raizulnasuha binti ab rashid

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


6.5.5 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

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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(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


Table 4.4 LQ model parameters for 15 MeV electron beam irradiation with


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


Table 4.8 MT model parameters for 6 MeV electron beam irradiation with


Table 4.9 MT model parameters for 15 MeV electron beam irradiation with


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


Table 4.12 RCR model parameters for 10 MV photon beam irradiation with


Table 4.13 RCR model parameters for 6 MeV electron beam irradiation with


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


Table 4.17 PLQ model parameters for 10 MV photon beam irradiation with


Table 4.18 PLQ model parameters for 6 MeV electron beam irradiation with


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


Table 4.23 2C model parameters for 15 MeV electron beam irradiation with


Table 4.24 KN model parameters for 6 MeV electron beam irradiation with


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


Table 4.27 Repair model parameters for 10 MV photon beam irradiation with


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


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


models...................................................................................... 120

Table 4.40 The dose enhancement factor (DEF) values for 6 MeV electron


models...................................................................................... 121

Table 4.41 The dose enhancement factor (DEF) values for 15 MeV electron


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


Table 4.45 The sensitization enhancement ratio (SER) values for 6 MeV




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


models...................................................................................... 132

Table 4.49 The radiation enhancement ratio (RER) values for 6 MV photon


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



models...................................................................................... 135

Table 4.52 The radiation enhancement ratio (RER) values for brachytherapy


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


Table 6.3 RCR model parameters for 150 MeV proton beam irradiation


xvii

Table 6.4 PLQ model parameters for 150 MeV proton beam irradiation with


Table 6.5 2C model parameters for 150 MeV proton beam irradiation with


Table 6.6 KN model parameters for 150 MeV proton beam irradiation with


Table 6.7 The dose enhancement factor (DEF) values for 150 MeV proton


models...................................................................................... 200


proton beam from cell survival curves of different radiobiology

models...................................................................................... 202



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


AuNPs...................................................................................... 244

xviii


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


AuNPs...................................................................................... 249


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


AuNPs...................................................................................... 253


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


AuNPs...................................................................................... 258


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 7.34 The dose enhancement factor (DEF) values for 81.5 keV



Table 7.35 The sensitization enhancement ratio (SER) values for 30 keV

synchrotron from cell survival curves of different radiobiology

models...................................................................................... 278



models...................................................................................... 279



models...................................................................................... 280

Table 7.38 The sensitization enhancement ratio (SER) values for 81.5 keV


models...................................................................................... 281

Table 7.39 The radiation enhancement ratio (RER) values for 30 keV









xx

Table 7.42 The radiation enhancement ratio (RER) values for 81.5 keV



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


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


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


Figure 4.8 The cell survival curves for 6 MeV electron beam irradiation with


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


and without AuNPs fitted using the MT model......................... 74






with and without AuNPs fitted using the MT model. ............... 77


without AuNPs using the MT model. ........................................ 78


and without AuNPs fitted using the RCR model....................... 80


and without AuNPs fitted using the RCR model....................... 81


and without AuNPs fitted using RCR model. ........................... 82


with and without AuNPs fitted using RCR model. ................... 83

xxiii


without AuNPs fitted using RCR model. .................................. 84


and without AuNPs fitted using the PLQ model ....................... 86


and without AuNPs fitted using the PLQ model. ...................... 87


and without AuNPs fitted using the PLQ model. ...................... 88


with and without AuNPs fitted using the PLQ model ............... 89


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


model. ........................................................................................ 93

Figure 4.28 The cell survival fraction curves for 15 MeV electron beam


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


and without AuNPs fitted using the repair model ..................... 98


and without AuNPs fitted using repair model. .......................... 99


and without AuNPs fitted using the repair model ................... 100


with and without AuNPs fitted using the repair model ........... 101


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



model and (f) KN model.......................................................... 106

Figure 4.39 The survival curves for 15 MeV electron beam according to




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


models...................................................................................... 119

Figure 4.42 Dose enhancement factor (DEF) quantified for 10 MV photon


models...................................................................................... 120

Figure 4.43 Dose enhancement factor (DEF) quantified for 6 MeV electron


models...................................................................................... 121

Figure 4.44 Dose enhancement factor (DEF) quantified for 15 MeV electron


models...................................................................................... 122

Figure 4.45 Dose enhancement factor (DEF) quantified for brachytherapy


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


models...................................................................................... 127

Figure 4.48 Sensitization enhancement ratio (SER) quantified for 6 MeV


models...................................................................................... 128



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


models...................................................................................... 132

Figure 4.52 Radiation enhancement ratio (RER) quantified for 10 MV photon


models...................................................................................... 133

Figure 4.53 Radiation enhancement ratio (RER) quantified for 6 MeV


models...................................................................................... 134



models...................................................................................... 135

Figure 4.55 Radiation enhancement ratio (RER) quantified for brachytherapy


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


Figure 4.59 The cells survival curves for HeLa and HCT116 cells under


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


with and without AuNPs fitted using MT model. ................... 192


with and without AuNPs fitted using RCR model. ................. 194


with and without AuNPs fitted using PLQ model. .................. 195

xxvii


with and without AuNPs fitted using 2C model. ..................... 196


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


Figure 6.14 Dose enhancement factor (DEF) quantified for 150 MeV proton


model. ...................................................................................... 199



models...................................................................................... 201



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


Figure 7.9 The cell survival curves for 81.5 keV irradiation with and without


Figure 7.10 The cell survival fraction curves for 30 keV irradiation with and

without AuNPs using MT model. ............................................ 240



Figure 7.12 The cell survival fraction curves for 81.5 keV irradiation with and



without AuNPs using RCR model. .......................................... 243




without AuNPs, using RCR model. ......................................... 245




without AuNPs fitted using PLQ model. ................................. 248






without AuNPs, fitted using PLQ model. ................................ 251


without AuNPs, fitted using the 2C model. ............................. 252



xxix






without AuNPs, fitted using the RM model. ........................... 257







Figure 7.29 The survival curves for 30 keV synchrotron beam according to






model, (b) MT model, (c) RCR model, (d) PLQ model, (e) 2C

model and (f) RM model. ........................................................ 263



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




Figure 7.33 Dose enhancement factor (DEF) quantified for 30 keV









xxx

Figure 7.36 Dose enhancement factor (DEF) quantified for 81.5 keV



Figure 7.37 Sensitization enhancement ratio (SER) quantified for 30 keV









Figure 7.40 Sensitization enhancement ratio (SER) quantified for 81.5 keV



Figure 7.41 Radiation enhancement ratio (RER) quantified for 30 keV









Figure 7.44 Radiation enhancement ratio (RER) quantified for 81.5 keV



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






xxxi


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.

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