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RADIOBIOLOGICAL MODELLING OF GOLD
NANOPARTICLES RADIOSENSITIZATION
EFFECTS IN CONVENTIONAL AND ADVANCED
RADIOTHERAPY TECHNIQUES
RAIZULNASUHA BINTI AB RASHID
UNIVERSITI SAINS MALAYSIA
2021
RADIOBIOLOGICAL MODELLING OF GOLD
NANOPARTICLES RADIOSENSITIZATION
EFFECTS IN CONVENTIONAL AND ADVANCED
RADIOTHERAPY TECHNIQUES
by
RAIZULNASUHA BINTI AB RASHID
Thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
June 2021
ii
ACKNOWLEDGEMENT
In the name of Allah swt the Most Gracious and the Most Merciful
Alhamdulillah, Praise to Allah for His endless blessings and strengths for me to
complete this thesis. First and foremost, I would like to express my deepest gratitude
to my supervisor, Dr. Wan Nordiana Wan Abd Rahman, for her invaluable guidance
during my candidature. Many thanks to my co-supervisor Profesor Dr. Khairunisak
Abdul Razak and Dr Mohd Hafiz Bin Mohd Zin for their support during the course of
my study. Special appreciations to all the staffs at the Department of Nuclear
Medicine, Radiotherapy and Oncology at the Hospital Universiti Sains Malaysia,
especially to Mdm. Norhayati Dollah and Mr Reduan Abdullah for being such a great
mentor during my data collection process. Deepest gratitude to Prof Moshi Geso for
beamtime arrangement at Australian Synchrotron. Special thanks also to Dr Andrew
Stevenson from Imaging and Medical Beamline and Dr. Stephanie Corde from Prince
of Wales Hospital with their assistance during data collection at Australian
Synchrotron. My proton beam irradiation would not be possible without the help from
Prof Ryohei Sasaki and Dr Hiroaki Akasaka from Kobe University Hospital, Dr.
Takahiro Tominaga from Hiroshima International University and staffs from Hyogo
Ion Beam Medical Centre. The research also indebted to Malaysian Ministry of Higher
Education for their financial support through FRGS grant (No:203/PPSK/6171160).
To my postgraduate colleagues: Belle, Emi, Hamizah, Afiq, Nashrul and Fazi, thanks
for all the positive experiences. Special gratitude to my family especially my beloved
mother, Zainab Hamat for the endless support, encouragement and countless love for
me especially during the challenging time of my doctorate study. Thank you.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENT ................................................................................ ii
TABLE OF CONTENTS ................................................................................. iii
LIST OF TABLES ......................................................................................... xiii
LIST OF FIGURES ....................................................................................... xxi
LIST OF ABBREVIATIONS .................................................................... xxxii
LIST OF SYMBOLS ................................................................................. xxxiv
ABSTRAK .................................................................................................. xxxvi
ABSTRACT .............................................................................................. xxxviii
CHAPTER 1 INTRODUCTION ................................................................ 1
1.1 Modern radiotherapy in cancer treatment .................................................. 1
1.2 Radiobiology in radiotherapy .................................................................... 3
1.2.1 Radiation induced cellular damage ............................................. 4
1.2.1(a) The time scale of radiation effects .............................. 4
1.2.1(a)(i) Physical phase ............................................ 4
1.2.1(a)(ii) Chemical phase ........................................ 4
1.2.1(a)(iii) Biological Phase ....................................... 5
1.2.1(b) DNA damage mechanism ........................................... 6
1.2.2 Radiation cell survival and damage ............................................. 8
1.2.2(a) Cell survival curves ..................................................... 8
1.3 Radiobiological model ............................................................................... 9
1.3.1 Application of the LQ model ....................................................... 9
1.3.1(a) The validity of the LQ model .................................... 11
iv
1.4 Problem statement and rationale of the study .......................................... 12
1.5 General objective ..................................................................................... 13
1.6 Specific objectives ................................................................................... 13
1.7 Thesis outlines ......................................................................................... 14
CHAPTER 2 LITERATURE REVIEW .................................................. 17
2.1 Nanomedicine in radiotherapy ................................................................. 17
2.2 Application of Gold Nanoparticles (AuNPs) in radiotherapy ................. 20
2.2.1 Characteristic and properties of AuNPs .................................... 21
2.2.2 Radiosensitization Effects by AuNPs ........................................ 23
2.2.2(a) Theoretical dose enhancement by high Z materials .. 23
2.2.3 Quantification of Radiosensitization Effects by AuNPs ........... 25
2.2.3(a) In-vitro ...................................................................... 25
2.2.3(b) In-vivo ....................................................................... 26
2.2.3(c) Monte Carlo .............................................................. 27
2.3 Mechanisms of AuNPs radiosensitization ............................................... 28
2.4 Pre-clinical application of AuNPs as a radiosensitizer in radiotherapy .. 31
2.4.1 Superficial and orthovoltage therapy......................................... 31
2.4.2 Megavoltage photon and electron beam radiotherapy ............... 32
2.4.3 Brachytherapy............................................................................ 33
2.4.4 Proton and carbon ion beam therapy ......................................... 33
2.5 Radiobiological model in clinical radiotherapy ....................................... 35
2.5.1 Linear quadratic model .............................................................. 35
2.5.2 Multi-target model ..................................................................... 36
2.5.3 Repairable conditionally repairable model ................................ 36
v
2.5.4 Pade’ linear quadratic model ..................................................... 37
2.5.5 Repair model ............................................................................. 38
2.5.6 Kavanagh-newman model ......................................................... 38
2.5.7 Two components model ............................................................ 39
CHAPTER 3 MATERIALS AND METHODS ....................................... 40
PART 1: CONVENTIONAL RADIOTHERAPY; PHOTON BEAM,
ELECTRON BEAM AND BRACHYTHERAPY ........................................ 40
3.1 Cell culture maintenance and protocol .................................................... 42
3.1.1 Cervical cancer cell lines (HeLa cells) preparation. .................. 42
3.2 Gold Nanoparticles (AuNPs) Preparations .............................................. 44
3.3 Gold nanoparticles (AuNPs) cellular uptake and localization ................. 45
3.3.1 Cellular uptake measurement of AuNPs for HeLa cells. .......... 45
3.3.2 Localization by light microscope observation for HeLa 116 cells.
................................................................................................... 45
3.4 Cytotoxicity assay for HeLa cells. ........................................................... 46
3.4.1 Presto blue assay........................................................................ 46
3.5 Cell survival study ................................................................................... 47
3.5.1 Irradiation with external beam radiotherapy (photon and electron
beams)........................................................................................ 47
3.5.1(a) Cells samples preparation ......................................... 47
3.5.1(b) Irradiation Setup ........................................................ 47
3.5.1(c) Radiation Dosimetry for photon and electron beams 49
3.5.2 Irradiation with HDR brachytherapy ......................................... 50
3.5.2(a) Cells samples preparation ......................................... 50
3.5.2(b) Brachytherapy irradiation setup ................................ 50
vi
3.5.2(c) Radiation dosimetry for brachytherapy .................... 51
3.6 Cell survival measurements ..................................................................... 51
3.6.1 Clonogenic assay ....................................................................... 51
3.7 Result Analysis ........................................................................................ 53
3.7.1 Radiobiological modelling ........................................................ 53
3.7.2 Methodologies for quantifying radiobiological enhancement
impact of AuNPs. ...................................................................... 54
3.7.2(a) Sensitisation Enhancement Ratio (SER) calculation 54
3.7.2(b) Radiation Enhancement Factor (REF) calculation ... 55
3.7.2(c) Radiation Enhancement Ratio (RER) calculation ..... 56
3.7.2(d) Dose Enhancement Factor (DEFxGy) calculation ...... 58
3.8 Statistical analysis .................................................................................... 59
CHAPTER 4 RESULTS ............................................................................ 60
4.1 Gold nanoparticles (AuNPs) cellular uptake into HeLa cells .................. 60
4.2 AuNPs localization inside HeLa cells ..................................................... 62
4.3 Gold nanoparticles (AuNPs) cytotoxicity on HeLa cells ........................ 63
4.4 Cell survival analysis using radiobiological models ............................... 66
4.4.1 Linear quadratic (LQ) model ..................................................... 67
4.4.2 Multi-target (MT) model ........................................................... 73
4.4.3 Repairable conditionally repairable (RCR) model .................... 79
4.4.4 Pade’ linear quadratic (PLQ) model .......................................... 85
4.4.5 Two-component (2C) model and Kavanagh & Newman (KN)
model ......................................................................................... 91
4.4.6 Repair model (RM).................................................................... 97
4.5 Comparison of cell survival analysis according to beam quality .......... 102
vii
4.5.1 Radiobiological parameters analysis ....................................... 109
4.6 Quantification of DEF, SER, RER and REF ......................................... 118
4.6.1 Dose Enhancement Factor (DEF) ............................................ 118
4.6.2 Sensitization Enhancement Ratio (SER) ................................. 124
4.6.3 Radiation enhancement ratio (RER) ........................................ 131
4.6.4 Radiation enhancement factor (REF) ...................................... 137
4.7 The influence of AuNPs sizes, concentrations and cell types on the
radiosensitization effects. ...................................................................... 138
4.7.1 Effect of AuNPs sizes. ............................................................. 138
4.7.2 Effect of AuNPs concentrations .............................................. 140
4.7.3 Effect of cell types ................................................................... 142
4.8 Radiation dose verification during samples irradiation ......................... 143
4.8.1 Megavoltage photon beams ..................................................... 143
4.8.2 Megavoltage electron beams ................................................... 147
4.8.3 Brachytherapy: Isodose distribution ........................................ 150
CHAPTER 5 DISCUSSION .................................................................... 152
5.1 Gold Nanoparticles (AuNPs) cellular uptake ........................................ 153
5.2 AuNPs localization in cells .................................................................... 154
5.3 Cytotoxicity of the gold nanoparticles (AuNPs) ................................... 156
5.4 Cell survival analysis using radiobiological models ............................. 159
5.5 Radiobiological parameters analysis ..................................................... 164
5.6 Quantification of DEF, SER, RER and REF ......................................... 167
5.7 Effect of AuNPs sizes ............................................................................ 169
5.8 Effect of AuNPs concentrations ............................................................ 173
viii
5.9 Effect of AuNPs on cell types ............................................................... 174
CHAPTER 6 PART II: ADVANCED RADIOTHERAPY; PROTON
BEAM THERAPY ........................................................................................ 176
6.1 Introduction............................................................................................ 176
6.1.1 Introduction to proton beam therapy ....................................... 176
6.2 Literature review .................................................................................... 179
6.3 Materials and Methods .......................................................................... 182
6.3.1 Cell samples preparation ......................................................... 182
6.3.2 Cellular uptake measurement of AuNPs for HCT 116 cells. .. 182
6.3.3 AuNPs localisation by light microscopy for HCT 116 cells. .. 183
6.3.4 Cytotoxicity assay for HCT 116 cells. .................................... 183
6.3.4(a) Presto blue assay ..................................................... 183
6.3.5 Proton Beam Irradiation .......................................................... 184
6.3.5(a) Cell survival measurements .................................... 186
6.3.5(b) Intracellular ROS measurements ............................ 186
6.3.5(c) Radiation dosimetry ................................................ 187
6.4 Results ................................................................................................... 188
6.4.1 Gold nanoparticles (AuNPs) cellular uptake into HCT 116
cells .......................................................................................... 188
6.4.2 Gold nanoparticles (AuNPs) localization for HCT 116 cells .. 189
6.4.3 AuNPs cytotoxicity test on HCT 116 cells.............................. 190
6.4.4 Cell survival analysis using radiobiological models ............... 191
6.4.4(a) Linear quadratic (LQ) model .................................. 191
6.4.4(b) Multi-target (MT) model ......................................... 192
6.4.4(c) Repairable conditionally repairable (RCR) model . 194
ix
6.4.4(d) Pade’ linear quadratic (PLQ) model ....................... 195
6.4.4(e) Two-component (2C) model ................................... 196
6.4.4(f) Kavanagh and Newman (KN) model ...................... 197
6.4.5 Quantification of DEF, SER and RER .................................... 199
6.4.5(a) Dose enhancement factor (DEF) ............................. 199
6.4.5(b) Sensitization enhancement ratio (SER) .................. 201
6.4.5(c) Radiation enhancement ratio (RER) ....................... 202
6.4.6 Reactive oxygen species (ROS) .............................................. 204
6.4.6(a) ROS measured with and without 1.9 nm of
AuNPs. .................................................................... 204
6.4.6(b) ROS comparison between different AuNPs sizes ... 205
6.4.6(c) Comparison between different beam types ............. 206
6.4.7 Proton beam dose verification during samples irradiation ...... 207
6.5 Discussion .............................................................................................. 209
6.5.1 Gold nanoparticles (AuNPs) cellular uptake in HCT 166
cells .......................................................................................... 209
6.5.2 Gold nanoparticles (AuNPs) localization for HCT 116 cells .. 210
6.5.3 Cytotoxicity of the gold nanoparticles (AuNPs) to the HCT 116
cells .......................................................................................... 211
6.5.4 Cell survival analysis using radiobiological models ............... 212
6.5.5 Quantification of DEF, SER and RER .................................... 215
6.5.6 Reactive Oxygen Species (ROS) Measurements .................... 216
6.5.6(a) ROS production with and without 1.9 nm of
AuNPs. .................................................................... 216
6.5.6(b) Effects of AuNPs sizes to ROS production ............ 217
6.5.6(c) Effects of beam types to ROS production ............... 219
x
CHAPTER 7 PART III: ADVANCED RADIOTHERAPY;
SYNCHROTRON MONOENERGETIC PHOTON BEAM THERAPY 222
7.1 Introduction............................................................................................ 222
7.1.1 Synchrotron Radiation ............................................................. 222
7.1.2 Synchrotron kilovoltage monoenergetic photon beam ............ 222
7.1.3 The exclusive characteristic of Synchrotron Light. ................. 224
7.1.4 Australian synchrotron ............................................................ 224
7.1.5 Synchrotron radiation applications .......................................... 225
7.1.6 Synchrotron radiation properties and application .................... 227
7.2 Literature Review .................................................................................. 228
7.3 Materials and Methods .......................................................................... 230
7.3.1 Mouse’s Mastocytoma (P815) cell lines preparation .............. 230
7.3.2 Gold nanoparticles (AuNPs) preparation ................................ 230
7.3.3 Cell survival study ................................................................... 230
7.3.4 Samples preparation for irradiation ......................................... 231
7.3.4(a) Cells samples preparation ....................................... 231
7.3.4(b) Irradiation Setup ...................................................... 231
7.3.4(c) Radiation Dosimetry ............................................... 232
7.3.5 Cell Survival Measurement ..................................................... 233
7.3.5(a) Viability Assay ........................................................ 233
7.3.6 Data analysis ............................................................................ 234
7.3.6(a) Radiobiological modelling ...................................... 234
7.3.6(b) Methodology for quantifying radiosensitization effects
of AuNPs. ................................................................ 234
7.4 Results ................................................................................................... 235
xi
7.4.1 Cell survival analysis using radiobiological models ............... 235
7.4.1(a) Linear quadratic (LQ) model .................................. 235
7.4.1(b) Multi-target (MT) model ......................................... 240
7.4.1(c) Repairable conditionally repairable (RCR) model . 243
7.4.1(d) Pade’ linear quadratic (PLQ) model ....................... 247
7.4.1(e) Two-component (2C) model ................................... 252
7.4.1(f) Repair model (RM) ................................................. 256
7.4.2 Comparison of cell survival analysis according to beam
quality. ..................................................................................... 261
7.4.3 Radiobiological parameters analysis ....................................... 261
7.4.4 Quantification of DEF, SER, RER and REF ........................... 272
7.4.4(a) Dose Enhancement Factor (DEF) ........................... 272
7.4.4(b) Sensitization Enhancement Ratio (SER). ............... 277
7.4.4(c) Radiation Enhancement Ratio (RER) ..................... 282
7.4.4(d) Radiation Enhancement Factor (REF) .................... 287
7.4.5 Evaluation on the influence of beam energies and AuNPs sizes on
the radiosensitization. .............................................................. 288
7.5 Discussion .............................................................................................. 294
7.5.1 Cell survival and radiobiological parameters analysis ............ 294
7.5.2 Quantification of DEF, SER, RER and REF ........................... 297
7.5.3 Influence of beam energies and AuNPs sizes on the
radiosensitization effects. ........................................................ 299
CHAPTER 8 CONCLUSION ................................................................. 301
8.1 Conventional beams radiotherapy ......................................................... 301
8.2 Proton beam therapy .............................................................................. 306
xii
8.3 Synchrotron monoenergetic photon beam therapy ................................ 307
8.4 Limitations ............................................................................................. 309
8.5 Future recommendations ....................................................................... 309
REFERENCES .............................................................................................. 311
APPENDICES
APPENDIX A TURNITIN SCREENING REPORT
APPENDIX B PUBLICATION LIST
APPENDIX C CONFERENCE PRESENTATIONS
APPENDIX D GOLD NANOPARTICLE SAMPLES
xiii
LIST OF TABLES
Page
Table 2.1 The characteristic and properties of AuNPs .............................. 21
Table 3.1 Radiobiological model equations with the parameters that
explained the intrinsic interactions. ........................................... 53
Table 4.1 LQ model parameters for 6 MV photon beam irradiation with and
without AuNPs. ......................................................................... 68
Table 4.2 LQ model parameters for 10 MV photon beam irradiation with
and without AuNPs.................................................................... 69
Table 4.3 LQ model parameters for 6 MeV electron beam irradiation with
and without AuNPs.................................................................... 70
Table 4.4 LQ model parameters for 15 MeV electron beam irradiation with
and without AuNPs.................................................................... 71
Table 4.5 LQ model parameters for brachytherapy irradiation with and
without AuNPs. ......................................................................... 72
Table 4.6 MT model parameters for 6 MV photon beam irradiation with and
without AuNPs. ......................................................................... 74
Table 4.7 MT model parameters for 10 MV photon beam irradiation with
and without AuNPs.................................................................... 75
Table 4.8 MT model parameters for 6 MeV electron beam irradiation with
and without AuNPs.................................................................... 76
Table 4.9 MT model parameters for 15 MeV electron beam irradiation with
and without AuNPs.................................................................... 77
Table 4.10 MT model parameters for brachytherapy irradiation with and
without AuNPs. ......................................................................... 78
Table 4.11 RCR model parameters for 6 MV photon beam irradiation with
and without AuNPs.................................................................... 80
Table 4.12 RCR model parameters for 10 MV photon beam irradiation with
and without AuNPs.................................................................... 81
Table 4.13 RCR model parameters for 6 MeV electron beam irradiation with
and without AuNPs.................................................................... 82
Table 4.14 RCR model parameters for 15 MeV electron beam irradiation
with and without AuNPs. .......................................................... 83
xiv
Table 4.15 RCR model parameters for brachytherapy irradiation with and
without AuNPs. ......................................................................... 84
Table 4.16 PLQ model parameters for 6 MV photon beam irradiation with
and without AuNPs.................................................................... 86
Table 4.17 PLQ model parameters for 10 MV photon beam irradiation with
and without AuNPs.................................................................... 87
Table 4.18 PLQ model parameters for 6 MeV electron beam irradiation with
and without AuNPs.................................................................... 88
Table 4.19 PLQ model parameters for 15 MeV electron beam irradiation
with and without AuNPs. .......................................................... 89
Table 4.20 PLQ model parameters for brachytherapy irradiation with and
without AuNPs. ......................................................................... 90
Table 4.21 2C model parameters for 6 MV photon beam irradiation with and
without AuNPs. ......................................................................... 92
Table 4.22 2C model parameters for 10 MV photon beam irradiation with
and without AuNPs.................................................................... 93
Table 4.23 2C model parameters for 15 MeV electron beam irradiation with
and without AuNPs.................................................................... 94
Table 4.24 KN model parameters for 6 MeV electron beam irradiation with
and without AuNPs.................................................................... 95
Table 4.25 KN model parameters for brachytherapy irradiation with and
without AuNPs. ......................................................................... 96
Table 4.26 Repair model parameters for 6 MV photon beam irradiation with
and without AuNPs.................................................................... 98
Table 4.27 Repair model parameters for 10 MV photon beam irradiation with
and without AuNPs.................................................................... 99
Table 4.28 Repair model parameters for 6 MeV electron beam irradiation
with and without AuNPs. ........................................................ 100
Table 4.29 Repair model parameters for 15 MeV electron beam irradiation
with and without AuNPs. ........................................................ 101
Table 4.30 Repair model parameters for brachytherapy irradiation with and
without AuNPs. ....................................................................... 102
Table 4.31 Linear quadratic model parameters for photon beam, electron
beam and brachytherapy. ......................................................... 111
xv
Table 4.32 Multi-target model parameters for photon beam, electron beam
and brachytherapy. .................................................................. 112
Table 4.33 Repairable conditionally repair model parameters for photon
beam, electron beam and brachytherapy. ................................ 113
Table 4.34 Pade’ linear quadratic model parameters for photon beam,
electron beam and brachytherapy. ........................................... 114
Table 4.35 Two-components model parameters for photon beam, electron
beam and brachytherapy. ......................................................... 115
Table 4.36 Repair model parameters for photon beam, electron beam and
brachytherapy. ......................................................................... 116
Table 4.37 Kavanagh and Newman model parameters for photon beam,
electron beam and brachytherapy. ........................................... 117
Table 4.38 The dose enhancement factor (DEF) values for 6 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 119
Table 4.39 The dose enhancement factor (DEF) values for 10 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 120
Table 4.40 The dose enhancement factor (DEF) values for 6 MeV electron
beam from cell survival curves of different radiobiology
models...................................................................................... 121
Table 4.41 The dose enhancement factor (DEF) values for 15 MeV electron
beam from cell survival curves of different radiobiology
models...................................................................................... 122
Table 4.42 The dose enhancement factor (DEF) values for brachytherapy
from cell survival curves of different radiobiology models. ... 123
Table 4.43 The sensitization enhancement ratio (SER) values for 6 MV from
cell survival curves of different radiobiology models. ............ 126
Table 4.44 The sensitization enhancement ratio (SER) values for 10 MV
from cell survival curves of different radiobiology models. ... 127
Table 4.45 The sensitization enhancement ratio (SER) values for 6 MeV
from cell survival curves of different radiobiology models. ... 128
Table 4.46 The sensitization enhancement ratio (SER) values for 15 MeV
from cell survival curves of different radiobiology models. ... 129
xvi
Table 4.47 The sensitization enhancement ratio (SER) values for
brachytherapy from cell survival curves of different radiobiology
models...................................................................................... 130
Table 4.48 The radiation enhancement ratio (RER) values for 6 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 132
Table 4.49 The radiation enhancement ratio (RER) values for 6 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 133
Table 4.50 The radiation enhancement ratio (RER) values for 6 MeV
electron beam from cell survival curves of different radiobiology
models...................................................................................... 134
Table 4.51 The radiation enhancement ratio (RER) values for 15 MeV
electron beam from cell survival curves of different radiobiology
models...................................................................................... 135
Table 4.52 The radiation enhancement ratio (RER) values for brachytherapy
from cell survival curves of different radiobiology models. ... 136
Table 4.53 The radiation enhancement factor (REF) values for photon and
electron beams from cell survival curves of multi-target
models...................................................................................... 137
Table 4.54 PLQ model parameter for irradiation with different sizes of
AuNPs...................................................................................... 139
Table 4.55 PLQ model parameter for irradiation with different concentration
of AuNPs. ................................................................................ 141
Table 4.56 PLQ model parameter for irradiation with different types of cells.
................................................................................................. 143
Table 4.57 Dose error for megavoltage photon beams radiotherapy ........ 146
Table 4.58 Dose error for megavoltage electron beams radiotherapy. ...... 149
Table 4.59 Isodose verification error for brachytherapy irradiation ......... 151
Table 6.1 LQ model parameters for 150 MeV proton beam irradiation with
and without AuNPs.................................................................. 192
Table 6.2 MT model parameters for 150 MeV proton beam irradiation with
and without AuNPs.................................................................. 193
Table 6.3 RCR model parameters for 150 MeV proton beam irradiation
with and without AuNPs. ........................................................ 194
xvii
Table 6.4 PLQ model parameters for 150 MeV proton beam irradiation with
and without AuNPs.................................................................. 195
Table 6.5 2C model parameters for 150 MeV proton beam irradiation with
and without AuNPs.................................................................. 196
Table 6.6 KN model parameters for 150 MeV proton beam irradiation with
and without AuNPs.................................................................. 197
Table 6.7 The dose enhancement factor (DEF) values for 150 MeV proton
beam from cell survival curves of different radiobiology
models...................................................................................... 200
Table 6.8 The sensitization enhancement ratio (SER) values for 150 MeV
proton beam from cell survival curves of different radiobiology
models...................................................................................... 202
Table 6.9 The radiation enhancement ratio (RER) values for 150 MeV
proton beam from cell survival curves of different radiobiology
models...................................................................................... 203
Table 6.10 Values of proton beam dose verification. ................................ 207
Table 7.1 The characteristic of synchrotron light .................................... 224
Table 7.2 LQ model parameters for 30 keV synchrotron irradiation with and
without AuNPs. ....................................................................... 236
Table 7.3 LQ model parameters for 40 keV irradiation with and without
AuNPs...................................................................................... 237
Table 7.4 LQ model parameters for 70 keV irradiation with and without
AuNPs...................................................................................... 238
Table 7.5 LQ model parameters for 81.5 keV irradiation with and without
AuNPs...................................................................................... 239
Table 7.6 MT model parameters for 30 keV irradiation with and without
AuNPs...................................................................................... 241
Table 7.7 MT model parameters for 40 keV irradiation with and without
AuNPs...................................................................................... 241
Table 7.8 MT model parameters for 81.5 keV irradiation with and without
AuNPs...................................................................................... 242
Table 7.9 RCR model parameters for 30 keV irradiation with and without
AuNPs...................................................................................... 244
Table 7.10 RCR model parameters for 40 keV irradiation with and without
AuNPs...................................................................................... 244
xviii
Table 7.11 RCR model parameters for 70 keV irradiation with and without
AuNPs...................................................................................... 245
Table 7.12 RCR model parameters for 81.5 keV irradiation with and without
AuNPs...................................................................................... 246
Table 7.13 PLQ model parameters for 30 keV irradiation with and without
AuNPs...................................................................................... 248
Table 7.14 PLQ model parameters for 40 keV irradiation with and without
AuNPs...................................................................................... 249
Table 7.15 PLQ model parameters for 70 keV irradiation with and without
AuNPs...................................................................................... 250
Table 7.16 PLQ model parameters for 81.5 keV irradiation with and without
AuNPs...................................................................................... 251
Table 7.17 2C model parameters for 30 keV irradiation with and without
AuNPs...................................................................................... 252
Table 7.18 2C model parameters for 40 keV irradiation with and without
AuNPs...................................................................................... 253
Table 7.19 2C model parameters for 70 keV irradiation with and without
AuNPs...................................................................................... 254
Table 7.20 2C model parameters for 81.5 keV irradiation with and without
AuNPs...................................................................................... 255
Table 7.21 RM model parameters for 30 keV irradiation with and without
AuNPs...................................................................................... 257
Table 7.22 RM model parameters for 40 keV irradiation with and without
AuNPs...................................................................................... 258
Table 7.23 RM model parameters for 70 keV irradiation with and without
AuNPs...................................................................................... 259
Table 7.24 RM model parameters for 81.5 keV irradiation with and without
AuNPs...................................................................................... 260
Table 7.25 Linear quadratic model parameters for synchrotron beam
irradiations. .............................................................................. 266
Table 7.26 Multi target model parameters for synchrotron beam irradiations.
................................................................................................. 267
Table 7.27 Repairable conditionally repair model parameters for synchrotron
beam irradiations. .................................................................... 268
xix
Table 7.28 Pade’ linear quadratic model parameters for synchrotron beam
irradiations. .............................................................................. 269
Table 7.29 Two-components model parameters for synchrotron beam
irradiations. .............................................................................. 270
Table 7.30 Repair model parameters for synchrotron beam irradiations. . 271
Table 7.31 The dose enhancement factor (DEF) values for 30 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 273
Table 7.32 The dose enhancement factor (DEF) values for 40 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 274
Table 7.33 The dose enhancement factor (DEF) values for 70 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 275
Table 7.34 The dose enhancement factor (DEF) values for 81.5 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 276
Table 7.35 The sensitization enhancement ratio (SER) values for 30 keV
synchrotron from cell survival curves of different radiobiology
models...................................................................................... 278
Table 7.36 The sensitization enhancement ratio (SER) values for 40 keV
synchrotron from cell survival curves of different radiobiology
models...................................................................................... 279
Table 7.37 The sensitization enhancement ratio (SER) values for 70 keV
synchrotron from cell survival curves of different radiobiology
models...................................................................................... 280
Table 7.38 The sensitization enhancement ratio (SER) values for 81.5 keV
synchrotron from cell survival curves of different radiobiology
models...................................................................................... 281
Table 7.39 The radiation enhancement ratio (RER) values for 30 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 283
Table 7.40 The radiation enhancement ratio (RER) values for 40 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 284
Table 7.41 The radiation enhancement ratio (RER) values for 70 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 285
xx
Table 7.42 The radiation enhancement ratio (RER) values for 81.5 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 286
Table 7.43 The radiation enhancement factor (REF) values for 30 keV, 40
keV, 70 keV and 81.5 keV synchrotron beams from cell survival
curves of multi-target model. .................................................. 287
xxi
LIST OF FIGURES
Page
Figure 1.1 The evolution of cancer radiotherapy treatments ........................ 2
Figure 1.2 The different levels of radiotherapy development. Reproduced
from Joiner et al., 2009. ............................................................... 3
Figure 1.3 The time-scale of effects of radiation interaction with biological
materials. ..................................................................................... 6
Figure 1.4 DNA damages mechanism of single and double strand breaks. . 7
Figure 1.5 Survival curve of HeLa cells proliferation versus dose (Puck and
Marcus, 1956). ............................................................................. 9
Figure 2.1 Nanomedicine in radiotherapy .................................................. 19
Figure 2.2 Energy dependence of the theoretical of DEF for several Au
aqueous mixtures (from bottom to top, the mass proportion of Au
in water, wAu range from 0.01 to 1. ........................................... 24
Figure 3.1 The research summary. .............................................................. 41
Figure 3.2 Cervical cancer (HeLa) cell lines .............................................. 42
Figure 3.3 Human colon carcinoma (HCT 116) cell lines .......................... 43
Figure 3.4 Photon beam irradiation setup. .................................................. 48
Figure 3.5 Electron beam irradiation setup. ................................................ 49
Figure 3.6 Brachytherapy irradiation setup. ............................................... 50
Figure 3.7 The flow of the process of clonogenic assay for cell survival
measurement. ............................................................................. 52
Figure 4.1 The uptake of AuNPs nanoparticles into HeLa cells as determined
by an increase in granularity (SSC). Incubation with (a) no
AuNPs (b) 1.9 nm AuNPs (c) 5 nm AuNPs (d) 15 nm
AuNPs........................................................................................ 61
Figure 4.2 AuNPs localization inside HeLa cells (100 µm
magnifications). ......................................................................... 62
Figure 4.3 The percentage of the cell’s viability at different AuNPs (1.9 nm)
concentrations versus incubation time (24, 48 and 72 hours). The
errors are a standard deviation from three independent
experiments (mean ± SD, n=3). ................................................. 63
xxii
Figure 4.4 The percentage of the cell’s viability versus different
concentrations of 5 nm AuNPs at 24, 48 and 72 hours incubation
time. The errors are a standard deviation from three independent
experiments (mean ± SD, n=3). ................................................. 65
Figure 4.5 The percentage of the cell’s viability versus different
concentrations of 15 nm AuNPs at 24, 48 and 72 hours incubation
time. The errors are a standard deviation from three independent
experiments (mean ± SD, n=3). ................................................. 66
Figure 4.6 The cell survival curves for 6 MVphoton beam irradiation with
and without AuNPs fitted using the LQ model. ........................ 68
Figure 4.7 The cell survival curves for 10 MV photon beam irradiation with
and without AuNPs fitted using the LQ model. ........................ 69
Figure 4.8 The cell survival curves for 6 MeV electron beam irradiation with
and without AuNPs fitted using the LQ model. ........................ 70
Figure 4.9 The cell survival curves for 15 MeV electron beam irradiation
with and without AuNPs fitted using the LQ model. ................ 71
Figure 4.10 The cell survival curves for brachytherapy irradiation with and
without AuNPs using the LQ model. ........................................ 72
Figure 4.11 The cell survival curves for 6 MV photon beam irradiation with
and without AuNPs fitted using the MT model......................... 74
Figure 4.12 The cell survival curves for 10 MV photon beam irradiation with
and without AuNPs fitted using the MT model......................... 75
Figure 4.13 The cell survival curves for 6 MeV electron beam irradiation with
and without AuNPs fitted using the MT model......................... 76
Figure 4.14 The cell survival curves for 15 MeV electron beam irradiation
with and without AuNPs fitted using the MT model. ............... 77
Figure 4.15 The cell survival curves for brachytherapy irradiation with and
without AuNPs using the MT model. ........................................ 78
Figure 4.16 The cell survival curves for 6 MV photon beam irradiation with
and without AuNPs fitted using the RCR model....................... 80
Figure 4.17 The cell survival curves for 10 MV photon beam irradiation with
and without AuNPs fitted using the RCR model....................... 81
Figure 4.18 The cell survival curves for 6 MeV electron beam irradiation with
and without AuNPs fitted using RCR model. ........................... 82
Figure 4.19 The cell survival curves for 15 MeV electron beam irradiation
with and without AuNPs fitted using RCR model. ................... 83
xxiii
Figure 4.20 The cell survival curves for brachytherapy irradiation with and
without AuNPs fitted using RCR model. .................................. 84
Figure 4.21 The cell survival curves for 6 MV photon beam irradiation with
and without AuNPs fitted using the PLQ model ....................... 86
Figure 4.22 The cell survival curves for 10 MV photon beam irradiation with
and without AuNPs fitted using the PLQ model. ...................... 87
Figure 4.23 The cell survival curves for 6 MeV electron beam irradiation with
and without AuNPs fitted using the PLQ model. ...................... 88
Figure 4.24 The cell survival curves for 15 MeV electron beam irradiation
with and without AuNPs fitted using the PLQ model ............... 89
Figure 4.25 The cell survival curves for brachytherapy irradiation with and
without AuNPs fitted using PLQ model .................................... 90
Figure 4.26 The cell survival fraction curves for 6 MV photon beam
irradiation with and without AuNPs fitted using the 2C
model. ........................................................................................ 92
Figure 4.27 The cell survival fraction curves for 10 MV photon beam
irradiation with and without AuNPs fitted using the 2C
model. ........................................................................................ 93
Figure 4.28 The cell survival fraction curves for 15 MeV electron beam
irradiation with and without AuNPs fitted using the 2C
model. ........................................................................................ 94
Figure 4.29 The cell survival fraction curves for 6 MeV electron beam
irradiation with and without AuNPs fitted using the KN
model. ........................................................................................ 95
Figure 4.30 The cell survival fraction curves for brachytherapy irradiation
with and without AuNPs fitted using the KN model. ................ 96
Figure 4.31 The cell survival curves for 6 MV photon beam irradiation with
and without AuNPs fitted using the repair model ..................... 98
Figure 4.32 The cell survival curves for 10 MV photon beam irradiation with
and without AuNPs fitted using repair model. .......................... 99
Figure 4.33 The cell survival curves for 6 MeV electron beam irradiation with
and without AuNPs fitted using the repair model ................... 100
Figure 4.34 The cell survival curves for 15 MeV electron beam irradiation
with and without AuNPs fitted using the repair model ........... 101
Figure 4.35 The cell survival curves for brachytherapy irradiation with and
without AuNPs fitted using the repair model .......................... 102
xxiv
Figure 4.36 The survival curves for 6 MV photon beam according to different
radiobiological models. Figure details with (a) LQ model, (b) MT
model, (c) RCR model, (d) PLQ model, (e) RM model and (f) 2C
model. ...................................................................................... 104
Figure 4.37 The survival curves for 10 MV photon beam according to
different radiobiological models. Figure details with (a) LQ
model, (b) MT model, (c) RCR model, (d) PLQ model, (e) RM
model and (f) 2C model. .......................................................... 105
Figure 4.38 The survival curves for 6 MeV electron beam according to
different radiobiological models. Figure details with (a) LQ
model, (b) MT model, (c) RCR model, (d) PLQ model, (e) RM
model and (f) KN model.......................................................... 106
Figure 4.39 The survival curves for 15 MeV electron beam according to
different radiobiological models. Figure details with (a) LQ
model, (b) MT model, (c) RCR model, (d) PLQ model, (e) RM
model and (f) 2C model. .......................................................... 107
Figure 4.40 The survival curves for brachytherapy according to different
radiobiological models. Figure details with (a) LQ model, (b) MT
model, (c) RCR model, (d) PLQ model, (e) RM model and (f) KN
model. ...................................................................................... 108
Figure 4.41 Dose enhancement factor (DEF) quantified for 6 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 119
Figure 4.42 Dose enhancement factor (DEF) quantified for 10 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 120
Figure 4.43 Dose enhancement factor (DEF) quantified for 6 MeV electron
beam from cell survival curves of different radiobiology
models...................................................................................... 121
Figure 4.44 Dose enhancement factor (DEF) quantified for 15 MeV electron
beam from cell survival curves of different radiobiology
models...................................................................................... 122
Figure 4.45 Dose enhancement factor (DEF) quantified for brachytherapy
from cell survival curves of different radiobiology models. ... 123
Figure 4.46 Sensitization enhancement ratio (SER) quantified for 6 MV
photon beam from cell survival curves of different radiobiology
models...................................................................................... 126
xxv
Figure 4.47 Sensitization enhancement ratio (SER) quantified for 10 MV
photon beam from cell survival curves of different radiobiology
models...................................................................................... 127
Figure 4.48 Sensitization enhancement ratio (SER) quantified for 6 MeV
electron beam from cell survival curves of different radiobiology
models...................................................................................... 128
Figure 4.49 Sensitization enhancement ratio (SER) quantified for 15 MeV
photon beam from cell survival curves of different radiobiology
models...................................................................................... 129
Figure 4.50 Sensitization enhancement ratio (SER) quantified for
brachytherapy from cell survival curves of different radiobiology
models...................................................................................... 130
Figure 4.51 Radiation enhancement ratio (RER) quantified for 6 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 132
Figure 4.52 Radiation enhancement ratio (RER) quantified for 10 MV photon
beam from cell survival curves of different radiobiology
models...................................................................................... 133
Figure 4.53 Radiation enhancement ratio (RER) quantified for 6 MeV
electron beam from cell survival curves of different radiobiology
models...................................................................................... 134
Figure 4.54 Radiation enhancement ratio (RER) quantified for 15 MeV
electron beam from cell survival curves of different radiobiology
models...................................................................................... 135
Figure 4.55 Radiation enhancement ratio (RER) quantified for brachytherapy
from cell survival curves of different radiobiology models. ... 136
Figure 4.56 Radiation enhancement factor (REF) quantified for photon and
electron beams from cell survival curves of multi-target
models...................................................................................... 137
Figure 4.57 The cells survival curves for 1.9, 5 and 15 nm AuNPs under
brachytherapy irradiation......................................................... 139
Figure 4.58. The cells survival curves for 1, 2 and 3 nm AuNPs under
brachytherapy irradiation......................................................... 141
Figure 4.59 The cells survival curves for HeLa and HCT116 cells under
brachytherapy irradiation......................................................... 142
Figure 4.60 Calibration curve for 6 MV photon beam ............................... 144
xxvi
Figure 4.61 Prescribed dose versus measured dose for 6 MV photon
beam ........................................................................................ 144
Figure 4.62 Calibration curve for 10 MV photon beam ............................. 145
Figure 4.63 Prescribed dose versus measured dose for 10 MV photon
beam ........................................................................................ 145
Figure 4.64 Calibration curve for 6 MeV electron beam. ........................... 147
Figure 4.65 Prescribed dose versus measured dose for 6 MeV electron
beam. ....................................................................................... 147
Figure 4.66 Calibration curve for 15 MeV electron beam. ......................... 148
Figure 4.67 Prescribed dose versus measured dose for 15 MeV electron beam.
................................................................................................. 148
Figure 4.68 Isodose distribution for brachytherapy irradiation of 1 Gy. .... 150
Figure 4.69 Isodose distribution for brachytherapy irradiation of 2 Gy . ... 150
Figure 4.70 Isodose distribution for brachytherapy irradiation of 4 Gy ..... 151
Figure 6.1 Depth dose distribution of proton spread-out bragg peak and other
types of radiotherapy beams .................................................... 178
Figure 6.2 The schematic diagram of the experimental set-up of cell
irradiation with proton beams. ................................................. 185
Figure 6.3 The experimental set-up of cell’s proton beam irradiation. .... 185
Figure 6.4 The uptake of AuNPs nanoparticles into HCT 116 cells (a)
without AuNPs and (b) with 1.9 nm AuNPs. .......................... 189
Figure 6.5 AuNPs localization inside HCT 116 cells (100
magnifications). ....................................................................... 189
Figure 6.6 The 1.9 nm AuNPs cytotoxicity on HCT 116 cell. ................. 190
Figure 6.7 The cell survival curves for 150 MeV proton beam irradiation
with and without AuNPs fitted using LQ model. .................... 191
Figure 6.8 The cell survival curves for 150 MeV proton beam irradiation
with and without AuNPs fitted using MT model. ................... 192
Figure 6.9 The cell survival curves for 150 MeV proton beam irradiation
with and without AuNPs fitted using RCR model. ................. 194
Figure 6.10 The cell survival curves for 150 MeV proton beam irradiation
with and without AuNPs fitted using PLQ model. .................. 195
xxvii
Figure 6.11 The cell survival curves for 150 MeV proton beam irradiation
with and without AuNPs fitted using 2C model. ..................... 196
Figure 6.12 The cell survival curves for 150 MeV proton beam irradiation
with and without AuNPs fitted using KN model..................... 197
Figure 6.13 The survival curves for 150 MeV proton beam according to
different radiobiological models. This figure details with (a) LQ
model, (b) MT model, (c) RCR model, (d) PLQ model, (e) KN
model and (f) 2C model. .......................................................... 198
Figure 6.14 Dose enhancement factor (DEF) quantified for 150 MeV proton
beam from cell survival curves of different radiobiology
model. ...................................................................................... 199
Figure 6.15 Sensitization enhancement ratio (SER) quantified for 150 MeV
proton beam from cell survival curves of different radiobiology
models...................................................................................... 201
Figure 6.16 Radiation enhancement ratio (RER) quantified for 150 MeV
proton beam from cell survival curves of different radiobiology
models...................................................................................... 203
Figure 6.17 The percentage of ROS production with and without 1.9 nm
AuNPs after irradiation with 150 MeV proton beam at 2 Gy
radiation dose........................................................................... 204
Figure 6.18 The percentage of ROS generation induced by 1.9 nm, 5 nm, and
15 nm AuNPs after irradiation with 150 MeV proton beam. .. 205
Figure 6.19 The comparison of ROS generation induced by 1.9 nm, 5 nm, and
15 nm AuNPs after irradiation with 150 MeV proton beam, 6 MV,
10 MV photon beam and 0.38 MeV gamma ray. .................... 206
Figure 6.20 Measured dose versus prescribed dose for proton beam
irradiation. ............................................................................... 208
Figure 7.1 Synchrotron work-flow and components of beam
production. ............................................................................... 223
Figure 7.2 Australian Synchrotron, Image obtained from Google map
satellite (10/9/2019). ................................................................ 225
Figure 7.3 Australian Synchrotron research fields. .................................. 225
Figure 7.4 Setup of cell irradiation with Australian Synchrotron beam
radiotherapy. ............................................................................ 231
Figure 7.5 Dosimetry setup for Synchrotron beam irradiation. Cell in PCR
tubes were located in front of Gafchromic EBT3 film. ........... 232
xxviii
Figure 7.6 The cell survival curves for 30 keV synchrotron irradiation with
and without AuNPs fitted using the LQ model. ...................... 236
Figure 7.7 The cell survival curves for 40 keV irradiation with and without
AuNPs fitted using the LQ model. .......................................... 237
Figure 7.8 The cell survival curves for 70 keV irradiation with and without
AuNPs fitted using the LQ model. .......................................... 238
Figure 7.9 The cell survival curves for 81.5 keV irradiation with and without
AuNPs fitted using the LQ model. .......................................... 239
Figure 7.10 The cell survival fraction curves for 30 keV irradiation with and
without AuNPs using MT model. ............................................ 240
Figure 7.11 The cell survival fraction curves for 40 keV irradiation with and
without AuNPs using MT model. ............................................ 241
Figure 7.12 The cell survival fraction curves for 81.5 keV irradiation with and
without AuNPs using MT model. ............................................ 242
Figure 7.13 The cell survival fraction curves for 30 keV irradiation with and
without AuNPs using RCR model. .......................................... 243
Figure 7.14 The cell survival fraction curves for 40 keV irradiation with and
without AuNPs using RCR model. .......................................... 244
Figure 7.15 The cell survival fraction curves for 70 keV irradiation with and
without AuNPs, using RCR model. ......................................... 245
Figure 7.16 The cell survival fraction curves for 81.5 keV irradiation with and
without AuNPs using RCR model. .......................................... 246
Figure 7.17 The cell survival fraction curves for 30 keV irradiation with and
without AuNPs fitted using PLQ model. ................................. 248
Figure 7.18 The cell survival fraction curves for 40 keV irradiation with and
without AuNPs fitted using PLQ model. ................................. 249
Figure 7.19 The cell survival fraction curves for 70 keV irradiation with and
without AuNPs fitted using PLQ model. ................................. 250
Figure 7.20 The cell survival fraction curves for 81.5 keV irradiation with and
without AuNPs, fitted using PLQ model. ................................ 251
Figure 7.21 The cell survival fraction curves for 30 keV irradiation with and
without AuNPs, fitted using the 2C model. ............................. 252
Figure 7.22 The cell survival fraction curves for 40 keV irradiation with and
without AuNPs, fitted using the 2C model. ............................. 253
xxix
Figure 7.23 The cell survival fraction curves for 70 keV irradiation with and
without AuNPs, fitted using the 2C model. ............................. 254
Figure 7.24 The cell survival fraction curves for 81.5 keV irradiation with and
without AuNPs, fitted using the 2C model. ............................. 255
Figure 7.25 The cell survival fraction curves for 30 keV irradiation with and
without AuNPs, fitted using the RM model. ........................... 257
Figure 7.26 The cell survival fraction curves for 40 keV irradiation with and
without AuNPs, fitted using the RM model. ........................... 258
Figure 7.27 The cell survival fraction curves for 70 keV irradiation with and
without AuNPs, fitted using the RM model. ........................... 259
Figure 7.28 The cell survival fraction curves for 81.5 keV irradiation with and
without AuNPs, fitted using the RM model. ........................... 260
Figure 7.29 The survival curves for 30 keV synchrotron beam according to
different radiobiological models. Figure details with (a) LQ
model, (b) MT model, (c) RCR model, (d) PLQ model, (e) RM
model and (f) 2C model. .......................................................... 262
Figure 7.30 The survival curves for 40 keV synchrotron beam according to
different radiobiological models. Figure details with (a) LQ
model, (b) MT model, (c) RCR model, (d) PLQ model, (e) 2C
model and (f) RM model. ........................................................ 263
Figure 7.31 The survival curves for 70 keV synchrotron beam according to
different radiobiological models. Figure details with (a) LQ
model, (b) RCR model, (c) PLQ model, (d) RM model, (e) 2C
model. ...................................................................................... 264
Figure 7.32 The survival curves for 81.5 keV synchrotron beam according to
different radiobiological models. Figure details with (a) LQ
model, (b) MT model, (c) RCR model, (d) PLQ model, (e) RM
model and (f) 2C model. .......................................................... 265
Figure 7.33 Dose enhancement factor (DEF) quantified for 30 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 273
Figure 7.34 Dose enhancement factor (DEF) quantified for 40 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 274
Figure 7.35 Dose enhancement factor (DEF) quantified for 70 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 275
xxx
Figure 7.36 Dose enhancement factor (DEF) quantified for 81.5 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 276
Figure 7.37 Sensitization enhancement ratio (SER) quantified for 30 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 278
Figure 7.38 Sensitization enhancement ratio (SER) quantified for 40 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 279
Figure 7.39 Sensitization enhancement ratio (SER) quantified for 70 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 280
Figure 7.40 Sensitization enhancement ratio (SER) quantified for 81.5 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 281
Figure 7.41 Radiation enhancement ratio (RER) quantified for 30 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 282
Figure 7.42 Radiation enhancement ratio (RER) quantified for 40 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 283
Figure 7.43 Radiation enhancement ratio (RER) quantified for 70 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 284
Figure 7.44 Radiation enhancement ratio (RER) quantified for 81.5 keV
synchrotron beam from cell survival curves of different
radiobiology models. ............................................................... 285
Figure 7.45 Radiation enhancement factor (REF) quantified for 30 keV, 40
keV, 70 keV and 81.5 keV synchrotron beams from cell survival
curves of multi-target model. .................................................. 287
Figure 7.46 The ratio of cells viability for control and treated cells with .. 289
Figure 7.47 The ratio of cells viability for control and treated cells with .. 289
Figure 7.48 The ratio of cells viability for control and treated cells with .. 290
Figure 7.49 The ratio of cells viability for control and treated cells with .. 290
Figure 7.50 The ratio of cells viability for control and treated cells with .. 291
Figure 7.51 The ratio of cells viability for control and treated cells with .. 291
xxxi
Figure 7.52 The ratio of cells viability for control and treated cells with .. 292
Figure 7.53 Combination of bar and line graph showing comparison of
experimental and theoretical DEF. .......................................... 293
Figure 8.1 Final results summary.............................................................. 308
xxxii
LIST OF ABBREVIATIONS
125I Iodine-125
192Ir Iridium-192
198Au Gold-198
2C 2-Components
Au Gold
AuNPs Gold nanoparticles
CO2 Carbon dioxide
DEF Dose Enhancement Factor
DMEM Dulbecco's Modified Eagle's medium
DNA Deoxyribonuclide acid
D-PBS Dulbecco's Phosphate-Buffered Saline
DSB Double strand breaks
EBT2 External beam therapy 2
EDTA Ethylenedinitrilotetraacetic acid
EPR Enhanced permeability and retention effect
FBS Fetal bovine serum
HDR High dose rate
IGRT Image guided radiotherapy
IMRT Intensity-modulated radiotherapy
KN Kavanagh-Newman
LINAC Linear accelerator
LET Linear energy transfer
xxxiii
LQ Linear Quadratic
MT Multi-target
NPs Nanoparticles
NTCP Normal tissue complication probability
PLQ Pade’ Linear Quadratic
TCP Tumor control probability
SER Sensitisation enhancement ratio
RCR Repairable Conditionally Repairable
REF Radiation Enhancement Factor
RER Radiation Enhancement Ratio
RM Repair Model
SSD Source to skin distance
SSB Single strand breaks
xxxiv
LIST OF SYMBOLS
a The initial mean number of damage events per unit dose
α The direct killing of cell “single hit” (linear component)
α1 The slope of a single-hit component
αn The slope of a compound multi-hit component
b The maximum amount of damage that can be repaired
β The impact of cell killing from “double hits” (quadratic component)
c The loss of repair due to more complex damage
cm2 Centimeter square
D Dose
D1 Dose at initial slope
Dq The size of the shoulder of the curve
D0 Dose at final slope
Gy Gray
g/mol Gram per mol
kV Kilovoltage
KO Linear rate
KOG Exponential rate
M Mega
MeV Megavoltage
mg/ml Miligram per mililiter
mM Milimol
Mol Mol
Molar Molarity
xxxv
MU/min Monitor Unit per Minutes
MV Mega voltage
n Number of targets / hit number
µl Microliter
γ The quantity lethal lesions
xxxvi
PEMODELAN RADIOBIOLOGI BAGI KESAN RADIOSENSITIVITI
PARTIKEL NANO EMAS DALAM TEKNIK KONVENSIONAL DAN
RADIOTERAPI TERMAJU
ABSTRAK
Keberkesanan strategi teknik radioterapi dengan nanoteknologi menjanjikan
penyelesaian dalam mengatasi permasalahan yang timbul dalam perawatan radioterapi
konvensional seperti kelemahan dalam memberi kesan radiasi secara selektif dan
bersasar terhadap tumor yang bersifat kalis radiasi. Partikel nano emas telah menjadi
bahan kajian yang pesat untuk bertindak sebagai penggalak kesan radiosensitiviti
dalam perawatan klinikal radioterapi. Meskipun terdapat banyak kajian yang telah
dijalankan, kepesatan penggunaan partikel nano emas sebagai pemeka sinaran
memerlukan pendekatan kajian yang sistematik dan mendalam untuk menilai secara
menyeluruh kesan radiobiologi terhadap rawatan radioterapi. Dalam penyelidikan ini,
kesan radiosensitiviti telah dikaji menggunakan pelbagai parameter experimen
terhadap rawatan radioterapi klinikal menggunakan tenaga radiasi yang berbeza
megavolt antaranya alur foton, elektron dan juga dos tinggi brakiterapi (bahan Iridium-
192). Selain radioterapi konventional, penggunaan rawatan radioterapi termaju seperti
alur proton dan kilovolt monoenergi foton ‘synchrotron radiation’ turut dikaji. Analisa
yang mendalam dengan menggunakan model-model radiobiologi dan cara-cara
mengkuantifikasinya juga turut diteliti secara spesifik. Pengaruh kesan radiosensitiviti
oleh partikel nano emas dari segi saiz, kepekatan dan jenis sel yang dirawat, beserta
pengkajian yang meliputi internalisasi, lokalisasi dan kesan toksiknya terhadap sel
turut di dikaji secara terperinci. Kebolehgunaan partikel nano emas untuk rawatan
xxxvii
radioterapi konvensional terbukti menghasilkan kesan radisensitiviti terutamanya
dengan dos tinggi brakiterapi, di mana ianya menunjukkan kesan lebih baik
berbanding radioterapi alur elektron dan foton. Impak radiosensitiviti yang berkesan
terbukti lebih besar dengan perawatan alur proton dan kilovolt monoenergi foton
‘synchrotron radiation’ yang bersepadanan dengan ramalan teori. Pengesahan melalui
model-model radiobiologi bertujuan untuk menghuraikan lekokan kemandirian sel
bergantung kepada jenis kualiti alur radiasi beserta cara-cara mengkuantifikasinya.
Berserta dengan itu, setiap model radiobiologi berserta parameter tersendiri turut
berperanan menjadi petunjuk yang menerangkan mekanisma kejadian yang
disebabkan oleh kesan radiosensitiviti. Saiz dan kepekatan partikel nano emas turut
mempengaruhi kesan radiosensitiviti, bergantung juga pada perbezaan jenis sel yang
dikaji. Sebelum melakukan kajian kesan radiosensitiviti radioterapi, pengunaan
partikel nano emas yang optima turut diambil kira dari segi kepekatan yang tidak
toksik dan intenalisasinya terhadap sel. Kesimpulannya, keberkesanan radisensitiviti
partikel nano emas boleh dicapai dengan menggunakan radioterapi klinikal megavot,
brakiterapi dan juga radioterapi termaju seperti terapi alur proton dan ‘synchrotron’
monoenergi foton radioterapi. Pemilihan secara terperinci model- model radiobiologi,
mampu memberi pemahaman tentang kesan radiobiologi sel yang teraruh dari partikel
nano emas dan juga piawaian cara-cara mengkuantifikasi amat penting untuk
memastikan kesesuaian parameter yang terbaik bagi rawatan klinikal partikel nano
emas. Cadangan yang jelas dan tertumpu amat penting dalam pengumpulan data
praklinikal bagi mempercepatkan peralihan kegunaan partikel nano emas ke peringkat
aplikasi klinikal kepada pesakit.
xxxviii
RADIOBIOLOGICAL MODELLING OF GOLD NANOPARTICLES
RADIOSENSITIZATION EFFECTS IN CONVENTIONAL AND
ADVANCED RADIOTHERAPY TECHNIQUES
ABSTRACT
Improvement of radiotherapy techniques with nanotechnology is a promising
strategy to overcome the limitations arises in conventional treatment such as lack of
eradication selectivity and radioresistance characteristic of targeted tumour. Gold
nanoparticles (AuNPs) is a subject of growing interest to induce radiosensitization
effects with potential application in clinical radiotherapy. Despite numerous
evidences, development and application of AuNPs as radiosensitizer require
systematic and comprehensive experimental approaches to fully evaluate the
radiobiological impact in radiotherapy. In this study, multiparametric investigations
on the radiosensitization effects were conducted using different energies of clinical
megavoltage photon, electron, and high-dose rate (HDR) gamma rays from Ir-192
source. In addition to conventional radiotherapy, advanced techniques using proton
beam and monoenergetic synchrotron photon beam of kilovoltage energies were also
employed. Comprehensive analysis using different radiobiological models and
quantification methods were specifically examined. Influence of AuNPs size,
concentration and types of cells on the radiosensitization effects were also elucidated
including internalization, localization and cytotoxicity of AuNPs. Applicability of
AuNPs for clinical conventional radiotherapy have been proven where
radiosensitization effects have been observed especially for HDR brachytherapy that
show better effects compare to electron and photon beam therapies. Substantially
bigger impact of AuNPs radiosensitization have been confirmed for proton beam and
xxxix
monoenergetic kilovoltage synchrotron photon beam in concurrently with theoretical
prediction. Validity of radiobiological models to describe the cell survival curve is
dependent on the beam quality as well as quantification methods. Radiobiological
models and their parameters also could be adopted as indicator to explain the
mechanistic events in the AuNPs’ radiosensitization effects. The AuNPs size and
concentration are found to influence the radiosensitization effects and different types
of cells exhibit different radiosensitivity responses. The radiosensitization impacts of
AuNPs with radiotherapy beams were considered with prior indication on the cellular
internalization of AuNPs and the non-toxic concentration for optimal AuNPs
application. As conclusion, effective radiosensitization by AuNPs could be achieved
for megavoltage clinical radiotherapy, brachytherapy and advanced radiotherapy such
as proton beam therapy and monoenergetic synchrotron beam radiotherapy. Precise
radiobiological characterization drawn from radiobiological models may provide
insight towards radiobiological impacts induced by AuNPs and standardization of
quantification methods is crucial for highlighting suitable parameters for AuNPs
clinical application. Clear directive recommendation from comprehensive preclinical
data provided in this study may expedite the clinical translation of AuNPs for human
application.
1
CHAPTER 1
INTRODUCTION
1.1 Modern radiotherapy in cancer treatment
Millions of people globally are dying with life-threatening cancer diseases for
decades. However, radiation therapy (RT) leads in cancer treatment by delivering
higher tumour doses while preserving healthy tissues aims for organ preservation, an
abiding principle of radiotherapy, thus resulting in increasing cancer cures with the
least morbidity. Radiotherapy aims to encompass all cancer cells with sufficient
therapeutic doses of radiation that aim on inhibition of cancer cells proliferating
capacity, eliminate the malignant cells, reduce the risk of recurrence while
simultaneously sparing surrounding normal tissues and ultimately to improve survival.
Enormous progress has been made on the development of precision RT
treatments using linear accelerators that pave towards current advanced technologies.
All efforts in understanding RT limitations and improved its ability to treat tumours
have been done through extensive research and development. Advances in
technologies specialized in radiation devices to improve RT such as dynamic multi-
leaf collimators, automated treatment planning, new imaging modalities, powerful
software operations and various treatment delivery methods help to achieve the
conformal radiation dose.
The improvement of radiotherapy techniques is routing through several
approaches. Firstly, the standard of prescribed dose and delivery at the target cancer
region were elevated. Secondly, introduction of the new method beyond the
conventional radiation techniques such as volumetric modulated arc therapy (VMAT)
and particles beam therapy. Thirdly, the combination of image-guidance and
radiotherapy enable dose monitoring delivery at daily treatment. Last and most
2
innovative outlook are exploring the radiobiological initiatives by manipulating the
nature of cancer cells behavior to become more radiosensitive towards the radiation
imposed (Joiner et al., 2009).
Figure 1.1 shows the evolution of the radiotherapy technique from standard
conventional treatment to conformal radiotherapy and currently the emerging of
particles beam therapy. A preclinical study on the synchrotron-based treatment is
currently undergoing and could be available for patients in the near future.
Figure 1.1 The evolution of cancer radiotherapy treatments
3
1.2 Radiobiology in radiotherapy
The theoretical and experimental radiobiology in radiotherapy offer strategies
and techniques from the most basic conventional therapy to specific treatment
directly on the manipulating radiobiological cellular environment. There are three
different levels on radiotherapy development as shown in Figure 1.2.
Figure 1.2 The different levels of radiotherapy development. Reproduced from
Joiner et al., 2009.
Radiotherapy development
Idea:
Conceptual basis for radiotherapy identifying
mechanism and processes that underlie the
response of tumours and normal tissues.
Examples: knowledge about
hypoxia, reoxygenation,
tumour cell repopulation and
mechanism of repair of DNA damage.
Treatment strategy :
Development of specific new
approaches in radiotherapy.
Examples: hypoxic cell sensitizers,
targated agents, high-linear energy transfer (LET) radiotherapy,
accelerated radiotherapy and
hyperfractionation.
Protocols:
Advice on the choice of schedules for
clinical radiotherapy. Example:
Conversion formulae for changes in
fractionation or dose rate, or advice on
whether to use chemotherapy
concurrently or sequentially with
radiation. Predicting the best treatment for the individual patient
(individualized radiotherapy)
4
1.2.1 Radiation induced cellular damage
Ionizing radiation causes atomic excitation and ionization of electron from the
molecules of cells that will induce biological damages and cell death. This effect
occurs in three phases which the physical phase, chemical phase and biological phase
as described in the following section.
1.2.1(a) The time scale of radiation effects
1.2.1(a)(i) Physical phase
This phase portrays the mechanisms between charged particles and the atoms
within the tissue composition. The light speed of electron takes about 10-18 second to
pass over the DNA strands and about 10-14 second to go across a mammalian cell. The
mechanism occurs when the external particles hit the orbital electrons resulting some
of the electron from atoms being expelled (ionization) and elevating others to higher
energy levels within an atom or molecule (ex citation ). The secondary electrons with
sufficient energies may excite or ionize other atoms near their path. For example, 1 Gy
of absorbed radiation dose will cause in excess of 105 ionizations within the volume
of every cell in diameter of 10 um.
1.2.1(a)(ii) Chemical phase
The chemical phase illustrates the process of atoms and molecules impaired
recurred with other cellular components in accelerated chemical interactions. The
formation of broken chemical bonds results from the processes of ionization and
excitation direct to the production of ‘free radicals’. These are highly potent with the
characteristics which were a highly reactive need to the restoration of electronic charge
equilibrium, which is accomplished within approximately 1 millisecond of radiation
exposure. The important characteristic of the chemical phase is the competition
between scavenging reactions, for example with sulphydryl compounds that
5
inactivated the free radicals and fixation reactions that lead to stable chemical changes
in biologically important molecules.
1.2.1(a)(iii) Biological Phase
The end stage of biological phase determines all the subsequent processes of
cell death. DNA lesions induced by radiation caused base damage, single-strand breaks
(SSBs) and double-strand breaks (DSBs) are aforethought to be most lethal. In the case
of SSBs damage, precision removal damaged base carried out by glycosylases,
forming damaged base or an abasic sites, which leads to fork collapse and DSB
formation.
DSBs are identified by specific protein regulator that works for cell apoptosis
responses or cell cycle arrest, thus stopping the proliferation of tumour cells with
damaged DNA and inhibiting tumorigenesis. The time–scale of the observable effects
of ionizing radiation may thus extend up to many years after exposure (Begg, Stewart
& Vens, 2011). Figure 1.3 shows the time-scale of effects of radiation interaction with
biological materials.
6
Figure 1.3 The time-scale of effects of radiation interaction with biological
materials.
1.2.1(b) DNA damage mechanism
Clusters of energy deposition events (ionization and excitations) occur at the
track of secondary electrons resulting in multiple closely-spaced lesions (multiply
damaged sites) within a range of 20 nm. This process has been recognized as important
for cell killing and in regard to the ability of cells to repair such lesions.
Irradiation generates damage in various patterns of lesions in DNA molecules
such as strand rupture either single or double strand breaks, base alteration, sugars
destruction and cross-links involving nuclear proteins such as histones and non-
histones form of dimers. Altogether the number of DNA lesions produced by
irradiation is about 100 distinct patterns. Alteration induced in the DNA of a cell by a
dose of 1-2 Gy is approximately: modified base by the formation of radical
7
hydroperoxide > 1000; single strand breaks (SSB) ~1000; double-strand breaks (DSB)
~40 (Wouters & Begg, 2009).
The indirect effect of strand break plays a prevalent role in cell killing, the most
frequent DNA lesion for in vitro study with the highest radiochemical yield. Single-
strand break (SSB) produced a large portion of hydroperoxide radicals. Following
breakage of the phosphodiester bond, the two strands separate.
Other experimental data show that low dose radiation produced early
radiosensitivity DSB, which is a critical indicator for cell killing. While for higher
doses, cell deaths are a consequence of unrepaired or mis-repaired DSB. Tremendous
effect is observed with high LET irradiation, resulting in the formation of complex
DSB lesions. Figure 1.4 shows the DNA damages mechanism of single and double
strand break.
Figure 1.4 DNA damages mechanism of single and double strand breaks.
8
1.2.2 Radiation cell survival and damage
The success of radiotherapy depends on the ability to cause damage to the
tumour’s cells. Quantification of the damage can be done by using the different
biological technique. However, clonogenic assay and cell survival curves are the basis
for understanding radiation cell survival and damages.
1.2.2(a) Cell survival curves
The first cell survival curve used for quantitative evaluation of radiobiological
was practised by Puck and Markus, 1956. Then in 2006, Nicolaas et al. had published
the protocol for clonogenic assay as the assessment for in-vitro techniques, based on
the ability of the cell considered to be survived then gives rise to a colony formation.
The colonies containing more than 50 cells were counted and depicted in graphically
by cell survival curve (Tubiana et al., 1990). Radiation sensitivity evaluation using this
protocol of different adherent cell type had become the gold standard for radiation
doses response.
The outcome of colonies formation will be presented in the so-called survival
curve, which is consists of survival fraction versus dose. The common pattern shows
the proportion of surviving cells (Sf) decreases as the dose (D) increases. The curve
was plotted in semi-logarithmic coordinates (log Sf as a function of D). This type of
plot emphasizes very small values of S at high doses but may sometimes less
appreciate survival after low doses. Thus, it is crucial to manipulate curves response
by presenting in mathematical functions. These are based on the hypothetical
mechanisms of cell lethality or acute damages, which are always emphasized by
radiobiological modelling (Tubiana et al., 1990). Figure 1.5 shows the survival curve
of HeLa cells proliferation versus dose (Puck and Marcus, 1956). The survival curve
9
for single cells of the HeLa cells line depicted the formation of the shoulder of the
curve is unequivocal and constitutes evidence for a multiple hit killing mechanism.
Figure 1.5 Survival curve of HeLa cells proliferation versus dose (Puck and
Marcus, 1956).
1.3 Radiobiological model
Radiobiological modelling is used for quantitative radiation biology that will
explain both dose-response and time-dose relationships.
1.3.1 Application of the LQ model
Radiobiological models primarily LQ model has been implemented over more
than a decade in clinical radiotherapy. The quantitative mathematical model has been
adequately simulated the shape of cell-survival curves for mammalian cells and was
applied to assess the optimum clinical outcome from conventional therapy to
sophisticated therapeutic approaches.
LQ model has been classified as the most common fitted radiobiological model
to the survival dose curve. The LQ model describes the relationship between total
isoeffective dose and the dose per fraction in fractionated radiotherapy. This model
10
forms the robust quantitative environment for considering the balance between acute
and late reactions (and effect on the tumour) as dose per fraction and total dose
changed. This is one of the most important developments in radiobiology application
to radiotherapy. In the present time, it is strongly recommended that LQ model should
always be used, with correctly chosen α/β ratio, to describe isoeffect dose relationships
at least over the range of doses per fraction between 1 and 5 Gy. The α/β ratio describes
the shape of the fractionation response: a low (0.5-6 Gy) is usually characteristic of
late-responding normal tissues and indicates a rapid increase of total dose, with
decreasing dose per fraction and a survival curve for the putative target cells that are
significantly curved. While for high ratio (7-20 Gy) represents the condition of the
early–responding normal tissues and rapidly-proliferating carcinomas; it indicates a
less curved cell-survival response for putative target cells. The LQ model is an
appropriate way to be used in clinical calculations and comparisons or in the change
in total dose for an altered dose per fraction either old or new treatments. For late
reactions, it is usually unnecessary to modify total dose in response to a change in
overall time, but for early reactions (and for tumour response) a correction for overall
treatment time should be included. Although the effect of time on biological effect is
complex, the simple linear correction has been shown to be of some values.
11
1.3.1(a) The validity of the LQ model
The question regarding the validity of the LQ approach can only be addressed
on an endpoint-by-endpoint basis. The LQ model has been applied to datasets from
quite a few clinical studies, and there has in many cases been good agreement between
the predicted and observed study outcome. This gives some confidence in using the
model to estimate the effects of changed dose fractionation in a situation where there
are clinical parameter estimates.
Data has supported that the implementation of the LQ model is reliable for dose
or dose per fraction ranges from about 1 Gy to 5 Gy. However, as the dose range of
the radiotherapy widens, the classical LQ model becomes less and less accurate
(Brahme, 2011). According to Andisheh, et al., (2013) LQ model is not suitable in the
high-dose region where it underestimates the surviving fraction in the high dose range.
Its parameters are essential but to describe the more complex treatment, a realistic
model is more desirable. Limitation of the LQ model in clinical radiotherapy could be
listed as follow:
1. LQ model does not accurately explain the observed (in-vivo) clinical data
2. LQ model was derived largely from in-vitro study and does neglect the side
effect irradiation on the neighbouring normal tissues.
3. LQ model does not consider the effect towards radioresistant cells which is
also known as cancer stem cells response. (Kirkpatrick et al. 2008).
12
1.4 Problem statement and rationale of the study
Ideally, the behavior shown by AuNPs as biocompatible radiosensitizer had
been proven by many studies. The highly productive of photoelectric interaction
between AuNPs and x-ray especially at kilovoltage energy caused simultaneously
increasing of secondary radiation beneficial to induce cell death significantly.
However, the nature of kilovoltage x-ray beam was very strongly attenuated by soft
tissues in which treatment limited only for superficial tumours. Therefore,
megavoltage radiation used in clinical radiotherapy will be more promising as it has
superior penetration that allows for greater dose uniformity and reduced the dose
delivered to surrounding healthy tissues. The application of AuNPs with megavoltage
radiotherapy could produce radiosensitization or dose enhancement as better as
kilovoltage x-ray beam (McMahon et al. 2011, Liu et al., 2018). Radiosensitization
effects have also been explored for advanced radiotherapy modalities using proton and
synchrotron-based therapy (Cho et al., 2016, Enferadi et al., 2017). However, the data
reported are still limited and identification on further parameters for quantifying
AuNPs radiosensitization effects is extremely important for clinical stage.
Therefore, the novelty of this study is to determine the radiosensitization
effects by AuNPs for different radiotherapy modalities: megavoltage photon beam,
electron beam and high-dose rate (HDR) gamma rays f rom Ir-192 source available in
HUSM (convebtional radiotherapy). For advanced radiotherapy, we had achanced to
investigate proton beam and monoenergetic synchrotron photon beam of kilovoltage
energies from our international research collaboration team. The radiosensitization
response is simultaneously manifested with various radiobiological model and
quantification methods. Such efforts had never been done from other reserachers in
full detail, thus the results obtained from this thesis will contribute to a more
13
comprehensive data in unravelling the mechanisms that influence the tissue’s
radiobiological response and cells’ reaction systems treated with AuNPs as
radiosensitizer across multiple radiotherapy beams. This is extremely important when
considering the wide range of various radiotherapy beams combined with AuNPs
treatments; as such huge data range will encompass a great understanding of the
complexity and specificity of identifying the ideal treatment selection and formulation
when optimizing the radiation dose given for clinical translations.
1.5 General objective
To characterize the radiosensitization effects by gold nanoparticles (AuNPs)
for advanced radiotherapy using multiple radiobiological models.
1.6 Specific objectives
1. To investigate the cytotoxicity, cellular uptake and intercellular localization of
AuNPs.
2. To quantify the radiosensitization effects by AuNPs for photon beam, electron
beam and HDR brachytherapy as well as proton beam and monoenergetic synchrotron
beam.
3. To analyze the validity of different radiobiological models and quantification
methods in characterizing the radiosensitization effects by AuNPs.
4. To evaluate parameters that influence the radiosensitization effects by AuNPs
such as cell types, beam energy, nanoparticles size and concentration.
14
1.7 Thesis outlines
The thesis is divided into two parts. Part 1 is the study on conventional clinical
radiotherapy which as follow photon beam, electron beam and HDR brachytherapy.
The study was conducted at Hospital Universiti Sains Malaysia, Health Campus,
Kubang Kerian, Kelantan.
Part II consists of the study conducted using specialized beam of the proton
beam and monochromatic synchrotron photon beam. The study on proton beam was
conducted at Hyogo Ion Beam Medical Center in Hyogo, Japan from the period of
2016-2018. The study using monochromatic synchrotron beam was conducted at
Australian Synchrotron in Clayton Melbourne Australia in which the beam time was
acquired in December 2015.
The chapters of the thesis:
Chapter 1: The introduction describes the modern radiotherapy approaches, the
specification on radiobiological effects and the innovation of nanomedicine
applications. The objectives of study are also emphasized.
Chapter 2: The literature review of the study details on gold nanoparticles application
with radiotherapy, the mechanism of the interactions and the pre-clinical test of
previous study.
Chapter 3: The methodology of the study was initially specified on cell culture
maintenance, gold nanoparticles preparation, cellular localization, uptake and
cytotoxicity assay protocol. The cell irradiations setup, radiation dosimetry and
clonogenic assay were illustrated. Radiobiological models applied for cell survival
curve and quantification methods of radiosensitization effects were then described.
15
Chapter 4: The description of the results begins with gold nanoparticles uptake,
localization and cytotoxicity. The cell survival analysis using different radiobiological
models were then presented. Linear Quadratic (LQ), Multi-Target (MT), Repairable
Conditionally Repair (RCR), Pade’ Linear Quadratic (PLQ), Repair (RM), Kavanagh
and Newman (KN) and Two Components (2C) Models are validated. The survival
curve of different beam was compared and analysed. Radiosensitization impact were
quantified using different methods (DEF, SER, RER, REF). The radiosensitization
effects due to AuNPs size, concentration and cell types are also examined.
Chapter 5: The findings of gold nanoparticles uptake, localization and cytotoxicity
was discussed. The detailed-on the radiosensitization impact for conventional
radiotherapy are explained. The influence of AuNPs sizes, concentrations and different
cells response were justified.
Chapter 6: Introduction and literature on proton beam therapy was briefly described.
The cell survival experimental work for proton beam was illustrated. The study was
also supported by additional measurement of reactive oxygen species (ROS) due to
AuNPs interaction with proton beam. All those findings were discussed in detail
accordingly.
Chapter 7: A brief introduction on synchrotron radiation production and its
applications is described. Literature review on previous challenge in overcome dose
limitation and optimization in radiotherapy were also discussed. In this study, the
experimental methods were explained, and results were analyzed using similar
radiobiological models approaches. The quantification of radiosensitization effects has
16
also been evaluated. The experimental data were also supported by theoretical dose
enhancement calculation. All the corresponding results were discussed.
Chapter 8: The conclusion of the study was reported by each main chapter,
respectively. Limitations and future recommendations were also discussed briefly.
17
CHAPTER 2
LITERATURE REVIEW
2.1 Nanomedicine in radiotherapy
Nanomedicine is an active research area that holds great future to intercede
with cancer treatment at the molecular level and deliver effective doses to targeted
cancer cells with improved selectivity and lessened toxicities towards neighbour
normal tissues. The core of nanomedicine is the development of nanoparticles,
materials that hold unique distinctions such as nanoscale size, high surface-to-volume
ratio and suitable physicochemical characteristics. Nanoparticles have wide range of
types are able to act as a multifunctional agent that incorporates with the specific
binding of drugs to targets in cancer cells or the tumour microenvironment,
simultaneous visualization of tumours using innovative imaging techniques, prolonged
drug-circulation times, controlled drug-release kinetics, and superior dose scheduling
for improved patient compliance. In an example, the carbon nanotubes have future
usefulness in cancer thermal ablation therapy, because of their electrical and thermal
conductivity. However, their properties such as length and diameter might cause
inflammatory and toxic effects (Markman et al., 2013).
Novel metal nanoparticles in particular gold nanoparticles are also capable as
a therapeutic agent, imaging contrast media and nanocarrier of such proteins, DNA or
RNA. The gold core is considered to be non-toxic and the therapeutic payload can be
forced to be released from the conjugate due to their photo-physical properties
(Webster et al., 2013). In radiotherapy, nanoparticles possess properties as a
radiosensitizer to enhance radiation dose to the target by increasing the probability of
radiation interaction and production of the secondary electron that will cause DNA
damage. Examples of nanoparticles based radiosensitizer that has the potential to be
18
applied in radiotherapy are metallic nanoparticles (Au, Bi, Pt, Gd), superparamagnetic
iron oxide nanoparticles, quantum dots and various hybrid nanoparticles.
Metallic nanoparticles composite a packed of metal particles which are
functionalized as scatter medium when interact with high energy radiation
(megavoltage) to produce secondary radiation. The secondary radiation directly
localized into tumours cells in large amount at a short distance. This mechanism gives
therapeutic radiation in tumours area at the same time despair neighbour of healthy
tissues. The concept of using high Z materials incorporated in cells was initially
investigated using iodine which presented higher efficiency for secondary radiation
production (Matsudaira et al., 1980). This study was also supported by a separate study
using gold films that indicated multifold and significant dose enhancement effect upon
irradiation (Regulla et al., 1998). Dose enhancement effects induced by high Z
materials are believed to be more efficient by resizing the gold or others types of metal
into nanoparticles.
Several types of metallic nanoparticles have been introduced as radiosensitizer
show similar significant dose enhancement effects when tested both in-vitro and in-
vivo. Metallic nanoparticles such as gadolinium, iron oxide, titanium and silver
nanoparticles provide similar outcome of dose enhancement or radiosensitization
effects (Tokumitsu et al., 2000; Xu et al., 2009; Khoei et al., 2014, Nakayama et al.,
2016 ). Superparamagnetic iron oxide nanoparticles for examples withhold special
properties of highly biocompatible while collaterally induced cytotoxic effects when
interact with radiation (Klein et al., 2013). In some cases, these nanoparticles are also
combined with chemotherapeutic agents or nanocomposite metal based like
(Fe2O4/Ag) for increased efficiency (Huang et al., 2012) (Zhao et al., 2012).
19
Other than metal-based nanoparticles, the non-metal nanoparticles also exhibit
potential to acts as radiosensitizer. Klein et al. (2012) have investigated the ultrasmall
uncapped and aminosilanized oxidized silicon nanoparticles to treat breast cancer cells
with X-rays. Quantum dots which made of semiconductor (CaF, LaF, ZnS or ZnO)
have been developed as photosensitizer capable in producing radicals upon absorption
of visible light. This type of light however gives less penetration and suitable
specifically for superficial cancer treatments (Shao et al., 2011).
Nanomedicine in general occupied various applications in radiotherapy.
Summary most of all classification of nanomedicine applied in elevating radiation
efficiency is presented in Figure 1.6.
Figure 2.1 Nanomedicine in radiotherapy
Surviving Targeting
20
2.2 Application of Gold Nanoparticles (AuNPs) in radiotherapy
The development of potential high Z materials as radiosensitizers in recent
years becomes more interesting. The basic principle of primary radiation collide with
high Z elements deposited secondary radiation locally surrounding targeted cancer
area may improve radiotherapy efficiency in treating malignant diseases. Pioneer
studies have proven the presence of iodine in mammalian cells in culture indeed
induced radiosensitization or dose enhancement effects that enhance cell killing when
combined with radiation. Subsequent studies with other types high Z matters had been
reported such as gold (Au), silver (Ag), hafnium oxide, platinum (Pt), gadolinium, iron
oxide (Fe3O4) and quantum dots (QDs). The most promising high Z elements which is
gold, tuned into nanoparticles scale given efficiency distribution throughout the
specific target. Gold nanoparticles (AuNPs) features of synthetic versatility, capable
for size, shape and surface characteristics modification.
Regulla et al. (2000) were first verified the dose enhancement effects using
monolayer culture of C3H/10T1/2 mouse embryo fibroblasts irradiated with 48 keV
X-ray on gold foil. This is followed by seminal work by Hainfeld et al. (2010) on nano-
small sized 1.9 nm AuNPs that were injected into the mice bearing SCCV11 squamous
cell carcinomas. The results show improvement in the survival days by the increment
from 53 days (-gold) to 76 days (+gold). The promising results of AuNPs are due to
the higher mass attenuation coefficient of AuNPs in which at 100 keV of photon
energy, gold could provide 2.7 times better sensitivity per unit weight than iodine and
gadolinium. This indication picturized the existence of AuNPs in the targeted tumour
may cause the dose enhancement effects in radiotherapy (Goswami et al., 2017).
Improvised gold nanoparticles i.e: AuNP-dual peptide has been investigated to show
that at 4 Gy, dose enhancement factor (DEF) up to 3.2 had been produced (Nicol et al.,
21
2017). AuNPs in combination with radiation also delayed the tumour growth around
10.2 days instead of 3.4 day with irradiation alone (Nicol et al., 2018).
Other promising study using multifunction AuNPs-formulation (Chitosan-
capped-AuNPs-Doxorubicin) with MCF-7 cells irradiated using 6 MV photon beam
exhibited potent radiosensitization effects up to 2 fold compared to irradiation only
(Fathy et al. 2018). Another latest investigation reported that 20 nm of AuNPs-PEG-
anti-human epidermal growth factor receptor type 2 (HER2) antibody showed a
significant dose enhancement at only 3 Gy of radiation dose (Hatoyama et al., 2019).
Shreds of evidence clearly potrayed the potential of the AuNPs application as
radiosensitizer on top of others different promising usage of AuNPs in many areas.
2.2.1 Characteristic and properties of AuNPs
Gold nanoparticles have been reported to show potential as radiosensitizers for
radiotherapy. This mainly due to its special characteristic and properties in Table 2.1.
Table 2.1 The characteristic and properties of AuNPs
Characteristics High-Z atomic number (Z=79), an ideal material for
radiosensitization reactions when combining with
radiotherapy (Hainfeld & Slatkin, 2008).
Gold being very inert, it is highly biocompatible which issues
need to be considered as in vivo inflammatory effect would
be resulted (Shukla et al., 2005).
Nanoparticles surface ratio able to enhance the effect of the
radiation over a large area of tumour thus eliminating the
need of the nanoparticles to be delivered to all the cells of the
tumour tissues (Jain et al., 2007).
22
Properties Nanoparticles are known to have low systemic clearance as
compared to low molecular contrast agents such as iodine
allowing the photosensitizing material enough time to get
absorbed into the tumour tissue (Longmire et al., 2008).
Nanoparticles are known to be well absorbed into the
systemic circulation, better permeation into the tumour tissue.
This along with lower clearance rate results in the enhanced
permeation and retention (EPR) effect (Iyer et al., 2006).
Better attaching target with antibodies or other targeting
moieties, given a good specifically delivered to the tumour
cells location (Hainfeld et al., 2008).
The feasible nano shape based on delivery requirements of
the tumours tissues (such as its size and location) so as to
achieve optimum delivery and effects (Chithrani et al., 2010).
Improving specific tissue pharmacokinetics, cause them easy
to image and quantify by using optimum dose level for best
result (Xia et al., 2016).
23
2.2.2 Radiosensitization Effects by AuNPs
2.2.2(a) Theoretical dose enhancement by high Z materials
A non-Monte Carlo method also known as a systematic analysis of mass
attenuation coefficient in different energies of photon had been simulated by Corde et
al. (2004) for dose enhancement produced by iodine. This theoretical model-based
calculation was also been used by Rahman et al. (2009) on AuNPs. The theoretically
expected dose enhancement ratio or factor was calculated from the variation of the
mass energy-absorption coefficient of the target due to the presence of high Z matter
such as AuNPs. The dose enhancement factor (DEF) calculation is shown in the
Equation 2.1:
𝐷𝐸𝐹 =(
𝜇𝑒𝑛𝜌
)𝐸
𝑤𝑎𝑡𝑒𝑟+ 𝐴𝑢
(𝜇𝑒𝑛
𝜌)
𝐸
𝑤𝑎𝑡𝑒𝑟 = 𝑤𝐴𝑢(
𝜇𝑒𝑛𝜌
)𝐸
𝐴𝑢+ (1− 𝑤𝐴𝑢)(
𝜇𝑒𝑛𝜌
)𝐸
𝑤𝑎𝑡𝑒𝑟
(𝜇𝑒𝑛
𝜌)
𝐸
𝑤𝑎𝑡𝑒𝑟 (2.1)
The mass-energy absorption coefficient for the considered compound were
simulated with monochromatic x-rays beam (energy: E) and in the fraction by weight
of Au in the mixture. Figure 2.1 shows the theoretical DEF for several Au aqueous
mixtures.
24
Figure 2.2 Energy dependence of the theoretical of DEF for several Au aqueous
mixtures (from bottom to top, the mass proportion of Au in water, wAu
range from 0.01 to 1.