn) block copolymers before hydrolysis of tBA groups
Figure 4.10 represents SEM images of samples after 7 and 14 days in SBF. The
PTFE film used in this study was not smooth, and SEM revealed scratch marks.
However, these topological features did not affect mineralisation since untreated
PTFE film did not mineralise in SBF for up to 2 weeks, in agreement with the
previous study.2 SEM images of all the P(FSm-b-AAn) attached surfaces showed no
mineral formation after 7 days in SBF. EDX spectra of these samples only showed
the carbon and fluorine. After 14 days, the modified samples showed very small
patchy mineral depositions.
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Chapter 4: In Vitro Mineralisation
E A
D
C
B F
G
H
Figure 4.10: SEM images of minerals formed on P(FSm-b-AAn) attached and untreated PTFE films after SBF immersion for 7 days: (A) sample 4A, (B) sample 4B, (C) sample 4C and (D) sample 4D (untreated PTFE), and 14 days: (E) sample 4A, (F) sample 4B, (G) sample 4C and (H) sample 4D.
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Chapter 4: In Vitro Mineralisation
Table 4.7: Atomic % of Ca and P from XPS survey scans and the Ca/P ratios.
After 7 days in SBF After 14 days in SBF a
Ca P Ca/P Ca P Ca/P
4A 1.3 0.6 2.2 0.9
0.9
0.8
0.5
0.7
0.6
1.8
1.3
1.3
4B 1.2 0.5 2.4 2.5
0.8
0.6
1.1
0.7
0.4
2.3
1.1
1.5
4C 1.2 0.5 2.4 0.9
0.9
1.0
0.5
0.3
0.4
1.8
3.0
2.5
4D 0 0 ― 0 0 ― a: three randomly chosen areas were analysed on each samples.
XPS was also used to identify any absorbed ions. Table 4.7 summarises atomic % of
Ca and P of the samples from XPS survey scans. Block copolymer attached PTFE
films (samples 4A-4C) showed ~1% calcium after 7 days in SBF. Small phosphorous
peaks were also identified from XPS survey scans of these samples. The Ca/P ratios
are also shown in Table 4.7. Calcium ions are most likely bound to the carboxylic
acid groups of the blocks as no mineral was observed on these samples from SEM.
For the samples after 14 days in SBF, three randomly chosen spots were analysed by
XPS, since SEM showed patchy mineralisation. The results showed small variations
in these three spots as expected. Compared to samples after 7 days, all samples
showed no significant increase in Ca % and P %, except one area of sample 4B
which had 2.5% Ca. Untreated PTFE (sample 4D) up to 14 days in SBF did not show
any calcium or phosphorous.
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Chapter 4: In Vitro Mineralisation
4.4 Discussion
4.4.1 SBF studies of PMAEP and PMOEP
In the present study, the mineralisation ability of a series of phosphate-containing
soluble polymers and cross-linked gels was investigated after 7 days immersion in
SBF. We have already shown that the different mineralisation outcomes of these
polymers are not simply due to the phosphate content, but a combination of factors,
including: polymer structure and phosphate distribution, as well as the degree of
cross-linking. This study also showed good correlation with previous mineralisation
studies of MAEP and MOEP-grafted ePTFE.
On the surfaces of the PMAEP and PMOEP gels, and the film obtained from soluble
PMAEP, thin layers of CaP mineral were observed. This tile-like morphology has
been observed previously to form on PMAEP-g-ePTFE.2 The Ca/P ratio from EDX
and FTIR analysis indicate that this mineral phase is possibly brushite
(CaHPO4·H2O). Brushite, which is a thermodynamically less stable phase, has been
proposed to be a precursor of the more stable HAP in an in vitro mineralisation
mechanism.53 On the PMOEP gel, a secondary growth, of presumably calcium-
deficient HAP (based on the Ca/P ratio and the crystal morphology), was observed.
A similar secondary growth has also been observed on PMAEP-g-ePTFE.2 Soluble
PMOEP cast on a glass surface formed large amounts of carbonated HAP which was
confirmed by FTIR. This is analogous to our earlier results, a sample where MOEP
was grafted onto ePTFE in methanol.15 Interestingly, PMOEP-g-ePTFE formed in
other solvents did not induce HAP nucleation, but rather different CaP phases, thus
suggesting that only the graft copolymer formed in methanol is structurally similar to
the soluble PMOEP of this study.
FTIR spectra of SBF-treated PMAEP samples also revealed the presence of
carboxylate groups. This indicates that large amounts of the PMAEP side chains
were hydrolysed at the C-O-C ester bond (shown as 2 in Scheme 4.3). Deprotonation
of these carboxyl groups occurs as a consequence of Ca2+ binding similar to that
observed previously for PAA-g-ePTFE 2 and for PAA gel in this study.
206
Chapter 4: In Vitro Mineralisation
O
OP O
OHOH
O*
R
n
O
OH
O
R
m
OHO
*
R
l
O
OP O
OHOH
O**
R
n
1
2
Scheme 4.3: Possible hydrolysis sites on the side-chain of the polymer and structure
of resulting polymer (R = H: MAEP, R = CH3: MOEP).
The effect of carboxylic acid groups on mineralisation was tested using a
poly(acrylic acid) (PAA) gel. Formation of similar tile-like CaP mineral layer on this
sample after 7 days immersion in SBF was evident from the SEM image. The FTIR
spectrum of this sample showed a broad phosphate band indicating a mixture of CaP
phases. The broad phosphate peak was also observed on the SBF-treated PAA-g-
ePTFE.2
The carboxylate band was not present in the ATR-FTIR spectra of anye PMOEP
samples. This indicates that the loss of phosphate in these polymers was due to the
cleavage at C-O-P linkage and not at the C-O-C linkage, which was the case in
PMAEP polymers. Therefore, it indicates that PMOEP contains some hydroxyl
groups. Another important observation is that since all PMOEP gels (samples 1C, 1D
and 1E) showed very similar mineralisation patterns, it can be assumed that these
gels are physically and chemically the same. This is especially supported by the fact
that the PEPDTA did not work as a RAFT agent for this monomer.
PMOEP samples generally showed better mineralisation compared to PMAEP.
Chirila et al.22 observed slightly better calcification of P(HEMA-co-MOEP)
compared to that of P(HEMA-co-MAEP) when these polymers were immersed in
SBF for up to 9 weeks, although PHEMA itself calcified much better. The difference
between MAEP and MOEP is that MOEP has an extra methyl group, which makes
this monomer more hydrophobic. The water solubility of MAEP is at least 30 % w/w
(in this study), whereas MOEP is only 4 % w/w. The effect of the hydrophobicity of
these polymers on mineralisation is not completely clear. A previous study of
207
Chapter 4: In Vitro Mineralisation
PMOEP-g-ePTFE has shown that the most hydrophobic surface had the best HAP
formation.15
However, the structures of PMAEP and PMOEP are further complicated by the fact
that this study showed that the hydrolysis of the side-chains in these polymers occurs
at different linkages. PMAEP polymers contain large amounts of carboxylic acids,
which can affect the mineral formation observed on PAA. On the other hand,
PMOEP does not contain this group, but has hydroxyl groups. The distribution of
phosphates in these polymers would also be different due to the cleavage of side-
chains.
Large amounts of carbonated HAP were only observed for the soluble PMOEP
sample despite the larger phosphate loss compared to that of the PMOEP gels. This
strongly supports the hypothesis that the flexibility of polymer chains, and hence the
degree of cross-linking, also influences mineralisation and not just the phosphate
content.
4.4.2 SBF Studies of Layer-by-Layer (LbL) Films
Layer-by-Layer films were fabricated using soluble PMAEP and PMOEP with PEI
on silicon wafers. Mineralisation of these films was investigated in SBF for up to 14
days. The PAA LbL film was also studied in SBF.
Both PMAEP and PMOEP films showed sparse and patchy CaP mineral formation
after 7 days in SBF. No further significant mineral growth was observed on these
materials after 14 days. The control PEI film and silicon wafer surface both showed
sparse and patchy calcium mineral formation after 7 days and CaP formation after 14
days. The PAA surface showed some Ca2+ uptake after 7 days in SBF but no
phosphorous was detected.
The PMAEP and PMOEP LbL films results are in contrast to the observed
mineralisation of the films obtained from soluble PMAEP and PMOEP films, which
yielded brushite-like minerals and a thick layer of HAP, respectively, in SBF after 7
days. The PAA gel also formed CaP minerals after 7 days in SBF, whereas the PAA
LbL film did not. AFM images of the PMAEP and PMOEP LbL films showed a
smooth topography indicating a uniform polymer film coverage (described in
208
Chapter 4: In Vitro Mineralisation
Chapter 3, Section 3.3.1), although the PMAEP film was slightly rough at the scales
analysed (1×1 μm, and 10×10 μm). Therefore, the patchy CaP formation cannot be
related to patchiness of the films.
It was evident from Infrared Reflection-Absorption Spectroscopy (IRRAS) analysis
of the PMOEP LbL film that a large amount of phosphate groups were interacting
with amine groups and, therefore, there were not many free phosphates available for
Ca2+ chelatation (described in Chapter 3, Section 3.3.1). This was also observed from
the XPS analysis of the PMAEP or PMOEP LbL films after 7 days in SBF.
Therefore, it can be concluded that LbL films do not contain enough free phosphate
groups to promote the nucleation of CaP mineral. These results also seem to support
the conclusion that the phosphates, or carboxylates, interacting with protonated
amines in the LbL films are incapable of chelating Ca2+.
For this study, only one layer of each polycation and polyanion was deposited, and
the layers are not expected to be uniformly distributed. There may be effects of
amines and hydroxyl groups from PEI and the substrate, respectively. In future work,
mineralisation of multi-layer films should be tested.
4.4.3 SBF Studies of Block Copolymers Coupled to Aminated Slides
Block copolymers consisting of PMAEP or PMOEP and PAAEMA fixed on
aminated slides (samples 3A-D) showed sparse, patchy CaP mineral formation after
up to two weeks in SBF. Although the control PAAEMA attached slide (sample 3E)
did not induce any CaP mineral formation, CaP was observed on the aminated slide
(sample 3F) after two weeks. This mineral phase had a distinctive morphology not
seen in any of the phosphate-containing samples. Although amine groups are not as
efficient at inducing CaP growth as phosphate and carboxylic acid groups, CaP
nucleation has previously been observed on amine terminated self assembled
monolayers.3
These results show that sparse mineralisation observed on the block-copolymer-
functionalized aminated slides is not due to the inhomogeneous dispersion of
phosphate groups, since the ToF-SIMS has shown a uniform distribution of these
groups (Chapter 3, Section 3.3.2). It is however possible that the total density of the
209
Chapter 4: In Vitro Mineralisation
phosphate groups on the surfaces may not be sufficient enough for mineral
formations. Another likely explanation is that ionic interactions between the
phosphate segment and the protonated amine groups leads to ion pairs that prevent
chelatation of calcium ions from the SBF solution. Although static ToF-SIMS
revealed that the most likely confirmation of sample 3A (P(MOEP-b-AAEMA)) on
the aminated slide was the PAAEMA segment coupled with the amine groups and
the PMOEP segments extending from the surface (Figure 4.10A), that analysis was
done under vacuum. Under wet conditions (e.g. in SBF) deprotonated phosphates
may very well interact with protonated amines on the slide and, hence, once again
prevent Ca2+ chelation (Figure 4.10B).
(A)
PAAEMA PMOEP
(B)
SBF
Figure 4.11: Proposed structures of a P(AAEMA-b-MOEP) block copolymer on an aminated slide (A) in vacuum and (B) in SBF solution.
The SBF results of both LbLs and coupled aminated slides support Matsuda’s
conclusion that the chelation of calcium ions by negatively charged groups plays an
important role in biomaterial mineralisation.3
4.4.4 SBF Studies of Fluorinated Amphiphilic Block Copolymers Adsorbed onto
PTFE Films
SEM images of P(FSm-b-AAn) adsorbed PTFE surfaces (samples 4A-C) did not
show any mineralisation after 7 days. However XPS revealed chelation of Ca2+ ions
by PAA segments as well as the presence of small amounts of phosphorous. Since
LbL films of PEI-PAA showed no phosphorous after 7 days in SBF, the free-
carboxylic acid groups on these modified films are better nucleators.
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Chapter 4: In Vitro Mineralisation
After 14 days, very small and patchy mineral formation was observed on all the
polymer adsorbed samples (samples 4A-C). These minerals were much smaller in
size compared to those formed on the phosphate-containing surfaces. The results
emphasises that carboxylic acid groups are not as efficient nucleators as phosphates.
It is also possible that the density of carboxylic acid groups may not have been high
enough to promote mineralisation.
In literature, carboxyl-terminated SAM surfaces and polymers containing -COOH
have shown an induction period (10 day for COOH-SAM) before apatite growth, and
this study correlates with this. Longer soaking time in SBF or pre-treatment with
Ca2+ ions may be required for these surfaces.
Nevertheless, it was evident that these adsorbed polymers were highly stable in SBF,
even up to two weeks with regular solution changes. Hence fluoropolymer surface
modification using block copolymers containing PFS is promising and further
investigation would be worthwhile.
211
Chapter 4: In Vitro Mineralisation
4.5 Conclusion
This study investigated the effects of polymeric features on mineralisation behaviour
in SBF using a series of phosphate-containing polymers, i.e. soluble and cross-linked
PMAEP and PMOEP. The effect of the carboxylic acid groups on mineralisation was
also investigated using poly(acrylic acid) (PAA) gels. The mineralisation of
fabricated surfaces (LbL films with soluble PMAEP and PMOEP polymers,
phosphate containing block copolymers attached to aminated slides, and carboxylic
acid-containing fluorinated block copolymers adsorbed to a PTFE substrate) were
studied.
Both soluble and cross-linked PMAEP samples showed the formation of brushite-
like minerals with tile morphology, whereas PMOEP gels showed secondary growth
of HAP on top of this mineral. The film obtained from soluble PMOEP showed large
amounts of HAP formation after 7 days in SBF. The FITR investigation also
revealed the presence of large amounts of carboxylic acid groups in PMAEP,
indicating cleavage of side-chains at C-O-C ester linkage. The PAA gel containing
carboxylic acid groups also formed a characteristic tile-like mineral layer in SBF
after 7 days. This leads to the conclusion that mineralisation is mainly influenced by
not only the nature of the monomer, but also by the structure of the polymer, as well
as the extent of cross-linking.
Both LbL films and block copolymers attached to aminated slides that contain
phosphate groups showed sparse and patchy mineralisation in SBF for up to two
weeks. Limited mineral growth on these samples highlights the importance of
accessible, ionic phosphate groups for calcium ion chelation and subsequent CaP
nucleation on materials designed for use in implantable materials. Fluorinated block
copolymers adsorbed PTFE films showed even smaller and patchier mineral
formation after two weeks in SBF compared to other fabricated surfaces containing
phosphate groups. This result emphasises that phosphate groups have stronger
tendency for CaP nucleation and mineralisation than the carboxylic acid groups, in
agreement with literature.
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Chapter 4: In Vitro Mineralisation
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Chapter 5: Overall Conclusions and Future Work
Chapter 5: Overall Conclusions and Future Work
Based on the observation that the incorporation of negatively-charged functional
groups, such as phosphate groups, have shown great promise for improving the bone-
bonding ability of polymeric materials by providing mineralisation nucleation sites,
the current study was aimed at investigating new approaches for the surface
modification of materials for biomedical applications. This was achieved using well-
defined functional polymers synthesised by controlled living radical polymerisation.
The in vitro mineralisation behaviour of a suite of phosphate-containing polymers
and functionalised surfaces containing phosphate and carboxylic acid groups was
subsequently studied.
5.1 Chapter 2
A suite of well-defined phosphate- and fluorine-containing polymers was
successfully synthesised using RAFT-mediated polymerisation. Despite the fact that
during the course of the investigation it was discovered that the MAEP and MOEP
monomers contain large amounts of diene impurities, it was still possible to prevent
cross-linking by limiting the molecular weight of the desired polymers. Since the
monomers also contain orthophosphoric acids, hydrolysis of the phosphate groups
were observed for both the gel and soluble polymers; with a more pronounced effect
shown for the soluble polymers. Moreover, both soluble and cross-linked PMAEP
homopolymers were found to contain large amounts of carboxylic acid groups
indicating hydrolysis at the C-O-C ester linkages. To immobilise these polymers onto
aminated slides, keto functionalities were successfully incorporated by block
copolymerisation with AAEMA. Well-defined fluorinated block copolymers were
synthesised using the RAFT technique for subsequent use as surfactants for the
surface modification of fluorinated bulk polymers. The RAFT-mediated
homopolymerisation of TFPA and TFPMA proceeded in a well-controlled manner,
whereas the molecular weights of PFS deviated significantly from the theoretical
value. This could be attributed to the self-initiation of FS and the long polymerisation
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times required. Chain extension of these fluorinated polymers with a range of
monomers, FS, tBA, AAEA or AAEMA was successfully carried out. Finally,
amphiphilic block copolymers containing carboxyl groups were synthesised by the
hydrolysis of tBA groups on the PtBA segments. The P(TFPMA-b-AAEMA) block
copolymer was reacted with small biomolecules (glycine or L-phenylalanyl glycine)
as a model system for the creation of biologically active surfactants.
5.2 Chapter 3
Three surface fabrication techniques were investigated: LbL assembly, coupling
reactions of keto-containing block copolymers onto aminated slides, and the
adsorption of fluorinated polymers onto PTFE. All of these techniques were found to
be robust for the polymers studied. An added advantage of these approaches is their
applicability to a wide range of materials. Surfaces containing carboxyl and
phosphate groups were produced by the LbL assembling of PEI and PAA, PMAEP
or PMOEP. The thickness of the PAA film was tuned by altering the pH of the
deposition solutions. AFM images showed that the topography of the PEI-PMAEP
LbL was patchy due to the presence of protonated carboxylate groups on the PMAEP
at low pH. On the other hand, the PEI-PMOEP LbL was found to be smooth.
Coupling reactions of the block copolymers consisting of PAAEMA and PMAEP or
PMOEP onto the aminated slides through reductive amination of the keto groups
with amines were successfully carried out. Regarding the conformation of the
attached block copolymer, ToF-SIMS revealed that the phosphate-containing
segments were more extended away from the surface whereas the PAAEMA
segments were attached to the slides. PFS was found to adsorb strongly onto PTFE in
the solvents studied, whereas PTFPA and PTFPMA were only adsorbed when DCM
was used as a solvent. The results were interpreted by considering the hydrophobicity
of the polymers, the solvent effects on the swelling of the PTFE and the solubility of
the polymers. P(FS-b-AA) with two PAA chain lengths were also adsorbed onto
PTFE. The contact angle measurements of these surfaces showed large hysteresis
indicating reorganisation of the flexible chains under the hydrated environment.
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5.3 Chapter 4
The mineralisation behaviour of several phosphate-containing polymers and
fabricated surfaces in SBF is discussed in this chapter. Both the soluble and gel
PMAEP polymers formed brushite-like minerals that were also found on the PAA
gel. The PMOEP gel showed a secondary growth of HAP on top of this initially
formed mineral phase. Large amounts of HAP formation was observed on the film
obtained from soluble PMOEP compared to the cross-linked gel, even though this
polymer had undergone greater loss of phosphate groups than the gel. This indicates
that the flexibility of the polymer chains affects mineralisation. Both LbL films and
block copolymers attached to aminated slides showed sparse and patchy
mineralisation in SBF even after two weeks. The P(FS-b-AA) adsorbed-PTFE
surface also showed only small amounts of mineral formation even after two weeks.
For all these systems the limited mineralisation could be due to the density of the
functional groups. In the case of LbL’s and the attached block copolymer, it can also
be a result of limited accessibility of ionic phosphate (or carboxylate) groups for
calcium chelation and subsequent mineralisation. For the attached block copolymer it
is possible that under wet conditions, deprotonated phosphates may very well interact
with protonated amines on the slide and, hence, once again prevent Ca2+ chelation. In
addition, for polymers containing carboxylic acid groups (i.e. PAA and hydrolysed
PMAEP) the limited mineralisation could also be due to the immersion time since
such groups have been shown to have some induction period before nucleation.
5.4 General Discussion
Most of the polymers synthesised in the current study were shown to be excellent
candidates for the surface modification of a range of polymeric materials. It was
demonstrated that with judicious choice of surface modification techniques, using
well-defined polymers, it is possible to produce surfaces that, at least under in vitro
conditions, respond differently to a biomineralisation environment. These results are
expected to find use in a wide range of biomaterials applications.
A number of key findings in the search for improved in vitro mineralisation of
surfaces were made. Firstly, the mobility of the polymer chains appeared to have a
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significant impact on the mineralisation outcome with the highly mobile chains
performing the best. Secondly, the availability of free phosphate groups to initially
chelate calcium ions was also found to be important and can explain the reason for
the pure outcome for the LbL assemblies and the block copolymer-attached slides. It
is possible that the density of the phosphate groups also influences the outcome
although this cannot be concluded definitely from the present data. Finally,
phosphate groups are known to exhibit stronger propensity for nucleation of CaP
than the carboxylate groups and this mineralisation study of homopolymers, LbLs
and flurosurfactants on PTFE supports this statement.
The surface modification techniques used in this study were successful in
immobilising well-defined polymers. Although LbL assembly has been previously
used in biomaterials science this study is unique in the use of well-defined
phosphate-containing polyelectrolytes. It is also the first to report the investigation of
the conformation of the attached block copolymers using ToF-SIMS. The use of
fluorinated surfactants synthesised by LRP in this area of science is novel and offers
a new direction to modifying fluoropolymer surfaces.
It is important to strongly stress that the both MAEP and MOEP monomers contain
large amounts of diene impurities and free orthophosphoric acid, and therefore the
polymerisation conditions will affect the hydrolysis of the phosphate groups as well
as preferential incorporation of dienes. Therefore, the obtained polymers must be
characterised solely for phosphate content as well as the state of the phosphates. The
contradictory mineralisation results of PMOEP-containing polymers in the literature
could be explained by this fact. This study supports the use of these monomers for
surface modifications of biomaterials to enhance bone bonding ability.
5.5 Future work
In order to optimise the syntheses developed in this thesis it would be advisable to
explore the possibility of developing a purification protocol for both the MAEP and
MOEP monomers. One approach would be to use solvent extraction to remove the
dienes and orthophosphoric acids. It would be expected that removal of the dienes
will further improve the mineralisation outcomes. The LbL and block copolymer
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attachment techniques are applicable to any surface that contains a variety of
functional groups and amine groups, respectively. These groups can be introduced
onto the biomaterial surface by techniques such as plasma treatment and aminolysis.
A search for alternate immobilisation techniques of phosphate-containing polymers
to ensure available free phosphate groups would also be useful. One possibility for
an immobilisation technique is by using the polymer end functional groups created
by the living radical polymerisation. These surfaces will allow us to investigate the
effect of chain length of the phosphate-containing polymers on mineralisation using
different molecular weight polymers.
Regarding with RAFT polymerisation of FS, further analyses of PFS would provide
more comprehensive arguments on the effect of the self-initiation on the deviation of
the molecular weights. These include using GPC with multiple angle laser light
scattering (MALLS) detectors to obtain the absolute molecular weights. It has also
been suggested to use electrospray ionization mass spectrometry (ESI-MS) to
identify the end groups before and after storage in THF and to confirm the UV-vis
spectrometry results.
Where the fluoropolymer surfactants are concerned, future work could include
investigation of the stability of the adsorbed fluorinated surfactants under applied
shear stress. Optimisation of the adsorption conditions is needed for chain
entanglements to occur which provide stable interlocking of the surfactants.
Parameters to be investigated include the chain length of the fluorinated segments,
the adsorption solvent, the washing solvent as well as the adsorption temperature.
The dependence of the PAA segment chain length on the mineralisation is another
factor warranting investigation.
Fluorinated block copolymers with carboxylic acid groups offer the possibility of
incorporating numerous biomolecules including cell adhesion peptides and growth
factors as well as phosphate groups. It is important in biomaterial applications to
investigate cell behaviour (e.g. attachment, growth and proliferation) on these
surfaces. The stability of the fluorinated surfactants under biological conditions is
also warranted.
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Since these adsorbed block copolymers are capable of reorganising depending on the
environmental conditions, LbL formation on top of these surfaces should provide
even more stable films. This will also allow the incorporation of biomolecules
through ionic interactions in addition to embedding them into LbLs produced from
biodegradable polymers.
The surface modification techniques presented in this study using well-defined
polymers with functional groups are suitable for use with a variety of materials and
hence in a wide range of applications. The study has also opened up the greater
opportunities to design precise surface properties in a controlled manner which
allows us to study the complicated process of mineralization as well as other
biological events such as protein adhesion and cell behaviour. Another advantage of
these modified surfaces is that further functionalisations such as the incorporation of
biomolecules to control specific cell and tissue responses can be carried out.
Finally, in vivo mineralisation tests using animal models would confirm results from
in vitro work which showed formation of different mineral phases on PMAEP and
PMOEP.
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