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Macromolecular Ruthenium Chemotherapeutics A UniqueApproach to Metastatic Cancer Treatment

Author:Blunden, Bianca

Publication Date:2014

DOI:https://doi.org/10.26190/unsworks/16865

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Macromolecular Ruthenium

Chemotherapeutics – A Unique Approach to Metastatic Cancer

Treatment

Bianca M. Blunden

A thesis submitted in fulfilment of the requirements for the degree of

Doctor Of Philosophy

Centre for Advanced Macromolecular Design

The University of New South Wales | School of Chemical Engineering

The Cooperative Research Centre for Polymers

August 2013

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Blunden

First name: Bianca Other name/s: Michelle

Abbreviation for degree as given in the University calendar: PhD

School: Chemical Engineering Faculty: Engineering

Title: Macromolecular ruthenium chemotherapeutics- A unique approach to metastatic cancer treatment

Abstract 350 words maximum: (PLEASE TYPE)

Novel macromolecular ruthenium chemotherapeutics were designed, synthesised and investigated in comparison to small drugs. Ruthenium possesses unique properties that have led to the development of exceptionally promising anticancer and antimetastatic therapeutics. The ruthenium(lll) and ruthenium(ll) drugs, NAMI-A [trans RuCI4(0MSO)Im]-lmH+ and RAPTA-C (RuCI2(p cymene)(PTA)], respectively, are two such candidates. The amplified benefit that can be gained by incorporating drugs into macromolecules has not previously been investigated for ruthenium agents. Two novel approaches to the synthesis of two types of rationally designed polymers, both containing ruthenium, were developed based on the ruthenium anticancer drugs NAMI-A and RAPTA-C. Both approaches utilised a distinct liganq of each drug. The first approach relied on the polymerisation of 4-vinyl imidazole via RAFT polymerisation. This created a macromolecular ligand for ruthenium(lll) to which a ruthenium precursor could be subsequently conjugated. The second approach used the inherent activity of the amide group on the PTA ligand of RAPTA-C to allow for conjugation to poly(2 iodoethyl methacrylate) via the substitution of the reactive halide. Two pathways were investigated to conjugate RAPTA-C to the polymer and both routes were tested using n-butyl iodide as a model compound. 1 0 and 20 NMR experiments were used to assess the conjugation and elucidate the superior pathway: a two-step reaction involving the conjugation of PTA via its reactive amine and subsequent complexation to form RAPT A C. Nano-sized carriers were designed to increase the cell uptake of these macromolecular drugs - micelles and nanotubes. Amphiphilic block copolymers incorporating NAMI-A or RAPTA-C were developed and self-assembled into micelles. RAPTA-C was also attached to cyclopeptide-polymer conjugates and selfassembled into nanotubes. A 1."5-times increase in cytotoxicity was found for NAMI-A micelles, and an outstanding 10-fold increase was found for both RAPTA-C micelles and nanotubes, when compared to the free drugs. An initial exploration into the antimetastatic activity revealed that the NAMI-A and RAPTA-C polymeric micelles significantly improved the inhibitory effects on both the migration and invasion of human breast cancer cells, indicating that it is highly probable that these nanoparticles will inhibit metastases in vivo. Thus, this work provides a substantial basis for the progression of these macromolecular chemotherapeutics into advanced biological studies.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only}.

····~········· · · · · ··· Signature

,, . 02· 11• ....... ~ .. ............. ':'f; ... .. . .... . Witness Date

The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.

FOR OFFICE USE ONLY Date of completion of requirements for Award:

THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS

“I have no special talent.

I am only passionately curious.”

– Albert Einstein

DECLARATIONS

ORIGINALITY STATEMENT

'I hereby declare that this submission is my own work and to the best of my knowledge it contains no

materials previously published or written by another person, or substantial proportions of material which

have been accepted for the award of any other degree or diploma at UNSW or any other educational

institution, except where due acknowledgement is made in the thesis. Any contribution made to the

research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the

thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to

the extent that assistance from others in the project's design and conception or in style, presentation and

linguistic expression is acknowledged.'

Bianca M. 8/unden Signed .... ~ .................. .. .................. . Date ..... J~~ .. ~.~f.:: . .l..4: .......... ................................... .

COPYRIGHT STATEMENT

'I hereby grant the University of New South Wales or its agents the right to archive and to make

available my thesis or dissertation in whole or part in the University libraries in all forms of media, now

or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights,

such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of

this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract

International.

I have either used no substantial portions of copyright material in my thesis or I have obtained

permission to use copyright material; where permission has not been granted I have applied/will apply

for a partial restriction of the digital copy of my thesis or dissertation.'

Bianca M. Blunden Signed .. ;RB.~:? .............. ....... ... ....... .. ... . Date .... J .. 4::.9.~?.: : .. !.~ .............................................. .

AUTHENTICITY STATEMENT

'I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version

of my thesis. No emendation of content has occurred and if there are any minor variations in formatting,

they are the result of the conversion to digital format.'

Bianca M. Blunden Signed ... ~~ ........ ................ : ........ . Date ..... t4::: .. ~.:J.~ ............................................. .

iii

ACKNOWLEDGEMENTS

Thank you to Martina Stenzel, my supervisor, for your constant positive attitude and

kindness – you have been a great inspiration to me.

To Donald Thomas, my co-supervisor, for your never-ending technical advice and

willingness to help – regardless of the hour.

And, to Hongxu Lu, for tirelessly completing all the biological work with a smile.

Thank you to all those (you know who you are) who contributed towards the

completion of my thesis – on an academic and personal level – for encouragement,

support and advice, for cups of tea and cake, a shoulder, a smile, a hug and many many

many good times of laughter and fun. Thank you for believing in me, even when I did

not. This is a superb PhD – in my opinion – and I could not have done it without you.

v

ABSTRACT

Novel macromolecular ruthenium chemotherapeutics were designed, synthesised and

investigated in comparison to small drugs. Ruthenium possesses unique properties that have

led to the development of exceptionally promising anticancer and antimetastatic therapeutics.

The ruthenium(III) and ruthenium(II) drugs, NAMI-A [trans-RuCl4(DMSO)Im]-ImH+ and RAPTA-C

[RuCl2(p-cymene)(PTA)], respectively, are two such candidates. The amplified benefit that can

be gained by incorporating drugs into macromolecules has not previously been investigated

for ruthenium agents.

Two novel approaches to the synthesis of two types of rationally designed polymers, both

containing ruthenium, were developed based on the ruthenium anticancer drugs NAMI-A and

RAPTA-C. Both approaches utilised a distinct ligand of each drug. The first approach relied on

the polymerisation of 4-vinyl imidazole via RAFT polymerisation. This created a

macromolecular ligand for ruthenium(III) to which a ruthenium precursor could be

subsequently conjugated. The second approach used the inherent activity of the amide group

on the PTA ligand of RAPTA-C to allow for conjugation to poly(2-iodoethyl methacrylate) via

the substitution of the reactive halide. Two pathways were investigated to conjugate RAPTA-C

to the polymer and both routes were tested using n-butyl iodide as a model compound. 1D

and 2D NMR experiments were used to assess the conjugation and elucidate the superior

pathway: a two-step reaction involving the conjugation of PTA via its reactive amine and

subsequent complexation to form RAPTA-C.

Nano-sized carriers were designed to increase the cell uptake of these macromolecular drugs –

micelles and nanotubes. Amphiphilic block copolymers incorporating NAMI-A or RAPTA-C were

developed and self-assembled into micelles. RAPTA-C was also attached to cyclopeptide-

polymer conjugates and self-assembled into nanotubes. A 1.5-times increase in cytotoxicity

was found for NAMI-A micelles, and an outstanding 10-fold increase was found for both

RAPTA-C micelles and nanotubes, when compared to the free drugs. An initial exploration into

the antimetastatic activity revealed that the NAMI-A and RAPTA-C polymeric micelles

significantly improved the inhibitory effects on both the migration and invasion of human

breast cancer cells, indicating that it is highly probable that these nanoparticles will inhibit

metastases in vivo. Thus, this work provides a substantial basis for the progression of these

macromolecular chemotherapeutics into advanced biological studies.

vii

PUBLICATIONS

Blunden, B. M., Chapman, R., Danial, M., Lu, H., Jolliffe, K. A., Perrier, S., & Stenzel, M. H.

Enhanced Chemotherapeutic Benefit of RAPTA-C by Conjugation to Cyclopeptide-Polymer Self-

Assembling Nanotubes. 2013, Submitted.

Blunden, B. M., Rawal, A., Lu, H. & Stenzel, M. H. Superior Chemotherapeutic Benefits from the

Ruthenium-Based Anti-Metastatic Drug NAMI-A through Conjugation to Polymeric Micelles.

Macromolecules, 2013, Accepted.

Blunden, B. M., Lu, H. & Stenzel, M. H. Enhanced Delivery of the RAPTA-C Macromolecular

Chemotherapeutic by Conjugation to Degradable Polymeric Micelles. Biomacromolecules,

2013, 14, 4177-4188.

Blunden, B. M., Thomas, D. S. & Stenzel, M. H. Macromolecular Ruthenium Complexes as Anti-

Cancer Agents. Polymer Chemistry, 2012, 3, 2964-2975.

Blunden, B. M., Thomas, D. S. & Stenzel, M. H. Analysis of Thiol-sensitive Core-cross-linked

Polymeric Micelles Carrying Nucleoside Pendant Groups using “On-line” Methods : Effect of

Hydrophobicity on Cross-linking and Degradation. Australian Journal of Chemistry, 2011, 64,

766–778.

ix

CONFERENCE PRESENTATIONS

Blunden, B. M., Lu, H. & Stenzel, M. H. Macromolecular Ruthenium(III) Chemotherapeutics.

Oral presentation at the 34th Australasian Polymer Symposium, Darwin, 7-10 July 2013.


Cancer Agents. Poster presentation at the Warwick Polymer Conference, Warwick, 8-12 July

2012.


Cancer Agents. Poster presentation at the 76th Prague Meeting on Macromolecules: Polymers

in Medicine, Prague, 1-5 July 2012.


Cancer Agents. Oral presentation at the 33rd Australasian Polymer Symposium, Hobart, 12-15

February 2012.

Blunden, B. M., Thomas, D. S. & Stenzel, M. H. Ruthenium Complexes Attached To Polymers As

Anticancer Agents. Oral presentation at the 32nd Australasian Polymer Symposium, Coffs

Harbour, 13-16 February 2011.

Blunden, B. M., Thomas, D. S. & Stenzel, M. H. Analysis of Thiol-Sensitive Core-Crosslinked

Polymeric Micelles Carrying Nucleoside Pendant Groups using ‘On-Line’ Methods: Effect of

Hydrophobicity on Crosslinking and Degradation. Poster presentation at the 32nd Australasian

Polymer Symposium, Coffs Harbour, 13-16 February 2011.

Blunden, B. M., Thomas, D. S. & Stenzel, M. H. Ruthenium Complexes Attached To Polymers As

Anticancer Agents. Oral presentation at the UWS Polymer Workshop, Sydney, November 2010.

Blunden, B. M., Zhang, L., & Stenzel, M. H. Thiol-Sensitive Polymeric Micelles Carrying

Nucleoside Pendant Groups. Oral presentation at the 30th Australasian Polymer Symposium,

Melbourne, 30 Nov – 4 Dec 2008.

1

TABLE OF CONTENTS

DECLARATIONS .............................................................................................................................................. i

ACKNOWLEDGEMENTS ................................................................................................................................ iii

ABSTRACT ..................................................................................................................................................... v

PUBLICATIONS ............................................................................................................................................. vii

CONFERENCE PRESENTATIONS .................................................................................................................... ix

TABLE OF CONTENTS ................................................................................................. 1

FIGURES ........................................................................................................................................................ 7

SCHEMES .................................................................................................................................................... 14

TABLES ........................................................................................................................................................ 15

ABBREVIATIONS ......................................................................................................................................... 17

SYMBOLS .................................................................................................................................................... 20

INTRODUCTION..................................................................................................................... 21

CHAPTER 1. LITERATURE REVIEW ................................................................................ 25

1.1. CANCER AND CHEMOTHERAPEUTICS...................................................................................26

1.1.1. Defining ‘Cancer’ .................................................................................................................. 26

1.1.2. Cancer Treatment ................................................................................................................. 28

1.2. METAL-BASED ANTICANCER COMPOUNDS ..........................................................................30

1.2.1. The Benchmark: Platinum-Based Anticancer Drugs ............................................................. 30

1.2.2. Advantages of Metal-centred & Organometallic Compounds ............................................. 31

1.2.3. Chemotherapeutic Metallopharmaceuticals ........................................................................ 32

1.3. RUTHENIUM CHEMISTRY .....................................................................................................34

1.3.1. Ligands .................................................................................................................................. 34

1.3.2. Ruthenium Compounds as Anti-Cancer Agents ................................................................... 37

1.4. RUTHENIUM COMPLEXES ....................................................................................................38

1.4.1. Ruthenium(IV) Complexes .................................................................................................... 39

1.4.2. Ruthenium(III) Complexes .................................................................................................... 40

1.4.3. Activation of Ruthenium Complexes .................................................................................... 46

1.4.4. Ruthenium(II) Complexes ..................................................................................................... 50

1.4.5. Other Ruthenium Compounds for Cancer Therapy .............................................................. 56

Table of Contents

2

1.5. DNA INTERACTIONS WITH RUTHENIUM.............................................................................. 57

1.5.1. DNA Damage .........................................................................................................................57

1.5.2. DNA Binding Modes ..............................................................................................................58

1.6. MACROMOLECULAR METAL COMPLEXES............................................................................ 61

1.7. POLYMERS AND ANTI-CANCER AGENTS .............................................................................. 62

1.7.1. Motivation ............................................................................................................................62

1.7.2. History of Polymeric Therapeutics ........................................................................................65

1.7.3. Design of Polymeric Agents ..................................................................................................66

1.7.4. Macromolecules in Clinical Evaluation .................................................................................68

1.8. POLYMERISATION ............................................................................................................... 69

1.8.1. Free Radical Polymerisation ..................................................................................................70

1.8.2. Reversible-Deactivation Radical Polymerisation ..................................................................70

1.8.3. Copolymerisation ..................................................................................................................71

1.8.4. Reversible Addition Fragmentation Chain Transfer Polymerisation (RAFT) .........................72

1.9. SELF-ASSEMBLED POLYMER NANOPARTICLES ..................................................................... 76

1.9.1. Polymeric Micelles ................................................................................................................76

1.9.2. Peptides ................................................................................................................................78

1.10. CONCLUSIONS ..................................................................................................................... 79

CHAPTER 2. RUTHENIUM(III) .......................................................................................... 81

2.1. SYNTHESES .......................................................................................................................... 84

2.1.1. Synthesis of 4-Vinyl Imidazole (VIm) .....................................................................................84

2.1.2. Polymerisation of 4-Vinyl Imidazole (PVIm) via RAFT Polymerisation ..................................84

2.1.3. Chain Extension of 4-Vinyl Imidazole with Poly(ethylene glycol) methyl ether acrylate

(PVIm-PPEGMEA) via RAFT Polymerisation ............................................................................................84

2.1.4. Synthesis of [DMSO2H][trans-RuCl4(DMSO)2] (Ru Precursor) ...............................................85

2.1.5. Synthesis of (ImH)[RuIII

Cl4(Im)(S-DMSO)] (NAMI-A) ..............................................................85

2.1.6. Synthesis of Macromolecular NAMI-A [P(NAMI-A)] .............................................................86

2.1.7. Synthesis of P(NAMI-A)-PPEGMEA .......................................................................................86

2.1.8. In Vitro Cytotoxicity Assay ....................................................................................................86

2.2. RESULTS AND DISCUSSION .................................................................................................. 87

2.2.1. Synthesis and Polymerisation of 4-Vinyl Imidazole ..............................................................87

2.2.2. Synthesis of NAMI-A .............................................................................................................90

Table of Contents

3

2.2.3. Synthesis of Macromolecular NAMI-A ................................................................................. 91

2.2.4. NAMI-A Block Copolymer ..................................................................................................... 95

2.2.5. Micellisation of Amphiphilic Block Copolymer ..................................................................... 99

2.2.6. In Vitro Cytotoxicity ............................................................................................................ 100

2.3. CONCLUSIONS ................................................................................................................... 103

CHAPTER 3. RUTHENIUM(II) ......................................................................................... 105

3.1. SYNTHESES ........................................................................................................................ 108

3.1.1. Synthesis of 2-Chloroethyl Methacrylate (CEMA) .............................................................. 108

3.1.2. Polymerisation of 2-Chloroethyl Methacrylate (PCEMA) via RAFT Polymerisation ........... 108

3.1.3. Polymer End-Group Modifications of PCEMA .................................................................... 108

3.1.4. Statistical Copolymerisation of N-(2-Hydroxypropyl) Methacrylamide and 2-Chloroethyl

Methacrylate (P(HPMA-CEMA)) via RAFT Polymerisation ................................................................... 109

3.1.5. Polymer End-Group Modifications of P(HPMA-CEMA) ...................................................... 109

3.1.6. Synthesis of Dichlororuthenium(II)(p-cymene)(1,3,5-triaza-7-phosphaadamantane)

(RAPTA-C).. .. ……………………………………………………………………………………………………………………………………110

3.1.7. Synthesis of a Low Molecular Weight Model Compound and Macromolecular Ruthenium

Complex… ………………………………………………………………………………………………………………………………………110

3.1.8. Synthesis of Macromolecular Ruthenium Complex: Copolymer-RAPTA-C via Route B ..... 111

3.1.9. Cytotoxicity Assay ............................................................................................................... 112

3.2. RESULTS & DISCUSSION ..................................................................................................... 112

3.2.1. Synthesis of PIEMA ............................................................................................................. 112

3.2.2. Model Reactions Using n-Butyl Iodide ............................................................................... 114

3.2.3. Route A: Synthesis of Dichlororuthenium(II) (p-cymene)(1,3,5-triaza-7-

phosphaadamantane) (RAPTA-C) and Reaction with Butyl Iodide ...................................................... 115

3.2.4. Route B: Reaction of PTA with n-Butyl Iodide and Subsequent Reaction with the RuCl2(p-

cymene) Dimer .................................................................................................................................... 118

3.2.5. Synthesis of Macromolecular Ruthenium Complex: Polymer-RAPTA-C ............................. 125

3.2.6. Statistical Copolymerisation of N-(2-Hydroxypropyl) Methacrylamide and 2-Chloroethyl

Methacrylate (P(HPMA-CEMA)) via RAFT Polymerisation ................................................................... 128

3.2.7. Cytotoxicity Assay ............................................................................................................... 131

3.3. CONCLUSIONS ................................................................................................................... 132

CHAPTER 4. POLYMERIC MICELLES ............................................................................ 135

4.1. SYNTHESES ........................................................................................................................ 138

Table of Contents

4

4.1.1. Synthesis of Dichlororuthenium(II)(p-cymene)(1,3,5-triaza-7-phosphaadamantane)

(RAPTA-C) ……………………………………………………………………………………………………………………………..……...138

4.1.2. Synthesis of Polylactide (PLA) MacroRAFT Agent .............................................................. 138

4.1.3. Synthesis of 2-Chloroethyl Methacrylate .......................................................................... 138

4.1.4. Polymerisation of 2-Hydroxyethyl Acrylate (HEA) with PLA MacroRAFT ........................... 138

4.1.5. Polymerisation of HEA and CEMA with PLA MacroRAFT ................................................... 139

4.1.6. Conjugation of RAPTA-C to (PLA-b-P(HEA-CEMA-F)) Copolymer ....................................... 140

4.1.7. Micellisation of Drug-loaded Copolymers ......................................................................... 141

4.1.8. In Vitro Cell Culture Assays ................................................................................................ 141

4.2. RESULTS AND DISCUSSION ................................................................................................ 143

4.2.1. Synthesis of PLA MacroRAFT Agent ................................................................................... 143

4.2.2. Amphiphilic Polymers ........................................................................................................ 145

4.2.3. RAPTA-C Conjugation ......................................................................................................... 151

4.2.4. Micellisation of Drug-Loaded Amphiphilic Polymers ......................................................... 154

4.2.5. In Vitro Cell Studies of Drug-Loaded Micelles .................................................................... 158

4.3. CONCLUSIONS ................................................................................................................... 165

CHAPTER 5. PEPTIDE TUBES ........................................................................................ 167

5.1. SYNTHESES ........................................................................................................................ 170

5.1.1. Synthesis of Cyclopeptides ................................................................................................ 170

5.1.2. Copolymerisation of HEA and CEMA via RAFT Polymerisation .......................................... 170

5.1.3. Copolymerisation of HEA and CEMA via RAFT Polymerisation using an Alkyne RAFT Agent

……………………………………………………………………………………………………………………………………..171

5.1.4. Conjugation of Polymers to Peptides................................................................................. 171

5.1.5. Conjugation of RAPTA-C to Cyclopeptide-Polymer Conjugates ......................................... 172

5.1.6. Self-Assembly of Tubes ...................................................................................................... 173

5.1.7. Crosslinked Nanotubes ...................................................................................................... 173

5.1.8. Cytotoxicity Assay .............................................................................................................. 173

5.2. RESULTS AND DISCUSSION ................................................................................................ 174

5.2.1. Copolymer Synthesis .......................................................................................................... 174

5.2.2. Cyclopeptide-Polymer Conjugation ................................................................................... 177

5.2.3. RAPTA-C Conjugation ......................................................................................................... 179

5.2.4. Self-Assembly of Tubes ...................................................................................................... 182

5.2.5. Crosslinked Nanotubes ...................................................................................................... 184

5.2.6. Cytotoxicity Evaluation ...................................................................................................... 185

Table of Contents

5

5.3. CONCLUSIONS ................................................................................................................... 188

CHAPTER 6. METASTATIC ASSESSMENT ................................................................... 189

6.1. METHODS .......................................................................................................................... 190

6.1.1. Tumour Cell Lines for In Vitro Tests.................................................................................... 190

6.1.2. Cell Viability Assay .............................................................................................................. 190

6.1.3. Migration Assay .................................................................................................................. 191

6.1.4. Invasion Assay .................................................................................................................... 191

6.2. RESULTS ............................................................................................................................ 192

6.2.1. Effect on Cell Viability ......................................................................................................... 194

6.2.2. Effects on Migration and Invasion ...................................................................................... 195

6.4. DISCUSSION ....................................................................................................................... 199

6.5. CONCLUSIONS ................................................................................................................... 200

CHAPTER 7. CONCLUSIONS & OUTLOOK ................................................................... 201

CHAPTER 8. MATERIALS & ANALYSES ....................................................................... 205

8.1. CHEMICALS ........................................................................................................................ 205

8.1.1. Ruthenium(III) NAMI-A Conjugation (Chapter 2) ............................................................... 205

8.1.2. Ruthenium(II) RAPTA-C Conjugation (Chapter 3) ............................................................... 206

8.1.3. Polymeric Micelles (Chapter 4) .......................................................................................... 206

8.1.4. Peptide Tubes (Chapter 5) .................................................................................................. 207

8.2. RAFT AGENT SYNTHESES ................................................................................................... 208

8.2.1. Cumyldiothiobenzoate (CDB, 1) ......................................................................................... 208

8.2.2. 2-(((dodecylthio)carbonothioyl)thio)-2-methylpropanoic acid (RAFT11, 2) ...................... 209

8.2.3. Benzyl (2-hydroxyethyl) carbonotrithoate (trithio, 3) ........................................................ 209

8.2.4. Butyl-trithiocarbonate Propanoic Acid (BTCPA, 4) ............................................................. 209

8.2.5. (prop-2-ynyl propanoate)yl Butyltrithiocarbonate (PPBTC, Alkyne RAFT, 5) ..................... 210

8.3. ANALYSIS TECHNIQUES ..................................................................................................... 210

8.3.1. Thin Layer Chromatography (TLC) ...................................................................................... 210

8.3.2. Size Exclusion Chromatography (SEC) ................................................................................ 210

8.3.3. Nuclear Magnetic Resonance (NMR) Spectrometry .......................................................... 211

8.3.4. Mass Spectrometry (MS) .................................................................................................... 212

Table of Contents

6

8.3.5. X-Ray Crystallography ........................................................................................................ 213

8.3.6. UV-Vis Spectrometry .......................................................................................................... 213

8.3.7. Fourier-Transform Near-Infrared (FT-NIR) Spectroscopy .................................................. 213

8.3.8. Dynamic Light Scattering (DLS) .......................................................................................... 214

8.3.9. Microscopy ......................................................................................................................... 214

8.3.10. Thermo Gravimetric Analysis (TGA) ................................................................................... 214

8.3.11. Elemental Analysis ............................................................................................................. 215

8.3.12. Microwave Reactions ......................................................................................................... 216

8.3.13. Fluorescence Spectrometry ............................................................................................... 216

REFERENCES ....................................................................................................................... 217

APPENDICES ........................................................................................................................ 235

Figures, Schemes & Tables

7

FIGURES

Figure 1-1: Loss of normal cell growth leading to the development of cancer. Adapted from the National

Cancer Institute website.32

...................................................................................................... 26

Figure 1-2: Five-year relative survival rate for selected cancers in Australia 2006-2010. Adapted from the

Australian Institute of Health report ‘Cancer in Australia’ 2012.1 .......................................... 27

Figure 1-3: Five year relative survival rate for selected cancers in Australia 1982-2010. Adapted from the

Australian Institute of Health report ‘Cancer in Australia’ 2012.1 .......................................... 27

Figure 1-4: The most promising ruthenium-based therapeutics were tested against a mammary

carcinoma model and show differing degrees of selectivity and toxicity towards metastases.

Toxic 6/10: 6 out of 10 treated mice died due to drug toxicity. Non selective cytotoxicity:

primary tumour and metastasis growth are inhibited in a similar way. Selectivity: metastasis

is inhibited more than primary tumour growth. High selectivity: metastasis is inhibited with

no or marginal effects on primary tumour growth. Adapted from Bergamo et al.62

............. 39

Figure 1-5: a) [trans-RuCl4(DMSO)Im]-Na

+ (NAMI) and b) [trans-RuCl4(DMSO)Im]

-ImH

+ (NAMI-A), where

Im = imidazole. ........................................................................................................................ 42

Figure 1-6: a) [RuCl4(Ind)2]-IndH

+ (KP1019) and b) [RuCl4(Im)2]

-ImH

+ (KP418), where Ind = indazole and Im

= imidazole. ............................................................................................................................. 46

Figure 1-7: Multiple cytotoxic routes of ruthenium anticancer agents. Chloride ligands are hydrolyses

upon entering the cell, due to the lower chloride concentration. This allows crosslinking of

DNA leading to apoptosis of the cell. Alternatively, ruthenium can mimic iron in the body by

binding to transferrin. It is then uptaken into the cell by transferrin receptors on the cell

surface. Adapted from Levina et al.46

..................................................................................... 49

Figure 1-8: Trend for an increase in cytotoxicity with an increase in the size of the arene ring system,

with X = Cl and PF6 as counterions. ......................................................................................... 52

Figure 1-9: a) [(Bip)RuCl(en)] (RM175) and b) [(p-cymene)RuCl(acac)], where Bip = biphenyl and acac =

acetylacetonate....................................................................................................................... 52

Figure 1-10: a) [RuCl2(toluene)(PTA)] (RAPTA-T), and b) [RuCl2(p-cymene)(PTA)]

(RAPTA-C), where PTA =

1,3,5-phosphaadamantane. .................................................................................................... 54

Figure 1-11: Passive targeting. A comparison of normal tissue vs. tumour tissue. Macromolecules are

more likely to accumulate and subsequently be retained in tumour tissue, due to increased

permeability and lack of a lymphatic drainage system. .......................................................... 64

Figure 1-12: Timeline of milestones in the emergence of anticancer polymer therapeutics. First drugs to

market are highlighted in yellow. GCSF: granulocyte colony-stimulating factor; PEG:

poly(ethylene glycol); SMANCS: styrene maleic anhydride-neocarzinostatin. Adapted from

Duncan.4 .................................................................................................................................. 65


8

Figure 1-13: The Ringsdorf136

Model: A rationale for the delivery of therapeutic drugs using polymer-

drug conjugates. Adapted from Larson.7 .................................................................................66

Figure 1-14: General structure of a Chain Transfer Agent (RAFT Agent). ...................................................72

Figure 1-15: Guidelines for the selection of R and Z groups for the polymerisation of the most common

families of monomers. R groups: fragmentation rates decrease from left to right. Z groups:

fragmentation rates increase and addition rates decrease from left to right. Dashed lines

indicate partial control over the polymerisation (i.e. control over the molecular weight

evolution but poor control over the polydispersity index.) MMA = methyl methacrylate, Sty =

styrene, MA = methyl acrylate, AM = acrylamide, VAc = vinyl acetate. Adapted from Moad et

al.168,177,178

................................................................................................................................73

Figure 1-16: Self-assembly of amphiphilic block copolymers into micelles. ...............................................78

Figure 2-1: Stack-plot of 1H NMR spectra. A: urocanic acid and B: 4-vinyl imidazole in DMSO-d6 at 25 °C.

The peak assignment corresponds to Scheme 2-2. .................................................................88

Figure 2-2: Stack-plot of 1H NMR spectra. A: poly(4-vinyl imidazole) and B: 4-vinyl imidazole in MeOD at

25 °C. The peak assignment corresponds to Scheme 2-2. .......................................................89

Figure 2-3: Water SEC trace if the polymerisation of 4-vinyl imidazole in acetic acid at 70 °C. [VIm] = 0.8

M, [VIm]:[RAFT]:[ACPA] = 200:1:0.25. .....................................................................................89

Figure 2-4: 1H NMR of (ImH)[Ru

IIICl4(Im)(S-DMSO)] (NAMI-A) in D2O at 25 °C. The very broad DMSO

ligand methyl peak at -15.2 is consistent with literature.74

....................................................91

Figure 2-5: UV-Vis spectrum in methanol at 25 ᵒC. The attachment of imidazole to RuIII

elicits a clear shift

in absorbance maxima from 375 nm to 400 nm. ....................................................................93

Figure 2-6: Stack-plot of 1H NMR spectra. A: poly(4-vinyl imidazole) (PVIm) and B: poly(vinyl imidazole)-

b-poly(poly(ethylene glycol) methyl ether acrylate) (PVIm-PPEGMEA) in MeOD at 25 °C. ....95

Figure 2-7: Water SEC trace of the polymerisation of poly(ethylene glycol) methyl ether acrylate in

methanol at 65 ᵒC using P(4-vinyl imidazole) macroRAFT agent (Mn,theo = 14 300 g.mol-1

).

[PEGMEA] = 0.4 M, [PEGMEA]:[PVIm]:[AIBN] = 50:1:0.2. Total reaction time = 4 hrs, xFT-NIR =

48 %, Mn,theo = 28 700 g.mol-1

. The eluent was a MilliQ water/acetic acid/methanol (54:23:23)

solution. ...................................................................................................................................96

Figure 2-8: Polymerisation of poly(ethylene glycol) methyl ether acrylate in methanol at 65 ᵒC usingP(4-

vinyl imidazole) macroRAFT agent, monitored via FT-IR. [PEGMEA] = 0.4 M,

[PEGMEA]:[PVIm]:[AIBN] = 50:1:0.2. .......................................................................................96

Figure 2-9: 1H saturation recovery experiment of NAMI-A, P(NAMI-A) and P(NAMI-A)-PPEGMEAvia solid-

state NMR. ...............................................................................................................................98

Figure 2-10: P(NAMI-A)-PPEGMEA Micelles prepared by dialysing MeOH solution against water. Sample

(a) was drop-loaded onto grid and air-dried. Samples (b) to (f) were drop-loaded onto grid,

air-dried and stained with Phosphotungstic Acid. Scale bar: d = 2 µm; c & f = 200 nm; a, b &

e = 100 nm. ........................................................................................................................... 100


9

Figure 2-11: Cytotoxicity of PVIm and PVIm-PPEGMEA against ovarian A2780 and OVCAR-3 and

pancreatic AsPC-1 cancer cell lines, after 72 hours, n = 4..................................................... 101

Figure 2-12: Cytotoxicity of NAMI-A and P(NAMI-A)-PPEGMEA against ovarian A2780 and OVCAR-3 and

pancreatic AsPC-1 cancer cell lines, after 72 hours. For P(NAMI-A)-PPEGMEA, the polymer

concentration at [Ru] = 449 and 224.5 µM is 10 and 5.0 µM, respectively. Mean ± SD, n = 4,

** significantly different p < 0.01, *** significantly different, p < 0.001. ............................. 102

Figure 3-1: Polymerisation of 2-chloroethyl methacrylate in 1,4-dioxane at 60 °C using CDB as RAFT

agent. [CEMA]= 2.5 M, [CEMA]:[RAFT]:[AIBN] = 300:1:0.5. .................................................. 113

Figure 3-2: 1H NMR spectra of PCEMA (bottom) and PIEMA (top) showing the shift in the chloroalkyl

peak at 3.73 to the iodoalkyl peak at 3.33. The peaks were monitored until > 98 %

conversion was achieved. ..................................................................................................... 114

Figure 3-3: 31

P NMR spectra showing the attempted synthesis of RAPTA-C in MeOH and CDCl3. The

reaction in CDCl3 produced two products that were not identified. .................................... 116

Figure 3-4: Stack-plot of 31

P NMR spectra. A: RAPTA-C, B: IBu-RAPTA-C, and C: Polymer-RAPTA-C in

DMSO-d6 at 25 °C. All spectra are representative of Route A. .............................................. 117

Figure 3-5: [1H-

31P] HMBC Spectrum of RAPTA-C and butylated RAPTA-C in DMSO-d6 at 25 °C. Both the

1H and

31P spectra are external projections. A shift in the cross-peaks assigned to the PTA

and p-cymene RAPTA-C complex ligands to the corresponding cross-peaks for the butylated

RAPTA-C complex ligands is evident. Alkylation of the PTA ligand causes a distinct difference

in the number of types of protons present in the PTA structure i.e. two types before and

four types after. .................................................................................................................... 118


P NMR spectra. A: PTA, B: RAPTA-C, C: IBu-PTA, D: IBu-RAPTA-C, E: Polymer-

PTA and F: Polymer-RAPTA-C, in DMSO-d6 at 25 °C. All spectra are representative of the

Route B synthesis. ................................................................................................................. 120

Figure 3-7: [1H-

13C] HMBC NMR Spectrum of n-butyl iodide + PTA in DMSO-d6 at 25 °C. Both the

1H and

13C spectra are external projections. A [

1H-

13C] HSQC was used in the identification of all

cross-peaks associated with the identified structures. Structural identification of compounds

corresponds to Scheme 3-3. A more detailed identification of the compounds is shown in

Appendix Figure A-2. ............................................................................................................. 121

Figure 3-8: [1H-

31P] HMBC NMR Spectrum of butylated PTA in DMSO-d6 at 25 °C.

1H and

31P spectra are

external projections. ............................................................................................................. 122

Figure 3-9: Initial PTA at 180.9 (bottom) shifted to Alkylated IBu-PTA at 214.1 (top). ............................ 122

Figure 3-10: [1H-

13C] HMBC NMR Spectrum of butylated PTA + RuCl2(p-cymene) Dimer in DMSO-d6 at 25

°C. 1H and

13C spectra are external projections. Structural identification of compounds

corresponds to Scheme 3-3. Note that unidentified correlations correspond to starting

materials that were previously identified in Figure 3-7. A more detailed identification of the

compounds is shown in Appendix Figure A-3. ...................................................................... 123


10

Figure 3-11: [1H-

15N] HMBC of RAPTA-C, butylated PTA and butylated RAPTA-C in DMSO-d6 at 25 °C.

Three distinctive 15

N chemical shifts were detected. No cross-peaks for protons associated

with positively charged 15

N were found, due to problems associated with peak detection of

charged nuclei. ..................................................................................................................... 124

Figure 3-12: Stack-plot of 1H spectra, showing the arene region. A: RuCl2(p-cymene) Dimer, B: RAPTA-C,

C: Polymer-RAPTA-C synthesised via Route A, D: Polymer-RAPTA-C synthesised via Route B.

The comparison shows that p-cymene dissociates from the complex during reaction. The

broad p-cymene peak produced via Route B provides evidence for the polymer-RAPTA-C

product. ................................................................................................................................ 126

Figure 3-13: The 31

P peak in PTA was monitored over time for each reaction. All 31

P peaks in a given

spectra were integrated and normalised to sum to 100. The fraction corresponding to each

product represents the conversion of one species to another. Since the complexation for all

experiments (model compound, polymer and copolymer) reached 100 % conversion in less

than one hour, no kinetic data was obtained and is not represented on this figure. .......... 127


P NMR spectra showing the synthesis steps of Copolymer-RAPTA-C. Solution

A: Copolymer + PTA. Solution B: Copolymer + PTA + RuCl2(p-cymene) Dimer. .................... 130

Figure 3-15: SEC traces for the RAPTA-C complex, Copolymer and Copolymer-RAPTA-C, in N,N-

dimethylacetamide at λ = 326 nm. The UV traces were normalised using the RI traces of the

same solution. ...................................................................................................................... 131

Figure 3-16: Cytotoxicity profile of Copolymer-RAPTA-C and RAPTA-C, after 72 hours, n = 3. ............... 132

Figure 4-1: Stack-plot of 1H NMR spectra. A: lactide and B: D,L-polylactide in DMSO-d6 at 25 °C. The peak

assignment corresponds to Scheme 4-2. .............................................................................. 144

Figure 4-2: SEC traces of the polymerisation of HEA with PLA MacroRAFT Agent in

N,N-dimethylacetamide at 50 ᵒC. [HEA]:[MacroRAFT]:[AIBN] = 400:1:0.2. ......................... 146

Figure 4-3: PLA-b-PHEA micelle sample was drop-loaded onto grid, air-dried and stained with

Phosphotungstic acid. Scale bar: a = 200 nm; b = 100 nm. .................................................. 146

Figure 4-4: DLS number average particle size distributions of PLA-b-PHEA, polymer A and polymer B

micelles analysed by DLS in water at 25 ᵒC, where ηPLA = 1.482. ......................................... 147

Figure 4-5: SEC traces of the polymerisation of HEA and CEMA with PLA MacroRAFT Agent in

N,N-dimethylacetamide at 50 ᵒC. [HEA]:[CEMA]:[F]:[MacroRAFT]:[AIBN] = 356:40:4:1:0.2.148

Figure 4-6: 1H NMR spectra of the polymerisation of HEA and CEMA with PLA MacroRAFT Agent in

DMSO-d6 at 25 ᵒC. [HEA]:[CEMA]:[F]:[MacroRAFT]:[AIBN] = 455:50:5:1:0.2. A: 1 hr, B: 3 hrs,

C: 5 hrs. ................................................................................................................................. 149

Figure 4-7: Polymer A before RAPTA-C conjugation. (a) to (c) Sample drop-loaded onto grid and air-dried.

(d) to (f) Sample drop-loaded onto grid, air-dried and stained with Phosphotungstic acid.

Scale bar: a, b, d, e = 200 nm; c & f = 100 nm. ..................................................................... 150


11

Figure 4-8: Polymer B before RAPTA-C conjugation. (a) & (b) Sample drop-loaded onto grid and air-dried.

(c) to (f) Sample drop-loaded onto grid, air-dried and stained with Phosphotungstic acid.

Scale bar: a = 500 nm; b = 2 µm; d, e = 100 nm; f = 50 nm. .................................................. 150

Figure 4-9: 1H Spectra showing the attachment of RAPTA-C to PLA-b-P(HEA-IEMA-F) in DMSO-d6 at 25 ᵒC.

A: Polymer before Finkelstein, B: Polymer after Finkelstein, C: PTA added to polymer in

solution, D: Dimer added to solution. ................................................................................... 152

Figure 4-10: 31

P Spectra showing the attachment of RAPTA-C to PLA-b-P(HEA-IEMA-F) polymer A via a

two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to polymer in solution,

Top: Dimer added to solution. Desired products are circled. ............................................... 153

Figure 4-11: 31

P Spectra showing the attachment of RAPTA-C to PLA-b-P(HEA-IEMA-F) polymer B via a

two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to polymer, Top:

Dimer added to solution. Desired products are circled. ....................................................... 153

Figure 4-12: The p-cymene region of the 1H spectra showing the attachment of RAPTA-C to PLA-b-

P(HEA-IEMA-F), in DMSO-d6 at 25 ᵒC. The broad polymer, residual unreacted

RuCl2(p-cymene) dimer and RAPTA-C p-cymene peaks are at 5.95 ppm, 5.86 ppm and

5.75 ppm, respectively. Bottom: Polymer A, Top: Polymer B. .............................................. 154

Figure 4-13: DLS number average particle size distributions of micelles A and B in water at 25 ᵒC, with

and without conjugated RAPTA-C. ........................................................................................ 155

Figure 4-14: Polymer A (a) to (c) Sample drop-loaded onto grid and air-dried. (d) to (f) Sample drop-

loaded onto grid, air-dried and stained with Phosphotungstic acid. Scale bar: d = 1 µm, a =

500 nm, b & e = 100 nm, c & f = 50 nm. ................................................................................ 156

Figure 4-15: Polymer B (a) to (d) Sample drop-loaded onto grid and air-dried. (e) to (h) Sample drop-

loaded onto grid, air-dried and stained with Phosphotungstic acid. Scale bar: a = 1 µm, d & f

= 200 nm, b & e = 100 nm, c & inset = 50 nm. ...................................................................... 157

Figure 4-16: Cytotoxicity profile of micelles self-assembled from polymers A and B before RAPTA-C

conjugation, after 72 hours, n = 4. ........................................................................................ 159

Figure 4-17: Cytotoxicity profile of RAPTA-C and micelles self-assembled from polymers A and B, against

ovarian carcinoma A2780 cells, after 72 hours, n = 4. .......................................................... 160


cisplatin-resistant ovarian carcinoma A2780cis cells, after 72 hours, n = 4. ........................ 160


ovarian carcinoma OVCAR-3 cells, after 72 hours, n = 4. ...................................................... 161

Figure 4-20: Confocal microphotographs of A2780 cells after incubation with micelles at 37 °C for three

hours. Polymers (green) were labelled with fluorescein. Cell nuclei (blue) were stained with

Hoechst 33342. Lysosomes (red) were stained with LysoTracker Red DND-99. Scale bar = 5

µm. ........................................................................................................................................ 162

Figure 4-21: Concentration of ruthenium per million A2780 cells measured by ICPMS, after digestion in

Aqua Regia. Mean ± SD, n = 3. *** significantly different, p < 0.001. ................................... 163


12

Figure 4-22: Colony formation of A2780 (A) and A2780cis (B) cells. Scale bar = 10 mm. ....................... 164

Figure 5-1: Polymerisation of 2-chloroethyl methacrylate and 2-hydroxyethyl acrylate in

N,N-dimethylacetamide at 60 °C using RAFT agent 4........................................................... 175


N,N-dimethylacetamide at 60 °C using RAFT agent 4. HEA:CEMA feed ratios

are A = 90:10, B = 80:20. ....................................................................................................... 175


N,N-dimethylacetamide at 60 °C using 4 as RAFT agent. [Monomer] = 2.4 M,

[HEA]:[CEMA]:[RAFT]:[AIBN] = 90:10:1:0.1. ......................................................................... 176


N,N-dimethylacetamide at 60 °C using RAFT agent 4. [Monomer] = 2.4 M,

[HEA]:[CEMA]:[RAFT]:[AIBN] = 80:20:1:0.1. ......................................................................... 176

Figure 5-5: 1H NMR spectra of the polymerisation of HEA and CEMA with RAFT Agent 5 in DMSO-d6 at 25

ᵒC. [HEA]:[CEMA]:[RAFT]:[AIBN] = 90:10:1:0.1. .................................................................... 177

Figure 5-6: Conjugation of P(HEA32-co-CEMA8) to boc-protected two-arm cyclopeptide in DMF:TFE = 3:1.

.............................................................................................................................................. 178

Figure 5-7: Conjugation of P(HEA58-co-CEMA10) to boc-protected two-arm cyclopeptide in DMF:TFE = 3:1.

.............................................................................................................................................. 178

Figure 5-8: IR of cyclopeptide and cyclopeptide-polymer conjugates. The disappearance of the azide

peak (inset) after conjugation of the polymer, and the appearance of the polymer carbonyl

and increasing C-H stretches due to the increase in molecular weight, are evident. .......... 179

Figure 5-9: 31

P Spectra showing the attachment of RAPTA-C to CP-P(HEA58-co-IEMA10) (Conjugate A) via a

two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to conjugate in

solution, Top: Dimer added to solution. Desired products are circled. ................................ 181

Figure 5-10: 31

P Spectra showing the attachment of RAPTA-C to CP-P(HEA32-co-IEMA8) (Conjugate B) via

a two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to conjugate in

solution, Top: Dimer added to solution. Desired products are circled. ................................ 181

Figure 5-11: The p-cymene region of the 1H spectra showing the attachment of RAPTA-C to CP-P(HEA-co-

CEMA), in DMSO-d6 at 25 ᵒC. The broad polymer, residual unreacted RuCl2(p-cymene) dimer

and RAPTA-C p-cymene peaks are at 5.95 ppm, 5.86 ppm and 5.75 ppm, respectively.

Bottom: Conjugate A, Top: Conjugate B. .............................................................................. 182

Figure 5-12: DLS intensity average particle size distributions of Conjugate A and B in water at 25 °C. .. 183

Figure 5-13: DLS number average particle size distributions of Conjugate A at 25 °C. Bottom: in DMSO.

Middle: DMF (10 %) was added to the solution and the solution re-analysed after 3 days.

Top: in water after dialysis. .................................................................................................. 183

Figure 5-14: Conjugate A. (a) and (b) CP-P(HEA58-(RAPTA-C-EMA)10)2. (c) to (f) Crosslinked CP-P(HEA58-

(RAPTA-C-EMA)10)2. Samples (a) to (d) were drop-loaded onto grid, air-dried and stained


13

with Osmium Tetroxide. Samples (e) and (f) were drop-loaded onto grid and air-dried. Scale

bar : b, d & f = 200 nm, a & e = 100 nm, c = 50 nm. .............................................................. 184

Figure 5-15: Conjugate B. Crosslinked CP-P(HEA32-(RAPTA-C-EMA)8)2. Samples (a) and (b) were drop-

loaded onto grid, air-dried and stained with Osmium Tetroxide. Samples (c) and (d) were

drop-loaded onto grid and air-dried. Scale bar : a, b & e = 200 nm, d = 100 nm, c = 50 nm.185

Figure 5-16: Cytotoxicity profile of cyclopeptide-polymer conjugate without RAPTA-C, after 72 hours, n =

4. ........................................................................................................................................... 187

Figure 5-17: Cytotoxicity profile of RAPTA-C and Conjugates A and B, against ovarian carcinoma A2780

cells, after 72 hours, n = 4. .................................................................................................... 187

Figure 5-18: Cytotoxicity profile of RAPTA-C and Conjugates A and B, against cisplatin-resistant ovarian

carcinoma A2780cis cells, after 72 hours, n = 4. ................................................................... 188

Figure 6-1: Viability of cells exposed to NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C Micelles A and

B at [Ru] = 5 µM. ................................................................................................................... 194

Figure 6-2: Effect of NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C Micelles A and B on the

chemotactic migration of cells through polycarbonate filters. MDA-MB-231, MCF-7 and CHO

cell were treated for one hour with the drugs where [Ru] = 5 µM. The cells were then

removed from the flasks, collected, re-suspended and seeded on the inserts of Transwell cell

culture chambers. Data represent cells that after 24 hrs have migrated and are present on

the lower surface of the filter. Data are the percent of variation vs. controls calculated from

the mean ± SD of one experiment performed in triplicate. *, significant difference, p < 0.05.

.............................................................................................................................................. 196

Figure 6-3: Effect of NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C Micelles A and B on the

haptotactic migration of cells through polycarbonate filters. MDA-MB-231, MCF-7 and CHO

cell were treated for one hour with the drugs where [Ru] = 5 µM. The cells were then

removed from the flasks, collected, re-suspended and seeded on the inserts of Transwell cell

culture chambers. Data represent cells that after 24 hrs have migrated and are present on

the lower surface of the filter. Data are the percent of variation vs. controls calculated from

the mean ± SD of one experiment performed in triplicate. *, significant difference, p < 0.05.

.............................................................................................................................................. 197

Figure 6-4: Effect of NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C Micelles A and B on the invasion

of cells through Matrigel®. MDA-MB-231, MCF-7 and CHO cells were treated for one hour

with the drugs where [Ru] = 5 µM. The cells were then removed from the flasks, collected,

re-suspended and seeded on inserts. Data represent cells that after 96 h have invaded and

are present on the lower surface of the filter. Data are the percent of variation vs. controls

calculated from the mean ± SD of one experiment performed in triplicate. *, significant

difference, p < 0.05. .............................................................................................................. 198

Figure 8-1: Structure of RAFT Agents. ...................................................................................................... 208


14

Figure A-1: IBu-RAPTA-C synthesised via Route 2 at 25ᵒC in DMSO-d6 was subsequently heated to 60 ᵒC

and monitored over time. Multiple side-products were observed. ..................................... 235

Figure A-2: Overlaid [1H-

13C] HMBC & [

1H-

13C] HSQC NMR Spectrum of Butyl Iodide + PTA in DMSO-d6 at

25 °C. Both the 1H and

13C spectra are external projections. ............................................... 236


13C] HMBC & [

1H-

13C] HSQC NMR Spectrum of Butylated PTA + RuCl2(p-cymene)

Dimer in DMSO-d6 at 25 °C. Both the 1H and

13C spectra are external projections. ............. 236

Figure A-4: Initial RAPTA-C at 465 (top) shifted to Alkylated IBu-RAPTA-C at 497 (bottom). .................. 237

SCHEMES

Scheme 1-1: Initiation of the RAFT process. a) The initiator fragments into radicals. b) The initiator

radical reacts with a monomer unit to form a polymerising radical.177

..................................73

Scheme 1-2: Pre-Equilibrium of the RAFT polymerisation process, as outlined by CSIRO.177

....................74

Scheme 1-3: Core Equilibrium of the RAFT polymerisation process, as outlined by CSIRO.177

The

intermediate radical is stabilised by the Z-group on the RAFT agent. .....................................74

Scheme 1-4: Propagation. a) A polymeric radical (P•

n) reacts with a monomer unit to extend the polymer

chain. b) The R-group radical (R•) also reacts with a monomer unit to form a polymerising

radical. .....................................................................................................................................74

Scheme 1-5: Formation of block copolymers via chain extension.27

..........................................................76

Scheme 2-1: Synthesis of the amphiphilic block copolymer P(NAMI-A)-PPEGMEA using 2-

(((dodecylthio)carbonothioyl)thio)-2-methylpropanoic acid RAFT Agent, and micellisation in

water. ......................................................................................................................................83

Scheme 2-2: Synthesis of 4-vinyl imidazole by decarboxylation of urocanic acid. Polymerisation of 4-vinyl

imidazole in acetic acid at 70 °C. [VIm] = 0.8 M, [VIm]:[RAFT]:[ACPA] = 200:1:0.25. The

corresponding 1H NMR assignment is shown in Figure 2-1 and Figure 2-2. ............................88

Scheme 2-3: Synthesis of [DMSO2H][trans-RuIII

Cl4(DMSO)2]. .....................................................................90

Scheme 2-4: Synthesis of (ImH)[RuIII

Cl4(Im)(S-DMSO)] (NAMI-A) and M-NAMI-A in acetone at room

temperature. P(NAMI-A) was prepared in ethanol. ................................................................92

Scheme 3-1: Synthesis of macromolecular ruthenium complex Polymer-RAPTA-C in DMSO-d6 at 80 °C

(Route A) and at 25 °C (Route B). ......................................................................................... 107

Scheme 3-2: Route A: Synthesis of dichlororuthenium(II)(p-cymene)(1,3,5-triaza-7-phosphaadamantane)

(RAPTA-C) and the subsequent reaction with n-butyl iodide. .............................................. 115


15

Scheme 3-3: Route B Synthesis. Addition of n-butyl iodide to PTA and the subsequent complexation to

give butylated RAPTA-C. The corresponding 1H structural assignment is shown in Figure 3-7

and Figure 3-10 top axes. ...................................................................................................... 119

Scheme 3-4: Synthesis of Macromolecular Ruthenium Complex Copolymer-RAPTA-C in DMSO-d6 at 25

°C. .......................................................................................................................................... 129

Scheme 4-1: Synthesis of amphiphilic polymers, end-group modification of chloride to iodide (Finkelstein

reaction), two-step conjugation of RAPTA-C and subsequent micellisation. ....................... 137

Scheme 4-2: Synthesis of polylactide macroRAFT agent via ring-opening polymerisation, in bulk at

240 ᵒC. ................................................................................................................................... 144

Scheme 5-1: Synthesis of cyclopeptide-polymer conjugates and attachment of RAPTA-C. .................... 169

TABLES

Table 1-1: Common ligands for the synthesis of metal complexes. ........................................................... 35

Table 1-2: Effects of NAMI and NAMI-A on tumours. Adapted from Bergamo et al.62

.............................. 44

Table 2-1: X-ray crystallography results were compared with literature values to confirm the synthesis of

[DMSO2H][trans-RuIII

Cl4(DMSO)2]. .......................................................................................... 90

Table 2-2: Thermogravimetric analysis of NAMI-A analogues. Samples were analysed using two different

atmospheres (nitrogen or oxygen) to degrade the samples. .................................................. 93

Table 2-3: Calculation to determine the number of unreacted imidazole polymer units and the number

of NAMI-A units. The number of elements in each polymer component was used to calculate

the total molecular weight for each element and then as a percentage of the total. These

values were compared to the elemental analysis results, with particular emphasis on Cl and

N since both unit types contain N but only NAMI-A contains Cl. From this, it was found that

49 % of polymer units were NAMI-A units. ............................................................................. 94

Table 2-4: IC50 values with respect to the polymer concentration, against ovarian A2780 and OVCAR-3

and pancreatic AsPC-1 cancer cell lines. ............................................................................... 101

Table 2-5: IC50 values with respect to the ruthenium concentration, against ovarian A2780 and OVCAR-3

and pancreatic AsPC-1 cancer cell lines. ............................................................................... 103

Table 3-1: Synthesis of low molecular weight model compound Butylated RAPTA-C and macromolecular

ruthenium complex Polymer-RAPTA-C. ................................................................................ 111

Table 4-1: Polymerisation of HEA with PLA MacroRAFT Agent in N,N-dimethylacetamide at 60 ᵒC.

[HEA]:[MacroRAFT]:[AIBN] = 400:1:0.2 ................................................................................. 139


16

Table 4-2: Polymerisation of HEA and CEMA with PLA MacroRAFT Agent in N,N-dimethylacetamide at 60

ᵒC. [HEA]:[CEMA]:[F]:[MacroRAFT]:[AIBN] = 455:50:4:1:0.2. ............................................... 140

Table 4-3: Micelles A and B analysed by DLS in water at 25 ᵒC, with and without conjugated RAPTA-C. 156

Table 4-4: Final ruthenium content in polymeric micelles determined using ICPOES and TGA analyses.158

Table 4-5: IC50 (µM) values of RAPTA-C and micelles self-assembled from polymers A and B, against

ovarian A2780, cisplatin-resistant ovarian A2780cis and ovarian OVCAR-3 cancer cell lines.

.............................................................................................................................................. 159

Table 4-6: Fluorescence intensity of polymer and micelle samples at concentrations used for microscopy

imaging i.e. prior to cell uptake. ........................................................................................... 162

Table 4-7: Surviving fraction of cancer cells after four hour exposure to Micelles and RAPTA-C. .......... 164

Table 5-1: End-group modification and conjugation of RAPTA-C to cyclopeptide-polymer conjugates. 173

Table 5-2: Final ruthenium content in cyclopeptide-polymer conjugates was determined using ICPOES.

.............................................................................................................................................. 186

Table 5-3: IC50 (µM) values of RAPTA-C and cyclopeptide-polymer conjugates A and B, against ovarian

A2780 and cisplatin-resistant ovarian A2780cis cancer cell lines. ....................................... 186

Table 6-1: Structures of the chemotherapeutic agents NAMI-A and RAPTA-C, and the amphiphilic block

copolymers that self-assemble into micelles. ...................................................................... 193

Table 6-2: IC50 (µM) values of NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C Micelles A and B, against

ovarian A2780 and OVCAR-3 cancer cell lines. ..................................................................... 199

17

ABBREVIATIONS

ACPA 4,4′-Azobis(4-cyanopentanoic acid)

AIBN Azobisisobutyronitrile

AM Acrylamide

ATRP Atom Transfer free Reversible Polymerisation

Ben Benzene

Bip Biphenyl

CDB Cumyl Diothiobenzoate

CEMA Chloroethyl Methacrylate

cisplatin cis-dichlorodiammine platinum(II)

CMC Critical Micelle Concentration

CMT Critical Micelle Temperature

COSY Correlation Spectroscopy

CSIRO Commonwealth Science and Industrial Research Organisation

CTA Chain Transfer Agent

CuAAc Copper-mediated Azide-alkyne Cycloaddition

Cym p-cymene

D2O Deuterium Oxide

DCM Dichloromethane

DHA 9,10-dihydroanthracene

DIPEA di(isopropyl)ethylamine / Hünig's Base

DLS Dynamic Light Scattering

DMAc N,N-Dimethylacetamide

DMF Dimethylformamide

DMSO Dimethyl Sulfoxide

DMSO-d6 Deuterated DMSO

DNA Dihydroboxy Nucelic Acid

EDTA Ethylenediaminetetraacetic Acid

en Ethylenediamine

EPR Enhanced Permeation and Retention

ERK Extracellular-signal-regulated Kinases

EtOH Ethanol

F fluorescein O-methacrylate

Abbreviations & Symbols

18

FDA Federal Drug Administration

FRP Free Radical Polymerisation

FT-IR Fourier Transform - Infra-Red

G2/M Cell Cycle Checkpoint

GCSF Granulocyte Colony-stimulating Factor

GSH Glutathione

GSSG Glutathione Disulfide

HBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

HCl Hydrochloric Acid

HEA 2-Hydroxyethyl Acrylate

HMBC Heteronuclear Multiple-bond Correlation Spectroscopy

HOBt 1-hydroxy-benzotriazole

HPMA N-(2-hydroxypropyl)methacrylamide

HSA Human Serum Albumin

HSQC Heteronuclear Single Quantum Coherence

hTf / Tf Human Transferrin / Transferrin

IBu Iodobutane

IC50 Concentration that achieves 50 % cell death.

ICPMS Inductively Coupled Plasma Mass Spectroscopy

ICPOES Inductively Coupled Plasma Optical Emission Spectroscopy

IEMA Iodoethyl Methacrylate

Im Imidazole

Ind Indazole

kp Propagation Rate Constant

KP1019 (IndH+)[RuIICl4(Ind)2]

KP418 [trans-RuCl4(Im)2]ImH

LFRP Living Free Radical Polymerisation

MA Methyl Acrylate

MacroRAFT Macromolecular RAFT Agent

MADIX Macromolecular Design via Interchange of Xanthate

MDR Multi-drug Resistance

MeOD Deuterated Methanol

MeOH Methanol

MMA Methyl Methacrylate

M-NAMI-A Methylated NAMI-A


19

MS Mass Spectrometry

MWCO Molecular Weight Cut-off

NaCl Sodium Chloride

NAMI [trans-RuCl4(DMSO)Im]-Na+

NAMI-A (ImH)[RuIII

Cl4(Im)(S-DMSO)]

NEt3 Triethylamine

NMP Nitroxide Mediated Polymerisation

NMR Nuclear Magnetic Resonance Spectrometry

NT Nanotube

O-DMSO Oxygen-bound DMSO

PBS Phosphine Buffer Solution

PDI Polydispersity Index

PDTA 1,2-propylenediamminetetraacetate

PDTA 1,2-propylenediamminetetraacetate

PEG Poly(ethylene glycol)

PEGMEA Poly(ethylene glycol) Methyl Ether Acrylate

PEO Poly(ethylene oxide)

PLA Poly(lactic acid)

PTA 1,3,5-triazaphosphaadamantane

r Reactivity Ratio

RAFT Reversible Addition Fragmentation chain Transfer polymerisation

RAPTA Ruthenium-Arene-PTA

RAPTA-C [Ruthenium dichloride(p-cymene)(PTA)]

RAPTA-T [Ruthenium dichloride(toluene)(PTA)]

RE Reticuloendothelial

RES Reticuloendethelial System

RM175 [Ruthenium chloride(biphenyl)(en)]

RNA Ribonucleic Acid

RT Room Temperature

Ru Ruthenium

Ru Precursor (DMSO2H)[trans-RuIII

Cl4(S-DMSO)2]

RuBen [RuIICl2(benzene)(DMSO)]

RuCl3.H2O Ruthenium Trichloride Hydrate

RuII Ru2+, Ruthenium(II)

RuIIi Ru3+, Ruthenium(III)


20

RuSal Ruthenium Salicylaldoxime

S-DMSO Sulphur-bound DMSO

S-DMSO Sulfur-bound DMSO

SEC Size Exclusion Chromatography

SLS Static Light Scattering

SMANCS Styrene Maleic Anhydride-neocarzinostatin

SnOct2 Tin(II) 2-ethylhexanoate / Stannous Octoate

Sty Styrene

TEM Transmission Electron Microscopy

TFA Trifluoroacetic Acid

TGA Thermogravimetric Analysis

THA 5,8,9,10-tetrahydroanthracene

THF Tetrahydrofuran

TLC Thin Layer Chromatography

Topo II Topoisomerase Type II

UV-Vis Ultraviolet-Visible

VAc Vinyl Acetate

VIm 4-Vinyl Imidazole

VPy N-Vinyl Pyrrolidone

SYMBOLS

µ Micro

ᵒC Degree Celcius

λ Wavelength

Å Angstrom

A Absorbance

21

INTRODUCTION

Cancer will impact the lives of every person in Australia, either directly as they suffer from the

disease themselves, or indirectly when friends, family or colleagues battle the disease. The

Australian Institute of Health and Welfare asserts that:

“In 2012, the risk of being diagnosed with cancer before the age of 85 is:

1 in 2 for males and 1 in 3 for females.”1

Countless researchers in multiple fields past and present have worked in the hope of

discovering a ‘magic bullet’ – a universal cure that will save the lives of millions. However,

many experts maintain that each individual cancer is its own separate disease, which makes a

single cure unlikely.2 Our current level of knowledge of cancer has drastically increased the

survival rate, due to the advances in early detection and prognosis. Significant advances have

been made in some areas such as: leukemia, breast cancer, and colon cancer. The survival

rates for these are high, especially when the disease is identified in the early stages.2 Cancers

that have few symptoms, are difficult to detect and metastasise (form secondary malignant

growths and spread throughout the body) are the most devastating due to the uncertain and

most often fatal prognosis.

“Years of research have shown that cancers are very diverse and undergo changes and

adaptations with time and in response to treatment (resistance). At some point, if left

unchecked almost all metastatic cancers become untreatable.”2

With the advances in our understanding of cancer (and in key fields such as chemistry and

biology) the research approach has shifted to a more targeted approach that seeks to

eradicate individual cancer cells, is able to treat metastases and has no side-effects.

Metal-based drugs became a central research focus after the discovery of cisplatin.1-3 They are

potent and clinically active, however their use is limited by an extensive number of side-effects

including: nephrotoxicity, cumulative neurotoxicity, ototoxicity (loss of hearing), and extreme

emetogenic (vomiting) potential.9 Second-generation metal-based drugs that aim at limiting

these side-effects include therapeutics based on ruthenium. These drugs are relatively new,

having only been investigated for the past 25 years.

Introduction

22

“Ruthenium compounds are second generation (post-platinum) transition metal

chemotherapeutics that possess unique properties granting them, at least in pre-

clinical studies, more selective entry into tumour cells with fewer toxic effects to normal

cells.”3

Numerous properties which make ruthenium compounds highly prospective for medicinal

applications are discussed in Chapter 1. However, since they are also small molecules, they are

not without limitations. The biodistribution of low molecular weight drugs can be altered

through polymer conjugation, enabling tumour-specific targeting and a reduction in toxicity.4

Thus, polymer therapeutics are increasingly being synthesised with the aim of better capturing

the remedial benefits that could be elicited from these small molecule drugs. Due to the

Enhanced Permeability and Retention (EPR) effect,5–8 the systemic toxicity of a drug is reduced

due to the preferential accumulation of the polymeric drug in cancerous tissue. A number of

macromolecules have entered clinical evaluation, beginning with the water-soluble HPMA

copolymer-doxorubicin conjugate (1994).7,8 Conjugates that have progressed to clinical trials

have predominantly used pre-approved drugs.7 However, polymer-drug conjugates have a

distinct pharmacokinetic profile, often differing from the free drug, due to the alternate route

of cellular uptake.8 Thus, investigations into other potential conjugates are also worthwhile.

“Further development of…ruthenium agents may rely on novel approaches including

rational combination strategies as well as identification of potential pharmacodynamic

biomarkers of drug activity aiding early phase clinical studies.”3

Until now, there has been no exploration into macromolecules containing ruthenium. It has

been demonstrated with other drugs, such as platinum-centred9 or gold-centred,10 that

encapsulation in, or conjugation to, a polymer matrix is advantageous. The surrounding

polymer protects the drug from the body’s immune system, increases solubility and often

increases cell uptake efficiency due to the cell entry process being altered from a diffusion

mechanism to endocytosis.11

This thesis is an initial exploration into the conjugation of ruthenium drugs to polymeric

entities, and an initial biological evaluation of the benefits. There are two promising

ruthenium-based families: Ru(III)-indazole/imidazole and Ru(II)-arene that have shown activity

against tumours but display only low toxicity to cancer cells.12,13 For the current research, the

most prospective drug from each group was chosen, these being NAMI-A and RAPTA-C.

Introduction

23

NAMI-A, investigated in Chapter 2, is a coordination compound that specifically targets tumour

metastases,3,14,15 preventing both development and growth.3,16,17 Its effect is independent of

the type of primary tumour or the stage of growth of metastases.3 It displays both anti-

angiogenic and anti-invasive properties on tumour cells and blood vessels16 and modifies

important parameters of metastasis such as tumour invasion, matrix metalloproteinases

activity and cell cycle progression.18

RAPTA-C, investigated in Chapter 3, is a ruthenium metallodrug that is weakly cytotoxic in vitro

but very selective and efficient on metastases in vivo.13,15,19–21 It is inactive against primary

tumours,22 but effective at reducing metastases combined with excellent clearance rates from

vital organs.16,23 It significantly inhibits the progression of cancer in animal models by reducing

the number and weight of solid metastases, with low general toxicity.22

Covalent attachment of a therapeutic agent to a selected polymer is the first step in the

development of macromolecular therapeutics and is a useful avenue to delay drug release

until it reaches a target site.24 However, a linear polymer chain is often not readily taken up by

cancer cells, giving IC50 values that are less favourable than those of the free drug.25 Polymers

present unique opportunities to develop a variety of architectures giving rise to drug carriers.

“The design and clinical development of new constructs (biodegradable polymers,

novel therapeutics-siRNA, peptides, etc.) that in many cases are targeting a widening

range of diseases shows an exciting future for this group of nano-sized medicines.”26

The creation of a drug carrier in the nano-size range enables fast endocytosis, a unique size

that leads to increased circulation times27 and has the potential to exploit the EPR effect.28 All

of which are important properties for therapeutic moieties. Chapters 4 and 5 investigate two

such nano-sized carriers, polymeric micelles and peptide-polymer nanotubes respectively, that

aim to improve cellular uptake of the macromolecules containing ruthenium.

Lastly, Chapter 6 evaluates the potential for activity of these macromolecular drugs toward

metastatic cancer – the ultimate aim of chemotherapeutics.

24

25

1 LITERATURE REVIEW

Ruthenium is a rare noble metal, not found in biological systems, with the ability to form

complexes with numerous ligands.29 It is a unique metal in the platinum group of transition

metals as three of its oxidation states are accessible under physiological conditions.5 It has a

partially filled 4d sub-shell which allows it to form complexes that are useful for a wide variety

of applications including catalysis, electronics, photochemistry, biosensors and anticancer

drugs.3

Notably, ruthenium anticancer drugs have shown specific activity against solid-tumour

metastases,30 which are the leading cause of death for cancer patients. Metastases are

extremely difficult to treat as they exist in multiple locations in the body, have low accessibility

to surgery and/or radiotherapy, and generally exhibit poor responsiveness to chemotherapy.31

Polymers have been shown to enhance the benefits that can be derived from anticancer

agents. The attachment of ruthenium complexes to macromolecules may increase their

benefits by increasing the circulation time in the body, decreasing their susceptibility to

degradation, decreasing recognition by the body’s immune system and targeting specific sites

within the body. This increases the dose that accumulates at a particular area and decreases

the side-effects due to the impact of the accumulation of anticancer drugs in unwanted

regions.

By designing macromolecular chemotherapeutics that are active on metastases, it is

anticipated that the prognosis for metastatic cancers can be drastically improved.

Chapter 1: Literature Review

26

1.1. CANCER AND CHEMOTHERAPEUTICS

1.1.1. Defining ‘Cancer’

Cancer is a term that encompasses all diseases that involve an uncontrolled division of

abnormal cells leading to a malignant growth (tumour) which can spread to other parts of the

body through the lymph and blood systems. Figure 1-1 shows an illustrative comparison of

healthy and unhealthy cell growth. Normal cells divide to produce new cells which replace old

or damaged cells that undergo apoptosis. Sometimes the genetic information contained within

a cell is damaged generating mutations that affect the normal cell growth and division. Thus,

unwanted new cells grow uncontrollably and old cells do not die as they should. The increased

amount of cells may form a mass called a tumour.32

Figure 1-1: Loss of normal cell growth leading to the development of cancer. Adapted from

the National Cancer Institute website.32

There are more than 100 types of cancer. Figure 1-2 indicates the current five-year survival

rate for selected cancers in Australia. Due to the advances in early detection and cancer


27

treatments, the patient survival rate has increased over the past three decades (Figure 1-3).

However, many types of cancer remain undetectable and untreatable.

Testis

Thyroid

Lip

Prostate

Melanoma of the skin

Breast (females)

Hodgkin lymphoma

Kidney

Non-Hodgkin lymphoma

Bowel

All cancers combined

Bladder

Brain

Unknown primary site

Liver

Lung

Mesothelioma

Pancreas

0 20 40 60 80 100

98

96

93

92

91

89

87

72

71

66

66

58

22

16

16

Five-Year Relative Survival / %

5

6

14

Figure 1-2: Five-year relative survival rate for selected cancers in Australia 2006 -2010.

Adapted from the Australian Institute of Health report ‘Cancer in Australia’ 2012.1

Pancreas Lung Bowel Prostate Breast All cancers

0

20

40

60

80

Fiv

e-Y

ear

Re

lative

Su

rviv

al /

%

Type of Cancer

1982-1987

2006-2010

Figure 1-3: Five year relative survival rate for selected cancers in Australia 1982 -2010.

Adapted from the Australian Institute of Health report ‘Cancer in Australia’ 2012.1


28

1.1.1.1. Metastasis

Metastasis, which is the leading cause of a reduced quality of life20 and for cancer death,33 is

the process by which cells spread to alternative locations and form secondary tumours. It

occurs at the latter stages of disease development and involves the leakage of cells from the

primary tumour and their reestablishment at secondary locations. Not all cells in the primary

tumour possess this ability and, as such, non-metastatic cancer cells have different gene

expression profiles.33 Metastasis depends on the ability of cells to escape/detach from the

primary lesion, degrade the extracellular matrix, migrate, colonise/invade, adhere to a new

organ and proliferate in a novel microenvironment.20,31 Although numerous mechanisms have

been proposed to explain the phenomenon, no theory has comprehensively explained all

biological observations. As such, developing cures for metastatic cancers is a very complex

task.

Chemotherapy is used to treat metastases due to their multiple locations and low accessibility

to surgery and/or radiotherapy.33 However, metastases are not responsive to many

chemotherapeutics, possibly due to their different proliferation kinetics.34 Cancers are very

diverse. They also change and adapt with time and in response to treatment (i.e. developing

resistance). Unfortunately, if left unchecked, almost all metastatic cancers become untreatable

at some stage.2 Once this process has occurred, the five-year survival rate decreases by

approximately half, depending on the type of cancer15 and thus this form is responsible for the

larger proportion of cancer deaths.

1.1.1.2. Cancer Causes and Risk Factors

There are a vast number of reasons that have been shown to cause or increase the risk of a

person developing cancer. They include chemicals, foods, genetics, hormones, infectious

agents, radiation, sunlight, tobacco, weight and lack of physical activity.32

1.1.2. Cancer Treatment

One of the main remedies for cancer is the treatment with synthetic drugs, known as

chemotherapy. These drugs, i.e. chemotherapeutic agents, can destroy, slow the growth

and/or prevent the spread of cancer cells to other parts of the body. Although they are


29

relatively potent and clinically active, a number of side-effects can limit their utility, including:

nephrotoxicity, cumulative neurotoxicity, ototoxicity (loss of hearing), and extreme

emetogenic (vomiting) potential.3

First generation chemotherapeutics focused on DNA as the primary target, whereby the drugs

would intercalate with DNA leading to apoptosis. However, the lack of selectively due to DNA

being the primary target results in the afore-mentioned side-effects.31 Furthermore, the use of

a single agent often fails to achieve complete cancer remission due to the development of

drug resistance in patients.35

1.1.2.1. Drug Resistance

Drug resistance is the reduction in effectiveness of a drug. Three types of resistance have been

identified:36–38

a. intrinsic drug resistance: the drug has no effect at all;

b. acquired drug resistance: normal response is observed at the beginning of the therapy,

which then diminishes in effect and often disappears completely;

c. multi-drug resistance (MDR): resistance to the employed chemotherapeutic agent and

simultaneous cross-resistance to a number of functionally and structurally different

hydrophobic drugs, with varied mechanisms of action.

The cellular mechanisms leading to MDR are not completely understood,38 but the

development of MDR modulators has been the focus of much research. They inhibit

transporter-mediated efflux so that an anticancer drug administered at the same time can

cause tumour cell death. Verapamil is an MDR modulator that has reached clinical trials and

been co-administered with ruthenium compounds, resulting in a significant improvement of

their toxicity to cancer cells.38,39

Although MDR modulators are beyond the scope of this thesis, they are interesting to highlight

since polymeric therapeutics may provide many of the same benefits, in terms of a delay or

inhibition of resistance. The body is less likely to develop resistance, and hence multi-drug

resistance to polymeric agents due to a decrease in recognition by the RES (see 1.7).


30

1.1.2.2. Classical versus Targeted Chemotherapy

Classical chemotherapy refers to drugs that impede the replication and mitotic processes of

tumour cells40 – the primary target being DNA.33 (It originated in the 1950’s when

chemotherapy was in its infancy). The general strategy is based on the knowledge that cancer

cells replicate their DNA more rapidly than normal cells and are therefore more susceptible to

DNA damage. However, this strategy also inflicts damage on healthy cells - especially those

that divide frequently; for example, hair follicles, bone marrow and cells lining the

gastrointestinal tract causing, often severe, side-effects.40

In recent years, with the advances in molecular oncology, research has shifted the focus to

targeted therapies that centre on specific signalling pathways that cancer cells depend on for

growth, proliferation, metastasis formation and angiogenesis.33 This strategy is contingent on

discovering unique and special features of particular cancer cells.40 The ruthenium complexes

discussed herein reside in this category.

1.2. METAL-BASED ANTICANCER COMPOUNDS

1.2.1. The Benchmark: Platinum-Based Anticancer Drugs

Cis-dichlorodiammine platinum(II) (cisplatin) and its analogues; for example, carboplatin and

oxaliplatin, are the most widely used anticancer drugs.40 Cisplatin was discovered in 196541,42

and is used to treat approximately 50-70 % of all cancer patients,5,43 usually in combination

with other drugs.16 It has shown efficacy in the treatment of testicular, ovarian, oropharyngeal,

bronchogenic, cervical and bladder carcinomas, lymphoma, osteosarcoma, melanoma and

neuroblastoma.16,39,44 Pre-clinical studies and clinical investigations provide evidence that DNA

is the biologic target for cisplatin. It forms irreversible adducts via the process of ligand

exchange,39 by binding genomic DNA in the cell nucleus forming stable intrastrand crosslinks

which then block replication and/or transcription. 33 The proposed mechanism of action is the

ability of cisplatin to bend DNA by crosslinking adjacent guanines, which causes a class of DNA

binding proteins to adhere to the site.45

However, cisplatin induces normal tissue toxicity, particularly to the kidney,39 producing many

of the aforementioned side-effects (see 1.1.2). Additionally, the development of acquired drug

resistance can occur in initially responsive disease types or be present as intrinsic drug


31

resistance in less-responsive disease types.39,40 It is also inactive against metastatic (secondary)

cancers.16

Due to the many drawbacks to platinum compounds, there is still a need for the development

of other drugs, with the focus now on developing drugs that interact differently with DNA,

have lower systemic toxicity (and fewer side-effects), have a broader range of activity, with

particular emphasis on metastatic cancer, can overcome inherent or acquired Cisplatin

resistance and are active towards tumours which are non-responsive to current

therapies.5,20,33,44,46

1.2.2. Advantages of Metal-centred & Organometallic

Compounds

Metal complexes have a metal centre, which is bound to various ligands.47 Organometallic

compounds typically have metal-to-carbon bonds.48 The intrinsic properties of transition-metal

ions, other than platinum, present the following advantages for the development of

chemotherapeutic agents:45,49

a. additional coordination sites for the attachment of alternative ligands;

b. changes in oxidation state, which can allow the development of prodrugs that are

activated by a change in oxidation state within the body;

c. alterations in ligand affinity and substitution kinetics, affecting complex stability, drug

activation and DNA interactions;

d. variety of available interactions i.e. H-bonding, π-stacking, coordinative bonds, special

recognition;

e. rigidity around the metal and flexibility in the ligands; and

f. photodynamic approaches to therapy, which may be clinically useful.

Allardyce et al5 classified various organometallics in pharmaceutical applications under the

following categories:

a. the ligands are labile and are displaced prior to reaching the diseased cell;

b. attaching organometallic groups to coordination complexes that have proven

therapeutic activity;

c. attaching organometallic groups to compounds of known biological function;


32

d. harnessing functional organometallic ligands that have well-defined properties in

organometallic chemistry which could be exploited in biological systems; and

e. water soluble organometallics based on radioactive elements.

Despite the differences between coordination and organometallic compounds, similar

activities of certain compounds suggests that similar modes of action occur.40

1.2.3. Chemotherapeutic Metallopharmaceuticals

Numerous second-generation metal-based drugs that aim at addressing the side-effects of

first-generation drugs have been investigated. Metal drugs may not be directly active, but

Dyson et al16 suggested that they may interact with proteins that control apoptosis, which

increases the activity of organic compounds that induce apoptosis. Titanocene dichloride was

the first organometallic anticancer agent to reach phase II clinical trials.49 Since then,

numerous organometallics and metal complexes have been proposed. The review by Clarke

et al45 presents an in-depth discussion of metal drugs, while a short summary to highlight the

main points of interest in this review, for each metal, is presented here.

1.2.3.1. Gallium

Gallium has been suggested as a treatment for osteoporosis, bone cancers and autoimmune

disease. Gallium nitrate has exhibited clinical activity against lymphoma, bladder carcinomas

and has an effect on intracellular signalling pathways. Ga3+ is a similar size to Fe3+ and thus may

mimic iron(III) naturally occurring in the body by binding to transferrin which is internalized in

a cell through transferrin receptors on the cell exterior. Gallium can also enter cells through

routes that do not require transferrin. The binding of Gallium to transferrin hinders DNA

synthesis by blocking Fe3+ uptake, which is important for antitumour therapy. Gallium is most

widely used to combat hypocalcaemia, which is often due to bone cancer.45

1.2.3.2. Rhodium

Rhodium complexes have shown good antitumour activity but toxic side-effects have

prevented their use. They may show similar activity to that of cisplatin, by binding to adjacent


33

guanines on DNA. Complexes have displayed the ability to inhibit DNA replication, bind to a

variety of proteins, including serum albumin, and irreversibly inhibit proteins with cysteine in

the active site. Monomeric rhodium complexes are active against several tumour cell lines and

show antineoplastic activity but have generally lower activity, possibly due to their inability to

be activated by reduction. Some rhodium metallo-intercalators have shown such remarkably

specific DNA binding, that new types of DNA-targeting agents have been suggested.45

1.2.3.3. Titanocenes & Metallocenes

Budotitane was the first non-platinum anticancer drug to enter clinical trials, as a therapy for

ascites and colorectal tumours.45 Titanocene dichloride is active against a varied range of

human carcinoma, including gastrointestinal and breast.49 Activity against platinum resistant

cell lines is indicative of a different mechanism of action. TiIV binds strongly to transferrin and

may move into cells through transferrin receptors.49 Titanium complexes were shown to have

dose-limiting toxic side-effects and formulation problems due to instability halted drug

evaluation.

Metallocene dihalide anticancer activity is dependent on the metal ion where active

complexes fall within a window of size and substitution reactivity of the metal ion.

Metallocene dichlorides have been shown to inhibit protein kinase activity. The hydrolysis of

the metal may determine the antitumour activity of the complex, as those forming neutral

species under physiological conditions may enter cells more easily to exert a toxic effect.45

1.2.3.4. Vanadium

Vanadate and vanadyl ions mimic the effect of insulin. The vanadyl ion binds proteins and

other cellular components at both oxygen and nitrogen sites. Transferrin appears to be

involved in its transport and metabolism. The anticancer activity of vanadium appears to be

linked to the peroxidase activity, possibly through oxidative damage. However, it is potentially

active through inhibiting enzymes involved in DNA metabolism.45


34

1.2.3.5. Tin

Generally the toxicity of tin compounds is considerably higher than cisplatin, but their low

solubility in aqueous media presents a significant problem. A balance between solubility and

lypophilicity is needed to optimize their efficacy. Organotin compounds affinity for protein

thiols may determine their biological activity. Some tin compounds have shown a toxic effect

selective to the immune system and some have been associated with the activation of nuclear

endonucleases connected with apoptotic DNA cleavage.45

1.3. RUTHENIUM CHEMISTRY

Ruthenium is the least abundant transition metal after rhodium (10-3 to 10-4 ppm). Ru is six-

coordinate with octahedral geometry.33 It has the largest range of oxidation states, from -2 in

[Ru(CO)4]2- to +8 in RuO4. These are accessible chemically and electrochemically. Most

inorganic chemistry occurs in the +2 and +3 states.18,50 The oxidation states II, III, and IV are all

accessible under physiological conditions, which makes ruthenium unique amongst the

platinum group metals.51 RuIII is the predominant oxidation state, as it is reported to be the

most stable.52 RuII and RuIV are readily accessible in the presence of biological reductants

(ascorbate and glutathione) or oxidants (oxygen and hydrogen peroxide). Two main oxidation

states are accessible in physiological conditions: Ru2+ (d6, diamagnetic) and Ru3+ (d5,

paramagnetic).33

Ruthenium complexes are redox-active, kinetically stable in numerous oxidation states and

have redox couples that are often reversible and relatively easy to prepare. They also execute

a number of organic and inorganic transformations due to their synthetic versatility, high

catalytic performance and high selectively.18 Hence, they have attracted much research

attention as they are useful in many fields of chemistry including: catalysis, electronics,

photochemistry, biosensors and as anticancer drugs.

1.3.1. Ligands

The choice of ligand for the synthesis of a metal complex is important as correlations between

the complex properties and the nature of the bound ligands are evident.49 There are many


35

factors that influence the choice of complex ligands, depending on the required outcome.

Ligands may be chosen to stabilise the transition metal in a less favourable oxidation state or

change the properties of the associative complex; for example, the solubility or boiling point.

Very few metal drugs reach the biological target without modification due to interactions with

macromolecules, such as proteins.15 The inherent properties of the ligand itself may also be

advantageous; they may present DNA-binding capabilities and selective toxicity. Generally,

heavy metals are toxic by binding to sulphur and nitrogen sites on proteins. This may interfere

with a number of modes of metabolism.45

Table 1-1 presents some common ligands that are used for constructing metal complexes and

coordination compounds.

Table 1-1: Common ligands for the synthesis of metal complexes.

Anionic Ligands Names Neutral Ligands Names

Br- bromo NH3 ammine

F- fluoro H2O aqua

O2-

oxo NO nitrosyl

OH- hydroxo CO carbonyl

CN- cyano O2 dioxygen

C2O42-

oxalato N2 dinitrogen

CO32-

carbonato C5H5N pyridine

CH3COO- acetato H2NCH2CH2NH2 ethylenediamine

Many ligands, for example the phosphorus-donor 1,3,5-triazaphosphaadamantane and the

nitrogen-donor imidazole, are used to impart particular important properties on ruthenium

complexes for medicinal applications.12

1.3.1.1. Phosphorus-donor Ligands

The chemistry of RuII complexes incorporating P-donor ligands has given rise to a versatile

organometallic reactivity.51 Phosphines can be easily modified by changing the organic

substituent, allowing fine-tuning of the electronic and steric properties of metal complexes.

Solubility in water can be enhanced by modifying the phosphine structure by introducing polar


36

substituents.53 An example of such a ligand is 1,3,5-triazaphosphaadamantane (PTA) (found in

the ruthenium anticancer drug RAPTA-C: see 1.4.4.2.1). The protonation of the PTA ligand

influences solubility. The protonated species has a higher solubility in water while the

deprotonated species has a higher solubility in hydrophobic solvents.5

1.3.1.2. Sulphur- and Oxygen-Donor Ligands

Dimethyl sulfoxide (DMSO) can form S-DMSO and O-DMSO linkage isomers, due to the

contrasting properties of the S and O atoms and depending on the nature and characteristics

of the transition metal ions. Most RuII complexes exhibit a great affinity for sulphur ligands.

However, RuIII species favour the binding of the O-donor site.18 Sulphide, thiolate and thioether

ligands display flexible electronic properties, making them unique.51 Sulfoxide ligands form

complexes that possess properties useful for radiosensitisers,51 in catalysis and

chemotherapy.18,52

1.3.1.3. Nitrogen-donor Ligands

RuIII complexes that have shown anticancer activity have been synthesised with imidazole,

indazole and ethylenediamine. RuII complexes have been synthesized53–60 with derivatives of

pyridine, pyrazine, pyrimidine, 2,2'-bipyridine, 4,4'-bipyridine, 1,10-phenanthroline, and

N-substituted thiosemicarbazide: N-methyl-isatin-3-thiosemicarbazone, isatin-3-(4-Cl-

phenyl)thiosemi-carbazone) and acetazolamide. Polypyridyl ligands have been shown to have

interesting spectroscopic, photochemical and electrochemical properties, which are useful in

areas such as photosensitisers for photochemical conversion of solar energy, molecular

electronic devices and as photoactive DNA agents for therapeutic purposes.18,51

1.3.1.4. Arenes & Chelating Ligands

Arenes are used to provide stability to complexes in a less stable oxidation state. They also

enhance the toxicity of the complex,14 and the aquation rate is modulated by variations in the

steric and electronic effects of the arene ligands.57 Chelating ligands influence the rate and

extent of aquation, the pKa of the aqua adduct and the rate and selectivity of binding to

nucleobases.12,61


37

1.3.2. Ruthenium Compounds as Anti-Cancer Agents

Interest in ruthenium complexes began when they showed therapeutic activity on tumours

which had developed a resistance to cisplatin or in which cisplatin was inactive. Non-cross-

resistance with cisplatin is credited to the nature of the interactions with the nucleic acid, the

arrangements of the adducts and how these differ from those formed by cisplatin.44

Ruthenium compounds bind to DNA forming predominantly interstrand crosslinks as opposed

to the intrastrand crosslinks favoured by cisplatin, due to the differing ligand geometry

between platinum and ruthenium.39 Consequently, the cancer cell lines that have developed

resistance to cisplatin by accelerating the rate of repair of intrastrand crosslinks are often

susceptible to ruthenium anticancer drugs.5

Ruthenium compounds are highly prospective for medicinal application as they also present

the following properties:

a. multiple oxidation states (II, III and IV), are accessible in biological fluids;3,5,14,15,40,51,62

b. favourable ligand-exchange kinetics3,5,15 with low toxicity;23,39,40,63,64

c. selective antitumour,3,21,62,65 especially antimetastatic (secondary) tumours,3,20,66

including cytotoxic and cytostatic activity67 in both in vivo and in vitro studies21 and

intrinsic angiostatic activity;68

d. multiple cytotoxic routes69 involving the competing processes of extracellular protein

binding (active transport), due to the ability to mimic iron3,5,13–16,23,38,39,70 and cellular

uptake (passive diffusion), or an association of these two processes,15,23,46 resulting in a

reduced chance of cancer cells developing a resistance mechanism to these drugs46,67

and lower toxicity;23

e. different molecular pathways involving the concurrent intercalation and covalent

binding with DNA22,29,44,46,59 and binding to extracellular sites inducing conformational

modifications that can have an antineoplastic effect;69

f. the ability to exchange with O- and N- donor molecules similarly to platinum drugs;62

g. octahedral geometry provides numerous synthetic opportunities for tuning the

biological activities by organising a wide range of ligands in the three-dimensional

space,71 the possibility to bind to nucleic acids62 and shows a differing interaction to

cisplatin;35 and


38

h. opportunity for the development of prodrugs, consisting of the inactive RuIII centre,15

which reduces to RuII in situ, due to the reducing environment caused by the low

oxygen content in solid tumours.62

As with all metal drugs, the activity of ruthenium compounds depends on both the oxidation

state of the metal centre and the associated ligands surrounding it.15,23

1.4. RUTHENIUM COMPLEXES

Numerous ruthenium complexes have been investigated for medicinal applications, over the

past 25 years. Successes include KP1019 which was developed for solid tumours and NAMI-A

which was prepared as an antimetastatic drug. Several RuII complexes; for example, RAPTA-C

and Ru-arene-en, containing an arene ligand are also under preclinical evaluation.35

Interestingly, the respective mechanisms of action of RuII and RuIII drugs are highly correlated,

but substantially different to cytotoxic platinum compounds.72 The main pharmacological

characteristics of the most promising drug candidates is presented by Bergamo et al.62

Figure 1-4 summarises the cytotoxic and antimetastatic effects for the key candidates currently

being investigated for preclinical and clinical trials. KP1019, RM175, RAPTA-T and NAMI-A are

discussed in sections 1.4.2.4, 1.4.4.1.1, 1.4.4.2, 1.4.2.3 respectively. Following is a description

of the main research interests, classifying them based on the oxidation state of the ruthenium

ion in the complexes.


39

KP1019 RM175 RAPTA-T NAMI-A0

20

40

60

80

100

120NH

N HN

HN

RuClCl

S

Cl

H3CO

CH3

Cl

HNN

NH

HN

RuClCl

ClCl

NHN

NH

RuCl

HNPF6

N

N

NP

RuCl

Cl

Toxic 6/10

High

SelectivitySelectivityNon-selective

Cytotoxicity

Inh

ibitio

n / %

Primary Tumour Lung Metastases

Non-selective

Cytotoxicity

Figure 1-4: The most promising ruthenium-based therapeutics were tested against a

mammary carcinoma model and show differing degrees of selectivity and toxicity towards

metastases. Toxic 6/10: 6 out of 10 treated mice died due to drug toxicity. Non selective

cytotoxicity: primary tumour and metastasis growth are inhibited in a similar way.

Selectivity: metastasis is inhibited more than primary tumour growth. High selectivity:

metastasis is inhibited with no or marginal effects on primary tumour growth. Adapted

from Bergamo et al.62

1.4.1. Ruthenium(IV) Complexes

Oxoruthenium(IV) complexes can cleave nucleic acids18 but polyaminopolycarboxylate

complexes are the only RuIV complexes tested for anticancer properties.15 These complexes are

six-coordinate, octahedral and highly water soluble. RuIV reduces to RuIII and further to RuII so

that both RuIII and RuII are present in vivo. Hence, the activation mechanism may be the same

as that of RuIII complexes, discussed in 1.4.2. These complexes have chloride ligands, as in

cisplatin, which dissociate to produce a number of reactive RuIII species. However, the metal

ion maintains its oxidation state as well as the 1,2-propylenediamminetetraacetate (PDTA)


40

ligand. The complex rapidly binds to albumin, aptotransferrin, or differric transferrin to

produce relatively stable adducts in which (PDTA)RuIII is probably bound at the protein

surface.73 RuIV polyaminopolycarboxylates are generally anionic, which is a minor disadvantage

as it increases the energy needed for binding to DNA. RuII complexes of this form have also

been investigated. It was found that although they can be stabilized with a variety of pi-

acceptor ligands, pi-bonding adequate to stabilize against autoxidation would likely eliminate

the formation of the n2-pyrimidine bond.45

1.4.2. Ruthenium(III) Complexes

Paramagnetic ruthenium(III) complexes are promising anticancer agents.52 This broad class of

multichloro RuIII antitumour agents differ from cisplatin by favouring interstrand rather than

intrastrand crosslinks.45 They have been termed ‘prodrugs’15 as they transform into the active

drug species by hydrolysis and redox reactions, with biologically occurring nucleophiles, in the

body18 (see 1.4.3). Two RuIII compounds have entered Phase II clinical trials. KP1019 (see

1.4.2.4) has been shown to have anticancer activity while NAMI-A (see 1.4.2.3) is an

antimetastatic agent.

1.4.2.1. Amine and Imine Complexes

A direct correlation between cytotoxicity and DNA binding has been identified in some

ruthenium am(m)ine anticancer compounds. Preferential binding to particular DNA bases has

also been observed and modification of the ruthenium centre from the +3 to +2 oxidation

state has been shown to elicit binding at specific DNA sites. Ammine, amine, and heterocyclic

complexes of ruthenium demonstrate inhibition of DNA replication, mutagenic activity and

induction of the SOS repair mechanism, binding to nuclear DNA, and reduction of RNA

synthesis.45 Evidence suggests that monoacido complexes can be active, however multichloro

compounds exhibit the best activity against primary tumours.45 Solubility can be enhanced by

increasing the number of chlorides or using dialkyl-sulfoxide analogues. Interestingly, some of

these do not show significant activity against primary tumours but are effective against tumour

metastases.20,45


41

1.4.2.2. Dimethyl Sulfoxide Complexes

RuII and RuIII DMSO complexes exhibit activity and are relatively nontoxic.45 DMSO allows

increased inertness in an aqueous environment due to the reinforcement of the Ru-DMSO

bond. The coordinated sulfoxide also helps in the reduction of the RuIII to RuII species as it

stabilizes this lower, and less stable, oxidation state. Analogous to platinum compounds, cis

and trans isomers of DMSO compounds, present different therapeutic effects.65 These

compounds also contain chloride ligands available for hydrolysis, and have similar chloride

substitution rates to cisplatin. Similarly to cisplatin, maintenance of the neutral complex in

blood most likely facilitates its crossing lipid membranes to enter the cell and the lower level of

intracellular chloride favours chloride loss and DNA binding. Thus, an analogous mechanism

might be expected. However, their activity against cisplatin-resistant strains suggests a

different overall mechanism of action.45 RuIII and RuII DMSO complexes show similar activities

and have redox potentials that are biologically accessible. Thus, it is likely that both oxidation

states are present in vivo and coordinate to biopolymers such as nucleic acids and

transferrin.59,71,73 Both cis and trans isomers cause a disruption of the DNA structure due to

crosslinking, however the trans isomer showed a five-fold higher content of ruthenium in DNA.

Ruthenium DMSO complexes have shown antimetastatic activity and may be important in the

treatment of metastatic cancer, which is particularly difficult to treat. They may be used to

minimise the growth of undetected micrometastases after surgery or radiotherapy.45

1.4.2.3. NAMI-A and Related Compounds

In [trans-RuCl4(DMSO)Im]-Na+ (NAMI) (Figure 1-5 a), ruthenium is coordinated to one imidazole

(Im) ligand, one DMSO ligand and bound to four chloride atoms. The complex is stabilized with

a sodium counterion forming a coordination compound. NAMI has been shown to interact

with the metastatic tumour rather than the tumour cells of a primary lesion.17 It has good

water solubility and is active against a broad range of tumours.45 It is structurally similar to

[trans-RuCl4(Im)2]-ImH+ (KP418) (see 1.4.2.4), but NAMI has a significantly higher

ruthenium(III/II) reduction potential. This is due to the pi-acceptor effect of the S-bound

DMSO, which also exerts a kinetic trans effect. Comparatively high concentrations (>100

microM) are needed to elicit a cytotoxic effect, which is dependent on the lipophilicity of the

complex and the presence of serum and plasma proteins.45


42

Of particular note is that:

a. only a small fraction of the NAMI reaches the tumour target;

b. its activity seems to be independent of its concentration in tumour cells; and

c. its mechanism of action does not involve DNA binding.17

NAMI may increase resistance to the formation of metastases, but this is not through an

enhanced antigenicity or an immunological response. A pronounced increase of the

extracellular matrix components in the tumour parenchyma and around tumour blood vessels,

has been observed, which probably hinders metastasis formation and blood flow to the

tumour.45 NAMI is the most selective antimetastatic agent of the NAMI-type group of

complexes but all of these complexes selectively interact with solid tumour metastases.

Anionic derivatives are highly water soluble, whereas neutral complexes are less so and

strongly dependant on the nature of the nitrogen ligand.52

The nature of the cation in the coordination complex proved to be important as the sodium

salt of NAMI was too unstable to enter clinical trials.5 Further progress into the development of

this type of complexes produced In [trans-RuCl4(DMSO)Im]-ImH+ (NAMI-A), which is analogous

to NAMI except that the coordination compound is formed with an imidazole counterion. The

replacement of Na+ with ImH+ provides the molecule with better chemical stability. The

molecule is more stable to air oxidation and avoids co-precipitation of DMSO with Na+ during

chemical preparation. It more closely resembles a ‘true drug’ as it has improved

pharmacological properties over NAMI i.e. it is more stable and is a reproducible solid.17,45

a)

b)

Figure 1-5: a) [trans-RuCl4(DMSO)Im]-Na

+ (NAMI) and b) [trans-RuCl4(DMSO)Im]

-ImH

+

(NAMI-A), where Im = imidazole.


43

NAMI-A74 (Figure 1-5 b) produces all the same biological effects as NAMI but the effect of

NAMI-A on metastasis number is significantly greater than that of NAMI (Table 1-2).17 The

unknown mechanism of action is unrelated to direct tumour cell cytotoxicity and DNA is not

the primary target of this compound in vivo.33 NAMI-A shows a dose-dependent66

antimetastatic effect, enters tumour cells by both passive diffusion and active transportation,69

and is devoid of immune toxicity at concentrations active on tumour cells.75 It has low

cytotoxicity and is inactive against primary tumours30,66 and thus failed the usual screens of

putative anticancer agents16 against a panel of 60 cell lines.33 However, it has been shown to

specifically target tumour metastases,3,14,15,66 preventing both development and growth3,16,17

and has significantly greater activity than cisplatin on these cell types.30,62 A series of biological

activities that influence cell functions are associated with metastasis control; for example, re-

adhesion, motility, and invasion of tumour cells.76 The NAMI-A effect appears to be

independent of the type of primary tumour or the stage of growth of metastases.3 It displays

the following features that may be relevant for its in vivo metastases inhibition:33

a. inhibition of tumour cell invasion and of matrix metallo proteinases;

b. upregulation of adhesion and downregulation of angiogenic activity;

c. ERK1/2 inhibition and caspase activation;

d. strong interaction with proteins, including albumin, transferrin and integrins.

NAMI-A also possesses both anti-angiogenic77 and anti-invasive properties on tumour cells and

blood vessels.16 It modifies important parameters of the metastasis such as tumour invasion,31

matrix metallo-proteinases activity and cell cycle progression.78 The mechanism by which

compounds reduce metastasis formation is unrelated to a direct tumour cell cytotoxicity,18 and

thus little is known about the exact mechanism. Analogous to NAMI, NAMI-A increases the

thickness of the connective tissue of the tumour capsule and around tumour blood

vessels.17,45,77 It efficiently binds to collagen and thus has shown the highest activity in the

lungs where its half-life of elimination is approximately eight times longer than that in the

primary tumour mass.16 Remarkably, renal toxicity by NAMI-A has been shown to be fully

reversible in mice.79


44

Table 1-2: Effects of NAMI and NAMI-A on tumours. Adapted from Bergamo et al.62

Effects of NAMI-A on preclinical models of sold metastasizing tumours.

Tumour type Main results

Lewis lung carcinoma Metastasis reduction up to 100 %

TS/A mammary adeno carcinoma Metastasis reduction up to 100 %

MCa mammary carcinoma Metastasis reduction up to 100 %

B16 melanoma Metastasis reduction up to 100 %

H460M2 human NSCLC xenograft Metastasis reduction up to 90 %

Effects of NAMI on experimental tumours

Tumour type Main results

Lewis lung carcinoma Metastasis reduction up to 100 %

Increased survival time

MCa mammary carcinoma Metastasis reduction up to 100 %

B16 melanoma Metastasis reduction up to 100 %

Increased survival time

Colon 26 Increase of life-span 53 %

Colon 38 Tumour reduction to 51 %

M5076 reticulum cell sarcoma Increase of life-span 40 %

P388 leukaemia Increase of life-span 28-73 %

P388/DDP leukaemia Increase of life-span 35-62 %

The DNA binding mode of NAMI-A is different to cisplatin. It undergoes a series of hydrolytic

processes in aqueous solution; the nature and rates being strongly pH-dependant.33,80 DMSO

allows increased inertness in an aqueous environment due to the reinforcement of the Ru-

DMSO bond. The coordinated sulfoxide also helps in the reduction of the RuIII to RuII species as

it stabilizes this lower and less stable oxidation state,52 since when DMSO is bound through

sulphur it is a fairly good pi-acceptor.33 The loss of chloride is catalysed by the reduction to RuII,

which is anticipated to occur under physiological conditions and is enhanced in vitro by traces

of biological reductants; for example, ascorbic acid or cysteine.45 The complex is readily

hydrolysed, particularly at physiological pH, but neither DMSO nor the nitrogen ligand are

readily replaced in aqueous solution.52 The interactions of NAMI-A with biologically relevant

molecules might involve different species formed through different hydrolysis pathways; for

example, hydrolysis of Cl or DMSO.80 These aquation products may be responsible for the

selective antimetastatic activity in vivo,80,81 however it is most likely due to the presence of

DMSO in the coordination sphere of its active metabolite(s).33


45

NAMI-A is much less toxic to healthy tissue than cisplatin.30 It does not modify cell growth, and

causes a transient cell cycle arrest of tumour cells, whereas cisplatin causes a dose-dependent

disruption of cell cycle phases and reduces cell proliferation.45 Co-administration with cisplatin

presented superior activity as the two drugs maintain their individual binding sites on plasma

proteins without any interference from each other.16

Several NAMI-A type monomeric and dimeric complexes have been developed which have a

similar RuIII nucleus coordinative environment and antimetastatic activity comparable to that

of NAMI-A. They are very effective on in vitro metastases despite having no effects on primary

tumour growth nor in vitro cytotoxicity.18

1.4.2.4. KP1019 and Related Compounds

In [RuCl4(Ind)2]-IndH+ (KP1019) (Figure 1-6 a) is coordinated to two indazole (Ind) ligands and

four chloride moieties. It has entered Phase II clinical trials.82 Initial testing and Phase I clinical

trials were conducted on a similar molecule containing two imidazole ligands (KP418) (Figure

1-6 b).83 However, KP1019 was found to be far less toxic, exhibited slightly higher antitumour

activity and overcame resistance. KP1019 has shown activity in vitro on colorectal cancer cell

lines and in vivo on chemoresistant colon carcinoma.62,82 The activity of KP1019 is superior to

that of 5-fluorouracil in experimental therapy of autochthonous colorectal carcinoma and a

broader spectrum of tumour activity is suggested by experiments on explanted human tumour

samples.82

This activity was determined to be due to the interaction with DNA and inhibition of DNA

synthesis due to a different mode of action on the nucleic acid machinery.62 Differences in

intracellular distribution patterns suggest that the major target is cytosolic rather than

nuclear84 and adduct formation with high molecular weight components is crucial for the

therapeutic effect of KP1019.85 Tumour inhibiting activity is further increased by the addition

of an excess of indazole, and aquation of the complex leads to mono- and di-aqua complexes

and possibly polynuclear complexes. Hydrolysis of compounds is pH-dependant, with

hydrolysis proceeding faster at higher pH.81,83 KP1019 is more stable against aquation and

hydrolysis than NAMI-A.46 Unlike NAMI compounds, it does not modify the metastatic cell

behaviour but its effects are limited to a cytotoxic reduction of cell population; i.e. a direct

antitumour effect by promoting apoptosis.3,62 However, the pharmacokinetics in vivo is similar

to that of NAMI-A. It is more cytotoxic after reduction, binds to transferrin and is uptaken into


46

the cell via receptor-mediated endocytosis,73 causing apoptosis via the mitochondrial

pathway.81,82

a)

b)

Figure 1-6: a) [RuCl4(Ind)2]-IndH

+ (KP1019) and b) [RuCl4(Im)2]

-ImH

+ (KP418), where Ind =

indazole and Im = imidazole.

1.4.3. Activation of Ruthenium Complexes

RuIV and RuIII are the inactive species of ruthenium, while RuII is the active species. Thus,

complexes synthesised with a RuIII or RuIV centre are termed ‘prodrugs’ as they are activated in

the body by being reduced to RuII. This may occur in the bloodstream or within the cell.

Activation of organometallic complexes towards substitution reactions may be triggered inside

cells by internal sources; for example, oxidation of a less labile ligand or chelate ring-opening,

and by external sources; for example, light of a particular wavelength.49

1.4.3.1. Activation by Hydrolysis

Metal containing anticancer drugs display multiple levels of complex interactions with cellular

DNA and proteins, often starting with the initial aquation of a particular ligand. Ruthenium

arene complexes that hydrolyse exhibit anticancer activity, while those that do not undergo

aquation show little to no activity, supporting the theory that complexes of this type bind to

biomolecules coordinatively to exert their cytotoxic activity.49 Many of these compounds may


47

have preferential interactions with specific DNA sequences and proteins, leading to inhibition

of essential cellular pathways, and thus providing anticancer activity.18

Ruthenium complexes are activated by an associative pathway leading to the exchange of

chloride ligands with water.67 This hydrolysis is an important step in the mechanism of action

and may be the rate determining step in DNA binding.63 Covalent modification of biological

targets occurs through the hydrated site of the ruthenium complex.67 A high concentration

(150 mM) of chloride ions outside the cell reduces the exchange of chloride ligands with water.

Inside the cell, the lower concentration (4-20 mM) of chloride ions increases hydrolysis (Figure

1-7).5,46 At physiologically-relevant concentrations (0.5-5 µM) and temperature (310 K), the

complexes exist in blood plasma as >89 % chloro complex. In the cell nucleus significant

amounts (45-65 %) of the more reactive aqua adducts form, together with smaller amounts of

the hydroxo-aqua complexes (9-25 %, pH 7.4).57,83 The pKa of the coordinated water molecule,

which is dependent on the nature of the arene and chelating ligand, determines whether the

more labile Ru-OH2 bond or the less reactive deprotonated Ru-OH form prevails in solution at a

given pH.49,57 The subsequent interaction with DNA may be similar to the cisplatin mechanism.

The hydrolysed species reacts with DNA, forming coordinative bonds to nitrogen atoms of the

nucelobases, which leads to a distorted DNA structure18 causing apoptosis.

1.4.3.2. Activation by Reduction of RuIII to RuII

The ‘activation by reduction’ mechanism has been proposed as a special feature of some

ruthenium species that provides selective toxicity, and lower general toxicity.40 Aligning with

this mechanism RuII compounds, when compared to RuIII compounds, generally showed higher

reactivity towards a given nucleotide.81 Firstly, a substitution reaction occurs through

hydrolysis, activating the complex. Then, the second substitution replaces the bound aqua

ligand by the target biomolecule.49 RuIII complexes reduce to RuII in vivo, and the molecule is

‘activated’. As reduction fills the dπ orbitals, those ligands that π-donate are no longer able to

do so and bind less strongly.45 A more labile species is produced, which is able to interact with

biological targets after dissociation of some ligands.52 The RuII species can then coordinate to

nucleic acids (Figure 1-7).44,52 Furthermore, the RuIII complexes remain in their relatively

inactive and unreactive RuIII oxidation state until they reach the tumour site. Reduction from

RuIII ‘prodrugs’ to RuII compounds is particularly efficient in tumour hypoxic environments.81

Tumour cells have a lower oxygen content and pH than healthy tissue, which reduces the

ruthenium to the more active RuII state.18,45 Tumour cells have a higher metabolism and thus


48

oxygen is quickly utilized. In rapidly growing tumours, the growth of new blood vessels (termed

neovascularization or angiogenesis) often fails to keep pace so that the tissue becomes hypoxic

or even anoxic. The primary energy source then becomes glycolysis. The production of lactic

acid tends to lower tumour pH, which may facilitate the release of ruthenium on histidine or

tyrosinate sites on transferrin. It also favours the reduction of complexes with a pH-dependant

reduction potential.45 RuII can also be oxidised back to the less active RuIII by molecular oxygen,

cytochrome oxidase, and other oxidants.45

1.4.3.3. Transferrin-Mediated Tumour Targeting

Tumour cells have an increased requirement for nutrients and a higher membrane

permeability and angiogenesis. Combined with the increased blood flow, both specific and

nonspecific uptake of metallopharmaceuticals results.45 Some cationic complexes may cross

the cell membrane by endocytosis after binding to anionic sites on the cell surface. Neutral

moieties may simply diffuse through the cell membrane. However, an alternative mechanism

is through transferrin receptor uptake. It is likely that the nonspecific uptake of ruthenium ions

into tumours transpires within a few hours since small ions are expelled fairly readily by the

kidneys. Transferrin-mediated uptake might extend over a longer time scale.45

The transferrins are a family of single-chain glycoproteins, typified by serum transferrin: the

iron transport protein in blood.71Transferrin transport is a receptor-mediated metal uptake

system where Fe3+, not Fe2+, is specifically recognised, along with a requisite synergistic anion.

When transferrin is loaded with two Fe3+ ions, it binds strongly to its receptor. It is then

internalised by cells, the iron is released and the protein is recirculated. Control of the metal

uptake and release can be achieved both thermodynamically and kinetically.70

In human serum, only about 30 % of transferrin is saturated with iron. This provides the

potential for binding to other metal ions. About 30 metal ions have been reported to do so,

with carbonate, oxalate, or other carboxylates as synergistic anions. Many heterometal-

transferrin complexes are also recognised by the transferrin receptor and many factors affect

the strength of the metal-binding including the pH and salt concentration. However, Fe3+ has a

higher affinity than any other metal ion for which the binding constant has been determined.70

It has been reported that RuIII complexes can mimic FeIII by binding to proteins such as serum

transferrin (hTf) and albumin (HSA).5,14 Multiacido RuIII complexes, in particular di- through

tetrachloro complexes, are conveyed in the blood by transferrin and albumin, with the major


49

portion adhering to the latter. Albumin can bind up to five (hydrolysed) [Cl4L2RuIII] which

results in a loss of structure in its helical domains.45 Thus, the uptake of RuIII by cells is

mediated by proteins and toxicity may be decreased as ruthenium is prevented from binding

or uptake until it has been delivered to the cells.45 Tracer studies with RuCl3 have shown

substantial Tf binding. In addition, Ru-Tf complexes themselves have revealed anticancer

activity.18 Injections of RuIII-Tf resulted in elevated tumour uptake of the metal.45 Since

heterometal-transferrin complexes are still recognised by Tf, Ru-Tf complexes may provide a

new family of less toxic and more effective antitumour agents by allowing targeted delivery of

metallodrugs via Tf-mediated endocytosis.45,71

Figure 1-7: Multiple cytotoxic routes of ruthenium anticancer agents. Chloride ligands are

hydrolyses upon entering the cell, due to the lower chloride concentration. This allows

crosslinking of DNA leading to apoptosis of the cell. Alternatively, ruthenium can mimic

iron in the body by binding to transferrin. It is then uptaken into the cell by transferrin

receptors on the cell surface. Adapted from Levina et al.46

Tumour cells, in comparison to normal cells, require a higher amount of iron due to their

rapidly dividing cells and thus comprise a higher number of transferrin receptors on the cell

surface.40,71 The cell-surface transferrin receptors bind ruthenium-loaded transferrin and the

complexes are endocytosed and transferred to acidic non-lysosomal compartments where


50

ruthenium is released. This creates an indirect drug targeting mechanism as the ruthenium

complex is accumulated selectively in cancerous cells.18 This not only reduces the general

toxicity of ruthenium drugs, but cancer cells are less likely to become resistant to drugs

employing multiple cytotoxic routes (Figure 1-7).46

1.4.4. Ruthenium(II) Complexes

The reduced RuII form of this metal ion has shown enhanced binding to DNA15 and thus further

research investigated air-stable complexes containing ruthenium in this active state.46 Initial

studies showed high solubility in water with very high stability against aquation, and inhibition

of several types of enzymes believed to be involved in cancer progression, or in the

development of resistance to drugs.46 Mechanisms of action of anticancer drugs with the RuII

centre are likely to be different from RuIII centred drugs, due to the activation-reduction and

iron-mimicking mechanisms being less applicable. RuII organometallic complexes bind to DNA

coordinatively and also by H-bonding and hydrophobic interactions produced by the

introduction of extended arene rings.44

1.4.4.1. Ruthenium(II)-Arene Complexes

Allardyce et al5identified that one of the main issues associated with the progression of

ruthenium coordination complexes into clinical trials is their instability and complex ligand

exchange chemistry. The arene, in this series of complexes, stabilises ruthenium in the +2

oxidation state and increases the lipophilicity of the complex.14,46,86 This may enhance

biomolecular recognition processes and assist cellular uptake of the compounds.12,39 There is a

direct correlation between in vitro cytotoxic activity and lipophilicity87,88 where increased

hydrophilicity of the arene ligand correlates with lower uptake while a more lipophilic arene

increases uptake at the expense of selectivity.76 A high degree of selectivity may be achieved

via kinetic effects through the choice of arene due to the π-π arene-base stacking interactions

(intercalation).59

Ru-arene complexes with monodentate ligands show low or no activity, while complexes with

a chelated ligand possess high activity.49 Reactions of complexes containing arenes (for

example, 5,8,9,10-tetrahydroanthracene (THA), biphenyl (Bip) and 9,10-dihydroanthracene

(DHA)) which can participate in π-π stacking are up to an order of magnitude quicker than


51

those comprising arenes (for example, p-cymene (Cym) and benzene (Ben)) which cannot.59

Kinetic effects such as this could influence the biological activity of this arene class of

complexes.59 The chosen arene also influences the rate of hydrolysis of the Ru-Cl bond, the

aciditiy of the resultant coordinated aqua ligand12 and the level of repair synthesis of

ruthenium-damaged DNA.44 The underlying antitumour activity of these RuII arene complexes

is different from that of cisplatin, which includes binding by intercalation with the arene.62 RuII-

arene complexes show excellent antiproliferative properties in vivo and in vitro.16,76

1.4.4.1.1. RM175 and Related Compounds

Initial studies of arene stabilized ruthenium complexes by Sadler and coworkers12,44,59,61,63

incorporated the bidentate N-donor ligand ethylenediamine (en) and the arene ligands Bip,

THA, DHA, Cym and Ben. The en ligand reduces the lability of the leaving group Cl-.57 Strong

cytotoxicity for cancer cells in vitro was associated with a parallel DNA interaction.3,39

Increasing overall hydrophobicity of the arene ligand resulted in a large increase in the growth

inhibitory activity.39 Furthermore, ruthenium complexes of the type [(n6-arene)RuCl(X)(Y)]

(where X,Y are monodentate or chelating ligands) were shown to be cytotoxic to cancer cells,

including cisplatin-resistant cell lines and the cytotoxicity increased with an increase in the size

of the arene ring system (Figure 1-8).5,39,57,59This may be due to the ability of the larger arenes

to intercalate with DNA, causing a further distortion of the DNA structure.12 Bicycle arenes also

displayed increased DNA damage than those with single ring systems.59 The influence of

chelate ligands on substitution reactions is dependent on the position of the chelate ligand

relative to the leaving group and also the nature of the central metal.57

Ethylenediamine RuII-arene complexes exhibit anticancer activity in vitro14 and in vivo39 and are

active against cisplatin-resistant cancer cells. Furthermore, activity has been found for

ethylenediamine,57,59 phosphino19 and aminoacidato RuII arene complexes (Figure 1-9 b).14,39,61


52

Figure 1-8: Trend for an increase in cytotoxicity with an increase in the size of the arene

ring system, with X = Cl and PF6 as counterions.

Stable bi-dentate chelating ligands (en), a more hydrophobic arene ligand (THA) and a single

ligand exchange centre (chloride) were associated with increased activity.39 Lastly, increased

hydrophobicity of the organometallic complex is associated with a higher level of intrinsic drug

potency,5 but also a greater degree of cross-resistance.39 RM175 (also known as ONCO4417)

(Figure 1-9 a) is a compound of this type that has a Bip arene, a chloride and en ligands. It has

shown in vivo activity, DNA damage at levels similar to cisplatin, non-cross-resistance

mechanisms with cisplatin and metastasis reduction.3,62 Anticancer complexes of the type [(n6-

arene)RuCl(en)]+ are also highly selective in their recognition or binding sites on nucleotides

and nucleosides.59

a)

b)

Figure 1-9: a) [(Bip)RuCl(en)] (RM175) and b) [(p-cymene)RuCl(acac)], where Bip = biphenyl

and acac = acetylacetonate.


53

1.4.4.1.2. Oxidation, Hydrolysis and Stability

Ruthenium does not readily undergo oxidation from the active oxidation state +2 to the

inactive oxidation state +3 as the arene ligand in this series provides a lipophilic side to the

complex.14,15,89 The choice of ligand can have a marked effect on the hydrolysis behaviour and

reactivity towards nucleobases.63

The arene ligand also stabilises the complex in water by resisting hydrolysis; for example in (n6-

C6H6)RuII.14 However, a pH-dependant mechanism5 for the activity of RuII-arene complexes has

been documented and reactions of the chloro RuII arene complexes with nucleotides proceed

via hydrolysis followed by rapid binding to the phosphate monoester group.21,59,81The chloro

and hydroxo adducts are less reactive than the aqua adducts and variations in the steric and

electronic effects of arene ligands modulate the aquation rate.57,59 These complexes exist

largely as the less reactive chloro complexes in blood plasma, while the percentage of aqua

species significantly increases in the cell cytoplasm and nucleus, where the chloride

concentrations are lower.21,46,57 The first hydrolysis is slower than the second and under

physiological conditions the second hydrolysis is extremely slow or not observed.81 Thus, only

minor quantities of the less reactive hydroxo species would occur at biological pH.57

The water-soluble chelated organometallic RuII complexes synthesized by Morris et al14 were

shown to have a potency comparable to that of the anticancer drug carboplatin. The

complexes underwent aquation which could be suppressed by the addition of sodium chloride.

Extracellular chloride concentrations tend to be higher than intracellular chloride levels and

thus aquation activates chloro RuII arene complexes inside cells prior to reactions with DNA

(see 1.4.3.1).21 Hydrolysis occurs via an associative pathway57 and can be the rate-limiting step

for interaction of these complexes with DNA bases so it should be possible to control both the

rate and extent of hydrolysis of organometallic RuII complexes by varying the arene,

monodentate (halide and other) ligand, and the chelate ligand.57

1.4.4.2. RAPTA Compounds

Ruthenium-Arene-PTA (RAPTA) compounds, characterised by Dyson and coworkers,40,64,90,91

contain the RuII centre, two labile chlorido ligands available for aquation, a lipophilic arene

ligand and the hydrophilic phosphine ligand 1,3,5-phosphaadamantane (PTA). They are weakly

cytotoxic to tumour cells in vitro, interacting with both DNA and proteins,15,91,92 display

antimicrobial activity,5 and typically not toxic to healthy cells,21 even after prolonged


54

exposure.3,16,23,76 Similarly to NAMI-A, RAPTA compounds failed the National Cancer Institute

screening process but extremely low general toxicity and excellent clearance rates were

observed.16,21

An example of these contains the toluene ligand and is termed RAPTA-T (Figure 1-10 a). This

compound has shown antimetastatic effects in vitro and in vivo, thought to be mediated

through interactions with extracellular matrix components and the cell surface, rather than

with DNA in the cell nucleus.3,20,46 It is less affected by detoxification mechanisms that afford

cisplatin-resistant cell lines’ protection against platinum-based drugs.93 It has also exhibited

pH-dependant DNA binding properties in vitro, and selectively reduces the growth of lung

metastases.16,20

a)

b)

Figure 1-10: a) [RuCl2(toluene)(PTA)] (RAPTA-T), and b) [RuCl2(p-cymene)(PTA)]

(RAPTA-C),

where PTA = 1,3,5-phosphaadamantane.

Another RAPTA compound, containing the p-cymene ligand is termed RAPTA-C (Figure 1-10 b).

It has shown similar activity to NAMI-A; high selectivity and promising anti-metastatic activity

both in vitro and in vivo,86 possibly due to a common putative target in the cell.94 Casini et al95

used mass spectrometry to show that RAPTA-C is considerably more reactive and

discriminatory towards protein targets than cisplatin. Allardyce et al5,64 found that the growth

of healthy cells was unaffected, but that it was toxic to hypoxic cells (those, such as cancer

cells, with slightly lower pH than normal cells).21 Further research indicated that it is inactive

against primary tumours,22 but effective at reducing metastases combined with excellent

clearance rates from vital organs.16,23


55

A predisposition towards protein binding may be responsible for the selective antimetastatic

effect of RAPTA-C.13 It effectively inhibits cell growth by triggering G2/M phase arrest and

apoptosis in cancer cells, and also slowing cell division.22 The complex binds to

oligonucleotides with loss of chloride, and sometimes the arene also.23,96 Interestingly, the loss

of the arene is not correlated with the strength of the ruthenium-arene bond.23 RAPTA-C

significantly inhibits the progression of cancer in animal models, by reducing the number and

weight of solid metastases, with low general toxicity.22 PTA is of critical importance in the toxic

mechanism of RAPTA-C, and is responsible for the selective drug action of RAPTA compounds

towards metastases.16 The protonated form of PTA is considered to be the active agent.21 It

promotes water solubility and facilitates the ability of the compound to cross the cell

membrane and exhibit pH-dependant DNA-damage,20 with greater binding at lower pH.5

1.4.4.2.1. 1,3,5-Triaza-7-Phosphaadamantane (PTA)

Organophosphines are one of the most common ancillary ligands used in organometallic

chemistry, as they can stabilize low metal oxidation states and are able to influence both steric

and electronic properties of catalytic compounds.53 A key advantage of using phosphines as

ligands is that they can be readily modified by changing the organic substituents. This property

provides an avenue to fine tune the electronic and steric properties of metal complexes.53

PTA and related species are important hydrophilic co-ligands in active biological transition

metal compounds.53 A degree of water solubility dependent on the nature of the arene but

provided by the PTA ligand assists administration and transport in the body.76,86 PTA is a cage

adamantane-like phosphine; an inert atmosphere is not required during synthesis as it is

neither air nor moisture sensitive in solid form. It is also stable when exposed to oxygen and

thermally stable up to 260 °C.53 It has a higher resistance to oxidation than other types of

water-soluble phosphines, which is important for the synthesis of RAPTA compounds. It is

soluble in water, methanol, ethanol, DMSO, acetone, chloroform and dichloromethane, but

less soluble in heavier alcohols such as 2-propanol or n-butanol and tetrahydrofuran. It is not

soluble in hydrocarbons such as toluene, benzene or hexane.53

The 31P chemical shifts of PTA in various solvents range from -104.4 ppm in solid state

to -96.2 ppm in aqueous solution, which may reflect the strong solvent interaction at the

nitrogen atoms.97 A highly complex 1H NMR spectra with multiple spin systems results from the

N-protonation or N-alkylation of PTA due to the decrease in molecular symmetry.53


56

Most of the distinctive features of PTA arise from its ability to form hydrogen bonds via the

three N-donor atoms in the adamantane ring.98 PTA also possesses the unique attribute that it

can be regioselectively protonated at nitrogen rather than at the phosphorus centre.53,97 This

property is important for the attachment of this ligand to polymers (see Chapter 1) and also

introduces a positive charge able to enhance the solubility in water.99PTA can be N-alkylated

using methyl iodide, ethyl iodide, (chloromethyl)benzene or diiodobutane in acetone or

methanol under refluxing conditions.97,100,101 The resulting alkyl salts PTA(R) (where R = alkyl)

are air and water stable, however as expected due to their ionic character, they are less

soluble in organic solvents than PTA. However, solubility in water is still maintained, which is

an important feature in the development of an anticancer drug. PTA forms the ammonium-

phosphine [PTA(H)]+ in water solutions with a pH lower than 6.5. The N-protonated and

N-alkylated PTA salts have been shown to be poorer nucleophiles than the parent PTA.53 It has

been suggested that once the RAPTA complexes bind to DNA, the pKa of the PTA ligand

increases to a value compatible with its protonation in the hypoxic environment. This exerts a

secondary hydrogen bonding interaction and the two interactions combined may induce a

greater toxicity that coordination alone.21

1.4.5. Other Ruthenium Compounds for Cancer Therapy

Simple ruthenium complexes of ammine and heterocyclic nitrogen ligands possess remarkable

immunosuppressant activity, which may be mediated by biological reductants.45

Preparations of the mixed-valent complex ‘ruthenium red’ have been used as a cytological

stain for over a century. Hence, its biological properties are well documented. It has an

incredible immunosuppressant activity and concentrates in tumours inhibiting tumour growth.

It also blocks Ca2+ transport in a number of biological systems, which may also have an effect

on tumours. However, according to Clarke et al,45 it is the dimeric impurity in ruthenium red

formulations that is responsible for the majority of the inhibition of Ca2+ uptake in

mitochondria.

Dinuclear ruthenium complexes have been examined as it was proposed that there would be

increased interchain binding which is more inhibitory to cell repair systems.102 They have a

notable chemical stability, high antimetastatic activity and behaviour comparable to that of

NAMI-A.18


57

Mixed dinuclear complexes, comprising platinum and ruthenium, have also been investigated

as they may release monomeric units in vivo or alternatively provide a novel mechanism of

action.

Ruthenium(II)-letrozole complexes have been synthesised with the aim of creating

multitasking drugs that are able to promote cell death by simultaneously targeting selected

signal transduction molecules: the ruthenium is incorporated to kill cancer cells and the

letrozole to inhibit the growth of surviving ones.35 The “strategy of using kinetically inert metal

centres as rigid structural scaffolds for the design of enzyme inhibitors leads to new

opportunities for creating highly potent molecular probes and ultimately drugs.”103

1.5. DNA INTERACTIONS WITH RUTHENIUM

DNA replication is a fundamental process in the development of cancer. Hence, DNA is a key

biological target for metal-based anticancer drugs. Diseased cells, as opposed to healthy cells,

are more likely to die as a result of DNA damage due to the modified cell control system.5 The

initial investigations of ruthenium drugs were undertaken as it was believed that they would

interact differently than cisplatin with DNA due to their differing geometries.35

1.5.1. DNA Damage

Autoxidation in air, disproportionation and hydrolysis create a dynamic system where DNA is

slowly, catalytically damaged. There are a variety of ways by which ruthenium can generate

these strand breaks in DNA, eventuating in cell death.45 DNA can be damaged by oxidizing

agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet

(UV) light and x-rays. UV light predominantly damages DNA by creating thymine dimers; cross-

links between adjacent pyrimidine bases in a DNA strand.104 Oxidants such as free radicals or

hydrogen peroxide induce several forms of damage, including base modifications, particularly

of guanosine, and double-strand breaks.105 The latter, being the most dangerous as they are

difficult to repair, can produce: point mutations, insertions, deletions from the DNA sequence

and chromosomal translocations.106 It has been estimated that 500 bases per day suffer

oxidative damage.107,108


58

Intercalators, usually aromatic and planar molecules, such as: ethidium, daunomycin,

doxorubicin and thalidomide, impose damage by moving into the space between two adjacent

base pairs.109 In order for the molecule to fit into the space, the bases must separate,

distorting the DNA strand by unwinding the double helix. Transcription and DNA replication

are inhibited by these structural changes causing toxicity and mutations. Hence, molecules of

this type are often carcinogens. Well-known examples include benzopyrene diol epoxide,

acridines, aflatoxin and ethidium bromide.110,111 However, due to their properties of inhibiting

DNA transcription and replication, they are also used in chemotherapy to inhibit rapidly-

growing cancer cells.112

1.5.2. DNA Binding Modes

An effective approach to the development of novel chemotherapeutic anticancer agents is the

design of DNA-binding agents that recognise specific sequences or structures and can modify

specific DNA functions such as replication and transcription.61 Site- and base- selective

recognition of nucleic acids can be successfully enhanced by metal coordination, combined

with; for example, hydrogen bonding, hydrophobic and electrostatic interactions.59 DNA-

binding moieties can be categorised according to the type of association with DNA; for

example, coordinative binding agents, groove-binders, intercalators, and phosphodiester

backbone-binders. Non-coordinative interactions depend on electrostatic interactions,

molecular recognition based on shape and size and hydrogen bonding.44 For instance, cationic

metal complexes have an electrostatic attraction to polyanionic nucleic acids and the rate of

RNA binding is strongly ionic-strength dependent.45 Furthermore, DNA sequences can be

labelled with metal ions and then used to recognise and cleave precise sites through base-

pairing and photochemical or redox attack.45

Cancer cell lines that have developed resistance to cisplatin by accelerating the rate of repair

of intrastrand crosslinks are often vulnerable to ruthenium anticancer drugs, since they form

interstrand crosslinks.5 The reactivity of the various binding sites of nucleobases toward

ruthenium at neutral pH decreased in the order G(N7)>I(N7)>I(N1), T(N3)>C(N3)>A(N7),A(N1).

Furthermore, pseudo-octahedral diamine RuII arene complexes are substantially more

discriminatory between G and A bases than their square-planar PtII counterparts.59 RuII and OsII

complexes form coordination bonds, in addition to H-bonds and intercalation between DNA

base pairs.44 They diffuse unchanged into the cell nucleus and undergo aquation, hydrolysis


59

and DNA binding. This results in cell death via apoptosis.46 RuII and RuIII complexes bind

strongly and selectively to guanine bases, forming monofunctional adducts on DNA oligomers,

with a strong preference for N7.5,12,14,61 In vivo and in vitro studies show that N7 binding is

stimulated by favourable arene-purine hydrophobic interactions.5,59 Hence, these complexes

may cause cytotoxicity by inhibiting cellular DNA synthesis,29 and the damage caused is more

difficult for enzymes to repair than DNA treated with cisplatin.12

1.5.2.1. Binding of Complexes with an Arene Ligand

For complexes containing an arene ligand, DNA binding is facilitated by the hydrophobic

interactions between the arene and DNA. Chen et al59 showed that ruthenium

ethylenediamine organometallic complexes are highly selective in their recognition of nucleic

acid bases and that the kind of coordinated arene has a dramatic effect on the binding kinetics.

Complexes have been synthesised with hydrogen-bond donor and acceptor chelating ligands.

DNA binding of the neutral donor N,N-chelating ligand (ethylenediamine) was compared with

the acceptor O,O-chelating ligand (acetylacetonate). For the complexes containing the donor

ligand there was exclusive binding to N7 of guanine in competitive nucleobase reactions and in

the absence of guanine, binding to cytosine or thymine but not adenine. In contrast, for the

acceptor ligand, the overall affinity for adenosine (N7 and N1 binding) was similar to that for

guanosine, however there was little binding to cytidine or thymidine.18,61

A direct correlation between cytotoxicity and the size of the arene was observed by

Allardyce et al5 (Figure 1-8). Both in vitro and in vivo data suggest that the most active

complexes contain the most hydrophobic arene ligands.5 RuII arene complexes of the type

[(n6-arene)Ru(en)Cl]+ bind to DNA oligonucleotides establishing monofunctional adducts.14,39,61

The rates of reaction in kinetic studies of cGMp with [(n6-arene)RuII(en)X)n+] (X = Cl- or H2O)

decreased in the order shown in Figure 1-8, suggesting that N7-binding is promoted by

favourable arene-purine hydrophobic interactions in the associative transition state.18,59 These

findings have shown that the diamine NH2 groups, the hydrophobic arene, and the chloride

leaving group all have important roles in the mechanism of detection of nucleic acids by

ruthenium arene complexes.59


60

1.5.2.2. Topoisomerases Type II

Genome integrity is maintained by a class of enzymes called topoisomerases, as they resolve

DNA intertwining. Type II enzymes are the most important for cell cycle progression and

survival of dividing cells as they play an important role in the replication, transcription,

recombination and segregation of chromosome pairs during cell division. Topoisomerase

(Topo) II is present as a major enzyme in the nuclei of rapidly dividing cells, especially in

neoplastic conditions.29 Research suggests that the antitumour activity of DNA-binding drugs

depends on their ability to interfere with the catalytic action of Topo II.29 Selectively poisoning

the Topo II enzyme could inhibit neoplastic cell proliferation and possibly induce apoptosis by

fragmenting DNA.29 RuIII complexes have the ability to form a ‘cleavage complex’ similar to that

of other Topo II poisons. Hence, in the presence of these drugs, the enzyme induces an

accumulation of permanent double strand breaks in DNA, which ultimately force the affected

cells to undergo apoptosis or necrosis.45

[RuIICl2(benzene)(dmso)] (RuBen) was shown to completely inhibit the DNA relaxation activity

of Topo II through the formation of a drug-induced cleavage complex, whereas a second

complex, ruthenium salicylaldoxime (RuSal) could not, suggesting that the difference in ligands

and their positioning around a metal atom may be the cause of Topo II poisoning.29 The

ruthenium atom could bind outside the DNA helix through ionic interactions or by covalent

bonding with the nucleotide bases.

RuBen could be categorized as a Topo II poison, which is a cleavable complex-forming, DNA-

binding but non-intercalating agent.29 Most poisons have large planar aromatic domains for

DNA binding and substituents for enzyme interaction. An intercalative mode of DNA binding is

not possible for RuBen because the benzene ring forms an organometallic bond with the

ruthenium atom. This inhibits pi-orbital stacking interactions of the aromatic ring with DNA

bases. The metal atom interacts covalently or noncovalently with DNA nucleotides and the

ligands form cross-links with the enzyme and prevent the DNA relegation step. This leads to

the formation of a stable drug-induced cleavage complex, which is a characteristic property of

most Topo II poisons.

Clarke et al45 pointed out that the hydrolysis of RuBen was not considered in this study and

that RuSal was thought to be a square planar complex and thus the species introduced into

solution may have been different to what was reported. However, regardless of the species in

solution, both complexes bound to DNA and similarly inhibited DNA replication and cell

proliferation. RuBen interfered with the DNA-stimulated ATPase activity of Topo II by


61

permitting DNA cleavage but preventing re-ligation, the formation of an enzyme-Ru-DNA

cleavage complex is suspected.45

1.5.2.3. Modulation of DNA Binding by Glutathione

Glutathione is an antioxidant and the most common cellular non-protein thiol. In cells, it

primarily exists in the reduced form (GSH) but is readily oxidized to the disulfide form (GSSG).

Glutathiones assist in protecting cells from reactive oxygen intermediates, UV radiation, and

heavy metal toxicity. GSH inhibits heavy metal binding to proteins and nucleic acids by

scavenging and sequestering the ions via coordination through its sulfhydryl. In selected cases,

GSH reduces metal ions, such as CrO42- and PtIV, to species that coordinate or else react with

DNA. Furthermore, GSH binding to PtII appears to contribute to cisplatin resistance in tumour

cells.45 The coordination of RuIII to DNA is facilitated by the reduction to the more substitution-

labile RuII. However, GSH coordinates to RuII, facilitating oxidation back to RuIII and inhibiting

DNA binding. But, adenine provides strong pi-binding sites for both RuII and RuIII, which may

account for ruthenium binding even at high concentrations of GSH.45

1.5.2.4. Inhibitors of Protein Kinases

Protein kinases are a huge superfamily of homologous proteins with 518 members in the

human genome.113 They regulate most aspects of cellular life and thus are one of the main

targets for anticancer drugs. Several ruthenium complexes have been developed that are

potent and selective inhibitors of protein kinases and thus shown that it is possible to

synthesise compounds possessing stabilities comparable to purely organic molecules.18

1.6. MACROMOLECULAR METAL COMPLEXES

Transition metals in macromolecules play an essential role in living systems, for example;

hemes as oxygen carriers, chlorophylls as energy conversion systems in photosynthesis, and

metalloenzymes for various catalytic reactions.114,115 Notably, vitamin B12 contains cobalt

bound directly to a carbon atom.12 Macromolecular metal complexes have been developed as

catalysts, membranes for water purification and photoresponsive moieties.


62

Wohrle114 defined four types of macromolecular metal complexes:

Type I. Covalent, coordination, or ionic binding of metal ions or complexes to linear or

cross-linked macromolecules.

Type II: A ligand is integrated into a linear or cross-linked macromolecule forming the

backbone.

Type III: The metal is integrated into the linear or cross-linked macromolecule forming

the backbone.

Type IV: The metal complex or metal nanoparticle is physically incorporated into the

macromolecule.

Incorporating metal complexes into macromolecules for therapeutic purposes opens a range

of possibilities to be explored.

1.7. POLYMERS AND ANTI-CANCER AGENTS

1.7.1. Motivation

The body is a superb machine as it quickly recognises and eliminates foreign entities. Thus,

free drugs that are administered are rapidly registered and removed from the body. However,

the glomerular filtration in the kidney has a molecular weight cut-off of approximately

50 000 g.mol-1 and thus polymer particles above this size have a higher circulation time.116

Polymers can also be self-assembled into drug carriers (see 1.9) and alterations can be made to

the surface of these particles to delay or inhibit recognition by the reticuloendethelial system

(RES), which can detect polymer particles and eliminate them from blood circulation.27

Poly(ethylene glycol) (PEG) is a biocompatible hydrophilic polymer that is known to

substantially enhance circulation time117 and has been attributed with ‘stealth

properties’.27,118,119 However, a major disadvantage of PEG is its nonbiodegradability,7 which

has led to the development of biodegradable polymers; for example polylactide.120 Other

polymers; for example, poly-N-vinylpyrrolidone and polyvinyl alcohol and also peptides and

polymer-lipid conjugates, decrease the overall immune response and inhibit RES uptake.121

40-60 % of drugs in development display inadequate bioavailability due to low water solubility.

Poor solubility generally results in a higher dosage to achieve the desired therapeutic outcome,

which can lead to toxicity problems.122 The aqueous solubility of hydrophobic drugs can be


63

radically enhanced by conjugation to,7,9 or encapsulation in,123,124 a hydrophilic polymer. The

drug dose administered to patients is often limited by the toxicity of the drug to other vital

organs.7 Polymers can be engineered to deliver a drug in a controlled manner at a target site,

such that the rate and duration of delivery can be therapeutically effective.7 This results in the

ability to administer higher drug concentrations to patients, while maintaining manageable

side-effects, or mitigating them altogether. The coupling of small-molecular weight drugs to

polymers also alters the biodistribution of the drugs following administration, favouring

accumulation in tumours.8

The delivery of a therapeutic to a solid tumour is regulated by physiological factors; for

example, tumour blood vasculature, lymphatic drainage and tumour interstitial fluid pressure,

and the physicochemical properties of the molecule; for example, surface characteristics

(charge and hydrophilicity) and particle size.121 Targeting tumour tissue relies on the ability to

effectively distinguish between healthy and cancerous vasculature. There are a number of

characteristics associated with tumour tissue, when compared to healthy tissue:121

a. distribution, density, length and diameter of tumour blood vessels are commonly more

heterogeneous, larger in size and more permeable;

b. lower average blood flow;

c. tumour vessels are leakier with pore sizes exceeding 100 nm; and

d. lack of a functional lymphatic drainage system, which hinders convection-mediated

transvascular and interstitial transport.

These properties allow for passive tumour targeting, which is shown by the EPR effect,

depicted in Figure 1-11. It displays the benefits of polymers with regards to the treatment of

solid tumours. The endothelial layer of blood vessels in diseased tissues is more porous to

large molecules. Contrasting with healthy tissue, cancerous tissue also does not have a

lymphatic drainage system. Thus, macromolecules are more likely to enter diseased tissue and

subsequently be retained.5,28


64

Figure 1-11: Passive targeting. A comparison of normal tissue vs. tumour tissue.

Macromolecules are more likely to accumulate and subsequently be retained in tumour

tissue, due to increased permeability and lack of a lymphatic drainage system.

The EPR effect has been found for compounds with a molecular weight greater than 40 KDa

but is negligible for smaller molecules, which readily redeploy to blood circulation via diffusion

and/or convection.121 EPR is also affected by tumour size, as it is superior in smaller

tumours.125,126 This may be due to the greater vessel density, which allows for more

extravasation, compared to larger tumours that contain larger avascular regions.121 “Limiting

the size of nanoparticles to less than 200 nm can promote extravasation as well as interstitial

transport.”121 The flexibility to tailor polymers with specific attributes; for example, molecular

weight, number and types of drug attached, targeting moieties and even bioresponsive

components8 makes them valuable drug delivery systems. In summary, the rationale for the

design of polymer-drug conjugates is as follows:4

Limit cellular uptake to the endocytic route.

Produce long-circulating conjugates, thereby aiding passive targeting through EPR, or

active targeting through the conjugation of targeting ligands (antibodies, peptides,

sugars).


65

1.7.2. History of Polymeric Therapeutics

Polymer therapeutics4,127 are nano-sized (5-100 nm) polymer-based pharmaceuticals that

include biologically active polymeric drugs,128 polymer-drug conjugates,129–131 polymer-protein

conjugates,132,133 polymeric micelles to which a drug is covalently bound134 and multi-

component polyplexes (containing covalent linkers) for gene and protein delivery.135 Figure

1-12 highlights the milestones in the development of polymeric therapeutics from 1950-2000.

Figure 1-12: Timeline of milestones in the emergence of anticancer polymer therapeutics.

First drugs to market are highlighted in yellow. GCSF: granulocyte colony -stimulating

factor; PEG: poly(ethylene glycol); SMANCS: styrene maleic anhydride -neocarzinostatin.

Adapted from Duncan.4


66

1.7.3. Design of Polymeric Agents

A major issue with polymer therapeutics is the variance in molecular weight, drug-loading and

the resulting conformation, in contrast to traditional small molecular weight therapeutics and

proteins that have well-defined chemical configurations. Thus, polymer-drug conjugates

should offer significant advantages in terms of efficacy and safety while also be simple and

cost-effective to synthesise.7

A “rational model for pharmacologically active polymers” was first proposed in 1975 by

Helmut Ringsdorf.136 This model (Figure 1-13) comprises a biocompatible polymer backbone

connected to three components:

a. a solubiliser, which is hydrophilic and ensures water solubility of the conjugate;

b. a drug, often bound to the polymer backbone via a linker; and

c. a targeting moiety, which transports the conjugate to a desirable location within the

body, and/or binds to a specific biological target.

Figure 1-13: The Ringsdorf136

Model: A rationale for the delivery of therapeutic drugs using

polymer-drug conjugates. Adapted from Larson.7

Polymers can be tailored with other specific properties including biocompatibility and

degradation under particular conditions. Biodegradable conjugates are advantageous in terms

of efficacy and safety, since the increased molecular weight can optimise the


67

pharmacokinetics, but biodegradation ensures elimination from the body post-treatment.7 The

primary objective of all therapeutic delivery systems is “the specific delivery of therapeutic

agents to their intended site of action in an attempt to improve efficacy and reduce toxicity.”7

Duncan4 highlighted the features that are needed in the design of a polymer-drug conjugate in

order to achieve this goal:

a. the polymer must be non-immunogenic, non-toxic and suitable for industrial-scale

manufacture;

b. the optimum molecular weight range for the polymer is 30 000 – 100 000 g.mol-1. It

should be high enough to ensure long circulation. However, for non-biodegradable

polymers it must be less than 40 000 gmol-1 to ensure renal elimination of the polymer

carrier after delivery of the drug. The higher polymer molecular weight limit ensures

that the conjugate will be small enough to extravasate into the tumour and enable

endocytic internalisation by all types of tumour cells;

c. an adequate drug payload in relation to its potency must be carried by the polymer.

d. the polymer-drug linker must be stable in transit to the tumour, but have an in-built

release mechanism of the drug at a desired rate once in the tumour cells;

e. the polymer must be able to access the correct intracellular compartment if the drug

exerts its effect through an intracellular pharmacological receptor; and

f. intracellular transfer and delivery of a drug out of the endosomal or lysosomal

compartment is essential for therapeutic activity and also may bypass mechanisms of

drug resistance that rely on membrane efflux pumps.

An ideal delivery system enables the conjugation of a targeting moiety and an active entity in a

simple chemical platform.8 Duncan26 subsequently described polymer conjugates with a three-

part design:

a. the polymer;

b. linker; and

c. payload, which may be drug or protein.

Targeting moieties and/or imaging agents may also be included. The chemistries associated

with all parts, as well as the molecular weight, polydispersity and architecture of the polymer

itself all have a significant impact on the safety and efficacy of the final therapeutic agent.26

The molecular weight and polymer structure, in particular, significantly impact the

pharmacokinetic profile and biological activity seen in patients, as shown by the comparison of

PEGASYS® to PEGIntron® in the treatment of Hepatitis C.137


68

1.7.4. Macromolecules in Clinical Evaluation

A number of macromolecules have entered clinical evaluation. A dextran-doxorubicin

conjugate was the first polymer-drug conjugate to be tested clinically,125 while the water-

soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate of covalently bound

doxorubicin was the first synthetic polymer-based anticancer conjugate to enter clinical trials

in 1994.7,8,125 Encouraging results of conjugates following these lead examples have

materialised from ongoing Phase I-III studies,26 including the HPMA copolymer platinate

Prolindac® with paclitaxel for ovarian cancer,138 OPAXIOTM with radiotherapy for oesophageal

cancer,139 poly(1-hydroxymethylethylene hydroxymethylformal) (Fleximer®)-camptothecin

conjugate XMT-1001 for advanced solid tumours,140 and PEG-irinotecan conjugate NKTR-102 in

breast141 and ovarian142 cancers. Polymer-conjugates are essentially macromolecular prodrugs,

typically comprising of three constituents:125

a. a natural, synthetic or pseudosynthetic water-soluble polymeric carrier generally of

molecular weight 10 000 - 100 000 gmol-1;

b. a biodegradable polymer-drug linkage (preferably one that is stable in the

bloodstream to ensure very low levels of free drug in plasma, thus reducing the

chance of bioactive drugs accumulating in healthy tissue); and

c. a bioactive antitumour compound.

Conjugates that have progressed to clinical trials have predominantly used pre-approved

drugs.7 However, polymer-drug conjugates have a distinct pharmacokinetic profile, often

differing from the lone drug, due to the alternate route of cellular uptake.8 Thus, investigations

into other potential conjugates are also of interest.

1.7.4.1. Polymeric Carriers with Covalently Bound Drugs

A variety of polymer-drug conjugates have been synthesised using water-soluble linear

polymers, including poly(N-vinyl-2-pyrrolidone),143,144 poly(vinyl alcohol),145 polyglutamic

acid146 and poly(malic acid).147 However, the two most widely explored chemistries are those

utilising poly(ethylene glycol) (PEG)148 and N-(2-hydroxypropyl)methacrylamide (HPMA).149 The

first clinically investigated water-soluble polymer-drug conjugate for cancer therapy was PK1,

which contains a HPMA copolymer backbone with the anticancer anthracycline antibiotic

doxorubicin attached.150 Numerous HPMA conjugates have since begun clinical


69

evaluation.125,149,151 Using hydrophilic polymers as the carrier can avoid rapid RE uptake (liver

and spleen) that is ordinarily seen for traditional nanoparticles and liposomes.125

Polymeric micelles, wherein the drug is covalently bound to hydrophobic units in the micelle

core, have been investigated. For example, docetaxel has been chemically conjugated to PEG-

b-poly(Ɛ-caprolactone) and then micellised.

1.7.4.2. Drugs Encapsulated in Polymeric Micelles

The potential of block copolymer micelles (see 1.9.1) was first recognised and investigated by

Kataoka152 and Kabanov153 and co-workers. The majority of these systems actually encapsulate

the drug, as opposed to covalently binding it.7 The perfect polymeric micelle should display

high drug loading ability, precise drug release, and appropriate biological compatibility and

stability.7 The characteristics and lengths of the hydrophilic and hydrophobic blocks are the

primary determinant of the physiochemical properties of the micelles.

The most commonly used hydrophilic polymer is PEG due to its highly hydrated nature,

widespread acceptance and availability, and ability to resist uptake by the RES (see 1.7.1). A

variety of hydrophobic blocks have been investigated. Micelle cores form spontaneously from

more hydrophobic unimers, while other less hydrophobic ones interact via electrostatic

interactions with hydrophobic drugs first, and then form micelles.154

Numerous hydrophobic drugs have been encapsulated in polymeric micelles, but the majority

of data on the biological activity of these systems has been attained from in vitro studies.155–157

To determine their potential as drug carriers, further in vivo data is necessary.7

1.8. POLYMERISATION

A polymer is a macromolecule containing repetitive units that are linked together via covalent

bonds. Polymerisation is the synthesis process performed to elucidate such a moiety. The

repeating units, termed monomers, may be identical giving a homopolymer, or different

leading to a copolymer.


70

1.8.1. Free Radical Polymerisation

Free Radical Polymerisation (FRP) is a method by which polymers can be synthesised. It

involves three main steps: initiation, propagation and termination.

Initiation is the creation of free radicals necessary to start the polymerisation process.

Initiators can be produced by thermal decomposition, photolysis, redox reactions, ionising

radiation, electrochemical processes, plasma, sonication, and the use of persulphates or

ternary initiators.

Propagation is the rapid reaction of a radical with a monomer unit and the subsequent

addition of further monomer units to generate a chain that ‘grows’ with the addition of more

monomer units.

Termination occurs when a radical reacts in a manner that prevents further propagation. The

most common method of termination is by coupling, where two radical species react with each

other forming a single molecule. Another method of termination is disproportionation. This

occurs when two radicals meet and exchange a proton, which gives two terminated chains,

one saturated and the other with a terminal double bond.158 Termination of the growth of one

polymer chain can occur when the polymer undergoes chain transfer to solvent, monomer,

polymers or added chain transfer agent. However, this process does not terminate the kinetic

chain length.

1.8.2. Reversible-Deactivation Radical Polymerisation

Reversible-Deactivation Radical Polymerisation,159 more commonly known (and referred to

here) as, Living Free Radical Polymerisation (LFRP) is a method of polymerisation that is

characterised by:160

a. a linear evolution of molecular weight with monomer to polymer conversion, and

time;

b. generated polymer chains that are long-lived and can be reactivated from a dormant

state; and

c. generated polymers possessing a narrow molecular weight distribution, typically

indicated by a PDI < 1.2.


71

First-order kinetic behaviour is also a characteristic of LFRP but does not deem a system to be

‘living’. It confirms the constant amount of radicals in the polymerisation system. From this

method, a variety of polymers with different structural characteristics can be synthesised such

as block, star, cyclic, and gradient polymers. It also provides a route to functional groups,

grafted polymers and modified surfaces. LFRP techniques include Nitroxide Mediated

Polymerisation (NMP),161 Atom Transfer free Radical Polymerisation (ATRP)162 and Reversible

Addition Fragmentation chain Transfer Polymerisation (RAFT).163

1.8.3. Copolymerisation

A copolymer, or heteropolymer, is a polymer derived from two, or more, monomers.164

Copolymerisation is the polymerisation of these monomers simultaneously, or sequentially, to

form a polymer that contains both starting species. The way two monomers interact to form a

copolymer system largely depends on the reactivity ratios (r) of the initial monomers. This

interaction produces three types of copolymerisation behaviour, defined by Equation 1-1.

Equation 1-1:

where kii are the rate coefficients.

Ideal copolymerisation occurs when r1r2 = 1. In this case, both monomers have the same

preference for adding to themselves as they do for adding to each other. Thus, the copolymer

composition is the same as the monomer feed ratio and there is random placement of

monomers along the polymer chain.

Alternating copolymerisation occurs when r1r2 = 0. In this case, both monomers have a

preference for adding to the other monomer, as opposed to adding to itself, and a series of

alternating monomers along the polymer chain is formed. Most copolymerisation systems lie

somewhere between ideal and alternating systems.

Block copolymerisation occurs when r1 > 1 and r2 > 1 and thus one monomer unit is consumed

completely before the second is incorporated into the polymer chain. This is rarely

encountered in a ‘normal’ free radical system. Hence, significant research has gone towards

developing a process whereby block copolymers can efficiently be made.165 RAFT has proven to

be an important synthesis technique in this regard.


72

1.8.4. Reversible Addition Fragmentation Chain Transfer

Polymerisation (RAFT)

Reversible Addition Fragmentation chain Transfer (RAFT) is a facile technique that can be used

in the synthesis of polymers with a range of architectures, including block copolymers.166 It was

discovered at CSIRO in 1998.167 Using this method to control polymerisations allows for the

synthesis of well-defined polymers displaying a narrow polydispersity with controlled chain

lengths and architectures.168 RAFT polymerisation can be used in solution, emulsion169,170 and

suspension124,171 polymerisations.

A successful RAFT polymerisation is a conventional free radical polymerisation of a monomer

that employs a Chain Transfer Agent (CTA), also known as a RAFT agent, which mediates the

polymerisation via a reversible chain-transfer process. The CTA contains a thiocarbonylthio

group, a Z-group for stability and an R-group which is the ‘leaving group’ during RAFT

polymerisation (Figure 1-14).

Figure 1-14: General structure of a Chain Transfer Agent (RAFT Agent).

The R and Z substituents influence the polymerisation reaction kinetics and the amount of

structural control.172 The S-R bond should be weak enough for fragmentation to occur while

the R-group radical that is formed must be stable and able to reinitiate polymerisation. The

thiocarbonylthio group ‘caps’ the polymerising chain and the Z-group determines how long this

radical will remain in a dormant state. Commonly used RAFT agents include dithioesters,167

dithiocarbamates,173 trithiocarbonates174,175 and xanthates.176 The CSIRO guidelines for the

selection of R and Z groups are illustrated in Figure 1-15.


73

Figure 1-15: Guidelines for the selection of R and Z groups for the polymerisation of the

most common families of monomers. R groups: fragmentation rates decrease from left to

right. Z groups: fragmentation rates increase and addition rates decrease from left to right.

Dashed lines indicate partial control over the polymerisation (i.e. control over the

molecular weight evolution but poor control over the polydispersity index.) MMA = methyl

methacrylate, Sty = styrene, MA = methyl acrylate, AM = acrylamide, VAc = vinyl acetate.

Adapted from Moad et al.168,177,178

The aim of RAFT is to engage the polymerising radical in an alternative process, in preference

to termination. A polymerisation is started through the introduction of a conventional free

radical initiator, which fragments in to radicals. The polymerising radical is formed in the

initiation process (Scheme 1-1).

Scheme 1-1: Initiation of the RAFT process. a) The initiator fragments into radicals. b) The

initiator radical reacts with a monomer unit to form a polymerising radical.177

There are two equilibriums in the RAFT process. During the pre-equilibrium (Scheme 1-2), the

RAFT agent is transformed into a polymeric RAFT agent as the polymeric radical joins the RAFT

agent and the R-group is detached as a radical.


74

Scheme 1-2: Pre-Equilibrium of the RAFT polymerisation process, as outlined by CSIRO.177

The core equilibrium (Scheme 1-3) is the process by which polymeric chains are activated and

deactivated. A polymeric radical attaches to the RAFT agent. The Z-group controls the

reactivity of the C=S bond and thus influences the rate of radical addition and fragmentation.

Scheme 1-3: Core Equilibrium of the RAFT polymerisation process, as outlined by CSIRO.177

The intermediate radical is stabilised by the Z-group on the RAFT agent.

This species (1 in Scheme 1-3) then lays dormant until another polymeric radical attaches to

the RAFT agent, forming the intermediate radical, which should be present at high

concentrations due to its slow fragmentation. The polymeric radical is then detached and again

propagates (Scheme 1-4). The monomer concentration determines the rate of polymerisation

(kp).

Scheme 1-4: Propagation. a) A polymeric radical (P•

n) reacts with a monomer unit to extend

the polymer chain. b) The R-group radical (R•) also reacts with a monomer unit to form a

polymerising radical.


75

1.8.4.1. Statistical Copolymers via RAFT

If two monomers are placed in a system with a RAFT agent and the reactivity ratios r1r2 ≠ 1 or

0, a statistical copolymer will be formed and the exact structure of the copolymer cannot be

regulated. Conventional free radical copolymerisation studies have been directed towards

copolymer compositions.179 Two models have been developed to describe copolymer

composition. The ‘terminal model’ assumes that the propagation reactivity is determined by

only the terminal unit.180 The ‘penultimate model’ assumes that propagation reactivity is

determined by both the terminal and penultimate units.181 For three copolymer systems, it

was shown that the RAFT process altered the copolymer composition compared to

conventional free radical copolymerisations by increasing the preference for the monomer

with the larger reactivity ratio.182

1.8.4.2. Block Copolymers via RAFT

Block copolymers are easily synthesised via the RAFT process by first synthesising a

macromolecular RAFT (macroRAFT) agent and then chain extending this moiety with a second

monomer (Scheme 1-5) i.e. initiating the polymerisation of a monomer in the presence of a

macroRAFT agent.

MacroRAFT agents are polymer chains whose end functionality is a thiocarbonylthio group as a

result of the RAFT process. Polymer blocks can only be generated from monomers with similar

reactivity’s. The result is the formation of A-B diblock copolymers.183 As with conventional

RAFT, an initiator splits into two radicals and begins the polymerisation of the second

monomer (B). When B attaches to the RAFT agent, polymer A leaves and propagates with B

monomers, hence forming a copolymer chain with two different blocks. The process is

repeated, extending both blocks, until termination occurs.


76

Scheme 1-5: Formation of block copolymers via chain extension.27

1.9. SELF-ASSEMBLED POLYMER NANOPARTICLES

A variety of different polymer architectures, including block, graft, star and statistical

copolymers, can be synthesised via the RAFT process.183 These polymers can then be self-

assembled into a variety of structures, including vesicles, micelles and rods, depending on the

morphology (for example, the lengths of the substituent polymers and chemical groups) of the

individual polymer. These structures can then be used to encapsulate and transport

therapeutic moeities into the body. Alternatively, drugs can be attached to the polymers,

creating macromolecular drugs of different architectures. The aim is to create a therapeutic

drug carrier that imparts both distribution and temporal control; i.e. the drug is carried to the

target site and slowly released.27

1.9.1. Polymeric Micelles

Larson et al7 defined micelles as “colloidal particles with a size of about 5-150 nm that consist

of self-assembled aggregates of amphiphilic molecules or surfactants.” Amphiphiles exist as

unimers in solution at low concentrations in aqueous media. The critical micelle concentration


77

(CMC) and temperature (CMT) determine the conditions at which micelles will form. As the

concentration is increased, the formation of aggregates is driven by a dynamic process which

sequesters hydrophobic regions. Thus, structures displaying a core surrounded by a hydrophilic

corona are formed. Lower CMC’s are associated with increased hydrophobicity and

hydrophobic block length.184,185

The dynamics of micellisation is an important consideration when evaluating the stability of a

drug delivery system, as the stability of the micelle is compromised upon injection of the

pharmaceutical formulation, due to the rapid decrease in concentration. The shape and size of

a micelle influences the biodistribution and drug loading capacity.27 “Micelles are small enough

(< 200 nm) to bypass filtration by interendothelial cell slits in the spleen, yet sufficiently large

(> 50 kDa) to avoid renal excretion.”9 Polymeric micelles are superior to low molecular weight

surfactant derived systems due to their increased stability,7 and other properties bestowed

due to their polymeric constituents (see 1.7.1). They present distinctive opportunities as drug

delivery vehicles as both the core and shell-forming blocks are tuneable; for example, stimuli

responsive linkages can be added, the core can be excavated after assembly and the size and

shape of the micelle can be engineered.186

Amphiphilic block copolymers can be easily synthesised via RAFT polymerisation. These diblock

copolymers, having both a hydrophilic and hydrophobic block, mainly form nanospheres,

nanocapsules, vesicles and micelles (Figure 1-16).187 A variety of alternative micellar

morphologies can be discovered by allowing amphiphilic block copolymers to spontaneously

assemble in aqueous media; for example, worm-like micelles which may present superior

benefits over traditional spherical micelles by increasing circulation time in the body and

decreasing drug accumulation in healthy tissue.186 The concentration and temperature at

which these polymers will form micelles or disintegrate in to unimers is unique to each block

copolymer.

The hydrophobic core is capable of physically encapsulating hydrophobic drugs or providing a

site for chemical attachment, while the hydrophilic shell provides good solubility and limits

detection by the RES (Figure 1-16). Micelles also present a unique size that leads to increased

circulation times of these carriers, and the synthesis by controlled radical methods offers the

potential for high surface functionality, which can be introduced at the end of the hydrophilic

block.27


78

Figure 1-16: Self-assembly of amphiphilic block copolymers into micelles .

Polymeric micelles have a remarkably low CMC (10-6 - 10-7 M) and their dissociation is

kinetically slow.188,189 However, they become unstable at high temperatures, at low

concentrations and under certain changes in solvent conditions,190 such as elevated

temperature, altered pH values or increased ionic strength.188 In order to prevent the

spontaneous dissociation and disintegration of the aggregate, physically and chemically

stabilised micelles have been developed.191 Micelle stability can be preserved by crosslinking of

the core,191–194 shell,195–197 core-shell interface or by polyelectrolyte complex formation.27 The

strengths and limitations of each method of crosslinking are highlighted by Stenzel.27

Micelle cross-linking strategies188 include radical chemistry,198–200 carbodiimide coupling,201,202

Michael addition, quarternisation, esterification, click chemistry,203 disulfide/thiol chemistry,204

polyelectrolyte complexation188 and photocrosslinking using short wavelength UV radiation.205

The crosslinking location and extent of crosslinking effects the physical and chemical

properties, stability, structure and applications of the resulting moiety.190

1.9.2. Peptides

Peptides are short chains of amino acids. They have been used as degradable linkers between

polymers and drugs125 such that drugs can be released at a target site. They have also been

shown to enhance the cell uptake of polymer-drug conjugates. For example, the cRGD peptide


79

targets αvβ3 integrins that are overexpressed on tumour vasculature and some tumour cells.

Toti et al206 showed that the cellular uptake of nanoparticles could be increased by 2-3-fold by

functionalizing nanoparticles with cRGD peptide.

Cyclopeptides are a distinctive type of peptide that can be self-assembled into hollow

nanotubular structures (nanotubes, NT’s), which are present in many natural and artificial

systems.207 Cyclopeptides containing 6-12 amino acids of alternating D and L chirality have

been shown to assemble into tubes through anti-parallel hydrogen bonding.207 Polymeric

chains can be grafted to these peptide NT’s preventing aggregation and improving

solubility.207,208

1.10. CONCLUSIONS

The benefit of incorporating therapeutic agents into macromolecules has been extensively

discussed previously.7,26,127,209,210 A number of metal-based drugs, including platinum9,211,212 and

gold,213 and their attachment to, or incorporation in, macromolecular entities has been

investigated. However, the benefit of integrating ruthenium based drugs3,46,62 into such

moieties has yet to be discovered.

Two ruthenium drugs, RAPTA-C (RuII) and NAMI-A (RuIII) were chosen due to their promising

anticancer activity and most notably their anti-metastatic activity. Although they are based on

ruthenium, from a structural and chemical viewpoint the compounds are very different. Their

oxidation states and ligand sets differ. RAPTA-C is an organometallic complex, whereas NAMI-A

is a traditional coordination compound. However, in vitro cell studies show distinct similarities.

Neither compound is active against primary tumours, but both reduce the number and weight

of metastatic cells. NAMI-A is slightly more effective, but the clearance of RAPTA-C is slightly

superior.21 This thesis is an initial exploration into the incorporation of these therapeutics into

polymers, in order to create macromolecular chemotherapeutics. It further explores the self-

assembly of these macromolecules into polymer nanoparticles and evaluates their anticancer

and anti-metastatic potential through initial biological experiments.

80

81

2 RUTHENIUM(III)

Over the past 25 years, numerous ruthenium complexes have been investigated for medicinal

applications. Two RuIII compounds have entered Phase II clinical trials: KP1019 has been shown

to have anticancer activity while NAMI-A is an antimetastatic agent. Antimetastatic agents are

extremely important in the treatment of cancer, since the majority of cancer patients die from

these secondary cancers.

NAMI-A has low cytotoxicity and is inactive against primary tumours. Accordingly, it failed the

usual screens of putative anticancer agents.16 However, it has been shown to specifically target

tumour metastases,3,14,15 preventing both development and growth,3,16,17 and has significantly

greater activity on these cell types, than benchmark drug cisplatin.62 The NAMI-A effect

appears to be independent of the type of primary tumour or the stage of growth of

metastases.3 It displays both anti-angiogenic, anti-invasive properties on tumour cells and

blood vessels,16 and modifies important parameters of the metastasis such as tumour invasion,

matrix metallo-proteinases activity and cell cycle progression.18

However, the hydrolytic stability of NAMI-A in phosphate buffer (pH 7.4) at 37 °C is a limiting

factor for administration since it has a half-life of less than 30 minutes. Thus, when given to

patients, NAMI-A is administered with a physiological concentration of sodium chloride to

enhance the stability in the infusion solution.81

It has previously been demonstrated that the therapeutic benefits of anticancer drugs, for

example platinum-centred9, gold-centred213 or ruthenium-centred,214,215 can be enhanced by

encapsulation in, or conjugation to, a polymer matrix. The surrounding polymer protects the

drug, increases solubility and often increases cell uptake efficiency due to the cell entry

process being altered from a diffusion mechanism to endocytosis.11 The creation of a drug

carrier in the nano-size range enables fast endocytosis. Micelles present a unique size that

Chapter 2: Ruthenium(III)

82

leads to increased circulation times,27 and the potential to exploit the EPR effect,28 which are

important properties for therapeutic moieties. Covalent attachment of a therapeutic agent is a

useful avenue to delay drug release until the micelle reaches a target site.24 Micelles

containing covalently bound drugs have been investigated, for example PEG-b-

poly(Ɛ-caprolactone) with chemically conjugated docetaxel,123 and a number of poly(ethylene

oxide) based polymers containing cisplatin.212,216–221

NAMI-A comprises an imidazole ligand and counterion. The imidazole ring is biocompatible,

antimicrobial, anti-inflammatory and can regulate blood pressure.222 It has many uses, which

often centre around its ability to bond to metals as a ligand, and its ability to hydrogen bond

with drugs and proteins.222 Imidazoles also play a vital role in the inhibition of post-

translational farnesylation - a key step for RAS proteins that influence cancer proliferation.222

Importantly, for this work, the imidazole ligand provides an avenue for polymerisation.

Poly(vinyl imidazole)s have long been investigated for their use as catalysts, pH-sensitive DNA

carriers,223 complex coacervates,224 non-viral gene delivery therapeutics,225,226 and as oxygen

transport membranes.222 Imidazolium salts are used to extract metal ions from aqueous

media,227 coat metal nanoparticles, dissolve carbohydrates, and create polyelectrolyte brushes

on surfaces.222 “Imidazole-based polymers readily associate with biological molecules through

hydrogen-bonding, and imidazolium analogues offer electrostatic interactions, aggregation,

and self-assembly.”222 Poly(N-vinyl imidazole) has been investigated as an agent to selectively

bind metal ions in order to isolate them from wastewater. It was found that complexation

occurred through the basic nitrogen atoms at position three of the imidazole ring.227 Thus, it is

proposed that a polymeric form of NAMI-A could also be synthesised using the reactive

nitrogen on the imidazole ring.

Imidazole-containing polymers are typically obtained using free radical methods, which does

not offer control of molecular weight and polymer architecture.226 Dual stimuli-responsive

block copolymers containing 1-vinylimidazolium monomers have been synthesised in a

controlled manner using the RAFT/MADIX process,228 and more recently Allen et al229

successfully polymerised 4-vinyl imidazole via RAFT, using a trithiocarbonate RAFT Agent and

glacial acetic acid as a distinctive solvent. The aim of this chapter is to explore a synthetic

avenue to a macromolecular version of NAMI-A, and also the synthesis of an amphiphilic block

copolymer incorporating NAMI-A, that will self-assemble into micelles (Scheme 2-1). 4-Vinyl

imidazole was polymerised via RAFT and the formation of NAMI-A was examined. The NAMI-A

micelles were then evaluated against different cancerous cell lines, and contrasted with the


83

cytotoxicity of NAMI-A. It is demonstrated for the first time that the conjugation of NAMI-A to

a nanoparticle can significantly enhance the drug performance.

Scheme 2-1: Synthesis of the amphiphilic block copolymer P(NAMI-A)-PPEGMEA using 2-

(((dodecylthio)carbonothioyl)thio) -2-methylpropanoic acid RAFT Agent, and micellisation in

water.

OlH 240°C

~~ 2.2 x 10-2 mbar ~ LNH Cl

\ Ru

Cl/ \ Cl

Ruthenium(lll) Trichloride

0 II

/s'-.....

DMSO

HCI,100 oc

LNH urocanic acid

MeOH

[DMS02H][trans-RuiiiCI4(DMSO)zl

4-vlnyllmldazole

l RAFT Agent, ACPA Acetic Acid, 70 oc

~· LNH

P(4-vinyl imidazole)

1 PEGMEA, AIBN MeOH, 65°C

~ LNH 7

0 Y z

PVIm-PPEGMEA

Mice lias


84

2.1. SYNTHESES

2.1.1. Synthesis of 4-Vinyl Imidazole (VIm)

4-vinyl imidazole (VIm) was synthesised following a modified literature procedure.229 Urocanic

acid (1.5 g, 1.1 x 10-2 mol) was heated to 240 °C under reduced pressure (2.2 x 10-2 mbar) for

six hours. The product distilled as a clear liquid, which solidified upon cooling to give VIm as a

waxy colourless solid (0.7 g, 66 %). 1H NMR (300.30 MHz, DMSO-d6, 25 °C): δ (ppm) = 12.12

(s, 1H, NH), 7.61 (s, 1H, Hd); 7.06 (s, 1H, Hc); 6.57 (dd, 1H, Hb); 5.60 (dd, 1H, Ha); 4.99 (dd, 1H,

Ha). 13C NMR (300.30 MHz, DMSO-d6, 25 °C): δ (ppm) = 136 (Cd); 135 (Cc); 128 (Cb); 120 (Cq);

110 (Ca).

2.1.2. Polymerisation of 4-Vinyl Imidazole (PVIm) via RAFT

Polymerisation

VIm was polymerised following a literature procedure.229 VIm (0.25 g, 2.7 x 10-3 mol), RAFT

Agent 2 (5.0 mg, 1.3 x 10-5 mol) and ACPA (0.9 mg, 3.3 x 10-6 mol) as initiator, were dissolved in

glacial acetic acid (3.3 mL) to give [VIm]:[RAFT]:[ACPA] = 200:1:0.25. The solution was

transferred to a 10 mL Schlenk vial and deoxygenated by four freeze-pump-thaw cycles, and

then placed in an oil bath at 70 °C. The polymerisation was stopped after 24 hours by cooling

the vial and opening to air. The solution was dialysed (MWCO = 3500 g.mol-1) against MilliQ

water and dried under vacuum to give poly(4-vinyl imidazole) (PVIm) as a pale yellow low

density solid (0.16 g). Total reaction time = 24 hrs, xNMR = 76 %, xMass = 65 %, Mn,theo =

14 300 g.mol-1. 1H NMR (300.30 MHz, MeOD, 25 °C): δ (ppm) = 7.52 (broad, 1H, Hd); 6.35

(broad, 1H, Hc); 2.40-1.90 (broad, 1H, Hb); 1.62 (broad, 2H, Ha).

2.1.3. Chain Extension of 4-Vinyl Imidazole with Poly(ethylene

glycol) methyl ether acrylate (PVIm-PPEGMEA) via RAFT

Polymerisation

PEGMEA (0.1 g, 2.3 x 10-4 mol), VIm MacroRAFT (40 mg, 4.6 x 10-6 mol) and AIBN (0.2 mg,

9.3 x 10-7 mol) as initiator, were dissolved in methanol (0.45 mL) to give

[PEGMEA]:[PVIm]:[AIBN] = 50:1:0.2. The solution was transferred to a 10 mL Schlenk vial and


85

deoxygenated by five freeze-pump-thaw cycles. The solution was subsequently transferred to

a 2mm FT-IR quartz cuvette in a glove-box, and placed in the preheated FT-IR cell at 65 °C. The

polymerisation was monitored over time and stopped after four hours by cooling the cuvette

and opening to air. The solution was dialysed (MWCO = 3500 g.mol-1) against MilliQ water and

dried under vacuum to give poly(4-vinyl imidazole)-b-poly(poly(ethylene glycol) methyl ether

acrylate) (PVIm-PPEGMEA) as a pale yellow rubbery solid (96 mg). Total reaction time = 4 hrs,

xFT-NIR = 48 %, xMass = 63 %, Mn,theo = 28 700 g.mol-1. 1H NMR (300.30 MHz, MeOD, 25 °C):

δ (ppm) = 7.54 (broad, 1H, Hd); 6.42 (broad, 1H, Hc); 4.23 (broad, 2H, Hg), 3.63 (broad, PEG),

3.53 (broad, 2H, Hh), 3.32 (broad, 3H, H1), 2.79 (broad, 1H, Hf), 2.40-1.90 (broad, 1H, Hb);

1.60 (broad, 4H, Ha & He).

2.1.4. Synthesis of [DMSO2H][trans-RuCl4(DMSO)2] (Ru

Precursor)

[DMSO2H][trans-RuCl4(DMSO)2] (Ru Precursor) was synthesised following a literature

protocol.52,230 Ruthenium trichloride hydrate (RuCl3.H2O) (1.5 g, mol) was combined with

dimethyl sulfoxide (DMSO) (7.0 mL) in a 100 mL round-bottom flask. Hydrochloric acid (HCl)

(32 %) (1.2 mL) was added and the solution heated to 80 °C for 20 minutes with vigorous

stirring. The solution turned a deep red colour and the temperature was increased to 100 °C

and kept at this temperature for 20 minutes, at which time the solution was an orange-red

colour. The solution was cooled to RT with stirring, and acetone (50 mL) and diethyl ether

(5 mL) were added. The solution was transferred into a conical flask and placed in a dark

cupboard to allow for crystal formation. Large red-orange crystals were washed with diethyl

ether, dried under vacuum, and confirmed by x-ray crystallography to be consistent with

literature (Table 2-1).230 1H NMR (600.13 MHz, D2O, 25 °C): δ (ppm) = 2.74 (s, 6H,

[DMSO2H]); -16.62 (very broad, 6H, [trans-RuCl4(DMSO)2]).

2.1.5. Synthesis of (ImH)[Ru IIICl4(Im)(S-DMSO)] (NAMI-A)

(ImH)[RuIIICl4(Im)(S-DMSO)] (NAMI-A) was synthesised following a literature procedure.74

[DMSO2H][trans-RuCl4(DMSO)2] (0.1 g, 1.8 x 10-4 mol) was crushed to a mustard-yellow powder

and suspended in acetone (2.0 mL) with stirring. Imidazole (50 mg, 7.4 x 10-4 mol) was then

slowly added while rapidly stirring. The reaction was left rapidly stirring for four hours, after


86

which time the product was filtered, washed with acetone (3.0 mL) and diethyl ether (3.0 mL),

and dried under vacuum, to give a mustard yellow-orange solid (54 mg, 65 %). The compound

was confirmed by 1H NMR to be consistent with literature. 1H NMR (600.13 MHz, D2O, 25 °C):

δ (ppm) = 8.71 (s, 1H, ImH); 7.50 (s, 1H, ImH); -3.43 (broad, 1H, Im); -5.13 (broad, 1H, Im); -6.54

(broad, 1H, Im); -15.1 (very broad, 6H, (S-DMSO).

2.1.6. Synthesis of Macromolecular NAMI-A [P(NAMI-A)]

In a typical preparation, [DMSO2H][trans-RuCl4(DMSO)2 (15 mg, 2.7 x 10-5 mol) was crushed to

an orange powder and dissolved in ethanol or methanol (1 mL). PVIm (10 mg, 1.1 x 10-4 mol)

was suspended in the same solvent (1 mL). The solutions were combined and stirred to give a

mustard yellow suspension. For cytotoxicity assays, the solution was diluted with water and

used immediately. For UV-Vis analysis, the solution was diluted with methanol. For elemental,

NMR and TGA analyses, the product was filtered, washed with diethyl ether (2 mL), and dried

under vacuum, to give a mustard yellow solid.

2.1.7. Synthesis of P(NAMI-A)-PPEGMEA

In a typical preparation, [DMSO2H][trans-RuCl4(DMSO)2 (4.6 mg, 7.9 x 10-6 mol) was crushed to

an orange powder and dissolved in methanol (1 mL). PVIm-PPEGMEA (5.1 mg, 3.2 x 10-5 mol)

was also dissolved in methanol (1 mL). The solutions were combined to give a bright yellow

solution and analysed via UV-Vis immediately. For cytotoxicity assays, the solution was slowly

diluted (one hour) and then dialysed (MWCO = 3500 g.mol-1) against MilliQ water for one hour

and used immediately. For solid-state NMR, the solution was vacuum-dried to give an orange

solid.

2.1.8. In Vitro Cytotoxicity Assay

Human ovarian carcinoma A2780 and OVCAR-3, and pancreatic AsPC-1 cells were cultured in

75 cm2 tissue culture flasks with RPMI 1640 medium supplemented with 10 % fetal bovine

serum, 4 mM glutamine, 100 U/mL penicillin, 100 µg.mL-1 streptomycin, 1 mM sodium

pyruvate at 37 °C under an atmosphere of 5 % CO2. After reaching 70 % confluence, the cells


87

were washed with phosphate buffered saline (PBS) and collected by trypsin/EDTA treatment.

The cells were seeded in 96-well cell culture plates at 4 000 cells per well and cultured at 37 °C

for one day. The medium in the cell culture plate was discarded and 100 μL fresh 2 ×

concentrated RPMI 1640 serum medium was added. The samples were added into the plate at

100 μL per well for 72 hours. Before loading onto the cells, solutions were sterilised by UV

irradiation for 15 minutes in a biosafety cabinet and then serially diluted (2 × dilution) with

sterile water and incubated for two hours at room temperature. The working concentration of

ethanol in the culture medium was adjusted to 1 v/v % for PVIm samples. 1 v/v % ethanol did

not show significant influence on the viability of A2780, OVCAR-3 and AsPC-1 cells.

The cell viability was measured using a WST-1 assay (Roche Diagnostics). This is a colorimetric

assay for the quantification of cell viability and proliferation that is based on the cleavage of a

tetrazolium salt (WST-1) by mitochondrial dehydrogenases in viable cells. Increased enzyme

activity leads to an increase in the amount of formazan dye, which is measured with a

microplate reader. After incubation for three days, the culture medium was removed and

100 µL fresh medium was added along with 10 µL WST-1. The plates were then incubated for

an additional four hours at 37 °C. After incubation, the absorbance of the samples against the

background control on a Benchmark Microplate Reader (Bio-Rad) was obtained at a

wavelength of 440 nm with a reference wavelength of 650 nm. Four wells under each

condition were used for the measurement to calculate the means and standard deviations. All

cytotoxicity data are reported as mean ± standard deviation. A two-tailed student’s t-test was

executed to reveal the statistical differences. A p-value less than 0.05 was considered

statistically significant.

2.2. RESULTS AND DISCUSSION

2.2.1. Synthesis and Polymerisation of 4-Vinyl Imidazole

Consistent with the observations by Allen et al229 the water soluble225 poly(N-vinyl imidazole)

could not be synthesised in a controlled manner since it forms a highly reactive and unstable

propagating radical due to the absence of resonance stabilisation. In contrast, 4-vinyl

imidazole (VIm) could be polymerised via RAFT, using a trithiocarbonate RAFT agent and glacial

acetic acid as a distinctive solvent, due to the increased radical stability. VIm was synthesised

and subsequently polymerised (Scheme 2-2) following a modified literature procedure.229


88

Urocanic acid was heated under reduced pressure to decarboxylate and distil as a clear liquid,

which solidified upon cooling to give a waxy colourless solid. The product was confirmed by

1H NMR (Figure 2-1). The monomer was subsequently polymerised via RAFT polymerisation

(Figure 2-2 and Figure 2-3) to give a colourless solid that was soluble in methanol, DMSO and a

water/ethanol mixture (9:1).

Scheme 2-2: Synthesis of 4-vinyl imidazole by decarboxylation of urocanic acid.

Polymerisation of 4-vinyl imidazole in acetic acid at 70 °C. [VIm] = 0.8 M,

[VIm]:[RAFT]:[ACPA] = 200:1:0.25. The corresponding 1H NMR assignment is shown in

Figure 2-1 and Figure 2-2.

Figure 2-1: Stack-plot of 1H NMR spectra. A: urocanic acid and B: 4 -vinyl imidazole in

DMSO-d6 at 25 °C. The peak assignment corresponds to Scheme 2-2.


89

Figure 2-2: Stack-plot of 1H NMR spectra. A: poly(4-vinyl imidazole) and B: 4-vinyl imidazole

in MeOD at 25 °C. The peak assignment corresponds to Scheme 2-2.

27 26 25 24 23 22 21 20 19 18 17

1 hour

2 hours

3 hours

4 hours

5 hours

6 hours

RT / mins

Conversion

Figure 2-3: Water SEC trace if the polymerisation of 4-vinyl imidazole in acetic acid at

70 °C. [VIm] = 0.8 M, [VIm]:[RAFT]:[ACPA] = 200:1:0.25.


90

2.2.2. Synthesis of NAMI-A

The Ru Precursor (Scheme 2-3) that is used in the synthesis of the antimetastatic compound

NAMI-A was synthesised following a literature procedure52,230 and confirmed by x-ray

crystallography to be consistent with literature (Table 2-1).230

Scheme 2-3: Synthesis of [DMSO2H][trans-RuII I

Cl4(DMSO)2].

Table 2-1: X-ray crystallography results were compared with literature values to confirm

the synthesis of [DMSO2H][trans-RuI I I

Cl4(DMSO)2].

Unit Cell Parameters Experimental Literature230

Crystal system monoclinic monoclinic

a (Å) 9.273 (1) 9.281 (5)

b (Å) 16.509 (3) 16.497 (9)

c (Å) 14.023 (3) 14.037 (8)

β (°) 100.79 (2) 100.811 (12)

Due to the unfavourable occurrence of two trans S-bonded DMSO’s in the precursor complex,

substitution of at least one of them with a stronger σ-donor ligand is a relatively simple task.230

Alessio et al52 reported that one DMSO ligand can rapidly be replaced in an organic solvent at

room temperature using a slight excess of a nitrogen ligand. Hence, NAMI-A (Scheme 2-4) was

synthesised following a literature procedure,74 characterised via NMR (Figure 2-4) and

confirmed to be consistent with literature due to the very broad DMSO methyl peak at -15.2.


91

Figure 2-4: 1H NMR of (ImH)[Ru

II ICl4(Im)(S-DMSO)] (NAMI-A) in D2O at 25 °C. The very broad

DMSO ligand methyl peak at -15.2 is consistent with literature.74

2.2.3. Synthesis of Macromolecular NAMI-A

A polymeric form of NAMI-A [P(NAMI-A)] was synthesised by combining PVIm (Mn,theo =

14 300 g.mol-1) and the Ru Precursor in ethanol or methanol. A methylated version of NAMI-A

(using 4-methyl imidazole) was also synthesised for comparison, since the polymeric form uses

4-vinyl imidazole. Following a literature procedure for the synthesis of NAMI-A,74 the imidazole

was added in a 4:1 excess to the Ru Precursor, to ensure complete conjugation of ruthenium

to imidazole, and the formation of imidazole counterions. (Previous research has shown that

the imidazole counterion imparts more favourable chemical properties on NAMI-A than other

counterions, for example Na+).17

Consistent with the synthesis of NAMI-A, the Ru precursor was first dissolved in the selected

solvent (methanol for P(NAMI-A) and acetone for M-NAMI-A) giving a bright orange solution.

The imidazole (PVIm or 4-methyl imidazole) was subsequently added while vigorously stirring,

immediately producing a yellow-orange suspended precipitate (Scheme 2-4). The solid was

washed and dried under vacuum. The UV-Vis absorbance maxima of the products in methanol

were compared (Figure 2-5). A clear shift from the Ru Precursor at 375 nm to NAMI-A, M-

NAMI-A and P(NAMI-A) at 400 nm, indicated complete substitution of one DMSO ligand with

an imidazole, producing the desired products.


92

Scheme 2-4: Synthesis of (ImH)[RuI II

Cl4(Im)(S-DMSO)] (NAMI-A) and M-NAMI-A in acetone

at room temperature. P(NAMI-A) was prepared in ethanol.


93

350 400 450 5000

1

2

3

4 [RuCl

4(DMSO)

2]

NAMI-A

M-NAMI-A

P(NAMI-A)

P(NAMI-A)-PPEGMEA

Ab

so

rba

nce

Wavelength / nm

Figure 2-5: UV-Vis spectrum in methanol at 25 ᵒC. The attachment of imidazole to RuII I

elicits a clear shift in absorbance maxima from 375 nm to 400 nm.

Thermogravimetric analysis was used to confirm the amount of ruthenium in each sample

(Table 2-2). A dry polymer sample was loaded onto a TGA pan and heated to 1000 °C at a rate

of 20 ᵒC.min-1. Since ruthenium oxidises to RuO2 at 600 °C, 75.9 % of the residual mass can be

ascribed to ruthenium, assuming that the residue is pure RuO2.

Table 2-2: Thermogravimetric analysis of NAMI-A analogues. Samples were analysed using

two different atmospheres (nitrogen or oxygen) to degrade the samples.

Sample Ru Precursor# NAMI-A M-NAMI-A P(NAMI-A)

Residue (%) using N2 21 23 21 13

Residue (%) using O2 24 29 23 21

Ru (%)* 18 22 17 16

Ru (% theo)a 18.16 22.11 20.83 -

*Ru fraction = (mass residue using O2 x 0.759) / initial mass #Ru Precursor = [DMSO2H][trans-RuCl4(DMSO)2]

acalculated from the structural formula


94

In addition, a dry polymer sample was submitted for solid-state elemental analysis. The

number of imidazole repeating units was calculated from the NMR conversion (n = 152). A

spread sheet was then constructed to determine the number of imidazole units that had

ruthenium attached, thus forming NAMI-A, assuming that for each imidazole with ruthenium

there is another imidazole that acts as a counterion. It was found that 49 % of the polymer

units were NAMI-A units (Table 2-3). This is consistent with the complete conjugation of all

added ruthenium since the Ru precursor was combined with a 4:1 excess of imidazole units

and two imidazole units form NAMI-A (one conjugated to ruthenium and the other forming the

positively charged counterion). The fraction of ruthenium calculated from TGA is also

consistent with the calculated percentage. The remaining error is due to the nature of polymer

entities and the variability between polymer chains. The resulting structure with the calculated

repeating units is depicted in Scheme 2-4.

Table 2-3: Calculation to determine the number of unreacted imidazole polymer units and

the number of NAMI-A units. The number of elements in each polymer component was

used to calculate the total molecular weight for each element and then as a percentage of

the total. These values were compared to the elemental analysis results, with particu lar

emphasis on Cl and N since both unit types contain N but only NAMI -A contains Cl. From

this, it was found that 49 % of polymer units were NAMI -A units.

Element C H N S Cl Ru O

Imidazole 5 5 2 NAMI-A 10 10 4 1 4 1 1

RAFT End-group 18 34 3 2 Element in P(NAMI-A)

a 1225 1241 483 82 318 79 81

Element Mw in P(NAMI-A)b 14711 1251 6763 2642 11256 8023 1302

Calculated Fraction of Mw (%)c 32 3 15 6 24 17 3

Elemental Analysis (%) 30 4 12 5 24 TGA (%)*

16

*See calculation in Table 2-2. aFraction of each element in P(NAMI-A) = (Imidazole x imidazole fraction x units in polymer) + (NAMI-A x NAMI-A

fraction x units in polymer) + RAFT. bMw of each element in P(NAMI-A) = a x molar mass of element.

cCalculated fraction of each element in polymer.


95

2.2.4. NAMI-A Block Copolymer

The P(NAMI-A) polymer is barely soluble in any solvent (including water). This low solubility in

water is advantageous as it provides an avenue to the formation of nanoparticles. One of the

primary motivations for incorporating anticancer drugs into polymers is to increase cell uptake

in the body. Since P(NAMI-A) is ‘insoluble’ in water, it can act as the hydrophobic component

of polymeric micelles, which have been shown to have better cell uptake than linear

polymers.25 Therefore, a suitable co-monomer, namely PEGMEA, was chosen and PVIm was

used as a macroRAFT agent and chain extended with PEGMEA (Figure 2-6). The monomodal

SEC trace (Figure 2-7) shows a clear shift in retention time, indicative of a change in molecular

weight. The absence of any low molecular weight shoulder indicates that the PVIm macroRAFT

is quite efficient in mediating the polymerisation of PEGMEA. Since the Mn values obtained

from the water SEC traces cannot be used due to the difference in the polymer and the PEO

calibration standards, the molecular weights used in further discussions were calculated using

conversions determined from FT-NIR (Figure 2-8).

Figure 2-6: Stack-plot of 1H NMR spectra. A: poly(4-vinyl imidazole) (PVIm) and B:

poly(vinyl imidazole)-b-poly(poly(ethylene glycol) methyl ether acrylate) (PVIm -PPEGMEA)

in MeOD at 25 °C.


96

24 22 20 18 16 14

PVIm-PPEGMEA

PVIm

RT / mins

Figure 2-7: Water SEC trace of the polymerisation of poly(ethylene glycol) methyl ether

acrylate in methanol at 65 ᵒC using P(4-vinyl imidazole) macroRAFT agent (Mn,theo =

14 300 g.mol-1

). [PEGMEA] = 0.4 M, [PEGMEA]:[PVIm]:[AIBN] = 50:1:0.2. Total reaction time

= 4 hrs, xFT-NIR = 48 %, Mn,theo = 28 700 g.mol-1

. The eluent was a MilliQ water/acetic

acid/methanol (54:23:23) solution.

0 1 2 3 40

10

20

30

40

50

60

Co

nve

rsio

n /

%

Time / hrs

Figure 2-8: Polymerisation of poly(ethylene glycol) methyl e ther acrylate in methanol at

65 ᵒC usingP(4-vinyl imidazole) macroRAFT agent, monitored via FT-IR. [PEGMEA] = 0.4 M,

[PEGMEA]:[PVIm]:[AIBN] = 50:1:0.2.


97

The Ru precursor was reacted with the copolymer P(NAMI-A)152-PPEGMEA19 in a similar fashion

to the homopolymer – the difference being that the copolymer remained soluble in methanol

after conjugation, and it could be dried and redissolved. UV-Vis was used to confirm the

complexation. Figure 2-5 shows a shift in the UV-Vis absorbance maxima, comparable to that

of NAMI-A and P(NAMI-A).

Solution NMR was used as a final analysis method to confirm the complex formation of

NAMI-A. However, this was not possible for the macromolecular forms of NAMI-A – neither

homopolymer nor copolymer. Paramagnetism of the RuIII nucleus combined with the inherent

broadening of polymer signals prevented a detailed solution NMR study. Solid-state NMR was

thus used to determine the homogeneity of NAMI-A within the P(NAMI-A) and P(NAMI-A)-

PPEGMEA polymers.

The presence of paramagnetic RuIII causes fast relaxation with concomitant line broadening

and paramagnetic contact shift in the 1H and 13C solid state NMR of these materials. As a result

using chemical shift to measure incorporation and bonding of the RuIII species is challenging.

An alternative method is to look at the spin-lattice relaxations of the 1H (T1H) where the

presence of a paramagnetic species would cause enhanced relaxation of the 1H nuclei. The T1H

was measured by a saturation recovery experiment where the equilibrium magnetisation was

first suppressed by a comb of saturation pulses and delays after which a recovery delay was

introduced for the 1H magnetization to recover. The total integrated area of the 1H NMR signal

(normalized against M0, the signal area for the fully recovered magnetization) was plotted

against the recovery time (ms) as seen in Figure 2-9. The data was fitted by a single or double

exponential of the form M/M0= 1– Aexp-t/T1A – Bexp-t/T1B, where A and B are the fractions of

slow and fast relaxing components with spin-lattice relaxation times of T1A ans T1B. It should be

noted that in the case of paramagnetic driven relaxation processes, the saturation recovery

behavior can be non-exponential (stretched exponential) particularly in the case of low natural

abundance NMR active nuclei such as 13C.231 However, in the case of nuclei such as 1H and in

the current materials with fast nuclear spin diffusion and moderate MAS (12 kHz), the

saturation recovery behavior is better described by a single or double exponential curve.

For example, the 1H saturation recovery of the neat NAMI-A complex is well fitted by a single

exponential with a T1H of 0.9 ms. The extremely fast T1H is consistent with the presence of the

paramagnetic RuIII in the solid material. In the case of P(NAMI-A), the overall fast spin lattice

relaxation of the 1H species, confirmed the attachment of a paramagnetic species, and thus it

can be inferred that ruthenium is still present as RuIII. However, the saturation recovery curve


98

of P(NAMI-A) followed a dual exponential behaviour. The faster relaxing component with a

very fast T1H relaxation time of 0.5 ms corresponded to 45 % of the polymer that is at an

average distance of ca. 1 nm from the RuIII complex, while the comparatively slower T1H

relaxation time of 12 ms corresponded to ca. 55 % of the polymer beyond nanometer

proximity of the RuIII complex. This is consistent with the elemental analysis and TGA results

which showed that approximately 50 % of the imidazole units formed NAMI-A.

Finally, the absence of any slow relaxing components with a T1H in the order of 100 ms to

several seconds, as would be expected for diamagnetic, amorphous, organic polymers, implies

that, on a conservative 10 nm scale, there is a uniform distribution of the RuIII complex within

the P(NAMI-A) polymer matrix.

Figure 2-9: 1H saturation recovery experiment of NAMI-A, P(NAMI-A) and P(NAMI-A)-

PPEGMEAvia solid-state NMR.

In the case of P(NAMI-A)-PPEGMEA the 1H spectrum of the copolymer yields two peaks - one

broad and one narrow. The broad component corresponds to the rigid P(NAMI-A) block

containing the RuIII complex with a fast T1H of ca. 1.5 ms relaxation. The narrow component

corresponds to the mobile PPEGMEA block which, as expected, has a distinctly longer


99

relaxation time (28 ms) than the P(NAMI-A) block. It is likely that the PPEGMEA block is

strongly coupled to the faster-relaxing proton pool of the P(NAMI-A) block accounting for a T1H

of ca. 28 ms. It is highly unlikely that the PPEGMEA contains RuIII since there are no binding

sites in this copolymer block to allow for the formation of the ruthenium complex.

Additionally, the UV-Vis spectrum (Figure 2-5) confirmed the attachment of the RuIII complex

to the imidazole units in the PVIm block, as shown by a clean shift of a single peak, and there

was no indication of RuIII interacting with the PPEGMEA block in the polymer. However,

approximately 6 % of the P(NAMI-A) block has a longer relaxation time of ca. 20 ms. We

hypothesize that this can be attributed to the region where both blocks are connected and

that the proximity to the PEG induces motional mobility to a fraction of the P(NAMI-A) which

can enhance the relaxation times. Considering the relatively small fraction of the P(NAMI-A)

block with the longer relaxation time, it is also possible that it has a lower RuIII concentration.

However, given the synthesis method, it is unlikely that the RuIII would distribute

heterogeneously within the PVIm copolymer block, since it is homogenous in the

homopolymer, on the 10 nm scale. Overall, these results are consistent with the distinct

PPEGMEA and P(NAMI-A) phases, where the predominant fraction of the PPEGMEA phase is

further from the paramagnetic centres.

In summary, these results clearly suggest the following:

a) The overall fast relaxation indicates that the complex is incorporated into the polymer;

b) The comparatively longer relaxation time (12 ms) indicates that on the 10 nm scale,

there is homogenous distribution of the complex;

c) The short relaxation time (0.5 ms) corresponds to the region within nanometer scale

proximity of the complex. It also indicates that the complex is dispersed within the

polymer and has not aggregated; and

d) The copolymer 1H spectrum consists of a broad and narrow component with very

different relaxation properties, indicative that RuIII is incorporated uniformly in a single

block of the polymer i.e. P(NAMI-A).

2.2.5. Micellisation of Amphiphilic Block Copolymer

After drug conjugation, the methanol solution was immediately dialysed against water to

remove methanol and simultaneously form micelles in solution. The dialysed yellow solution

was analysed using TEM (Figure 2-10) to determine micelle size and homogeneity. TEM shows


100

micelles to be a reasonable size, although a size distribution is evident. They are round in

shape, but probably slightly distorted due to the drying process for TEM preparation.

The unstained sample in Figure 2-10 (a) appears black due to the conjugation of ruthenium,

which has a high electron density. Closer inspection of Figure 2-10 (a) reveals the darker

appearance of the centre of the micelle due to the location of ruthenium in the core of the

micelle.

Figure 2-10: P(NAMI-A)-PPEGMEA Micelles prepared by dialysing MeOH solution against

water. Sample (a) was drop-loaded onto grid and air-dried. Samples (b) to (f) were drop-

loaded onto grid, air-dried and stained with Phosphotungstic Acid. Scale bar: d = 2 µm; c &

f = 200 nm; a, b & e = 100 nm.

2.2.6. In Vitro Cytotoxicity

The NAMI-A polymeric micelles were tested against ovarian A2780 and OVCAR-3, and

pancreatic AsPC-1 cancer cell lines, and compared with the drug NAMI-A. The homopolymer

and block copolymer prior to drug conjugation were also tested for toxicity (Table 2-4, Figure

2-11). PVIm was found to be toxic, which makes P(NAMI-A) an undesirable therapeutic agent,

since the toxicity may be derived from the polymer itself. This can be attributed to the positive


101

charges of the imidazole units, as positively charged polymers have previously shown

toxicity.232

Table 2-4: IC50 values with respect to the polymer concentration, against ovarian A2780

and OVCAR-3 and pancreatic AsPC-1 cancer cell lines.

IC50 ([P] µM) A2780 AsPC-1 OVCAR-3

PVIm 0.37 0.51 0.70

PVIm-PPEGMEA > 15.37 > 15.37 > 15.37

P(NAMI-A)-PPEGMEA* 9.77 8.86 9.65

*Polymer concentrations at IC50 in Table 2-5.

102

103

104

0

20

40

60

80

100

120

A2780 PVIm-PPEGMEA

A2780 PVIm

AsPC-1 PVIm-PPEGMEA

AsPC-1 PVIm

Ovcar-3 PVIm-PPEGMEA

Ovcar-3 PVIm

Su

rviv

al R

ate

/ %

Polymer Concentration / nM

Figure 2-11: Cytotoxicity of PVIm and PVIm-PPEGMEA against ovarian A2780 and OVCAR-3

and pancreatic AsPC-1 cancer cell lines, after 72 hours, n = 4.


102

The copolymer, PVIm-PPEGMEA, was completely nontoxic to all cell lines up to a concentration

of 10 µM. This was surprising since the copolymer still contains unmodified PVIm. The reason

for this complete lack of toxicity can be attributed to the morphology of the block copolymer.

The homopolymer PVIm is insoluble in water, which leads to the micelle formation of PVIm-

PPEGMEA. The sudden disappearance of apparent cytotoxicity has been observed earlier when

a cationic polymer formed the core of a crosslinked micelle resulting in the loss of toxicity in

this assay.198 A statistically significant decrease in the IC50 values for P(NAMI-A)-PPEGMEA

micelles compared with NAMI-A at the same ruthenium concentration, was found for all cell

lines (Table 2-5, Figure 2-12). The micelles were found to be ~1.5 times better than NAMI-A at

inhibiting cancer cell growth, across the tested cell lines. Most notably, it was active on the

highly aggressive pancreatic cancer cell line.

449 µM 224.5 µM 449 µM 224.5 µM 449 µM 224.5 µM0

20

40

60

80

100

120

Ovcar-3AsPC-1

Su

rviv

al R

ate

/ %

P(NAMI-A)-PPEGMEA NAMI-A

A2780

**

**

**

**

***

***

Figure 2-12: Cytotoxicity of NAMI-A and P(NAMI-A)-PPEGMEA against ovarian A2780 and

OVCAR-3 and pancreatic AsPC-1 cancer cell lines, after 72 hours. For P(NAMI-A)-PPEGMEA,

the polymer concentration at [Ru] = 449 and 224.5 µM is 10 and 5.0 µM, respectively.

Mean ± SD, n = 4, ** significantly different p < 0.01, *** significantly different, p < 0.001.


103

Table 2-5: IC50 values with respect to the ruthenium concentration, against ovarian A2780

and OVCAR-3 and pancreatic AsPC-1 cancer cell lines.

IC50 ([Ru] µM) A2780 AsPC-1 OVCAR-3

NAMI-A Literature > 50085

- -

Precursor Complex 165.5 271.7 -

NAMI-A 595.6 601.7 737.8

P(NAMI-A)-PPEGMEA 438.7 397.6 433.4

2.3. CONCLUSIONS

An amphiphilic block copolymer capable of self-assembling into polymeric micelles was

recognised as an appropriate drug carrier for NAMI-A. A suitable method for the synthesis of a

macromolecular NAMI-A drug was identified – the polymerisation of vinyl imidazole and

subsequent addition of a ruthenium(III) precursor complex. This macromolecular drug was

chain extended with the water soluble biocompatible PEGMEA and self-assembled into

micelles. On average, across the tested cell lines, a 1.5 times increase in toxicity was found for

the NAMI-A block copolymer micelles when compared to the NAMI-A molecule. Chapter 6

entails an assessment of the metastatic effects of this macromolecular NAMI-A

chemotherapeutic.

104

105

3 RUTHENIUM(II)

The ruthenium(II) group of anticancer remedies

include the [RuCl2(arene)(PTA)] (RAPTA) series of

complexes, which contain an RuII centre, a chosen

arene ligand and the phosphine ligand 1,3,5-triaza-

7-phosphaadamantane (PTA).46 The arene provides

a hydrophobic (lipophilic) face for the complex and

also stabilises the RuII centre12,96 to ensure that the

complex does not readily undergo oxidation to

RuIII.14 The arene selection affects both the rate of

hydrolysis of the Ru-Cl bond, the acidity of the

resulting coordinated aqua ligand12 and can also influence the degree of repair synthesis of

ruthenium-damaged DNA.44 A trend for an increase in arene size corresponding to increased

cytotoxicity, due to better intercalation and further DNA distortion, has also been found.12,57,59

The chloride ligands provide a site for hydrolysis,49,81,83 occurring by an associative pathway,57

and hence Ru activation.44,49,63,83 Aquation occurs inside cells,3 prior to reactions with DNA,

since extracellular chloride concentrations are higher than intracellular concentrations.12,14,21,46

The PTA ligand results in water-solubility,44,46,53,99 due to its ability to form hydrogen bonds via

the three N-donor atoms,98 and is important for the toxic mechanism of the complexes.96

One of the most promising anticancer agents in this series contains the p-cymene ligand and is

termed RAPTA-C. It is a ruthenium metallodrug that is weakly cytotoxic in vitro but very

selective and efficient on metastases in vivo.13,15,19–21 RAPTA-C has shown high selectivity

towards cancer cells,5,38,91 inactivity against primary tumours,22 and metastasis process-

inhibiting properties.16,81 It is effective at reducing metastases combined with excellent

clearance rates from vital organs.16,23 The complex binds to oligonucleotides with loss of

Chapter 3: Ruthenium(II)

106

chloride, and sometimes the arene also.23,96 A predisposition towards protein binding may be

responsible for the selective antimetastatic effect of RAPTA-C,13 as it effectively inhibits cell

growth by triggering G2/M phase arrest and apoptosis in cancer cells, and also slows cell

division.22 RAPTA-C significantly inhibits the progression of cancer in animal models, by

reducing the number and weight of solid metastases, with low general toxicity,22 and has also

shown pH-dependent binding properties81 and DNA damage.21,49 PTA, which can be protonated

at low pH to form the ammonium phosphine [PTA(H)]+,53 is of critical importance in the toxic

mechanism of RAPTA-C.5

However, RAPTA-C is limited by the high dose required for an effective treatment. The

promising developments of this drug could be further enhanced by encapsulation in, or

conjugation to, a polymer matrix, as the surrounding polymer protects the drug, increases

solubility and often increases cell uptake efficiency due to the cell entry process being altered

from a diffusion mechanism to endocytosis.11

The aim of this chapter is to explore synthetic avenues to conjugate RAPTA-C to a polymer.7

Two different routes were explored. The common feature of both pathways is the utilization of

the inherent activity of the nitrogen groups of the PTA molecule as a site for alkylation53,99,233

by the polymer. The first route examines the synthesis of the complex and subsequent

conjugation to the polymer. The second examines the attachment of PTA to the polymer and

the subsequent complexation to give the Polymer-RAPTA-C macromolecule (Scheme 3-1).


107

Scheme 3-1: Synthesis of macromolecular ruthenium complex Polymer-RAPTA-C in

DMSO-d6 at 80 °C (Route A) and at 25 °C (Route B).


108

3.1. SYNTHESES

3.1.1. Synthesis of 2-Chloroethyl Methacrylate (CEMA)

Methacryloyl chloride (14 g, 1.4 x 10-1 mol), 2-chloroethanol (10 g, 1.2 x 10-1 mol) and NEt3

(21 mL) were dissolved in THF (70 mL) and reacted for 1 hour while cooling the solution in an

ice-bath. The product was extracted with diethyl ether, to give a pale yellow liquid. 1H NMR

(300.17 MHz, CDCl3, 25 °C): δ (ppm) = 5.78 (s, 1H); 5.23 (s, 1H); 4.01 (t, 2H); 3.33 (t, 2H); 1.57

(s, 3H).

3.1.2. Polymerisation of 2-Chloroethyl Methacrylate (PCEMA)

via RAFT Polymerisation

CEMA (2.2 g, 1.5 x 10-2 mol), CDB (14 mg, 5.0 x 10-5 mol) as RAFT Agent and AIBN (4.0 mg,

2.5 x 10-5 mol) as initiator, were dissolved in 1,4-dioxane (2 mL) to give [CEMA]:[CDB]:[AIBN] =

300:1:0.5. The solution was deoxygenated by nitrogen purging for 30 minutes and placed in an

oil bath at 60 °C. The polymerisation was stopped after eight hours by placing the vial in an ice

bath. The solution was precipitated, washed with diethyl ether, and dried under vacuum to

give poly(2-chloroethyl methacrylate) (PCEMA) as a pink solid (2.0 g, 92 %). Total reaction time

= 8 hrs, Mn,theo = 41 000 g.mol-1, Mn,SEC = 40 000 g.mol-1, PDI = 1.17.

3.1.3. Polymer End-Group Modifications of PCEMA

To cleave the RAFT end-group, a literature procedure was followed.234 PCEMA (0.5 g,

1.3 x 10-5 mol) and AIBN (41 mg, 2.5 x 10-4 mol) were dissolved in toluene (10 mL). The solution

was degassed by N2 bubbling and heated to 80 °C for 2.5 hours. The solution was cooled and

the product precipitated by the dropwise addition of the solution into cold hexane. The

polymer was filtered and dried to give a white powder (Mn = 42 800 g.mol-1, PDI = 1.17).

To convert the chloride end-group to an iodide end-group, the Finkelstein method was used.235

PCEMA (0.3 g, 2.0 x 10-3 mol) and NaI (0.4 g, 2.8 x 10-3 mol) were dissolved in anhydrous

acetone (20 mL). The solution was refluxed for 72 hours and cooled. The polymer was


109

precipitated in water and freeze-dried, to give poly(2-iodoethyl methacrylate) (PIEMA) as a

white powder (Mn = 40 000 g.mol-1, PDI = 1.13).

3.1.4. Statistical Copolymerisation of N-(2-Hydroxypropyl)

Methacrylamide and 2-Chloroethyl Methacrylate

(P(HPMA-CEMA)) via RAFT Polymerisation

CEMA (0.3 g, 2.0 x 10-3 mol) dissolved in 1,4-dioxane (2 mL) and HPMA (2.6 g, 1.8 x 10-2 mol),

CDB (18 mg, 6.7 x 10-5 mol) as RAFT Agent and AIBN (6.0 mg, 3.4 x 10-5 mol) as initiator,

dissolved in DMF (9 mL) were combined, to give [HPMA]:[CEMA]:[CDB]:[AIBN] = 270:30:1:0.5.

The solution was deoxygenated by nitrogen purging for 30 minutes and placed in an oil bath at

60 °C. The polymerisation was stopped after eight hours by placing the vial in an ice bath. The

solution was precipitated, washed with diethyl ether, and dried under vacuum to give

poly((2-hydroxypropyl) methacrylamide)-(2-chloroethyl methacrylate)) (P(HPMA-CEMA)) as a

pink solid (0.5 g, 17 %). Total reaction time = 8 hrs, Mn,theo(NMR) = 37 600 g.mol-1, Mn,SEC =

21 300 g.mol-1, PDI = 1.15, [HPMA]:[CEMA] = 4:1 (calculated from NMR).

3.1.5. Polymer End-Group Modifications of P(HPMA-CEMA)

To cleave the RAFT end-group, a literature procedure was followed.234 P(HPMA-CEMA) (0.5 g,

2.2 x 10-5 mol) and AIBN (72 mg, 4.4 x 10-4 mol) were dissolved in DMAc (10 mL). The solution

was degassed by N2 bubbling and heated to 80 °C for 2.5 hours. The solution was dialysed

against water and freeze-dried to obtain a solid. The procedure was repeated, the solution

dialysed and freeze-dried to give a pale pink solid (Mn,SEC = 21 600 g.mol-1, PDI = 1.15).

To convert the chloride end-group to an iodide end-group, the Finkelstein method was used.158

P(HPMA-CEMA) (0.4 g, 2.6 x 10-3 mol) and NaI (0.5 g, 3.6 x 10-3 mol) were dissolved in

anhydrous acetone (25 mL) and DMAc (5 mL). The solution was refluxed for 48 hours, dialysed

against water and freeze-dried to obtain a solid. The procedure was repeated, the solution

dialysed and freeze-dried to give poly((2-hydroxypropyl methacrylamide)-(2-iodoethyl

methacrylate)) (P(HPMA-IEMA)) as a pale pink powder (Mn,SEC = 21 700 g.mol-1, PDI = 1.19).


110

3.1.6. Synthesis of Dichlororuthenium(II)(p-cymene)(1,3,5-

triaza-7-phosphaadamantane) (RAPTA-C)

RAPTA-C was synthesised following a literature protocol.5,19 RuCl2(p-cymene) dimer (0.2 g,

3.3 x 10-4 mol) and PTA (0.1 g, 6.6 x 10-4 mol) were dissolved in methanol (70 mL). The solution

was refluxed under argon for three hours. Red-orange crystals were grown from

DCM/Heptane in a glove-box and the compound confirmed by x-ray crystallography to be

consistent with literature.19 1H NMR (500.17 MHz, DMSO-d6, 25 °C): δ (ppm) = 5.74 (dd, 4H);

4.46 (s, 6H); 4.10 (s, 6H); 2.55 (m, 1H); 1.87 (s, 3H); 1.05 (d, 6H). 31P NMR (300.17 MHz,

DMSO-d6, 25 °C): δ(ppm) = -33.40 (RuCl2(p-cymene)(PTA); -30.80

(RuCl(OH)(p-cymene)(PTA); -29.01 (RuCl(H2O)(p-cymene)(PTA).

3.1.7. Synthesis of a Low Molecular Weight Model Compound

and Macromolecular Ruthenium Complex

3.1.7.1. Route A: Iodated Compound + RAPTA-C (Table 3-1)

The iodated compound and RAPTA-C were dissolved in DMSO-d6 in a glove-box. The solution

was monitored via NMR over time.

3.1.7.2. Route B: Iodated Compound + PTA + RuCl2(p-cymene) Dimer

(Table 3-1)

The iodated compound and PTA were dissolved in DMSO-d6 in a glove-box. The solution was

monitored over time via NMR. The RuCl2(p-cymene) dimer was subsequently added and the

solution monitored over time via NMR .


111

Table 3-1: Synthesis of low molecular weight model compound B utylated RAPTA-C and

macromolecular ruthenium complex Polymer-RAPTA-C.

Synthesis of Butylated

RAPTA-C

Synthesis of Polymer-

RAPTA-C

Route A Route B Route A Route B

n-Butyl Iodide 4.0 mg

2.2 x 10-5

mol

5.0 mg

2.6 x 10-5

mol

PIEMA 9.0 mg

3.8 x 10-5

mol

9.0 mg

3.8 x 10-5

mol

RAPTA-C 10 mg

2.2 x 10-5

mol

18 mg

3.8 x 10-5

mol

PTA 4.0 mg

2.6 x 10-5

mol

6.0 mg

3.8 x 10-5

mol

RuCl2(p-cymene) Dimer 8.0 mg

1.3 x 10-5

mol

24 mg

3.8 x 10-5

mol

DMSO-d6 0.8 mL 1.0 mL 1.0 mL 1.0 mL

Temperature 25 °C 25 °C 25 °C, 80 °C 25 °C

3.1.8. Synthesis of Macromolecular Ruthenium Complex:

Copolymer-RAPTA-C via Route B

3.1.8.1. Solution A: P(HPMA-IEMA) + PTA

P(HPMA-IEMA) (26 mg, 1.5 x 10-4 mol) and PTA (8.0 mg, 5.1 x 10-5 mol) were dissolved in

DMSO-d6 (1.2 mL), in a glove-box, to give a clear solution. The solution was split into two equal

parts (solutions A and B). Solution A was monitored via NMR over time at 25 °C.

3.1.8.2. Solution B: P(HPMA-IEMA) + PTA + RuCl2(p-cymene) Dimer

Solution B was left in the glove-box. After seven days, RuCl2(p-cymene) dimer (8.0 mg,

1.3 x 10-5 mol) was subsequently added and the orange solution measured via NMR at 25 °C.

Solutions A and B were purified by dialysis (MWCO = 3.5 kDa) against MilliQ water.


112

3.1.9. Cytotoxicity Assay

Dr. Vien Huynh kindly completed the following assay. The Sulforhodamine B (SRB) assay

established by the U.S. National Cancer Institute for rapid, sensitive, and inexpensive screening

of antitumour drugs in microplates was employed to screen the cytotoxicity of Copolymer-

RAPTA-C and RAPTA-C.236 Human ovarian cancer cells (OVCAR-3) diluted in 100 L of RPMI

1640 medium (2 mM L-glutamine, 1.5 g.L-1 sodium bicarbonate, 10 mM HEPES, 4.5 g.L-1

glucose, 1 mM sodium pyruvate) were seeded into the wells with 3000 cells/well. The

microtiter plates were left for 24 hours at 37 °C and then exposed to various doses of

copolymer-RAPTA-C or RAPTA-C and incubated for 72 hours. Cell cultures were fixed with TCA

(10 %, w/v) and incubated at 4 °C for one hour. The wells were then washed five times with

tap water to remove TCA, growth medium and low molecular weight metabolites. Plates were

air dried and then stored until use. TCA-fixed cells were stained for 30 minutes with

0.4 % (w/v) SRB dissolved in 1 % (v/v) acetic acid. At the end of the staining period, SRB was

removed and cultures were quickly rinsed five times with 1 % (v/v) acetic acid to remove

unbound dye. Subsequently, the cultured plates were air dried until no conspicuous moisture

was visible before bound dye was shaken in 100 L of 10 mM Tris base for five minutes. The

absorbance at 570 nm of each well was measured using microtiter plate reader Scanning

spectrophotometer (BioTek's PowerWave HT Microplate Reader and KC4 Software). Each

sample was replicated three times.

3.2. RESULTS & DISCUSSION

3.2.1. Synthesis of PIEMA

The attachment of RAPTA-C (Scheme 3-1) to a polymer required the design of a suitable

polymeric scaffold that allowed post modification in an efficient and orthogonal manner.

Alkylhalogenide was identified as a reactive group that can undergo nucleophilic substitution

of halogenides with amines. RAPTA-C contains PTA that allows the formation of quaternary

ammonium cations (Scheme 3-1). Therefore, 2-chloroethyl methacrylate (CEMA) was

synthesised and polymerized using RAFT polymerization, with CDB as the controlling agent.

The successful polymerisation of CEMA was confirmed by 1H NMR by assessing the

disappearance of the vinyl peaks. The polymerisation was well-controlled with a linear


113

molecular weight evolution and a narrow polydispersity (Figure 3-1). The polymer used in

subsequent reactions had Mn,SEC = 40 000 g.mol-1 , PDI = 1.17.

0 10 20 30 40 50 60 700

10000

20000

1.0

1.5

2.0

Mn,SEC

Mn,theo(NMR)

Mn /

g.m

ol-1

Conversion / %

PD

I

Figure 3-1: Polymerisation of 2-chloroethyl methacrylate in 1,4-dioxane at 60 °C using CDB

as RAFT agent. [CEMA]= 2.5 M, [CEMA]:[RAFT]:[AIBN] = 300:1:0.5.

The reactivity of the pendant chloride functionality, on the polymer, was tested by attempting

the reaction with PTA (Scheme 3-1, Route B). Poly(2-chloroethyl methacrylate) and PTA were

dissolved in DMAc and the mixture was monitored via NMR. Only a nominal conversion

(shown by the shift in the 31P chemical signal) was achieved. Issues with solubility and solvents

were also encountered during purification attempts. To address the low reactivity and to

prevent any possible side reactions several steps were implemented. Initially, the RAFT end-

groups were cleaved from the polymer using the Perrier technique234 to eliminate the

possibility of crosslinking through these groups. In addition, to increase the reactivity of the

polymer towards nucleophilic substitution with PTA, the chloride functionalities were

converted to iodide groups using the Finkelstein reaction with NaI.67 This substitution could be

monitored using 1H NMR since the chloroethyl peak and iodoethyl peak had different chemical

shifts (Figure 3-2). Poly(2-iodoethyl methacrylate) (PIEMA) was then reacted with PTA. The


114

reaction was monitored and confirmed by 31P NMR, confirming the increased reactivity of

PIEMA compared to PCEMA. However, a detailed analysis of the resulting compound was

impossible, due to a combination of factors, such as the complex NMR spectra which was

difficult to analyse due to very broad peaks combined with the loss of splitting patterns.

Figure 3-2: 1H NMR spectra of PCEMA (bottom) and PIEMA (top) showing the shift in the

chloroalkyl peak at 3.73 to the iodoalkyl peak at 3.33. The peaks were monitored until

> 98 % conversion was achieved.

3.2.2. Model Reactions Using n-Butyl Iodide

A thorough investigation of the reaction of the polymer with either RAPTA-C (Route A) or PTA

(Route B) was still speculative, since broadening of NMR signals prevented a confident peak

assignment. Subsequently, a model compound was developed that could assist in assessing

which route was more efficient. The reaction with n-butyl iodide instead of PIEMA was

identified as a suitable substitute that could facilitate NMR analysis. The initial investigation on

the polymer used DMAc as the solvent with a DMSO-d6 capillary to allow for NMR analysis. The

resulting 1H NMR spectra had a large array of broad signals, inherent in polymers, overlapped

with large solvent peaks, making interpretation extremely difficult. Further investigations,

including the model compound and the polymer, employed DMSO-d6 as the solvent,

eliminating the large DMAc peaks. No distinct differences in chemical shifts were found

between the DMAc and DMSO-d6 experiments and thus it is suggested that there is no

significant solvent interaction even though DMSO is regarded as a good complexing ligand.


115

Two routes were investigated. Route A involved the addition of n-butyl iodide to RAPTA-C in

degassed DMSO-d6. Route B involved the reaction of n-butyl iodide and PTA, and the

subsequent addition of the RuCl2(p-cymene) dimer, in degassed DMSO-d6.

3.2.3. Route A: Synthesis of Dichlororuthenium(II)

(p-cymene)(1,3,5-triaza-7-phosphaadamantane)

(RAPTA-C) and Reaction with Butyl Iodide

RAPTA-C was synthesised, following a literature procedure (Scheme 3-2) and characterised by

1H NMR and x-ray crystallography. Crystals for x-ray analysis were grown from a DCM/Heptane

layered solution in the glove-box. They diffracted strongly and spots were sharp, indicating

good quality single crystals. Crystals gave the orthorhombic crystal system with Primitive

Lattice Cell volume 4283 A3, where a = 13.198, b = 15. 472 and c = 20.973 Å. These unit cell

parameters were compared with literature values and found to be in good agreement with the

crystal data.19,64 It was found that the choice of solvent for this reaction is very important.

Reaction in chloroform led to two products while methanol resulted in a single product at high

yield (Figure 3-3).

Scheme 3-2: Route A: Synthesis of dichlororuthenium(II)(p-cymene)(1,3,5-triaza-7-

phosphaadamantane) (RAPTA-C) and the subsequent reaction with n-butyl iodide.


116

Figure 3-3: 31

P NMR spectra showing the attempted synthesis of RAPTA-C in MeOH and

CDCl3. The reaction in CDCl3 produced two products that were not identified.

The hydrolysis of the complex, where the two chloride ligands are replaced by one hydroxide

and one water ligand, was observed by mass spectrometry and UV-Vis and shown to be

consistent with literature.83 Mass spectrometry measurements in dry methanol and a

methanol/water (50:50) solution gave m/z values of 465 (Appendix, Figure A-4 top) and 428

(Appendix, Figure A-4 bottom) for the non-hydrolysed and hydrolysed complexes, respectively.

The isotopic pattern was indicative of ruthenium. UV-Vis absorption maxima at λ = 346 nm (A =

0.407 nm) in DMSO and λ = 326 nm (A = 0.434) in water were obtained. The complex in water

was in fact the diaqua-complex with the chloride ligands replaced by water ligands. Addition of

water to the complex solution in DMSO led to the complete hydrolysis of the chloride ligands,

observable by the shift in the absorption maximum, in less than one hour.

The reaction between PTA and the RuCl2(p-cymene) dimer was monitored via 31P NMR, in

DMSO-d6 (Figure 3-4), which showed that the initial PTA chemical shift at -103 ppm migrated

to -33 ppm. Minor product peaks, attributed to the hydrolysis products, were observed at -31

and -29 ppm. Residual RuCl2(p-cymene) dimer was still present in the product, but this

compound could not be separated and removed due to identical solubilities.


117


P NMR spectra. A: RAPTA-C, B: IBu-RAPTA-C, and C: Polymer-

RAPTA-C in DMSO-d6 at 25 °C. All spectra are representative of Route A.

The reaction between n-butyl iodide and RAPTA-C was followed using 31P NMR, in DMSO-d6.

Figure 3-4 shows that the initial 31P RAPTA-C peak at -33 ppm shifted to -20 ppm after

12 hours. The solution was then analysed using 1H NMR. Importantly, a peak attributable to

the protons attached to the carbon previously iodated, and now attached to nitrogen, was

identified. This peak was shown to have an interesting splitting pattern. 2D NMR was also used

to analyse the resulting compound. As expected, the [1H-31P] HMBC (Figure 3-4) shows

correlations between both the PTA protons and arene protons and the phosphorous peaks of

RAPTA-C and the butylated RAPTA-C product. Two types of protons are distinguishable in the

RAPTA-C complex. After alkylation, multiple types are displayed in the 1H spectra (Figure 3-5

top axis) but only four types show cross-peaks with 31P. This allowed for the determination of

the chemical shifts for each of the protons in the PTA cage. Most interesting is that the

p-cymene ligand is correlated to phosphorous through the ruthenium centre, shown by the

diagonal splitting pattern of the p-cymene cross-peaks in Figure 3-5.

Conversion of RAPTA-C to IBu-RAPTA-C using equimolar amounts in DMSO at ambient

temperatures reached 60 % after 12 hours. Extended reaction times were not possible since

the p-cymene ligand dissociated from the complex when the solution was monitored over

longer time periods. The two products obtained were the targeted product and unreacted

RAPTA-C next to a small amount of hydrolysed product.


118

Figure 3-5: [1H-

31P] HMBC Spectrum of RAPTA-C and butylated RAPTA-C in DMSO-d6 at

25 °C. Both the 1H and

31P spectra are external projections. A shift in the cross-peaks

assigned to the PTA and p-cymene RAPTA-C complex ligands to the corresponding cross-

peaks for the butylated RAPTA-C complex ligands is evident. Alkylation of the PTA ligand

causes a distinct difference in the number of types of protons present in the PTA structure

i.e. two types before and four types after.

3.2.4. Route B: Reaction of PTA with n-Butyl Iodide and

Subsequent Reaction with the RuCl2(p-cymene) Dimer

The alternative route employs a two-step procedure, which is outlined in Scheme 3-3. The

reaction between n-butyl iodide and PTA was analysed by 31P NMR. Figure 3-6 shows that the

initial PTA peak at -103 ppm shifted to -84 ppm, consistent with 31P literature values for PTA

alkylation.53 The reaction went to completion after only one hour and all the PTA was

consumed (Figure 3-6). The 1H NMR spectrum of the final solution (Figure 3-7 top axis) shows

the same peak, as identified post-reaction in route A, attributable to the protons attached to

the carbon previously iodated, and now attached to nitrogen. Consistent with the reaction

product from route A, this peak was shown to have an interesting splitting pattern. A

systematic homo-decoupling study combined with a [1H-1H] COSY experiment elucidated that

this was second-order 1H-1H coupling. A small impurity present in the n-butyl iodide was also

shown to be unaffected by the reaction with PTA. Furthermore, the [1H-13C] HMBC, shown in

Figure 3-7, allowed for the complete identification of the intermediary product of route B. A


119

small amount of unreacted n-butyl iodide was also identified, which may be due to

experimental error when trying to weigh such a small amount into an NMR tube. The

[1H-31P] HMBC for this intermediary solution allowed for the identification of all protons in the

PTA cage (Figure 3-8). Both 1H and 31P coupling is evident. The coupling patterns are indicative

of the attachment of the butyl chain to a nitrogen group in PTA. The integrations confirm that

only mono-alkylation occurs. Lastly, the compound was analysed via MS, confirming the

predicted molecular weight (Figure 3-9).

Scheme 3-3: Route B Synthesis. Addition of n-butyl iodide to PTA and the subsequent

complexation to give butylated RAPTA-C. The corresponding 1H structural assignment is

shown in Figure 3-7 and Figure 3-10 top axes.


120


P NMR spectra. A: PTA, B: RAPTA-C, C: IBu-PTA, D: IBu-RAPTA-C,

E: Polymer-PTA and F: Polymer-RAPTA-C, in DMSO-d6 at 25 °C. All spectra are

representative of the Route B synthesis.


121

Figure 3-7: [1H-

13C] HMBC NMR Spectrum of n-butyl iodide + PTA in DMSO-d6 at 25 °C. Both

the 1H and

13C spectra are external projections. A [

1H-

13C] HSQC was used in the

identification of all cross-peaks associated with the identified structures. Structural

identification of compounds corresponds to Scheme 3-3. A more detailed identification of

the compounds is shown in Appendix Figure A-2.

The RuCl2(p-cymene) dimer was subsequently added to the intermediary solution and the

reaction analysed by 31P NMR. The 31P peak at -84 ppm shifted to -20 ppm (Figure 3-6). 100 %

conversion was achieved after one hour (Figure 3-13). The compound was analysed via MS,

showing an expected molecular weight change (Appendix Figure A-4) and a characteristic

splitting pattern due to the seven isotopes of ruthenium.57

Repeated experiments found that RAPTA-C, identified at -33 ppm (Figure 3-4), was also

produced as a product, if residual PTA remained after the initial reaction. This is interesting

since the formation of RAPTA-C was previously thwarted by a change in solvent (Figure 3-3).

Furthermore, the RAPTA-C complexation required increased energy (reflux conditions) to

proceed. Thus, it is suspected that DMSO-d6 may act as a catalyst for this reaction. The final

product of route B and the intermediary product, butylated PTA, were identified in the

[1H-13C] HMBC (Figure 3-10).


122

Figure 3-8: [1H-

31P] HMBC NMR Spectrum of butylated PTA in DMSO-d6 at 25 °C.

1H and

31P

spectra are external projections.

Figure 3-9: Initial PTA at 180.9 (bottom) shifted to Alkylated IBu-PTA at 214.1 (top).


123

Figure 3-10: [1H-

13C] HMBC NMR Spectrum of butylated PTA + RuCl2(p-cymene) Dimer in

DMSO-d6 at 25 °C. 1H and

13C spectra are external projections. Structural identification of

compounds corresponds to Scheme 3-3. Note that unidentified correlations correspond to

starting materials that were previously identified in Figure 3-7. A more detailed

identification of the compounds is shown in Appendix Figure A-3.

In summary, the reaction between n-butyl iodide and PTA consumed all of the PTA in the

system, after only one hour. The subsequent reaction with RuCl2(p-cymene) dimer was almost

instantaneous and complete in less than one hour with 100 % of PTA now acting as a ligand for

ruthenium.

Further evidence for the success of the Route B synthesis was shown with a [1H-15N] NMR

spectrum (Figure 3-11). Distinct 15N chemical shifts were found for each step in the synthesis.

No cross-peaks for protons associated with the positively charged nitrogen group were found.

It is suspected that this is due to issues with peak detection of charged nuclei.


124

Figure 3-11: [1H-

15N] HMBC of RAPTA-C, butylated PTA and butylated RAPTA-C in DMSO-d6

at 25 °C. Three distinctive 15

N chemical shifts were detected. No cross-peaks for protons

associated with positively charged 15

N were found, due to problems associated with peak

detection of charged nuclei.

The lessons learned from the model reaction provide a robust set of tools for the analysis of

the reaction between the polymer and RAPTA-C via routes A and B. The following conclusions

can be drawn from the preliminary study:

PTA is alkylated at a nitrogen group, while the phosphorous group is not involved.

PTA only reacts with one nitrogen so will not act as a crosslinker by involving two

nitrogens.

All reactions proceed at ambient temperature, which is possibly due to the catalytic

activity of DMSO.

The reaction between RAPTA-C and n-butyl iodide (Route A) is slow and incomplete

(~60 % after 12 hours). An extended reaction time can lead to the labilisation of the

p-cymene ligand.


125

Alkylation of all the PTA in the system (Route B) was complete in less than one hour.

Formation of the complex as a result of the reaction between PTA and the

RuCl2(p-cymene) dimer (Route B) reached 100 % conversion in less than one hour.

Formation of the complex as a result of the reaction between PTA and

RuCl2(p-cymene) dimer is complete in less than one hour and the reaction with

RuCl2(p-cymene) dimer reached 100 % conversion in less than one hour. Both

pathways give the same product and can possibly be used interchangeably, albeit

route A is slower and prone to p-cymene ligand loss.

3.2.5. Synthesis of Macromolecular Ruthenium Complex:

Polymer-RAPTA-C

3.2.5.1. Route A: PIEMA + RAPTA-C

The reaction between PIEMA and RAPTA-C was followed by 31P NMR. In contrast to the model

compound the reaction did not proceed, shown by the unchanging RAPTA-C 31P peak. The

solution was subsequently heated to 80 °C and the RAPTA-C peak shifted to -18 ppm (Figure

3-4), consistent with that found for the model compound reaction. According to 31P NMR, 75 %

conversion was achieved after 12 hours. At first glance, the reaction using the polymer led to

similar results, but higher temperatures were required to achieve the same outcome.

However, further analysis of the 1H spectra revealed that the p-cymene ligand dissociated from

the complex during the synthesis (Figure 3-12, Spectrum C). This is possibly due to the heat

applied to the reaction in order for it to proceed. The loss of p-cymene was already observed

in the model reaction at ambient temperature after an extended time, but now that elevated

temperature is required to accelerate the reaction the loss of the ligand is even more

pronounced.


126

Figure 3-12: Stack-plot of 1H spectra, showing the arene region. A: RuCl2(p-cymene) Dimer,

B: RAPTA-C, C: Polymer-RAPTA-C synthesised via Route A, D: Polymer-RAPTA-C synthesised

via Route B. The comparison shows that p-cymene dissociates from the complex during

reaction. The broad p-cymene peak produced via Route B provides evidence for the

polymer-RAPTA-C product.

3.2.5.2. Route B: PIEMA + PTA + RuCl2(p-cymene) Dimer

The reaction between PIEMA and PTA was analysed by 31P NMR at ambient temperature.

Figure 3-6 shows that the initial PTA peak at -103 ppm shifted to -84 ppm, consistent with that

found for the model compound reaction. 55 % conversion was achieved after two days (Figure

3-13). The Polymer-PTA peak was broad, due to the inherent rigidity of polymers. To confirm

this reasoning, the sample was heated to 50 °C since if it were not a polymer then heating

would cause the peak to sharpen. The peak remained broad even at the elevated temperature.


reaction analysed by 31P NMR. The peak at -84 ppm shifted to -18 ppm, also consistent with

the model compound reaction (Figure 3-6). 100 % conversion was achieved after one hour


127

(Figure 3-13). Also consistent with the model compound reaction, it was found that residual

unreacted PTA converted to RAPTA-C on addition of the RuCl2(p-cymene) dimer. Nucleophilic

substitution of the alkyliodide with PTA was found to be much slower in the polymer

compared with the model compound (Figure 3-13).

0 10 20 30 40 50 60 70 80 900

20

40

60

80

100 IBu + PTA

Polymer + PTA

Copolymer + PTA

Convers

ion

/ %

Time / hrs

Figure 3-13: The 31

P peak in PTA was monitored over time for each reaction. All 31

P peaks in

a given spectra were integrated and normalised to sum to 100. The fraction corresponding

to each product represents the conversion of one species to another. Since the

complexation for all experiments (model compound, polymer and copolymer) reached

100 % conversion in less than one hour, no kinetic data was obtained and is not

represented on this figure.

This may be due to the inaccessibility of the iodide groups, or shielding effect of the polymer,

due to the large polymer backbone. Attaching PTA groups to the polymer also introduces

positive charges along the polymer, making it less susceptible to the introduction of further

PTA groups, due to the repulsive force between like-charged species. Repulsion of these

charges leads to an entropically unfavourable chain stretching. Therefore, the number of

pendant PTA groups along the polymer chain is limited. 31P NMR evidence was heavily relied

on for the determination of polymer products, since polymer 1H NMR spectra are difficult to


128

interpret due to broad overlapping peaks and loss of indicative splitting patterns. However, the

chemical shifts of the broad polymer peaks were consistent with the chemical shifts in the

model compound. Distinctive regions could also be drawn upon for more information. In

contrast to Route A, the broad p-cymene peak produced via Route B provides evidence for the

polymer-RAPTA-C product. Incorporation within the polymer reduces the mobility of the

species, causing peak broadening (Figure 3-12, Spectrum D).

In summary, route B was clearly identified as the preferred pathway. In contrast to the

complete reaction between n-butyl iodide and PTA, the reaction between PTA and the

polymer only reaches a maximum of approximately 50 %.

3.2.6. Statistical Copolymerisation of N-(2-Hydroxypropyl)

Methacrylamide and 2-Chloroethyl Methacrylate

(P(HPMA-CEMA)) via RAFT Polymerisation

In contrast to the advantageous water solubility of the RAPTA-C drug, a disadvantage of the

final macromolecular drug is the absence of water solubility. As a macromolecular drug, the

product is expected to dissolve in aqueous solution, which can be addressed by preparing a

copolymer based on PIEMA and PHPMA, which is a FDA approved water-soluble polymer.

The successful copolymerisation of CEMA and HPMA was confirmed by 1H NMR and SEC

analysis. A 9:1 HPMA:CEMA feed ratio produced a 4:1 output ratio, and thus it is reasonable to

suggest that a gradient statistical copolymer is produced. Reactivity ratios in literature of MMA

and HPMA indeed suggest a preference for the HPMA consumption.237 The incorporation of

HPMA in the polymer increased water solubility but decreased the polymer’s susceptibility to

RAFT-group cleavage and the Finkelstein reaction, possibly due to the hydrophilic nature of

HPMA. Thus, the reaction times for these steps were doubled.


129

Scheme 3-4: Synthesis of Macromolecular Ruthenium Complex Copolymer -RAPTA-C in

DMSO-d6 at 25 °C.

Only Route B was employed for further modification. The reaction between P(HPMA-IEMA)

and PTA was analysed by 31P NMR. Consistent with the homopolymer and model compound,

the initial PTA peak at -103 ppm shifted to -84 ppm (Figure 3-14). 53 % conversion was

achieved after four days (Figure 3-13). It is interesting to note that the conversion of the PTA

conjugation did not increase. A possible reason might be the gradient structure of the polymer,

meaning that IEMA groups are concentrated at one end of the polymer and steric hindrance is

still a factor.


reaction analysed by 31P NMR (Figure 3-14). The 31P peak at -84 ppm shifted to -18 ppm, also

consistent with the model compound and homopolymer reactions (Figure 3-6). The product

peak was elucidated after one hour and 100 % conversion of attached PTA to RAPTA-C

complex was achieved. This was calculated by integrating all of the 31P peaks in the final

solution mixture and normalising them to add to 100 (Figure 3-14). The peak corresponding to

the copolymer represented 54 % of the total peak integration before and after complexation.


130

Also consistent with the homopolymer and model compound reactions, it was found that

residual unreacted PTA produced RAPTA-C on addition of the RuCl2(p-cymene) dimer.

Complete conversion of PTA to RAPTA-C is also shown in Figure 3-14.


P NMR spectra showing the synthesis steps of

Copolymer-RAPTA-C. Solution A: Copolymer + PTA. Solution B: Copolymer + PTA +

RuCl2(p-cymene) Dimer.

The solutions were purified by dialysis against water, to remove residual PTA (Solution A) and

RAPTA-C (Solution B). Both the Copolymer-PTA and Copolymer-RAPTA-C were water soluble,

yielding clear and orange solutions respectively. The UV-Vis absorption maximum at λ =

326 nm was assigned to the RAPTA-C complex and could now be used to test the attachment

of RAPTA-C to the polymer. Further analysis of the final product was carried out using SEC with

a UV-Vis detector, which was set to λ = 326 nm, the absorption maximum of the hydrolysed

RAPTA-C complex. The polymer prior to the reaction absorbs slightly at this wavelength and is

therefore visible using the UV-Vis detector. SEC confirmed that the RAPTA-C complex was

attached to the polymer, and that no residual unbound complex remained in the system

(Figure 3-15). The absence of intensity in the lower molecular weight range (higher retention

time) shows that all free RAPTA-C was successfully removed during dialysis. In addition, the

area under the curve of RAPTA-C alone was compared to the area of the polymer taking into

account the UV-Vis intensity of the polymer prior to the reaction with RAPTA-C. The resulting


131

intensity of the polymer confirms the NMR results that approximately 50 % of all available

iodide groups have reacted. SEC provided the last piece of the puzzle by showing that after the

successful RAPTA-C conjugation, the resulting polymer has a monomodal distribution and

crosslinking products are absent. In conclusion, we have obtained a polymer with

hydroxypropyl methacrylate units, iodoethyl methacrylate units and RAPTA-C ethyl

methacrylate units, of the following structure: P(HPMA172-IEMA44-(RAPTA-C-EMA)44).

20 30 40 50

0.01

0.02

0.03

0.04

0.36

0.37

0.38

0.39

0.40 P(HPMA-IEMA)

P(HPMA-IEMA)-RAPTA-C

RAPTA-C

Norm

alis

ed U

V R

esponse (

326 n

m)

RT / mins

Figure 3-15: SEC traces for the RAPTA-C complex, Copolymer and Copolymer-RAPTA-C, in

N,N-dimethylacetamide at λ = 326 nm. The UV traces were normalised using the RI traces

of the same solution.


A cytotoxicity profile for the Copolymer-RAPTA-C product was compared with that of the free

RAPTA-C drug (Figure 3-16). The IC50 value of RAPTA-C was similar to the values cited in

literature.91 The polymer bound RAPTA-C, although active, had a lower toxicity when


132

correlated to the RAPTA-C concentration. This is not unexpected considering that the polymer

diffuses slowly across the cell membrane and the hydrodynamic diameter is too small to

activate endocytosis.238 Further work in Chapters 4 and 5 targets the improved cellular uptake

to enhance toxicity. It is hypothesised that the cytotoxicity of Copolymer-RAPTA-C can be

much improved through the conjugation of a cell uptake transporter, for example a protein or

peptide, or by incorporating the polymer into a nano-sized structure that is known to undergo

fast and efficient endocytosis.

1 10 100 10000

20

40

60

80

100

120

Copolymer RAPTA-C

RAPTA-C

Ce

ll V

iab

ility

/ %

RAPTA-C Concentration / M

Figure 3-16: Cytotoxicity profile of Copolymer-RAPTA-C and RAPTA-C, after 72 hours, n = 3.

3.3. CONCLUSIONS

Nucleophilic substitution of halides with amines was identified as an appropriate mechanism

for the conjugation of RAPTA-C to a suitably designed polymeric scaffold (synthesized from 2-

chloroethyl methacrylate), since RAPTA-C contains a PTA ligand that is susceptible to

alkylation. A representative model compound was first synthesised and characterised using

NMR analyses. The information gained from this study provided a set of tools that could then

be used to assess the successful attachment of the complex to a chosen polymer and

copolymer. Two routes to attachment were investigated. The first route involved the synthesis


133

of the complex and subsequent attachment to an iodated compound. The second route

involved the attachment of the PTA ligand and subsequent complexation to form RAPTA-C. The

second route was found to be more favourable since the first caused the labilisation of the

arene ligand. It was found that the PTA alkylation (i.e. attachment to the polymer) was the

limiting step. The subsequent complexation went to completion in less than one hour. This

two-step synthetic pathway provides a facile route to attach RAPTA drugs to polymers.

Following from this work, Chapters 4 and 5 focus on improving cellular uptake and thus the

toxicity of this macromolecular chemotherapeutic.

134

135

4 POLYMERIC MICELLES

To exploit the therapeutic benefits that can be harnessed from RAPTA-C, the drug should be

protected from degradation before reaching the target, and passively delivered to cancer cells.

Chapter 3 showed that RAPTA-C can be successfully attached to polymers, creating

macromolecular ruthenium drugs, without losing their activity.214 However, a linear polymer

chain is often not readily taken up by cancer cells, giving IC50 values that are less favourable

than those of the free drug25 – accounting for the reduced activity of the macromolecule

synthesised in Chapter 3.

The creation of a drug carrier in the nano-size range enables fast endocytosis. Micelles present

a unique size that leads to increased circulation times,27 and the potential to exploit the EPR

effect,28 which are important properties for therapeutic moieties. Covalent attachment of a

therapeutic agent is a useful avenue to delay drug release until the micelle reaches a target

site.24 Micelles, wherein a drug is covalently bound have been investigated, for example PEG-b-

poly(Ɛ-caprolactone) with chemically conjugated docetaxel,123 and a number of poly(ethylene

oxide) based polymers containing cisplatin.212,216–221 Water soluble metalla-cages have been

investigated as carriers for ruthenium drugs, and shown to greatly improve the cytotoxicity of

the free drug - thought to be due to the EPR effect.54

The advantages of nano-sized drug carriers are evident and they have been successfully used

with an array of drugs. The drug is often physically encapsulated or – when conjugated to the

polymer chain – can be released again upon a trigger or by simple hydrolysis. A range of

polymer-drug conjugates are available that rely on the concept of having a cleavable linker

between polymer and drug.151 However, the polymer-RAPTA-C conjugate reported in Chapter

3 is unlikely to degrade into its components and is therefore termed a macromolecular drug.

Macromolecular drugs need to be sufficiently agile to reach their intracellular target, but this

Chapter 4: Polymeric Micelles

136

prerequisite cannot be fulfilled by a nano-sized carrier that is, for example, unlikely to enter

the nucleus. It is therefore essential to design a carrier that can take on a nano-sized micellar

structure, but is also able to degrade into smaller polymer chains over time to increase the

activity of the macromolecular drug.

Degradable polymers based on lactic acid have been used in numerous medical applications,

including: sutures, implantable medical devices, dental applications, scaffolds and stents;

because they degrade in the body by simple hydrolysis of the ester backbone to safe and non-

toxic compounds, which are excreted by the kidneys or eradicated as carbon dioxide and water

via well-documented biochemical pathways.120 Polylactide (PLA) was thus chosen as a suitable

hydrophobic polymer since it is already used in treatments for prostate cancer due to its

biodegradability.4

Reversible Addition Fragmentation Chain Transfer Polymerisation (RAFT),167,168,239,240 in

combination with ring-opening polymerisation,241 provide mechanisms to create diblock

copolymers that can self-assemble into stable micelles,190 and possess PLA as a degradable

block to ensure the release of the macromolecular drug (Scheme 4-1).

In this chapter, 2-chloroethyl methacrylate (CEMA) as the reactive building block for RAPTA-C

conjugation was copolymerised with 2-hydroxyethyl acrylate (HEA) due to its hydrophilic

nature, such that it would form the shell of the micelle and the ruthenium drug could be

distributed within the shell. The central question of this chapter is whether the therapeutic

benefits of RAPTA-C macromolecules can be further enhanced by incorporating them into

degradable micelles, thus reducing the effective dose.


137

Scheme 4-1: Synthesis of amphiphilic polymers, end-group modification of chloride to

iodide (Finkelstein reaction), two-step conjugation of RAPTA-C and subsequent

micellisation.

Micelles

SnOct:!

__ X14_:_·Xc_a_. Ha\fa~J~~~A(~ 0 0

D,L·Iactide )=o =i=o DMAc 60'C

- ~ so Y.to-\d0~s)ls

OH 0 x

1. Nal, Acetone, 70 'C

2. DMSO, 25 'C

<~;"p ~__J

PTA

Water

9 CI CI2 ' I I I Ru---R~'CI I .0

Cl

RuCI2(p-cymene) dlmer

0 + 0

( ( OH Cl


138

4.1. SYNTHESES

4.1.1. Synthesis of Dichlororuthenium(II)(p-cymene)(1,3,5-

triaza-7-phosphaadamantane) (RAPTA-C)

See Chapter 3 for the synthesis procedure of RAPTA-C.

4.1.2. Synthesis of Polylactide (PLA) MacroRAFT Agent

Polylactide (PLA) was synthesised following a modified literature procedure.241 Lactide (15 g,

1.0 x 10-1 mol) was combined with RAFT Agent 3 (0.3 g, 1.1 x 10-3 mol) and stirred under

vacuum at 120 ᵒC for six hours. The flask was purged with argon and then, dried and degassed

SnOct2 (58 mg, 1.4 x 10-4 mol) in dry toluene (1 mL), was added to the yellow mixture. The

temperature was increased to 140 ᵒC for two hours. The mixture became a golden liquid which

solidified to a glassy mass upon cooling. DCM was added to the cooled solid and left to dissolve

overnight. The DCM solution was then added dropwise to an excess of chilled methanol, while

stirring. The polymer was redissolved in methanol and re-precipitated before being dried

under vacuum to give a glassy yellow solid (10 g, 67 %). 1H NMR (300.17 MHz, DMSO-d6, 25 °C):

δ (ppm) = 5.17 (m, 1H, Ha), 1.45 (m, 3H, Hb). 13C NMR (300.17 MHz, DMSO-d6, 25 °C): δ (ppm) =

168 (CO); 72 (Cb); 15 (Ca). Total reaction time = 2 hrs, Mn,theo = 14 000 g.mol-1, Mn,UV-Vis =

26 000 g.mol-1, Mn,SEC = 15 000 g.mol-1, PDI = 1.39.

4.1.3. Synthesis of 2-Chloroethyl Methacrylate

See Chapter 3 for the synthesis procedure of this monomer.

4.1.4. Polymerisation of 2-Hydroxyethyl Acrylate (HEA) with

PLA MacroRAFT

Hydroxyethyl acrylate (HEA) (1.2 g, 1.0 x 10-2 mol), PLA MacroRAFT (0.5 g, 2.5 x 10-5 mol) and

AIBN (0.8 mg, 5.0 x 10-6 mol) as initiator, were combined in DMAc (8.0 mL) to give

[HEA]:[MacroRAFT]:[AIBN] = 400:1:0.2. The solution was sealed with a rubber septum,


139

deoxygenated by purging with argon for one hour, and placed in an oil bath at 60 °C. Aliquots

were removed with a degassed needle at progressive time intervals. The polymer was

precipitated into cold diethyl ether, dissolved in DCM/MeOH and re-precipitated, before being

dried under vacuum to give polylactide-b-(poly(hydroxyethyl acrylate) (PLA-b-PHEA) as a pale

yellow solid (Table 4-1).

Table 4-1: Polymerisation of HEA with PLA MacroRAFT Agent in N,N-dimethylacetamide at

60 ᵒC. [HEA]:[MacroRAFT]:[AIBN] = 400:1:0.2

Time (hrs) XHEA (%) PLA:PHEA (NMR) Mn,SEC (g.mol-1

)

1 22 1:0.4 20 500

4 65 1:1.1 30 100

7 79 1:1.3 29 800

X = monomer conversion

4.1.5. Polymerisation of HEA and CEMA with PLA MacroRAFT

Hydroxyethyl acrylate (HEA) (0.6 g, 5.3 x 10-3 mol), chloroethyl methacrylate (CEMA) (89 mg,

6.0 x 10-4 mol), fluorescein O-methacrylate (F) (24 mg, 6.0 x 10-5 mol), PLA MacroRAFT (0.3 g,

1.5 x 10-5 mol) and AIBN (0.5 mg, 3.0 x 10-6 mol) as initiator, were combined in DMAc (5.0 mL)

to give [HEA]:[CEMA]:[F]:[MacroRAFT]:[AIBN] = 356:40:4:1:0.2. The solution was sealed with a

rubber septum, deoxygenated by purging with argon for one hour, and placed in an oil bath at

60 °C. Aliquots were removed with a degassed needle at progressive time intervals. The

polymer was precipitated into cold diethyl ether, dissolved in DCM and re-precipitated, before

being dried under vacuum to give polylactide-b-(poly(hydroxyethyl acrylate-co-chloroethyl

methacrylate-co-fluorescein O-methacrylate)) (PLA-b-P(HEA-CEMA-F)) as pale yellow solids.

1H NMR (300.17 MHz, DMSO-d6, 25 °C): δ (ppm) = 5.18 (PLA, 1H); 4.74 (HEA, OH); 4.23 (CEMA,

2H); 4.01 (HEA, 2H); 3.80 (CEMA, 2H); 3.54 (HEA, 2H); 2.26 (HEA, 1H); 2-1.58 (HEA, 2H & CEMA,

2H); 1.45 (PLA, 3H); 1.00 (CEMA, 1H) (Table 4-2).


140

Table 4-2: Polymerisation of HEA and CEMA with PLA MacroRAFT Agent in

N,N-dimethylacetamide at 60 ᵒC. [HEA]:[CEMA]:[F]:[MacroRAFT]:[AIBN] = 455:50:4:1:0.2.

Polymer Time

(hrs)

XHEA

(%)

XCEMA

(%)

PLAxx-P(HEAyy-CEMAzz)

(NMR)

Mn,SEC

(g.mol-1

) PDI

A 3 64 37 PLA347-HEA291-CEMA19 28 000 1.34

B 5 99 99 PLA347-HEA450-CEMA50 30 500 1.38

X = monomer conversion

4.1.6. Conjugation of RAPTA-C to (PLA-b-P(HEA-CEMA-F))

Copolymer

Following the procedure detailed in Chapter 3, RAPTA-C was attached via a three-step

procedure (Scheme 4-1).

4.1.6.1. Finkelstein

Polymers were transferred to a 50 mL or 100 mL round-bottomed flask, fitted with a reflux

condenser, and dissolved in dry acetone (A: 3 mL, B: 5 mL) with vigorous stirring and heat

(60 ᵒC). Sodium iodide (A: 59 mg, B: 75 mg) was subsequently added and the solution turned

cloudy. The solution was refluxed at 70 ᵒC for 3 to 5 days, at which time the solution was

cloudy and yellow. It was cooled and precipitated in diethyl ether and dried under vacuum to

give a pale yellow solid: polylactide-b-(poly(hydroxyethyl acrylate-co-iodoethyl methacrylate-

co-fluorescein O-methacrylate)) (PLA-b-P(HEA-IEMA-F)). 1H NMR (300.17 MHz, DMSO, 25 °C):

δ (ppm) = 5.18 (PLA, 1H); 4.74 (HEA, OH); 4.23 (IEMA, 2H); 4.01 (HEA, 2H); 3.40 (IEMA, 2H);

3.54 (HEA, 2H); 2.26 (HEA, 1H); 2-1.58 (HEA, 2H & IEMA, 2H); 1.45 (PLA, 3H); 1.00 (IEMA, 1H).

4.1.6.2. Attachment of PTA to Copolymer

The polymer and PTA (8.0 mg, 5.1 x 10-5 mol) were combined in an NMR tube, sealed with a

rubber septum, evacuated and filled with argon. Degassed DMSO-d6 (1 mL) was transferred to

the NMR tube using a degassed gas-tight needle. The clear solution was analysed via 1H and 31P

NMR until conversion from PTA to PTA-Polymer was stable.


141

4.1.6.3. RAPTA-C Complexation

The ruthenium dimer (13 mg, 2.0 x 10-5

mol) was subsequently dissolved in the DMSO solution

and the orange solution re-analysed via 1H and 31P NMR.

4.1.7. Micellisation of Drug-loaded Copolymers

Typically, 0.5 mL of the DMSO solution, where [polymer] = 10 mg.mL-1, was diluted with 1.5 mL

DMF. 3.5 mL MilliQ water was then added using a syringe pump, at a rate of 0.2 mL.hr-1. The

deep red-orange solution was subsequently dialysed against MilliQ water (MWCO =

3500 g.mol-1) to give a pale orange solution, which was diluted to 10 mL. Samples were

analysed using DLS, TEM, ICPOES and TGA and used for subsequent cell studies.

4.1.8. In Vitro Cell Culture Assays

Dr. Hongxu Lu kindly completed the following cell culture assays. Human ovarian carcinoma

A2780, A2780cis and OVCAR-3 cells, and human pancreatic carcinoma AsPC-1 cells were

cultured in 75 cm2 tissue culture flasks with RPMI 1640 medium supplemented with 10 % fetal

bovine serum, 4 mM glutamine, 100 U/mL penicillin, 100 µg.mL-1 streptomycin, 1 mM sodium

pyruvate at 37 °C under an atmosphere of 5 % CO2. After reaching 70 % confluence, the cells

were washed with phosphate buffered saline (PBS) and collected by trypsin/EDTA treatment.

The cell suspension was used for the evaluation of cellular responses.

4.1.8.1. Cytotoxicity Evaluation

A2780, A2780cis and OVCAR-3 cells were used for cytotoxicity analyses. The cells were seeded

in 96-well cell culture plates at 4 000 cells per well and cultured at 37 °C for one day. Solutions

were sterilised by UV irradiation for one hour in a biosafety cabinet and then serially diluted

(2 × dilution) with sterile water and incubated for two hours. The medium in the cell culture

plate was discarded and 100 μL fresh 2 × concentrated RPMI 1640 serum medium was added.

The samples were added into the plate at 100 μL per well for 72 hours. The cell viability was

measured using a WST-1 assay (Roche Diagnostics). This is a colorimetric assay for the

quantification of cell viability and proliferation that is based on the cleavage of a tetrazolium

salt (WST-1) by mitochondrial dehydrogenases in viable cells. Increased enzyme activity leads


142

to an increase in the amount of formazan dye, which is measured with a microplate reader.

After incubation for three days, the culture medium was removed and 100 µL fresh medium

was added along with 10 µL WST-1. The plates were then incubated for an additional four

hours at 37 °C. After incubation, the absorbance of the samples against the background control

on a Benchmark Microplate Reader (Bio-Rad) was obtained at a wavelength of 440 nm with a

reference wavelength of 650 nm. Four wells under each condition were used for the

measurement to calculate the means and standard deviations.

4.1.8.2. Colony Formation Assay

Unlike the cell proliferation assay, the colony formation assay measures the productive

integrity of the cells following withdrawal of drug treatment. The assay was performed as

described by Franken et al242 with some modifications. 40 000 cells were seeded to each well

of a 24-well tissue culture plate and incubated overnight. Samples of micelles A, micelles B and

RAPTA-C were added to the cells such that the Ru concentration of each well was 25 μM. After

a four hour incubation, the cells were washed thrice with PBS and collected by trypsin

treatment. Single cell suspension was then plated in six well plates with fresh PRMI 1640

medium (400 cells per well), and the medium was changed every three days. After seven days

of incubation, the cells were washed thrice with PBS and incubated with a staining solution for

one hour. The staining solution is an aqueous solution with 0.5 % crystal violet and 2.5 %

glutaraldehyde. The plates were washed five times with water and air-dried. The colony over

50 cells was counted using Image J. The data were calculated based on Equation 4-1 and

Equation 4-2.

Equation 4-1: ( )

Equation 4-2: ( )

4.1.8.3. Laser Scanning Confocal Microscopy

A2780 cells were seeded in 35 mm Fluorodish (World Precision Instruments) at a density of

60 000 per dish and cultured for three days with RPMI 1640 medium supplemented with 10 %

fetal bovine serum. Micelle solution was loaded to the cells at a working concentration of

100 µg.mL-1 polymer and incubated at 37 °C for designed periods for three hours. After

incubation, the cells were washed thrice with PBS, stained with 2.0 µg.mL-1 Hoechst 33342


143

(Invitrogen) for five minutes and washed thrice with PBS. Finally, the cells were stained with

100 nM LysoTracker Red DND-99 (Invitrogen) for one minute. The dye solution was quickly

removed and the cells were gently rinsed with PBS. The dishes were mounted in PBS and

observed under a laser scanning confocal microscope.

4.1.8.4. Cellular Uptake of Ruthenium

A2780 cells were seeded in six well plates and incubated at 37 °C with 5 % CO2 for one day

prior to micelle treatment. During treatment, the medium was replaced with incubation media

containing either micelles A, micelles B, or RAPTA-C in RPMI-1640 at 37 °C for six hours. The

cell monolayer was washed thrice with PBS and treated with trypsin/EDTA to detach the cells.

The cells were pelleted by centrifugation for 10 minutes. The pellet was rinsed with MilliQ

water, frozen at -80 °C and lyophilised. The ruthenium content in the cell pellets was

determined using TGA.


4.2.1. Synthesis of PLA MacroRAFT Agent

A polylactide macroRAFT agent (PLA) was polymerised via ring-opening polymerisation (ROP)

using a reactive RAFT agent as initiator, following a modified literature procedure241 (Scheme

4-2). The structure was confirmed by 1H NMR (Figure 4-1) and the molecular weight of PLA was

determined using the signals of the RAFT functionality compared to the PLA signals. The

random distribution of L- and D-units along the polymer backbone impedes the orientation of

the chains into crystalline domains, thus yielding an amorphous glassy polymer.120 The length

of the polymer was controlled by the concentration of the RAFT Agent.

A number of issues were encountered during this synthesis procedure, and thus it was

repeated until a satisfactory product was obtained for further work. Undetectable traces of

impurities, for example water, can disrupt the reaction.120 The ROP is initiated via the hydroxyl

group of the RAFT agent, using tin(II) 2-ethyl-hexanoate (SnOct2) as a catalyst since it has FDA

approval as a food additive.120 The catalyst is necessary to increase the molecular weight of the

polymer.243 If there is any water present in the reaction vessel then it will also initiate

polymerisation, producing PLA without the RAFT end-group. To dry the lactide monomer and

RAFT agent, the temperature of the oil bath was maintained at 120 ᵒC. However, during this


144

process, some monomer crystallised in the upper parts of the flask and a heat-gun was used to

melt it back into the bulk molten mixture. Thus, it is possible that a small proportion may have

polymerised during the drying step via initiation with water. Subsequently, the catalyst was

added and the temperature increased to 140 ᵒC, in order to initiate the ROP.

Scheme 4-2: Synthesis of polylactide macroRAFT agent via ring-opening polymerisation, in

bulk at 240 ᵒC.

Figure 4-1: Stack-plot of 1H NMR spectra. A: lactide and B: D,L-polylactide in DMSO-d6 at

25 °C. The peak assignment corresponds to Scheme 4-2.

UV-Vis was used to confirm the RAFT agent end-group concentration in the polymer and

calculate the molecular weight (see 4.1.2). The concentration of trithiocarbonate groups in the


145

macroRAFT agent was calculated using the absorption maximum from the RAFT agent at

427 nm (ϵ = 53 L.mol.cm-1) in chloroform. The molecular weight of the macroRAFT agent was

derived from Equation 4-3.

Equation 4-3: ( )

where msample is the weight of polymer sample (in g), ϵ427 nm is the molar absorptivity at

λ = 427 nm (in L mol cm-1), d is the length of the pathway (in cm), V is the volume (in L) of the

sample, and A is the measured absorbance.

The theoretical molecular weight was calculated using the molar ratio between lactide and

RAFT agent and a maximum conversion of 100 %. The theoretical value was close to the value

given by SEC, but half that calculated from the UV absorbance. The discrepancy between the

theoretical and UV experimental value is indicative of a less than 100 % efficiency of initiation

of the hydroxyl initiator,241 producing longer than expected polymer chains with the terminal

RAFT agent end functionality. Any free (unreacted) RAFT agent was removed during

purification.

4.2.2. Amphiphilic Polymers

PLA was first chain extended with HEA as a proof of concept for chain extension and also to

test the micellisation of the system. The successful chain extension indicated by a monomodal

distribution and clear shift in molecular weight (Figure 4-2) further confirms that the ROP was

successful. However, some low molecular weight tailing in the SEC trace could be due to free

polylactide produced during the drying step. TEM images (Figure 4-3) proved the formation of

micelles, and the size was consistent with DLS measurements (Figure 4-4).


146

10000 100000 1000000

PLA MacroRAFT

PLA-PHEA

log (MW / g·mol-1)

Conversion

Figure 4-2: SEC traces of the polymerisation of HEA with PLA MacroRAFT Agent in

N,N-dimethylacetamide at 50 ᵒC. [HEA]:[MacroRAFT]:[AIBN] = 400:1:0.2.

Figure 4-3: PLA-b-PHEA micelle sample was drop-loaded onto grid, air-dried and stained

with Phosphotungstic acid. Scale bar: a = 200 nm; b = 100 nm.


147

1 10 100 1000 10000

Diameter / nm

Polymer A

Polymer B

PLA-PHEA

Figure 4-4: DLS number average particle size distributions of PLA-b-PHEA, polymer A and

polymer B micelles analysed by DLS in water at 25 ᵒC, where ηPLA = 1.482.

Subsequently, PLA was chain extended with a mixture of HEA, CEMA and 1 % fluorescein

O-methacrylate (Figure 4-5). Incorporation of the fluorescing monomer was deemed necessary

to monitor the cell uptake. The consumption of both monomers was monitored over time

using 1H NMR analyses (Figure 4-6, Table 4-2). Since the methacrylate was incorporated into

the polymer faster than the acrylate, the hydrophilic block has a slight gradient structure with

CEMA slightly enriched at the periphery of the resulting micelle. The molecular weight of the

polymer increased linearly with monomer conversion. The molecular weight distribution (PDI)

remained narrow, although slight broadening due to low molecular weight tailing can be

observed at higher conversions (Table 4-1, Figure 4-5).


148

10000 100000 1000000

PLA MacroRAFT

PLA-P(HEA-CEMA-F)

log (MW / g·mol-1)

Conversion

Figure 4-5: SEC traces of the polymerisation of HEA and CEMA with PLA MacroRAFT Agent

in N,N-dimethylacetamide at 50 ᵒC. [HEA]:[CEMA]:[F]:[MacroRAFT]:[AIBN] = 356:40:4:1:0.2.

Two polymers, with differing hydrophilic (HEA-CEMA) block lengths, were isolated for further

modification (labelled Polymer A and B in Table 4-2). Prior to modification, the polymers were

dissolved in DMSO/DMF (1:2) and then water was added slowly and the mixture dialysed

against water to remove DMSO and DMF. The dialysed solutions were analysed using DLS

(Figure 4-4) and TEM (Figure 4-7 and Figure 4-8) to determine micelle size and homogeneity.

The DLS trace indicates that the micelle size is not uniform, consistent with TEM images that

show the presence of smaller micelles.


149

Figure 4-6: 1H NMR spectra of the polymerisation of HEA and CEMA with PLA MacroRAFT

Agent in DMSO-d6 at 25 ᵒC. [HEA]:[CEMA]:[F]:[MacroRAFT]:[AIBN] = 455:50:5:1:0.2. A: 1 hr,

B: 3 hrs, C: 5 hrs.


150

Figure 4-7: Polymer A before RAPTA-C conjugation. (a) to (c) Sample drop-loaded onto grid

and air-dried. (d) to (f) Sample drop-loaded onto grid, air-dried and stained with

Phosphotungstic acid. Scale bar: a, b, d, e = 200 nm; c & f = 100 nm.

Figure 4-8: Polymer B before RAPTA-C conjugation. (a) & (b) Sample drop-loaded onto grid

and air-dried. (c) to (f) Sample drop-loaded onto grid, air-dried and stained with

Phosphotungstic acid. Scale bar: a = 500 nm; b = 2 µm; d, e = 100 nm; f = 50 nm.


151

4.2.3. RAPTA-C Conjugation

The chloride end-groups of the CEMA monomer were converted to iodide groups using the

Finkelstein reaction.235 The 1H NMR signal corresponding to the CH2 group alpha to Cl at

3.80 ppm shifted upfield to 3.40 ppm when the chloride was substituted with iodide. This

signal could be monitored and integrated relative to the adjoining CH2 signal at 4.23 ppm,

which did not move (Figure 4-9). The reaction was stopped when complete halogen

substitution had been achieved.

RAPTA-C was then attached to the polymer using the method detailed in Chapter 3. PTA was

initially bound via a substitution reaction of iodide with nitrogen. Ruthenium dimer was

subsequently added, resulting in the complexation formation of RAPTA-C. The reaction was

followed using 1H (Figure 4-9) and 31P NMR (Figure 4-10 and Figure 4-11). The 31P chemical shift

of the initial PTA peak at -103 ppm shifted to -84 ppm (Figure 4-10 and Figure 4-11, bottom

spectra) as the alkylation of PTA induces a deshielding of the 31P signal.99 The subsequent

complexation induces a very large deshielding of the corresponding signal, shown by the peak

shift to -18 ppm (Figure 4-10 and Figure 4-11, top spectra). These values were consistent with

the conjugation to the homopolymer in Chapter 3. As found previously, the Polymer-PTA and

Polymer-RAPTA-C peaks were broad, due to the inherent rigidity of polymers. Furthermore,

the residual unreacted PTA underwent a complexation reaction producing RAPTA-C on

addition of the RuCl2(p-cymene) dimer, shown by the 31P chemical shift at -103 ppm moving to

-33 ppm.

A highly complex 1H NMR spectra with multiple spin systems results from the N-alkylation of

PTA due to the decrease in molecular symmetry.53 Due to the combined characteristic

broadening from polymer entities, the 1H spectra are difficult and tedious to interpret.

However, the p-cymene region for the reactions provided further evidence for the conjugation

reaction as the peaks for the residual unreacted RuCl2(p-cymene) dimer, RAPTA-C and the

broad peak for the conjugated RAPTA-C are evident (Figure 4-12). Also consistent with Chapter

3, the first step was found to be the rate-limiting step in the conjugation. Peak ‘e’ in Figure 4-9

no longer appears at 4.23 ppm after the addition of PTA, indicating the complete conversion of

all iodide units to PTA. All conjugated and unconjugated PTA was subsequently consumed

during the RAPTA-C complexation, giving both free RAPTA-C and conjugated RAPTA-C.


152

Figure 4-9: 1H Spectra showing the attachment of RAPTA-C to PLA-b-P(HEA-IEMA-F) in

DMSO-d6 at 25 ᵒC. A: Polymer before Finkelstein, B: Polymer after Finkelstein, C: PTA

added to polymer in solution, D: Dimer added to solution.


153

Figure 4-10: 31

P Spectra showing the attachment of RAPTA-C to PLA-b-P(HEA-IEMA-F)

polymer A via a two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to

polymer in solution, Top: Dimer added to solution. Desired products are circled.

Figure 4-11: 31

P Spectra showing the attachment of RAPTA-C to PLA-b-P(HEA-IEMA-F)

polymer B via a two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to

polymer, Top: Dimer added to solution. Desired products are circled.


154

Figure 4-12: The p-cymene region of the 1H spectra showing the attachment of RAPTA-C to

PLA-b-P(HEA-IEMA-F), in DMSO-d6 at 25 ᵒC. The broad polymer, residual unreacted

RuCl2(p-cymene) dimer and RAPTA-C p-cymene peaks are at 5.95 ppm, 5.86 ppm and

5.75 ppm, respectively. Bottom: Polymer A, Top: Polymer B.

Both polymers, A and B, led to similar results. The resulting structures had a polymer

composition of PLA347-b-P(HEA291-(RAPTA-C-EMA)19) (Polymer A) and

PLA347-b-P(HEA450-(RAPTA-C-EMA)50) (Polymer B).

4.2.4. Micellisation of Drug-Loaded Amphiphilic Polymers

After drug conjugation, the DMSO solutions were diluted with DMF and then water was added

slowly and the mixture dialysed to remove DMSO, DMF and unreacted RAPTA-C. The dialysed

pale orange solutions were analysed using DLS (Figure 4-4 and Table 4-3) and TEM (Figure 4-14

and Figure 4-15) to determine micelle size and homogeneity. The high PDI in the DLS trace

indicates that the micelle size is not uniform, consistent with TEM images that show the

presence of smaller micelles. The size distribution found by DLS (Figure 4-13) was larger than

the images obtained by TEM, since micelles swell in solution and contract when dried.


155

However, the large differences between diameters obtained from TEM and DLS cannot be

explained by hydration alone and may be indicative of substantial aggregation in solution

(Table 4-3).

1 10 100 1000 10000

Diameter / nm

Micelles A

Micelles B

Micelles A (with RAPTA-C)

Micelles B (with RAPTA-C)

Figure 4-13: DLS number average particle size distributions of micelles A and B in water at

25 ᵒC, with and without conjugated RAPTA-C.

The tailing in the DLS size distribution can be attributed to aggregation of micelles, as shown in

Figure 4-14 (b) and Figure 4-15 (b). This aggregation was found to be more prominent in

micelle B samples, clearly indicated by the Z-average and PDI (Table 4-3). This may influence

the cytotoxic effect and cell uptake displayed by each of the micelle samples. Interestingly, the

average micelle size increases after RAPTA-C conjugation but the PDI decreases indicating

better and more homogenous micelle formation. TEM images clearly show the core-shell

structure of the micelles, for both stained and unstained samples. Since no core-shell

distinction was evident for PLA-b-PHEA micelle samples (Figure 4-3), this provides further

evidence that RAPTA-C is indeed conjugated and located in the shell of the micelle. The shell


156

appears darker than the core in the stained samples, due to the higher electron density of

ruthenium. However, the converse is evident for the unstained samples, where the core

appears darker.

Table 4-3: Micelles A and B analysed by DLS in water at 25 ᵒC, with and without conjugated

RAPTA-C.

Z-average

(nm)

from DLS PDI

Zeta

Potential

(mV)

Diameter (nm)

from TEM

Micelles A (without RAPTA-C) 160.7 0.439 100

Micelles B (without RAPTA-C) 235.5 0.601 150

Micelles A (with RAPTA-C) 251.8 0.332 20.8 ± 0.65 50

Micelles B (with RAPTA-C) 554.5 0.370 18.0 ± 0.45 70

Figure 4-14: Polymer A (a) to (c) Sample drop-loaded onto grid and air-dried. (d) to (f)

Sample drop-loaded onto grid, air-dried and stained with Phosphotungstic acid. Scale bar:

d = 1 µm, a = 500 nm, b & e = 100 nm, c & f = 50 nm.


157

Figure 4-15: Polymer B (a) to (d) Sample drop-loaded onto grid and air-dried. (e) to (h)

Sample drop-loaded onto grid, air-dried and stained with Phosphotungstic acid. Scale bar:

a = 1 µm, d & f = 200 nm, b & e = 100 nm, c & inset = 50 nm.

ICPOES and TGA were used to determine the final ruthenium content in each sample, and

found to be consistent (Table 4-4) within the same order of magnitude. TGA analyses were

conducted in two ways. Firstly, a sample of the micelle solution used for cell studies was added

to the TGA pan and heated until dry. It was subsequently analysed via TGA to determine the

ruthenium concentration in the sample. Secondly, a sample of the micelle solution was

lyophilised and then analysed via TGA to determine the % ruthenium content in the dry

sample. The TGA results were consistent and also consistent with ICPOES results. ICPOES

analyses were also conducted in two ways: with and without digestion using Aqua Regia. From

the TGA analysis of RAPTA-C, it was found that RuCl3 is the final residue since ruthenium in

RAPTA-C is only 21 %, while the weight residue of 44 % equates to RuCl3 (101.07 g.mol-1).


158

Table 4-4: Final ruthenium content in polymeric micelles determined using ICPOES and TGA

analyses.

ICPOES TGA

Sample [Ru] (mmol.L-1

)a [Ru] (mmol.L

-1)b [Ru] (mmol.L

-1)c Ru (%)

c Ru (%)

d

Polymer A 5.0 x 10-2

6.7 x 10-2

3.4 x 10-2

5.2 1.5

Polymer B 1.2 x 10-1

1.2 x 10-1

1.0 x 10-1

11 10

RAPTA-C - - - - 44

aICPOES without digestion.

bICPOES with Aqua Regia digestion.

a,bICPOES results in mg.L

-1 were converted to mmol.L

-1 using the molecular weight of Ru = 101.07 g.mol

-1.

cTGA solution analysis gave Ru weight in mg and was converted to mmol.L

-1 using the molecular weight of RuCl3 =

207.42 g.mol-1

.

dTGA solid analysis: Ru weight is % of total solid analysed.

4.2.5. In Vitro Cell Studies of Drug-Loaded Micelles

Micelles self-assembled from polymers A and B were tested against ovarian A2780, cisplatin-

resistant ovarian A2780cis and ovarian OVCAR-3 human cancer cell lines and compared with

the drug RAPTA-C. The toxicity of the polymer prior to drug conjugation was in addition

investigated and found to be non-toxic at concentrations up to 250 µg.mL-1 (Figure 4-16).

The micelles demonstrated a significantly lower (~10x) IC50 value than the free drug against all

cell lines (Table 4-5, Figure 4-17, Figure 4-18 and Figure 4-19). Micelles A displayed the highest

toxicity against all cell lines, with a marked difference between the two micelle samples on

A2780cis (Table 4-5).


159

100

101

102

0

20

40

60

80

100

120

140

A2780 Polymer A

A2780 Polymer B

A2780cis Polymer A

A2780cis Polymer B

Ovcar-3 Polymer A

Ovcar-3 Polymer B

Su

rviv

al R

ate

/ %

Ru Concentration / M

Figure 4-16: Cytotoxicity profile of micelles self -assembled from polymers A and B before

RAPTA-C conjugation, after 72 hours, n = 4.

Table 4-5: IC50 (µM) values of RAPTA-C and micelles self-assembled from polymers A and B,

against ovarian A2780, cisplatin-resistant ovarian A2780cis and ovarian OVCAR-3 cancer

cell lines.

IC50 (µM) A2780 A2780cis OVCAR-3

RAPTA-C Literature 353 ± 1494 >20054 -

RAPTA-C 271 266 300

Micelles A 15 24 46

Micelles B 51 101 61


160

10-1

100

101

102

103

0

20

40

60

80

100

120

RAPTA-C

Polymer A

Polymer B

A2

78

0 S

urv

iva

l R

ate

/ %


Figure 4-17: Cytotoxicity profile of RAPTA-C and micelles self-assembled from polymers A

and B, against ovarian carcinoma A2780 cells , after 72 hours, n = 4.

10-1

100

101

102

103

104

0

20

40

60

80

100

120

RAPTA-C

Polymer A

Polymer B

A2780cis

Surv

ival R

ate

/ %



and B, against cisplatin-resistant ovarian carcinoma A2780cis cells , after 72 hours, n = 4.


161

10-1

100

101

102

103

104

0

20

40

60

80

100

120

RAPTA-C

Polymer A

Polymer B

OV

CA

R-3

Surv

ival R

ate

/ %



and B, against ovarian carcinoma OVCAR-3 cells, after 72 hours, n = 4.

Micelles assembled from polymer A had the highest toxicity. This increased toxicity of micelles

A compared to micelles B cannot be immediately explained, since the toxicity is evaluated at

the same ruthenium concentration. Higher toxicities are often associated with higher cell

uptake of the drug delivery system, as it has recently been demonstrated with a drug delivery

system for cisplatin.9 The uptake of the drug carrier was therefore monitored using confocal

fluorescence microscopy. The micelles composed from polymers A and B were incubated with

A2780 for three hours. The cell nuclei (blue) and lysosomes (red) were subsequently stained to

help identify the location of the green fluorescent micelles (Figure 4-20). The micelles were

found to be co-localized with the lysosomes, indicative of an endocytic pathway. Upon initial

inspection the green fluorescence seems to be more intense inside the cells when employing

polymer A as the drug carrier. Since, the initial fluorescence of the micelle samples at the

concentrations used for confocal imaging is statistically similar (Table 4-6), it can be inferred

that micelles prepared from polymer A induce a better cell uptake.


162

Figure 4-20: Confocal microphotographs of A2780 cells after incubation with micelles at 37

°C for three hours. Polymers (green) were labelled with fluorescein. Cell nuclei (blue) were

stained with Hoechst 33342. Lysosomes (red) were stained with LysoTracker Red DND-99.

Scale bar = 5 µm.

Table 4-6: Fluorescence intensity of polymer and micelle samples at concentrations used

for microscopy imaging i.e. prior to cell uptake.

Fluorescence Intensity Micelle A Micelle B Polymer A Polymer B

Average 90.49 92.14 48.65 56.94

Standard Deviation (n = 3) 0.63 0.38 1.34 0.84

Although the confocal fluorescent studies give a first indication of a better uptake of micelles A,

a more quantitative approach is necessary. Toxicity can be related directly to the amount of

drug that enters the cells. The final amount of ruthenium internalised in A2780 cells was

analysed by ICPMS (Figure 4-21). Micelles had a significantly higher uptake than RAPTA-C. Also

consistent with the increased cytotoxicity, micelles prepared from polymer A had a

significantly higher uptake than micelles prepared from polymer B.


163

Micelle A Micelle B RAPTA-C0

5

10

15

20

* * *

* * *

[Ru

] /

mill

ion c

ells

/ n

g

* * *

Figure 4-21: Concentration of ruthenium per million A2780 cells measured by ICPMS, after

digestion in Aqua Regia. Mean ± SD, n = 3. *** significantly different, p < 0.001.

A colony formation assay was used to evaluate the effects of cell regrowth after exposing them

to ruthenium. Cells were incubated with a solution of 25 M RAPTA-C and the equivalent

amount of drug in the micelle for four hours. The cells were subsequently washed to remove

the micelles and the drug and plated in six well plates at a very low density for seven days to

allow colony formation (Figure 4-22). Fewer colonies were formed by both cell lines after

treatment with micelles A at a Ru concentration of 25 μM. However, micelles B and RAPTA-C

didn’t show the same effect. The colony formation assay was also assessed via the surviving

fraction, which is the ratio of colonies formed after treatment and the number of cells seeded

that can become colonies. The surviving fraction in Table 4-7 reveal that 25 μM Ru from

micelles A inhibit the colony formation. There is no obvious inhibition effect of micelles B and

RAPTA-C (Ru at 25 μM) on the colony formation ability. These results, thus, suggest micelle A

treatment is highly effective in suppressing the colony-forming ability of A2780 and A2780cis

cells.


164

Figure 4-22: Colony formation of A2780 (A) and A2780cis (B) cells. Scale bar = 10 mm.

Table 4-7: Surviving fraction of cancer cells after four hour exposure to Micelles and

RAPTA-C.

Ru (µM) A2780 A2780cis OVCAR-3 AsPC-1

Micelle A 25 0.36 0.33 0.88 1.28

Micelle B 25 1.04 0.91 1.15 1.23

RAPTA-C 25 1.08 0.90 1.31 1.21

It is evident that both micelles enhance the performance of RAPTA-C due to better cell uptake.

Micelles A, prepared from PLA347-P(HEA291-CEMA19), enhance this effect significantly compared

to micelles B, prepared from (PLA347-P(HEA450-CEMA50). The key is the better uptake of micelles

A compared to micelles B. Micelles B are slightly larger, but this size increase is not

pronounced enough to explain the difference. However, a remarkable difference between

both micelles is the strong tendency of micelles B to aggregate. This is potentially due to the

longer hydrophilic water-soluble blocks which result in more star-like micelles, possibly

promoting entanglements between micelles. It is also possible that the higher loading of


165

RAPTA-C per polymer chain, leads to a reduced solubility, thus enhancing aggregation. The

strong aggregation leads to the appearance of larger particles, which are known to have a

reduced cellular uptake.

4.3. CONCLUSIONS

An amphiphilic block copolymer, polylactide-b-poly(hydroxyethyl acrylate-co-chloroethyl

methacrylate), capable of self-assembling into polymeric micelles was identified as an

appropriate drug carrier for RAPTA-C. Following from Chapter 3, it was known that RAPTA-C

could be attached to a polymer moiety via nucleophilic substitution of an available halogen

with an amide in the PTA ligand. A series of water-soluble biodegradable block copolymers

were designed incorporating the CEMA monomer to allow for conjugation of RAPTA-C in the

shell of the micelle. Two of these polymers were used to test the RAPTA-C conjugation,

micellisation and subsequent cytotoxicity and cell uptake of these self-assembled structures.

Confocal microscopy images confirmed cell uptake of the micelles into the lysosome of the

cells, indicative of an endocytic pathway. On average, across the tested cell lines, a remarkable

10-fold increase in toxicity was found for the macromolecular drugs when compared to the

RAPTA-C molecule. Furthermore, the cell uptake of ruthenium was analysed and a significant

increase was found for the micelles compared to RAPTA-C. Notably, micelles prepared from

the polymer containing fewer HEA units had the highest cytotoxicity, the best cell uptake of

ruthenium and were highly effective in suppressing the colony-forming ability of cells. This may

due to better and increased homogeneity of micelle formation, and decreased aggregation of

these micelles. Chapter 6 entails a detailed assessment of the metastatic effects of the

macromolecular RAPTA-C chemotherapeutics.

166

167

5 PEPTIDE TUBES

Chapter 3 proved that RAPTA-C can be successfully conjugated to a polymer, creating a

macromolecular chemotherapeutic. However, since linear chains are often not readily taken

up by cancer cells,25 it is important to transport these drugs into the cell using an appropriate

sized carrier that will subsequently disassemble to release the small polymer-drug conjugates.

Nanotubes (NTs) are therefore an ideal candidate for drug delivery applications – they are in

the nano-size range and disassemble upon disruption of the hydrogen-bonding holding them

together.

Hollow nanotubular structures are present in many natural and artificial systems.244,245

Cyclopeptides containing 6-12 amino acids of alternating D and L chirality have been shown to

assemble into tubes through anti-parallel hydrogen bonding.246 They are stable against

proteases due to their cyclic structure and the incorporation of di-amino acid residues, but

disassemble under specific (acidic) conditions (for example, in TFA). The extended hydrogen

bonding allows for the formation of rigid tube-like structures, which typically extend over

100 nm. This property cannot be achieved using polymers alone, since they do not have the

required rigidity. Rod-like structures present an additional advantage since they can span a

large area on cells and provide multivalent drug display and/or release.247

Polymeric chains can be grafted to these peptide NT’s preventing aggregation and improving

solubility.207,208 Grafting polymeric chains also gives a degree of length control248,249 as well as

functionality from the polymer.250,251 RAPTA-C can be incorporated into these polymer arms

and since disruption of the hydrogen bonding results in the disassembly of the nanotubes, the

RAPTA-C macromolecules should be released upon entering the cell. Therefore, NT’s provide

an interesting ‘natural’ scaffold in the nano-size range that may allow the transport of RAPTA-C

to target sites.

Chapter 5: Peptide Tubes

168

Reversible Addition Fragmentation Chain Transfer Polymerisation (RAFT),167,168,239,240 in

combination with copper catalysed azide-alkyne cycloaddition (CuAAC),252 provide mechanisms

to create cyclopeptide-polymer conjugates that can self-assemble into NT’s. Poly(2-

hydroxyethyl acrylate) (PHEA) can improve the solubility of such conjugates, and the resulting

nanotubes, in polar solvents.249 Moreover, as shown in Chapter 4, HEA can be copolymerised

with 2-chloroethyl methacrylate (CEMA) providing a conjugation avenue for RAPTA-C.214

Following from Chapter 3, it was known that RAPTA-C could be attached to this copolymer via

nucleophilic substitution of the available halogen with an amide in the PTA ligand.

Furthermore, polymers synthesised with an alkyne RAFT agent can be conjugated to

cyclopeptides via the amide group using CuAAc.

In this chapter, the synthesis of a novel nano-scale drug carrier containing the ruthenium

therapeutic RAPTA-C was investigated. This involved the synthesis of a two-arm cyclopeptide,

the conjugation of a copolymer, consisting of HEA and CEMA, and the subsequent conjugation

of RAPTA-C (Scheme 5-1). The cytotoxicity of these entities was evaluated to determine their

potential as chemotherapeutic agents.


169

Scheme 5-1: Synthesis of cyclopeptide-polymer conjugates and attachment of RAPTA-C.

DMF TFE CuS04

Na-ascorbate 100 •c

1. Nal, Acetone, 70 •c 2. PTA, DMSO, 25 •c 3. RuCI2(p-cymene) dimer


170

5.1. SYNTHESES

5.1.1. Synthesis of Cyclopeptides

Dr. Robert Chapman kindly synthesised the boc-protected two-arm cyclopeptides at Sydney

University.207 Linear peptide H2N-L-Trp(Boc)-D-Leu-L-Lys(N3)-D-Leu-L-Trp(Boc)-D-Leu-LLys(N3)-

D-Leu-OH (1.7 g, 1.2 mmol) was dissolved in DMF (1.0 L) under a N2 atmosphere, and cooled to

0 ᵒC. HBTU (1.5 equiv.), HOBt (1.5 equiv.) and DIPEA (3 equiv.) were dissolved in DMF (20 mL)

and added dropwise to the mixture. The mixture was stirred at room temperature and

monitored by TLC. After 72 hours, a further 1.5 equiv. of HBTU and HOBt, with DIPEA

(3 equiv.), were added. After a total of 84 hours, the solution was concentrated to near

dryness and triturated from MeOH to yield a white precipitate. The precipitate was washed

with MeOH five times to yield the Boc protected cyclopeptide (0.8 g, 46 %). Boc groups were

removed by dissolving the protected peptide in TFA/triisopropanol/thioanisole/water

(85:5:5:5), and stirring for two hours. Concentration of the mixture, followed by trituration

with MeOH gave the desired product. Linear Peptide: 1H-NMR (300 MHz, d-TFA, 25 °C):

δ (ppm) = 4.53-4.96 (m, 7H, 7 α- CH); 4.24-4.40 (m, 1H, 1 α-CH); 3.35 (br, 8H, 4 N3-CH2); 1.35-

2.20 (m, 36H, 12 Lys CH2 & 4 Leu CH & 4 Leu CH2); 0.95 (br, 24H, 8 Leu CH3). m/z (ESI)

1087.8 ([M+H]+, calc = 1087.7). Cyclopeptide: 1H NMR (300 MHz, d-TFA, 25 °C): δ (ppm) = 4.60-

4.97 (m, 8H, α-CH); 3.35 (m, 8H, N3-CH2); 1.34-2.21 (m, 36H, Lys CH2 & Leu CH); 0.97 (br s,

24H, Leu CH3). m/z (APCI) = 1069.53 ([M+H]+, calc = 1069.69). IR (ATR, ZnSe) cm-1: 3277, 2950-

2850, 2098, 1687, 1624, 1541 (br).

5.1.2. Copolymerisation of HEA and CEMA via RAFT

Polymerisation

2-hydroxyethyl acrylate (HEA) (1.8 g, 1.5 x 10-2 mol), 2-chloroethyl methacrylate (CEMA) (0.3 g,

1.7 x 10-3 mol), RAFT agent 4 (40 mg, 1.7 x 10-4 mol) and AIBN (2.8 mg, 1.7 x 10-6 mol) as

initiator, were combined in DMAc (5.0 mL) to give [HEA]:[CEMA]:[RAFT]:[AIBN] = 90:10:1:0.1.

The solution was sealed with a rubber septum, deoxygenated by purging with argon for

30 minutes, and placed in an oil bath at 60 °C. Samples were taken over time with a degassed

needle for analysis via SEC and NMR. After 16 hours, the solution was precipitated into cold

diethyl ether, dissolved in MeOH and re-precipitated, before being dried under vacuum to give

poly(hydroxyethyl acrylate-co-chloroethyl methacrylate) P(HEA-CEMA) as a yellow rubbery


171

solid (0.6 g, 98 %). A second copolymerisation using the same protocol, but with

[HEA]:[CEMA]:[RAFT]:[AIBN] = 80:20:1:0.1, gave poly(hydroxyethyl acrylate-co-chloroethyl

methacrylate) P(HEA-CEMA) as a yellow rubbery solid (0.6 g, 98 %). 1H NMR (300.17 MHz,

DMSO, 25 °C): δ (ppm) = 4.74 (HEA, OH); 4.23 (CEMA, 2H); 4.01 (HEA, 2H); 3.80 (CEMA, 2H);

3.54 (HEA, 2H); 2.26 (HEA, 1H); 2-1.58 (HEA, 2H & CEMA, 2H); 1.00 (CEMA, 1H).

5.1.3. Copolymerisation of HEA and CEMA via RAFT

Polymerisation using an Alkyne RAFT Agent

2-hydroxyethyl acrylate (HEA) (0.7 g, 6.1 x 10-3 mol), chloroethyl methacrylate (CEMA) (0.1 g,

6.7 x 10-4 mol), RAFT agent 5 (19 mg, 6.7 x 10-5 mol) and AIBN (1.0 mg, 6.7 x 10-6 mol) as

initiator, were combined in DMAc (2.0 mL) to give [HEA]:[CEMA]:[RAFT]:[AIBN] = 90:10:1:0.1.

The solution was sealed with a rubber septum, deoxygenated by purging with argon for one

hour, and placed in an oil bath at 60 °C. After three hours the solution was precipitated into

cold diethyl ether, dissolved in MeOH and re-precipitated, before being dried under vacuum to

give poly(hydroxyethyl acrylate-co-chloroethyl methacrylate) P(HEA-CEMA) as a yellow

rubbery solid (0.4 g, 50 %). 1H NMR (300.17 MHz, DMSO, 25 °C): δ (ppm) = 4.74 (HEA, OH); 4.23

(CEMA, 2H); 4.01 (HEA, 2H); 3.80 (CEMA, 2H); 3.54 (HEA, 2H); 2.26 (HEA, 1H); 2-1.58 (HEA, 2H

& CEMA, 2H); 1.00 (CEMA, 1H).

5.1.4. Conjugation of Polymers to Peptides

Dr. Robert Chapman kindly assisted with the following procedure at Sydney University. P(HEA-

CEMA) (0.2 g, 3.1 x 10-5 mol), copper sulphate pentahydrate (14 mg, 5.8 x 10-5 mol) and DMF

(3.0 ml) were combined. Cyclopeptide (Fmoc-Lys(N3)-OH) (16 mg, 1.5 x 10-5 mol), sodium

ascorbate (10 mg, 5.8 x 10-5 mol) and TFE (1.0 mL) were combined in a separate vial and

sonicated for 10 minutes. Both solutions were then combined to give a green solution and

placed in a microwave reactor at 100 ᵒC for 15 minutes. DMF was removed from the resulting

brown solution by vacuum, the product dissolved in MeOH and a small amount of precipitate

removed by centrifugation. The product was purified by prep-SEC using a Sephadex LH60

column and analysed by SEC and IR. Residual copper was subsequently removed by the

addition of cuprisorb resin in a DMF/DMSO solution of the product for four weeks. IR (ATR,

ZnSe) cm-1: 3276, 2092, 1728, 1687, 1628, 1541 (br).


172

5.1.5. Conjugation of RAPTA-C to Cyclopeptide-Polymer

Conjugates

Consistent with the procedure detailed in Chapter 3, RAPTA-C was attached via the following

three-step procedure:

5.1.5.1. Finkelstein reaction

Conjugates were transferred to a 50 mL round-bottomed flask, fitted with a reflux condenser.

Dry acetone was added with a small amount of dry DMF to solubilise the conjugate and the

solution vigorously stirred at 70 ᵒC. Sodium iodide was subsequently added and the solution

turned cloudy. The solution was refluxed at 70 ᵒC for three to five days, at which time the

solution was cloudy and yellow. It was cooled and precipitated in diethyl ether and dried under

vacuum to give a pale yellow solid: cyclopeptide-(poly(hydroxyethyl acrylate-co-iodoethyl

methacrylate))2 (CP-P(HEA-IEMA)2). 1H NMR (300.17 MHz, DMSO, 25 °C): δ (ppm) = 4.74 (HEA,

OH); 4.23 (IEMA, 2H); 4.01 (HEA, 2H); 3.40 (IEMA, 2H); 3.54 (HEA, 2H); 2.26 (HEA, 1H); 2-1.58

(HEA, 2H & IEMA, 2H); 1.00 (IEMA, 1H).

5.1.5.2. Attachment of PTA to Copolymer

The Conjugate and PTA were combined in an NMR tube, sealed with a rubber septum,

evacuated and filled with argon. Degassed DMSO-d6 was transferred to the NMR tube using a

degassed gas-tight needle. The clear solution was analysed via NMR until conversion from PTA

to PTA-Polymer was stable. 31P NMR (600.13 MHz, DMSO, 25 °C): δ (ppm) = -84 (conjugated

PTA); -103 (unconjugated PTA).

5.1.5.3. RAPTA-C Complexation

The ruthenium dimer was subsequently dissolved in the DMSO conjugate solution and the

orange solution re-analysed via 31P NMR. 31P NMR (600.13 MHz, DMSO, 25 °C): δ (ppm)

= -18 (conjugated RAPTA-C); -33 (unconjugated RAPTA-C).


173

Table 5-1: End-group modification and conjugation of RAPTA-C to cyclopeptide-polymer

conjugates.

Polymer Code Finkelstein RAPTA-C Attachment

Acetone NaI PTA Ruthenium Dimer DMSO-d6

Conjugate A 10 mL 0.1 g 10 mg

6.4 x 10-5

mol

15 mg

2.5 x 10-5

mol 1.0 mL

Conjugate B 2.0 mL 20 mg 0.5 mg

2.0 x 10-4

mol

1.0 mg

1.6 x 10-3

mol 0.5 mL

5.1.6. Self-Assembly of Tubes

Typically, 0.5 mL of the cyclopeptide-polymer conjugate (~ 1 mg.mL-1) in DMSO was diluted

with 1.5 mL MilliQ water using a syringe pump, at a rate of 0.15 mL hr-1. The deep red-orange

solution was subsequently dialysed against MilliQ water (MWCO = 3500 g.mol-1) to give a

cloudy solution. The solution was sonicated for one hour and vortex stirred before analysis by

DLS and ICPOES. It was then used for the cytotoxicity evaluation.

5.1.7. Crosslinked Nanotubes

Cyclopeptide-polymer conjugates were crosslinked following a modified literature

procedure.248 1.8 mL crosslinking stock solution (2.0 µL dibutylin dilaurate, 15 µL tolylene

diisocyanate, 4.0 mL anhydrous DMSO) was added dropwise to 0.2 mL of the DMSO conjugate

solutions A and B with vigorous stirring. The mixture was stirred for 24 hours over molecular

sieves (4 Å). The brown cloudy solution was subsequently dialysed against MilliQ water to give

white-grey cloudy solutions which were analysed via TEM.


Dr. Hongxu Lu kindly completed the following assay. A2780, A2780cis and OVCAR-3 cells were

used for cytotoxicity analyses. The cells were seeded in 96-well cell culture plates at 4 000 cells

per well and cultured at 37 °C for one day. Solutions were sterilised by UV irradiation for one


174

hour in a biosafety cabinet and then serially diluted (2 × dilution) with sterile water and

incubated for two hours. The medium in the cell culture plate was discarded and 100 μL fresh

2 × concentrated RPMI 1640 serum medium added. The samples were added into the plate at

100 μL per well for 72 hours. The cell viability was measured using a WST-1 assay (Roche

Diagnostics). This is a colorimetric assay for the quantification of cell viability and proliferation

that is based on the cleavage of a tetrazolium salt (WST-1) by mitochondrial dehydrogenases in

viable cells. Increased enzyme activity leads to an increase in the amount of formazan dye,

which is measured with a microplate reader. After incubation for three days, the culture

medium was removed and 100 µL fresh medium was added along with 10 µL WST-1. The

plates were then incubated for an additional four hours at 37 °C. After incubation, the

absorbance of the samples against the background control on a Benchmark Microplate Reader

(Bio-Rad) was obtained at a wavelength of 440 nm with a reference wavelength of 650 nm.

Four wells under each condition were used to calculate the means and standard deviations.


5.2.1. Copolymer Synthesis

Poly(2-hydroxyethyl acrylate) (PHEA) has previously been shown to improve the solubility of

cyclopeptide conjugates, and the resulting nanotubes, in polar solvents such as water.249 HEA

has also previously been copolymerised with 2-chloroethyl methacrylate (CEMA).215

Copolymerisation with CEMA provides a conjugation avenue for the ruthenium anticancer

therapeutic RAPTA-C [Rutheniumdichloride(p-cymene)PTA], through a substitution reaction of

the halogen with the PTA (1,3,5-triazaphosphaadamantane) ligand.214 Thus, the synthesis of

the copolymer and conjugation to a cyclopeptide provides an avenue to a water-soluble nano-

scale drug carrier. HEA and CEMA were first polymerised using the model RAFT Agent 4 to

determine the kinetics of the polymerisation system and the consumption of HEA and CEMA

over time. Two systems with feed ratios of 90:10 and 80:20 (HEA:CEMA) were investigated.

The consumption of both monomers was monitored over time using 1H NMR analyses. Since

the methacrylate was incorporated into the polymer faster than the acrylate, the polymer has

a slight gradient structure (Figure 5-1). The molecular weight of the polymer increased linearly

with monomer conversion (Figure 5-2) and the molecular weight distribution (PDI) remained

narrow (Figure 5-2, Figure 5-3, Figure 5-4).


175

0 20 40 60 80 1000

20

40

60

80

100

Feed HEA:CEMA 90:10

Feed HEA:CEMA 80:20

HE

A / %

Conversion / %

0 2 4 6 8 10 12 14 16

Time / hrs


N,N-dimethylacetamide at 60 °C using RAFT agent 4.

0.0 0.2 0.4 0.6 0.8 1.00

5000

10000

1.0

1.2

1.4

1.6

Polymer A

Mn(SEC)

Mn(theo)

Polymer B

Mn(SEC)

Mn(theo)

Mn /

g.m

ol-1

Conversion / %

PD

I


N,N-dimethylacetamide at 60 °C using RAFT agent 4. HEA:CEMA feed ratios

are A = 90:10, B = 80:20.


176

100 1000 10000 100000

P(HEA-CEMA)

log (MW / g·mol-1)

Conversion


N,N-dimethylacetamide at 60 °C using 4 as RAFT agent. [Monomer] = 2.4 M,

[HEA]:[CEMA]:[RAFT]:[AIBN] = 90:10:1:0.1.

100 1000 10000 100000

P(HEA-CEMA)

log (MW / g·mol-1)

Conversion


N,N-dimethylacetamide at 60 °C using RAFT agent 4. [Monomer] = 2.4 M,

[HEA]:[CEMA]:[RAFT]:[AIBN] = 80:20:1:0.1.


177

Subsequently, HEA and CEMA were copolymerised using RAFT agent 5 (Figure 5-5), containing

an alkyne R-group, in order to obtain a statistical copolymer with an alkyne terminal

functionality for further attachment to the cyclopeptide via the azide-alkyne click reaction. The

polymerisation proceeded as expected, similarly to the polymerisation with RAFT agent 4, to

give polymers with narrow molecular weight distributions (Figure 5-6 and Figure 5-7).

Figure 5-5: 1H NMR spectra of the polymerisation of HEA and CEMA with RAFT Agent 5 in

DMSO-d6 at 25 ᵒC. [HEA]:[CEMA]:[RAFT]:[AIBN] = 90:10:1:0.1.

5.2.2. Cyclopeptide-Polymer Conjugation

Two polymers [P(HEA32-co-CEMA8) and P(HEA58-co-CEMA10)] were isolated for further work.

These were coupled to the boc-protected two-arm cyclopeptide using the copper-mediated

azide-alkyne cycloaddition (CuAAC) method.252 SEC traces show a clear shift for both

conjugates (Figure 5-6 and Figure 5-7). The high molecular weight broadening is indicative of a

small amount of aggregation of the conjugates into dimers and trimers during analysis.207


178

100 1000 10000 100000 1000000

P(HEA32

-CEMA8)

CP-P(HEA32

-CEMA8)

2

log (MW / g·mol-1)

Figure 5-6: Conjugation of P(HEA32-co-CEMA8) to boc-protected two-arm cyclopeptide

in DMF:TFE = 3:1.

100 1000 10000 100000 1000000

P(HEA58

-CEMA10

)

CP-P(HEA58

-CEMA10

)2

log (MW / g·mol-1)

Figure 5-7: Conjugation of P(HEA58-co-CEMA10) to boc-protected two-arm cyclopeptide in

DMF:TFE = 3:1.


179

Complete coupling of the polymer to the peptide is further evidenced by the FTIR spectrum

which shows no residual azide at 2095 cm-1 (Figure 5-8). The appearance of the polymer

carbonyl and increasing C-H stretches due to the increase in molecular weight, are evident.

4000 3500 3000 2500 2000 1500 1000

Azide

Cyclopeptide

CP-P(HEA32

-co-CEMA8)

2

CP-P(HEA58

-co-CEMA10

)2

Wavenumber / cm-1

Polymer C=O

2100

Figure 5-8: IR of cyclopeptide and cyclopeptide-polymer conjugates. The disappearance of

the azide peak (inset) after conjugation of the polymer, and the appearanc e of the polymer

carbonyl and increasing C-H stretches due to the increase in molecular weight, are evident.

5.2.3. RAPTA-C Conjugation

The two conjugates (labelled Conjugate A and Conjugate B in Table 5-1) underwent the

Finkelstein reaction235 to convert the chloride end-groups of the CEMA monomer to iodide

end-groups. This was necessary to increase the reactivity of the halogen to facilitate the

reaction with PTA.214 The 1H NMR signal corresponding to CH2-Cl at 3.80 ppm shifted upfield to

3.40 ppm when the chloride was substituted with iodide. This signal could be monitored

relative to the adjoining CH2 signal at 4.23 ppm, which did not move.

RAPTA-C was then attached to the polymer using the method detailed in Chapter 3.214 PTA was

initially bound via a substitution reaction of iodide with nitrogen. Ruthenium dimer was


180

subsequently added, resulting in the complexation formation of RAPTA-C. The reaction was

followed using 31P NMR (Figure 5-9 and Figure 5-10). The 31P chemical shift of the initial PTA

peak at -103 ppm shifted to -84 ppm (Figure 5-9 and Figure 5-10, bottom spectra) as the

alkylation of PTA induces a deshielding of the 31P signal.99 The subsequent complexation

induces a very large deshielding of the corresponding signal, shown by the peak shift

to -18 ppm (Figure 5-9 and Figure 5-10, top spectra). These values were consistent with the

conjugation to the homopolymer in Chapter 3.214 As found previously, the Polymer-PTA and

Polymer-RAPTA-C peaks were broad, due to the inherent rigidity of polymers. Furthermore,

the residual unreacted PTA underwent a complexation reaction producing RAPTA-C on

addition of the RuCl2(p-cymene) dimer, shown by the 31P chemical shift at -103 ppm moving to

-33 ppm.

A highly complex 1H NMR spectra with multiple spin systems results from the N-alkylation of

PTA due to the decrease in molecular symmetry.53 Due to the combined characteristic

broadening from the conjugates, the 1H spectrum is difficult to interpret. However, the

p-cymene region for the reactions provided further evidence for the conjugation reaction as

the peaks for the residual unreacted RuCl2(p-cymene) dimer, RAPTA-C and the broad peak for

the conjugated RAPTA-C are evident (Figure 5-11). Also consistent with Chapter 3,214 the first

step was found to be the rate-limiting step in the conjugation. All conjugated and

unconjugated PTA was consumed during the RAPTA-C complexation, giving both free RAPTA-C

and conjugated RAPTA-C. Small side-products evident in the 31P spectra, apart from the

phosphorous oxide peak previously observed, are most likely the hydrolysis products due to

the exchange of the chloride ligands with hydroxide and water.

Assuming complete conversion of IEMA units to RAPTA-C, the following two conjugate

products were obtained: CP-P(HEA58-co-(RAPTA-C-EMA)10) (Conjugate A) and CP-P(HEA32-co-

(RAPTA-C-EMA)8) (Conjugate B).


181

Figure 5-9: 31

P Spectra showing the attachment of RAPTA-C to CP-P(HEA58-co-IEMA10)

(Conjugate A) via a two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to

conjugate in solution, Top: Dimer added to solution. Desired products are circled.

Figure 5-10: 31

P Spectra showing the attachment of RAPTA-C to CP-P(HEA32-co-IEMA8)

(Conjugate B) via a two-step one-pot synthesis, in DMSO-d6 at 25 ᵒC. Bottom: PTA added to

conjugate in solution, Top: Dimer added to solution. Desired products are circled.


182

Figure 5-11: The p-cymene region of the 1H spectra showing the attachment of RAPTA-C to

CP-P(HEA-co-CEMA), in DMSO-d6 at 25 ᵒC. The broad polymer, residual unreacted

RuCl2(p-cymene) dimer and RAPTA-C p-cymene peaks are at 5.95 ppm, 5.86 ppm and 5.75

ppm, respectively. Bottom: Conjugate A, Top: Conjugate B.

5.2.4. Self-Assembly of Tubes

After drug conjugation, the DMSO solutions were diluted with water and the mixture dialysed

to remove DMSO, unconjugated RAPTA-C and phosphorous oxide. Self-assembly of the

conjugates into nanotubes (NT’s) was investigated using DLS and TEM. Since DLS assumes

particles to be spherical, no information about the structure could be obtained. However,

some trends were evident. DLS measurements of both conjugates showed large sizes and

broad distributions (Figure 5-12).

DLS of conjugate A in both DMSO and water indicated large aggregates, which dramatically

decreased in size after the addition of DMF. This is indicative of the disassembly of the

aggregates, consistent with the dissociation of NT’s due to the disruption of hydrogen bonding

(Figure 5-13) in a more competitive solvent i.e. DMF. However, the strength of the aggregation

is remarkable – unexpectedly even in DMSO aggregates are still evident. There are two

considerations for the self-assembly of NT’s – a balance between steric hindrance of the

polymer arms and protection of the hydrogen bonding peptide core. This balance is dependent

on the solvent used and polymer employed.248


183

1 10 100 1000 10000

Diameter / nm

Conjugate A

Conjugate B

Figure 5-12: DLS intensity average particle size distributions of Conjugate A and B in water

at 25 °C.

0.1 1 10 100 1000 10000

Diameter / nm

DMSO

DMSO/DMF

Water

Figure 5-13: DLS number average particle size distributions of Conjugate A at 25 °C.

Bottom: in DMSO. Middle: DMF (10 %) was added to the solution and the solution re -

analysed after 3 days. Top: in water after dialysi s.


184

5.2.5. Crosslinked Nanotubes

The dynamic nature of NT’s formed from cyclopeptides, results in difficulties when attempting

to obtain TEM images, as they dissociate when they are loaded on to TEM grids due to the

disruption of the hydrogen bonding holding them together. Thus, conjugates A and B were

crosslinked using the OH functionality of the HEA units, to ‘lock in’ the structure. TEM images

of the crosslinked conjugates confirmed the formation of NT’s (Figure 5-14 and Figure 5-15).

Crosslinking between tubes is evidenced by the large overlap and stacking of tubes, and also

by the cloudiness of the crosslinked solutions. Large (~300 nm) spherical impurities can also be

seen in the TEM images, with a higher concentration in the conjugate B sample – the

conjugate with shorter polymer arms.

The polymer system used in this work has not been previously investigated. However,

Chapman et al248 observed that cyclopeptides with PHEA arms of DP30 were insoluble in

water, whereas at DP100 they were soluble in water. Conjugate B has shorter arms (DP40)

compared with conjugate A (DP68). A lower solubility of conjugate B in water may account for

the large impurities if decreased solubility results in increased aggregation of the sample.

Figure 5-14: Conjugate A. (a) and (b) CP-P(HEA58-(RAPTA-C-EMA)10)2. (c) to (f) Crosslinked

CP-P(HEA58-(RAPTA-C-EMA)10)2. Samples (a) to (d) were drop-loaded onto grid, air-dried

and stained with Osmium Tetroxide. Samples (e) and (f) were drop -loaded onto grid and

air-dried. Scale bar : b, d & f = 200 nm, a & e = 100 nm, c = 50 nm.


185

Figure 5-15: Conjugate B. Crosslinked CP-P(HEA32-(RAPTA-C-EMA)8)2. Samples (a) and (b)

were drop-loaded onto grid, air-dried and stained with Osmium Tetroxide. Samples (c) and

(d) were drop-loaded onto grid and air-dried. Scale bar : a, b & e = 200 nm, d = 100 nm, c =

50 nm.

5.2.6. Cytotoxicity Evaluation

The final ruthenium concentration in samples from conjugates A and B, after dialysis, was

determined by ICPOES and used to evaluate the cytotoxicity of the conjugates. The solution

colour clearly indicated that conjugate A contained a higher amount of ruthenium than

conjugate B and this was confirmed by ICPOES to be a 10-fold difference (Table 5-2). However,

the lower ruthenium concentration may be due to a lower overall material content and not a

lower drug conjugation. Due to the numerous steps involved in the synthesis of these systems,

it is likely that the final conjugate mass is inaccurate. Since NMR analyses clearly showed that

the conjugation proceeded in both systems, it is suggested that the overall concentration of

peptide tubes in sample B is lower.


186

Conjugates were tested against ovarian A2780 and cisplatin-resistant ovarian A2780cis cancer

cell lines and compared with the drug RAPTA-C. The toxicity of the conjugate prior to drug

conjugation was also tested and found to be non-toxic at concentrations up to 590 µg.mL-1

(Figure 5-16). A significant decrease (~10x) in the IC50 value for Conjugate A was found for both

cell lines, compared with RAPTA-C (Table 5-3, Figure 5-17, Figure 5-18). Conjugate B was not

found to have any effect. This is likely due to the lower ruthenium sample concentration

resulting in a maximum concentration below the IC50 value (Table 5-3).

Table 5-2: Final ruthenium content in cyclopeptide-polymer conjugates was determined

using ICPOES.

Ru Concentration (mmol.L-1

)

Conjugate A 5 x 10-2

Conjugate B 5 x 10-3

Table 5-3: IC50 (µM) values of RAPTA-C and cyclopeptide-polymer conjugates A and B,

against ovarian A2780 and cisplatin-resistant ovarian A2780cis cancer cell lines.

IC50 (µM) A2780 A2780cis

RAPTA-C Literature 353 ± 1494

>20054

RAPTA-C 271 266

Conjugate A 15 22

Conjugate B >2.5 >2.5


187

101

102

103

0

20

40

60

80

100

120

140

A2780 CP-P(HEA-CEMA)2

A2780cis CP-P(HEA-CEMA)2

Ovcar-3 CP-P(HEA-CEMA)2

Surv

ival R

ate

/ %

Conjugate Concentration / nM

Figure 5-16: Cytotoxicity profile of cyclopeptide-polymer conjugate without RAPTA-C, after

72 hours, n = 4.

101

102

103

104

105

106

107

0

20

40

60

80

100

120

RAPTA-C

Conjugate A

Conjugate B

A2

78

0 S

urv

iva

l R

ate

/ %

Ru Concentration / nM

Figure 5-17: Cytotoxicity profile of RAPTA-C and Conjugates A and B, against ovarian

carcinoma A2780 cells, after 72 hours, n = 4.


188

101

102

103

104

105

106

107

0

20

40

60

80

100

120

RAPTA-C

Conjugate A

Conjugate B

A278

0cis

Surv

iva

l R

ate

/ %

Ru Concentration / nM

Figure 5-18: Cytotoxicity profile of RAPTA-C and Conjugates A and B, against cisplatin -

resistant ovarian carcinoma A2780cis cells , after 72 hours, n = 4.

5.3. CONCLUSIONS

Cyclopeptide nanotubes were identified as an appropriate drug carrier for RAPTA-C. A

copolymer containing HEA (for solubility) and CEMA (for conjugation) was designed to increase

solubility of the peptides and allow for conjugation of RAPTA-C. The copolymer was prepared

and successfully conjugated to self-assembling cyclopeptides. The drug was also successfully

conjugated without disrupting the self-assembly of the conjugates into nanotubes, shown by

DLS and TEM. Two conjugates containing copolymers of differing lengths were synthesised and

used to test the RAPTA-C conjugation, nanotube formation and subsequent cytotoxicity of

these moieties. A 10-fold increase in cytotoxicity was found for conjugate A, compared with

the RAPTA-C. The final ruthenium concentration in conjugate B was below the threshold to

elucidate its cytotoxicity. It would be worthwhile to synthesise a larger quantity of material

and re-evaluate this conjugate for cytotoxic effects.

189

6 METASTATIC ASSESSMENT

The majority of cancer patients die from metastatic (secondary) cancer as the current

therapies are often inadequate at treating cancer in this developed stage. The development of

chemotherapeutics that specifically target and treat metastases is key to the eradication of

cancer as a disease. Organometallic complexes and coordination compounds synthesised with

a ruthenium centre are distinctive as they have not only shown promise as anticancer

therapeutics but many are active, and sometimes selectively active, on tumour metastases.

Chapters 2-5 showed that the cytotoxicity and cell uptake of two ruthenium drugs, NAMI-A

and RAPTA-C, can be enhanced through the synthesis of macromolecular versions of these

chemotherapeutics and incorporation of them into nanoparticles (micelles).

However, the evaluation of their potential by cytotoxicity assays only determined whether the

therapeutics are active on cancerous cells. To determine their antimetastatic potential, a more

detailed analysis is needed, which is most often conducted in animal models. However, it has

been established by Sava and co-workers that, for NAMI-A-type compounds, in vitro inhibition

of tumour invasion correlates with the in vivo inhibition of metastasis formation.30,33,78

Bergamo et al20 subsequently developed a series of in vitro assessments, that mimic in vivo

processes, to evaluate the interference of RAPTA-T on some of the steps of the metastatic

progression. These experiments can be used to screen compounds for antimetastatic activity

prior to testing the compounds in vivo. Based on this work, the potential antimetastatic

activity of these macromolecular ruthenium chemotherapeutics has been preliminarily

explored.

Chapter 6: Metastatic Assessment

190

6.1. METHODS

Dr. Hongxu Lu kindly performed the following assays for this Chapter.

6.1.1. Tumour Cell Lines for In Vitro Tests

The highly invasive MDA-MB-231 human breast cancer cell line was obtained from the

Australia Cell Bank. The MCF-7 human breast cancer cell line was kindly supplied by the Lowy

Cancer Research Centre (UNSW, Sydney, Australia). The non-tumourigenic Chinese hamster

ovary (CHO) cells were kindly supplied by St. George Hospital (Sydney. Australia). The three cell

lines were cultured in 75 cm2 tissue culture flasks with a complete medium composed of

Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % FBS, 4 mM glutamine,

100 U.mL-1 penicillin, 100 µg.mL-1 streptomycin, 1 mM sodium pyruvate and 1 % MEM non-

essential amino acid at 37 °C under an atmosphere of 5 % CO2. After reaching 70 % confluence,

the cells were washed with PBS and collected by trypsin/EDTA treatment. The cells were

seeded in 6-well cell culture plates and incubated for one day before drug treatment. The

ruthenium concentration in all samples was 5 µM.

6.1.2. Cell Viability Assay

The effects on the cell viability were evaluated by a WST-1 assay. The cells were seeded on

96-well plates at 4 000 cells/well and incubated for 24 hours. The samples were loaded to the

cell with the complete medium and cultured for 72 hours at 37 ºC in the incubator. The

viability was measured by the WST-1 assay as described previously in Chapter 5.

Cells without sample treatment were used as controls. Each assay was done in triplicate. Data

represent means ± SD. A one way analysis of variance was used to reveal the statistical

difference among different groups. A Dunnett’s post hoc test was used to compare the

difference between samples and the control. A p-value less than 0.05 was considered a

statistically significant difference.


191

6.1.3. Migration Assay

The migratory ability resulting from a haptotactic or a chemotactic stimulus was measured in

24-well Millicell® hanging cell culture inserts (Millipore, Billerica, MA, USA) by the methods of

Bergamo et al with some modifications.20 In the haptotaxis assay the lower surface of a

polyethylene terephthalate (PET) filter (8-µm pore size) was coated with 10 µg.mL-1 fibronectin

and left in a humidified cell culture chamber at 4 ˚C overnight, then washed with PBS before

cell seeding. In the chemotaxis assay, inserts were used without coating. Cells were treated for

one hour with all the samples in the complete medium. Then the cells were washed with PBS

three times, collected by trypsinisation and centrifugation, and re-suspended in serum-free

medium supplemented with 0.1 % w/v bovine serum albumin (BSA). A 0.2 mL cell suspension

at 5 x 105 cells.mL-1 was seeded in the upper compartment of each insert. The lower

compartment was filled with serum free medium supplemented with 0.1 % w/v BSA and with

complete medium for the haptotaxis and the chemotaxis assay, respectively. Cells were

incubated at 37 ˚C for 24 hours, and then the cells on the upper surface of the filters were

removed with a cotton swab. The migrated cells in the lower surface were washed with PBS,

trypsinised with 0.3 mL trypsin/EDTA and counted by a hemacytometer.

6.1.4. Invasion Assay

24-well Millicell® hanging cell culture inserts (8-µm pore size) were coated with 50 µL of

1 mg.mL-1 Matrigel (diluted in serum-free DMEM medium; BD Sciences, Franklin Lakes, NJ) and

air dried overnight at room temperature. The filters were reconstituted with serum free

medium immediately before use. 0.5 × 105 cells in 0.2 mL serum free medium (with 0.1 % BSA)

were added to the upper chamber and the lower compartment was filled with complete

medium. The cells were allowed to invade for 48 hours at 37 ºC in a CO2 incubator. Then the

cells on the upper surface of the filters were removed with a cotton swab. The invading cells

on the lower surface were washed with PBS, trypsinised with 0.3 mL trypsin/EDTA and counted

by a hemacytometer.


192

6.2. RESULTS

Bergamo et al20 designed a series of in vitro experiments that simulate the main steps of

metastatic progression: detachment from the primary tumour, degradation of the extracellular

matrix, ability to migrate, invade and adhere to a new organ. The migration and invasion

assays were chosen to assess the antimetastatic potential of the ruthenium conjugated

nanoparticles RAPTA-C Micelles A and B, and NAMI-A Micelles, in comparison to the drugs

RAPTA-C and NAMI-A.

The effects of RAPTA-C and NAMI-A and the micelles incorporating these drugs (Table 6-1)

were evaluated against three cell lines characterised by differing degrees of malignancy

(MDA-MB-231 > MCF-7 > CHO).


193

Table 6-1: Structures of the chemotherapeutic agents NAMI-A and RAPTA-C, and the

amphiphilic block copolymers that self-assemble into micelles.

Chemotherapeutic Name Structure

RAPTA-C

NAMI-A

NAMI-A Micelles

Dh = ~100 nm

P(NAMI-A)152-b-PPEGMEA19

RAPTA-C Micelles

Dh = ~100 nm

A: PLA347-b-

P(HEA291-(RAPTA-C-EMA)19

B: PLA347-b-

P(HEA450-(RAPTA-C-EMA)50


194

6.2.1. Effect on Cell Viability

A ruthenium concentration of 5 µM was chosen as a suitable concentration to be used for the

following experiments since the cell viability was unaffected by the compounds at this

ruthenium concentration. Figure 6-1 shows that the ruthenium compound induces only slight

and non-significant oscillations (± 10 %) in cell viability as compared to the relevant controls.

This is necessary in order to exclude the interference of the cytotoxicity of the compounds in

the migration and invasion assays. Using the same ruthenium concentration across different

drug types also allowed for a direct comparison of effects.

CHO MCF-7 MDA-MB-231

0

10

20

30

40

50

60

70

80

90

100

110

Su

rviv

al R

ate

/ %

NAMI-A Micelles NAMI-A RAPTA-C

RAPTA-C Micelles A RAPTA-C Micelles B

Figure 6-1: Viability of cells exposed to NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C

Micelles A and B at [Ru] = 5 µM.


195

6.2.2. Effects on Migration and Invasion

The influence of all drugs on the migration process of the three cell lines MDA-MB-231

(invasive cancerous), MCF-7 (non-invasive, cancerous) and CHO (non-cancerous) was

evaluated when a chemical (chemotaxis) and a contact (haptotaxis) stimulus was applied to

promote cell movement (Figure 6-2 and Figure 6-3). The effect on the invasive ability of these

cells through Matrigel® was also assessed (Figure 6-4). Notably, the macromolecular drugs

assembled into micelles had a greater inhibitory effect on both the migration and invasion of

cells, than the comparative drugs NAMI-A and RAPTA-C.

6.2.2.1. Migration

In the case of the RAPTA-C drugs, the greatest inhibition was achieved with MDA-MB-231 cells.

MCF-7 cells also show a significantly reduced migratory ability. In contrast, there is little or no

effect on the CHO cells, with respect to the controls. The inhibitory effect on the chemotactic

migration was slightly better than the haptotactic migration (Figure 6-2 and Figure 6-3).

In the case of the NAMI-A drugs, the chemotactic and haptotactic migration of both the MDA-

MB-231 and MCF-7 cells was inhibited more than that of the CHO cells. However, no

statistically significant effect was observed with respect to the controls (Figure 6-2 and Figure

6-3).

6.2.2.2. Invasion

In the case of the RAPTA-C drugs, the invasive ability of MDA-MB-231 and MCF-7 cells was

significantly inhibited, whereas CHO cells were the least affected or unaffected, with respect to

the controls. Micelle A and B had differing inhibition effects on the invasive and non-invasive

cells, with Micelle A having a greater inhibitory effect on MCF-7 cells while Micelle B had the

greatest inhibitory effect on MDA-MB-231 cells (Figure 6-4).

In the case of the NAMI-A drugs, the invasive ability of both the MDA-MB-231 and MCF-7 cells

was inhibited more than that of the CHO cells, with respect to the controls. A statistically

significant inhibitory effect was observed for the invasive MDA-MB-231 cell line (Figure 6-4).


196


-100

-75

-50

-25

0

25

*

*

*

**

Mig

ratin

g C

ells

% V

ari

an

ce

vs C

on

tro

l

NAMI-A Micelles

NAMI-A

RAPTA-C Micelles A

RAPTA-C Micelles B

RAPTA-C

Figure 6-2: Effect of NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C Micelles A and B on

the chemotactic migration of cells through polycarbonate filters. MDA-MB-231, MCF-7 and

CHO cell were treated for one hour with the drugs where [Ru] = 5 µM. The cells were then

removed from the flasks, collected, re -suspended and seeded on the inserts of Transwell

cell culture chambers. Data represent cells that after 24 hrs have migrated and are present

on the lower surface of the filter. Data are the percent of variation vs. controls calculated

from the mean ± SD of one experiment performed in triplicate. *, significant difference,

p < 0.05.


197


-100

-75

-50

-25

0

25

**

**

Mig

ratin

g C

ells

% V

ari

an

ce

vs C

on

tro

l

NAMI-A Micelles

NAMI-A

RAPTA-C Micelles A

RAPTA-C Micelles B

RAPTA-C


the haptotactic migration of cells through polycarbonate filters. MDA-MB-231, MCF-7 and

CHO cell were treated for one hour with the drugs where [Ru] = 5 µM. The cells were then

removed from the flasks, collected, re-suspended and seeded on the inserts of Transwell

cell culture chambers. Data represent cells that after 24 hrs have migrated and are present

on the lower surface of the filter. Data are the percent of variation vs. controls calculated

from the mean ± SD of one experiment performed in triplicate. *, significant difference,

p < 0.05.


198


-100

-75

-50

-25

0

25

*

**

*

*

*

Inva

din

g C

ells

% V

ari

an

ce

vs C

on

tro

l

NAMI-A Micelles

NAMI-A

RAPTA-C Micelles A

RAPTA-C Micelles B

RAPTA-C


the invasion of cells through Matrigel®. MDA-MB-231, MCF-7 and CHO cells were treated

for one hour with the drugs where [Ru] = 5 µM. The cells were then removed from the

flasks, collected, re-suspended and seeded on inserts. Data represent cells that after 96 h

have invaded and are present on the lower surface of the filter. Data are the percent of

variation vs. controls calculated from the mean ± SD of one experiment performed in

triplicate. *, significant difference, p < 0.05.


199

6.4. DISCUSSION

All the tested drugs display cell type specificity, characterised by a more pronounced effect on

tumour cells, than on the non-tumourigenic CHO cells.

The RAPTA-C micelles have a stronger inhibitory effect on the migration of the highly invasive

MDA-MB-231 cells, in comparison to MCF-7 non-invasive cells, indicating that these

macromolecular drugs interfere more selectively with tumour cells with the highest inclination

to invade and metastasise. However, the same trend is only present for Micelles B in the

invasion assay. The reason that Micelles A have a stronger effect on MCF-7 cells in the invasion

assay cannot be explained and would be interesting to explore further. It may indicate that

micelles could be tailored towards specific cell types with differing degrees of malignancy.

The NAMI-A micelles did not display any statistically significant effect for the migration assay,

although they had a more pronounced inhibitory effect than NAMI-A. However, since the

NAMI-A micelles significantly inhibited the invasion of the highly invasive MDA-MB-231 cells, it

can be inferred that these drugs also interfere more selectively with tumour cells with the

highest inclination to invade and metastasise.

Table 6-2: IC50 (µM) values of NAMI-A, NAMI-A Micelles, RAPTA-C and RAPTA-C Micelles A

and B, against ovarian A2780 and OVCAR-3 cancer cell lines.

IC50 (µM) A2780 OVCAR-3

RAPTA-C Literature 353 ± 1494

-

RAPTA-C 271 300

RAPTA-C Micelles A 15 46

RAPTA-C Micelles B 51 61

NAMI-A Literature > 50085

-

NAMI-A 596 738

NAMI-A Micelles 439 433

The concentration chosen allowed the direct comparison of data across RAPTA-C and NAMI-A

drugs. However, all the cytotoxicity data in previous studies of both the RAPTA-C and NAMI-A

analogues has shown that NAMI-A drugs consistently yield higher IC50 values (Table 6-2). Thus,


200

it is probable that the ruthenium concentration used in these assays was insufficient to clearly

show the inhibitory effects that the NAMI-A drugs may possess. It would be beneficial to re-

evaluate the NAMI-A micelles at a higher ruthenium concentration.

6.5. CONCLUSIONS

The incorporation of the ruthenium drugs NAMI-A and RAPTA-C into polymeric nanoparticles

improved the inhibitory effects on both the migration and invasion of cancer cells. Since the

inhibition of the in vitro migration and invasion of cells can be correlated with the in vivo

inhibition of metastasis formation, it can be inferred that NAMI-A and RAPTA-C micelles have

an improved antimetastatic ability in comparison to the free drugs NAMI-A and RAPTA-C.

Further work is necessary to better understand the differences in the inhibitory effects and

antimetastatic potential of these new chemotherapeutics.

201

7 CONCLUSIONS & OUTLOOK

Ruthenium possesses unique properties that have led to the development of exceptionally

promising anticancer and antimetastatic therapeutics. The amplified benefits, for example

increasing cytotoxicity while reducing undesirable side-effects, that can be elicited by

incorporating drugs into macromolecules has not previously been investigated for ruthenium

agents. Drawing on the benefits that can be elicited from interdisciplinary research, this thesis

integrated inorganic chemistry, polymer chemistry, and biological experiments to complete

the aims of this project:

(a) To develop a synthetic approach for the design of macromolecular ruthenium

therapeutics;

(b) To incorporate ruthenium containing macromolecules into polymer architectures able

to self-assemble into nano-sized drug carriers;

(c) To evaluate the potential benefits as anticancer therapeutics of (a) and (b); and

(d) To determine the potential antimetastatic activity of (a) and (b).

To this end, two novel and facile approaches to the synthesis of two types of macromolecules

containing ruthenium were developed, based on the ruthenium anticancer drugs NAMI-A and

RAPTA-C. The first approach relied on the polymerisation of 4-vinyl imidazole – an analogue of

imidazole which is a ligand of the NAMI-A molecule. The second approach used the inherent

activity of the amide group on the PTA ligand of RAPTA-C to allow for an efficient conjugation

to a rationally designed polymer. Subsequently, amphiphilic polymers were designed,

incorporating the afore-mentioned macromolecular species, in order to prepare micelles.

Furthermore, a convergent approach was employed for the preparation of cyclopeptide-

polymer nanotubes. The different drug conjugates, NAMI-A and RAPTA-C containing micelles,

as well as the cyclopeptide-polymer conjugates were evaluated for cytotoxic activity. The most

Chapter 7: Conclusions & Outlook

202

promising candidates, namely the micelles containing NAMI-A and RAPTA-C, were also

evaluated for antimetastatic activity.

Polymerisation of 4-vinyl imidazole was recognised as an efficient strategy to develop a

macromolecule incorporating NAMI-A. Due to the reactivity of the amine in the imidazole ring,

conjugation of a ruthenium precursor, to form the NAMI-A drug, was exceptionally efficient. A

1.5 times increase in cytotoxicity was found for NAMI-A containing micelles when compared to

free NAMI-A – a favourable result for a drug that requires a comparatively high administration

concentration relative to other chemotherapeutics. As with the NAMI-A molecule, the major

obstacle of the macromolecule was the instability of the compound – since hydrolysis of the

chloride ligands causes labilisation of the DMSO ligand.

Macromolecular therapeutics based on the ruthenium(II) drug RAPTA-C were distinctively

more stable in water, which from an administration point of view makes them more attractive

as anticancer compounds. Nucleophilic substitution of halides with amines was identified as an

appropriate mechanism for conjugation of RAPTA-C to a suitably designed polymeric scaffold,

since RAPTA-C contains a PTA ligand that is susceptible to alkylation. Poly(2-chloroethyl

methacrylate) [PCEMA] was prepared via RAFT polymerisation and its reactivity increased

using the Finkelstein reaction, to give poly(2-iodoethyl methacrylate) [PIEMA] which served as

a suitably reactive scaffold to attach RAPTA-C. Two pathways were employed to conjugate

RAPTA-C to the polymer and both routes were tested using n-butyl iodide as a model

compound. 1D and 2D NMR experiments were used to follow conjugation and elucidate the

superior pathway: a two-step reaction involving the conjugation of PTA and subsequent

complexation to form RAPTA-C.

Two pathways to the development of RAPTA-C nano-sized drug carriers were chosen. Both

relied on the copolymerisation of 2-hydroxyethyl acrylate (HEA) (for water solubility) and 2-

chloroethyl methacrylate (CEMA) (for efficient conjugation of RAPTA-C). Firstly, an amphiphilic

block copolymer capable of self-assembling into polymeric micelles, and also possessing a

core-forming biodegradable polylactide (PLA) block was designed. This design protected

RAPTA-C during transport to the target site, at which time the degradable PLA block was

destroyed and the macromolecule containing RAPTA-C released. Secondly, a ‘natural’ scaffold

was chosen. The HEA-CEMA copolymer was conjugated to cyclopeptides that self-assembled

into nanotubes. This also afforded protection to RAPTA-C and offers a potential release

mechanism since nanotubes can disassemble under specific (acidic) conditions due to the

disruption of the hydrogen bonding keeping them together.

Chapter 7: Conclusions & Outlook

203

An outstanding 10-fold increase in cytotoxicity was found for both of these new entities,

micelles and nanotubes containing RAPTA-C, when compared to the free drug RAPTA-C. This is

a remarkable increase that warrants further biological investigations. The micelles would,

however, be the preferred system due to the relatively simpler and cheaper synthetic

procedure. The cell uptake of the RAPTA-C containing micelles into the lysosomes of cells was

confirmed, indicative of an endocytic pathway. Future work could involve a more in-depth

study of this pathway, the interaction of the macromolecular drug with the cell and the effect

of the degradation of the micelle on drug release and cell interaction.

The unique attribute of the most promising ruthenium therapeutics, and of both NAMI-A and

RAPTA-C, is their remarkable activity on metastatic cancers. Thus, an initial exploration into the

antimetastatic activity of the ruthenium macromolecular drugs synthesised in this thesis was

key in determining their potential. The incorporation of the ruthenium drugs NAMI-A and

RAPTA-C into polymeric nanoparticles significantly improved the inhibitory effects on both the

migration and invasion of the highly invasive MDA-MB-231 and poorly invasive MCF-7 human

breast cancer cells, in comparison to RAPTA-C and NAMI-A. Since the inhibition of the in vitro

migration and invasion of cancer cells can be correlated with the inhibition in vivo of

metastasis formation, it is highly probable that these nanoparticles will inhibit metastases in

vivo.

This thesis provides concrete evidence that macromolecules containing ruthenium, when

incorporated into advanced polymer architectures, display outstanding increases in

cytotoxicity making them superior chemotherapeutics. Furthermore, in vitro analyses of their

antimetastatic potential indicate that these entities are extremely promising antimetastatic

therapeutic candidates for in vivo studies. Having successfully prepared novel superior

macromolecular ruthenium based therapeutics for the treatment of cancer, this work provides

a substantial basis for the progression of these macromolecular chemotherapeutics into

advanced biological studies and the evaluation of them against animal models.

204

205

8 MATERIALS & ANALYSES

The chemicals, experimental protocols and analysis techniques used in this thesis are

presented in this chapter and referred to in chapters 2 to 5.

8.1. CHEMICALS

Unless otherwise specified, all chemicals were reagent grade and used as received.

8.1.1. Ruthenium(III) NAMI-A Conjugation (Chapter 2)

Urocanic acid (99 %, Aldrich), 4,4'-azobis(cyanopentanoic acid) (ACPA; 98 %, Fluka), glacial

acetic acid (>99.5 % , Aldrich), methanol (MeOH; HPLC grade, APS), ethanol (EtOH; absolute,

Fluka), dimethylsulfoxide (DMSO; >98.9 %, Ajax Finechem), hydrochloric acid (HCl; 37 %,

Aldrich), acetone (HPLC grade, Aldrich); diethyl ether (99 %, Ajax Finechem), imidazole (>95 %,

Fluka), ruthenium trichloride hydrate (RuCl3.H2O; Aldrich), poly(ethylene glycol) methyl ether

acrylate (PEGMEA; Aldrich). 2,2-azobis(isobutyronitrile) (AIBN; 98 %, Fluka) was purified by

recrystallisation from methanol. MilliQ water was obtained from an UltraPure water

purification system. The RAFT agent 2-methyl-2-(((undecylthio)carbonothioyl)thio)propanoic

acid (2) was prepared according to a literature procedure.253

Chapter 8: Materials & Analyses

206

8.1.2. Ruthenium(II) RAPTA-C Conjugation (Chapter 3)

Ethanol (absolute, Fluka), methanol (MeOH; HPLC grade, APS), methacryloyl chloride (97 %,

Lancaster), 2-chloroethoanol (> 99 %, Aldrich), triethylamine (NEt3; > 9 %, Aldrich), N-(2-

hydroxypropyl) methacrylamide (HPMA; Polysciences Inc.), tetrahydrofuran (THF; 99.7 %,

Ajax), toluene (>99.4 %, Ajax), N,N-dimethylacetamide (DMAc; 99.9 %, Aldrich),

N,N-dimethylformamide (DMF; 99 %, Aldrich) dimethylsulfoxide (DMSO; >98.9 %, Ajax

Finechem), silica gel (DAVISIL), 2,6-di-tert-butyl-4-methylphenol (BHT; 98 %, Aldrich),

deuterated water (D2O; Cambridge Isotope Laboratories), 4,4'-azobis(cyanopentanoic acid)

(ACPA; 98 %, Fluka), ethyl acetate (EtOAc; 95 %, Ajax Finechem), n-hexane (95 %, Ajax

Finechem), sodium iodide (NaI; > 99.5 %, Aldrich), dichlororuthenium(II)(p-cymene) dimer

(RuCl2(p-cymene) Dimer; 97 %, Aldrich), 1,3,5-triaza-7-phosphaadamantane (PTA; 97 %,

Aldrich), diethyl ether (99 %, Ajax Finechem), petroleum ether (BR 40–60 °C; 90 %, Ajax

Finechem), magnesium sulfate (MgSO4; 70 %, Ajax Finechem), 1,4-dioxane (99 %, Aldrich), n-

butyl iodide (IBu; 99 %, Aldrich) were used without any further purification. Acetone (HPLC

grade, Aldrich) was dried for 24 hours over 3 Å molecular sieves, activated under vacuum at

100 °C for 12 hours. Deuterated dimethylsulfoxide-d6 (DMSO-d6; Cambridge Isotope

Laboratories) was degassed by five freeze-pump-thaw cycles. 2,2-azobis(isobutyronitrile)

(AIBN; 98 %, Fluka) was purified by recrystallisation from methanol. The RAFT agent

cumyldithiobenzoate (CDB, 1) was prepared according to literature procedure.254

8.1.3. Polymeric Micelles (Chapter 4)

Methanol (MeOH; HPLC grade, APS), methacryloyl chloride (97 %, Lancaster), 2-chloroethoanol

(> 99 %, Aldrich), triethylamine (NEt3; > 9 %, Aldrich), fluorescein O-methacrylate (F; 97 %,

Aldrich), toluene (>99.4 %, Ajax), N,N-dimethylacetamide (DMAc; 99.9 %, Aldrich),

N,N-dimethylformamide (DMF; 99 %, Aldrich) dimethylsulfoxide (DMSO; >98.9 %, Ajax

Finechem), silica gel (DAVISIL), ethyl acetate (EtOAc; 95 %, Ajax Finechem), n-hexane (95 %,

Ajax Finechem), dichloromethane (DCM; >99.8 %, Aldrich), sodium iodide (NaI; > 99.5 %,

Aldrich), dichlororuthenium(II)(p-cymene) dimer (RuCl2(p-cymene) Dimer; 97 %, Aldrich), 1,3,5-

triaza-7-phosphaadamantane (PTA; 97 %, Aldrich), diethyl ether (99 %, Ajax Finechem), tin(II)

2-ethyl-hexanoate (SnOct2; 95 %, Aldrich), 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide; Aldrich)

were used without any further purification. 2-hydroxyethyl acrylate (HEA; 96 %, Aldrich) was

destabilised by passing over basic aluminium oxide. Acetone (HPLC grade, Aldrich) was dried


207

for 24 hours over 3 Å molecular sieves, activated under vacuum at 100 °C for 12 hours.

Deuterated dimethylsulfoxide-d6 (DMSO-d6; Cambridge Isotope Laboratories) was degassed by

bubbling with argon for 30 minutes. 2,2-azobis(isobutyronitrile) (AIBN; 98 %, Fluka) was

purified by recrystallisation from methanol. The monomer 2-chloroethyl methacrylate

(CEMA)214 was synthesised according to a literature procedure. MilliQ water was obtained

from an UltraPure water purification system. The RAFT agent benzyl (2-hydroxyethyl)

carbonotrithioate was prepared according to a literature procedure.241

8.1.4. Peptide Tubes (Chapter 5)

Methanol (HPLC grade, APS), methacryloyl chloride (97 %, Lancaster), 2-chloroethoanol (>

99 %, Aldrich), triethylamine (NEt3; > 9 %, Aldrich), N,N-dimethylacetamide (DMAc; 99.9 %,

Aldrich), N,N-dimethylformamide (DMF; 99 %, Aldrich) dimethylsulfoxide (DMSO; >98.9 %, Ajax

Finechem), silica gel (DAVISIL), ethyl acetate (EtOAc; 95 %, Ajax Finechem), n-hexane (95 %,

Ajax Finechem), dichloromethane (DCM; >99.8 %, Aldrich), sodium iodide (NaI; > 99.5 %,

Aldrich), dichlororuthenium(II)(p-cymene) dimer (RuCl2(p-cymene) Dimer; 97 %, Aldrich), 1,3,5-

triaza-7-phosphaadamantane (PTA; 97 %, Aldrich), diethyl ether (99 %, Ajax Finechem), Hünig's

base (di(isopropyl)ethylamine, DIPEA; Aldrich), trifluoroacetic acid (TFA; Aldrich), sulfuryl

chloride (Aldrich) were used without any further purification. Fmoc-Lys-OH, Fmoc-Trp(Boc)-

OH, Fmoc-D-Leu-OH, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium (HBTU) and 1-

hydroxy-benzotriazole (HOBt) were obtained from Novabiochem and used as supplied. 2-

hydroxyethyl acrylate (HEA; 96 %, Aldrich) was destabilised by passing over basic aluminium

oxide. Acetone (HPLC grade, Aldrich) was dried for 24 hours over 3 Å molecular sieves,

activated under vacuum at 100 °C for 12 hours. Deuterated dimethylsulfoxide-d6 (DMSO-d6;

Cambridge Isotope Laboratories) was degassed by bubbling with argon for 30 minutes. 2,2-

azobis(isobutyronitrile) (AIBN; 98 %, Fluka) was purified by recrystallisation from methanol.

The monomer 2-chloroethyl methacrylate (CEMA)214 was synthesised according to a literature

procedure. Cuprisorb resin was obtained from KCPC at Sydney University. MilliQ water was

obtained from an UltraPure water purification system. Butyl-trithiocarbonate propanoic acid

(4) was obtained from Dulux and used as supplied. The RAFT agent (prop-2-ynyl propanoate)yl

butyltrithiocarbonate (5) was prepared following a literature procedure.208


208

8.2. RAFT AGENT SYNTHESES

Figure 8-1: Structure of RAFT Agents.

8.2.1. Cumyldiothiobenzoate (CDB, 1)

Cumyldithiobenzoate was prepared according to a literature procedure.254 Benzyl chloride

(12.6 g) was added dropwise over one hour to a round bottomed flask containing elemental

sulfur (6.4 g), 25 % sodium methoxide solution in methanol (40 g) and methanol (40 g). The

resulting brown solution was refluxed at 80°C overnight. After cooling to room temperature,

the mixture was filtered to remove the white solid (sodium chloride) and then methanol

removed via rotary evaporation. The resulting brown solid was re-dissolved in distilled water

(100 mL), and washed three times with diethyl ether (3 x 50 mL). Diethyl ether (50 mL) was

added to the solution and the two phase mixture acidified with 32 % aqueous HCl until the

aqueous layer lost its characteristic brown colour and the top layer was deep purple. The

etherous (purple, dithiobenzoic acid) layer was extracted, before deionised water (120 mL) and

1.0 M NaOH (240 mL) were added to extract sodium dithiobenzoate as the aqueous layer. This

washing process was repeated twice to give a final solution of sodium dithiobenzoate.

Potassium ferricyanide (13 g, 0.04 mol) was dissolved in deionised water (200 mL). Sodium

dithiobenzoate solution (140 mL) was transferred to a conical flask equipped with a magnetic

stirrer bar. Potassium ferricyanide solution was then added dropwise to the sodium

dithiobenzoate over one hour under vigorous stirring. The red precipitate was filtered and

washed with deionised water until the washings become colourless. The solid was dried in

vacuo at room temperature overnight and the product recrystallised from ethanol.


209

A solution of AIBN (5.8 g, 0.02 mol) and the recrystallised bis(thiobenzoyl) disulfide (4.3 g,

0.01 mol) in ethyl acetate (80 mL) was refluxed at 80 °C for 18 hours. After removal of the

volatiles in vacuo the crude product was purified by silica gel column chromatography with

ethyl acetate/n-hexane (2:3) as eluent to give 2-cyanoprop-2-yl dithiobenzoate as a red liquid.

On cooling to -20 °C, the product became a red solid.

8.2.2. 2-(((dodecylthio)carbonothioyl)thio)-2-

methylpropanoic acid (RAFT11, 2)

2-(((dodecylthio)carbonothioyl)thio)-2-methylpropanoic acid was kindly prepared by Andrew

Gregory according to a literature procedure.253

8.2.3. Benzyl (2-hydroxyethyl) carbonotrithoate (trithio, 3)

Benzyl (2-hydroxyethyl) carbonotrithoate was prepared according to a literature procedure.241

2-Mercaptoethanol (20 mL, 0.23 mol) was added to a solution of potassium hydroxide (13 g,

0.23 mol) in water (250 mL). After the dropwise addition of carbon disulfide (30 mL), the

orange solution was stirred for five hours. The mixture was then heated with benzyl bromide

(39.6 g, 0.23 mol) for 12 hours at 80 °C. After cooling, the water phase was extracted by the

addition of chloroform (300 mL, then 2 x 100 mL). The combined organic layers were dried

over anhydrous magnesium sulfate. After evaporation of the solvent, the remaining product

was dissolved in dichloromethane and run through a column containing aluminum oxide. The

solvent was removed under reduced pressure. The product was purified by gel column

chromatography with a hexane/ethyl acetate (7:3) as the eluent to yield a yellow liquid.

1H NMR(CDCl3): 3.59 (t, 2H,CH2-O), 3.85 (t, 2H, CH2-S), 4.62 (s, 2H, CH2-Ph), 7.31 (m, 5H, Ph).

13C NMR (CDCl3): 41.5 (CH2-O), 43.9 (CH2-S), 62.7 (CH2-Ph), 129.2-136.8 (Ph), 137.1 (Ph), 225.9

(CdS).

8.2.4. Butyl-trithiocarbonate Propanoic Acid (BTCPA, 4)

BTCPA was kindly provided by Robert Chapman at The University of Sydney who was given it by

Algi Serelis at Dulux.


210

8.2.5. (prop-2-ynyl propanoate)yl Butyltrithiocarbonate

(PPBTC, Alkyne RAFT, 5)

PPBTC was kindly prepared by Raphael Barbey at The University of Sydney.255

Butyltrithiocarbonate propanoic acid (BTCPA) (1.0 g, 4.3 mmol) was dissolved in DCM (50 mL)

and cooled in an ice bath. To this was added propargyl alcohol (1.2 g, 22 mmol), 1-[3-

(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 1.7 g, 8.6 mmol) and

N,N-dimethylaminopyridine (DMAP, 0.5 g, 4.4 mmol). After reaction for two hours at 0 °C and

then for 16 hours at room temperature, the excess reagents were removed by washing with

water (5 x 20 ml). The organic fraction was dried over MgSO4, concentrated, and passed over a

silica pad with Toluene/EtOAc (9:1). The solvent was evaporated to give 5 as a yellow oil (1.1 g,

3.1 mmol, 72 %). 1H NMR (200 MHz, CDCl3) ppm: 0.86 (t, J = 7.2 Hz, 3H), 1.36 (sext., J = 7.0

Hz, 2H), 1.49-1.72 (m, 5H), 2.42 (t, J = 2.5 Hz, 1H), 3.29 (t, 2H), 4.66 (d, J = 10.8 Hz, 2H), 4.78 (q,

J = 7.4 Hz, 1H).

8.3. ANALYSIS TECHNIQUES

8.3.1. Thin Layer Chromatography (TLC)

Elution of monomer or RAFT agent was tracked using TLC plates. A small drop of sample was

placed on a TLC silica-plate, allowed to absorb the eluent and then viewed under UV-light.

Sample stains were marked for later referral.

8.3.2. Size Exclusion Chromatography (SEC)

8.3.2.1. DMAc SEC with RI and UV-Vis Detectors

SEC was performed using a Shimadzu modular system comprising a DGU-12A degasser,

LC-10AT pump, SIL-10AD automatic injector, CTO-10A column oven, RID-10A refractive index

detector, and a SPD-10A Shimadzu UV-Vis detector. A 50 × 7.8 mm guard column and four 300

× 7.8 mm linear columns (500, 103, 104, and 105 Å pore size, 5 μm particle size) were used for

the analyses. N,N-dimethylacetamide (DMAc) (HPLC grade, 0.05 % w/v of 2,6-dibutyl-4-

methylphenol (BHT), 0.03 % w/v of LiBr) with a flow rate of 1 mL.min–1 and a constant


211

temperature of 50 °C was used as the mobile phase with an injection volume of 50 μL. The

samples were filtered through 0.45 μm filters. The unit was calibrated using commercially

available linear polystyrene standards (0.5 – 1000 kDa, Polymer Laboratories). Chromatograms

were processed using Cirrus 2.0 software (Polymer Laboratories).

8.3.2.2. DMF SEC with RI Detector (KCPC, The University of Sydney)

SEC was performed on a Varian GPC50 with differential refractive index (DRI) detection.

Retention times and dispersities of the polymers and conjugates were determined relative to a

set of narrow molecular weight polystyrene standards (1 – 1600 kDa). DMF with 0.1 % (wt) LiBr

was taken as the eluent at a flowrate of 0.7 mL min-1 at 50 °C. Samples were prepared by

filtration of a solution of 2 – 4 mg.mL-1 polymer through a 0.45 µm PTFE filter. Water (1 % v/v)

was taken as a flowrate marker.

8.3.2.3. Water SEC

SEC was implemented using a Shimadzu modular system comprising a DGU-12A degasser, LC-

10AT pump, SIL-10AD automatic injector, CTO-10A column oven and a RID-10A refractive index

detector. An Agilent aquagel-OH guard column and two linear columns (Agilent PL aquagel-OH

Mixed-H and Mixed-M 8 μm particle size) were used for the analyses. A MilliQ water/acetic

acid/methanol (54:23:23) solution with a flow rate of 0.8 mL min–1 and a constant temperature

of 30 °C was used as the mobile phase with an injection volume of 100 μL. The samples were

filtered through 0.45 μm filters. The unit was calibrated using commercially available linear

polyethylene oxide standards (0.5 – 1000 kDa, Polymer Laboratories). Chromatograms were

processed using Cirrus 2.0 software (Polymer Laboratories).

8.3.3. Nuclear Magnetic Resonance (NMR) Spectrometry

8.3.3.1. Solution-State NMR

NMR general characterisation analyses were conducted using a Bruker Avance DPX 300

spectrometer (1H, 300.2 MHz). Further characterisation was conducted using a Bruker DMX

600 MHz spectrometer (1H, 600.13 MHz) with a QNP probe and an Avance III 500 MHz

spectrometer (1H, 500.13 MHz) with a TBI probe. The 600 MHz and 500 MHz spectrometer

parameters were: 8390 Hz sweep width, 1.95 s acquisition time, 2 s recycle delay; 6009 Hz

sweep width, 5.45 s acquisition time, 5 s recycle delay; respectively. The pulse program


212

WATERGATE was used for water suppression.256 Samples were analysed in deuterated

solvents. All chemical shifts are stated in ppm (δ) relative to tetramethylsilane (δ = 0 ppm),

referenced to the chemical shifts of residual solvent resonances (1H and 13C). 31P spectra were

calibrated to H3PO4 (85 %) (δ = 0 ppm).

8.3.3.2. Solid-State NMR

Dr. Aditya Rawal kindly performed the solid-state analyses at the Nuclear Magnetic Resonance

Facility, UNSW. The solid-state NMR measurements were carried out on a Bruker Avance III

300 MHz spectrometer (1H, 299.8 MHz). The samples were center packed in 4 mm zirconia

rotors with Kel-F® caps and spun up to 12 kHz in a Bruker 4 mm H-X double resonance wide

bore solids probe head with variable temperature capability. The 1H NMR T1H measurements

were done using a saturation recovery experiment involving a saturation comb made of fifty to

a hundred delays of 30 s to 1 ms duration each. The recycle delays were set to 100 ms with

256 to 512 transients detected for signal averaging.

8.3.4. Mass Spectrometry (MS)

All samples were analysed using a Thermo Finnigan LCQ Deca quadrupole ion trap mass

spectrometer (Thermo Finnigan, San Jose, CA) equipped with an atmospheric pressure

ionization source operating in the nebulizer-assisted electrospray mode used in the positive

ion mode. Mass calibration was performed using caffeine, Met-Arg-Phe-Ala acetate salt

(MRFA, Sigma-Aldrich, 90 %), and Ultramark 1621 in the m/z range 195–1822. All spectra were

acquired within the m/z range of 150–2000. Typical instrumental parameters were a spray

voltage of 4.5 kV, a capillary voltage of 44 V, a capillary temperature of 275 °C, and a flow rate

of 5 μL min−1. Nitrogen was used as the sheath gas (flow: 50 % maximum), and helium was

used as the auxiliary gas (flow: 5 % maximum). Approximately 35 scans were signal averaged

to obtain the final mass spectrum.

Samples were analysed in methanol or water/methanol (1:1). All data were processed using

the Xcalibur™ software included with Thermo Finnigan products. Theoretical molecular

weights were calculated using the exact mass for the most abundant peak in any given isotopic

pattern. Molecular weights were calculated using the following values: C12 = 11.9994516, H1 =

1.0072766, O16 = 15.9943662, Cl17 = 34.9683043, N15 = 14.0025256, P15 = 30.9732131 and Ru44

= 95.9070496.


213

8.3.5. X-Ray Crystallography

Dr. Mohan Bhadbhade kindly performed the x-ray crystallography analyses at the Solid State &

Elemental Analysis Unit, UNSW. Crystals were grown from a DCM/Heptane layered solution in

a glove-box. The single crystal of the compound for analysis was mounted on a Bruker APEXII

CCD single crystal diffractometer equipped with graphite monochromated MoK radiation.

8.3.6. UV-Vis Spectrometry

Samples in Chapter 2 were analysed using a Varian Cary 300 UV-Vis Spectrophotometer, fitted

with a single cell chamber. Samples were analysed at concentrations of 1-5 mg.mL-1 in MeOH

and water, from 200 – 800 nm at a scan rate of 600 nm.min-1.

Samples in Chapter 3 were analysed using a UV-1700 PharmaSpec Shimadzu spectrometer,

fitted with two cell chambers. Samples were analysed at concentrations of 0.50, 0.25, 0.13,

0.06 and 0.03 mg.mL-1 (where 0.1 < A < 1) in DMSO and water, against a reference cell

containing either DMSO or water.

8.3.7. Fourier-Transform Near-Infrared (FT-NIR) Spectroscopy

For Chapter 2, FT-NIR spectroscopy was used to determine monomer conversions by following

the decrease of the vinylic stretching overtone of the monomer. A Bruker IFS 66\S Fourier

transform spectrometer equipped with a tungsten halogen lamp, a CaF2 beam splitter, and a

liquid nitrogen cooled InSb detector was used. The sample was placed in a FT-NIR quartz

cuvette (2 mm) and polymerised at 65 °C. Traces were elaborated with OPUS software.

For Chapter 5, FT-IR was conducted at KCPC (The University of Sydney) on a Varian Scimitar

800 FT-IR in DRIFTS (diffuse reflectance infrared fourier transform spectroscopy) mode, against

a KBr background.


214

8.3.8. Dynamic Light Scattering (DLS)

Particle sizes were determined using a Malvern Zetasizer Nanoseries Nano-ZS instrument

equipped with a 4 mV He-Ne laser operating at λ = 632 nm. Solution concentrations were

~1 mg.mL-1 polymer in MilliQ water at 25 °C. Five measurements were averaged for each

sample.

8.3.9. Microscopy

8.3.9.1. Transmission Electron Microscopy (TEM)

TEM micrographs were obtained using a JEOL1400 transmission electron microscope,

consisting of a dispersive x-ray analyser interfaced to the column and a Gatan CCD facilitating

the acquisition of digital images. It was operated at an accelerating voltage of 80 kV. Samples

were prepared by casting a 1 mg.mL-1 polymer solution onto a copper grid. Negative staining

using uranyl acetate or phosphotungstic acid, or positive staining using osmium tetroxide, was

employed.

8.3.9.2. Laser Scanning Confocal Microscopy

Confocal microscope images were obtained using a laser scanning confocal microscope system

(Zeiss LSM 780) equipped with a Diode 405-30 laser, an argon laser and a DPSS 561-10 laser

(excitation and absorbance wavelengths: 405 nm, 488 nm and 561 nm, respectively)

connected to a Zeiss Axio Observer.Z1 inverted microscope (oil immersion × 100 /1.4 NA

objective). The ZEN2011 imaging software (Zeiss) was used for image acquisition and

processing.

8.3.10. Thermo Gravimetric Analysis (TGA)

Thermal decomposition properties of products were recorded using a TG5000

Thermogravimetric Analyzer. The sample (< 1 mg) was placed on a thermo-balance and heated

from 25 ᵒC to 600 ᵒC at 20 ᵒC min-1 and held isothermally for 10 minutes under an air or

nitrogen atmosphere.


215

8.3.11. Elemental Analysis

8.3.11.1. Inductively Coupled Plasma Optical Emission Spectrometry

(ICPOES)

Dr. Dorothy Yu kindly performed the ICPOES analyses at the Solid State & Elemental Analysis

Unit, UNSW. The Perkin-Elmer Optima DV7300 inductively coupled plasma optical emission

spectrometer (ICPOES) (Perkin-Elmer, Norwalk, CT, U.S.A.) was used for quantitative

determinations of ruthenium. All experiments were carried out using the following

parameters: incident ratio frequency power of 1300 W, plasma gas flow of 15 L.min−1, auxiliary

argon flow of 0.5 L.min−1, pump rate of 0.5 mL.min-1. The nebulizer gas flow was adjusted to

maximize ion intensity at 0.70 L.min−1, as indicated by the mass flow controller. The sample

introduction system employed was a Burgener PEEK Mira Mist nebuliser with cyclonic spray

chamber. Samples were viewed in axial mode at a height of 15 mm above the load coil. The

element/mass detected was 44Ru using 0, 0.1, 1 and 10 mg.mL-1 standards diluted from

certified 1000 mg.L-1 standards, and 1 ppm Yittium was added online as an internal standard to

correct for the loss of analyte during sample preparation or sample inlet. A total of three

replicates were measured at a normal resolution. Samples were analysed with and without

prior digestion with Aqua Regia solution at 90 °C for one hour.

8.3.11.2. Inductively Coupled Plasma Mass Spectrometry (ICPMS)

Dr. Dorothy Yu kindly performed the ICPMS analyses at the Solid State & Elemental Analysis

Unit, UNSW. The Perkin-Elmer NexION 300D inductively coupled plasma mass spectrometer

(ICPMS) (Perkin-Elmer, Norwalk, CT, U.S.A.) was used for quantitative determinations of

ruthenium. All experiments were carried out using the following parameters: incident ratio

frequency power of 1400 W, plasma gas flow of 15 L.min−1, auxiliary argon flow of 1.2 L.min−1,

nebulizer gas flow of 0.98 L.min−1. The nebulizer gas flow was adjusted to maximize ion

intensity at 0.4 mL.min−1, as indicated by the mass flow controller. The sample introduction

system employed was a SeaSpray U-Series nebuliser with ESI Quartz Peltier Cooled cyclonic

spray chamber (PC3). The element/mass detected was 44Ru using 0.2, 1, 10 and 100 µg.L-1

standards prepared in 2 % Aqua Regia, and Rh102.905

used as an internal standard. A total of

three replicates were measured at a normal resolution. Samples were analysed after digestion

with Aqua Regia solution at 80 °C for one hour.


216

8.3.11.3. Solid-state Elemental Analysis

Polymer samples were sent to The Campbell Microanalytical Laboratory at the University of

Otago in New Zealand for elemental analysis.

8.3.12. Microwave Reactions

Microwave reactions were performed in a CEM Discover SP microwave reactor. The initial

power input was set to 200 W and the 5 mL vessel used was cooled using N2 throughout the

reaction to maximize the microwave power input, which averaged 50 W.

8.3.13. Fluorescence Spectrometry

The fluorescence intensities of micelles were measured under a Cary Eclipse Fluorescence

Spectrophotometer (Agilent Technologies). The excitation and emission wavelengths were 490

nm and 512 nm, respectively. The experiments were carried out in triplicate. The standard

deviation was calculated from three independent measurements.

217

REFERENCES

1. Australian Institute of Health and Welfare. Cancer in Australia: in brief 2012. Cancer 72, 1–27 (2012).

2. Abou-gharbia, M. & Childers, W. E. Discovery of Innovative Therapeutics: Today’s Realities and Tomorrow's Vision. Part 1. Criticisms Faced by the Pharmaceutical Industry. J. Med. Chem. 56, 5659–5672 (2013).

3. Antonarakis, E. S. & Emadi, A. Ruthenium-based chemotherapeutics: are they ready for prime time? Cancer Chemother. Pharm. 66, 1–9 (2010).

4. Duncan, R. Polymer conjugates as anticancer nanomedicines. Nat. Rev. Cancer 6, 688–701 (2006).

5. Allardyce, C. S., Dorcier, A., Scolaro, C. & Dyson, P. J. Development of organometallic (organo-transition metal) pharmaceuticals. Appl. Organomet. Chem. 19, 1–10 (2005).

6. Miyata, K., Christie, R. J., Kataoka, K. & James Christie, R. Polymeric micelles for nano-scale drug delivery. React. Funct. Polym. 71, 227–234 (2011).

7. Larson, N. & Ghandehari, H. Polymeric conjugates for drug delivery. Chem. Mater. 24, 840–853 (2012).

8. Satchi-Fainaro, R., Duncan, R. & Barnes, C. M. Polymer Therapeutics for Cancer: Current Status and Future Challenges. Adv. Polym. Sci. 193, 1–65 (2006).

9. Huynh, V. T., Quek, J. Y., de Souza, P. L. & Stenzel, M. H. Block copolymer micelles with pendant bifunctional chelator for platinum drugs: effect of spacer length on the viability of tumor cells. Biomacromolecules 13, 1010–23 (2012).

10. Pearson, S., Allen, N. & Stenzel, M. H. Core-shell particles with glycopolymer shell and polynucleoside core via RAFT: From micelles to rods. J. Polym. Sci., Part A Polym. Chem. 47, 1706–1723 (2009).

11. Savić, R., Eisenberg, A. & Maysinger, D. Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle-cell interactions. J. Drug Target. 14, 343–55 (2006).

12. Peacock, A. F. A. & Sadler, P. J. Medicinal organometallic chemistry: designing metal arene complexes as anticancer agents. Chem. Asian J. 3, 1890–9 (2008).

13. Wu, B. et al. A ruthenium antimetastasis agent forms specific histone protein adducts in the nucleosome core. Chem. Eur. J. 17, 3562–6 (2011).

14. Morris, R. R. E. et al. Inhibition of cancer cell growth by ruthenium (II) arene complexes. J. Med. Chem. 44, 3616–21 (2001).

References

218

15. Allardyce, C. S. & Dyson, P. J. Ruthenium in Medicine : Current Clinical Uses and Future Prospects. Platin. Met. Rev. 45, 62–9 (2001).

16. Dyson, P. J. & Sava, G. Metal-based antitumour drugs in the post genomic era. Dalt. Trans. 35, 1929–33 (2006).

17. Sava, G. et al. Pharmacological control of lung metastases of solid tumours by a novel ruthenium complex. Clini. Exp. Metastasis 16, 371–9 (1998).

18. Kostova, I. Ruthenium complexes as anticancer agents. Curr. Med. Chem. 13, 1085–1107 (2006).

19. Allardyce, C. S., Dyson, P. J., Ellis, D. J. & Heath, S. L. [Ru(η6-p-cymene)Cl2(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane): a water soluble compound that exhibits pH dependent DNA binding providing selectivity for diseased cells. Chem. Commun. (Cambridge, U. K.) 37, 1396–7 (2001).

20. Bergamo, A., Masi, A., Dyson, P. J. & Sava, G. Modulation of the metastatic progression of breast cancer with an organometallic ruthenium compound. Int. J. Oncol. 33, 1281–1289 (2008).

21. Scolaro, C. et al. In vitro and in vivo evaluation of ruthenium(II)-arene PTA complexes. J. Med. Chem. 48, 4161–71 (2005).

22. Chatterjee, S., Kundu, S., Bhattacharyya, A., Hartinger, C. G. & Dyson, P. J. The ruthenium(II)-arene compound RAPTA-C induces apoptosis in EAC cells through mitochondrial and p53-JNK pathways. J. Biol. Inorg. Chem. 13, 1149–55 (2008).

23. Scolaro, C. et al. Influence of Hydrogen-Bonding Substituents on the Cytotoxicity of RAPTA Compounds. Organometallics 25, 756–65 (2006).

24. Ambade, A. V, Savariar, E. N. & Thayumanavan, S. Dendrimeric micelles for controlled drug release and targeted delivery. Mol. Pharm. 2, 264–72 (2005).

25. Barz, M., Luxenhofer, R., Zentel, R. & Kabanov, A. V. The uptake of hydroxypropyl methacrylamide based homo, random and block copolymers by human multi-drug resistant breast adenocarcinoma cells. Biomaterials 30, 5682–90 (2009).

26. Duncan, R. Polymer therapeutics as nanomedicines: new perspectives. Curr. Opin. Biotechnol. 22, 492–501 (2011).

27. Stenzel, M. H. RAFT polymerization: an avenue to functional polymeric micelles for drug delivery. Chem. Commun. (Cambridge, U. K.) 44, 3486–503 (2008).

28. Matsumura, Y. & Maeda, H. A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs. Cancer Res. 46, 6387–92 (1986).

29. Gopal, Y. N., Jayaraju, D. & Kondapi, A. K. Inhibition of topoisomerase II catalytic activity by two ruthenium compounds: a ligand-dependent mode of action. Biochem. 38, 4382–8 (1999).

References

219

30. Bergamo, A. et al. In vitro cell cycle arrest, in vivo action on solid metastasizing tumors, and host toxicity of the antimetastatic drug NAMI-A and cisplatin. J. Pharm. Exp. Ther. 289, 559–64 (1999).

31. Bergamo, A. & Sava, G. Ruthenium complexes can target determinants of tumour malignancy. Dalt. Trans. 36, 1267–72 (2007).

32. NCI at NIH. Defining Cancer. What is Cancer (2012). at <http://www.cancer.gov/cancertopics/cancerlibrary/what-is-cancer/>

33. Bratsos, I., Jedner, S., Gianferrara, T. & Alessio, E. Ruthenium Anticancer Compounds: Challenges and Expectations. Chimia (Aarau). 61, 692–7 (2007).

34. Mangiapia, G. et al. Ruthenium-based complex nanocarriers for cancer therapy. Biomaterials 33, 3770–82 (2012).

35. Castonguay, A., Doucet, C., Juhas, M. & Maysinger, D. New ruthenium(II)-letrozole complexes as anticancer therapeutics. J. Med. Chem. 55, 8799–806 (2012).

36. Hill, B. Drug resistance. Int. J. Oncol. 9, 197–203 (1996).

37. Salvà Lacombe, P., García Vicente, J. A. A., Costa Pagès, J. & Lucio Morselli, P. Causes and problems of nonresponse or poor response to drugs. Drugs 51, 552–70 (1996).

38. Vock, C. a et al. Development of ruthenium antitumor drugs that overcome multidrug resistance mechanisms. J. Med. Chem. 50, 2166–75 (2007).

39. Aird, R. E. et al. In vitro and in vivo activity and cross resistance profiles of novel ruthenium (II) organometallic arene complexes in human ovarian cancer. Br. J. Cancer 86, 1652–7 (2002).

40. Han Ang, W. & Dyson, P. J. Classical and Non-Classical Ruthenium-Based Anticancer Drugs: Towards Targeted Chemotherapy. Euro. J. Inorg. Chem. 20, 4003–18 (2006).

41. Rosenberg, B., Van Camp, L. & Krigas, T. Inhibition of Cell Division in Escherichia coli by Electrolysis Products from a Platinum Electrode. Nature 205, 698–9 (1965).

42. Rosenberg, B., Vancamp, L., Trosko, J. E. & Mansour, V. H. Platinum Compounds: a New Class of Potent Antitumour Agents. Nature 222, 385–6 (1969).

43. Romero-Canelón, I., Salassa, L. & Sadler, P. J. The contrasting activity of iodido versus chlorido ruthenium and osmium arene azo- and imino-pyridine anticancer complexes: control of cell selectivity, cross-resistance, p53 dependence, and apoptosis pathway. J. Med. Chem. 56, 1291–300 (2013).

44. Pizarro, A. M. & Sadler, P. J. Unusual DNA binding modes for metal anticancer complexes. Biochimie 91, 1198–211 (2009).

45. Clarke, M. J., Zhu, F. & Frasca, D. R. Non-Platinum Chemotherapeutic Metallopharmaceuticals. Chem. Rev. 99, 2511–34 (1999).

http://www.cancer.gov/cancertopics/cancerlibrary/what-is-cancer/

References

220

46. Levina, A., Mitra, A. & Lay, P. A. Recent developments in ruthenium anticancer drugs. Metallomics 1, 458–70 (2009).

47. Basolo, F. & Johnson, R. C. Coordination Chemistry. (Whitstable Litho Ltd, 1986).

48. Cotton, F., Wilkinson, G., Gaus, P. & Bryant, R. Basic inorganic chemistry. 1–708 (Wiley, 1987). at <http://www.getcited.org/pub/102545063>

49. Pizarro, A., Habtemariam, A. & Sadler, P. in Med. Organomet. Chem. (Jaouen, G. & Metzler-Nolte, N.) 32, 21–56 (Springer Berlin Heidelberg, 2010).

50. King, R. B. & Johnson, M. Encyclopedia of Inorganic Chemistry. by RB King, John Wiley Sons, New York 4, 1896—915 (John Wiley & Sons Ltd, 1994).

51. Sabo-Etienne, S. & Grellier, M. in Encycl. Inorg. Chem. 4757–75 (John Wiley & Sons Ltd, 2006). at <http://dx.doi.org/10.1002/0470862106.ia208>

52. Alessio, E. et al. Synthesis and characterization of two new classes of ruthenium(III)-sulfoxide complexes with nitrogen donor ligands (L): Na[trans-RuCl4(R2SO)(L)] and mer, cis-RuCl3(R2SO)(R2SO)(L). The crystal structure of Na[trans-RuCl4(DMSO)(NH3)] · 2DMSO, Na[trans-RuCl. Inorg. Chim. Acta 203, 205–17 (1993).

53. Phillips, A. D., Gonsalvi, L., Romerosa, A., Vizza, F. & Peruzzini, M. Coordination chemistry of 1,3,5-triaza-7-phosphaadamantane (PTA). Coord. Chem. Rev. 248, 955–93 (2004).

54. Furrer, M. A., Schmitt, F., Wiederkehr, M., Juillerat-Jeanneret, L. & Therrien, B. Cellular delivery of pyrenyl-arene ruthenium complexes by a water-soluble arene ruthenium metalla-cage. Dalt. Trans. 41, 7201–11 (2012).

55. Batista, A. A. et al. Ruthenium complexes containing tertiary phosphines and imidazole or 2,2′-bipyridine ligands. Inorg. Chim. Acta 230, 111–7 (1995).

56. Bennett, M. A. & Smith, A. K. Arene Ruthenium (II) Complexes formed by Dehydrogenation of Cyclo-hexadienes with Ruthenium (III) Trichloride. J. Chem. Soc., Dalt. Trans. 2, 233–41 (1974).

57. Wang, F. et al. Kinetics of aquation and anation of ruthenium(II) arene anticancer complexes, acidity and X-ray structures of aqua adducts. Chem. Eur. J. 9, 5810–20 (2003).

58. Sundberg, R. J., Bryan, R. F., Taylor, I. F. & Taube, H. Nitrogen-Bound and Carbon-Bound Imidazole Complexes of Ruthenium Ammines’. J. Am. Chem. Soc 96, 381–92 (1974).

59. Chen, H., Parkinson, J. A., Morris, R. E. & Sadler, P. J. Highly selective binding of organometallic ruthenium ethylenediamine complexes to nucleic acids: novel recognition mechanisms. JACS 125, 173–86 (2003).

60. McCormick, F. B., Cox, D. D. & Gleason, W. B. Synthesis, structure, and disproportionation of labile benzeneruthenium acetonitrile (.eta.6-C6H6)Ru(CH3CN)2Cl+ salts. Organometallics 12, 610–2 (1993).

http://www.getcited.org/pub/102545063

http://dx.doi.org/10.1002/0470862106.ia208

References

221

61. Fernández, R., Melchart, M., Habtemariam, A., Parsons, S. & Sadler, P. J. Use of chelating ligands to tune the reactive site of half-sandwich ruthenium(II)-arene anticancer complexes. Chem. Eur. J. 10, 5173–9 (2004).

62. Bergamo, A., Gaiddon, C., Schellens, J. H. M., Beijnen, J. H. & Sava, G. Approaching tumour therapy beyond platinum drugs: status of the art and perspectives of ruthenium drug candidates. J. Inorg. Biochem. 106, 90–9 (2012).

63. Peacock, A. F. A. et al. Tuning the reactivity of osmium(II) and ruthenium(II) arene complexes under physiological conditions. J. Am. Chem. Soc 128, 1739–48 (2006).

64. Allardyce, C. S., Dyson, P. J., Ellis, D. J., Salter, P. A. & Scopelliti, R. Synthesis and characterisation of some water soluble ruthenium(II)–arene complexes and an investigation of their antibiotic and antiviral properties. J. Organomet. Chem. 668, 35–42 (2003).

65. Sava, G., Pacor, S., Zorzet, S., Alessio, E. & Mestroni, G. Antitumour properties of dimethylsulphoxide ruthenium (II) complexes in the Lewis lung carcinoma system. Pharmacol. Res. 21, 617–28 (1989).

66. Cocchietto, M., Zorzet, S., Sorc, A. & Sava, G. Primary tumor, lung and kidney retention and antimetastasis effect of NAMI-A following different routes of administration. Invest. New Drugs 21, 55–62 (2003).

67. Ratanaphan, A., Temboot, P. & Dyson, P. J. In vitro ruthenation of human breast cancer suppressor gene 1 (BRCA1) by the antimetastasis compound RAPTA-C and its analogue CarboRAPTA-C. Chem. Biodivers. 7, 1290–302 (2010).

68. Nowak-Sliwinska, P. et al. Organometallic ruthenium(II) arene compounds with antiangiogenic activity. J. Med. Chem. 54, 3895–902 (2011).

69. Frausin, F. et al. Tumour cell uptake of the metastasis inhibitor ruthenium complex NAMI-A and its in vitro effects on KB cells. Cancer Chemother. Pharm. 50, 405–11 (2002).

70. Sun, H., Li, H. & Sadler, P. J. Transferrin as a metal ion mediator. Chem. Rev. 99, 2817–42 (1999).

71. Guo, W. et al. Transferrin serves as a mediator to deliver organometallic ruthenium(II) anticancer complexes into cells. Inorg. Chem. 52, 5328–38 (2013).

72. Guidi, F. et al. The molecular mechanisms of antimetastatic ruthenium compounds explored through DIGE proteomics. J. Inorg. Biochem. 118, 94–9 (2013).

73. Pongratz, M. et al. Transferrin binding and transferrin-mediated cellular uptake of the ruthenium coordination compound KP1019, studied by means of AAS, ESI-MS and CD spectroscopy. J. Anal. At. Spectrom. 19, 46 (2004).

74. Mestroni, G., Alessio, E. & Sava, G. Salts of anionic complexes of RU (III), as antimetastatic and antineoplastic agents. US Pat. 6,221,905 14 (2001).

References

222

75. Bacac, M. et al. Cocultures of metastatic and host immune cells: selective effects of NAMI-A for tumor cells. Cancer Immunol. Immunother. 53, 1101–10 (2004).

76. Scolaro, C. et al. Tuning the hydrophobicity of ruthenium(II)-arene (RAPTA) drugs to modify uptake, biomolecular interactions and efficacy. Dalt. Trans. 36, 5065–72 (2007).

77. Vacca, A. et al. Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A. Br. J. Cancer 4, 993–8 (2002).

78. Alessio, E., Mestroni, G., Bergamo, A. & Sava, G. Ruthenium antimetastatic agents. Curr. Top. Med. Chem. 4, 1525–35 (2004).

79. Vadori, M. et al. Features and full reversibility of the renal toxicity of the ruthenium-based drug NAMI-A in mice. J. Inorg. Biochem. 118, 21–7 (2013).

80. Bacac, M. et al. The hydrolysis of the anti-cancer ruthenium complex NAMI-A affects its DNA binding and antimetastatic activity: an NMR evaluation. J. Inorg. Biochem. 98, 402–12 (2004).

81. Groessl, M., Hartinger, C. G., Dyson, P. J. & Keppler, B. K. CZE-ICP-MS as a tool for studying the hydrolysis of ruthenium anticancer drug candidates and their reactivity towards the DNA model compound dGMP. J. Inorg. Biochem. 102, 1060–5 (2008).

82. Hartinger, C. G. et al. From bench to bedside--preclinical and early clinical development of the anticancer agent indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J. Inorg. Biochem. 100, 891–904 (2006).

83. Scolaro, C., Hartinger, C. G., Allardyce, C. S., Keppler, B. K. & Dyson, P. J. Hydrolysis study of the bifunctional antitumour compound RAPTA-C, [Ru(eta6-p-cymene)Cl2(pta)]. J. Inorg. Biochem. 102, 1743–8 (2008).

84. Heffeter, P. et al. Intracellular protein binding patterns of the anticancer ruthenium drugs KP1019 and KP1339. J. Biol. Inorg. Chem. 15, 737–48 (2010).

85. Groessl, M., Zava, O. & Dyson, P. J. Cellular uptake and subcellular distribution of ruthenium-based metallodrugs under clinical investigation versus cisplatin. Metallomics 3, 591–9 (2011).

86. Renfrew, A. Ruthenium(II) Arene Compounds as Versatile Anticancer Agents. Chimia (Aarau). 63, 217–9 (2009).

87. Renfrew, A. K., Juillerat-Jeanneret, L. & Dyson, P. J. Adding diversity to ruthenium(II)–arene anticancer (RAPTA) compounds via click chemistry: The influence of hydrophobic chains. J. Organomet. Chem. 696, 772–9 (2011).

88. Hadjiliadis, N. & Sletten, E. Metal Complex-DNA Interactions. (Blackwell Publishing Ltd, 2009).

89. Zelonka, R. & Baird, M. Reactions of benzene complexes of ruthernium (II). J. Organomet. Chem. 35, 43–6 (1972).

References

223

90. Dyson, P. J., Ellis, D. J. & Laurenczy, G. G. Minor modifications to the ligands surrounding a ruthenium complex lead to major differences in the way in which they catalyse the hydrogenation of arenes. Adv. Synth. Catal. 345, 211–5 (2003).

91. Casini, A. et al. Reactivity of an antimetastatic organometallic ruthenium compound with metallothionein-2: relevance to the mechanism of action. Metallomics 1, 434–41 (2009).

92. Renfrew, A. K. et al. Influence of Structural Variation on the Anticancer Activity of RAPTA-Type Complexes: ptn versus pta. Organometallics 28, 1165–72 (2009).

93. Wolters, D. A., Stefanopoulou, M., Dyson, P. J. & Groessl, M. Combination of metallomics and proteomics to study the effects of the metallodrug RAPTA-T on human cancer cells. Metallomics 4, 1185–96 (2012).

94. Casini, A. et al. Rationalization of the inhibition activity of structurally related organometallic compounds against the drug target cathepsin B by DFT. J. Magn. Reson. 39, 5556–63 (2010).

95. Casini, A. et al. Exploring metallodrug-protein interactions by mass spectrometry: comparisons between platinum coordination complexes and an organometallic ruthenium compound. J. Biol. Inorg. Chem. 14, 761–70 (2009).

96. Dorcier, A. et al. Binding of Organometallic Ruthenium(II) and Osmium(II) Complexes to an Oligonucleotide: A Combined Mass Spectrometric and Theoretical Study †. Organometallics 24, 2114–23 (2005).

97. Fisher, K. J., Alyea, E. C. & Shahnazarian, N. A 31P NMR Study of the Water Soluble Derivatives OF 1,3,5-Triaza-7-Phosphaadamantane (PTA). Phosphorus. Sulfur. Silicon Relat. Elem. 48, 37–40 (1990).

98. Bolaño, S. et al. PGSE NMR Studies on RAPTA Derivatives: Evidence for the Formation of H-Bonded Dicationic Species. Organometallics 27, 1649–52 (2008).

99. Servin, P. et al. Grafting of water-soluble phosphines to dendrimers and their use in catalysis: positive dendritic effects in aqueous media. Dalt. Trans. 38, 4432–4 (2009).

100. Pruchnik, F. P. & Smoleński, P. New rhodium(I) water-soluble complexes with 1-alkyl-1-azonia-3,5-diaza-7-phospha-adamantane iodides and their catalytic activity. Appl. Organomet. Chem. 13, 829–36 (1999).

101. García-Fernández, A., Díez, J., Gamasa, M. P., Lastra, E. & García-ferna, A. Facile modification of 1,3,5-triaza-7-phosphatricyclo[3.3.1.1(3,7)]decane phosphanes coordinated to ruthenium(II). Inorg. Chem. 48, 2471–81 (2009).

102. Bergamo, A. et al. Distinct effects of dinuclear ruthenium(III) complexes on cell proliferation and on cell cycle regulation in human and murine tumor cell lines. J. Pharm. Exp. Ther. 305, 725–32 (2003).

103. Williams, D. S. S. et al. Switching on a signaling pathway with an organoruthenium complex. Angew. Chem. Int. Ed. 44, 1984–7 (2005).

References

224

104. Douki, T., Reynaud-Angelin, A., Cadet, J. & Sage, E. Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation. Biochem. 42, 9221–6 (2003).

105. Cadet, J. et al. Hydroxyl radicals and DNA base damage. Mutat. Res. Mol. Mech. Mutagen. 424, 9–21 (1999).

106. Valerie, K. & Povirk, L. F. Regulation and mechanisms of mammalian double-strand break repair. Oncogene 22, 5792–812 (2003).

107. Shigenaga, M. K., Gimeno, C. J. & Ames, B. N. Urinary 8-hydroxy-2’-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc. Natl. Acad. Sci. U. S. A. 86, 9697–701 (1989).

108. Cathcart, R., Schwiers, E., Saul, R. L. & Ames, B. N. Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage. Proc. Natl. Acad. Sci. U. S. A. 81, 5633–7 (1984).

109. Stephens, T. D., Bunde, C. J. . & Fillmore, B. J. Mechanism of action in thalidomide teratogenesis. Biochem. Pharmacol. 59, 1489–99 (2000).

110. Ferguson, L. R. & Denny, W. A. The genetic toxicology of acridines. Mutat. Res. 258, 123–60 (1991).

111. Jeffrey, A. M. DNA modification by chemical carcinogens. Pharmacol. Ther. 28, 237–72 (1985).

112. M. F. Brana, B. S. P., M. Cacho, B. S. P., A. Gradillas, B. S. P., B. de Pascual-Teresa, B. S. P. & A. Ramos, B. S. P. Intercalators as Anticancer Drugs. Curr. Pharm. Des. 7, 1745–1780 (2001).

113. Horiuchi, K. Y. Y., Wang, Y., Diamond, S. L. L. & Ma, H. Microarrays for the functional analysis of the chemical-kinase interactome. J. Biomol. Screen. 11, 48–56 (2006).

114. Wöhrle, D. Macromolecular Metal Complexes: Materials for Various Applications. Angew. Chem. Int. Ed. 44, 7500–7502 (2005).

115. Lippard, S. J. & Berg, J. M. Principles of Bioinorganic Chemistry. (University Science Books, 1994).

116. Seymour, L. W., Duncan, R., Strohalm, J. & Kopecek, J. Effect of molecular weight (Mw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal, and intravenous administration to rats. J. Biomed. Master. Res. 21, 1341–58 (1987).

117. Stolnik, S., Illum, L. & Davis, S. S. Long circulating microparticulate drug carriers. Adv. Drug Deliv. Rev. 16, 195–214 (1995).

118. Rösler, A., Vandermeulen, G. W. M. & Klok, H.-A. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Adv. Drug Deliv. Rev. 53, 95–108 (2001).

References

225

119. Nishiyama, N. & Kataoka, K. Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery. Pharmacol. Ther. 112, 630–48 (2006).

120. Bendix, D. Chemical synthesis of polylactide and its copolymers for medical applications. Polym. Degrad. Stab. 59, 129–135 (1998).

121. Li, Y., Wang, J., Wientjes, M. G. & Au, J. L.-S. Delivery of nanomedicines to extracellular and intracellular compartments of a solid tumor. Adv. Drug Deliv. Rev. 64, 29–39 (2012).

122. Votano, J. R. J. et al. Prediction of aqueous solubility based on large datasets using several QSPR models utilizing topological structure representation. Chem. Biodivers. 1, 1829–41 (2004).

123. Mikhail, A. S. & Allen, C. Poly(ethylene glycol)-b-poly(epsilon-caprolactone) micelles containing chemically conjugated and physically entrapped docetaxel: synthesis, characterization, and the influence of the drug on micelle morphology. Biomacromolecules 11, 1273–80 (2010).

124. Gregory, A. & Stenzel, M. H. Complex polymer architectures via RAFT polymerization: From fundamental process to extending the scope using click chemistry and nature’s building blocks. Prog. Polym. Sci. 37, 38–105 (2012).

125. Duncan, R. Development of HPMA copolymer-anticancer conjugates: clinical experience and lessons learnt. Adv. Drug Deliv. Rev. 61, 1131–48 (2009).

126. Sat, Y. N., Burger, A. M., Fiebig, H. H., Sausville, E. A. & Duncan, R. Comparison of vascular permeability and enzymatic activation of the polymeric prodrug HPMA copolymer–doxorubicin (PK1) in human tumour xenografts. In Proc. Am. Assoc. Cancer Res. Vol. 90, 41 (1999).

127. Duncan, R. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2, 347–60 (2003).

128. Donaruma, L. G. Synthetic biologically active polymers. Prog. Polym. Sci. 4, 1–25 (1975).

129. Duncan, R. Drug-polymer conjugates: potential for improved chemotherapy. Anticancer. Drugs 3, 175–210 (1992).

130. Kopecek, J. HPMA copolymer–anticancer drug conjugates: design, activity, and mechanism of action. Euro. J. Pharma. Biopharm. 50, 61–81 (2000).

131. Duncan, R. in Biomed. Asp. Drug Target. (Muzykantov, V. R. & Torchilin, V. P.) 239 (Springer, 2002). at <http://books.google.com/books?hl=en&lr=&id=EI155610wCsC&pgis=1>

132. Harris, J. M. & Chess., R. B. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discov. 214–221 (2003).

http://books.google.com/books?hl=en&lr=&id=EI155610wCsC&pgis=1

References

226

133. Pasut, G., Guiotto, A. & Veronese, F. Protein, peptide and non-peptide drug PEGylation for therapeutic application. Expert Opin. Ther. Patents 14, 859–894 (2004).

134. Yokoyama, M. et al. Characterization and Anticancer Activity of the Micelle-forming Polymeric Anticancer Drug Adriamycin-conjugated Poly ( ethylene glycol ) -Poly ( aspartic acid ) Block Copolymer. Cancer Res. 50, 1693–1700 (1990).

135. Pack, D. W., Hoffman, A. S., Pun, S. & Stayton, P. S. Design and development of polymers for gene delivery. Nat. Rev. Drug Discov. 4, 581–93 (2005).

136. Ringsdorf, H. Structure and properties of pharmacologically active polymers. J. Polym. Sci. Polym. Symp. 51, 135–53 (2007).

137. Aghemo, A., Rumi, M. G. & Colombo, M. Pegylated interferons alpha2a and alpha2b in the treatment of chronic hepatitis C. Nat. Rev. Gastroenterol. Hepatol. 7, 485–94 (2010).

138. Nowotnik, D. P. & Cvitkovic, E. ProLindac (AP5346): a review of the development of an HPMA DACH platinum Polymer Therapeutic. Adv. Drug Deliv. Rev. 61, 1214–9 (2009).

139. Dipetrillo, T. et al. Neoadjuvant paclitaxel poliglumex, cisplatin, and radiation for esophageal cancer: a phase 2 trial. Am. J. Clin. Oncol. 35, 64–7 (2012).

140. Sausville, E. A. et al. Phase I study of XMT-1001 given IV every 3 weeks to patients with advanced solid tumors. J. Clin. Oncol. (2010).

141. Awada, A. A. et al. Significant Efficacy in a Phase 2 Study of NKTR-102 , a Novel Polymer Conjugate of Irinotecan , in Patients With Pre-Treated Metastatic Breast Cancer ( MBC ). San Antonio Breast Cancer Symp. 2010 (2010).

142. Vergote, I. B. et al. Phase II study of NKTR-102 in women with platinum-resistant/refractory ovarian cancer. J. Clin. Oncol. 28, (2010).

143. Kaneda, Y. et al. The use of PVP as a polymeric carrier to improve the plasma half-life of drugs. Biomaterials 25, 3259–66 (2004).

144. Kamada, H. et al. Antitumor Activity of Tumor Necrosis Factor-α Conjugated with Polyvinylpyrrolidone on Solid Tumors in Mice. Cancer 60, 6416–20 (2000).

145. Yasukawa, T. et al. Targeted Delivery of Anti–Angiogenic Agent TNP-470 Using Water-Soluble Polymer in the Treatment of Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 40, 2690–6 (1999).

146. Chipman, S. D., Oldham, F. B., Pezzoni, G. & Singer, J. W. Biological and clinical characterization of paclitaxel poliglumex (PPX, CT-2103), a macromolecular polymer-drug conjugate. Int. J. Nanomed. 1, 375–83 (2006).

147. Ljubimova, J. Y. et al. Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery. Nanomed. 3, 247–65 (2008).

References

227

148. Pasut, G. & Veronese, F. M. PEG conjugates in clinical development or use as anticancer agents: an overview. Adv. Drug Deliv. Rev. 61, 1177–88 (2009).

149. Kopecek, J. & Kopecková, P. HPMA copolymers: origins, early developments, present, and future. Adv. Drug Deliv. Rev. 62, 122–49 (2010).

150. Vasey, P. A. et al. Phase I Clinical and Pharmacokinetic Study of PK1 [N-(2-Hydroxypropyl ) methacrylamide Copolymer Doxorubicin]: First Member of a New Class of Chemotherapeutic Agents −− Drug-Polymer Conjugates. Clin. Cancer Res. 5, 83–94 (1999).

151. Duncan, R. & Vicent, M. J. Do HPMA copolymer conjugates have a future as clinically useful nanomedicines? A critical overview of current status and future opportunities. Adv. Drug Deliv. Rev. 62, 272–82 (2010).

152. Yokoyama, M. et al. Preparation of micelle-forming polymer-drug conjugates. Bioconj. Chem. 3, 295–301 (1992).

153. Kabanov, A. V., Vinogradov, S. V., Suzdaltseva, Y. G. & Alakhov, V. Y. Water-Soluble Block Polycations as Carriers for Oligonucleotide Delivery. Bioconjug. Chem. 6, 639–43 (1995).

154. Chandran, T., Katragadda, U., Teng, Q. & Tan, C. Design and evaluation of micellar nanocarriers for 17-allyamino-17-demethoxygeldanamycin (17-AAG). Int. J. Pharm. 392, 170–7 (2010).

155. Talelli, M., Rijcken, C. J. F., van Nostrum, C. F., Storm, G. & Hennink, W. E. Micelles based on HPMA copolymers. Adv. Drug Deliv. Rev. 62, 231–9 (2010).

156. Krimmer, S. G., Pan, H., Liu, J., Yang, J. & Kopeček, J. Synthesis and characterization of poly(ε-caprolactone)-block-poly[N-(2-hydroxypropyl)methacrylamide] micelles for drug delivery. Macromol. Biosci. 11, 1041–51 (2011).

157. Soga, O. et al. Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery. J. Control. Release 103, 341–53 (2005).

158. Clayden, J., Greeves, N. & Warren, S. Organic Chemistry. (Oxford University Press, 2000).

159. Jenkins, A. D., Jones, R. G. & Moad, G. Terminology for reversible-deactivation radical polymerization previously called “controlled” radical or “living” radical polymerization (IUPAC Recommendations 2010). Pure Appl. Chem. 82, 483–91 (2010).

160. Barner-Kowollik, C., Quinn, J. F., Morsley, D. R. & Davis, T. P. Modeling the reversible addition-fragmentation chain transfer process in cumyl dithiobenzoate-mediated styrene homopolymerizations: Assessing rate coefficients for the addition-fragmentation equilibrium. J. Polym. Sci., Part A Polym. Chem. 39, 1353–65 (2001).

161. Rizzardo, E. & Solomon, D. H. On the Origins of Nitroxide Mediated Polymerization (NMP) and Reversible Addition-Fragmentation Chain Transfer (RAFT). Aust. J. Chem. 65, 945–69 (2012).

References

228

162. Wang, J. & Matyjaszewski, K. Controlled/“living” radical polymerization. atom transfer radical polymerization in the presence of transition-metal complexes. J. Am. Chem. Soc. 117, 5614–5615 (1995).

163. Stenzel, M. in Handb. RAFT Polym. (Barner-Kowollik, C.) 73 (CAMD, UNSW, 2008).

164. Odian, G. Principles of Polymerization. 1–832 (Wiley-Interscience, 2004).

165. Peppas, N. A. & Odian, G. Principles of polymerization. J. Control. Release 22, 294 (1992).

166. Moad, G., Rizzardo, E. & Thang, S. H. Living Radical Polymerization by the RAFT Process. Aust. J. Chem. 58, 379 (2005).

167. Chiefari, J. et al. Living Free-Radical Polymerization by Reversible Addition-Fragmentation Chain Treansfer: The RAFT Process. Macromolecules 31, 5559–62 (1998).

168. Moad, G., Rizzardo, E. & Thang, S. H. Living Radical Polymerization by the RAFT Process - A Second Update. Aust. J. Chem. 62, 1402–72 (2009).

169. Ting, S. R. S., Min, E. H., Zetterlund, P. B. & Stenzel, M. H. Controlled/Living ab Initio Emulsion Polymerization via a Glucose RAFT stab: Degradable Cross-Linked Glyco-Particles for Concanavalin A/Fim H Conjugations to Cluster E. coli Bacteria. Macromolecules 43, 5211–21 (2010).

170. Zhang, L., Katapodi, K., Davis, T. P., Barner-Kowollik, C. & Stenzel, M. H. Using the reversible addition–fragmentation chain transfer process to synthesize core-crosslinked micelles. J. Polym. Sci., Part A Polym. Chem. 44, 2177–94 (2006).

171. Nguyen, T. L. U., Eagles, K., Davis, T. P., Barner-Kowollik, C. & Stenzel, M. H. Investigation of the influence of the architectures of poly(vinyl pyrrolidone) polymers made via the reversible addition–fragmentation chain transfer/macromolecular design via the interchange of xanthates mechanism on the stabilization of suspension polym. J. Polym. Sci., Part A Polym. Chem. 44, 4372–83 (2006).

172. Sigma-Aldrich. RAFT: Choosing the Right Agent to Achieve Controlled Polymerization. RAFT Polym. (2013). at <http://www.sigmaaldrich.com/materials-science/polymer-science/raft-polymerization.html>

173. Destarac, M., Charmot, D., Franck, X. & Zard, S. Z. Dithiocarbamates as universal reversible addition-fragmentation chain transfer agents. Macromol. Rapid Commun. 21, 1035–9 (2000).

174. Thang, S. H., Chong, (Bill)Y.K., Mayadunne, R. T. A., Moad, G. & Rizzardo, E. A novel synthesis of functional dithioesters, dithiocarbamates, xanthates and trithiocarbonates. Tetrahedron Lett. 40, 2435–8 (1999).

175. Mayadunne, R. T. A. et al. Living Polymers by the Use of Trithiocarbonates as Reversible Addition−Fragmentation Chain Transfer (RAFT) Agents: ABA Triblock Copolymers by Radical Polymerization in Two Steps. Macromolecules 33, 243–5 (2000).

http://www.sigmaaldrich.com/materials-science/polymer-science/raft-polymerization.html



References

229

176. Francis, R. & Ajayaghosh, A. Minimization of Homopolymer Formation and Control of Dispersity in Free Radical Induced Graft Polymerization Using Xanthate Derived Macro-photoinitiators. Macromolecules 33, 4699–704 (2000).

177. Moad, G., Rizzardo, E. & Thang, S. H. Living Radical Polymerization by the RAFT Process—A First Update. Aust. J. Chem. 59, 669–92 (2006).

178. Moad, G., Rizzardo, E. & Thang, S. H. Radical addition–fragmentation chemistry in polymer synthesis. Polymer (Guildf). 49, 1079–1131 (2008).

179. Dubé, M., Penlidis, A., Reilly, P. P. M. & Dube, M. A. A systematic approach to the study of multicomponent polymerization kinetics: the butyl acrylate/methyl methacrylate/vinyl acetate example. IV. Optimal Bayesian design of emulsion terpolymerization experiments in a pilot plant reactor. J. Polym. Sci., Part A Polym. Chem. 34, 811–31 (1996).

180. Merz, E., Alfrey, T. & Goldfinger, G. Intramolecular reactions in vinyl polymers as a means of investigation of the propagation step. J. Polym. Sci. 1, 75–82 (1946).

181. Fukuda, T., Ma, Y. Y. D. & Inagaki, H. Free-Radical Copolymerization. 3. Determination of Rate Constants of Propagation and Termination for the Styrene/Methyl Methacrylate System. A Critical Test of Terminal-Model Kinetics. Macromolecules 18, 17–26 (1985).

182. Feldermann, A. et al. Reversible addition fragmentation chain transfer copolymerization: influence of the RAFT process on the copolymer composition. Polymer (Guildf). 45, 3997–4007 (2004).

183. Stenzel, M. H. in RAFT Polym. (CAMD, UNSW, 2007).

184. Torchilin, V. P. Structure and design of polymeric surfactant-based drug delivery systems. J. Control. Release 73, 137–72 (2001).

185. Kabanov, A. V, Batrakova, E. V & Alakhov, V. Y. Pluronic® block copolymers as novel polymer therapeutics for drug and gene delivery. J. Control. Release 82, 189–212 (2002).

186. Joralemon, M. J., McRae, S. & Emrick, T. PEGylated polymers for medicine: from conjugation to self-assembled systems. Chem. Commun. (Cambridge, U. K.) 46, 1377–93 (2010).

187. Letchford, K. & Burt, H. A review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes. Euro. J. Pharma. Biopharm. 65, 259–69 (2007).

188. Read, E. S. & Armes, S. P. Recent advances in shell cross-linked micelles. Chem. Commun. (Cambridge, U. K.) 43, 3021–35 (2007).

189. Kwon, G. et al. Micelles based on AB block copolymers of poly(ethylene oxide) and poly(.beta.-benzyl L-aspartate). Langmuir 9, 945–9 (1993).

References

230

190. O’Reilly, R. K., Hawker, C. J. & Wooley, K. L. Cross-linked block copolymer micelles: functional nanostructures of great potential and versatility. Chem. Soc. Rev. 35, 1068–83 (2006).

191. Smith, A. E., Xu, X. & McCormick, C. L. Stimuli-responsive amphiphilic (co)polymers via RAFT polymerization. Prog. Polym. Sci. 35, 45–93 (2010).

192. Guo, A., Liu, G. & Tao, J. Star Polymers and Nanospheres from Cross-Linkable Diblock Copolymers. Macromolecules 29, 2487–93 (1996).

193. Tao, J., Stewart, S., Liu, G. & Yang, M. Star and Cylindrical Micelles of Polystyrene- block -poly(2-cinnamoylethyl methacrylate) in Cyclopentane. Macromolecules 30, 2738–45 (1997).

194. Henselwood, F. & Liu, G. Water-Soluble Porous Nanospheres. Macromolecules 31, 4213–4217 (1998).

195. Skey, J. & O’Reilly, R. K. Synthesis of chiral micelles and nanoparticles from amino acid based monomers using RAFT polymerization. J. Polym. Sci., Part A Polym. Chem. 46, 3690–702 (2008).

196. Xu, X., Smith, A. E., Kirkland, S. E. & McCormick, C. L. Aqueous RAFT Synthesis of pH-Responsive Triblock Copolymer mPEO−PAPMA−PDPAEMA and Formation of Shell Cross-Linked Micelles. Macromolecules 41, 8429–35 (2008).

197. Thurmond, K. B., Kowalewski, T. & Wooley, K. L. Water-Soluble Knedel-like Structures : The Preparation of Shell-Cross-Linked Small Particles. J. Am. Chem. Soc 31, 7239–40 (1996).

198. Zhang, L. et al. Shell-cross-linked micelles containing cationic polymers synthesized via the RAFT process: toward a more biocompatible gene delivery system. Biomacromolecules 8, 2890–901 (2007).

199. Deshpande, M. C. et al. Influence of polymer architecture on the structure of complexes formed by PEG–tertiary amine methacrylate copolymers and phosphorothioate oligonucleotide. J. Control. Release 81, 185–199 (2002).

200. Tan, J. F., Ravi, P., Too, H. P., Hatton, T. A. & Tam, K. C. Association behavior of biotinylated and non-biotinylated poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate). Biomacromolecules 6, 498–506 (2005).

201. Thurmond, K. B., Kowalewski, T. & Wooley, K. L. Shell Cross-Linked Knedels: A Synthetic Study of the Factors Affecting the Dimensions and Properties of Amphiphilic Core-Shell Nanospheres. J. Am. Chem. Soc 119, 6656–65 (1997).

202. Remsen, E. E., Thurmond, K. B. & Wooley, K. L. Solution and Surface Charge Properties of Shell Cross-Linked Knedel Nanoparticles. Macromolecules 32, 3685–9 (1999).

203. Joralemon, M. J., O’Reilly, R. K., Hawker, C. J. & Wooley, K. L. Shell click-crosslinked (SCC) nanoparticles: a new methodology for synthesis and orthogonal functionalization. J. Am. Chem. Soc 127, 16892–9 (2005).

References

231

204. Ma, J. et al. Synthesis and Solution-state Assembly or Bulk State Thiol-ene Crosslinking of Pyrrolidinone- and Alkene-functionalized Amphiphilic Block Fluorocopolymers: From Functional Nanoparticles to Anti-fouling Coatings. Aust. J. Chem. 63, 1159 (2010).

205. Saito, K., Ingalls, L. R., Lee, J. & Warner, J. C. Core-bound polymeric micellar system based on photocrosslinking of thymine. Chem. Commun. (Cambridge, U. K.) 43, 2503–5 (2007).

206. Toti, U. S., Guru, B. R., Grill, A. E. & Panyam, J. Interfacial Activity Assisted Surface Functionalization : A Novel Approach To Incorporate Maleimide Functional Groups and cRGD Peptide on Polymeric Nanoparticles for Targeted Drug Delivery. Mol. Pharm. 7, 1108–17 (2010).

207. Chapman, R., Jolliffe, K. A. & Perrier, S. Modular design for the controlled production of polymeric nanotubes from polymer/peptide conjugates. Polym. Chem. 2, 1956–63 (2011).

208. Chapman, R., Jolliffe, K. A. & Perrier, S. Synthesis of Self-assembling Cyclic Peptide-polymer Conjugates using Click Chemistry. Aust. J. Chem. 63, 1169 (2010).

209. Duncan, R., Connors, T. A. & Meada, H. Drug targeting in cancer therapy: the magic bullet, what next? J. Drug Target. 3, 317–9 (1996).

210. Venditto, V. J. & Szoka, F. C. Cancer nanomedicines: so many papers and so few drugs! Adv. Drug Deliv. Rev. 65, 80–8 (2013).

211. Karim, K. J. A. Polymeric Platinum Conjugated Drug for Cancer Treatment. 1–196 (2012).

212. Bontha, S., Kabanov, A. V & Bronich, T. K. Polymer micelles with cross-linked ionic cores for delivery of anticancer drugs. J. Control. Release 114, 163–74 (2006).

213. Pearson, S., Scarano, W. & Stenzel, M. H. Micelles based on gold-glycopolymer complexes as new chemotherapy drug delivery agents. Chem. Commun. (Cambridge, U. K.) 48, 4695–7 (2012).

214. Blunden, B. M., Thomas, D. S. & Stenzel, M. H. Macromolecular ruthenium complexes as anti-cancer agents. Polym. Chem. 3, 2964–75 (2012).

215. Blunden, B. M., Lu, H. & Stenzel, M. H. Enhanced delivery of the RAPTA-C macromolecular chemotherapeutic by conjugation to degradable polymeric micelles. Biomacromolecules 14, 4177–88 (2013).

216. Cabral, H., Nishiyama, N., Okazaki, S., Koyama, H. & Kataoka, K. Preparation and biological properties of dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt)-loaded polymeric micelles. J. Control. Release 101, 223–32 (2005).

217. Cabral, H., Nishiyama, N. & Kataoka, K. Optimization of (1,2-diamino-cyclohexane)platinum(II)-loaded polymeric micelles directed to improved tumor targeting and enhanced antitumor activity. J. Control. Release 121, 146–55 (2007).

References

232

218. Nishiyama, N. et al. Preparation and Characterization of Self-Assembled Polymer−Metal Complex Micelle from cis -Dichlorodiammineplatinum(II) and Poly(ethylene glycol)−Poly( α ,β-aspartic acid) Block Copolymer in an Aqueous Medium. Langmuir 15, 377–83 (1999).

219. Nishiyama, N. & Kataoka, K. Preparation and characterization of size-controlled polymeric micelle containing cis-dichlorodiammineplatinum(II) in the core. J. Control. Release 74, 83–94 (2001).

220. Nishiyama, N. et al. Novel cisplatin-incorporated polymeric micelles can eradicate solid tumors in mice. Cancer Res. 63, 8977–83 (2003).

221. Matsumoto, A., Matsukawa, Y., Suzuki, T. & Yoshino, H. Drug release characteristics of multi-reservoir type microspheres with poly(dl-lactide-co-glycolide) and poly(dl-lactide). J. Control. Release 106, 172–80 (2005).

222. Anderson, E. B. & Long, T. E. Imidazole- and imidazolium-containing polymers for biology and material science applications. Polymer (Guildf). 51, 2447–54 (2010).

223. Hakamatani, T., Asayama, S. & Kawakami, H. Synthesis of alkylated poly(1-vinylimidazole) for a new pH-sensitive DNA carrier. Nucleic Acids Sym. Se. 52, 677–8 (2008).

224. Srivastava, A., Waite, J. H., Stucky, G. D. & Mikhailovsky, A. Fluorescence Investigations into Complex Coacervation between Polyvinylimidazole and Sodium Alginate. Macromolecules 42, 2168–76 (2009).

225. Asayama, S., Sekine, T., Kawakami, H. & Nagaoka, S. Design of aminated poly(1-vinylimidazole) for a new pH-sensitive polycation to enhance cell-specific gene delivery. Bioconj. Chem. 18, 1662–7 (2007).

226. Green, M. D. et al. Tailoring macromolecular architecture with imidazole functionality: A perspective for controlled polymerization processes. Euro. Polym. J. 47, 486–96 (2011).

227. Pekel, N. & Gu, O. Investigation of complex formation between poly ( N-vinyl imidazole ) and various metal ions using the molar ratio method. Colloid. Polym. Sci. 573, 570–3 (1999).

228. Yuan, J., Schlaad, H., Giordano, C. & Antonietti, M. Double hydrophilic diblock copolymers containing a poly(ionic liquid) segment: Controlled synthesis, solution property, and application as carbon precursor. Euro. Polym. J. 47, 772–81 (2011).

229. Allen, M. H., Hemp, S. T., Smith, A. E. & Long, T. E. Controlled Radical Polymerization of 4-Vinylimidazole. Macromolecules 45, 3669–76 (2012).

230. Alessio, E. et al. Synthesis, molecular structure, and chemical behavior of hydrogen trans-bis(dimethyl sulfoxide)tetrachlororuthenate(III) and mer-trichlorotris(dimethyl sulfoxide)ruthenium(III): the first fully characterized chloride-dimethyl sulfoxide-ruthenium(III) comp. Inorg. Chem. 30, 609–18 (1991).

References

233

231. Panich, A. M. & Furman, G. B. Nuclear spin–lattice relaxation and paramagnetic defects in carbon nanomaterials. Diam. Relat. Mater. 23, 157–161 (2012).

232. Lv, H., Zhang, S., Wang, B., Cui, S. & Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control. Release 114, 100–9 (2006).

233. Legrand, F.-X. et al. Aqueous rhodium-catalyzed hydroformylation of 1-decene in the presence of randomly methylated β-cyclodextrin and 1,3,5-triaza-7-phosphaadamantane derivatives. Appl. Catal., A. 362, 62–6 (2009).

234. Perrier, S. S., Takolpuckdee, P. & Mars, C. A. Reversible addition-fragmentation chain transfer polymerization: End group modification for functionalized polymers and chain transfer agent recovery. Macromolecules 38, 3–7 (2005).

235. Xiang, W. et al. In situ quaterizable oligo-organophosphazene electrolyte with modified nanocomposite SiO2 for all-solid-state dye-sensitized solar cell. Electrochim. Acta 54, 4186–91 (2009).

236. Skehan, P. et al. New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. JNCI 82, 1107–12 (1990).

237. Deshpande, A., Deshpande, D. D. & Babu, G. N. Effect of temperature on the monomer reactivity ratios of 2-hydroxypropyl methacrylate with methyl acrylate and methyl methacrylate. Polym. Bull. 6, 1–6 (1981).

238. Kim, Y., Pourgholami, M. H., Morris, D. L. & Stenzel, M. H. Effect of cross-linking on the performance of micelles as drug delivery carriers: a cell uptake study. Biomacromolecules 13, 814–25 (2012).

239. Moad, G. & Thang, S. H. RAFT Polymerization: Materials of The Future, Science of Today: Radical Polymerization – The Next Stage. Aust. J. Chem. 62, 1379 (2009).

240. Perrier, S. & Takolpuckdee, P. Macromolecular design via reversible addition-fragmentation chain transfer (RAFT)/xanthates (MADIX) polymerization. J. Polym. Sci., Part A Polym. Chem. 43, 5347–93 (2005).

241. Hales, M., Barner-Kowollik, C., Davis, T. P. & Stenzel, M. H. Shell-cross-linked vesicles synthesized from block copolymers of Poly(D,L-lactide) and Poly(N-isopropyl acrylamide) as thermoresponsive nanocontainers. Langmuir 20, 10809–17 (2004).

242. Franken, N. a P. et al. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–9 (2006).

243. Sin, L. T., Rahmat, A. R. & Rahman, W. A. W. A. Polylactic Acid: PLA Biopolymer Technology and Applications. null null, 341 (William Andrew, 2013).

244. Chapman, R., Danial, M., Koh, M. L., Jolliffe, K. A. & Perrier, S. Design and properties of functional nanotubes from the self-assembly of cyclic peptide templates. Chem. Soc. Rev. 41, 6023–41 (2012).

245. Bong, D. T., Clark, T. D., Granja, J. R. & Ghadiri, M. R. Organic Nanotubes. Angew. Chem. Int. Ed. 40, 988–1011 (2001).

References

234

246. Brea, R. J., Reiriz, C. & Granja, J. R. Towards functional bionanomaterials based on self-assembling cyclic peptide nanotubes. Chem. Soc. Rev. 39, 1448–56 (2010).

247. Webber, M. J., Kessler, J. a & Stupp, S. I. Emerging peptide nanomedicine to regenerate tissues and organs. J. Intern. Med. 267, 71–88 (2010).

248. Chapman, R., Koh, M. L., Warr, G. G., Jolliffe, K. A. & Perrier, S. Structure elucidation and control of cyclic peptide-derived nanotube assemblies in solution. Chem. Sci. 4, 2581 (2013).

249. Chapman, R., Warr, G. G., Perrier, S. & Jolliffe, K. A. Water-soluble and pH-responsive polymeric nanotubes from cyclic peptide templates. Chem. Eur. J. 19, 1955–61 (2013).

250. Couet, J., Samuel, J. D. J. S., Kopyshev, A., Santer, S. & Biesalski, M. Peptide-polymer hybrid nanotubes. Angew. Chem. Int. Ed. 44, 3297–301 (2005).

251. Ten Cate, M. G. J., Severin, N. & Börner, H. G. Self-Assembling Peptide−Polymer Conjugates Comprising (d-alt-l)-Cyclopeptides as Aggregator Domains. Macromolecules 39, 7831–38 (2006).

252. Poon, C. K., Chapman, R., Jolliffe, K. A. & Perrier, S. Pushing the limits of copper mediated azide–alkyne cycloaddition (CuAAC) to conjugate polymeric chains to cyclic peptides. Polym. Chem. 3, 1820–26 (2012).

253. Skey, J. & O’Reilly, R. K. Facile one pot synthesis of a range of reversible addition-fragmentation chain transfer (RAFT) agents. Chem. Commun. (Cambridge, U. K.) 44, 4183–5 (2008).

254. Oae, S., Yagihara, T. & Okabe, T. Reduction of semipolar sulphur linkages with carbodithioic acids and addition of carbodithioic acids to olefins. Tetrahedron 28, 3203–16 (1972).

255. Barbey, R. & Perrier, S. A Facile Route to Functional Hyperbranched Polymers by Combining Reversible Addition–Fragmentation Chain Transfer Polymerization, Thiol–Yne Chemistry, and Postpolymerization Modification Strategies. ACS Macro Lett. 2, 366–70 (2013).

256. Piotto, M., Saudek, V. & Sklenář, V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661–5 (1992).

235

APPENDICES

Figure A-1: IBu-RAPTA-C synthesised via Route 2 at 25ᵒC in DMSO-d6 was subsequently heated to

60 ᵒC and monitored over time. Multiple side-products were observed.

Appendices

236


13C] HMBC & [

1H-

13C] HSQC NMR Spectrum of Butyl Iodide + PTA in

DMSO-d6 at 25 °C. Both the 1H and

13C spectra are external projections.


13C] HMBC & [

1H-

13C] HSQC NMR Spectrum of Butylated PTA +

RuCl2(p-cymene) Dimer in DMSO-d6 at 25 °C. Both the 1H and

13C spectra are external projections.

Appendices

237

Figure A-4: Initial RAPTA-C at 465 (top) shifted to Alkylated IBu-RAPTA-C at 497 (bottom).

Macromolecular Ruthenium Chemotherapeutics A ... - UNSWorks

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