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University of Wollongong Thesis Collection 2017+ University of Wollongong Thesis Collections

2017

Investigating the properties and application of tantalum pentoxide nanostructures for cancer radiotherapy

Ryan S. Brown University of Wollongong

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Recommended Citation Brown, Ryan S., Investigating the properties and application of tantalum pentoxide nanostructures for cancer radiotherapy, Doctor of Philosophy thesis, School of Physics, University of Wollongong, 2017. https://ro.uow.edu.au/theses1/137

Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] INVESTIGATING THE PROPERTIES AND APPLICATION OF TANTALUM PENTOXIDE NANOSTRUCTURES FOR CANCER RADIOTHERAPY

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

DOCTOR OF PHILOSOPHY

from

UNIVERSITYOF WOLLONGONG

by

RYAN S. BROWN Bachelor of Medical Radiation Physics Advanced (Class 1 Honours)

SCHOOLOF PHYSICS,FACULTY OF ENGINEERINGAND INFORMATION SCIENCES 2017 Abstract

In this thesis, a new material is investigated for application in radiation medicine. In particular, to the fields of dose-enhancement radiotherapy (DERT) and UV pro- tection. The material developed was tantalum pentoxide (elemental composition

T a2O5) in the form of ceramic nanostructured particles (NSPs). Prior to this study, no literature existed on the application of T a2O5 NSPs to DERT or UV protection, highlighting the novelty and significance of the results presented in this thesis. A series of toxicity studies revealed T a2O5 NSPs to be non-toxic over time periods of up to 14 days, concentrations up to 500 µg/ml and multiple cell lines. Efficacy of radiosensitisation was quantified using the sensitisation enhancement ratio, with a maximum SER of 1.46 measured in a 10 MV X-ray photon beam with a concentra- tion of 500 µg/ml on 9L cells. This enhancement not only compares well with other compounds currently utilised in DERT, but in some cases exceeds the efficacy at this beam energy, notably in the case of gold (Au) nanoparticles (NPs). Furthermore, radiosensitisation was observed to be dependent on beam energy and NSP morphol- ogy, although at higher NSP concentrations no significant change in enhancement was observed. This lack of enhancement was attributed to a phenomenon not yet observed in DERT, referred to as the ”shell effect”.

The first introduction of T a2O5 NSPs for UV protection applications was investi- gated using UV-visible absorbance spectroscopy, as well as cellular experiments with non-cancerous MDCK cells. All results were compared to commercial-grade zinc oxide (ZnO) NPs, which are currently utilised in sunscreen and cosmetic products

ii Abstract iii

for UV protection. T a2O5 NSPs were found to be non toxic to MDCK cells, as well as weak reactive oxygen species (ROS) generators, in stark contrast to ZnO NPs.

Most significant, T a2O5 NSPs showed strong absorbance over the entire UV region, much higher than that measured for ZnO NPs. The degree of UV protection provided by each NP was quantified using the in vitro sun protection factor (SPF), yielding a value of 12.12 for T a2O5 NSPs, and 1.28 for ZnO NPs. Analysis using the critical wavelength and UVA to UVB ratio showed both NPs were strong broad-spectrum protectors.

The first two chapters establish the motivations and scope of the study, as well as cur- rent literature and theory pertinent to NPs and their application to nanomedicine and radiotherapy. Chapter 3 gives a description of general experimental methodologies commonly used throughout the study, with particular emphasis on NP preparation and cell line experiments.

Chapters 4 and 5 focus on physical and biological characterisation of T a2O5 NSPs, and their interactions with multiple cell lines. Physical characterisation experiments probe the morphology, absorbance and scattering properties of the NSPs. Biological characterisation experiments explore toxicity and cellular uptake of the NSPs with cancerous 9L and non-cancerous MDCK cell lines primarily.

Chapters 6 and 7 represent the culmination of this work, where by the effectiveness of T a2O5 NSPs as radiosensitisers and UV protectors is evaluated and quantified. Explanations of the phenomena observed are presented with reference to the char- acterisation results detailed in chapters 4 and 5, in conjunction with theories and literature reviewed in chapter 2. Chapter 8 summarises the main findings of this thesis work, and proposes further avenues for experimentation in the future. Statement of Originality

This is to certify that the work described in this thesis is entirely my own, except where due reference is made in the text.

No work in this thesis has been submitted for a degree to any other university or institution.

Signed

Ryan S. Brown 31st March, 2017

iv Acknowledgments

This project has been a journey of hard work and perseverance culminating in the exciting results obtained. Needless to say, I would have been unable to achieve any of this without the guidance of several people. Firstly, I would like to thank Assoc. Prof. Michael Lerch and Prof. Anatoly Rosenfeld, for assigning me such an inter- esting and promising project, as well as for imparting their own knowledge onto me throughout the course of my Honours and PhD years, as well as the staff at IHMRI for their constant guidance.

Secondly, I would like to thank Dr. Moeava Tehei who provided constant guidance, invaluable experience and worked tirelessly to aid in achieving my goals.

Next, Dr. Stephanie Corde-Tehei for taking time out of her own usual activities to make herself available on weekends for me to complete the irradiation experiments and for providing great insight into the project itself.

To Sianne Oktaria and Adam Briggs, and all other PhD and Masters students in the T.N.T group, without whom this project would never have come to fruition. All aided in extensive, and often tedious, lab work, while Adam provided me with the support, ability and will to complete this postgraduate project, and made my undergraduate degree a lot of fun (along with Dr. Lee Coates). May we continue to enjoy ourselves in the future field of Medical Physics and eventually achieve our goal of working

v Acknowledgments vi together again.

I thank all the physics staff at STGCCC, most notably my supervisor Andrew Howie and director Anna Ralston, for providing me with the time, support and motivation to push through the end of my thesis and achieve this incredible goal of completion while working full-time in an incredible job.

An immense thank you must go to my parents who provided constant support and tolerated me through thick and thin, love you both.

Finally, my beautiful girlfriend Kayla, all the thanks in the world must go to you for constantly providing me with inspiration and motivation to complete my project, es- pecially during peak times of stress and despair. Your beautiful nature and example, as well as that of all my other close friends and family, have helped shape the person I am today, and in turn, the quality and success of this thesis project. Acknowledgments vii Publications

1. Briggs, A., Corde, S., Oktaria, S., Brown, R., Rosenfeld, A., Lerch, M., ... and Tehei, M. (2013). Cerium oxide nanoparticles: influence of the high-Z component revealed on radioresistant 9L cell survival under X-ray irradiation. Nanomedicine: Nanotechnology, Biology and Medicine, 9(7), 1098-1105.

2. Brown, R., Tehei, M., Oktaria, S., Briggs, A., Stewart, C., Konstantinov, K., ... and Lerch, M. (2013). Enhancing the sensitivity of in vitro 9L cells exposed to X-ray radiation fields using Ta2O5 nanoparticles. Australasian Physical and Engineering Sciences in Medicine, 36(1), 65-141. [Conference paper]

3. Brown, R., Tehei, M., Oktaria, S., Briggs, A., Stewart, C., Konstantinov, K., ... and Lerch, M. (2014). High-Z Nanostructured Ceramics in Radiotherapy: First Ev- idence of Ta2O5-Induced Dose Enhancement on Radioresistant Cancer Cells in an MV Photon Field. Particle and Particle Systems Characterization, 31(4), 500-505.

4. Brown, R., Corde, S., Oktaria, S., Rosenfeld, A., Lerch, M., ... and Tehei, M. (2016). Nanostructures, concentrations and energies: An ideal equation to extend therapeutic efficiency on radioresistant 9L tumour cells using Ta2O5 ceramic nanos- tructured particles. Biomedical Physics and Engineering Express, 3(1).

5. Brown, R., Corde, S., Oktaria, S., Rosenfeld, A., Lerch, M., ... and Tehei, M. (2016). An overview of Ta2O5 ceramic nanostructured particles in dose enhance- ment radiotherapy. Australasian Physical and Engineering Sciences in Medicine, 39;1045-1190. [Conference paper]

6. Brown, R., Corde, S., Cardillo, D., Oktaria, S., Rosenfeld, A., Lerch, M., ... and Tehei, M. (2017). First evidence of broad-spectrum UV protection by Ta2O5 nanos- tructured ceramics. Biomedical Physics and Engineering Express.

The author of this thesis work had major contributions to both the writing of the publications listed above, as well as collection and analysis of the data presented in the publications themselves. In particular, the publications where the candidate is listed as the first author represent significant contributions in the sense that: the manuscript was drafted and prepared by the candidate, all comments made by co- Acknowledgments viii authors were collated and applied by the candidate, the candidate was involved in and responsible for the majority of primary data collection as part of a multidisciplinary team, the candidate was responsible for facilitating and discussing results as part of a multidisciplinary analysis, and in general, the candidate was responsible for managing experiments related to the application of T a2O5 NSPs to cancer therapy. Acknowledgments ix Conference Oral Presentations

All presentations described hereafter are oral presentations detailing content related to the projects contained in this thesis.

1. Radiation 2012 Conference, Australian Institute of Nuclear Science and Engi- neering (AINSE), Lucas Heights, NSW, Australia, 17th February 2012, Enhancing the sensitivity of in vitro 9L cells exposed to X-ray radiation fields using T a2O5 nanoparticles.

2. 2012 Engineering and Physical Sciences in Medicine (EPSM) Conference, Gold Coast, Australia, 2nd December 2012, Tantalum Pentoxide Ceramic Nanoparticles: Novel Non-toxic Radiosensitisers of a Radioresistant Tumor Model.

3. The 20th Australian Institute of Physics (AIP) Congress and 37th Australian Con- ference on Optical Fibre Technology (ACOFT), UNSW, Sydney, Australia, 9th De- cember 2012, The dose enhancement effect revealed on 9L glioma cells in X-ray photon fields utilising novel T a2O5 nanoparticles.

4. Prince of Wales Hospital (POWH) Radiation Oncology Meeting, Randwick, NSW, Australia, 29th November 2013, An overview of the properties and application of T a2O5 nanoparticles as radiosensitisers on gliosarcoma cells in MV and kV X-ray photon fields.

5. Centre for Medical Radiation Physics (CMRP) PhD Seminar, University of Wol- longong (UOW), NSW, Australia, 20th June 2014, High-Z Nanostructured Ceramics in Radiotherapy: First Evidence for the Application of T a2O5 in Dose Enhancement Radiotherapy.

6. Cancer and Drug Discovery Group (CDDG) Seminar, Illawarra Health and Med- ical Research Institute (IHMRI), UOW, NSW, Australia, 2nd April 2015, High-Z Nanostructured Ceramics in Radiotherapy: The Dependence of Radiosensitisation on Concentration, Beam Energy and Particle Morphology for T a2O5 in Dose En- hancement Radiotherapy. Acknowledgments x

7. Targeted Nanotherapies (TNT) Group Meeting Presentation, CMRP, UOW, NSW, Australia, 16th July 2015, An Update on the Application of High-Z Nanostructured Ceramics in Radiotherapy: Physical Characterisation, Biological Characterisation and Dependency Assessment for T a2O5 in Dose Enhancement Radiotherapy.

8. MedPhys 2015, University of Sydney (USYD), NSW, Australia, 8th December 2015, Nanostructures, concentrations and energies: An ideal equation to extend ther- apeutic efficiency on radioresistant 9L tumour cells using T a2O5 ceramic nanostruc- tured particles.

9. St George Cancer Care Centre (STGCCC) Continuing Professional Development (CPD) Seminar, St George Hospital, Kogarah, NSW, Australia, 1st February 2016, An Introduction to the Application of T a2O5 Nanostructured Particles to Dose En- hancement Radiotherapy.

10. STGCCC CPD EPSM Preparation Seminar, St George Hospital, Kogarah, NSW. Australia, 10th October 2016, An overview of T a2O5 ceramic nanostructured parti- cles in dose enhancement radiotherapy.

11. 2016 EPSM Conference, Sydney, Australia, 6th November 2016, An overview of T a2O5 ceramic nanostructured particles in dose enhancement radiotherapy. Contents

1 Introduction 1

1.1 Background ...... 1

1.2 Thesis Overview ...... 5

1.2.1 Objectives ...... 5

1.2.2 Aims ...... 5

2 Literature Review 7

2.1 Introduction ...... 7

2.2 Nanoparticles ...... 8

2.2.1 Introduction to Nanotechnology, Nanomaterials and Nanopar- ticles ...... 8

2.2.2 Properties of Nanoparticles ...... 10

2.2.3 Mechanisms of Interaction Between Nanoparticles and Cells 17

2.3 Properties and Applications of Tantalum Oxides ...... 24

2.3.1 Properties of Tantalum ...... 24

2.3.2 Clinical Applications For Tantalum ...... 25

2.3.3 Properties and Applications of Tantalum Oxides ...... 28

2.3.4 Application of Tantalum Oxide Nanoparticles to Nanomedicine 32

2.3.5 Characterisation Methods for T a2O5 NPs ...... 35

2.4 Radiotherapy and Radiobiology of Cancer ...... 36

xi CONTENTS xii

2.4.1 Introduction ...... 36

2.4.2 Introduction to Radiobiology of Cancer Cells ...... 36

2.4.3 Dose-Enhancement in Radiation Therapy ...... 48

2.4.4 Dose-enhancement with Nanomaterials ...... 51

2.4.5 Cell Survival Curves ...... 52

3 Experimental Details and General Methodology 54

3.1 Introduction ...... 54

3.2 Synthesis and Storage of T a2O5 NSPs ...... 54

3.3 Suspension and Sonication of T a2O5 NSPs in Solution ...... 57

3.4 Cell Line Characteristics ...... 59

3.5 Maintenance and Sampling of the Cell Culture ...... 62

3.6 Clonogenic Assay Technique ...... 64

3.7 Statistical Analysis ...... 66

4 Physical Characterisation of T a2O5 Nanostructured Particles 67

4.1 Introduction ...... 67

4.2 X-ray Diffraction ...... 68

4.2.1 Theory ...... 68

4.2.2 Method ...... 70

4.2.3 Results and Discussion ...... 70

4.3 Scanning Electron Microscopy ...... 72

4.3.1 Theory ...... 72

4.3.2 Method ...... 73

4.3.3 Results and Discussion ...... 74

4.4 High-Resolution Transmission Electron Microscopy ...... 77 CONTENTS xiii

4.4.1 Theory ...... 77

4.4.2 Method ...... 80

4.4.3 Results and Discussion ...... 80

4.5 Dynamic Light Scattering ...... 82

4.5.1 Theory ...... 82

4.5.2 Method ...... 84

4.5.3 Results and Discussion ...... 84

4.6 UV-vis Spectrophotometry ...... 86

4.6.1 Theory ...... 86

4.6.2 Method ...... 88

4.6.3 Results and Discussion ...... 88

5 Biological Characterisation of Cells to T a2O5 Nanostructured Particles 90

5.1 Introduction ...... 90

5.2 Cell Viability Assessment- 14 Days ...... 91

5.2.1 Method ...... 91

5.2.2 Results and Discussion ...... 92

5.3 Cytotoxicity Assessment- 6 Hours ...... 93

5.3.1 Method ...... 93

5.3.2 Results and Discussion ...... 94

5.4 Cytotoxicity Assessment- 1 and 2 Day Exposures ...... 96

5.4.1 Method ...... 96

5.4.2 Results and Discussion ...... 96

5.5 Cytotoxicity Assessment- 24 Hours and Maximum Concentration . 97

5.5.1 Method ...... 97

5.5.2 Results and Discussion ...... 98 CONTENTS xiv

5.6 Cytotoxicity Assessment- 24 hours and Multiple Cell Lines . . . . . 99

5.6.1 Method ...... 99

5.6.2 Results and Discussion ...... 99

5.7 FACS Flow Cytometry ...... 101

5.7.1 Method ...... 101

5.7.2 Results and Discussion ...... 102

5.8 Confocal Microscopy ...... 104

5.8.1 Method ...... 104

5.8.2 Results and Discussion ...... 104

6 Radiosensitisation Experiments using T a2O5 NSPs 107

6.1 Introduction ...... 107

6.2 Investigating the Radiosensitising Effect of T a2O5 TNSPs in an MV Photon Field ...... 108

6.2.1 Introduction ...... 108

6.2.2 Method ...... 112

6.2.3 Results and Discussion ...... 113

6.2.4 Conclusion ...... 115

6.3 Investigating the Dependency of Radiosensitisation in T a2O5 NSPs on Beam Energy, NSP Morphology and NSP Concentration . . . . . 116

6.3.1 Introduction ...... 116

6.3.2 Methods ...... 120

6.3.3 Results ...... 122

6.3.4 Discussion ...... 124

6.3.5 Conclusion ...... 131

7 UV Protection Experiments using T a2O5 Nanostructured Particles 133 CONTENTS xv

7.1 Introduction ...... 134

7.2 Methods ...... 136

7.2.1 T a2O5 NSP and ZnO NP production ...... 136

7.2.2 Cell line and culture ...... 136

7.2.3 X-ray Diffraction ...... 137

7.2.4 High-Resolution Transmission Electron Microscopy . . . . 137

7.2.5 Clonogenic cell survival assay ...... 137

7.2.6 Toxicity assessment ...... 138

7.2.7 Flow cytometry analysis ...... 138

7.2.8 UV-visible absorption spectroscopy ...... 139

7.2.9 SPF analysis ...... 140

7.3 Results and Discussion ...... 141

7.3.1 Characterisation of T a2O5 NSPs and ZnO NPs ...... 141

7.3.2 Cytotoxicity of T a2O5 NSPs and ZnO NPs to MDCK cells . 144

7.3.3 ROS-generative capacity of T a2O5 NSPs and ZnO NPs in MDCK cells ...... 146

7.3.4 UV absorption properties of T a2O5 NSPs and ZnO NPs . . 149

7.3.5 SPF analysis ...... 153

7.4 Conclusion ...... 155

8 Conclusions and Future Work 157

Bibliography 163 List of Figures

1.1 The proportion of patients contracting different types of tumours, as well as the proportion who receive RT [1]...... 2

1.2 A Varian TrueBeam LINAC commonly utilised by clinical depart- ments in EBRT treatments, including IMRT, VMAT and SBRT [2]. . 3

2.1 The branches of constituent terms used to describe different types of nanomaterials, as set by the ISO [3]...... 8

2.2 Illustration of the relationship between surface area and particle size [4]...... 10

2.3 A graphical abstract depicting the free radical scavenging mecha- nism exhibited by cerium oxide nanoparticles irradiated in an X-ray photon field for radiation protection applications. The beam energy- dependent nature of protection and enhancement is highlighted in this particular study [5]...... 11

2.4 Classification features of nanostructures [4]...... 12

2.5 TEM images of cerium oxide NPs in solution before sonication [6], note the presence of significant agglomeration. The scale bar at the bottom of the image represents 100 nm...... 15

2.6 TEM images of cerium oxide NPs after sonication portraying degree of dispersion [6], note the reduction in aggregate size due to sonica- tion. The scale bar at the bottom of the image represents 120 nm. . . 16

2.7 Cellular uptake of CeO2 NPs in A549 cells as a function of their associated zeta potential, indicating that increased uptake occurs for negative surface charges [7]...... 18

xvi LIST OF FIGURES xvii

2.8 Intracellular Au NP concentration, as an indicator of cellular uptake, illustrated graphically as a function of Au NP size, showing that op- timal uptake occurs for 50 nm Au NPs [8]...... 19

2.9 Basic physiological structure of a typical cell, including various or- ganelles and molecules [9]...... 20

2.10 The process of internalisation by phagocytosis in three steps [10]. . 22

2.11 The microstructure of tantalum in its porous form, with pores dis- played ranging from 400 to 600 microns in size [11]...... 25

2.12 Tantalum-based implants used in orthopaedics [11]...... 27

2.13 T1-weighted axial spin-echo images of a mouse implanted with a subcutaneous hepatic tumour, with images taken before administra- tion of MRI contrast material and at various times after, showing an increase in contrast-enhancement over time [12]...... 29

2.14 Cellular uptake as a function of NP radius for iron oxide NPs in 9L glioma cells with two typical minimum/maximum radii used for comparison and optimal radii for uptake derived for each curve based on the peak maximum value [13]...... 31

2.15 A graphic displaying the particle dynamics in the SFE process, not- ing the production, growth and encapsulation phases [14]...... 34

2.16 The 3 phases of effects for biological systems exposed to radiation over time [15]...... 37

2.17 The therapetic index applied to the situation where chemotherapy has been added to radiotherapy [15]...... 38

2.18 The 6 hallmarks of cancer, representing the biological and organising capabilities of neoplastic disease [16]...... 40

2.19 An illustration of the differences between SSB and DSB in DNA [17]. 42

2.20 The cell survival curves on the left portray the dependence of ra- diosensitivity on phase in the cell cycle, and coupled with image on the right, it is evident that surviving fraction can vary by up to a fac- tor of 100 in the G2/M phase compared to the latter part of the S phase [18]...... 44

2.21 Cell survival curves for mammalian cells exposed to X-rays under oxic and hypoxic environments, with the associated OER calculated and shown [19]...... 45 LIST OF FIGURES xviii

2.22 Hypoxic fraction as a function of tumour life, displaying the reoxy- genation effect following radiation exposure [19]...... 46

2.23 Photon cross-section as a function of energy for various interactions in lead [20]. K(n and e) refers to pair production through interaction with the nuclear and electron fields, T refers to photoelectric effect, and coherent/incoherent scattering is also shown...... 49

2.24 A survival curve illustrating the degree of dose-enhancement for Au NPs [21]...... 53

3.1 A schematic diagram of an autoclave commonly used in laboratory sterilisation processes, similar to that used in this work [22]. . . . . 56

3.2 A fine tip ultrasound sonicator [23], used in dispersing NPs before experimentation...... 58

3.3 9L cell growth over time, modelled using an exponential fit, values shown represent the mean of 3 experiments and errors reported are ± 1 standard deviation [24]...... 60

3.4 MDCK cell growth over time, modelled using an exponential fit, val- ues shown represent the mean of 3 experiments and errors reported are ± 1 standard deviation [24]...... 61

3.5 MCF-7 cell growth over time, modelled using an exponential fit, val- ues shown represent the mean of 3 experiments and errors reported are ± 1 standard deviation [24]...... 62

3.6 A graphic of the square grids used to count cells on the haemocy- tometer, performed under a microscope [25]...... 63

3.7 A image taken of a stained petri dish as part of a clonogenic assay, note formation of numerous visible colonies...... 65

4.1 Typical XRD patterns for T a2O5 NSPs, with the image on the left showing an amorphous form, while the patterns on the right show crystalline T a2O5 NSPs annealed at different temperatures [26]. . . 69

4.2 XRD patterns illustrating various peaks of reflection, and their cor- responding Miller indices, for TNSPs and PNSPs. The patterns for the respective phases correlate well with that observed in the litera- ture [26], as discussed in the introduction...... 71

4.3 A typical SEM image detailing the degree of aggregation exhibited by T a2O5 NPs [26]...... 73 LIST OF FIGURES xix

4.4 A SEM image taken of T a2O5 TNSPs in PBS solution without soni- cation...... 75

4.5 A SEM image taken of T a2O5 TNSPs in PBS solution with sonication. 75

4.6 A high magnification SEM image taken of T a2O5 PNSPs in PBS solution with sonication...... 76

4.7 A schematic diagram outlining the primary components of a typical TEM [27]...... 78

4.8 A HRTEM image of crystalline mesoporous T a2O5 NPs [28]. . . . 79

4.9 HRTEM images of the TNSPs (left) and PNSPs (right) captured at a scale of 50 nm...... 80

4.10 The optical configuration and experimental set up for the Malvern Zetasizer Nano ZS [29]...... 83

4.11 HRTEM image and (inset) DLS particle size distribution spectrum for LPD-synthesised T a2O5 NPs [30]...... 85

4.12 DLS size distribution spectrum for the T a2O5 PNSPs...... 86

4.13 DLS size distribution spectrum for the T a2O5 TNSPs...... 87

4.14 The experimental set up and components of a typical UV-vis spec- trophotometer used for UV absorption spectroscopy measurements [31]...... 88

4.15 UV-vis absorption spectrum for the T a2O5 PNSPs, showing a distin- guished peak in the UVC region...... 89

5.1 A graph of normalised cell count against time for determination of cell viability and cytotoxicity over a 14 day period. Values are nor- malised to the day 14 maximum cell count value...... 92

5.2 Cell survival fractions for a variety of concentrations following 6 hour exposure of the cells to the TNSP...... 95

5.3 Cell survival fractions for a variety of concentrations following 6 hour exposure of the cells to the TNSP...... 97

5.4 Cytotoxicity of TNSPs and PNSPs to 9L cells following a 24 hour period of exposure...... 98 LIST OF FIGURES xx

5.5 Cytotoxicity of PNSPs to 9L, MDCK and MCF-7 cells following a 24 hour period of exposure...... 100

5.6 Cytotoxicity of CeO −2 NPs to 9L, MDCK and MCF-7 cells follow- ing a 24 hour period of exposure [32]...... 101

5.7 FSC vs. SSC histograms representative of 9L cellular uptake of TNSPs and PNSPs at 0, 50 and 500 µg/ml following 24 hours ex- posure...... 102

5.8 FSC vs. SSC histograms representative of MDCK cellular uptake of PNSPs at 0, 50 and 500 µg/ml following 24 hours exposure. . . . . 103

5.9 The Leica confocal laser scanning microscope, with associated laser systems and image analysis software. This was the same apparatus utilised in experimental measurements [24]...... 105

5.10 9L cells treated with TNSPs (left column) and PNSPs (middle col- umn) at concentrations of 50 and 500 µg/ml. The untreated sample is represented in the top right hand column, and the bottom right hand panel is a zoomed region from the adjacent panel containing 9L cells treated with PNSPs at 500 µg/ml. The red arrows indicate endosome-encapsulated PNSPs around the nucleus of the cell. . . . 106

6.1 Cell survival curves for 9L cell cultures irradiated with 10 MV X- rays showing the dose modifying response of T a2O5 TNSPs. Sur- viving fractions were normalised to non-irradiated control cells, av- erages taken from a sample size of 3 and errors given ± 1 standard deviation from the mean...... 113

6.2 Cell survival curves for 9L cell cultures irradiated with 150 kVp X- rays showing the dose modifying response of tantalum pentoxide TNSPs at concentrations of 50 and 500 µg/ml...... 123

6.3 Cell survival curves for 9L cell cultures irradiated with 150 kVp X- rays showing the dose modifying response of tantalum pentoxide PNSPs at concentrations of 50 and 500 µg/ml...... 124

6.4 Cell survival curves for 9L cell cultures irradiated with 10 MV X- rays showing the dose modifying response of tantalum pentoxide TNSPs at concentrations of 50 and 500 µg/ml...... 125

6.5 Cell survival curves for 9L cell cultures irradiated with 10 MV X- rays showing the dose modifying response of tantalum pentoxide PNSPs at concentrations of 50 and 500 µg/ml...... 126 LIST OF FIGURES xxi

7.1 XRD patterns illustrating various peaks of reflection, and their cor- responding Miller indices, for T a2O5 and ZnO NPs...... 142

7.2 HRTEM images of a) T a2O5 NSPs and b) ZnO NPs, displaying the surface morphology of each substance...... 144

7.3 MDCK cell viability, or surviving fraction, following 24 hours expo- sure to 0, 50 µg/ml T a2O5 NSPs and ZnO NPs. SFs are normalised to the control (0 µg/ml) and errors given ± 1 standard deviation from the mean...... 145

7.4 Relative fluorescence of DCF in MDCK cells, following 24 hours exposure to 0, 50 µg/ml T a2O5 NSPs and ZnO NPs. Values are normalised to the control (0 µg/ml) and errors given ± 1 standard deviation from the mean...... 147

7.5 UV-vis spectra showing the absorption coefficient as a function of wavelength for ZnO NPs and T a2O5 NSPs. The wavelength of max- imum absorption for each sample is also shown...... 150

7.6 UV-vis transmission spectra for ZnO NPs and T a2O5 NSPs. . . . . 151

7.7 Plot of (αhν)2 as a function of hν for determination of the band gap energy for ZnO NPs and T a2O5 NSPs...... 152 List of Tables

4.1 Mean crystallite size, unit cell parameters, crystal structure and phase summarised for the TNSPs and PNSPs...... 72

6.1 Alpha and beta parameters for the 10 MV experiments with and with- out the presence of the NSP showing a modified response of the cells to the radiation...... 114

6.2 Alpha, beta and SER for the TNSPs and PNSPs at 0, 50 and 500 µg/ml, as well as 150 kVp and 10 MV. Errors are given ± 1 standard devia- tion from the mean...... 127

7.1 Comparison of crystal structure and agglomeration parameters for ZnO and T a2O5 NSPs...... 143

7.2 The wavelength of maximum absorption, extinction coefficient and energy gap parameters for ZnO and T a2O5 NSPs...... 151

7.3 The in vitro SPF, UV protection factors, UVA:UVB ratio and critical wavelength parameters for ZnO and T a2O5 NSPs...... 153

xxii List of Abbreviations

AINSE Australian Institute of Nuclear Science and Engineering EPSM Engineering and Physical Sciences in Medicine AIP Australian Institute of Physics ACOFT Australian Conference on Optical Fibre Technology POWH Prince of Wales Hospital CMRP Centre for Medical Radiation Physics UOW University of Wollongong CDDG Cancer and Drug Discovery Group IHMRI Illawarra Health and Medical Research Institute TNT Targeted Nanotherapies USYD University of Sydney STGCCC St George Cancer Care Centre CPD Continuing Professional Development RT Radiation Therapy TV Tumour Volume EBRT External Beam Radiotherapy HDR High Dose Rate LDR Low Dose Rate LINAC Linear Accelerator IMRT Intensity-Modulated Radiotherapy VMAT Volumetric Modulated Arc Therapy SBRT Stereotactic Body Radiation Therapy NPs Nanoparticles

xxiii List of Abbreviations xxiv

DERT Dose Enhancement Radiotherapy NSPs Nanostructured Particles UV Ultraviolet

T a2O5 Tantalum Pentoxide MDCK Madin-Darby Canine Kidney ROS Reactive Oxygen Species FACS Fluorescence-Activated Cell Sorting kV kilovoltage MV Megavoltage PEG Polyethylene Glycol SPIONs Superparamagnetic Iron Oxide Nanoparticles DNA Deoxyribonucleic Acid ASTM American Society for Testing and Materials MRI Magnetic Resonance Imaging PMMA Polymethyl Methacrylate LDLs Low-density Lipoproteins PDT Photodynamic Therapy BBB Blood-brain Barrier CT Computed Tomography SFE Sodium Gas-phase Combustion Synthesis and Encapsulation Process OLEA Oleic Acid XRD X-ray Diffraction SEM Scanning Electron Microscopy TEM Transmission Electron Microscopy UV-vis Ultraviolet-visible Spectrophotometry DLS Dynamic Light Scattering MRT Microbeam Radiotherapy LET Linear Energy Transfer DSB Double-Strand Breaks SSB Single-Strand Breaks List of Abbreviations xxv

OER Oxygen Enhancement Ratio DER Dose Enhancement Ratio PAT Photon Activation Therapy SER Sensitisation Enhancement Ratio DEF Dose Enhancement Factor BRT Binary Radiation Therapy NEXT Nanoparticle Enhanced X-ray Therapy LQM Linear Quadratic Model ISEM Institute for Superconducting and Electronic Materials TNSP Thermal Nanostructured Particle PNSP Precipitation Nanostructured Particle BSC Biosafety Cabinet PBS Phosphate Buffered Saline ECACC European Collection of Authenticated Cell Cultures ATCC American Type Culture Collection DMEM Dulbeccos Modied Eagles Medium FBS Fetal Bovine Serum PE Plating Efficiency SF Surviving Fraction FWHM Full-Width at Half-Maximum HRTEM High-resolution Transmission Electron Microscopy PDI Polydispersity Index LPD Liquid Phase Deposition FSC Forward Scatter SSC Side Scatter SPION Superparamagnetic Iron Oxide Nanoparticles LRE Local Radiation Environment HBSS Hank’s Balanced Salt Solution RBE Radiobiological Effectiveness SSD Source-surface Distance List of Abbreviations xxvi

SPF Sun Protection Factor MFI Mean Fluorescence Intensity UPF Ultraviolet Protection Factor CIE International Commission on Illumination JCPDS Joint Committee on Powder Diffraction Standard HCP Hexagonal Close-packed Chapter 1

Introduction

1.1 Background

Cancer, almost unarguably, remains the predominant threat to human health with one of the highest incidence and mortality rates of any disease throughout history. It is estimated in Australia that, by current trends, more than 50 percent of the Australian population will be diagnosed with some form of cancer by the age of 85 [33]. On a worldwide scale, cancerous disease accounts for approximately 1 in 8 deaths [34]. Despite significant research efforts, an effective cure for all cancers is still elusive, making current treatment methods the best line of defense. The primary pillars of any cancer treatment regime involve one, or more, of the following: surgery, radiotherapy and chemotherapy. In general, surgery is the most commonly used treatment method across all tumour types, with tumour excision substantially reducing disease-related symptoms and probability of recurrence, especially in the case of non-metastatic disease. This is closely followed by radiotherapy, or radiation therapy (RT), as the next most effective means of cancer treatment, with over 50% of patients receiving RT at some point as part of the radiation management plan [35]. Figure 1.1 is an excerpt from Delaney et al. that details the proportion of patients receiving RT for different sites, and highlights the significant part it plays in head and neck cancers that are difficult to treat, such as gliomas [1].

1 Introduction 2

Figure 1.1 The proportion of patients contracting different types of tumours, as well as the proportion who receive RT [1].

Chemotherapy, as a form of systemic therapy, is still effective at treating disease, however many of these chemotherapeutic agents present significant toxicities to nor- mal tissues, which has lead to the emergence of targeted cytotoxic and radiosensi- tising agents. The aim of these targeted agents is to increase local tumour control while minimising damage to normal tissues. This idea of improving local tumour control is one being embraced by new techniques in RT. This is achieved practically by increasing the dose to the tumour volume (TV), while minimising the dose to the surrounding normal tissues.

RT is comprised of two main branches, namely external beam radiotherapy (EBRT) and brachytherapy. Brachytherapy can be high dose rate (HDR) or low dose rate Introduction 3

(LDR), and involves the insertion of a radioactive source directly into, or in close proximity of, the tumour. Due to the high dose gradients surrounding brachytherapy sources, and much lower nominal photon energies involved relative to EBRT, high doses can be delivered to the tumour whilst achieving effective normal tissue spar- ing. This is a technique that uses the concept of local tumour control well, and is used effectively to treat tumours surrounded by critical structures, notably prostate cancer, although this technique will not be explored deeply in this study.

EBRT utilises high energy photons, produced in a linear accelerator (LINAC) to treat the tumour (see Figure 1.2). New techniques currently being implemented clinically that involve beam modulation and highly accurate field shaping include intensity- modulated radiotherapy (IMRT), volumetric modulated arc therapy (VMAT) and stereotactic body radiotherapy (SBRT). These advanced methods of dose delivery revolve around improving the underlying goal of RT, i.e., increasing the controlled dose to the TV while reducing the dose to the normal tissues.

Figure 1.2 A Varian TrueBeam LINAC commonly utilised by clinical departments in EBRT treatments, including IMRT, VMAT and SBRT [2]. Introduction 4

Although these methods have shown considerable success, there still remains bar- riers to treatment that conventional EBRT treatment methods find difficult to over- come. Glioblastomas, as well as other head and neck cancers, are an example of this, with significant barriers to treatment due to the location, often surrounded by critical structures, as well as their biology, often exhibiting radioresistance, hypoxia and metastasis [36]. It is for this reason that new methods must be explored, and one particular method for increasing local dose involves the use of nanoparticles (NPs) or drugs, in conjunction with EBRT, called dose enhancement radiotherapy (DERT). Au NPs have shown significant promise in the field [37] [38], however this work will pioneer the use of a new compound, namely tantalum pentoxide (T a2O5) NPs or nanostructured particles (NSPs). Two samples of T a2O5 NSPs were prepared for experimentation, differing in their production methods, with the intent of examin- ing how morphological differences in these NSPs would affect various aspects of radiosensitisation. This study largely focuses on the application of T a2O5 NSPs to DERT, however throughout the characterisation phase of experimentation, it was found that these NSPs contained properties favorable for ultraviolet (UV) absorption, therefore, a second application to UV protection will also be explored. Introduction 5

1.2 Thesis Overview

1.2.1 Objectives

The metaphysical objective of cancer therapy is to cure the patient of their disease, using a variety of treatment methods, while minimising damage sustained by the patient. Radiotherapy, as one of the primary treatment methods, is more specific in this goal, endeavoring to maximise the dose to the tumour, while minimising the dose to non-cancerous tissues. DERT is a relatively new technique that aims to enhance the effectiveness of radiotherapy through the use of radiosensitising compounds, such as chemotherapy drugs and NPs, that increase local tumour control. Some of the physical and biological properties associated with NPs, and their interaction with human tissues and radiation, also make them useful in other fields. For this reason, this work will focus primarily on the application of T a2O5 NSPs to cancer therapy and aim to fulfill the following fundamental objectives:

• Investigate the application of T a2O5 NSPs to DERT

• Investigate the application of T a2O5 NSPs to UV protection

1.2.2 Aims

In exploring the application of T a2O5 NSPs to DERT and UV protection, this thesis describes numerous experiments that were conducted as part of a comprehensive research plan designed to achieve this goal. The individual aims of these experiments are as follows:

• Effectively synthesise two forms of T a2O5 NSPs with differing morphological characteristics for experimental investigation and comparative purposes.

• Characterise the physical properties of T a2O5 NSPs, including crystallite and morphological parameters. Introduction 6

• Establish protocols and optimum parameters for suspension and use of the NSPs in cellular experiments, including the use of 9L and MDCK cells.

• Evaluate the cytotoxic effects of the NSPs to a variety of cells and analyse the effect of NSP concentration and exposure time on cytotoxicity.

• Examine biological interactions between T a2O5 NSPs and cells, notably inter- nalisation and reactive oxygen species (ROS) generation, using fluorescence- activated cell sorting (FACS) flow cytometry and confocal microscopy.

• Determine the degree of dose enhancement provided by T a2O5 NSPs to 9L and Madin-Darby canine kidney (MDCK) cells in kilovoltage (kV) and mega- voltage (MV) X-ray photon fields.

• Investigate the effects of NP concentration, beam energy and NP morphology

on the degree of radiosensitisation provided by T a2O5 NSPs to 9L cells.

• Analyse the UV absorption properties of T a2O5 NSPs and evaluate their ap- plicability to UV protection. Chapter 2

Literature Review

2.1 Introduction

Radiotherapy (often combined with chemotherapy and surgery) remains one of the foremost techniques for cancer treatment worldwide and, as such, demands signifi- cant research and development. The primary aim of RT is to maximise dose to the tumour while minimising dose to the normal tissues. Recently developed techniques in the field of RT, such as IMRT and SBRT, have revolved around improving local tu- mour control, however normal tissue complications still remain an issue. A relatively new technique being pioneered is drug-enhanced radiotherapy, where certain mate- rials can be mobilised in the form of a drug to be introduced in close proximity to the cancerous tumour cells. Materials can be chosen to create a local dose-enhancing effect in the presence of radiation so that tumour cells can be destroyed more effec- tively and healthy tissue spared, this technique is called DERT. We will be investi- gating evidence for the use of T a2O5 NSPs as dose-enhancers, or radiosensitisers as they are also referred to. In order to understand the mechanisms of interaction that makes DERT possible, it is necessary to first consider the properties of NPs them- selves, and their interactions with biological systems before applying these principles to nanomedicine and radiotherapy.

7 Literature Review 8

2.2 Nanoparticles

2.2.1 Introduction to Nanotechnology, Nanomaterials and Nanopar- ticles

Nanotechnology refers to the manipulation of matter and development of technol- ogy in the nanoscale. Although commercial applications of nanotechnology and widespread interest in research have been relatively new developments, historically the origins of nanotechnology can be traced back much further. One of the first docu- mented scientific reports regarding nanotechnology was published by Michael Fara- day where he discovered a method for the synthesis of colloidal gold particles [39]. In 1959, Richard Feynman gave, what is arguably considered, the first public talk about the principles of nanotechnology. Although he may not have been aware at the time, this talk, ”There’s plenty of room at the bottom”, where he discussed the ma- nipulation of atoms and molecules at the atomic scale, would inspire substantial re- search and lay the foundations for the field of nanotechnology as we see it today [40].

Figure 2.1 The branches of constituent terms used to describe different types of nanomateri- als, as set by the ISO [3]. Literature Review 9

Nanotechnology is an area of science concerned with the study of particles on the atomic scale and the use of nanomaterials for various applications. The term ”nano- materials” is quite broad, however, it refers to all materials with structural compo- nents in the sub-micron region; while ”nanoparticles” appears to be a subsidiary of this encapsulating all particles in the sub-micron region with 3 dimensions in the nanoscale [4]. In this thesis, the term ”nanostructured particles” (NSPs) will be used to describe the ceramic compounds produced for experimentation. This term en- capsulates materials with at least one dimension in the nanoscale, or whose basic structure is comprised of nanomaterials. Many of these terms are often used inter- changeably (see Figure 2.1 for a tree diagram illustrating the relationships between the various terms), however in this work the different types of particles will be dis- tinguished accordingly [41].

Nanomaterials are currently being used successfully in cosmetics, drug delivery, electronic devices and therapeutics [42]. Perhaps the most exciting branch, how- ever, is nanomedicine where nanomaterials, among other techniques, are used in the prevention and treatment of disease. Current research being conducted in this area includes the use of: quantum dots as photosensitisers for cancer therapy [43]; cerium oxide nanoparticles as free-radical scavengers for radiation protection [44]; and gold nanoparticles as dose enhancers for radiotherapy [21], among a vast array of others. Research into the use of nanoparticles in radiotherapy, especially dose-enhancement, is still being pioneered; although, promising results have been seen with gold, pacli- taxel, silicon and zinc oxide quantum dots [43]. We will see though that the potential use of, and research into, nanoparticles for the various applications discussed is re- lated to their unique properties and behaviours exhibited. Literature Review 10

2.2.2 Properties of Nanoparticles

Indicative of their name, nanoparticles refer to structures with 3 dimensions of the or- der 1-100nm [42], although the literature will often generally use the term to describe any structures with dimensions in the nanoscale. The most distinguishing character- istics of nanoparticles is the unique properties they develop at the nanoscale, which often bear no resemblance to those of the bulk materials they are derived form. These physical and chemical property changes primarily occur due to the diverse arrange- ment, spacing and structure of surface atoms [45]. Some papers [46] also note the contribution of quantum effects, in the form of quantum confinement, in changing properties from bulk to nanomaterials.

The massive reduction in size to the nanoscale for nanoparticles results in a high particle number per unit mass and a much larger surface area (see Figure 2.2). This increase in the ratio of surface area to volume drastically increases the chemical reactivity (it has been noted that movement from micro to nano-scale can increase reactivity 1000 times) [4].

Figure 2.2 Illustration of the relationship between surface area and particle size [4]. Literature Review 11

Quantum effects are most notably observed in the case of quantum dots (engineered nanostructures), which are synthesised with sizes less than the Bohr radius (approx- imately 1-5 nm) to take advantage of quantum confinement effects [47]. Quantum confinement of electrons gives rise to a quantised energy spectrum, magnetic mo- ments and changing ability to transfer electrical charge [4]. Applications of nanoma- terials in the cosmetics and sunscreen industries have been observed for zinc oxide and titanium dioxide NPs primarily, which are utilised as UV blockers as a result of their strong UV absorption properties in the UVA and UVB wavelengths [48] [49]. Another property exploited by particles on the nanoscale is superparamagnetism, a unique form of magnetism where magnetisation can vary depending on temperature. Superparamagnetic iron oxide NPs, or SPIONs as they are commonly referred to in the field, are currently being used in MRI contrast, and other diagnostic imaging applications [50].

Figure 2.3 A graphical abstract depicting the free radical scavenging mechanism exhibited by cerium oxide nanoparticles irradiated in an X-ray photon field for radiation protection ap- plications. The beam energy-dependent nature of protection and enhancement is highlighted in this particular study [5]. Literature Review 12

Photoluminescence and florescence are optical properties that are enhanced and ex- ploited by quantum dots in the form of carbon and gold NPs, as well as silicon NPs for the purposes of cellular and in vivo imaging [51] [52] [53]. Finally, mixed valence state, or the ability to change oxygen vacancies, are other examples of prop- erties that may exist on the nanoscale, specifically in the case of cerium oxide NPs, which utilise this property to exhibit a free-radical scavenging effect (illustrated in Figure 2.3), which makes them useful in radiation protection applications [6].

These generalised property changes differ between different types of NPs, even those of the same elemental and atomic composition. This necessitates a classification system that specifies the various physical and chemical aspects of NPs that help shape the specialised properties they may exert.

Figure 2.4 Classification features of nanostructures [4].

This is particularly important when considering the toxicity of NPs, which is strongly dependent on morphology, and free nanoparticles (as opposed to fixed nanostruc- Literature Review 13 tures) are able to move around the body with ease, in which case toxic characteristics are a significant issue [54] [55].

It is evident based on the vast array of properties and types of NPs in existence that there are multiple ways to classify NPs, however, Buzea et al. proposes four main features that may be used to describe nanoparticles, namely dimensionality, morphol- ogy, composition, uniformity and agglomeration.

Dimensionality refers to an aspect ratio that gives the number of dimensions in the nanoscale (between one and three). Free nanoparticles, such as gold, cerium oxide and iron oxide, fall into the three-dimensional category with all dimensions in the nanoscale. Materials with lower order dimensionality include gold thin films and nanorods, all utilised in the field of DERT, along with spherical free Au NPs [56] [57]. Dimensionality is one way of describing the size, aspect ratio and type of nano- material under classification and, as such, can be useful in characterising properties, such as internalisation and potential applications.

Morphology takes into account aspect ratio, sphericity and flatness. In general terms, this classification criterion is primarily focused on describing the shape of the NP. Tube-like nanostructures represent high-aspect ratio materials, such as Au nanowires utilised in electromechanical devices for their extremely thin size in one dimension (less than 2 nm) and ultra-high tensile strength [58]. Free spherical NPs represent low-aspect ratio NPs, for example hollow spherical silica NPs used in drug delivery applications [59]. The low aspect ratio and spherical shape is advantageous mor- phology for internalisation and transfer throughout the body by metabolic processes, which is highly desirable in drug delivery applications, validating reasons for their use in this field. Generally, throughout the literature NP morphology is utilised as a broader term describing features including: crystallinity, size, agglomeration and shape. These terms will be discussed in more detail in the ”Physical characterisation Literature Review 14 of NPs” section, and are directly relevant to the experiments performed in this work. Although some of these terms are encompassed in the present definition, this classi- fication system separates agglomeration into an entirely separate sub-field, based on the premise that changes in agglomeration can drastically impact the properties of the material, demanding a classification section on its own.

The composition of NPs can vary from pure materials consisting of a single type of atom, to conglomerations of nanomaterials. NPs consisting of a single type of material are usually synthesised artificially, as opposed to being found naturally, and include the majority of materials used in DERT, including Au NPs, Pt NPs and Gd NPs. Conglomerations of materials, containing different types of atoms, are com- monly used in drug delivery systems, especially for the case of cytotoxic drugs, chemotherapy drugs are for materials to be transported in the body that may not be biocompatible. Quantum dots have been loaded with the chemotherapy drug pa- clitaxel in anti-cancer drug therapy [60] and Au NPs have been coated with polyethy- lene glycol (PEG) to improve their biocompatibility in DERT [61].

Uniformity and agglomeration refers to the state of the NPs, whether they exist as suspensions, powders, free particles or agglomerates, which is inadvertently deter- mined by their chemistry and electromagnetic properties. The degree of agglomera- tion for a NP can have dramatic effects on the behaviours it exhibits. In some cases, large agglomerations of NPs lose their unique characteristics and take on the be- haviours of the larger particles they now resemble [4]. Reier et al. did a comparative study on the electrochemical surface characteristics for select NPs and their corre- sponding bulk material counterparts and revealed distinct differences. For Pt NPs, they measured an increase in oxophilic nature compared to the bulk material [62]. For experiments, such as those carried out in the current study, where the success of the experiment is directly dependent on the properties exhibited by the NP in its free state it can be important to ensure the particles remain dispersed to avoid aggregation Literature Review 15 and also to ensure homogeneous concentrations of the NP in suspension. This has been done in the past through the use of a sonicator, in which ultrasonic waves pass through the particles in suspension and re-disperse them [6] (Figures 2.5 and 2.6).

Figure 2.5 TEM images of cerium oxide NPs in solution before sonication [6], note the presence of significant agglomeration. The scale bar at the bottom of the image represents 100 nm. Literature Review 16

Figure 2.6 TEM images of cerium oxide NPs after sonication portraying degree of dispersion [6], note the reduction in aggregate size due to sonication. The scale bar at the bottom of the image represents 120 nm.

Evidently, the increased reactivity with matter (especially cells) and surface area on the nanoscale, in association with the unique relatively unexplored characteristics of some NPs makes them perfect candidates for experimentation. Because their size can be made comparable to that of cellular pathways in the body they show great potential in nanomedicinal applications, such as DERT. The NP characteristics discussed in this section are important to define as they directly influence biological interactions with cells, including cytotoxicity and internalisation. Therefore, once a NP has been physically characterised, it is prudent to characterise its biological interactions on a cellular level, which relies on a firm understanding of the mechanisms involved in NP-cell interactions. Literature Review 17

2.2.3 Mechanisms of Interaction Between Nanoparticles and Cells

Cellular interactions with NPs depend on their chemistry, shape, surface properties and size; therefore, because of the unique nature of these properties it is not known exactly how all NPs behave. The two primary biological interactions to consider between NPs and cells are toxicity and uptake. These two processes are strongly dependent on the NP properties described previously, as well as properties unique to the host cells. Because the nature of these processes can be unique to the cells and NPs in question, as well as dependent on numerous other unknown biological factors, the research regarding this topic is incomplete. This is why most studies will perform their own physical and biological characterisation of the cells and NPs be- fore embarking on further experimentation, as will be performed in this study. This section will, however, describe the foundations of NP-cell interactions, as well as some processes commonly observed in DERT and UV protection.

NPs may engage in cellular uptake via two main processes, namely binding to the cell membrane followed by internalisation [63]. Attraction, and subsequent attach- ment, to the cell membrane can occur if the NPs are functionalised with receptors on their outer surface to specifically target the cell, however, without this function- ality, it appears to be an electrostatic interaction dependent on several properties, including: surface charge, shape and size of the NPs themselves. Certain types of NPs have shown increased uptake with modified negative surface charge, measured in the form of the zeta potential, which causes bonding to cationic membrane sites and subsequent internalisation by endocytosis [7]. In particular, this is evidenced by

CeO2 NPs, which demonstrated increased cellular uptake in lung adenocarcinoma cells (A549) when a negative zeta potential was applied (see Figure 2.7). This was achieved by modification of the pH of the NP solution [7]. The idea that negative surface charges are favourable for cellular uptake in metal oxide NPs is further sup- ported in the case of superparamagnetic iron oxide NPs (SPIONs), which showed increased affinity for attraction to the cellular membrane [64]. Therefore, modifica- Literature Review 18 tion of surface charge may be effective in increasing cellular uptake.

Figure 2.7 Cellular uptake of CeO2 NPs in A549 cells as a function of their associated zeta potential, indicating that increased uptake occurs for negative surface charges [7].

Size and shape of the NPs are morphological features known to influence the effi- ciency of uptake [65]. In DERT, a study investigated the dependency of uptake in Hela mammalian cells with Au NPs in the size range of 10-100 nm [8]. Au NPs ap- proximately 50 nm in size exhibited the strongest uptake characteristics (Figure 2.8), suggesting the existence of an optimum size for NP uptake, which may or may not be a unique value for different types of NPs (a comparative study across several types of NPs is required to ascertain the nature of this). This was especially significant as it was widely held that the smaller NPs could be manufactured, the more effec- tive cellular uptake would be [66]. A comparison was also made between spherical and rod-shaped Au NPs, which showed an increase in cellular uptake of 500% for spherical NPs relative to rod-shaped NPs, establishing that shape plays an important part in effectiveness of internalisation and that spherical NPs may be effectively in- ternalised at a greater efficiency than other shapes due to their reduced membrane Literature Review 19 wrapping time [8].

Figure 2.8 Intracellular Au NP concentration, as an indicator of cellular uptake, illustrated graphically as a function of Au NP size, showing that optimal uptake occurs for 50 nm Au NPs [8].

Because NP size influences the efficiency and mechanisms of cellular uptake, it also affects the final resting location of the NP within the cell. There are a number of pos- sible resting locations including inside the nucleus, outside the cellular membrane or within the majority of physiological subcellular components [67] [68]. Relatively large particles, approximately 10 microns in size, commonly seen in NPs with ten- dencies for aggregation, have been shown to localise in cytoplasmic vacuoles. NPs in the 10-100 nm range generally localise in organelles, especially mitochondria [4] (see Figure 2.9 for cellular anatomy), while even smaller NPs (less than 10 nm) can be very unpredictable in their localisation due to differences in their method for up- take. The exact size threshold is not known, although, it is hypothesised that NPs comparable to, or less than, the size of the cells themselves (approximately 2 nm for deoxyribonucleic acid (DNA), 4 nm for globular proteins and 7 nm for cell mem- branes [69]) may have improved efficiency of uptake due to internalisation through Literature Review 20 ion channels or pores in the cell membrane [70].

Figure 2.9 Basic physiological structure of a typical cell, including various organelles and molecules [9].

Internalisation, the process where by the NPs penetrate the cell and engage in sub- cellular localisation, can occur along several pathways. Passive uptake occurs when electrostatic forces, intermolecular forces or steric (particle properties synthesised to promote interaction with cell receptors) interactions allow the particle to enter the cell. Although this process may resemble normal phagocytosis, it may not result Literature Review 21 in the production of a typical phagosome-encapsulating vesicle around the NP [71]. Because the cell is deprived of this extra protection the NP is free to interact with various organelles and other components, sometimes in a harmful way. Depending on whether the NP is subject to normal phagocytosis or not, it can be found in an array of different places inside the cell, including the nucleus, where it has the poten- tial to cause DNA damage [4]. Active uptake for NPs is commonly achieved through endocytosis, the process where by macromolecules meet the cellular membrane and transport into the cell is facilitated by enclosure of the macromolecule in a mem- brane vesicle. There are various forms of endocytosis, however the most relevant for nanomaterials in DERT is phagocytosis (see Figure 2.10 for visualisation of the steps involved in the process), where solid materials are internalised by specialised cells forming vesicles [65]. These interactions have been observed for Au NPs [72] and iron oxide NPs [73], however the size and localisation of these vesicle-surrounded materials within the cell cannot be specifically stated for all nanomaterials as it de- pends on a vast array of factors, including: NP type, morphology, cell type, and mechanism of uptake.

The vast majority of NPs and compounds used in DERT would undergo internali- sation via endocytotic pathways (including phagocytosis), however this presents a potential issue as endosome-encapsulated nanomaterials have been shown to localise around the perinuclear region, not inside the nucleus, and the encompassed material finds it difficult to enter the cytosol [74]. Historically, evidence in DERT (as shown for Au, Bi and Pt NPs [75]) has suggested that the optimal localisation region for NPs is within the nucleus, where the DNA of the cell resides, so that maximum damage can be inflicted on the critical structures of the cell and maximise the probability of cell death [76]. Literature Review 22

Figure 2.10 The process of internalisation by phagocytosis in three steps [10].

A study utilised functionalised Au nanostars to overcome the endocytotic localisa- tion problem by targeting nucleolin, a phosphoprotein in the nucleus and cytoplasm of cancer cells known to transport anticancer ligands to the nucleus [77]. The study showed increased apoptosis and reduced cell viability in HeLa cervical cancer cells, and was able to visualise the damage to the nucleus caused by the nanoconstructs using confocal microscopy [78], supporting the importance for targeting the nucleus in DERT. More recent studies though have been exploring the possibility of targeting other regions of the cell for optimising damage in DERT, and comparing results to the historical standard, i.e., the nucelus. McNamara et al. used Monte Carlo simula- tions on Au NPs in a compartmentalised human cell model to show that when the Au NPs were localised in the cytosol, the energy deposition to the mitochondrial volume was greater than that for the nuclear volume, indicating that dose enhancement and cell death still effectively occurred as a result of deformation to the mitochondria, suggesting that cell structures other than the nucleus can prove to be effective targets for DERT [79]. Literature Review 23

As much as internalisation is an important biological process to characterise, espe- cially from an efficacy point of view in DERT, toxicity is a biological interaction that must be considered, primarily to ensure the safety of the patient, and to decipher possible physiological effects that may arise from significant toxicities. There are several types of toxicity that may occur in interactions between NPs and cells, in- cluding: cytotoxicity, phototoxicity and genotoxicity [80]. Cytotoxicity refers to any general toxicity incurred by the cell, as a result of the presence and interaction of the NP with the cell. Possible mechanisms of cytotoxicity, intrinsic to the NP itself, can involve chemical reactions where the release of ions damages the cell; surface catal- ysed reactions leading to ROS generation; or stress caused by NP morphology. In the case of tantalum NPs, all of these mechanisms are possible, although cytotoxicity resulting from the chemical release of ions is unlikely to cause significant toxicity as tantalum ions have a low probability of combining with biomolecules to cause dam- age [81]. Phototoxicity refers to toxic interactions that arise from irradiation of the NP, which may stimulate ROS production, namely superoxide anion, singlet oxygen and hydroxyl radicals in the case of metal oxide NPs [82]. This has been observed in the case of ZnO and TiO2 NPs, where photoreactivity and phototoxicity has been shown to be significant, resulting in cell damage in UV protection applications [83]. Finally, genotoxicity refers to the specific case where toxicity mechanisms directly lead to DNA damage. From a safety perspective, and minimising damage to normal tissues, this is of particular concern as any toxicity mechanisms that directly dam- age the DNA of a cell have the highest probability of inducing cell death. In the

field of UV protection, genotoxicity has been reported in the cases of ZnO and TiO2 NPs due to both intrinsic and photo-induced toxicity interactions [84]. Evidently, the physiological risk presented by NP-induced cytotoxicity necessitates comprehensive physical and biological characterisation be performed, especially in the application of NPs to UV protection and DERT. Literature Review 24

2.3 Properties and Applications of Tantalum Oxides

2.3.1 Properties of Tantalum

The potential use of tantalum (Ta) in medical applications is based upon several im- portant properties. Tantalum has a relatively high atomic number (Z=73) and has a high temperature threshold for maintaining mechanical properties (1000 ° C) [85]. It is known to be chemically stable, resistant to corrosion and highly ductile, which has lead to its use as a biomaterial in human implantation. Acceptable host response over a long period of time has seen the American Society for Testing and Materials (ASTM) form a consensus for the use of standard tantalum in bioimplantation [86]. In vitro studies and in vivo studies with human dermal fibroblasts and micrometer- size tantalum particles have shown no growth inhibition. On a cellular level, particles of this size were seen to undergo phagocytosis and furthermore, presented low toxi- city [87].

There seems to be a consensus among the literature that suggests tantalum bulk ma- terial is very non-toxic and even on the microscale presents little to no cytotoxicity to cells [86] [87] [11]. A study involving the effect of metal carboranes on cancer cells, including MCF-7, concluded for a tantalum compound that it was significantly toxic towards tumor cells [54]. This may have been due to the presence of any number of elements in the compound, including chlorine, or due to complex interactions of the compound overall; however, this still ceases to portray that tantalum is in any way toxic towards healthy human tissue. Further studies involving porous forms of tantalum [88] (see Figure 2.11) showed no inflammation in rat tissue following im- plantation suggesting that even in different forms tantalum remains non-toxic and biocompatible [11]. Literature Review 25

Figure 2.11 The microstructure of tantalum in its porous form, with pores displayed ranging from 400 to 600 microns in size [11].

Having established that pure tantalum metal is biologically and chemically inert both in vitro and in vivo, exhibiting properties of low toxicity and low solubility, the ele- ment may be considered as a potential candidate for nanomedical applications. How- ever, it is prudent to first examine the current medical applications of tantalum, for which the literature suggests there are well established uses.

2.3.2 Clinical Applications For Tantalum

The biocompatibility of tantalum makes it useful in a number of medical applica- tions. It has been used successfully in orthopaedic surgery as porous tantalum has been shown to be similar to, and in some aspects better than, cortical and cancellous bone [89]. Furthermore, it has been used in cartilage resurfacing and as a bone graft substitute [90]. Figure 2.12 exemplifies the many uses of tantalum in orthopaedic surgery. Properties of chemical and erosion resistance have also seen tantalum used in the field of dentistry [91].

Tantalum displays a variety of applications in diagnostic radiology, adding to its flex- Literature Review 26 ibility across the field of medicine. It represents an excellent radiographic marker with its high density allowing for almost complete radiation attenuation [92]. As a powder, it has been used as a contrast material in X-ray imaging of the lungs [93]. Additionally, in vivo studies have been performed that give evidence to its potential use as a radiopacifier in the detection of blockages in blood vessels [94].

Historically, tantalum has been used since the 1940’s in repairing cranial fractures and vascular clips [95]. This is important with the emergence of Magnetic Reso- nance Imaging (MRI) as an affluent diagnostic imaging modality, which uses strong magnetic fields (3T systems are becoming common) to image the body. Any pa- tient with ferromagnetic implants cannot be imaged or the object will be ripped from the body causing serious injury to the patient. Tantalum has been shown to be non- ferromagnetic for fields up until 1.5T [96], which means it may be used surgically in patients without revoking their privilege to be scanned in an MRI. It has also been observed to produce minimal artefacts in MRI images [97]. Many of the studies re- ferring to the use of bulk Ta in radiology applications are relatively old (1940-1990), and while Ta is still used in these clinical applications, the lack of recent literature is due to a shift in focus towards investigating different forms of Ta (nano-sized, com- binations with other compounds), many of which will be discussed in sections 2.3.3 and 2.3.4. Literature Review 27

Figure 2.12 Tantalum-based implants used in orthopaedics [11].

In the context of radiotherapy, and choosing a suitable material for DERT applica- tions, this is also a significant advantage. The rapid development of MRI has made it the gold standard in image quality, providing anatomical and physiological infor- mation unmatched by any other modality, which has led to recent interest in the field of radiation oncology for cancer centres purchasing their own MRI machines, separate to diagnostic departments, for their own planning and imaging purposes in cancer therapy. Furthermore, research into MRI-linacs, where radiation treatment is guided in real-time by MRI, is another example of the future applications MRI has in radiation oncology [98]. As a result of this, it would be a significant advantage thera- peutically if the material used for DERT was compatible with MRI so that inter- and intra-fraction imaging could be performed on the patient, regardless if the material was present in their system or not.

Evidently, based on the many applications of tantalum across widespread medical fields, we can conclude that it shows great promise in future nanomedical clinical applications because of its low toxicity, chemical resistance and biocompatibility, Literature Review 28 among other emerging trends in the field of radiation oncology. Having considered the properties and applications of the bulk tantalum metal, we will now consider the combination of tantalum with other elements as a compound that can be made more effective and versatile in therapeutic applications.

2.3.3 Properties and Applications of Tantalum Oxides

The combination of metals with oxides has further enhanced their properties in sev- eral ways, especially in the field of medicine. Oxide layers that form on metals in the process of oxidation give a natural protective coating that may provide extra pro- tection for cells in biomedical applications [91]. More importantly, it has been seen that amorphous oxide NPs have been able to form platforms, or linkages, that allow them to be combined with certain drugs, such as heparin, in a process known as drug- loading [99]. In addition to this, a study involving drug delivery using silicon dioxide microparticles found that the material polymethyl methacrylate (PMMA) became ad- herent to the particle’s surface by means of a linkage to the oxide layer [100]. This linkage mechanism associated with the oxide layer allows the grafting of particles to a drug, such as cisplatin, or to biological molecules, including proteins or lipids that may be used to target specific tumour receptors in a process known a function- alisation. If tantalum oxide was to be harnessed for use in radiotherapy, it could be introduced into the body by means of a drug, necessitating a linkage such as this. Therefore, the formulation of tantalum oxide possesses potential for use in radiother- apy. Literature Review 29

Figure 2.13 T1-weighted axial spin-echo images of a mouse implanted with a subcutaneous hepatic tumour, with images taken before administration of MRI contrast material and at various times after, showing an increase in contrast-enhancement over time [12].

The idea of functionalising a material for potential use as a drug-delivery system is generally based on the modification of surface properties of the material, which we have shown is aided by the presence of an oxide layer. Specific ligands and bio- logical molecules can be attached to the NP so that it will follow specific metabolic pathways and eventually settle in the required region around or inside the tumour. The molecules of choice are specific to different tumor cell types, because of cell- specific characteristics and variation in tumour receptors, and can be used to target structures on the cellular membrane, as well as inside the cell nucleus. Low-density lipoproteins (LDLs) have already shown an ability to transport high-Z NPs, and other photosensitisers, to tumor cell membranes in photodynamic therapy (PDT) [101]. In fact, the use of the LDL receptor pathway for transporting nanomaterials to tumour cells is well-established in the literature, due to the fact that certain tumours have overexpressed LDL receptors for the provision of cholestrol in membrane synthe- Literature Review 30 sis [102]. Gadolinium-loaded LDLs have been used as MRI-contrast agents both in vitro and in vivo, as shown in Figure 2.13, with the NPs successfully targeting the tumour cells and accumulating in the area of interest [12]. Additionally, albumin NPs have been successfully transported across the blood-brain barrier (BBB) by linkage to apolipoproteins [103]. This would be very desirable in NPs designed for the treat- ment of brain tumours, such as gliomas, where drug efficacy is directly linked to successful transport across the BBB and into the tumour- a known challenge in suc- cessful drug treatments of these types of tumours.

Having considered functionalisation and successful targeting of the tumour cells by the NP, internalisation is then a consideration as optimal localisation of the NP for DERT would be within the cell nucleus or, at least in the perinuclear region (beyond the cell membrane) in close proximity to the nucleus and other organelles (as dis- cussed previously). Internalisation by endocytosis is then desirable and likely for the majority of NPs, especially when functionalised with LDLs [104]; however, the probability of uptake is dependent on the size of the NP primarily, which must ap- proximate the size of the cellular channels. The exact number varies between cell types and throughout the scientific community; however, numbers from 1-50 nm seem to be commonly quoted for cancer cells [105]. A study of size-dependent en- docytosis for Au NPs on glycoproteins showed that 45 nm Au NPs were internalised by endocytosis, while 75 nm Au NPs localised on the surface [106]. Literature Review 31

Figure 2.14 Cellular uptake as a function of NP radius for iron oxide NPs in 9L glioma cells with two typical minimum/maximum radii used for comparison and optimal radii for uptake derived for each curve based on the peak maximum value [13].

A theoretical approach to the NP size dependence of endocytosis for iron oxide NPs in 9L glioma cells showed optimal uptake to occur at approximately 30 nm (see Fig- ure 2.14) for which cellular uptake reached a maximum value of 2301, and to cease entirely at a theshold value of approximately 50-60 nm [13]. Further measurements with keratinocytes and cerium NP uptake have shown the channels for entry to be approximately 10nm [107]; however, they may dilate to 26nm during internalisation, allowing larger NPs to enter also. The methods of functionalisation discussed would introduce the NP to a suitable spot around the cancer cell; although, there are meth- ods that can be used to target the DNA of the nucleus specifically, which would be ideal for our purposes. Incorporation of ethidium ligands has been used in associ- ation with Au NPs to target DNA [108] and methods, such as this, could be used effectively in conjunction with tantalum oxide for functionalisation. Literature Review 32

2.3.4 Application of Tantalum Oxide Nanoparticles to Nanomedicine

There are currently minimal applications of tantalum oxide NPs around the world, and especially in the field of nanomedicine. This means the literature regarding their current use is scarce; however, this presents great opportunity for research and de- velopment. Currently, metal oxide NPs are being used in dermatology in the form of cosmetics and sunscreen with zinc oxide and titanium dioxide NPs dictating the majority of market share [109] [110]. It is also apparent that NPs of different types (metal, metal oxides, drug compounds, quantum dots, ceramics) are being used clin- ically to treat diseases and cancers, including Alzheimer’s disease [111], lung carci- noma [112], and gliomas [113]. In terms of the current uses of tantalum NPs, across all fields, it appears they are primarily used as anodes in the telecommunication and computer industries. The pentoxide layer that forms honours the NP with its capaci- tance, a fundamental intrinsic property to its function. Capacitance and particle size exhibit an inversely proportional relationship so that minimising the size of the tan- talum oxide crystallites is paramount to improving their performance [114].

Applications of tantalum oxide NPs to medicine are few, however, T a2O5 NPs have been used successfully as injectable bone substitute materials [115] [116], while also being used as radio opaque resins in tooth fillers [117], both biomedical applications indicative of the biocompatibility of this material. The literature suggests that current research surrounding T a2O5 NPs is restricted in nanomedicine to diagnostic appli- cations, with in vitro and in vivo studies being performed utilising tantalum oxide NPs as image contrast agents (due to their documented radio opaque properties) in X-ray, computed tomography (CT) and MR imaging modalities [118] [119]. Clearly though, the minimal applications of these NPs in therapeutics allows for great re- search opportunity. The cost of sourcing bulk materials for NP production is another advantage of Ta over other metals used in nanomedicine, such as gold and cerium. Ta has an average cost of 120 USD/kg, much cheaper than gold at 40 000USD/kg and cerium at 600 USD/kg [120]. With Australia home to one of the most abundant Literature Review 33 tantalum reserves in the world [121], this could prove an important asset for the coun- try’s economic and industrial future, while ensuring widespread use of the NP if it is proven to be clinically effective and production can be developed in a cost-effective and sustainable way.

The production of NPs is made difficult due to a number of possible complications. Their high surface reactivity means it is more difficult to produce non-oxide nano- materials due to the natural tendency to stabilise and recombine with oxygen and thus require minimal exposure [122]. This does not mean production of oxide-based nanomaterials are without their own challenges, notably isolation from atmospheric oxygen and moisture, as these can represent significant risk of contamination [123]. Evidently, both sterilisation and control of NP characteristics (morphology) are im- portant elements to consider in their production and, although there are a small range of methods for production, one particular method that incorporates both features well is the sodium gas-phase combustion synthesis and encapsulation process (SFE). It can be used to produce raw tantalum NP powder according to the equation [14]:

T aCl5 + 5Na + inert => T a + 5NaCl + inert (2.1)

The NPs are produced and encapsulated by the salt (Figure 2.15), which allows their morphology (particle size) to be controlled and minimises the likelihood of contam- ination. Literature Review 34

Figure 2.15 A graphic displaying the particle dynamics in the SFE process, noting the pro- duction, growth and encapsulation phases [14].

Another simpler method of production involves the use of oleic acid (OLEA) solu- tion as a surfuctant and tantalum ethoxide with the presence of argon gas in a vacuum at 120 ° C from which T a2O5 precipitates. This is then heated in a furnace at approx- imately 700-800 ° C in air to form NPs [26]. As we will show later though, methods such as those presented here often lead to large aggregates, particularly in the case of

T a2O5, due to the lack of forced dispersion as the NPs aggregate to reduce surface reactivity [124].

Finally, a production method developed by Kominami et al. has been successfully used to synthesise beta phase T a2O5 NPs of various sizes, ranging from 3-60 nm, for the purpose of photocatalytic experimentation [125]. This solvothermal reaction involves combining tantalum pentabutoxide with toluene in water and heating to a temperature of 200-300 ° C, from which T a2O5 NPs precipitate. Temperature of annealing was chosen based on previous solvothermal production reactions for T iO2

NPs [126] [127]. Production of one form of T a2O5 NSPs utilised in this thesis work is based on the solvothermal precipitation reaction described in the previous study for reasons of resource-availability and the advantage that particle-size and aggrega- tion control of T a2O5 appears to be quite high for this particular synthesis method (a more detailed discussion of this can be found in Chapter 3). Literature Review 35

2.3.5 Characterisation Methods for T a2O5 NPs

For any given type of NP, composition and morphology can vary significantly be- tween different batches depending on elements, such as production method, anneal- ing temperature, post-production treatment (sonication or drying) and storage [128] [129]. Therefore, it is necessary to perform physical characterisation of certain NP features, such as phase, crystal structure, particle size and aggregation. This can be done using a variety of techniques, including: X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), ultraviolet- visible spectrophotometry (UV-vis), photoluminescence and cathodoluminescence measurements, Raman spectroscopy, dynamic light scattering (DLS) measurements and radiopacity. All of these techniques are utilised in physical characterisation of the NPs described in this thesis work, with more detailed descriptions of the tech- niques found in Chapter 4, along with associated experimental results. Considering the workflow for research in DERT, once physical and biological characterisation of the NP, and associated cells for experimentation, have been performed it is then possible to embark on radiological experimentation for the purpose of quantifying the enhancement effect provided by the NPs. This requires a firm understanding of radiobiology and not only the interactions of radiation with the NP, but also interac- tions of radiation with cancerous and non-cancerous tissues. Literature Review 36

2.4 Radiotherapy and Radiobiology of Cancer

2.4.1 Introduction

Radiotherapy involves the treatment of cancerous cells with radiation in an effort to eradicate the tumour. In an ideal setting, one would hope to deliver all of the pre- scribed dose to the TV itself, whilst sparing any surrounding healthy tissue. The most common form of treatment today is conventional radiotherapy where high en- ergy photon beams are used to irradiate the tumour; however, this delivers a sig- nificant amount of dose to surrounding healthy tissue, harming the patient. EBRT techniques, such as IMRT, SBRT and VMAT have been developed and used clin- ically to aid in minimising dose to healthy tissue; while Microbeam Radiotherapy (MRT) is also being developed [130], although the problem still remains. One new technique being pioneered is that of DERT where a foreign particle is introduced into the tumour prior to irradiation with the intention of selectively increasing the absorbed dose to the tumor alone [131]. The mechanisms dictating this are based on a fundamental understanding of the principles behind radiation damage in cancer cells.

2.4.2 Introduction to Radiobiology of Cancer Cells

Radiobiology studies the effects of ionising radiation on the body, particularly on the cellular level. Radiation therapy is based on the phenomenon, whereby, the radiation beam interacts with the body as it passes through and transfers some of its energy to surrounding cells, hopefully causing targeted cancer cell death [132]. Biologi- cal damage, and possibly cell death, occurs when the beam directly (or indirectly) ionises the component atoms of DNA [133].

When a biological system is exposed to radiation, an array of cascading events takes place, eventually culminating in various degrees of biological damage. The chronol- ogy of these events can be separated into 3 main phases, namely physical, chemical Literature Review 37 and biological phases (shown in Figure 2.16).

Figure 2.16 The 3 phases of effects for biological systems exposed to radiation over time [15].

The physical phase describes the very initial stages of exposure where by primary photons and charged particles interact, through ionisation and excitation reactions, with tissue atoms and lead to cascades of secondary electrons and future events. The chemical phase represents the intermediate stages, where by free radicals created from the reactions in the physical phase go on to react with cellular components leading to further damage in the restoration of electronic equilibrium. The biolog- ical phase follows, encompassing the early period of events following the chemical phase, and then all subsequent events, that may span many years. Enzyme reactions are initiated after chemical damage, repair processes begin as the symptoms of nor- mal tissue damage are observed (early effects), cell proliferation occurs for all cell types, and finally, late effects are observed [134].

It is important to remember that in radiation therapy both cancerous and normal tis- sues are exposed to radiation, and the effects of both need to be taken into account in planning and treatment regimes. The response of cancerous tissue to radiation Literature Review 38 is usually expressed as regression, where tumour size decreases due to cell death outweighing proliferation and repair; regrowth, where the tumour increases in size; and local control, where tumour regrowth does not occur within the lifespan of the patient [135]. Normal tissue response varies depending on dose, and can range in severity from mild skin irritations to severe health conditions, and change over time according to whether they are normal or late effects. Generally, these can be min- imised using dose fractionation, where hyperfractionation allows the same dose to be delivered to the tumour over an increased number of treatments, allowing recovery of normal tissues [136].

Figure 2.17 The therapetic index applied to the situation where chemotherapy has been added to radiotherapy [15]. Literature Review 39

Because radiation treatment damages both normal and cancerous tissues, it is es- sential to the success of the treatment, i.e., to cure the patient, that the damage to cancerous tissue significantly outweighs damage to normal tissues. This concept is described by the therapeutic index, defined as the tumour response for a fixed level of normal tissue damage [137]. In the same sense, any changes to a treatment regime, such as the addition of a drug or radiosensitiser as would be the case in DERT, need to be assessed in terms of the additional tumour control and normal tissue toxicities that are introduced as a result. The therapeutic index is a good way of quantifying the complex interplay of these factors, and assessing the potential benefits of treatment modifications, or a new treatment entirely (this concept is illustrated graphically in Figure 2.17).

There are multiple cellular recovery processes that occur as a result of radiation dam- age, and consequently, a number of factors that can be utilised in radiation treat- ment regimes to influence and exploit cellular recovery to maximise tumour control and minimise normal tissue complications. Recovery from sublethal damage occurs when the prescribed dose is split into two fractions delivered hours (or more) apart, with the extended time between fractions allowing for cellular repair to occur, mak- ing them more resistant to the second fraction than if the total dose was delivered in one fraction [138]. Similar to this case, the dose rate effect allows for increased cel- lular recovery when reduced to a low level, with time between pulses increased, and less clustered and overall damage for each cell. Finally, the most commonly exploited clinical radiotherapy recovery process is fractionation, which involves splitting the total dose into daily doses over a period of approximately 4-6 weeks, depending on the total prescribed dose. This leads to a sparing effect for tissues, as the 24 hour period between fractions allows for increased cellular repair [139]. Cellular repair itself has been noted to consist of several components, exponential in nature with half lives of minutes and hours [19]. Literature Review 40

Although there are several processes of cellular recovery that result from various as- pects of radiation treatment, the biological factors that dictate normal and cancerous tissue response to radiotherapy, which clinically represents fractionated radiotherapy, may be summarised by the 5 R’s of radiotherapy, originally proposed by Withers et al. [140]. These factors are used to describe tissue response, in terms of whether they may become more radioresistant or radiosensitive following treatment. The first factor is repair, discussed previously, and referring to the period of time immediately after exposure where cellular repair mechanisms are engaged. Reassortment refers to the influence of the cell cycle, in that following the first fraction, the majority of surviving cells will align and be in the resistant S phase. Repopulation occurs naturally as tumour cells proliferate over the course of treatment. Reoxygenation is an effect particularly relevant to hypoxic cells, which show resistance to treatment, although following exposure will become more oxygenated and thus less radioresis- tant. Radiosensitivity is the final factor, and refers to the inherent differences between tumours, their intrinsic characteristics, and response to treatment, which culminates in varied radiosensitivity between different tumour types [141].

Figure 2.18 The 6 hallmarks of cancer, representing the biological and organising capabilities of neoplastic disease [16]. Literature Review 41

The reason for the use of radiation in the treatment of cancer is inextricably linked to several defining characteristics of cancer cells. They usually have a high degree of vascularity, providing increased blood supply and nutrients for growth. Conse- quently, they have a higher reproductive rate and increased break-down of glucose for energy. When normal cells reproduce, they cease growth when coming into close proximity of each other, on the other hand, cancer cells continue to grow regard- less of contact and will often protrude into other components of the body. One of the major problems with cancer cells is their ability to proliferate indefinitely, which means they are hard to control and create space problems in the body that often im- pair normal organ functions [142]. Cellular differentiation is the process where a cell’s characteristics undergo rapid change to create a more specialised cell; how- ever, cancer cells are relatively undifferentiated and can even change over time into a less mature state. The high rate of cellular division and undifferentiated nature of cancer cells happens to make them more radiosensitive. Evidently, this shows how radiation can be particularly effective in killing cancer cells [133].

Perhaps the most comprehensive review of cancerous disease is provided by the pub- lications of Hanahan and Weinberg in 2000 and 2011 [142] [16], where a logical framework was proposed for the identification and understanding of the biology of cancerous disease, known as the hallmarks of cancer (Figure 2.18). The 6 biological capabilities, representing the hallmarks of cancer, include: sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis, and resisting cell death. Far greater detail on the mechanisms and biochemistry of cancer, including more detailed de- scriptions of the hallmarks can be found in these review articles, however this lies outside the scope of this thesis.

Effective cell death is caused by damage to the DNA. This can occur through di- rect or indirect ionisation of the composite atoms in the double helix structure that Literature Review 42

Figure 2.19 An illustration of the differences between SSB and DSB in DNA [17]. is DNA (containing nucleotides with sugar, phosphate and base pairs). Indirect ion- isation occurs when water molecules are ionised in the process of radiolysis, since cells are approximately 80% water, creating highly reactive free radicals (OH− and H+). These are known as ROS and are actually quite effective in chemically dam- aging DNA. Direct ionisation occurs when the beam directly ionises atoms in the DNA, causing direct damage; however, this occurs less frequently than the indirect case [143].

The energy transferred to matter in general by ionising radiation is described by the absorbed dose D. This is determined by the energy (E) deposited in a medium per unit mass of the medium (m) and, consequently, has S.I units J/Kg or Gy [144].

E D = (2.2) m

Linear Energy Transfer (LET) is a measure of the energy deposition in a medium Literature Review 43 by local interactions. It is generally concerned with energy transfer by means of secondary particles and can be restricted to include only particles (usually electrons) with energies below a certain threshold value. Low energy secondary electrons (delta electrons) are a form of high LET radiation that can be particularly effective in killing cancer cells. As the restriction approaches infinity, LET approaches linear collisional dT stopping power (total energy transfer per unit length, − dx ) [145].

Damage can be in the form of double-strand breaks (DSB) or single-strand breaks (SSB). In any given nucleus exposed to 1 Gy of radiation, approximately 20000 ioni- sations will take place leading to 1000 SSBs and 40 DSBs [146]. High LET radiation is generally linked to greater incidence of DSB, as is the case with low energy elec- trons, which create clustered damage and, evidently this causes effective cell death. Low LET radiation, such as gamma radiation, can produce single electrons that cause SSB and only damage the cell, and are far less effective in leading to eventual cell death (both cases are shown in figure 2.19). Many SSB in close succession can lead to eventual cell death; however, DSB are far more effective in tumor control and this is why dose-enhanced radiotherapy is being researched extensively [133].

Radiation damage sustained by cells also varies depending on their stage in the cell cycle. This cycle represents the life of a cell and is comprised of: the M phase where cells are born in the processes of mitosis and cytokinesis, the G1 phase being an intermediate period of cell growth, the S phase where DNA synthesis occurs, and finally the G2 phase where additional cell growth eventuates before the cycle begins again (Figure 2.20 graphically displays the chronology of the cycle, as well as the radiation effects during each phase of the cycle) [147]. It is important to note from a therapeutic point of view that cells in the S phase tend to be the most radioresistant, and cells in the M and G2 phases are the most sensitive as they have little time to re- pair damage before embarking on mitosis again [148]. Evidently, knowledge of the cell cycle can be exploited to maximise the radiosensitivity of cells before radiation Literature Review 44 is delivered, there by maximising therapeutic effect.

Figure 2.20 The cell survival curves on the left portray the dependence of radiosensitivity on phase in the cell cycle, and coupled with image on the right, it is evident that surviving fraction can vary by up to a factor of 100 in the G2/M phase compared to the latter part of the S phase [18].

Cellular response to radiation exposure, or radiosensitisation, is not only dependent on stage in the cell cycle, but also on oxygen content. The oxygen effect refers to the phenomenon where the presence of oxygen in tissues increases the dose received upon radiation exposure due to increased free radical generation and, consequently, increased DNA damage [149]. This dose-modifying effect is quantified by the oxy- gen enhancement ratio (OER), defined as the ratio of radiation doses in anoxic and oxic conditions, delivering the same biological end point [150] (illustrated graph- ically in Figure 2.21). For the majority of cellular tissues, the OER provided for X-ray irradiation is approximately 3 [151]. Literature Review 45

Figure 2.21 Cell survival curves for mammalian cells exposed to X-rays under oxic and hypoxic environments, with the associated OER calculated and shown [19].

Although normal tissues are generally considered well oxygenated, the oxygen effect has a significant impact in radiotherapy of cancerous tissues, in particular, hypoxic tissues. Tumour angiogenesis can lead to inadequate blood vessel development in certain regions of tumours, leading to oxygen deprivation, and consequently, hy- poxic tumour cells [152]. Hypoxic cell presence is especially important to consider in high single fraction dose treatments, such as hypofractionated SBRT, as the high Literature Review 46 dose will ablate the oxic cells, making the surviving cells primarily hypoxic in na- ture [153]. As stipulated in the 5 R’s of radiotherapy, reoxygenation refers to the biological response of tissues following irradiation where by they become more oxy- genated and, therefore, more radiosensitive by virtue of the oxygen effect [154]. Consequently, this makes fractionated radiotherapy particularly effective in treating hypoxic tumours, with reoxygenation of tumour cells occurring directly after each fraction (Figure 2.22), allowing for an increased number of the total hypoxic cells in the tumour to be killed each treatment.

Figure 2.22 Hypoxic fraction as a function of tumour life, displaying the reoxygenation effect following radiation exposure [19].

Radiation experiments utilise a range of different types of cancer cells, and it must be noted that the effects quoted in vitro may differ significantly to in vivo results. It is, therefore, beneficial to minimise this gap as much as possible. In the case of dose-enhancement, 9L gliosarcoma cells would be very suitable as they are a known radioresistant cell [155]. If dose-enhancement can be observed in the case of a ra- dioresistant cell, it would not only prove useful in the treatment of hypoxic tumors but also in the treatment of less radioresistant tumor cells. 9L cells have shown to require high doses for tumor control, doses of 25 Gy and even 40 Gy in one fraction Literature Review 47 have been reported [155]. This clearly exceeds doses recommended for minimal nor- mal tissue complication (clinically, 2 Gy/fraction is commonly used [156]). Values that are commonly used to describe radiosensitivity and resistance respectively are α and β, which for glioblastomas have been quoted at approximately 0.24 Gy−1 and 0.029 Gy−2 [157] respectively. These cells will form the basis for experimentation and investigation into the phenomenon of dose-enhancement. Literature Review 48

2.4.3 Dose-Enhancement in Radiation Therapy

Dose-enhanced radiotherapy is based on the principle of introducing a high-Z (high- atomic number) particle into close proximity with the tumor and irradiating to obtain an increase in radiosensitivty due to interactions of the primary beam with the foreign material producing secondary particles and scattering events that cause additional damage to the tumor itself. The reason for the use of high-Z materials over other elements is due to the Z-dependence of the cross section for interactions (the cross section is approximately proportional to Z2 and Z4 for pair production and photo- electric effect respectively) . When the photon beam interacts with the particles the chief interactions include: photoelectric effect, Compton effect and pair production. These all dominate to varying degrees as a function of incident photon beam energy (as shown in figure 2.23).

There are two main regions of incident beam energy that we will be concerned with, i.e., the MeV and keV regions. The keV region of irradiation has already been probed with a number of elements. This form of therapy has often been referred to as ”Pho- ton Activation Therapy” (PAT), mainly due to the fact that beam energies of approx- imately 100 keV are used to irradiate high Z elements and in this energy region the photoelectric effect dominates interactions. Localised dose-enhancement has been observed mainly due to production of high-LET auger electrons, which are very ef- fective at causing clustered DNA damage and efficient cell death [158].

Common ways of quantifying the degree of dose-enhancement are by the Dose En- hancement Ratio (DER, ratio of doses giving a prescribed surviving fraction), Sen- sitisation Enhancement Ratio (SER, DER defined for 10 % cell survival) and Dose Enhancement Factor (DEF, ratio of survival fractions defined at 2 Gy) [159]. DEFs of 1.83 and 1.64 have been quoted for Iodine and Gadolinium from irradiation of 140 kV X-rays [160]; however, other papers have quoted numbers ranging from 1-2.5 depending on the type of measure, but more importantly depending on the con- Literature Review 49

Figure 2.23 Photon cross-section as a function of energy for various interactions in lead [20]. K(n and e) refers to pair production through interaction with the nuclear and electron fields, T refers to photoelectric effect, and coherent/incoherent scattering is also shown. centration of the particle and beam energy. A large degree of consensus seems to be that the optimum energy for dose-enhancement is around or just above the K-edge of the element itself. Here, production of secondary electrons may be maximised caus- ing direct damage to the DNA or indirect damage through ROS production if within range (quoted to be 4-300 nm depending on the type of radical [161]. An optimal energy of 50 keV has been suggested for iodine compounds where the dose to the medium and dose to the water is at its greatest difference, just above the K-edge of 33 keV [159], giving an SER of 2.03.

Radiosensitisation has also been observed in the case of platinum, which in some cases has exhibited a cytotoxic effect to the cancer cells also with irradiation around Literature Review 50 the K-edge (78 keV) [158]. When the particle is incorporated as a compound or drug, as is the case with many Gd and I compounds, it is often referred to as ”Bi- nary Radiation Therapy” (BRT). As of yet, minimal research has been conducted into the dose-enhancing effects of the MeV region of radiation; however, we might expect that Compton interactions and pair production would dominate interactions (providing photons meet the required 1.022 MeV) resulting in higher energy sec- ondary particles with lower LET, but higher range. This leaves an interesting avenue for investigation though, as does the NP avenue, which is only just being pioneered. Literature Review 51

2.4.4 Dose-enhancement with Nanomaterials

In section 2.2.3, it was stated that NPs can be synthesised with a size approximating that of cellular pathways, allowing them to settle in a number of possible locations including: close proximity to the cell, around the cellular membrane causing a dis- ruption, or (most effectively) within the cell following internalisation. In the case of X-ray based dose-enhancement, the majority of extra cellular damage is caused by the production of low energy high LET Auger electrons; however, these may only travel micrometres before being absorbed, so the effect is maximised when the par- ticle is placed within range of the DNA [162]. This is why high-Z NPs possess great potential in the field of radiosensitisation.

Several experiments have been conducted using Au NPs with sizes as small as 1.9 nm, concentrations from 10-100 µg/ml and even higher, showing DERs of approximately 2 in most cases [21]. It has also been shown though that the degree of enhancement is dependent on particle size and particle concentration, necessitating an optimum to be found for each [162] [21] [8].

Titania NPs doped with lanthanides have recently shown a reduction in cancer cell proliferation of up to 75 % primarily through generation of photon-activated ROS in a process called photodynamic therapy. The 50 nm particles were shown to inflict optimal damage [161].

Other studies with lipid nanocapsules have shown enhancement [163], paclitaxel- loaded NPs have exhibited an SER of 1.4 on MCF-7 cells [164], and there have also been some promising results with quantum and carbon dots in the field called Nanoparticle Enhanced X-ray Therapy (NEXT) [43]. Literature Review 52

2.4.5 Cell Survival Curves

Some of the first experiments utilising cell survival curves involved measurement of the effects of X-rays on mammalian cells [165]. This also detailed the now com- mon practise of cultivating cells in a clonogenic assay before staining and counting colonies. Fertil et al. established that different types of tumor cells have an intrin- sic radiosensitivity, a concept now well known and utilised in the ”Linear Quadratic Model” (LQM) [166]. Then, the first utilisation of the LQM with survival curves and binary radiation treatment occurred when Towle et al. performed experiments testing the effect of heat on radiosensitivity [167].

Cell survival is now commonly modelled according to the LQM [156]:

S = exp(−(αD + βD2)) (2.3)

Here, S represents the cell survival probability and D the total absorbed dose in Gy. The constants α and β represent the degree of radiosensitivity and radioresistance respectively, and dictate the slope of the survival curve in the initial and final stages respectively. They are also each indicative of cell kill due to single hits, and cell kill due to more than one hit.

Figure 2.24 shows a typical example of a survival curve shown as a semi-logarithmic plot of S against D. Curves illustrating irradiation of cells with and without the NP allow for a comparison to be made, such as the SER, which compares D at 10 % survival. This is the method we will be utilising in measuring the degree of dose- enhancement provided by T a2O5 NSPs at a variety of beam energies. Although there is limited literature pertaining to T a2O5 NPs and their application in nanomedicine, we see that there is much evidence to suggest they are strong candidates for use in DERT. Literature Review 53

Figure 2.24 A survival curve illustrating the degree of dose-enhancement for Au NPs [21]. Chapter 3

Experimental Details and General Methodology

3.1 Introduction

This chapter describes the materials, general methodology and facilities associated with the work in this thesis. Features of NSP production, preparation and storage are outlined, as well as characteristics of cells utilised in experimentation. The tech- niques outlined here are only general, in the sense that they apply to preparatory phases, or form a component of multiple experiments outlined in this thesis. For more detailed explanations of the methodology behind specialised experiments, see the methodology sections of chapters 4, 5, 6 and 7.

3.2 Synthesis and Storage of T a2O5 NSPs

All T a2O5 NSPs were synthesised by Dr. Konstantinov at the Institute for Super- conducting and Electronic Materials (ISEM), Innovation Campus, UOW. For the purposes of experimentation, two different forms of T a2O5 NSPs were synthesised, with different procedures, so as to create materials that were atomically similar in composition, yet different in morphology (as will be discussed in chapter 4), and to

54 Experimental Details and General Methodology 55 investigate the effect these differences would have in radiotherapy and UV protection applications. For simplicity, each NSP is referred to with an acronym according to its production method. They are the T a2O5 thermal NSP (TNSP) and T a2O5 precipita- tion NSP (PNSP). The term NSP is used to describe the materials, as opposed to NP, as a result of the morphology of each material, which has tendencies for aggregation and consequently, not all dimensions are in the nanoscale, making the term NSP a more accurate description of the material structure.

T a2O5 TNSPs were synthesised using a thermal oxidation reaction. This process involved solid state thermal oxidation of Ta metal foil with purity 99.99 %. The foil was placed in an alumina crucible prior to heating at 800 ° C for 1 hour, which leads to complete oxidation and formation of T a2O5 nano powder.

T a2O5 PNSPs were synthesised using a precipitation-based chemical reaction utilis- ing T a(OEt)5(Sigma-Alrich 99.99 %) and an ethoxide decomposition reaction with water. This method was derived from the article written by Kominami et al. [125].

Two drops (1 mL) of T a(OEt)5 was first added to a small amount of ethanol (15 mL) and mixed carefully to avoid disrupting the surface of the ethanol. Excess deionised water was added resulting in a white fine precipitate that was filtered out and washed in an Eppendorf model 5702 centrifuge. The filtered tantalum hydroxide powder was finally placed in a furnace and heated for 8 hours at 700 ° C, where by thermal de- composition occurs and T a2O5 nanocrystallites form.

Drying of the NSPs was necessary as any interaction with moisture caused violent agglomeration to form a hard compound that was of no use. An aluminium crucible was wiped free of debris and the NSP added before being placed into a furnace at 140 ° C for at least 2 hours. The final destination 50 ml falcon tube was placed in a drying oven at 70 ° C to prevent moisture transfer following the drying process. Upon completion, the NSP was gently stirred with a sterile spatula in the crucible before Experimental Details and General Methodology 56 being promptly added to the pre-heated falcon tube. The lid was kept on, secured with parafilm, double contained with silicon crystals and placed inside a desiccator to prevent moisture absorption (quantitative weight analysis can be performed to en- sure no moisture absorption).

Since the NSPs were to be later used in combination with cells, it was essential they be sterilised before use to eliminate introduction of contamination in any form into the cell culture. Sterilisation involved the use of an autoclave, such as the one shown in Figure 3.1.

Figure 3.1 A schematic diagram of an autoclave commonly used in laboratory sterilisation processes, similar to that used in this work [22].

Pre-heated glass vials were used for sterilisation and subsequent storage of the NSPs. A beaker containing the glass vial and material was placed inside the autoclave and set for a ”dry run” at 121 ° C for 45 minutes. Upon completion, the vials were sealed with parafilm in a container filled with silica and placed in a desiccator. Lids were Experimental Details and General Methodology 57 only removed, following this process, inside a Biosafety Cabinet (BSC) or Cytotoxic Cabinet to ensure sterile conditions were maintained.

3.3 Suspension and Sonication of T a2O5 NSPs in So- lution

Suspension of the NSPs in solution was necessary to ensure a homogeneous distribu- tion of the desired concentration of NSPs amongst the cells. The amount of material required was measured and weighed using electronic scales and filter paper before being placed in a glass vial. The vial containing material was then sterilised accord- ing to the previous procedure. The material was then transferred to a 15 ml falcon tube and the volume made up using Phosphate Buffered Saline (PBS) (without cal- cium and magnesium ions), all inside a BSC under sterile conditions. This allowed for a variety of concentrations to be tested.

It is well known that NPs suffer from a degree of agglomeration, which is why soni- cation (using a fine tip probe sonicator as shown in figure 3.2) is necessary to disperse the NSPs as much as possible into smaller aggregates and achieve a more homoge- neous distribution in solution. Experimental Details and General Methodology 58

Figure 3.2 A fine tip ultrasound sonicator [23], used in dispersing NPs before experimenta- tion.

The fine tip probe and surroundings in the casing were cleaned using ethanol to min- imise the risk of contamination. The 15 ml falcon tube containing the NSP was placed inside a larger 120 ml specimen jar (half filled with water) to create the effect of an ultrasonic bath, where the solution can be sonicated in a non-sterile environ- ment whilst maintaining sterility, as the lid need not be opened. This jar set up was placed in an ice filled bucket to reduce heat build-up and elevated on a stand so that the fine tip probe could be submerged at least 20 mm into the water. Care was taken to ensure the tip did not touch the sides as this would weaken the ultrasonic waves and minimise the dispersion effect. The material was sonicated for 30 minutes total in 10 minute intervals to minimise heat build up, with the duty control set to constant Experimental Details and General Methodology 59 and strength set to a maximum of 7.

These procedures were followed in all experiments utilising T a2O5 NSPs, that re- quired the material to be in solution, and where a homogenous distribution was pre- ferred.

3.4 Cell Line Characteristics

The cell lines employed for experimentation in this work included: the rodent-derived 9L gliosarcoma, the MDCK, and human breast MCF-7 carcinoma. 9L and MDCK cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC), and MCF-7 cells from the American Type Culture Collection (ATCC). All cell lines were maintained, stored and underwent biological experimentation at IHMRI, UOW.

The 9L cell line is a rat brain-derived glioma [168], fibroblast-like in morphol- ogy, adherent in growth mode, and a known radioresistant cell line [169], compa- rable in radioresistivity to both human glioma cells [157] and other rat tumour cell lines [170]. It is a well-established animal tumour model for cancer research stud- ies [169]. Knowledge of the mean population doubling time is essential for cor- rect characterisation of the cell line, as well as planning of future experiments and seeding numbers. The mean doubling time was measured by Oktaria et al. to be 32-34 hours [24], based on exponential growth assays (results shown in Figure 3.3), which compared well with literary values of 31.2 hours [171]. Experimental Details and General Methodology 60

Figure 3.3 9L cell growth over time, modelled using an exponential fit, values shown repre- sent the mean of 3 experiments and errors reported are ± 1 standard deviation [24].

The MDCK cell line is a canine-derived non-cancerous kidney cell line, originally developed by Madin and Darby [172]. It is an adherent cell line, epithelial in mor- phology [173], and partially chosen for its relative ease in cellular maintenance compared to other primary cultures. It was primarily chosen in this work as a non- cancerous comparison model to compliment the 9L cell line, and is well established in the literature as a non-cancerous epithelial cell model [174], as well as a good model for studying permeation of the BBB [175]. The mean population doubling time was experimentally measured by Oktaria et al. to be 19 hours (Figure 3.4), comparing well with the literary value of 21 hours [176]. Experimental Details and General Methodology 61

Figure 3.4 MDCK cell growth over time, modelled using an exponential fit, values shown represent the mean of 3 experiments and errors reported are ± 1 standard deviation [24].

The MCF-7 cell line is a human-derived breast adenocarcinoma cell line [177], ad- herent in growth mode, epithelial in morphology and a known radiosensitive cell line, especially in comparison to 9L cells [178]. It was chosen as a radiosensitive comparison cell line, and is a well established breast cancer tumour model in the literature [179]. Oktaria et al. experimentally determined the mean population dou- bling time to be 25-27 hours (based on exponential growth studies shown in Figure 3.5, correlating well with the literary reported value of 24 hours [180]. Experimental Details and General Methodology 62

Figure 3.5 MCF-7 cell growth over time, modelled using an exponential fit, values shown represent the mean of 3 experiments and errors reported are ± 1 standard deviation [24].

3.5 Maintenance and Sampling of the Cell Culture

All adherent cells required ”splitting” into a subculture on a weekly basis to avoid culture death. This was done by bringing the cells into suspension and transferring an appropriate amount to another flask. Cells were checked for level of confluence first, solutions warmed to 37 ° C and processes carried out in a BSC. The T75cm2 flask containing the cells had the existing spent complete Dulbeccos modied ea- gles medium (DMEM) with sodium pyruvate (supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicilin and Streptomycin) removed, PBS (without Ca and Mg ions) was added twice and removed to wash the inner surface of the flask contain- ing the cell monolayer, Trypsin-EDTA was added to suspend the cells, and then they were placed in an incubator for 5 minutes. The flask was then tapped gently to re- move adherent cells and checked under the microscope, before approximately 1 mil- lion cells were added to the new labelled T75 flask with fresh medium and a vented Experimental Details and General Methodology 63 cap. The cells in their T75 flask were stored inside a humidified incubator at 37 ° C

2 and 5% (v/v) CO2. Cell concentration was maintained at 20000-40000 cells/cm and passaged at 90% confluence for passage numbers up to 30. Cells were also tested for mycoplasma contamination.

Counting of the cells was performed via a sampling technique. A small sample of cells were added to an Eppendorf tube and vortexed to achieve a homogeneous dis- tribution in solution. 10 µl of cell suspension was pipetted onto a Neubauer haemo- cytometer (as shown in Figure 3.6) under a cover slip so that the number of cells in 10 grids could be counted under a microscope.

Figure 3.6 A graphic of the square grids used to count cells on the haemocytometer, per- formed under a microscope [25]. Experimental Details and General Methodology 64

This represents the number of cells per µl of solution, allowing the volume for any number of cells to be calculated, as well as the total number of cells inside the orig- inal flask. At times, dilution with Trypan Blue was necessary, in which case the number of cells counted was multiplied by the dilution factor. The advantage of this technique is that it is easy to ensure only live cells are counted as these show up white in the dye, while dead cells are stained blue as the solution enters the damaged cell membrane. This sampling technique was utilised whenever counting of cells was necessary.

3.6 Clonogenic Assay Technique

The clonogenic assay, originally developed by Puck and Markus in 1956 [165], is a robust technique commonly employed for the evaluation of cell survival in vitro following exposure to radiation or cytotoxic agents [181] [182]. Clonogenic assays were used to assess cell survival in this work and completed with cells plated in trip- licate for a control (no NSP), and range of tantalum pentoxide NSPs concentrations, up to 500 µg/ml (see chapters 5 and 6 for specific concentrations based on experi- mental variables). The 9L cells were brought to 90% confluence, tantalum pentoxide NSP solution prepared and added to the cell culture medium; then once exposure time had been reached the cells were split into 100 mm petri dishes with 10 ml c- DMEM and incubated for 15 doubling times. The initial seeding number of cells (N) split for each concentration was obtained by increasing a reference number (1500) by the death rate (r%) obtained from the toxicity measured over 6 hours.

r N = (1 + ) ∗ 1500 (3.1) 100

The reference number of 1500 cells was chosen based on previous experiments using Experimental Details and General Methodology 65

9L cells by Oktaria et al. [24]. The 3 seeding numbers used for each concentration were then N, 3N and 9N. Following this, each dish was washed with 5 ml PBS (with calcium and magnesium ions) and stained with 5 ml of staining solution (25% crystal violet and 75% ethanol). Colonies consisting of no less than 50 cells [183] were counted (n) and compared with inital seeding values (I) to obtain the plating efficiency (PE) (see Figure 3.7).

n PE = (3.2) I

The surviving fraction (SF) could then be determined by comparing the control PE

(PEc) with the PE of the group containing the NSP (PEx).

PE SF = x (3.3) PEc

Figure 3.7 A image taken of a stained petri dish as part of a clonogenic assay, note formation of numerous visible colonies. Experimental Details and General Methodology 66

3.7 Statistical Analysis

Cell survival experiments were repeated three times to ensure reliability and mea- sured in triplicate for each sample. All values are expressed as the mean of 3 mea- surements with the experimental uncertainty given as one standard deviation. The LQM was fitted to cell survival data using KaleidaGraph software. The fit of the data was weighted according to the error for each dose in the determination of the radiobiological constants, α and β. Statistical analysis of surviving fraction data was performed using a two-tailed Student’s t-test under the assumption of equal variance. A P value less than, or equal to, 0.05 was considered statistically significant. Chapter 4

Physical Characterisation of T a2O5 Nanostructured Particles

4.1 Introduction

This chapter is concerned with describing the techniques utilised in physical charac- terisation of T a2O5 NSPs. For the vast majority of experiments, both the TNSPs and PNSPs were studied concurrently, to compare and contrast property differences aris- ing from their respective synthesis methods. Physical and biological characterisation are both required to fully understand all aspects of NP safety and efficacy, however this chapter will focus on the physical aspects, while the following chapter 5 will cover the biological aspects of characterisation. Physical characterisation techniques employed include morphological and optical absorption, and scattering, methods.

The motivation for these experiments stems from necessity, with regard to the nature of the field of nanomedicine, and the novelty of the T a2O5 NSPs presented in this work. Nanomedicine is a rapidly evolving field, with new nanomaterials and nanos- tructures being developed, and the scope of applications expanding, constantly. Con- sequently, it is necessary to characterise the properties and behaviours of NPs to com- prehensively understand their safety and efficacy in therapeutic applications [184]. Physical properties, such as morphology and photon absorption/scattering, are par-

67 Physical Characterisation of T a2O5 Nanostructured Particles 68 ticularly important attributes to understand for the application of NPs to DERT and UV protection. They allow for early predictions of potential therapeutic benefits and biological consequences, even before in vitro and in vivo experiments are performed, and are essential in the interpretation of biological experimental results.

4.2 X-ray Diffraction

4.2.1 Theory

XRD is the process whereby X-rays of a known wavelength (λ) are elastically scat- tered from the material in question in order to determine information regarding crys- tal composition and structure. The incident beam interacts with the electron cloud of each atom in the crystal lattice according to Bragg’s law [185]:

nλ = 2dsinθ (4.1)

Here, d is the interplanar spacing, n the order of diffraction and 2θ the diffraction angle. The Laue conditions relate d with the lattice parameter a and the Miller indices (h,k,l) of each plane. Using these two equations (4.1 and 4.2) and measurements taken of the material using a diffractometer, crystal structure may be obtained [185].

a d = √ (4.2) h2 + k2 + l2

It should be noted that equation 4.2 relates the interplanar spacing and lattice param- eter for a simple cubic structure only, and requires modification for differing crystal structures with more complex planes of reflection. Furthermore, particle size (t) can be determined through peak analysis of the experimentally determined diffraction pattern and Scherrer’s formula [186]: Physical Characterisation of T a2O5 Nanostructured Particles 69

Kλ t = (4.3) βcosθ

Here, K is a constant based on crystal geometry, β the full-width at half-maximum (FWHM) and θ the Bragg angle.

Figure 4.1 Typical XRD patterns for T a2O5 NSPs, with the image on the left showing an amorphous form, while the patterns on the right show crystalline T a2O5 NSPs annealed at different temperatures [26].

XRD patterns for T a2O5 NSPs are well represented in the literature because of the ease with which phase, structure and crystallite size may be extracted from the re- sulting patterns. There are several production methods that can be employed in syn- thesising these NSPs, including polycondensation, thermal oxidation, decomposition and chemical precipitation [187]. Although some of these methods are quite similar, NSP morphology can vary substantially because of small changes in the produc- tion, for example the chemicals used for precipitation, the annealing temperature, and cooling time [26]. XRD patterns have been obtained for T a2O5 NSPs prepared using similar methods to those employed in this work, yielding both amorphous and crystalline materials (Figure 4.1), with crystallite sizes ranging from several nanome- tres to hundreds of micrometres [188] [189]. An interesting observation has been that heating the NSPs past 750 ° C tends to impart a phase change from hexagonal Physical Characterisation of T a2O5 Nanostructured Particles 70 to orthorhombic, perhaps a consequence of mechanisms driving particle size reduc- tion [26].

4.2.2 Method

Samples of T a2O5 TNSPs and PNSPs were independently placed in a GBC MMA X-ray diffraction system so that crystal structure, phase, mean crystallite size and unit cell parameters could be determined from the resulting diffraction pattern. Each sample was placed onto a quartz plate with ethanol before being placed inside the diffractometer. The intensity of Cu K-alpha X-ray reflections (generated with an ac- celerating voltage of 40 kVp ad cathode current 30 mA) was monitored as a function of the diffraction angle, varying between 20 and 80 degrees. This generated a diffrac- tion pattern, from which the dominant phase and crystal structure could be identified by comparison with the XRD database. Mean crystallite size (t), sometimes referred to as mean particle size, was determined by selecting the most intense diffraction peak free of doublets, from which θ and β could be found for use in Scherrer’s equa- tion [190]:

Kλ t = (4.4) βcosθ

The constants used were K=0.89 and λ=1.5418 Angstroms, based on past experi- ments performed with the X-ray diffractometer.

4.2.3 Results and Discussion

Diffraction patterns were constructed for each NSP, shown in Figure 4.2, with each curve containing peaks of similar shape, intensity and position, indicative that the

NSP samples are of similar atomic composition, namely T a2O5. Closer inspection of the TNSP pattern revealed the existence of additional smaller peaks, compared to the PNSP pattern, signifying a structural difference between the samples despite their Physical Characterisation of T a2O5 Nanostructured Particles 71 similar chemical composition. Peak analysis and database comparison of the diffrac- tion patterns revealed the TNSPs to be orthorhombic beta phase (JCPDS:25-0922), while the PNSPs are hexagonal delta phase (JCPDS:19-1299). This structural dif- ference is fundamentally linked to the production methods employed for each NSP, where by the annealing temperature of 700° C produces hexagonal phase T a2O5 for the PNSPs, as opposed to 800° C for the TNSPs, which results in orthorhombic formation. This is supported in the literature, where the temperature threshold for orthorhombic transformation in T a2O5 NPs has been shown to be 750° C [26]. The most notable feature of this transformation is the splitting of (1,1,0) hexagonal phase peak into (0,22,0) and (2,15,1) orthorhombic phase peaks [191], clearly evidenced in Figure 4.2.

Figure 4.2 XRD patterns illustrating various peaks of reflection, and their corresponding Miller indices, for TNSPs and PNSPs. The patterns for the respective phases correlate well with that observed in the literature [26], as discussed in the introduction.

Additionally, the unit cell parameters were derived for each sample (data summarised Physical Characterisation of T a2O5 Nanostructured Particles 72

Table 4.1 Mean crystallite size, unit cell parameters, crystal structure and phase summarised for the TNSPs and PNSPs.

Type of T a2O5 Size (nm) a (A)˚ b (A)˚ c (A)˚ Structure Phase TNSP 56 6.2 40.29 3.89 Orthorhombic Beta PNSP 45.5 3.62 3.62 3.88 Hexagonal Delta in Table 6.1) with a = b characteristic of the hexagonal structure in the PNSPs [192]. Mean crystallite size (t) was calculated by means of Scherrer’s Equation [190] and analyis of an appropriate peak (0,0,1), proving the TNSPs (56 nm) to be larger than the PNSPs (45.5 nm). With regards to DERT, these sizes are quite large compared to Au NPs, which commonly employ 1.9 nm [21] and even 1.4 nm [193], suggesting their ability to be internalised by cells may be undermined. However, a study into NP size-dependency of internalisation has revealed 50 nm to be the optimal size for internalisation in Au NPs [8], suggesting damage to cells will be maximised and val- idating our choice to investigate NSPs of this size. Confocal microscopy results in chapter 4 will confirm this.

4.3 Scanning Electron Microscopy

4.3.1 Theory

SEM utilises an electron microscope in producing high resolution images. A high en- ergy electron beam (of the order 10 keV typically) is produced via thermionic emis- sion before the electrons interact with sample atoms allowing images to be formed from the production of back-scattered electrons, secondary electrons and character- istic X-rays. A raster scan technique samples a rectangular area, and because most properties are power dependent, a high degree of magnification can be made possi- ble [194]. Physical Characterisation of T a2O5 Nanostructured Particles 73

Figure 4.3 A typical SEM image detailing the degree of aggregation exhibited by T a2O5 NPs [26].

SEM has been used effectively in the characterisation of T a2O5 NPs, particularly with respect to the degree of aggregation. Samples have shown significant agglom- erations with sizes in the order of micrometres, which may necessitate dispersion before any useful application is attempted [195] [26].

4.3.2 Method

T a2O5 NSPs were observed using SEM in order to determine agglomeration, ob- serve surface morphology, and determine the extent to which sonication disperses the NSPs. Two samples each of T a2O5 TNSPs and PNSPs were prepared in PBS solution (concentration 50µg/ml, similar to what was used in radiation experiments) with one sample sonicated and the other non-sonicated. These were dried, mixed Physical Characterisation of T a2O5 Nanostructured Particles 74 with ethanol and pipetted onto an aluminium tray into 6 holes (3 samples of each per hole). This was placed inside the electron microscope allowing surface morphology to be observed and an assessment to be made on the effectiveness of sonication in dispersing the NSPs (several images were captured).

4.3.3 Results and Discussion

T a2O5 NSPs were prepared in solution with and without sonication and samples analysed using SEM. Images taken of the TNSP samples (shown in Figures 4.4 and 4.5) revealed some large aggregates to be present before sonication, approximately 200 microns in size. After sonication, a global reduction in aggregate size was ob- served with the largest aggregate approximately 20 microns in size, establishing that sonication is indeed effective in globally reducing the size of large aggregates by a factor of 10. The effectiveness of sonication observed here correlates well with that reported in the literature. Rzigalinski et al. investigated the dispersive effects of sonication on 7 nm CeO2 NPs, observing similar degrees of agglomeration in unsonicated samples and similar factors of reduction in agglomerate size upon son- ication [6]. The drying process was known to cause a degree of aggregation in the NSP samples [196], so the actual size of aggregates in solution may in fact be smaller than what was observed; however, this means our results are at least conservative in nature. Physical Characterisation of T a2O5 Nanostructured Particles 75

Figure 4.4 A SEM image taken of T a2O5 TNSPs in PBS solution without sonication.

Figure 4.5 A SEM image taken of T a2O5 TNSPs in PBS solution with sonication.

Although morphological characterisation of both NSPs will be performed with high- Physical Characterisation of T a2O5 Nanostructured Particles 76 resolution TEM (HRTEM), a high magnification SEM image was taken of the PNSP (Figure 4.6) for comparative purposes with HRTEM, and to probe the high quality limits of SEM. The image itself reveals remarkable detail in the morphology of the PNSP, clearly displaying constituent crystallites of the larger aggregate particle, and defining grain boundaries. The crystallites appear stacked, and a degree of agglomer- ation is evident, perhaps exacerbated by the preparation process, and appear mostly spherical in shape, with a size roughly correlating with that determined in XRD; however, these results will be confirmed by HRTEM in the following section.

Figure 4.6 A high magnification SEM image taken of T a2O5 PNSPs in PBS solution with sonication.

Literary results regarding characterisation of T a2O5 NSPs is thin, although Huang et al. utilised the method to visualise agglomeration in amorphous T a2O5 NSPs [26]. This is due to the fact that HRTEM is widely considered the ”gold standard” for mor- phological characterisation [197], because of its superior resolution and high quality Physical Characterisation of T a2O5 Nanostructured Particles 77 images relative to SEM, hence we will be employing HRTEM for the accurate mor- phological characterisation of T a2O5 NSPs.

4.4 High-Resolution Transmission Electron Microscopy

4.4.1 Theory

High-resolution transmission electron microscopy (HRTEM) involves the use of a transmission electron microscope (TEM) in generating high resolution images of ob- jects (Figure 4.7 details the schematic of a typical TEM), with high magnifications, on the atomic scale. The mechanism of image generation is based on the electron gun generating an electron beam through thermionic emission, and lenses collimate the beam at the specimen to be imaged. The electrons pass through the sample, un- dergoing absorption and scatter interactions, before being transmitted through the specimen, after which the projector lenses are used to focus the electrons onto a flu- orescent screen for the image to be formed (more sophisticated image detection may be used as well, such as charge-coupled devices) [198]. Physical Characterisation of T a2O5 Nanostructured Particles 78

Figure 4.7 A schematic diagram outlining the primary components of a typical TEM [27]. Physical Characterisation of T a2O5 Nanostructured Particles 79

HRTEM is frequently used in nanotechnology and nanomedicine, among other fields, for the investigation and characterisation of not only physical nanomaterials, but also biological materials. This is due to the unmatched resolution with which images may be provided, as low as 0.05 nm [199], allowing for extreme detail to be seen on the atomic scale, including crystal structure and lattice defects. HRTEM is commonly used in DERT and UV protection applications for the characterisation of NPs, in de- termining morphological information such as aggregation, surface properties, shape, crytallinity and particle size. T a2O5 NPs have been characterised in the literature us- ing HRTEM [28] [26] (see Figure 4.8), however independent measurements should always be performed in any study due to possible variations in production methodol- ogy that may influence morphological characteristics of the resultant NPs, especially features like crystallite size, aggregation and structure.

Figure 4.8 A HRTEM image of crystalline mesoporous T a2O5 NPs [28]. Physical Characterisation of T a2O5 Nanostructured Particles 80

4.4.2 Method

Morphological parameters- crystallinity, size, agglomeration and shape- were inves- tigated using a JEOL (2011) 200 kVp High-Resolution Transmission Electron Mi- croscope. Samples of both T a2O5 TNSPs and PNSPs were independently placed on a cuvette before being analysed using HRTEM and the morphology of each NSP identified and compared.

4.4.3 Results and Discussion

Figure 4.9 HRTEM images of the TNSPs (left) and PNSPs (right) captured at a scale of 50 nm.

High quality images acquired (Figure 4.9) indicated that both NSPs are highly crys- talline in nature, possessing mean crystallite sizes in agreement with that derived us- ing Scherrer’s formula. The tendency for agglomeration is seen to differ significantly between the two samples, with the TNSPs exhibiting larger aggregates approximately Physical Characterisation of T a2O5 Nanostructured Particles 81

400 microns in maximum size, while the PNSPs are globally much more dispersed with maximum aggregates of 100 microns. This aggregation does add uncertainty to the determination of mean crystallite size, and mean aggregate size, hence the use of multiple methods for size confirmation, and taking an average over at least 100 particles. It must be noted that the sample preparation process causes the NSPs to agglomerate, so that the actual aggregate size in solution is much less. This differ- ence in particle aggregation may be attributed to their respective production methods, where by the higher reaction temperature in thermal decomposition of the TNSP re- sults in larger crystallites, with increased size distribution, and larger aggregates. Comparitively, the precipitation reaction utilises a lower reaction temperature, and involves dispersion treatment so that the crystallites are less likely to aggregate in an attempt to reduce the total surface energy of the material. These differences in the methods of synthesis directly affect the NSP (crystallite) morphology, there by influencing aggregate morphology as well. The TNSPs consist of larger crystallites, higher in aspect ratio, irregular in shape and less dispersed leading to aggregates which are larger and less dispersed than their PNSP counterparts.

The two NSPs are also morphologically dissimilar in terms of shape. The TNSPs display an irregular shape while the PNSPs are distinctly spherical. Based on the smaller crystallite size and resistance to aggregation, we would naturally expect the PNSPs to be more easily internalised than the TNSPs. The less obvious fact, though, is that the spherical nature of the PNSPs also increases their degree of internalisa- tion. It has been shown for Au that spherical NPs portray preferential uptake to other shapes, such as nanorods, in HeLa cells. In this particular study, endocytosis was identified as the mechanism of internalisation, with low aspect ratio spherical NPs being preferentially internalised due to greater availability of cell receptor bonding sites for serum proteins, which initiate endocytosis [8]. Evidently, we not only ex- pect the PNSPs to be more easily internalised than their thermal counterparts, there by imparting greater damage to the cells, but we may also expect the internalisation mechanism to be endocytosis. This will be confirmed, however, through FACS flow Physical Characterisation of T a2O5 Nanostructured Particles 82 cytometry and confocal microscopy.

4.5 Dynamic Light Scattering

4.5.1 Theory

Dynamic light scattering (DLS) is an optical technique used to measure the size dis- tribution of particles in suspension, with a resolution of less than a nanometre [200]. The principle of measurement is based on analysing the random movement of par- ticles in suspension. As they collide with one another they undergo Brownian mo- tion, the speed of which depends on particle size and viscosity of the liquid. A laser projects light through the sample (experimental setup and schematic of the Malvern Zetasizer shown in Figure 4.10), and as the scattering intensity fluctuates over time due to particle motion the translational diffusion coefficient (D) can be determined and hydrodynamic diameter (DH ) ascertained from the Stokes-Einstein equation [201]:

kT D = (4.5) 3πηDH

In this equation, k is Boltzmann’s constant, T is the temperature of the sample and η the viscosity.

The scattered light intensity fluctuations are dependent on particle size, with greater fluctuation indicative of smaller particle sizes. The correlator continuously monitors these signals to construct the correlation function unique to that sample. The correla- tion function is modelled based on an exponential decay equation depending on the experimentally determined D. Cumulants analysis is applied to determine mean par- ticle size and polydispersity index (PDI). The PDI is dimensionless number, ranging from 0 to 1, that is a relative indicator of the broadness in particle size distribution. It is calculated by applying a Gaussian function to the size distribution and using Physical Characterisation of T a2O5 Nanostructured Particles 83

Figure 4.10 The optical configuration and experimental set up for the Malvern Zetasizer Nano ZS [29].

the standard deviation (σ) along with the mean particle diameter (ZD) in equation 4.6 [202].

σ2 PDI = 2 (4.6) ZD

It is important to note that PDI values greater than 0.7 indicate broad distribution, 0.08 to 0.7 medium or average distribution, and less than 0.08 very narrow distribu- tion [203].

While DLS is a useful technique for measuring and quantifying the degree of particle dispersion, it seems to be not well represented in the literature for characterisation Physical Characterisation of T a2O5 Nanostructured Particles 84

of T a2O5 NPs, or even for the majority of materials in nanomedicine. This may be due to the fact that particle dispersion, and size are often analysed with confocal microscopy and HRTEM, and while quantification of the size distribution is useful, usually direct observation of particle morphology is acceptable. Nonetheless, some measurements do exist, with Nakata et al. synthesising T a2O5 NPs using a liquid phase deposition (LPD) method [30]. They were able to determine the mean parti- cle size and PDI using DLS (Figure 4.11) and found them to be 4.5 nm and 0.025, indicating the particles were small compared to other synthesis methods and near monodispersed, reflected in the size distribution spectrum. This may be due to the fact that DLS measurements rely on the particle suspension being well dispersed, as the presence of large aggregates or flocculants can distort the average results and increase the uncertainty in particle size measurements. This is why it is often recom- mended to filter samples before analysis, and perform sonication or vortexing of the sample immediately prior to measurement to increase dispersion.

4.5.2 Method

T a2O5 TNSPs and PNSPs each had their particle size distribution spectrum mea- sured, along with mean particle diameter and PDI, using the Malvern Zetasizer Nano ZS and the technique of DLS. Samples of each NSP were prepared according to the preparation and suspension protocols in chapter 2 at concentrations of 1 mg/ml, and underwent sonication and vortexing directly before measurement according to the relevant protocols in chapter 2 also. The samples were filtered through ultra-fine filter paper to eliminate any particles greater than 10 microns that may disrupt the results and distort scattering measurements. Samples of each NSP were placed into a well plate using a pipette, and all samples were measured in triplicate to ensure reliability.

4.5.3 Results and Discussion

The spectrum for the PNSP reveals a single intense peak at 1411 nm, and an aver- age particle diameter of 2450 nm. These values are somewhat larger than expected, Physical Characterisation of T a2O5 Nanostructured Particles 85

Figure 4.11 HRTEM image and (inset) DLS particle size distribution spectrum for LPD- synthesised T a2O5 NPs [30]. especially when compared to the literature, however correlate with the aggregation observed in HRTEM images, which showed aggregates in the order of 10 microns. A PDI of 0.49 was calculated, indicative that the PNSP sample contains particles with a medium level of size distribution.

The spectrum for the TNSP varied significantly from that of the PNSP, revealing a spectrum with 3 intense peaks at 512 nm, 224 nm and 5604 nm. Although, it is likely the largest 5 micron peak is part of the micron-sized aggregates not filtered out by the filter paper in the initial preparatory steps. The mean particle diameter was 615 nm, Physical Characterisation of T a2O5 Nanostructured Particles 86

Figure 4.12 DLS size distribution spectrum for the T a2O5 PNSPs. lower than that measured for the PNSPs. The primary difference though, and perhaps the most meaningful result to compare, is that of the PDI, which was 0.852, a factor of 2 higher than that for the PNSPs, placing the TNSPs in the broad spectrum range of particle size distribution. This was expected based on observations made in HRTEM that the TNSPs are much more aggregated than the PNSPs, and irregular in shape, leading to increased heterogeneity in particle size and a broad size distribution.

4.6 UV-vis Spectrophotometry

This section will present the raw UV-vis spectral data for PNSPs, however only a brief analysis will be given, as a more thorough evaluation of the results may be found in chapter 7.

4.6.1 Theory

Ultraviolet-visible (UV-vis) spectrophotometry, or spectroscopy, refers to the mea- surement of light spectra in the ultraviolet and visible wavelength regions of the Physical Characterisation of T a2O5 Nanostructured Particles 87

Figure 4.13 DLS size distribution spectrum for the T a2O5 TNSPs. electromagnetic spectrum. It is based on the principle of optical absorbance, with molecules in the sample undergoing electronic transitions as they are excited from the ground state to a higher energy level. The longer the wavelength absorbed of an incoming photon, the smaller the gap in energy levels between the initial and final states of the excited electron. The experimental set up and measurement principles for a spectrophotometer (shown in Figure 4.14) involve a deuterium/tungsten lamp projecting light through a monochromator, which splits the light into constituent wavelengths with intensity I0, before it is transmitted through the measured sam- ple at intensity I [204]. The transmitted light is detected by a photomultiplier tube or diode array. The transmittance (T) is defined as:

I T = (4.7) I0

The absorbance can then be derived from the transmittance according to equation 4.8: Physical Characterisation of T a2O5 Nanostructured Particles 88

A = −log(T ) (4.8)

Figure 4.14 The experimental set up and components of a typical UV-vis spectrophotometer used for UV absorption spectroscopy measurements [31].

4.6.2 Method

Assessment of the UV absorption properties of T a2O5 PNSPs was performed using a Shimadzu UV-3600 UV-vis spectrophotometer in the wavelength range of 200- 800 nm and quartz cuvettes (1ml) with path length `=1 cm. NSP samples were dispersed in ethanol with concentration c=0.05 g/L, and underwent 60 minutes of sonication in an ultrasonic water bath to ensure maximum dispersion of the NSPs in the sample and reduce aggregation. Immediately prior to measurement, samples were mixed in a vortex to ensure the mixture was homogeneous.

4.6.3 Results and Discussion

The absorption curve for T a2O5 NSPs displays significant absorption over the entire UV range (Figure 4.15). It has a defined maximum absorption peak around the lower limit of the UVB range (269 nm) that continuously decreases across the UVA and visible wavelengths, correlating well with spectra observed in the literature [205].

The significant degree of absorbance observed for this material, particularly in the UV region, makes it an ideal candidate for UV protection applications. This includes acting as a UV filter for sunscreens, cosmetics and textiles. Further testing is required Physical Characterisation of T a2O5 Nanostructured Particles 89

Figure 4.15 UV-vis absorption spectrum for the T a2O5 PNSPs, showing a distinguished peak in the UVC region. to determine suitability for UV protection applications, including protection factor analysis, ROS generation, and interactions with non-cancerous cells, in particular, any carcinogenic properties. A more detailed analysis, including application of the Beer-Lambert law, and comparisons to ZnO NPs, can be found in the UV applications sections in chapter 7. Chapter 5

Biological Characterisation of Cells to T a2O5 Nanostructured Particles

5.1 Introduction

This chapter describes the techniques and results associated with biological charac- terisation of T a2O5 NSPs, and their interactions with cells in vitro. This chapter compliments the previous chapter 3 (physical characterisation), in characterising the properties and behaviours of T a2O5 NSPs in their entirety. For the following exper- iments, both T a2O5 TNSPs and PNSPs are explored, and their respective properties compared, and results regarding biological interactions linked to their physiochemi- cal origins. Multiple cell lines are explored, including radioresistant 9L gliosarcoma, non-cancerous MDCK, and radiosensitive MCF-7 cells; although, the predominant focus of experimentation is on 9L cells. This stems from the fact that 9L cells form the foundations of radiation experiments, ultimately revealing the radiosensitising benefits of T a2O5 NSPs in DERT, and as a result, requiring the most comprehen- sive characterisation. Secondary to this are characterisation experiments involving MDCK cells, although still very significant and paramount for non-cancerous cell comparisons in DERT. Additionally, MDCK characterisation will form the basis for interpretation of UV protection results and applicability. The techniques explored in this chapter include: cytotoxicity, internalisation, ROS generation and cellular inter-

90 Biological Characterisation of Cells to T a2O5 Nanostructured Particles 91 actions.

Similar to the motivations for physical characterisation experimentation, biological characterisation is essential in the field of nanomedicine for accurately predicting the safety and efficacy of nanomaterials in clinical therapeutic applications [206]. In particular, it reveals the cytotoxic component of drug-assisted cancer therapy, and any potential toxicity to non-cancerous tissues. These behaviours are both signifi- cant considerations of DERT, where NPs such as Au have demonstrated cytotoxic tendencies [207], and UV protection, where any toxicity is undesirable and NPs such as ZnO have exhibited both cytotoxicity and phototoxicity [208].

5.2 Cell Viability Assessment- 14 Days

5.2.1 Method

Cells were counted each day over a 14 day period to determine cell viability and the level of toxicity T a2O5 TNSPs posed to the 9L cells over an extended period of time. A solution of the TNSP was prepared in accordance with the sterilisation, solution and sonication protocols in chapter 2. 10000 cells were transferred to 28 60 mm petri dishes (1 for each day in duplicate) to make a volume of 5.97 ml (cells and DMEM) before 30 µl of TNSP suspension was added to give a concentration of 2 µg/ml in 6 ml of solution (performed according to the splitting and sampling protocols). 28 petri dishes were also prepared in a similar way without the TNSP. Each day 4 dishes were counted (duplication allowed an average to be taken), which allowed the number of viable cells to be graphed as a function of time and a cytotox- icity assessment was made by comparing the control and material-containing dishes. Values were normalised to the maximum cell count at day 14 in the control group. This small concentration was chosen initially to gauge the effects of small amounts of the material on cells, however as confidence was gained in sample preparation Biological Characterisation of Cells to T a2O5 Nanostructured Particles 92 methods (section 3.3) and toxicity results were accurately and reliably known for small concentrations, the limits of concentration-dependent toxicity were tested, and the results are presented in section 5.5.

5.2.2 Results and Discussion

Viable 9L cells were counted each day over 14 days for a control set of petri dishes and a set containing 2 µg/ml concentration of T a2O5 TNSPs. This allowed % nor- malised cell counts to be graphed over time (as shown in Figure 5.1) and by compar- ison of the control and TNSP curves a cytotoxicity assessment could be made.

Figure 5.1 A graph of normalised cell count against time for determination of cell viability and cytotoxicity over a 14 day period. Values are normalised to the day 14 maximum cell count value.

The curves follow an exponential trend in accordance with the exponential prolif- eration of cells over time. We see the 2 curves strongly correlate with each other, not deviating by more than 2-3 % over the 14 day period. Error bars represent one standard error from the mean and do not exceed 5 % at any time. The data suggests that TNSPs are non-toxic to 9L cells. The data from 0-3 days was neglected due Biological Characterisation of Cells to T a2O5 Nanostructured Particles 93 to unreliable results, with an insufficient number of cells existing for sampling, pri- marily caused by a low seeding number. These results were later obtained using the clonogenic assay technique, and will be detailed in the following sections.

5.3 Cytotoxicity Assessment- 6 Hours

5.3.1 Method

A typical time between implantation of the NSP and irradiation is 6 hours based on the experimental protocol used, so a toxicity assessment was to be made over this time period using the clonogenic assay method. As a secondary objective, a suit- able seeding number of cells had to be found for radiation experiments, i.e., one that would give a suitable amount of colonies for survival analysis following irradiation (not too many that colonies could not be distinguished, however not too few that cells could not proliferate).

Four different concentrations of the TNSP were constructed for cytotoxic compari- son, namely 2, 10, 30 and 50 µg/ml; as well as a control, giving a total of 5 con- centration conditions (dried, sterilised, sonicated, dissolved according to previous protocols in chapter 2). The cells were grown in a T75cm2 flask before being split into 5 T12.5cm2 flasks for experimentation. Past experiments by Sianne Oktaria showed the doubling time of the 9L cells to be approximately 36 hours, which meant 500000 cells were seeded per flask to become confluent in 72 hours. Once conflu- ent, the TNSP was added to the 4 flasks and all 5 flasks placed inside the incubator for 6 hours. The results of toxicity following 6 hour exposure were noted for each concentration and used to modify initial seeding estimates for the respective concen- trations in the clonogenic assay (a higher degree of toxicity means more cells need to be added initially in the clonogenic assay to ensure a suitable level of proliferation). Biological Characterisation of Cells to T a2O5 Nanostructured Particles 94

The flasks were split into petri dishes, colonies allowed to grow, before fixing and staining was performed, all according to the clonogenic assay protocol in chapter 3. The reference number of 1500 cells was obtained from previous experiments using 9L cells by Oktaria et al. [24]. The 3 seeding numbers used for each concentration were then N, 3N and 9N.

5.3.2 Results and Discussion

4 different concentrations (2, 10, 30 and 50 µg/ml) and a control were used to assess the cytotoxicity of the TNSP to the 9L cells over a 6 hour period. Prior to plating, the 5 flasks were sampled and showed a cell death rate varying from 6 % to 14 %, which is relatively non-toxic; although the clonogenic assay was used to give more accurate results based on cell viability and clonogen formation, while assessing the suitability of 3 different seeding numbers for each concentration (N, 3N, 9N). Initial seeding numbers were modified according to the cell death rate, as per the protocol in chapter 2. Biological Characterisation of Cells to T a2O5 Nanostructured Particles 95

Figure 5.2 Cell survival fractions for a variety of concentrations following 6 hour exposure of the cells to the TNSP.

Analysis of the dishes following staining showed all 3N and 9N dishes to be over- populated, while N gave suitable numbers of colonies (approximately 100-150), por- traying that N=1500 cells was a good reference number for radiation experiments.

Counting of the colonies allowed survival fraction as a function of concentration to be plotted and normalised to the control (as shown in Figure 5.2). The graph con- veys a relatively constant trend, with the majority of points lying within 1 standard error from the mean, and a maximum deviation of 8 %. Values slightly higher than 1 are due to natural variation in colony numbers, as a result of cell proliferation. This illustrates that the TNSP is relatively non-toxic to the cells over a 6 hour period, with approximately 100 % survival, even in the case of the highest concentration 50 µg/ml, which allowed us to employ the use of this concentration in radiation ex- periments. Once confidence was gained in experimental technique, and radiosensiti- sation results were successfully confirmed for 50 µg/ml (results shown in chapter 6), Biological Characterisation of Cells to T a2O5 Nanostructured Particles 96 higher concentrations were probed to ascertain whether they could be appropriately used in radiation experiments to enhance the sensitising effect without compromis- ing cytotoxicity These cytotoxicity results are presented in section 5.5 and radiation experiments at higher concentrations in chapter 6.

5.4 Cytotoxicity Assessment- 1 and 2 Day Exposures

5.4.1 Method

Cytotoxicity was tested in a similar way to the 6 hour exposure method, utilising the clonogenic assay technique. The motivation for testing 1 and 2 day exposure was to probe whether more time would allow the NSP to interact with the cells com- pletely and possibly increase the dose enhancement effect. For each day of exposure, a clonogenic assay was plated in triplicate for a control (no NSP) and 50 µg/ml con- centration of T a2O5 TNSPs (totalling 12 petri dishes).

5.4.2 Results and Discussion

Clonogenic assays were used to determine the cytotoxicity of the TNSP (50µg/ml) to the 9L cells after 1 and 2 days exposure. Following counting of the colonies for both the 1 and 2 day assays, survival fraction data showed 0% toxicity after 1 day and 8% toxicity after 2 days (Figure 5.3). This again shows that the TNSP is relatively non-toxic, especially over this period of time, to be conservative though, we used an exposure time of 24 hours in the radiation experiments. Biological Characterisation of Cells to T a2O5 Nanostructured Particles 97

Figure 5.3 Cell survival fractions for a variety of concentrations following 6 hour exposure of the cells to the TNSP.

5.5 Cytotoxicity Assessment- 24 Hours and Maximum Concentration

5.5.1 Method

To assess the toxicity of TNSPs and PNSPs to 9L cells, NSP concentrations of 0 (2 controls), 50, 100, 200 and 500 µg/ml were exposed to 9L cells for a period of 24 hours prior to plating. 9L cells were brought to 90 % confluence inside T12.5 cm2 flasks (BD Falcon™) before exposure to the NSP. Solutions of TNSPs and PNSPs were prepared according to the NSP suspension and sonication protocols in chapter 3, and the appropriate volumes corresponding to each examinable concentration added to the cells. Once the desired exposure time had been reached, cells were plated in triplicate according to the clonogenic cell survival assay technique in chapter 3. Following plate staining and colony counting, surviving fractions were obtained for Biological Characterisation of Cells to T a2O5 Nanostructured Particles 98 each NSP at each concentration, and normalised to the control group (0 µg/ml).

5.5.2 Results and Discussion

Figure 5.4 Cytotoxicity of TNSPs and PNSPs to 9L cells following a 24 hour period of exposure.

Maximum toxicity values of approximately 20 % and 30 % were measured (Figure 5.4) for TNSPs and PNSPs respectively. Significant drops in cell colonies and satu- ration occured at 100 µg/ml for both NSPs. Additionally, both NSPs exhibited 80 % SF at 100 µg/ml, which is exactly the same SF as 1.9 nm Au NPs tested at the same concentration on MDA-231-MB (breast cancer) cells (more sensitive than 9L) [21]. This suggests they are, at a minimum, comparable to Au NPs in terms of cytotoxicity, which are already used clinically in DERT and widely considered non-toxic [209]. Moreover, there is considerable support in the literature to suggest toxicity values as high as 50 % may be considered non-toxic [210], supporting the fact that both

T a2O5 NSPs may be considered relatively non-toxic at the highest concentrations tested. These results formed the basis for the concentrations utilised in radiation Biological Characterisation of Cells to T a2O5 Nanostructured Particles 99 experiments.

5.6 Cytotoxicity Assessment- 24 hours and Multiple Cell Lines

5.6.1 Method

To assess the toxicity of PNSPs to multiple cell lines, including: 9L, MDCK and MCF-7 cells, NSP concentrations of 0 (2 controls), 50, 100, 200 and 500 µg/ml were exposed to the cells for a period of 24 hours prior to plating. All cells were brought to 90 % confluence inside T12.5 cm2 flasks (BD Falcon™) before exposure to the PNSP. Solutions of PNSPs were prepared according to the NSP suspension and son- ication protocols in chapter 3, and the appropriate volumes corresponding to each examinable concentration added to the cells. Once the desired exposure time had been reached, cells were plated in triplicate according to the clonogenic cell survival assay technique in chapter 3. Following plate staining and colony counting, sur- viving fractions were obtained for each NSP at each concentration, and normalised to the control group (0 µg/ml). These were plotted against each other for toxicity comparison.

5.6.2 Results and Discussion

Maximum toxicity values of 30%, 52% and 70% were observed for 9L, MDCK and MCF-7 cell lines respectively (Figure 5.5). All cell lines followed similar trends of a sharp increase in toxicity at 100 µg/ml before saturation of the curve to a relatively constant toxicity value up to the maximum concentration tested (500 µg/ml).

These results correlate well with those reported in a similar experiment by Briggs et al. [32]. In this experiment, the same cell lines were tested, with the same exposure time and concentration range, however the materials used for cell exposure were CeO − 2 NPs. The results are presented in Figure 5.6, and it is evident that the same overall trend is observed, including a sharp increase in toxicity at 100 µg/ml. The Biological Characterisation of Cells to T a2O5 Nanostructured Particles 100

Figure 5.5 Cytotoxicity of PNSPs to 9L, MDCK and MCF-7 cells following a 24 hour period of exposure. maximum toxicity values for 9L and MCF-7 cells of 30% and 75% correlate well with the results presented in this work. The toxicity of MDCK cells was similarly observed to be in between the values for the two other cell lines, although lower in the work of Briggs et al., reporting a value of 70% at 500 µg/ml. Although there are no in vitro toxicity results for T a2O5 NSPs in the literature, the trend of varying toxicity with different cell lines for the same NP type has been observed for Au NPs in DERT applications. A study by Pan et al. tested the size dependent toxicity effects of Au NPs on 4 different cell lines, and noted high toxicities for smaller particle sizes (1-2 nm), as well as varying toxicity accross the cell lines, and a sharp increase in toxicity at a specific threshold concentration value [207]. It was suggested that mechanisms of toxicity could be due to interactions of the material with the cellular membrane, or even within the cell, if NPs are internalised by endocytosis. Biological Characterisation of Cells to T a2O5 Nanostructured Particles 101

Figure 5.6 Cytotoxicity of CeO − 2 NPs to 9L, MDCK and MCF-7 cells following a 24 hour period of exposure [32].

5.7 FACS Flow Cytometry

5.7.1 Method

9L cells and MDCK cells were both separately exposed to T a2O5 TNSPs and PNSPs at concentrations of 0, 50 and 500 µg/ml for 24 hours. Trypsinisation was used to de- tach the cells before being placed in a centrifuge at 1500 rpm and 4° C for 5 minutes and rinsing twice with cold PBS (without calcium and magnesium). Flow cytometric measurements and analyses were performed using a Becton-Dickinson fluoresence- activated cell sorting (FACS) flow cytometer (BD LSR II; BD Biosciences, Franklin Lakes, USA). Each analysed sample contained a minimum of 10,000 cells where cell doublets and aggregates were gated out using a two parameter histogram of FL2-Area versus FL2-Height. Data analysis was performed using the FACSDiva software, as- sessing both forward (FSC) and side scatter (SSC) intensities. The median cell SSC Biological Characterisation of Cells to T a2O5 Nanostructured Particles 102 values were employed as a marker of cellular granularity, which is proportional to particle uptake and internalisation [211].

5.7.2 Results and Discussion

The magnitude of FSC and SSC values are representative of cell diameter (size) and granularity respectively, with changes in cellular granularity (mean SSC) propor- tional to the degree of internalisation [212]. Data for the 9L cell line was presented in the form of a histogram with SSC graphed as a function of FSC abscissa (Figure 5.7).

Figure 5.7 FSC vs. SSC histograms representative of 9L cellular uptake of TNSPs and PNSPs at 0, 50 and 500 µg/ml following 24 hours exposure.

For all concentrations tested in the 9L cell line, we see no significant difference in the mean SSC values when comparing the two NSPs themselves [211], suggesting both NSPs are internalised to a similar degree. When examining each NSP on its own though, however, and comparing to the control, we see significant increases in the degree of internalisation as the concentration of the NSP increases. Although this Biological Characterisation of Cells to T a2O5 Nanostructured Particles 103 dependency has been rarely tested explicitly, it is not unheard of, with bismuth ferrite

(BiFeO3) NPs [212] and superparamagnetic iron oxide NPs (SPION) [213] exhibit- ing similar trends. For both NSPs, we see an approximate 10% increase in the mean SSC values at 50 µg/ml, and at 500 µg/ml we observe a substantial increase in the mean SSC of approximately 300%. These results suggest that at low concentrations the majority of the NSPs remain external to the 9L cell membrane as aggregates, while at higher concentrations a greater portion of the NSPs are internalised by the 9L cells.

Figure 5.8 FSC vs. SSC histograms representative of MDCK cellular uptake of PNSPs at 0, 50 and 500 µg/ml following 24 hours exposure.

Examination of the mean SSC values for PNSPs on MDCK cells (Figure 5.8) shows a 40% and 270% increase at 50 and 500 µg/ml respectively. These values are sim- ilar to, and within random variations of, the values measured for internalisation in 9L cells. This suggests similar portions of the NSPs are internalised in each case, possibly by similar endocytotic mechanisms of uptake, and a non-cell specific nature Biological Characterisation of Cells to T a2O5 Nanostructured Particles 104 of uptake for these particular NSPs. Further testing needs to be performed to val- idate these claims, however confocal microscopy experiments (section 5.8) proved useful in illuminating the mechanism of uptake, and observation of general interac- tions between the NSPs and the cells. Having been satisfied that the results for PNSP exposure on MDCK cells were similar to that for 9L cells, and that little difference was seen in uptake between the two types of NSPs on 9L cells, it was decided to not repeat the experiment for TNSP-exposure of MDCK cells in order to conserve the material for future experiments.

5.8 Confocal Microscopy

5.8.1 Method

Confocal microscopy was used to observe and assess the interactions between T a2O5 NSPs and 9L cells on a cellular level, using the high-resolution and magnification of the confocal microscope. 9L cells were brought to 90 % confluence and exposed to TNSPs and PNSPs at concentrations of 0 (control), 50 and 500 µg/ml for a period of 24 hours. Light and fluorescence microscopy images were obtained using a Leica confocal laser scanning microscope (Leica TCS SP5 Advcanced System UVVIS-IR and X1-Port Access with SMD FCS and CO2 incubation chamber, Germany) and the accompanying image manager software Leica Application Suite Advanced Fluores- cence (LAS AF, v.2.6.17314, Germany) located at IHMRI, Wollongong, Australia (shown in Figure 5.9).

5.8.2 Results and Discussion

Confocal microscope images were captured detailing the interaction of the TNSPs and PNSPs with 9L cells at concentrations of 0 (control), 50 and 500 µg/ml.

Analysis of the resultant images (Figure 5.10) revealed that both NSPs show a speci- ficity to the 9L cells at all concentrations, forming aggregates and being attracted to the cellular membrane. This aggregation of the NSPs is more pronounced at the Biological Characterisation of Cells to T a2O5 Nanostructured Particles 105

Figure 5.9 The Leica confocal laser scanning microscope, with associated laser systems and image analysis software. This was the same apparatus utilised in experimental measurements [24]. highest concentration tested (500 µg/ml). Comparing the two NSPs, we see the TNSPs are more prone to aggregation, forming much larger clusters around the 9L cells compared to the PNSPs, for all particle concentrations. On the other hand, the PNSPs show little to no aggregation and are of uniform size. Despite the lack of aggregation, clustering of the PNSPs is still observed around the cellular mem- brane and nucleus due to an intrinsic attraction of the PNSPs to the 9L cells. We also observe the presence of endosome-encapsulated NSPs (indicated by red arrows in Figure 5.10), particularly in the 500 µg/ml cases, suggesting the internalisation mechanism activated here is endocytosis. This is similar to Au NPs, which have been shown to exhibit clustering inside the cytoplasm of HeLa cells, and through the use of confocal imaging, were observed to localise in lysosomes [214]. Evidently, the images suggest the PNSPs are again superior to the TNSPs in terms of particle morphology (aggregation), specificity and internalisation. However, based on the Biological Characterisation of Cells to T a2O5 Nanostructured Particles 106

Figure 5.10 9L cells treated with TNSPs (left column) and PNSPs (middle column) at concentrations of 50 and 500 µg/ml. The untreated sample is represented in the top right hand column, and the bottom right hand panel is a zoomed region from the adjacent panel containing 9L cells treated with PNSPs at 500 µg/ml. The red arrows indicate endosome- encapsulated PNSPs around the nucleus of the cell. significant increase in specificity and clustering from 50 to 500 µg/ml for both NSPs, it would be logical to expect greater enhancement than what was observed in radia- tion experiments (chapter 6), although an explanation for this phenomenon is offered and supported by the results presented in section 6.3, along with the introduction of a new effect known as the ”shell effect”. Chapter 6

Radiosensitisation Experiments using T a2O5 NSPs

6.1 Introduction

This chapter represents the culmination of the majority of the work presented in this thesis, where by, the radiosensitising abilities of T a2O5 NSPs are quantified in an X- ray photon field, with in vitro cell cultures. Physical and biological characterisation of the T a2O5 NSPs is utilised in the interpretation of the results, in order to describe primary interactions between the radiation and the NSPs, as well as secondary inter- actions between the NSPs and the cells. Mechanisms of dose enhancement are ex- plored, and literary comparisons made with well-established nanomaterials in DERT, such as Au NPs. Radiation experiments utilise both T a2O5 TNSPs and PNSPs to gauge the effects of particle morphology on dose enhancement, as well as the de- pendency on beam energy (150 kVp and 10 MV) and NSP concentration (0, 50 and 500 µg/ml).

The motivation for applying T a2O5 NSPs to DERT originated with the constant need for more effective treatment methods in radioresistant cancers, such as glioblastomas. Current radiotherapy treatment methods for brain cancers, such as SBRT, can be ef- fective in arresting tumour growth, however often result in significant dose to critical

107 Radiosensitisation Experiments using T a2O5 NSPs 108 peripheral structures (brain). DERT presents an effective advantage to current treat- ment methods, in that, nanomaterials may be introduced into the tumour, allowing for significant local dose enhancement to the tumour, without increased dose to sur- rounding normal tissues, allowing for increased tumour control and reduced normal tissue complications. Au NPs currently dominate research in the field of DERT, however have presented issues with significant toxicity to both cancerous and non- cancerous cells [207], and are hard to functionalise in their raw NP form without significant modifications. T a2O5 NSPs present promise in their high-Z nature, in- herent low toxicity and biocompatibility, as well as their oxide layer providing the means for functionalisation [100]. The treatment of brain cancers with radiosensitis- ers, especially hypoxic glioblastomas, requires the material to cross the BBB in order to provide effective enhancement to the tumour target. This is a known challenge for nanomaterials in DERT [215], primarily due to difficulties in functionalisation, however combinations with ligands and proteins, such as LDL [216], have allowed nanomaterials to cross the BBB, and techniques such as this one could be employed to make T a2O5 NSPs effective radiosensitisers for radioresistant cancers.

6.2 Investigating the Radiosensitising Effect of T a2O5 TNSPs in an MV Photon Field

6.2.1 Introduction

The treatment of aggressive radioresistant tumors, such as some gliomas, is ex- tremely difficult with the established methods of cancer treatment being almost inef- fective in the delivery of local tumor control [217]. One well accepted animal tumor model often used to investigate ways to improve radiation treatment outcomes of such tumors is the 9L cell line [218]. The 9L cell line is derived from gliosarcoma rat-brain cancer cells possessing known radioresistant characteristics, with a radia- tion dose as high as 40 Gy in one fraction required for effective tumor control [155]. Radiosensitisation Experiments using T a2O5 NSPs 109

Radiotherapy is a common form of cancer treatment with over 50% of cancer patients receiving some form of radiotherapy as part of their cancer management plan, and a significant fraction of these patients undergo external megavoltage (MV) photon beam therapy. Interaction of the primary beam with tissue is dominated by Comp- ton scattering and pair production processes (provided the photon energy meets the required 1.022 MeV to initiate pair production); with the photoelectric absorption process also playing a minor role [219]. These processes lead to energy deposition within the tissues via the resulting high energy secondary electrons that have low LET [220]. The low LET nature of these secondary particles is one reason why, to date, there has been little research published on dose enhancement effects in MV ra- diotherapy. Dose enhancement at MV energies has not been fully investigated in the literature because generally cells are irradiated with 6 MV beams where the Z depen- dency of the interaction of photons with matter is extremely low. This dependency is paramount in facilitating dose enhancement [161]. As such, irradiation at kVp en- ergies may appear more appealing for research into dose enhancement; however, it should be noted that at higher MV energies- 10 MV and greater- the Z-dependency is more prominent [156]. In light of the fact that external MV photon beam therapy represents a very common radiotherapy modality for cancer treatment, and given the recent explosion in research of NPs for diagnostic and therapeutic procedures, it is important to improve current knowledge of the impact of NPs in the context of dose enhancement at MV energies.

DERT is a potential technique to improve MV radiotherapy of radioresistant tumors. DERT involves the introduction of high-Z material into close proximity to the tu- mor, and leads to a effective increase in the radiosensitivity of the tumor when ex- posed to the radiation field. The radiation-related increase in sensitivity is due to a change in the local radiation environment (LRE) bought about by the interaction of the local radiation field with the high-Z material. Charged particles (in the form of pair-production positrons and electrons, Compton scattered electrons, photoelectrons and/or Auger electrons) and free radicals result from the above interaction, some of Radiosensitisation Experiments using T a2O5 NSPs 110 which can have high LET, making the LRE more lethal. The increased lethality of the LRE causes additional damage to the tumor. Purely from a radiation physics per- spective, the use of high-Z materials is ideal for DERT due to the Z-dependence of the mass attenuation coefficient, which is approximately proportional to Z and Z3 for pair production and photoelectric effect respectively [221].

Current studies reporting the use of nanomaterials for dose enhancement have utilised gadolinium-based NPs [222], paclitaxel-loaded NPs [164] and quantum dots [43]. Some of the most promising experiments have been conducted using gold NPs of crystallite sizes as small as 1.4 nm [193] in concentrations up to 500 µg/ml [223],which have lead to DERs of approximately 2 for T98G human glioblastoma cells exposed to gold NPs when quantified using the clonogenic assay technique [21]. The degree of dose enhancement is dependent on particle size [224] and particle concentration [21], necessitating an optimum to be found for each.

One possible way to realize DERT is through the use of ceramic NSPs, such as cerium oxide ceramic NSPs, which have been previously reported as free radical scavengers and radioprotectors [225], although ceramic NSPs have not yet been in- vestigated as radiosensitisers. These NSPs are commonly engineered with a crys- tallite size approximating that of cellular pathways to internalisation. Depending on the size of the NSPs and the level of any associated aggregation, the adminis- tered NSPs could then settle in a number of locations including close proximity to the cell, around the cellular membrane or within the cell via a variety of internali- sation processes [7]. For larger NSPs (10-100 nm in diameter) with aggregates of 10-100 microns, and in the context of MV radiotherapy, only low energy secondary electrons originating from close to the surface of the NSP will contribute to localized dose enhancement, due to their short range in tissue (0.1-10 nm) [223]. Low energy secondary electrons originating from deeper within such NSPs will not contribute to dose enhancement if they do not escape the NSP. The effective range of these elec- Radiosensitisation Experiments using T a2O5 NSPs 111 trons in the high-Z NSP is less than that in tissue. On the other hand, higher energy photoelectrons, Compton scattered electrons, and electrons (and positrons) related to pair production originating from the NSPs would be expected to form an integral part of enhanced dose in MV radiotherapy. Their greater range allows for significantly more of these electrons to escape the NSP causing cellular damage via direct or indi- rect damage, therefore effectively increasing the radiosensitisation of the otherwise radioresistant tumor tissue.

The level of radiosensitisation is measured by the degree of dose enhancement and is defined in the literature in several ways. The DER, the ratio of doses giving a prescribed end point, the SER, the DER defined for a 10% end point and the DEF, the ratio of end points defined at 2 Gy are all commonly used [159].

T a2O5 is one of many unexplored ceramic compounds that contain potentially desir- able characteristics for DERT that have not yet been tested in any widespread nanoth- erapy application. Tantalum metal has a relatively high atomic number (Z=73) which is advantageous for dose enhancement; is a known biocompatible material, having been used in orthopaedic surgery [89] and consequently, is non-toxic to healthy cells. Furthermore, its combination with oxygen is based on the notion that the oxide may provide a linkage for the NSP to be functionalised and made to target tumor cells [226]. T a2O5 NSPs themselves have been used in radiation imaging applica- tions in the form of tooth fillers [117] and bone substitutes [227] [116], where no toxicity to non-cancerous tissues was reported. Therefore, they have added potential stemming from their functionalisation ability and high-Z nature for use in the field of imaging [228], emphasising the research opportunities it presents as a theranostic material.

In this thesis work, we have developed a process to engineer T a2O5 TNSPs with a controlled size distribution, as described in section 3.2. Ultimately, we hope to be Radiosensitisation Experiments using T a2O5 NSPs 112 able to systematically isolate the contribution to cell death from the different mech- anisms (interactions of the NSP with the cells) mentioned above. The experiments described in the following section (6.2) focus on NSPs with a size distribution of 50- 70 nm to investigate the dose enhancement related effects of high-Z NSPs using 9L gliosarcoma cells exposed to T a2O5 TNSPs and irradiated with 10 MV X-rays from a clinical LINAC. AAs shown in the results section (6.2.3), we have measured a SER of 1.33 through utilisation of the clonogenic assay technique, which is comparable to published gold NP data [229] and highlights the exciting potential of T a2O5 NSPs in DERT of radioresistant tumors. In addition, and more importantly, we observe a significant change in the shape of the survival curve for 9L cells exposed to the

T a2O5 NSPs and irradiated, indicative of a change in the quality of the LRE.

6.2.2 Method

To determine the effectiveness of T a2O5 NSPs as radiosensitisers, 9L cells exposed (50 µg/ml) and unexposed to the NSP for 24 hours were irradiated to an absorbed dose of between 1 and 8 Gy using 10 MV X-rays. All radiation experiments were car- ried out at the radiation oncology department at the Prince of Wales Hospital (Rand- wick, NSW, Australia) using an Elekta Axesse TM LINAC (Elekta AB, Kungstens- gatan,Stockholm, Sweden). The cells were irradiated in T12.5 cm2 Falcon flasks completely filled with Hank’s Balanced Salt Solution (HBSS) in a single fraction at a dose rate of 600 MU/min and at room temperature. Flasks were irradiated horizon- tally in a 30x30x10 cm3 solid water phantom, with the cell mono layer situated at a depth of 2.5 cm from the beam entrance. No air bubbles were present inside the flask so as to ensure electronic equilibrium conditions were present at the depth of the cells. Post-irradiation clonogenic assays were used to assess cell clonogenic sur- vival following radiation experiments. Following 15 doubling times of incubation, dishes were fixed, stained and counted to determine surviving fractions. Survival curves were fit according to the LQM using the associated error bars of each point as weighting factors. The LQM [156] describes cell surviving fraction (SF) mathemat- Radiosensitisation Experiments using T a2O5 NSPs 113 ically as a function of absorbed dose (D).

2 SF (D) = e−αD−βD (6.1)

The parameters α (Gy−1) and β (Gy−2) are indicative of cell radiosensitivity and repair effectiveness respectively. We were able to extrapolate these values based on the fit for each survival curve and compare experiments (with and without the NSP) to quantify the degree of dose enhancement by calculation of the SER (the ratio of doses giving 10% surviving fraction on the cell survival curves).

6.2.3 Results and Discussion

Figure 6.1 Cell survival curves for 9L cell cultures irradiated with 10 MV X-rays showing the dose modifying response of T a2O5 TNSPs. Surviving fractions were normalised to non- irradiated control cells, averages taken from a sample size of 3 and errors given ± 1 standard deviation from the mean.

The resultant survival curves (Figure 6.1) showed that cells containing the NSP were subject to a higher degree of damage, illustrated by the lower surviving fractions for each radiation dose in comparison to the control. Furthermore, the linear fit of Radiosensitisation Experiments using T a2O5 NSPs 114

Table 6.1 Alpha and beta parameters for the 10 MV experiments with and without the presence of the NSP showing a modified response of the cells to the radiation.

NSP Concentration α(Gy−1) β(Gy−2) 0 µg/ml 0.19 ± 0.05 0.012 ± 0.008 50 µg/ml 0.38 ± 0.03 N/A the survival curves in a semi-log scale demonstrated an essential increase in alpha and absence of beta (as shown in Table 6.1, where the N/A beta value refers to the absence of a beta component in the TNSP linear curve). This is indicative of not only an increase in cell radiosensitivity, but also radiation damage similar to that ob- served in cells irradiated with high LET charged particles or neutrons, representative of a change in the quality of radiation damage to the cells. The SER calculated from these survival curves is 1.33 ± 0.07, confirming a significant degree of dose enhance- ment. The change in radiation quality and high degree of radiosensitisation observed here may be a consequence of the large nanocrystallite size. Brun et al. [230] found that the largest gold NPs tested (92 nm) gave the highest dose enhancement factor (3), although the mechanism was not fully explained. We propose that the larger crystallites and their associated aggregates may be partially absorbing energy from secondary electrons enhancing the low energy part of their energy spectra giving them a higher LET, leading to the increased lethality observed in our experiments, despite the fact we irradiate with a 10 MV X-ray photon beam.

Another mechanism explaining this behavior involves more effective electron backscat- tering on aggregates of T a2O5 NSPs leading to dose enhancement on a T a2O5 to water interface, a phenomenon well known in radiation therapy with MV photon beams, notably in the case of titanium hip prostheses [231]. This dose enhancement is a maximum at the NSP surface and propagates (for a distance larger than the size of the NSP) outward from the NSP surface producing a ”dose enhancement tail”, which affects many cells surrounding the NSP [56]. With increasing size and Z of Radiosensitisation Experiments using T a2O5 NSPs 115 the NSP aggregates and decreasing electron energy, the backscattering mechanism increases suggesting that the ”dose enhancement tail” is produced mostly by lower energy electrons that additionally can be associated with an increase in Radiobiolog- ical Effectiveness (RBE). This mechanism may also be contributing significantly to the effects observed for larger NSPs and their aggregates. Further investigation of this effect will be modelled with Monte Carlo simulations.

There is currently no published research regarding T a2O5 NSPs in DERT how- ever, gold NPs have achieved SERs of 1.29 and 1.16 for 6 MV and 15 MV X-ray beams respectively; in the presence of less radioresistant MDA-MB-231 breast can- cer cells [229]. Given these results are based on the use of much smaller (1.9 nm) and concentrated (up to 100 µg/ml) NSPs with the ability to be internalised by the target cells, the results obtained for larger T a2O5 NSPs on more radioresistant cells are ex- tremely interesting; especially seeing the 10 MV case already exceeds that quoted for gold at 6 MV. This, coupled with the superior non-toxic nature of tantalum pentoxide NSPs over gold NPs, suggests they may possess intrinsic properties that allow them to exceed gold in the field of dose enhancement.

6.2.4 Conclusion

In conclusion, it is clear that more effective treatments of radioresistant cancers is an ongoing and important focus of much international research and development. DERT, through the application of specifically engineered ceramic NSPs, represents an innovative approach, by which a commonly available form of current cancer treat- ment may be improved, leading to more effective treatment outcomes. In this pioneer work we have demonstrated the potential of tantalum pentoxide NSPs to increase the radiosensitivity of the very radioresistant 9L cell line to MV radiation therapy. Our in vitro experiments showed that the NSPs were nontoxic yet led to significant sensitisa- tion enhancement. The increased sensitisation appears as both an increased cell death Radiosensitisation Experiments using T a2O5 NSPs 116 and an increased lethality of the local radiation environment. Further refinement and optimisation of T a2O5 NSPs in terms of particle size, aggregation and concentration may reveal a future novel treatment for radioresistant tumors. These aspects will be explored in the following sections.

6.3 Investigating the Dependency of Radiosensitisation in T a2O5 NSPs on Beam Energy, NSP Morphology and NSP Concentration

6.3.1 Introduction

Cancer represents the foremost incurable disease and single greatest threat to human longevity of not only contemporary times, but also historically and for the foresee- able future. Currently, cancerous disease accounts for 1 in 8 deaths worldwide [34] with over 50% of patients [232] utilising some form of radiotherapy in their treat- ment plan. This method relies on X-rays in irradiating the target volume, however, it is difficult to maximise damage to the tumor volume, increasing tumor control probability, while sparing healthy tissue. One solution is based on artificially in- creasing local energy deposition on the tumor itself, effectively sparing surrounding non-cancerous tissue. Known as DERT, this technique involves the introduction of high-Z atoms into close proximity to the tumor which, following exposure to the local radiation field, increase the selective damage and killing of the tumor cells. This radiation-induced increase in radiosensitivity is facilitated by the production of charged particles and ROS, which are created when incoming photons interact with target high-Z atoms. The use of high-Z atoms are favourable due to the increased probability of photon interactions, which stems from the Z-dependence of the mass attenuation coefficient. The mass attenuation coefficient due to the photoelectric ef- τ Z 3 fect ( ρ ) is approximately proportional to ( E ) , where Z is the atomic number of the absorbing medium and E is the energy of the incident photon. Furthermore, the mass

κ 2 attentuation coefficient due to pair production ( ρ ) exhibits a Z dependence [221]. Radiosensitisation Experiments using T a2O5 NSPs 117

DERT is especially promising for the treatment of aggressive radioresistant tumors and tumor cells, such as the gliosarcoma 9L cell line utilised in this study [218].

High-Z materials used in DERT have been dominated by metal NPs in the form of gold, platinum and gadolinium. Au NPs have emerged as a leading contender in the field, with initial in vivo studies by Hainfeld et al. revealing DERs up to 6 us- ing 1.9 nm Au NP concentrations of 7 mg/g (Au to tumor mass ratio) and 250 kVp irradiation on mice containing subcutaneous EMT-6 mammary carcinomas [37]. Al- though popularity for research has revolved around kilovoltage (kVp) energy X-rays, recent studies by Jain et al. have generated SERs of 1.29 and 1.16 on MDA-MB-231 cells treated with 12 µM 1.9 nm Au NPs using 6 MV and 15 MV X-ray beams, re- spectively [38].

Ceramic NPs are now emerging as potential alternatives to metal NPs in DERT. Ini- tially, ceramic NPs in the form of CeO2 were used as radioprotectors [225], however, it has recently been shown that the influence of the high-Z component at kVp energies creates a dose enhancement effect, when sensitisation outweighs the protective free radical scavenging effect [5]. Following this, we revealed T a2O5 NSPs as the first nanoceramics to provide effective radiosensitisation on radioresistant tumor cells. In this recent study (results described in section 6.2), T a2O5 NSPs were shown to be non-toxic to 9L cells and yielded an SER of 1.33 with a 10 MV X-ray beam [233]. We postulated that this effective enhancement at MV energies could be attributed to secondary electrons produced through photoelectric interactions and pair production, with scattering on NSP aggregates leading to increased RBE. We expected that these NSP aggregates would be less effective at kVp energies due to the absorption of the short range secondary electrons produced by the comparatively larger aggregates. Higher concentrations were also predicted to be less effective owing to the increase in aggregation and, therefore aggregate size associated with higher concentrations of nanoparticles. To investigate these hypotheses, experiments were performed at kVp Radiosensitisation Experiments using T a2O5 NSPs 118 energies, as well as MV energies, at different concentrations utilising the original aggregative T a2O5 NSP (TNSP), as well as a new T a2O5 NSP with reduced ten- dency for aggregation (PNSP). The biological and physical mechanisms underlying changes in these factors were additionally examined, with respect to their effect on radiosensitisation.

The main interactions of concern between the incoming photon X-ray beam and the target NSPs leading to dose enhancement include: the photoelectric effect, Comp- ton scattering and pair production, with each process more prominent depending on the incident beam energy. At 10 MeV energies, the photoelectric effect is less likely than the other processes, only occuring approximately 18% of the time [219], and the secondary electrons produced are consequently high energy in nature, with low LET. LET is a measure of the average energy deposited locally to the absorbing medium dE per unit length ( dx ) for a charged particle along its track [144], and is particularly important to consider for this study as particles with high LET represent preferable increases in the RBE of the local radiation environment. At keV energies, the photo- electric effect is the dominating interaction process, with the highest cross-sectional probability, and not only results in the production of photoelectrons, but can also lead to Auger cascades. These Auger electrons have an LET of 10-25 keV/µm [234], representing high LET particles, which are particularly desirable in DERT applica- tions. In the context of this study, short range particles such as these will be unable to penetrate NSP aggregates, and will only contribute to dose enhancement if pro- duced in close proximity to the cell, unimpeded by any aggregates. Compton scat- tering is significant at both MeV and keV energies, however because this process has no Z-dependence it does not largely contribute to high-Z enhancement. Because pair production has a minimum threshold photon energy of 1.022 MeV, the process is non-existent at keV energies, and has a cross section that increases with energy above the threshold. The dominance of this process at MeV energies means the sec- ondary electrons produced are high energy in nature, therefore having low LET of approximately 0.2 keV/µm for kinetic energies of 1-10 MeV [235]. Consequently, Radiosensitisation Experiments using T a2O5 NSPs 119 the high range of these secondary electrons means they are able to penetrate several micron-thick layers of NSP aggregates before damaging the cell.

This work presents an in-depth analysis into the dependencies of radiosensitisation on beam energy, particle morphology and particle concentration for T a2O5 NSPs. The 9L gliosarcoma rat-brain cell line was chosen for all experiments due to its known aggressive and radioresistant characteristics [218], under the premise that if radiosensitisation may be proven in the most radioresistant tumors, this will consti- tute the minimum therapeutic capabilities of the NSP, which can only be more effec- tive in less radioresistant tumors. Analysis of multiple beam energies is essential with MV energies signifying not only the majority of beam treatments, but also the most relevant area of the energy spectrum to treat the majority of cancers. The kVp range is of particular interest, since this represents the most intensely researched energy in DERT and leads to the production of high LET electrons creating more damage. The 150 kVp beam was chosen for both its clinical relevance and to maximise the dose enhancement to the NSP doped medium relative to a water equivalent medium, based on the formalism described by Corde et al. [159]. The 150 kVp beam produces an ef- fective energy of 65 keV, which lies close to, yet above the effective energy required to maximise dose enhancement in T a2O5 (33 keV). Investigation into kVp energies also lays the foundations for future experimentation with emerging cancer treatment modalities using synchrotron radiation. The synchrotron allows for the production of a monochromatic keV radiation beam accurately tuned to the optimal energy for absorption in the material with high dose rate, having the potential to drastically in- crease radiosensitisation. Additionally, investigation into the effect of increasing NP concentration on SER is again important, since it is widely thought that this relation- ship should increase the SER, however Butterworth et al. has shown that depending on the cell line, increasing the concentration of the NP may increase, decrease or, in fact, not change the SER [21], although no mechanism for explaining this effect was suggested. Two morphologically different phases of T a2O5 NSPs were prepared by thermal oxidation- referred to as TNSPs- and precipitation- referred to as PNSPs- Radiosensitisation Experiments using T a2O5 NSPs 120 reactions. We were able to demonstrate significant radiosensitisation at 150 kVp and 10 MV for the optimised version of the T a2O5 NSPs (PNSPs), culminating in a maximum SER of 1.46. Finally, the effect of increasing the NP concentration proved statistically insignificant on DERT at both beam energies and NSP types. Interest- ingly, this lack of enhancement was attributed to a phenomenon not yet observed in DERT, and explained by the formation of layers by the NSP, which may be more or less efficient in cell damage under an X-ray field depending on their thickness, for a given concentration of NSPs.

6.3.2 Methods

6.3.2.1 Cellular irradiation with NSPs

All radiation experiments were carried out at the radiation oncology department at the Prince of Wales Hospital (Randwick, NSW, Australia). Beam energies of 10 MV and 150 kVp were investigated using an Elekta Axesse™ LINAC (Elekta AB, Kung- stensgatan, Stockholm, Sweden) and a Nucletron Oldelft Therapax DXT 300 Series 3 Orthovoltage unit (Nucletron B.V., Veenendaal, The Netherlands), respectively. The 9L cell culture was brought to confluence prior to irradiation and exposed to the tan- talum pentoxide NSPs for 24 hours at concentrations of 50 and 500 µg/ml. For MV experiments, 9L cells were irradiated in T12.5 cm2 flasks (BD Falcon™) completely filled with HBSS so no air bubbles were present inside the flask to ensure electronic equilibrium conditions were present at the depth of the cells. For kVp experiments, cells were contained and irradiated in 6 mm of medium to maximise the accuracy in delivering the prescribed dose to the cell monolayer. All doses (1, 2, 3, 5 & 8 Gy) were delivered in single fractions at room temperature with dose rates of 5 and 0.7 Gy/min for 10 MV and 150 kVp X-ray photon beam energies, respectively. Tis- sue culture flasks were irradiated horizontally surrounded by a 30x30x10 cm3 solid water phantom to ensure backscattering effects were taken into account and human physiological conditions accurately modelled, with the cell monolayer situated at a source-surface distance (SSD) of 100 cm and 50 cm for 10 MV and 150 kVp beam energies, respectively. Unirradiated control samples (with and without tantalum pen- Radiosensitisation Experiments using T a2O5 NSPs 121 toxide NSPs) were utilised and handled under the same conditions as the irradiated samples.

6.3.2.2 Cell survival analysis

Survival curves were fit according to the LQM using the associated error bars of each point as weighting factors. The LQM [156] describes cell SF mathematically as a function of absorbed dose (D).

2 SF (D) = e−αD−βD (6.2)

The parameters α (Gy−1) and β (Gy−2) are indicative of cell radiosensitivity and repair effectiveness respectively. We were able to extrapolate these values based on the fit for each survival curve and compare experiments (with and without the NSP) to quantify the degree of dose enhancement by calculation of the SER (the ratio of doses giving 10% surviving fraction on the cell survival curves).

6.3.2.3 Sensitisation enhancement ratio

We measured the degree of dose enhancement by means of the SER, defined as the ratio of doses giving 10% surviving fraction on the cell survival curves. Given dose (D) as a function of surviving fraction (SF) on two curves, namely the control curve with no NSP (Dc(SF)) and the curve containing the NSP (Dx(SF)), the SER can be mathematically expressed as:

D (0.1) SER = c (6.3) Dx(0.1)

6.3.2.4 Statistical Analysis

Cell survival experiments were repeated three times to ensure reliability and mea- sured in triplicate for each sample. All values are expressed as the mean of 3 mea- surements with the experimental uncertainty given as one standard deviation. The Radiosensitisation Experiments using T a2O5 NSPs 122

LQ model was fitted to cell survival data using KaleidaGraph software. The fit of the data was weighted according to the error for each dose in the determination of the radiobiological constants, α and β. Statistical analysis of surviving fraction data was performed using a two-tailed Student’s t-test under the assumption of equal variance. A P value less than, or equal to, 0.05 was considered statistically significant.

6.3.3 Results

6.3.3.1 The dependency of radiosensitisation on particle morphology, NSP con- centration and beam energy for TNSPs and PNSPs on 9L cells in X-ray fields

Radiation experiments were performed using the clonogenic assay technique to as- sess the influence of particle morphology, NSP concentration and beam energy on sensitisation of 9L cells by TNSPs and PNSPs, with resultant survival curves (Fig- ures 6.2, 6.3, 6.4 and 6.5) fit according to the LQM.

Alpha and beta parameters were extrapolated based on SF data, and SERs calcu- lated based on comparison of 10% survival doses (Table 6.2). In all cases, cells containing either of the NSPs demonstrated lower SFs in comparison to the control, indicative of greater induced damage to the cells. Comparing results for each NSP, we see the PNSP exhibits significant sensitisation, far greater than that of the TNSP, for irradiation at 150 kVp and both concentrations examined (50 µg/ml, p=0.0274 and 500 µg/ml, p=0.0126), suggesting a morphological difference in the PNSPs gives them superior sensitisation ability at kVp beam energies. Similarly, we ob- serve greater sensitisation by the PNSP at 10 MV for both concentrations tested, however the difference in SERs between the two NSPs proves to be statistically in- significant (50 µg/ml, p=0.6274 and 500 µg/ml, p=0.3531). At 10 MV, the effects of differing morphology, attributed to the TNSPs and PNSPs, on cell damage become less pronounced due to the long range (centimetres) of secondary Compton scattered electrons, as well as electrons and positrons originating from pair production, that are produced. These long range, low LET (0.2 keV/µm) particles produce uniform dose enhancement across the cell culture independent of NSP distribution, but rather Radiosensitisation Experiments using T a2O5 NSPs 123

Figure 6.2 Cell survival curves for 9L cell cultures irradiated with 150 kVp X-rays show- ing the dose modifying response of tantalum pentoxide TNSPs at concentrations of 50 and 500 µg/ml. dependent on the total NSP concentration.

Analysing the effect of NSP concentration on sensitisation, we see insignificant in- creases in the SERs for the TNSP of approximately 3% at both beam energies tested (150 kVp, p=0.5655 and 10 MV, p=0.2879). The PNSP exhibits a slightly higher in- crease of 8 % at both beam energies used (150 kVp, p=0.2017 and 10 MV, p=0.1551).

Finally, we observe significant beam energy dependence of sensitisation for the TNSPs, with approximate 25 % increases in cell death from 150 kVp to 10 MV at both concentrations tested (50 µg/ml, p=0.0274 and 500 µg/ml, p=0.063). In contrast, the PNSPs exhibit a weaker dependence with an approximate increase of 3 % from Radiosensitisation Experiments using T a2O5 NSPs 124

Figure 6.3 Cell survival curves for 9L cell cultures irradiated with 150 kVp X-rays show- ing the dose modifying response of tantalum pentoxide PNSPs at concentrations of 50 and 500 µg/ml.

150 kVp to 10 MV at both concentrations tested (50 µg/ml, p=0.6274 and 500 µg/ml, p=0.6585). This again supports our hypothesis that morphological differences in the two NSPs give rise to varying degrees of sensitisation for any given beam energy and concentration. Evidently, the results imply that the PNSPs are superior radiosensitis- ers to their TNSP counterparts, not only in terms of the magnitude of sensitisation, but also in consistency of delivering effective radiosensitisation across varying beam energies and nanocrystallite concentrations.

6.3.4 Discussion

Significant radiosensitisation was observed in the majority of radiation experiments indicated by increased cell death of treated cells compared to their respective un- treated control cells, with the exception of 150 kVp irradiation of TNSPs on 9L cells. Radiosensitisation Experiments using T a2O5 NSPs 125

Figure 6.4 Cell survival curves for 9L cell cultures irradiated with 10 MV X-rays show- ing the dose modifying response of tantalum pentoxide TNSPs at concentrations of 50 and 500 µg/ml.

A lack of enhancement is observed at this beam energy (SER50=1.07, p=0.0561 and

SER500=1.10, p=0.1318) due to the existence of large TNSP aggregates, shown to be as large as 400 microns through HR-TEM imaging. At 150 kVp, the photoelectric ef- fect and Compton scattering will dominate the majority of photon beam interactions, producing photoelectrons, Auger cascades and Compton scattered electrons. The av- erage energy of these photoelectrons, as well as the Compton scattered electrons, is of the order of 50 keV or less, giving them a maximum range of 10 microns. On the other hand, the average energy of Auger electrons is much less, in the order of 10 eV, making them very high LET particles with a much smaller range of a few nanome- tres [223]. Evidently, the range of these secondary electrons is much less than the size of the aggregates themselves, proving that the majority are absorbed by the NSP aggregates before depositing their energy to cells, with only minimal contributions Radiosensitisation Experiments using T a2O5 NSPs 126

Figure 6.5 Cell survival curves for 9L cell cultures irradiated with 10 MV X-rays show- ing the dose modifying response of tantalum pentoxide PNSPs at concentrations of 50 and 500 µg/ml. to enhancement provided by interactions with atoms on the surface of the aggregates.

In contrast, irradiation of PNSPs on 9L cells at the same beam energy (150 kVp) re- veals significant sensitisation (SER50=1.33, p=0.0147 and SER500=1.43, p=0.0113) due to morphological differences in the two NSPs. HR-TEM and XRD established that the PNSP crystallites are of spherical nature and smaller size (45.5 nm) compared to the TNSPs, both favourable characteritics for increased internalisation. Further- more, FACS flow cytometry confirmed the existence of higher uptake of PNSPs in 9L cells. The ability to be more effectively internalised means a higher proportion of the PNSPs are localised within the cell, making these particles more lethal. High LET secondary electrons with very small range, such as Auger electrons, as well as ROS Radiosensitisation Experiments using T a2O5 NSPs 127

Table 6.2 Alpha, beta and SER for the TNSPs and PNSPs at 0, 50 and 500 µg/ml, as well as 150 kVp and 10 MV. Errors are given ± 1 standard deviation from the mean.

−1 −2 Beam Energy and Type of T a2O5 NSP Concentration(µg/ml) α(Gy ) β(Gy ) SER0,SER50,SER500 150 kVp TNSP 0 0.30 ± 0.04 N/A 1 50 0.32 ± 0.05 N/A 1.07 ± 0.03 500 0.33 ± 0.02 N/A 1.10 ± 0.07 150 kVp PNSP 0 0.30 ± 0.04 N/A 1 50 0.40 ± 0.01 N/A 1.33 ± 0.07 500 0.43 ± 0.01 N/A 1.43 ± 0.08 10 MV TNSP 0 0.19 ± 0.05 0.012 ± 0.008 1 50 0.38 ± 0.03 N/A 1.33 ± 0.07 500 0.40 ± 0.03 N/A 1.40 ± 0.07 10 MV PNSP 0 0.19 ± 0.05 0.012 ± 0.008 1 50 0.39 ± 0.03 N/A 1.36 ± 0.07 500 0.43 ± 0.03 N/A 1.46 ± 0.07 are able to deposit their energy before absorption by the bulk material, delivering single and double-strand breaks to the DNA of the cell. In addition to size and shape, HR-TEM images show the PNSPs to exist with far less aggregation (maximum size of 100 microns) and increased uniformity. Though the most interesting aspect of these NSPs is shown through the confocal microscope images, which demonstrate that both NSPs show a specificity to the cells, forming clusters around the cellular membrane and nucleus. Furthermore, this effect is exasperated at higher concentra- tions (500 µg/ml). We establish that the NSPs are congregating around the cells in concentric layers, effectively forming a shell around the cell. The inner layers of this shell generate low energy photoelectrons and Auger electrons, as well as ROS, that create a high LET radiation environment within the shell and lead to effective overkill of the encapsulated cell [236]. The outer layers absorb any secondary electrons orig- inating from within the outer layers themselves since the range of these electrons is less than the thickness of the shell. However, significant localised dose enhancement is induced in cells surrounding the shell through lateral scattering of electrons. Using the “Local Effect Model” for analysis [237], and considering a cubic volume of in- teraction surrounding each shelled cell, we see that electrons originating from beam interactions with the media are backscattered off the shell creating a sharp dose peak at the surface of the shell that drops off quickly with increasing distance from the Radiosensitisation Experiments using T a2O5 NSPs 128 shell. Photoelectons resulting from interactions with the shell may also be forward scattered, creating a similar dose distribution that maximises at the shell surface and drops off quickly towards the edge of the volume of interest. Formation of these shell structures results in a global reduction in the number of individual cells surrounded by the NSP (non-uniform distribution), as well as an increase in the distance between adjacent shell structures. At 150 kVp, the dose tails are quite small, leading to signif- icant enhancement with the PNSP, owing to the existence of smaller more dispersed shells in comparison to the TNSP, since a higher portion of cells are exposed to lo- calised scattering effects.

The difference in SERs for each NP concentration exhibits an unexpected trend, con- trary to the literature, where by we note no statistically significant change in the SERs with increasing concentration. Butterworth et al. [21] established that the degree of radiosensitisation increases with increasing concentration of Au NPs on T98G and AGO-1522B cell lines. The expected increased enhancement is based on the princi- ple that higher concentrations of the NP will increase the number of NPs surrounding 9L cells, there by generating a higher number of secondary electrons and ROS, cul- minating in increased damage to the cells. Given the large increase in NP concentra- tion (factor of 10) we would expect a large change in the SER; especially given the 50 % increase in the DEF observed for a factor of 10 increase in Au NP concentration (10 µg/ml to 100 µg/ml) on T98G cells [21]. The lack of additional enhancement at higher NSP concentrations is a direct consequence of the “shell effect”, where by the existence of shells creates a non-uniform distribution of the NSPs among the cells, and increasing the NSP concentration only increases the thickness of the NSP shells, not the number of cells encapsulated by shells. At 150 kVp, the short dose tails result in similar enhancement for both concentrations on both NSPs. Slight increases in en- hancement are due to the reduced distance between adjacent shell structures, owing to the increase in shell thickness at higher concentrations. Radiosensitisation Experiments using T a2O5 NSPs 129

We see large increases again in radiosensitisation for irradiation of NSPs on 9L cells with 10 MV X-rays. The increase in SER for the TNSPs between 150 kVp and 10 MV is massive, with the high energy part of the spectrum primarily responsible for the improvement in enhancement. The TNSP aggregates still exist here absorb- ing the lower energy part of the spectrum, however higher energy photoelectrons, Compton scattered electrons and pair-produced electrons also exist here, with parti- cle energies of a few MeVs travelling cms in the bulk material before being absorbed. These long range particles are able to deposit their energy in the cells, however while traversing the bulk material a significant portion will undergo interactions with the NSP aggregate atoms, losing energy and eventually becoming low energy particles with high LET just prior to energy deposition in the cell, giving them enhanced dam- aging ability. This increase in particle lethality contributes to the linearity observed in the semi-log survival curve. Additionally, scattered electrons originating from in- teractions with the primary beam exhibit an energy dependent spectrum, where by effective backscattering off TNSP aggregates leads to dose enhancement at the NSP surface propagating radially outwards leading to a “dose enhancement tail” [233]. These enhancement mechanisms combine to achieve the significant amount of sen- sitisation observed at 50 µg/ml.

Irradiation of PNSPs at 10 MV provides the greatest radiosensitisation of all parame- ters tested (SER500=1.46, p=0.0076), however similar in magnitude to the TNSPs at this beam energy. This is essentially due to the production of high energy particles, limited not only to photoelectrons (scattered into shelled cells or out of the cells) and Compton scattered electrons, but also pair-produced electrons and positrons [219]. The enhancement effect provided by these particles will be independent of the NSP type, due to the long range associated with their energies, and is uniform throughout the cell culture. Electrons resulting from the low energy region of the photon energy spectrum scattered from the shell will induce overkill damage to the cells, as a result of their high LET [236]. Furthermore, forward and backscattered electrons create ra- dial dose tails, that are a maximum at the shell surface and fall off quickly extending Radiosensitisation Experiments using T a2O5 NSPs 130 out. These effects combine to produce similar enhancement in the two NSP types, with slightly increased enhancement observed in the PNSPs due to their more dis- persed morphology, and more efficient backscattered tail effect. Again, increasing the concentration to 500 µg/ml leads to an insignificant change in SER as a result of the “shell effect”, where by the shell thickness increases, leading to similar size dose tails as the 50 µg/ml case.

The idea of NSPs forming clusters, or shells, and its effect on the radiosensitisation of radioresistant cells in radiobiological experiments is entirely novel, however, the influence of NP clustering has been considered before in previous Monte Carlo stud- ies. Zygmanski et al. [238] investigated the effects of NP clustering and changes in particle morhpology on the dose enhancement ratio (DER) for Au NPs in a wa- ter phantom. They were able to show that clusters of NPs produced higher DERs over a greater radial distance (several microns) than single Au NPs (approximately 100 nm), which correlates well with the phenomena observed in this study, and the establishment of a shielding effect, despite the obvious differences in experimental set up. Furthermore, a new study has emerged that considers the important contri- bution to dose enhancement from scattered photoelectrons of intermediate energies, rather than just low energy high LET Auger electrons, while also considering the distribution of NPs in the cells, and that the DNA of the nucleus does not need to be the sole target of radiation in achieving effective cell death. This particular study analyses the effects of Au NPs on MDA-MB-231 human breast cancer cells using radiobiological assays and GEANT4 studies, and uses the LEM as well as a bio- physical model, to achieve good agreement between simulated and radiobiological results [239], highlighting the need to use models such as these in DERT, and the importance of considering the distribution of the NPs in the cells.

There have been Monte Carlo studies published recently, as part of a collaboration in our TNT group at UOW, that use the GEANT4 package to simulate dose enhance- Radiosensitisation Experiments using T a2O5 NSPs 131

ment to cells for the case of T a2O5 NSPs, there by supporting the radiobiological results presented in this investigation. The 100 nm size T a2O5 NSPs were shown to provide significant enhancement in the 5 to 50 MeV energy range for proton ther- apy [240]. Furthermore, in a separate study, T a2O5 NSPs were shown to provide significant enhancement in microbeam radiation therapy. They observe an energy dependent-sensitisation in the 70-100 keV energy range for synchrotron-produced microbeams and confirm effective sensitisation when the cells are surrounded by shells or clusters of NSPs [241]. Finally, a simulation was performed that investi- gated the effect of T a2O5 NSP distribution in cells on the dose enhancement pro- duced in a 150 kVp X-ray radiation environment [236]. This particular experiment complements the kVp section of the results presented in this chapter well as it essen- tially represents a direct Monte Carlo comparison of our radiobiological data. The 5 µm NSP shells were simulated, and the dependence of radiosensitisation on par- ticle concentration and morphology confirmed. The lack of enhancement at higher concentrations due to NSP shell formation confirms the hypotheses, results and ex- planations of the radiobiological data presented in this thesis work.

6.3.5 Conclusion

This particular investigation highlights the multiple factors influencing radiosensiti- sation, and how they may be optimised to maximise the effect with non-toxic ceramic

T a2O5 NSPs. The PNSPs were synthesised with spherical shape, smaller particle size and less tendency for aggregation compared to the TNSPs, and yielded a higher SER than the TNSPs at all concentrations and beam energies tested. For both NSPs and all beam energies, the SER demonstrated insignifcant changes in value for vary- ing NSP concentration. However, enhancement was impeded by a shielding mecha- nism referred to as the “shell effect”. This effect involves the attraction of the NSPs towards the 9L cells resulting in the formation of layered shells of NSPs around in- dividual cells. The natural affinity of numerous NSPs to be drawn to a particular cell results in a global reduction in the number of cells surrounded by the NSP, and an in- crease in the distance between adjacent shell structures surrounding their respective Radiosensitisation Experiments using T a2O5 NSPs 132 cells. Finally, enhancement increased with beam energy, exhibiting a proportional relationship contrary to trends observed in the literature for other NPs, owing to the existence of aggregates in the NSPs. A maximum SER of 1.46 was achieved with irradiation of 500 µg/ml PNSPs on 9L cells at a beam energy of 10 MV. This work represents a comparative compilation of the many dependencies dose enhancement relies on, and how they may be optimised for maximum effectiveness. Future experi- ments will look at more sophisticated production techniques for further optimisation of NSP morphology, while looking at possible functionalisation and combination with other molecules, such as MTX for increased cell death and PEG for improved biocompatibility, eventually culminating in in vivo experimentation. Monte Carlo studies have been performed in collaboration with this work to examine the physical mechanisms underlying the dose enhancement observed here, including investigation into the unexpected shielding mechanism, known as the “shell effect”. These have confirmed many of the findings presented in this chapter, in particular the impor- tance of the NSP distribution in determining radiosensitisation, and the dependence on NSP morphology and concentration for kVp X-rays. Optimisation of the NSP concentration will be investigated and different cell lines will be probed to look for a differential effect that may further improve DERT. Chapter 7

UV Protection Experiments using T a2O5 Nanostructured Particles

This chapter continues on the theme of the thesis by investigating radiation appli- cations related to T a2O5 NSPs, however in this case the NSPs are assessed as a radiation protection material. This chapter presents the UV protection properties as- sociated with T a2O5 NSPs, and evaluates their applicability to the cosmetics and sunscreen protection industries. For the experiments described in this chapter, only

T a2O5 PNSPs are examined, i.e., without any reference to the TNSP, therefore, they are referred to with the general term T a2O5 NSPs. This particular investigation is a unique part of this work in that measurements of a second entirely different NP are included. ZnO NP measurements are included in all experiments, provided with thanks by another member of the TNT team Dean Cardillo, for comparative purposes. The ZnO NPs utilised in this study are commercially bought and used in current sun- screens as UV absorbers, are well researched and characterised in the literature, and consequently, represent the perfect benchmark for comparing with the UV protective properties of T a2O5 NSPs. This study covers physical and biological characterisa- tion of both NPs, using non-cancerous MDCK cells as a surrogate for normal tissue, and culminates in sun protection factor (SPF) analysis for the purpose of quantifying the UV protection benefits of both NPs.

133 UV Protection Experiments using T a2O5 Nanostructured Particles 134

The idea and motivation for applying T a2O5 NSPs to the field of UV protection was stimulated during routine physical characterisation measurements. Optical mea- surements, in the form of UV-vis spectrophotometry were performed as part of NSP characterisation for DERT applications, when significant photon absorption in the UV wavelengths was observed. This result, along with the inherent biocompatibility and low toxicity (confirmed as part of biological characterisation) of T a2O5 NSPs sparked interest in application to UV protection. There is no published literature on the application of T a2O5 NSPs to UV protection in cosmetics, sunscreens or textiles, establishing the novelty of the results presented here.

7.1 Introduction

Australia has one of the highest rates of skin cancer in the world, with skin cancer representing 80% of all diagnosed cancers each year, and 2 in 3 people will be diag- nosed with skin cancer by the age of 70 [242]. Skin cancers are primarily formed as a result of the biological interaction between UV (ultraviolet) radiation (100-400 nm) and the skin. UV radiation can be separated into 3 primary bands or wavelength ranges, namely UVC (100-290 nm), UVB (290-320 nm) and UVA (320-400 nm). Fortunately, molecular interactions between UV radiation and the ozone layer allow the majority of UVC and some UVB radiation to be absorbed before reaching the surface of the Earth. For this reason, sunscreens are designed to protect against both UVA and UVB radiation effects, and this is commonly implemented through the in- corporation of inorganic UV fillers. While many fillers can be used, two of the most common nanoparticle (NP) UV fillers are ZnO and TiO2, which are preferred due to their strong UV absorption characteristics, photostability and transparency in sun- screen [243] [244]. Other NPs currently being researched for their UV protection applications in sunscreen include CeO2 [245] and hematite [246], while ZnO [247],

MgO and Al2O3 [248], are all also used for UV protection in the textiles industry. UV Protection Experiments using T a2O5 Nanostructured Particles 135

It is well established that ZnO and TiO2 NPs are effective UV attenuators, however, they have also been shown to exhibit significant cytotoxicity, in particular genotoxic- ity, as well as phototoxicity [208] [249] [250]. This is chiefly due to reactive oxygen species (ROS) production, which is extremely undesirable in UV protection appli- cations, as this in itself leads to tissue damage and possible carcinogenesis. As a result, the need for safer materials with low toxicity and high UV absorbance has arisen in the sunscreen and cosmetics industries, which given the potential seen in various NPs, has sparked interest in investigation of new NPs. Tantalum pentoxide nanostructured particles (T a2O5 NSPs) have not yet been investigated in any UV protection applications, although have shown significant promise in the areas of dose enhancement radiotherapy [233], bone substitution [116] and capacitance [251]. The properties that have made them successful in these fields are applicable to use in UV protection applications, including low cytotoxicity, biocompatibility and signifi- cantly large band gap energy of 3.8 eV [205]. While T a2O5 NSPs have been shown to significantly increase the production of secondary electrons and ROS in X-ray radiation fields for the purpose of dose enhancement in radiotherapy [233], in this investigation, it would be advantageous for there to be minimal ROS production for application to UV protection. The difference here will be that excitation should oc- cur through UV and visible wavelengths, rather than X-ray wavelengths of radiation. Experiments will be performed to assess the intrinsic, or visible light-induced, ROS production capacity of the T a2O5 NSPs, however one limitation of the investigation will be that UV-stimulated ROS production cannot be evaluated. This will form the basis of future experimentation, although should be taken into account when inter- preting the results presented.

In this study, we assess the UV protection properties associated with T a2O5 NSPs and offer a comparison of the same measurements performed on commercial-grade ZnO NPs, similar to those currently used in sunscreen applications and research. Characterisation of the NPs was performed using XRD and HRTEM, while MDCK cells, were used in cytotoxicity experiments and ROS assessment. Finally, UV-vis UV Protection Experiments using T a2O5 Nanostructured Particles 136 spectroscopy was employed to quantify the UV absorbance of each sample, and the in vitro SPF method was applied for evaluation of UV protective efficacy.

7.2 Methods

7.2.1 T a2O5 NSP and ZnO NP production

Tantalum pentoxide NSPs were synthesised by a precipitation-based chemical reac- tion utilising T a(OEt)5(Sigma-Alrich 99.99%) and an ethoxide decomposition re- action with water. This method is described in detail in section 3.2, as the synthesis method for PNSPs. Furthermore, drying, sterilisation, suspension and sonication of the NSPs was carried out according to the methods described in sections 3.2 and 3.3.

ZnO NPs were purchased from a commercial supplier (Symrise) and characterised to ensure they were of adequate quality and similarity to those currently incorporated in commercial sunscreens, textiles and associated research.

7.2.2 Cell line and culture

Cellular experiments employed a culture of Madin-Darby Canine Kidney (MDCK) cells derived from normal kidney cortex cells of a cocker spaniel [172] [173]. The cells were maintained in a T75cm2 flask with completed Dulbecco’s Modified Ea- gle Medium (c-DMEM from GIBCO, supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin and Streptomycin) and incubated at 37° C and 5% (v/v)

CO2. MDCK cells are an adherent, epithelial, well-characterised cell line, known for their stable clonogen production and well established as an in vitro cell model for assessment of drug absorption, metabolism and toxicology [252]. UV Protection Experiments using T a2O5 Nanostructured Particles 137

7.2.3 X-ray Diffraction

Characterisation of the NPs involved the use of X-ray diffraction (XRD) for deter- mination of phase, structure, particle size and unit cell parameters. Samples of both NPs were independently placed in a GBC MMA X-ray diffraction system using Cu K-alpha radiation with wavelength λ = 1.5418 A,˚ accelerating voltage 40 kVp and cathode current 30 mA; in order to determine information regarding crystal structure and mean particle size (t). Mean crystallite size (t) was determined through utilisa- tion of Scherrer’s Equation (7.1) [190], where k=0.89 is a geometric shape constant, B is the full-width at half-maximum (FWHM) of the most intense primary phase diffraction peak, and θ is the Bragg diffraction angle in radians.

kλ t = (7.1) Bcosθ

The agglomeration number (N) was also calculated as a relative indicator of each

NP’s tendency for aggregation, where R is the radius of the NP, NA is Avagadro’s number, ρ is the density of the NP material, and M is the molecular weight of the NP [253].

4 ρ N = πR3N (7.2) 3 A M

7.2.4 High-Resolution Transmission Electron Microscopy

Morphological parameters- crystallinity, size, agglomeration and shape- were inves- tigated using a JEOL (2011) 200 kVp High-Resolution Transmission Electron Mi- croscope (HRTEM).

7.2.5 Clonogenic cell survival assay

Clonogenic assays [165] were used to assess cell survival and completed with MDCK cells plated in triplicate for a control (no NP) and 50 µg/ml concentration of T a2O5 UV Protection Experiments using T a2O5 Nanostructured Particles 138

NSPs and ZnO NPs. This was performed according to the method described in sec- tion 3.6.

7.2.6 Toxicity assessment

To assess the cytotoxicity of T a2O5 NSPs and ZnO NPs to MDCK cells, NP con- centrations of 0 and 50 µg/ml were exposed to MDCK cells for a period of 24 hours prior to plating. MDCK cells were brought to 90 % confluence inside T12.5 cm2

flasks (BD Falcon™) before exposure to the NP. Solutions of T a2O5 NSPs and ZnO

NPs were prepared according to the “T a2O5 NSP and ZnO NP production” protocol, and the appropriate volumes corresponding to each examinable concentration added to the cells. Once the desired exposure time had been reached, cells were plated in triplicate according to the “Clonogenic cell survival assay” technique described in section 3.6. Following plate staining and colony counting, surviving fractions were obtained for each NP at each concentration, and normalised to the control group (0 µg/ml).

7.2.7 Flow cytometry analysis

Quantification of the change in ROS levels due to the presence of the NP in MDCK cells was performed using a Becton-Dickinson fluorescence-activated cell sorting (FACS) flow cytometer (BD LSR II; BD Biosciences, Franklin Lakes, USA), in as- sociation with the oxidation-sensitive compound 2’-7’-dichlorofluorescein diacetate

(DCF) [254]. MDCK cells were brought to 90% confluence and exposed to T a2O5 NSPs and ZnO NPs at concentrations of 0, 50 µg/ml for 24 hours. Trypsinisation was used to detach the cells before being placed in a centrifuge at 1500 rpm and 4° C for 5 minutes and rinsing twice with cold PBS (without calcium and magne- sium). 10 µM DCF solution was added to the cell suspension, and allowed to sit under dark conditions for 30 minutes. Each analysed sample contained a minimum of 10,000 cells where cell doublets and aggregates were gated out using a two pa- rameter histogram of FL2-Area versus FL2-Height. A negative control sample was used initially as a reference for voltage adjustment to attain an optimal distribution UV Protection Experiments using T a2O5 Nanostructured Particles 139 of forward and side scatter intensities. Data analysis was performed using the FACS- Diva software, assessing forward scatter (FSC), side scatter (SSC) and counts vs. 515/20 nm intensities. Excitation and detection photon wavelengths of 480 nm and 515 nm were employed respectively. The mean fluorescence intensity (MFI) values were extracted using the software and normalised to the control values (without the NP) to yield relative fluorescence [255].

7.2.8 UV-visible absorption spectroscopy

Assessment of the UV absorption properties of T a2O5 NSPs and ZnO NPs was per- formed using a Shimadzu UV-3600 UV-vis spectrophotometer in the wavelength range of 200-800 nm and quartz cuvettes (1ml) with path length `=1 cm. NP samples were dispersed in ethanol with concentration c=0.05 g/L, and underwent 60 minutes of sonication in an ultrasonic water bath to ensure maximum dispersion of the NPs in the sample and reduce aggregation. Immediately prior to measurement, samples were mixed in a vortex to ensure the mixture was homogeneous. Absorbance (A in a.u) data was collected for each NP, and converted to the absorption coefficient (α in cm−1) with equation 7.3, where ρ is the density of the NP sample.

2.303 ∗ 103 ∗ A ∗ ρ α = (7.3) ` ∗ c

The absorbance data was used to calculate the transmittance (T) according to the following relation:

A = −log(T ) (7.4)

The Beer-Lambert Law was used to calculate the extinction coefficient ( in Lg−1cm−1) in equation 7.5.

A = c` (7.5) UV Protection Experiments using T a2O5 Nanostructured Particles 140

Finally, the band gap energy (Eg in eV) for direct transitions (n = 2) was determined for each material using the following equation 7.6:

2 (αhν) = B(hν − Eg) (7.6)

In this equation, hν represents the incident photon energy and B is a constant depen- dent on the intrinsic properties of the material [205]. Plotting (αhν)2 as a function of hν yields a sigmoidal curve, and by extrapolating the linear segment of the curve to the x-axis the direct transition Eg can be obtained for each material.

7.2.9 SPF analysis

The in vitro Sun Protection Factor (SPF), or Ultraviolet Protection Factor (UPF) as it is also referred to, was calculated according to the equation developed by Diffey and Robson [256].

R 400E(λ)S(λ) dλ. SPF = 290 (7.7) R 400 290 E(λ)S(λ)T (λ) dλ.

Note that the SPF equation integrates over the UVA and UVB ranges, so that it rep- resents a quantitative indicator of the protective ability of the NP over the entire effective UV range. The effectiveness of the materials in the specific UV bands was evaluated using the UVA PF and UVB PF.

R 400E(λ)S(λ) dλ. UV AP F = 320 (7.8) R 400 320 E(λ)S(λ)T (λ) dλ.

R 320E(λ)S(λ) dλ. UVBPF = 290 (7.9) R 320 290 E(λ)S(λ)T (λ) dλ.

In these equations, E(λ) represents the relative erythemal spectral effectiveness de- fined by the International Commission on Illumination (CIE) [257], S(λ) is the solar UV Protection Experiments using T a2O5 Nanostructured Particles 141 spectral irradiance (in W m−2nm−1), and T(λ) is the spectral transmittance of the material. The UVA/UVB ratio (UVA/UVB) was used as an indicator of the broad-spectrum UV protective ability of each NP. It is the ratio of the mean absorbances in the UVA (320-400 nm) and UVB (290-320 nm) wavelength ranges [258], where A(λ) is the absorbance.

R 400 320 A(λ) dλ. R 400 320 dλ. UV A/UV B = R 320 (7.10) 290 A(λ) dλ. R 320 290 dλ.

Finally, the critical wavelength (λc) was used as another measure of broad-spectrum protective ability, using the integral of the absorbance spectrum over the entire UV wavelength range [259].

Z λc Z 400 A(λ) dλ. = 0.9 ∗ A(λ) dλ. (7.11) 290 290

7.3 Results and Discussion

7.3.1 Characterisation of T a2O5 NSPs and ZnO NPs

Samples of T a2O5 NSPs and ZnO NPs were analysed using XRD to determine the nature and size of their crystal structure, with the resultant diffraction patterns shown in Figure 7.1.

Both spectral patterns contain well-defined peaks, indicative of highly crystalline nanoscale materials. Comparison of the peak positions and intensities with the Joint

Committee on Powder Diffraction Standard (JCPDS) database revealed the T a2O5 NSPs to be hexagonal delta phase (JCPDS: 19-1299) while the ZnO NPs were typical of the wurtzite hexagonal close-packed (HCP) phase structure (JCPDS: 36-1451). The Miller indices were derived for each peak, based on their structural identification, UV Protection Experiments using T a2O5 Nanostructured Particles 142

Figure 7.1 XRD patterns illustrating various peaks of reflection, and their corresponding Miller indices, for T a2O5 and ZnO NPs.

and for the T a2O5 pattern, we note the lack of doublet formation. Doublet formation, particularly in the case of the (1,1,0) peak, is indicative of orthorhombic formation [191], which is known to occur for annealing temperatures greater than 750° C [26].

Since the T a2O5 NSPs were annealed to 700° C, we would expect them to remain in the hexagonal phase, which is confirmed by the presence of singular defined peaks in the diffraction pattern measured. Peak broadening can be observed in the ZnO NP diffraction peaks, and is especially significant when compared to the pattern for

T a2O5 NSPs. This is typically a particle size effect, known to become increasingly pronounced as crystallite size decreases [260], and is indicative that the ZnO NPs are smaller in size compared to the T a2O5 NSPs, a notion confirmed by calculation of the mean crystallite size (data shown in Table 7.1).

The XRD pattern, along with the derived lattice parameters, crystal structure and JCPDS identification number for the commercially purchased ZnO NPs match those UV Protection Experiments using T a2O5 Nanostructured Particles 143

Table 7.1 Comparison of crystal structure and agglomeration parameters for ZnO and T a2O5 NSPs.

Type of NP Size (nm) a (A)˚ c (A)˚ Phase Structure Card Number Agglomeration Number ZnO 14 ± 2 3.25 5.21 Wurtzite (HCP) JCPDS:36-1451 (6.0 ± 0.9)∗104

5 T a2O5 PNSP 46 ± 7 3.62 3.88 Hexagonal Delta JCPDS:19-1299 (5.5 ± 0.8)∗10

used in several published studies involving industrial and cosmetic applications for commercially available ZnO NPs [261] [262] [245], establishing the nature and qual- ity of the ZnO NPs utilised in this study.

The agglomeration numbers calculated for each NP establish that the T a2O5 NSPs have an agglomeration number an order of magnitude higher than the ZnO NPs, in- dicative of their increased tendency for agglomeration. The degree of agglomeration and crystallite size is further examined in HRTEM analysis (Figure 7.2).

The HRTEM images produced show the distribution of T a2O5 NSPs and ZnO NPs, noting the exhibition of fringes, indicative of maxima and minima regions of in- teraction. Both NPs display a narrow particle size distribution, good crystallinity, and crystallite sizes in agreement with those derived from XRD (approximately

15 nm and 50 nm for T a2O5 NSPs and ZnO NPs respectively). The small discrep- ancy in particle measurements, and slightly smaller values obtained in XRD anal- ysis, can be attributed to the differences in the respective criteria for measurement in each method. HRTEM size calculations are based on visible grain boundaries, while XRD uses planar reflections in the crystalline region to determine size, which carries tighter restrictions and results in smaller particle sizes relative to the first method [263]. The particle sizes used in this study, particularly for ZnO, are com- monly utilised in several UV protection studies and commercially available products. A size dependent relationship with UV absorbance has been shown for ZnO NPs, with the most effective range noted to be 14-40 nm [264], validating the choice of particle sizes used in this study. UV Protection Experiments using T a2O5 Nanostructured Particles 144

Figure 7.2 HRTEM images of a) T a2O5 NSPs and b) ZnO NPs, displaying the surface morphology of each substance.

Both NPs show some tendency for agglomeration, with T a2O5 NSPs more predis- posed to larger agglomerates, correlating with agglomeration number calculations above. Furthermore, both NPs are spherical in shape, however it has been shown for ZnO NPs that changing the shape of the NPs (rods, spherical, petals) has a neg- ligible effect on antibacterial and UV-blocking properties [265]. Finally, both NPs contain clearly defined particle boundaries that are clearly touching, establishing the crystallites are in physical contact with one another.

7.3.2 Cytotoxicity of T a2O5 NSPs and ZnO NPs to MDCK cells

Cytotoxicity of the NPs to non-cancerous MDCK cells was explored in vitro, as an indicator of response when applied to the human body in vivo, where a cytotoxic re- UV Protection Experiments using T a2O5 Nanostructured Particles 145 sponse of normal tissues to the NPs is undesirable. Exposure period of 24 hours and NP concentration of 50 µg/ml were both chosen based on typical literary compar- ative studies and commercial sunscreens utilising ZnO NPs. Results are presented graphically in Figure 7.3, where T a2O5 NSPs and ZnO NPs display toxicities of 9 % and 44 % respectively.

Figure 7.3 MDCK cell viability, or surviving fraction, following 24 hours exposure to 0, 50 µg/ml T a2O5 NSPs and ZnO NPs. SFs are normalised to the control (0 µg/ml) and errors given ± 1 standard deviation from the mean.

These results suggest T a2O5 NSPs are non-toxic to MDCK cells, supporting their claim for potential use in sunscreen applications. Other published studies exist cor- roborating this result, namely the application of T a2O5 NSPs as radiosensitisers to glioblastoma cells, where the same exposure time was used, and similar toxicity re- ported at 50 µg/ml, however low toxicity was reported at concentrations as high as 500 µg/ml [233]. Another study reported tantalum oxide NPs to be non-toxic at con- centrations as high as 2.4 mg/ml, an extreme concentration, both in vivo and in vitro, UV Protection Experiments using T a2O5 Nanostructured Particles 146 in rat and mammalian cells for image contrast applications in CT [266].

The high toxicity reported here for ZnO NPs correlates well with toxicities observed in the literature [267], and the general concern shown for the commercial use of ZnO NPs as UV protectors in sunscreens, given their controversial cytotoxic tendencies. A study compared the cytotoxicity of several commercial ZnO-based sunscreens to human keratinocytes over a range of exposure periods and NP concentrations, and found for 24 hours exposure and 40 µg/ml the toxicity to be approximately 50 %, however at 80 µg/ml the toxicity increased drastically to over 90%, noting possible cytostatic activity [245]. There are various possible mechanisms for cytotoxicity, although literature suggests ROS-induced oxidative stress is the likely cause of cell death. This has been demonstrated for several organisms, and the most harmful nor- mal tissue effects have been observed in the 4-20 nm size range. Although Zn2+ ion dissociation leading to cellular membrane stress is noted as another possible cytotoxic mechanism, ROS-induced stress has proven to be the primary contribu- tor [268]. Taccola et al. showed similar high toxicities for ZnO NPs prepared in a similar fashion to this study [269] and Demir et al. established the presence genotox- icity for ZnO NPs in both human and mammalian cell cultures [249], highlighting the inherent risk of carcinogenesis. Therefore, it seems apparent that ZnO NPs dis- play significant cytotoxic properties to multiple cell lines and organisms, correlating well with the results presented in this study for morphologically similar commercial ZnO NPs examined under similar experimental conditions.

7.3.3 ROS-generative capacity of T a2O5 NSPs and ZnO NPs in MDCK cells

The productive capacity of T a2O5 NSPs and ZnO NPs for generating ROS in MDCK cells was evaluated using the MFI of oxidation-sensitive DCF. Cells were exposed for 24 hours to each NP, at concentrations of 0 and 50 µg/ml. The MFI was normalised to the control to yield relative fluorescence, depicted graphically in Figure 7.4. UV Protection Experiments using T a2O5 Nanostructured Particles 147

Figure 7.4 Relative fluorescence of DCF in MDCK cells, following 24 hours exposure to 0, 50 µg/ml T a2O5 NSPs and ZnO NPs. Values are normalised to the control (0 µg/ml) and errors given ± 1 standard deviation from the mean.

Exposure of the cells to T a2O5 NSPs at 50 µg/ml resulted in an insignificant change in relative fluorescence of 14%, indicating that the NSPs are weak ROS generators. This could be expected based on the inherent biocompatibility of these NSPs in the literature, and the low cytotoxicity reported in this study. As evident for ZnO NPs, high cytotoxicities can sometimes be an indicator of ROS being produced, leading to cell death.

Exposure to ZnO NPs at 50 µg/ml showed a significant change in relative fluores- cence of 250%, indicative that ZnO NPs are strong ROS generators, especially rel- ative to T a2O5 NSPs, which correlates with current trends being reported in the literature. It is interesting to note that while the ZnO NPs contain less O atoms per molecule than the T a2O5 NSPs, they exhibit greater toxicity. While this corre- UV Protection Experiments using T a2O5 Nanostructured Particles 148 lates with expected trends in the literature as discussed, the exact difference in the mechanisms of ROS production are not clear. One possible explanation may lie in the band gap energy, which for ZnO NPs is approximately 3 eV, within the visible light spectrum, and less than the band gap energy of T a2O5 NSPs, which is 3.2 eV (see section Table 7.2), lying within the UV spectrum. The lower band gap energy of ZnO NPs may explain the increased production of electrons, as they are excited to the conduction band in the presence of visible light, contributing to ROS production and oxidative stress in the cells. Specific FACS flow cytometry measurements using DCF as an indicator for the presence of ROS generated from ZnO NPs are lacking [270], despite widespread consensus of their oxidative potential (generally through other means), although this particular method has been verified in the case of silica NPs with similar size and morphology to the NPs used in this study. Exposure times of 3 and 24 hours were employed with NP concentrations of 25-200 µg/ml, establishing time and concentration dependencies of ROS generation, with a maximum increase in relative fluorescence of 320% for 24 hours exposure and 200 µg/ml [271]. Con- sidering the evidence for ROS generation in commercial ZnO NPs, Taccola et al. has used flow cytometry to measure intracellular ROS generation in mesenchymal stem cells, showing significant ROS production and resultant cytotoxicity with ZnO NPs at concentrations as low as 15 µg/ml, although noted the existence of a significant increase in ROS production at a threshold NP concentration of 20 µg/ml [269].

It has been established that the commercial ZnO NPs utilised in this study exhibit significant intrinsic ROS generative potential, much greater than the T a2O5 NSPs, resulting in high NP-induced cytotoxicity, however it is important to distinguish be- tween intrinsic NP produced ROS and UV-stimulated ROS, which lead to phototox- icity. Both are undesirable in sunscreen applications, and although phototoxicity is not explored specifically in this study, it is relevant to note that ZnO NPs have also exhibited significant UV-induced phototoxicity [208]. UV Protection Experiments using T a2O5 Nanostructured Particles 149

7.3.4 UV absorption properties of T a2O5 NSPs and ZnO NPs

In analysing the UV protection potential of a material, it is important to first consider not only the wavelength range the material most effectively absorbs in, but where this lies within the UV spectrum. The UV section of the electromagnetic spectrum is comprised of UVA (320-400 nm), UVB (290-320 nm) and UVC (100-290 nm) bands of radiation. Fortunately, UVC and a significant portion of UVB radiation is filtered by the ozone layer before reaching the lower atmosphere. Consequently, strong ab- sorption in the UVA and UVB ranges are of particular importance in UV protector materials.

UV-vis absorption spectra were acquired for each NP in the range 200-800 nm, with results shown graphically in Figure 7.5.

The absorption curve for T a2O5 NSPs displays significant absorption, especially when compared to the ZnO NP curve (approximately 10 times more intense in the UV peak), over the entire UV range. It has a defined maximum absorption peak around the lower limit of the UVB range (269 nm) that continuously decreases across the UVA and visible wavelengths, correlating well with spectra observed in the lit- erature [205]. The absorption curve for ZnO NPs is significantly less intense than the curve for T a2O5 NSPs, exhibiting a flatter maximum peak (241 nm) in the UVA and UVB regions, largely arising from exciton interactions, before decreasing in the visible region. The results shown here correlate well with ZnO NPs of simi- lar morphology (14 nm) found in the literature exhibiting similar curve shapes and trends [263] [208]. Interestingly, absorption has been shown to depend on particle size, increasing between 15 and 40 nm. Furthermore, inherent particle absorption increases up to 70 nm, where the NPs become opaque to UV radiation. As particle size increases past 70 nm, absorption decreases again due to a reduction in particle density [264]. UV Protection Experiments using T a2O5 Nanostructured Particles 150

Figure 7.5 UV-vis spectra showing the absorption coefficient as a function of wavelength for ZnO NPs and T a2O5 NSPs. The wavelength of maximum absorption for each sample is also shown.

Examination of the transmittance curve for each NP (Figure 7.6) shows that T a2O5 NSPs transmit significantly less UV radiation than their ZnO counterparts, confirm- ing what was observed with the absorbance curves.

T a2O5 NSPs transmit approximately 10 and 7 times less radiation in the UVB and UVAranges respectively compared to ZnO NPs. This is a very significant result since ZnO NPs are generally considered strong broad spectrum protectors, with their high UVA/UVB ratio, and more commonly incorporated into commercial sunscreens as primary UVA attenuators, in conjunction with T iO2 NPs as UVB attenuators [208].

These results suggests T a2O5 NSPs could serve as viable substitutes for these NPs, offering superior UV absorption properties. UV Protection Experiments using T a2O5 Nanostructured Particles 151

Figure 7.6 UV-vis transmission spectra for ZnO NPs and T a2O5 NSPs.

The band gap energy for direct transitions was determined for each NP by extrapola- tion of the curves, shown in Figure 7.7, to the x-axis.

The energy gap for each material, along with the wavelength of maximum UV ab- sorption and associated extinction coefficient, are summarised in Table 7.2.

Table 7.2 The wavelength of maximum absorption, extinction coefficient and energy gap parameters for ZnO and T a2O5 NSPs.

−1 −1 Type of NP λmax (nm)  (Lg cm ) Eg (eV) ZnO 241 ± 3 2.39 ± 0.03 3.08 ± 0.05

T a2O5 PNSP 269 ± 3 15.3 ± 0.2 3.2 ± 0.05

The experimentally determined energy gap for T a2O5 NSPs was 3.2 eV. There is UV Protection Experiments using T a2O5 Nanostructured Particles 152

Figure 7.7 Plot of (αhν)2 as a function of hν for determination of the band gap energy for ZnO NPs and T a2O5 NSPs.

considerable controversy regarding the true energy gap value for T a2O5, known to be as high as 4.2 eV for the bulk material, generally increasing as the material be- comes more amorphous due to structural disorder and lattice defects [272]. Consid- ering structural differences, the energy gap for bulk orthorhombic beta phase T a2O5 is approximately 3.8 eV, the same as its NP form [205]. The bulk value for hexago- nal delta phase T a2O5 is 3 eV [272], and assuming the NP form is again similar, this correlates well with the experimentally determined value found for the hexagonal

T a2O5 NSPs utilised in this study.

The energy gap value for ZnO NPs was found to be 3.08 eV, correlating well with values in the literature approximating 3.2 eV, although depending on size and struc- ture, can be as high as 3.4 eV [264]. It is also noted that the zinc-blende bulk material exhibits a slightly higher energy gap than the wurtzite structure [208]. UV Protection Experiments using T a2O5 Nanostructured Particles 153

The band gap values determined here correlate well with past studies, and both lie within the useful UV range of absorption. The slightly higher value for T a2O5 NSPs represents a blue shift towards shorter wavelengths in the UV range relative to ZnO, making it a more useful UV attenuator.

7.3.5 SPF analysis

The SPF obtained for ZnO NPs (1.28, all values tabulated in Table 7.3) was slightly lower than expected, with values of 2-3 expected based on the literature [246], al- though values can vary depending on the emulsion type used, concentration of NPs in the sample, and NP morphology. Despite this, for comparative purposes, the SPF for ZnO NPs is within the range we would expect, and satisfies expectations for other UV protection indicators. The similar values obtained for the UVA and UVB PFs is typical of ZnO NPs, resulting in their UVA/UVB ratio being near unity, correlating with the value calculated in this study (0.954).

Table 7.3 The in vitro SPF, UV protection factors, UVA:UVB ratio and critical wavelength parameters for ZnO and T a2O5 NSPs.

Type of NP SPF UVAPF UVBPF UVA/UVB λc (nm) ZnO 1.28 1.28 1.28 0.954 375 ± 1

T a2O5 PNSP 12.12 8.56 11.52 0.791 386 ± 1

The SPF found for T a2O5 NSPs (12.12) is significantly higher than that found for commercial-grade ZnO NPs using the same testing methods, as well as those found for the UVA and UVB PFs- a very significant and novel result given that there exists no published literary study measuring these factors for T a2O5 NSPs. A study by Truffault et al. measured the SPF and UVA PFs for several types of NPs applica- ble to sunscreen applications and yielded values of 9.07 and 8.69 for hematite, 3.33 and 3.04 for ZnO, and 10.55 and 4.88 for TiO2 NPs [246]. Comparing these val- UV Protection Experiments using T a2O5 Nanostructured Particles 154

ues to the experimentally determined values for T a2O5 NSPs in this study (12.12,

8.56 and 11.52 for SPF, UVA and UVB PFs respectively), we see that T a2O5 NSPs offer significant UV protection capabilities, much higher than many NPs currently being researched, and used in commercial sunscreen applications as UV fillers. The European Commission, along with other international governing organisations, have dictated that the UVA PF must be at least 1/3 of the overall SPF, which has been subsequently adopted by the Australian and New Zealand Standards for sunscreens

(AS/NZS 2604:2012) [273]. Not only do the results for T a2O5 NSPs meet this ex- pectation (as do ZnO NPs), but also similar expectations set for the UVA/UVB ratio and critical wavelength. According to the Boots star rating system (a 5 star sys- tem), the UVA/UVB ratio is separated into classes with ZnO NPs ranked highly with class 5, and T a2O5 NSPs ranked moderately at class 3 [258]. A more effective measure though of broad-spectrum protection is the critical wavelength, which if ex- ceeds 370 nm suggests the material is a broad-spectrum protector with significant UVA absorption capability, as first proposed by the European Commission [258]. As expected ZnO NPs satisfy the criterion with a critical wavelength of 375 nm, how- ever T a2O5 NSPs with a value of 386 nm actually exceed that of their counterparts. This is logical, and actually a more meaningful result when trying to interpret broad- spectrum protection since this indicator takes into account the intensity of absorption at all wavelengths, where as the UVA/UVB ratio only measures the relative intensity in one UV band compared to the other.

The in vitro SPF testing method is a validated method for SPF measurement hav- ing been used extensively in UV protection studies for both sunscreen [274] and textile [247] applications. It is listed as a potential component for sunscreen pro- tection validation in international standard documents from organisations, including the European Commission, and more notably, in the Australian regulatory guidelines for sunscreens [275], although usually carries more stringent criterion for analysis. There also exist studies comparing the effectiveness of the in vitro and in vivo SPF methods directly on a variety of sunscreens, containing ZnO and TiO2 NPs among UV Protection Experiments using T a2O5 Nanostructured Particles 155 other ingredients [276], concluding that the in vitro method is a convenient and con- sistent measure of SPF, however the in vivo method does yield more accurate results compared to the claimed SPF value and replicates true conditions for sunscreen ap- plication more fully [277]. Considering this, we can be sure that the results presented here are valid and accurate, however future experiments will involve testing T a2O5 NSPs with different emulsions, and in conjunction with different sunscreens, while also including in vivo SPF measurements to further confirm the efficacy of these NPs.

7.4 Conclusion

This study reports on the UV protection-related properties on commercial-grade ZnO

NPs and novel T a2O5 NSPs for sunscreen applications. There currently exists no published data on the application of T a2O5 NSPs to UV protection, especially in the form of SPF measurements. The materials were initially characterised using XRD and HRTEM, establishing the nanoscale morphology of each material, and that the ZnO NPs utilised in this study were morphologically similar to those used in previ- ous studies, and currently in use in UV-protective sunscreen applications. Toxicity testing performed on MDCK cells using the clonogenic assay technique revealed that the T a2O5 NSPs had insignificant toxicity, while the ZnO NPs had significant cyto- toxicity, up to 44%, as expected from the literature. ROS testing using DCF revealed that the T a2O5 NSPs were weak ROS-generators, with an insignificant change in MFI, while the ZnO NPs were strong ROS generators with a 250% increase in MFI.

UV-vis experiments revealed that T a2O5 NSPs had significant absorbance in the UV range, a factor of 10 more intense than ZnO NPs, with an energy gap of 3.2 eV, simi- lar to that of ZnO (3.08 eV), but slightly higher and in the effective UV range. In vitro

SPF measurements showed that T a2O5 NSPs had an SPF of 12.12, UVA PF of 8.56 and UVB PF of 11.52, all significantly higher than ZnO NPs (1.28 for all factors). Although the UVA/UVB ratio was strongest for ZnO as a known broad-spectrum protector, the critical wavelength for T a2O5 NSPs (386 nm) was higher than that for ZnO NPs (375 nm), while both satisfied the minimum 370 nm criterion set by the UV Protection Experiments using T a2O5 Nanostructured Particles 156

European Commission for broad-spectrum UV protection. The authors note that al- though the in vitro SPF measurements are well-established, reliable, consistent and accurate methods for SPF determination, they are still unable to perfectly replicate application in reality in the same way that in vivo measurements are able to, and this will form part of future experimentation. Chapter 8

Conclusions and Future Work

This thesis work examines the application of a new material to radiation medicine, and in particular, nanomedicine. The proposed ceramic material, T a2O5 NSPs, is produced, characterised and investigated for applications to DERT, UV protection and IGRT. The literature review presented in chapter 2 highlighted the fact that

T a2O5 NSPs had not yet been investigated for the applications explored in this study, establishing the novelty and significance of the results reported by this study. Based on the scope of this work, and the review conducted, the objectives and experimental aims detailed in chapter 1 were formulated and successfully addressed throughout the study.

Chapter 3 describes the synthesis methods for each of the NSPs tested in this work, including T a2O5 TNSPs and PNSPs. The cell lines used for biological and radiobi- ological testing- including 9L, MDCK and MCF-7- were characterised, and general experimental methods described. This satisfied the material production/suspension and cellular characterisation experimental aims in section 1.2.2.

Chapter 4 focuses on the physical characterisation of T a2O5 TNSPs and PNSPs, us- ing a vast array of equipment and methodologies. XRD established that the mean crystallite size of the TNSPs was greater than the PNSPs. SEM and HRTEM pro-

157 Conclusions and Future Work 158 vided morphological information about the NSPs, revealing the PNSPs to be much more dispersed than the TNSPs, with smaller aggregate sizes. DLS provided infor- mation about the mean particle size for each NSP type, and showed that the TNSPs contain a much broader spectrum of particle sizes, in comparison to the PNSPs, as measured by the PDI, likely due to the aggressive aggregation of the TNSP and ir- regular particle shapes. The final UV-vis experiment established that the PNSPs pos- sessed strong absorption characteristics, particularly in the UV region, where maxi- mum absorption was noted at 269 nm. This was the stimulus for the UV protection investigation described in chapter 7.

Chapter 5 details biological studies of the interactions of T a2O5 NSPs with in vitro cell lines, primarily cancerous radioresistant 9L gliosarcoma cells. Toxicity studies showed that the NSPs displayed insignificant toxicity over long periods of time (up to 14 days). Both NSP types were also shown to present minimal toxicity over the clin- ically and experimentally relevant time of 24 hours, up to a maximum concentration of 500 µg/ml. FACS flow cytometry measurements established that the NSPs were significantly internalised by both 9L and MDCK cells, up to 300% at 500 µg/ml, and that the effect was NSP concentration-dependent. Confocal microscopy images revealed the presence of NSPs encapsulated by endosomes, and that both NSP types demonstrated a specificity to the cells, forming distributions of concentric shells. The thickness of these shells increased at higher NSP concentrations, and the PNSP shells were much more dispersed than the TNSP shells, consisting of aggregates much smaller in size. The results described in this chapter, along with chapter 4, formed the basis for interpretation of the results described in chapter 6 (radiation experi- ments). Of particular importance was the observation of NSP clustering, specificity and shell formation around 9L cells.

Chapter 6 focuses on the radiosensitising applications of T a2O5 NSPs in DERT. Sig- nificant sensitisation of radioresistant 9L cells was achieved for both NSPs, at both Conclusions and Future Work 159

150 kVp and 10 MV X-ray beam energies. A maximum SER of 1.46 was measured for the PNSPs at 500 µg/ml in a 10 MV beam. The smaller particle size and more dis- persed nature of the PNSPs compared to the TNSPs led to overall higher SERs for all beam energies and concentrations tested, indicative of preferential particle morphol- ogy for DERT. While beam energy-dependent sensitisation was observed, the degree of radiosensitisation was found to be independent of NSP concentration (statistically insignificant differences observed for both NSPs at both beam energies). This result was attributed to the shell effect, where by the NSPs have an affinity to the 9L cells forming concentric shells, the thickness of which depends on the NSP concentration applied. Increasing the concentration was not observed to increase the distribution of the NSP in the sample, nor the proportion of cells exposed to secondary electrons, but merely to increase the thickness of the shells, resulting in similar SERs at 500 µg/ml compared to the lower 50 µg/ml NSP concentration. These results correlated well with published Monte Carlo simulation studies carried out in collaboration with this study.

Chapter 7 carries on from the UV-vis experiment conducted in chapter 4 to evalu- ate the application of T a2O5 PNSPs to UV protection, by comparing them to com- mercially available ZnO NPs. Toxicity assessments of both NPs to non-cancerous

MDCK cells at 50 µg/ml showed that T a2O5 PNSPs are non-toxic, in accordance with previous 9L experiments in chapter 5, while the ZnO NPs exhibit significant toxicity to MDCK cells, unfavourable for UV protection applications. Flow cytome- try performed with DCF established that the ZnO NPs were strong ROS-generators while the T a2O5 PNSPs were weak in comparison, a factor of 2.5 less in relative flu- orescence. UV absorption measurements showed that the T a2O5 PNSPs exhibited much higher absorption over the entire UV-vis spectral range relative to ZnO NPs.

The in vitro SPF factor of 12.12 calculated for T a2O5 PNSPs was significantly larger than that of the Zno NPs (1.28), indicative of much greater capacity for UV protec- tion. While ZnO NPs are well known as broad-spectrum UV protectors, the critical wavelength and UVA:UVB ratio were used to establish that the T a2O5 PNSPs may Conclusions and Future Work 160 also be considered broad-spectrum protectors, making them novel candidates for UV protection applications.

Inspection of the results presented demonstrates that all aims and objectives outlined in chapter 1 were met in this thesis work. While the results and theories discussed are sound and well supported, there are still some aspects that would benefit from fur- ther investigation for completeness. FACS flow cytometry measurements were com- pleted for PNSPs on MDCK cells, however unlike the 9L results, the experiments were not carried out for TNSPs because of time and resource limitations. Confocal microscopy experiments were performed on 9L cells, however not for MDCK cells, which would be an interesting investigation to see whether the same NSP affinity and shell clustering is observed for a different non-cancerous cell line. Any differences observed would support the notion that these effects are cell-specific, and may indi- cate a preferential radiobiological specificity of the NSPs to some types of cancerous cells, beneficial for clinical radiotherapy applications. Finally, UV-vis spectroscopy was performed for PNSPs, but not TNSPs, and this information would be useful for comparative purposes to observe how the UV protective ability may depend on mor- phological NSP features, such as particle size and aggregation.

Future work for this project would be highly flexible, based on the numerous prop- erties and potential applications of T a2O5 NSPs, and could follow the avenues of radiosensitisation, UV protection or IGRT. In radiosensitisation, radiation experi- ments could be carried out at even higher NSP concentrations to see if the shell effect changes nature or if sensitisation is improved. Much smaller crystallite sizes, in the range of 1-10 nm could be manufactured and tested, similar to the sizes used for Au NPs. PEG-coated particles could be considered for experimentation to see if aggregation and biocompatibility can be improved, especially for smaller NPs, which are more likely to aggregate and pose toxicity concerns. Radiosensitisation results would benefit from experiments performed on additional cell lines, including Conclusions and Future Work 161 radiosensitive cancerous cells (MCF-7) and non-cancerous cells (MDCK), to deter- mine whether the degree of radiosensitisation changes as it should, or if preferential sensitisation of cancerous cells is observed relative to non-cancerous cells. The ulti- mate aim would be to progress to in vivo experimentation on rats to gauge how the NSP interacts in a physiological environment, whether toxicity changes, and if the degree of radiosensitisation differs from in vitro results.

Considering the field of UV protection, different NSP morphologies could be studied to assess the effect on UV absorption and the SPF factor. Experiments could be per- formed on mixtures of the material with cream, or inclusion with fabrics, as is used in the sunscreen and textiles industries. Finally, determination of the in vivo SPF would be ideal to confirm the significant protective ability of the material, and would allow direct comparisons to be made to other commercial products in a physiological context.

Investigation of T a2O5 NSPs to IGRT would be a novel route to take in establishing the theranostic properties of this material. Being a high-Z material with strong ab- sorption characteristics, it is likely to offer excellent contrast in both CT and planar kV scans. This is especially relevant clinically, as if the NSP was to be used ther- apeutically, and possessed a high HU number to give good soft tissue contrast, the NSP could be injected during CT simulation for patient treatment to assist the radia- tion oncologists in contouring the planned target volume (PTV). Furthermore, since it is common practice to perform planar kV or CBCT images on linear accelerators prior to patient treatment to confirm setup position, the localisation of the NSP in the tumour would make matching of the treatment image to the reference (planned) CT much more accurate. There is much potential for experimentation here, including characterisation of the imaging properties of the NSP, as well as in vitro and in vivo experimentation. Conclusions and Future Work 162

While there are many possibilities for further experimentation here, this is only made possible by the foundational experiments outlined in this thesis work. The novelty and significance of the results presented is undeniable, especially when compared to the current literature. The potential for T a2O5 NSPs to be applied to various fields of radiotherapy and radiation protection makes it an exciting prospect for future study. Bibliography

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