The Effect of Magnetic Fields and Magnetic Nanoparticles on Cell Viability and Neurite Outgrowth of SH-SY5Y Neuroblastoma Cells

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The Effect of Magnetic Fields and Magnetic Nanoparticles on Cell Viability and Neurite Outgrowth of SH-SY5Y neuroblastoma cells

Enad Abed Mahmood Alabed

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Recommended Citation Alabed, Enad Abed Mahmood, The Effect of Magnetic Fields and Magnetic Nanoparticles on Cell Viability and Neurite Outgrowth of SH-SY5Y neuroblastoma cells, thesis, School of Biological Sciences, University of Wollongong, 2018. https://ro.uow.edu.au/theses1/279

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The Effect of Magnetic Fields and Magnetic Nanoparticles on Cell Viability and Neurite Outgrowth of SH-SY5Y neuroblastoma cells

Enad Abed Mahmood Alabed

This thesis is presented as part of the requirements for the conferral of the degree:

Master of Philosophy in Biological Sciences

Supervisors Dr. Lezanne Ooi Dr. Md Shahriar Hossain Prof. Jung Ho Kim A/Prof Konstantin Konstantinov

University of Wollongong School of Biological Sciences March 2018

This work is © copyright by Enad Abed Mahmood Alabed, 2018. All Rights Reserved. No part of this work may be reproduced, stored in a retrieval system, transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of the author or the University of Wollongong.

Ethical and Biosafety issues The biosafety and ethical issues considered in this research follow the instructions for working with hazardous chemical guidelines contained in the laboratory disposal guidelines, the biosafety manual, Illawarra health and medical research institute (IHMRI) induction handbook, the Australian institute of innovative materials (AIIM) induction handbook, the operating manual of facilities, the working after hours work guidelines in IHMRI and AIIM, as well as risk assessment and completing biosafety and genetic modified organism (GMO) quizzes.

Acknowledgments

First of all, I like to give thanks to Allah for giving me the power, health, and courage to complete my studies. Many thanks also to my supervisors Dr. Lezanne Ooi and Dr. Md Shahriar Hossain for their continuous support and encouragement. Special thanks to my Father and Mother for their lifelong support and encouragement, I shall always remember their assistance and loving kindness. Special thanks also to the love of my life, my wife, who has been beside me in all the challenging situations I have experienced. Many thanks also to my children; my sweetheart Soolaf, the lovely Abed Alrahman, superhero Mustafa, and cute Mohammed for their enthusiasm and consistent support. This also includes my brothers, sisters, relatives and friends who have been praying for success in this study for a master degree, despite their often difficult circumstances in Iraq. Many thanks also to my friends Kadhim, Nezar, and others who live in Australia for their encouragement and support. Finally, I wish to thank the Higher Committee for Education Development in Iraq (HCED Iraq) for financial support during this study.

Abstract

While there is an increasing interest in using electromagnetic fields and nanomaterials to boost neuronal differentiation and/or protect neurons from oxidative stress, little is known about effects of their cells in vitro. We investigated the effect of external magnetic fields (alternating current (AC) and direct current (DC) MF) on the neuronal viability, differentiation, and neurite outgrowth of SH-SY5Y neuroblastoma human cells in vitro. This study indicated that low frequency and fields with weak strength AC MF, and fields with high strength DC MF improved the efficiency of retinoic acid mediated neuronal differentiation without any adverse effects on neuronal viability; as shown by increased length of neurites in SH-SY5Y neuroblastoma cells. The cell viability has not changed after AC MF exposure, and it had no real adverse effect on cell confluency after DC MF exposure; in fact, even without retinoic acid, AC and DC MF promoted neurite outgrowth in low serum conditions. This study has therefore identified a simple and cost-effective method for differentiating SH-SY5Y cells without expensive reagents. We also describe a novel DC MF source that is simple to apply and very efficient, in fact our results suggest that this DC MF system is better than AC MF with regards to the viability and differentiation of SH-SY5Y cells. This research is a new way of promoting neurite outgrowth in a commonly used neuronal-like cell line model. We also tested two different nanomaterials to assess their ability to protect SH- SY5Y cells. We first tested the cytotoxic effect of oleic acid – coated and uncoated iron oxide (Fe3O4) NPs, and the cytotoxic impact of uncoated Yttrium oxide (Y2O3) NPs on the viability of neuroblastoma cells in vitro. The anti-oxidant impact of Yttrium oxide NPs was explored by checking whether or not it could reduce the oxidative stress induced by H2O2 in vitro on SH-SY5Y cells. This study also indicated that Yttrium oxide NPs could work as free radical scavengers to reduce the oxidative stress induced by hydrogen peroxide in SH-SY5Y cells in culture, however the protective effects were complex and depended on the concentration of nanoparticles and hydrogen peroxide used. This research provides a first step in understanding the effects that external magnetic fields and nanoparticles have on neuronal-like cells in culture. This combination of NPs guided by external magnetic field (AC or DC) should be considered in future research to exploit all the features of these nanomaterials and magnetic fields in neuronal differentiation and survival.

Contents

The Effect of Magnetic Fields and Magnetic Nanoparticles on Cell Viability and Neurite Outgrowth of SH-SY5Y neuroblastoma cells ...... I Declaration ...... III Ethical and Biosafety issues ...... IV Acknowledgments ...... V Abstract ...... VI Chapter 1 ...... 1 Introduction ...... 1 1.1 Electromagnetic fields (EMF) ...... 1 1.2 Effects of ELF-EMF on cells in vitro ...... 1 1.2.1 Neurite Outgrowth and Differentiation of various cell models after applying ELF- EMF ...... 1 1.2.2 The Differentiation of Human Mesenchymal Stem Cells on Graphene-Based Substrates is improved by Applying (ELF-EMF)...... 2 1.2.3 Short term and long term impact of (ELF-EMF) exposure on Alzheimer’s disease (AD) ...... 3 1.3 Magnetic nanoparticles ...... 4

1.3.1 Fe3O4 nanoparticles ...... 6 1.3.2 Size of MNPs ...... 8 1.3.3 Biomedical applications of MNPs ...... 8

1.3.4 Biomedical application of Fe3O4 NPs ...... 10 1.3.5 Neuronal Regeneration through Magnetic Nanoparticles...... 11 1.3.6 Cytotoxicity of iron oxide nanoparticles (IONPs) on Cell Lines ...... 12 1.4 SH-SY5Y cells ...... 14 1.5 Aims ...... 14 Chapter 2 ...... 15 SH-SY5Y cell culture ...... 15 The impact of AC and DC MF on Cell viability ...... 15 The cytotoxicity test of homemade and commercial MNPs ...... 15 The effect of AC and DC MF on Neuronal differentiation ...... 16 Magnetic Exposure System ...... 17 TEM images of the homemade and commercial MNPs ...... 18 Dynamic Light scattering test (DLS test) on homemade MNPs ...... 18 VII

Dynamic Light scattering test (DLS test) on commercial MNPs...... 19 Statistics ...... 19 Chapter 3 ...... 20 No significant effect of DC MF on cell viability ...... 20 Significant impact of AC EMF on cell viability ...... 20 The influence of combined DC / AC MF on cell viability ...... 21 The effects of AC and DC MF on neurite outgrowth ...... 22 Comparison of the effect of DC and AC MF on neurite outgrowth ...... 32 Chapter 4 ...... 33

The impact of homemade and commercial MNPs (Fe3O4 NPs) on cell viability ...... 33 TEM images of homemade and commercial MNPs ...... 33 DLS test of homemade and commercial MNPs ...... 35 Cytotoxicity assessment of both types of MNPs ...... 37 Homemade MNPs ...... 37 Commercial MNPs ...... 37 Chapter 5 ...... 39 Discussion ...... 39 5.1 AC and DC MF ...... 39

5.2 Fe3O4 NPs ...... 41 5.4 Future direction of the research ...... 43 5.5 Conclusion ...... 44 References ...... 45 Appendix (Preliminary Collaborative Study on application of Yttria ceramic nanoparticles for ROS scavenging) ...... 52 Introduction ...... 52

1.1 Yttrium oxide nanoparticles (Y2O3 NPs) ...... 52 1.2 Oxidative stress ...... 52 1.3 Detoxification of ROS ...... 53 1.4 Advanced Multifunctional Materials for site-specific free radical scavenging ...... 54 1.5 Aim ...... 55 2. Experimental works and design ...... 56 SH-SY5Y cell culture ...... 56

The cytotoxicity test of homemade Y2O3 NPs ...... 56

The cytotoxicity test of hydrogen peroxide (H2O2) ...... 56

VIII

Free radical scavenging test of Y2O3 NPs ...... 57 TEM images of the homemade and commercial MNPs ...... 57 Statistics ...... 57 3. Results ...... 58 The impact of antioxidant NPs on SH-SY5Y viability ...... 58

TEM images of homemade Yttrium oxide Y2O3 NPs: ...... 58

Cytotoxicity of Y2O3 NPs ...... 58

Cytotoxicity of H2O2 ...... 59

Free radical scavenging test of Y2O3 NPs ...... 60 4. Discussion ...... 62 5. Future direction of the research ...... 63 6. Conclusion ...... 64

List of figures Figure 1.1: (Guduru, 2013) shows advantages and disadvantages of using magnetic nanoparticles for various applications……………………………….……………………….…. 9 Figure 2.1: Experimental design and schematic diagram of the neurite outgrowth experiments……………………………………………….………………………….………….16 Figure 2.2: Magnetic exposure system holder designed using the Solid Material software….. 18 Figure 3.1: (A) Cell viability was assessed by AlamarBlue assay to investigate the impact of 100 mT of DC MF. (B) cell viability test was also carried out to investigate the impact of treating the cells with 1 mT, 100 Hz of AC MF for 4 hours/day over 3 days.……………………………………………………………...………………..…………… 21 Figure 3.2: (A) Cell viability was assessed by AlamarBlue assay to investigate the impact of both 100 mT of DC, and 1 mT, 100 Hz of AC for 2 and 4 hours on cell viability. (B) Cell viability was also tested by AlamarBlue assay to explore the effect of 100 mT of DC, and 1 mT, 100 Hz of AC for 30, 60 and 90 minutes on the cell viability.………………………….....….. 22 Figure 3.3: Impact of AC and DC MF treatment for 4 hours/day over 2 days on the neurite outgrowth of SH-SY5Y cells in the presence and absence of RA…………………………..… 25 Figure 3.4: IncuCyte images demonstrating the effects of treating SH-SY5Y cells for 4 hours/day over 2 days with AC and DC MF on neurite outgrowth in the presence and absence of RA……………………………………………………………………………………………… 27 Figure 3.5: The impact of AC and DC MF treatment for 4 hours / day for 3 days on the neurite outgrowth of SH-SY5Y cells in the presence and absence of RA…………………………..… 29 Figure 3.6: IncuCyte images illustrating the impact of treating SH-SY5Y cells for 4 hours / day over 3 days with DC and AC MF on neurite outgrowth in the presence and absence of RA..... 31 Figure 4.1: TEM image of homemade MNPs....……………………………………………… 34

Figure 4.2: (A – C) TEM images of the commercial Fe3O4 NPs. (D) particles size distribution of Fe3O4 NPs which indicate that the high ratio of Fe3O4 NPs is around 20 nm……..……...… 35 Figure 4.3: DLS test of homemade MNPs which indicate the particle size distribution in distilled water………………………………………………………………………………….. 36 Figure 4.4: DLS test of commercial MNPs which indicate the particle size distribution in ethanol absolute……………………………………………………………..…………………. 36 Figure 4.5: The cytotoxicity of serial dilution of homemade MNPs……………………….…. 37 Figure 4.6: The cytotoxicity of narrow range of commercial MNPs concentrations…………. 38 Figure 4.7: The cytotoxicity of wide range of commercial MNPs concentrations………….... 38

Figure A.1: The cytotoxicity of Y2O3 NPs on cell viability………………………………….. 59

Figure A.2: The cytotoxicity of H2O2 on cell viability………………………………………. 59 Figure A.3: The anti-oxidant impact of three different concentrations (5, 15, and 25 µg / ml) of

Y2O3 NPs for the oxidative stress induced by three different concentrations (0.025, 0.05, and

0.075 mM) of H2O2……………………………………………………………………………. 61

List of tables Table 1.1: (Polak and Shefi, 2015) The most important factors in the studies, classified on the basis of activity, kind of NPs, coating, use, type of activity, and toxicity are given below:…………………………………………………………………………………. 13 Table 3.1: Comparison between the fold change in neurite length of MF compared to control in the 4 hours / day for 2 days and 4 hours / day for 3 days conditions when RA is present and absent………………………………………………..…………………. 32 Table 3.2: Comparison between the fold change in neurite branch points of MF compared to control in the 4 hours / day for 2 days and 4 hours / day for 3 days conditions when RA is present and absent……………………………………………. 32

Chapter 1

Introduction

1.1 Electromagnetic fields (EMF) Globally, a question has arisen regarding the use of electromagnetic field (EMF) on brain development, but since avoiding exposure to extremely low-frequency electromagnetic field (ELF-EMF) is impossible because electricity is used everywhere, it is known as the extremely low frequency (ELF) spectrum because its frequency ranges from about 50/60 Hz. External magnetic fields are divided into three levels depending on their frequency, that is < 300 Hz, 300 Hz – 10 MHz, and 10 MHz – 300 GHz. Since ELF is less than 300 Hz, the range of intermediate frequency (IF) is between 300 Hz and 10 MHz, and the range of radiofrequency (RF) is between 10 MHz and 300 GHz (Wang and Zhang, 2017). Although it is almost impossible to avoid exposure to EMF in modern societies, its effect on neurogenesis and neuronal function remains unclear, which means that understanding the impact of EMF on neurons is an important task. Moreover, a major challenge within the field of cellular neuroscience is generating cells that faithfully recapitulate neuronal functions.

1.2 Effects of ELF-EMF on cells in vitro

1.2.1 Neurite Outgrowth and Differentiation of various cell models after applying ELF-EMF Very little is known about on how ELF-EMF exposure affects the embryonic neural stem cells (NSC) cellular division and proliferation, how ELF-EMF influences embryonic neurogenesis, or how hippocampal neurogenesis in adult mice be enhanced by exposure to ELF-EMF. The impact of ELF-EMF on neural stem cells NSCs has been researched by Ma and co-authors who exposed NSCs to ELF-EMF (50 Hz, 1mT) for 4 hours per day over 1, 2, and 3 days; they found that exposing cells to ELF-EMF increased the proliferation and maintenance of NSC (Ma et al., 2016). Moreover, after

1 exposure to ELF-EMF, the expression of proneural genes NeuroD and Ngn1 increased; they are important for neuronal differentiation and neurite outgrowth. The exposure of ELF-EMF for NSC has improved coupled intracellular Ca+2 by up-regulating the gene expression of TRPC1 (Ma et al., 2016). Eliminating this up-regulation of proneural genes and promoting neuronal differentiation and neurite outgrowth induced by ELF- EMF occurred when the TRPC1 expression was silenced (Ma et al., 2016). This result suggests that promoting neuronal differentiation coupled with the neurite outgrowth of NSCs via up-regulation, the expression of TRPC1 and proneural genes (NeuroD and Ngn1) occurs when they are exposed to ELF-EMF. These outcomes help us to understand how exposure to ELF-EMF affects the development of embryonic brains (Ma et al., 2016). Another important procedure in brain development is neurite outgrowth which includes the projection formation and maturation of neuronal fibre connections known as synapses (Ma et al., 2016). Moreover, ELF-EMF (50Hz, 1mT) increases the growth and division of cells in various cell models such as human neuroblastoma (IMR32) and rat pituitary (GH3) cells (Grassi et al., 2004), HL-60 leukemia cells, rat-1 fibroblasts and WI-38 diploid fibroblasts (Wolf et al., 2005), and also promote the cellular division of NSCs (Cuccurazzu et al., 2010, Sherafat et al., 2012).

It has been reported that pulsed EMF can also increase the growth of neurites with respect to the direction of EMF in different cell models. For instance, the average length of neurites of PC12 rat pheochromocytoma cells increased by ELF-EMF exposure in the direction of EMF (Zhang et al., 2006), while the neurite outgrowth of dorsal root ganglia (DRG) also increased in the direction of +EMF (Macias et al., 2000).

Blackman and co-workers tested the impact of an electric field and an AC magnetic field (AC MF) on the neurite outgrowth of PC-12D rat pheochromocytoma cells and found that whilst 0.2 - 115 μV/m of the electric field had no effect, 2.2 and 4 mT, 50 Hz of AC MF stimulated neurite outgrowth (Blackman et al., 1993b).

1.2.2 The Differentiation of Human Mesenchymal Stem Cells on Graphene-Based Substrates is improved by Applying (ELF-EMF). Functioning as a remarkable tool for the growth, differentiation, and fate conversion of cells, whereas graphene is a non-cytotoxic biocompatible substance whose properties enable it to be manipulated for regenerative medicine and tissue engineering as a 2 scaffold for biological tissues. Graphene has probable applications for nerve regeneration owing to the substrate support of neuronal differentiation of stem cells (Lee et al., 2015), but its application needs improvement because the mechanism for differentiation by graphene substrates is still unclear. The biological efficiency of neuronal differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs) grown on a graphene-coated substrate was improved synergistically when exposed to very low frequency electromagnetic fields (ELF-EMF; 50 Hz, 1mT). Lee and co-workers found that the expression for 170 genes changed considerably (fold change ≥ 1.4) with or without EMF when used with graphene substrate, compared to glass only control. They divided these genes into 4 groups depending on their function: (i) Neurogenesis; (ii) Differentiation; (iii) Extracellular matrix; (iv) cell migration. Each group contains several genes. We mentioned one gene from each group that changed considerably, for instance, NR4A2 (Nuclear receptor subfamily 4, group A, member 2) belongs to neurogenesis , EDNRB (Endothelin receptor type B) considers one of the differentiation group, CTSG (cathepsin G) belongs to the extracellular matrix group, and USP6 (dual specificity phosphatase 6) is considered as part of the cell migration group (Lee et al., 2015). Lee and co-authors demonstrated that changes in global gene expression profiles could enhance neurogenesis and result in an up-regulation of cell adhesion molecules; this requires an intracellular calcium influx and an activated focal adhesion kinase signaling pathway that is stimulated by producing extracellular matrix protein. These results could form the basis for a very useful therapeutic strategy in regenerative medicine (Lee et al., 2015).

1.2.3 Short term and long term impact of (ELF-EMF) exposure on Alzheimer’s disease (AD) EMF technology has undergone intensive development and utilisation in recent times so as a consequence, the health effects of EMF have also been the focus of research. Owing to their potential influence on the induction of Alzheimer’s disease (AD), very low-frequency electromagnetic fields (ELF-EMF) have also been studied with increasing interest. To study the interaction between ELF-EMF exposure and memory degeneracy in rats, Zhang and co-authors selected 20 healthy male Sprague-Dawley (SD) rats and divided them into two random groups with a sample size of 10. The animals were then subjected to a sham exposure or exposed to 100 μT /50 Hz ELF-EMF and then various tests such as, (i) ELISA assays to assess the Amyloid-beta (Aβ) 3 content in the cortex, hippocampus, and plasma; (ii) the Morris water maze (MWM) to assess any alteration in cognition and memory; and (iii) hematoxylin and eosin (H & E) staining to assess the morphology of neurons. The results indicated that apart from a lack of difference in body weight compared to the control group, other indications included: (i) no real alterations in the Aβ; (ii) no malfunction in memory and cognition compared to the sham exposure group; and (iii) no histological alteration in neuronal morphology in the ELF-EMF exposure group. These results indicated that exposure to 100 μT/50 Hz ELF-EMF did not influence the cognition and memory of the animals, and it did not alter their neuron morphology or expression of Aβ (Zhang et al., 2015). To uncover the potential influence and underlying mechanism of ELF-EMF exposure on living organisms, more extensive research should to be undertaken. Alzheimer’s disease (AD) is part of the spectrum of neurodegenerative illnesses that could possibly be induced by low-frequency magnetic field (LF-MF). To determine the influence of long term exposure to LF-MF (50 Hz, 1mT) with the onset of the AD and amyotrophic lateral sclerosis (ALS) in established mouse models, Liebl and co-authors undertook extensive research on APP23 mice and mice expressing mutant Cu/Zn- superoxide dismutase (SOD1), respectively. APP23 transgenic mice is considered to be a mouse model of Alzheimer diseases (Van Dam et al., 2014). The learning disorder of the APP23 mice did not worsen after continuous exposure for 16 months, and LF-MF continuous exposure for 8 and 10 months had no effect on the propagation of disease and survival of SOD1G93A or SOD1G85R mice, respectively. Furthermore, continuous exposure to LF-MF on SOD1 mice did not increase their protein aggregation and glial activation. Moreover, the same magnetic field did not change the Aβ and APP gene level of APP23 transgenic mice. These results and the biochemical assessment of protein aggregation, glial activation, and levels of toxic protein species indicate that the cellular phenomena involved in the pathogenesis of AD or ALS was not influenced by LF-MF (Liebl et al., 2015).

1.3 Magnetic nanoparticles Magnetic nanoparticles (MNPs) can be manipulated with assistance from an external magnetic field; without a magnetic field MNPs seem to be characterised by para- magnetism, whereas within a magnetic field they seem to be characterised by super- para-magnetism. Although NPs become magnetised after exposure to an external

4 magnetic field (EMF), they do not exhibit remanence (permanent magnetisation once the magnetic field is turned off), superparamagnetic iron oxide NPs (SPIONs) are given priority over other MNPs (Chomoucka et al., 2010). When the surfaces of SPIONs are modified appropriately, they exhibit, (i) stability against aggregation at physiological pH within a large range of concentration and ionic strength, (ii) reactive areas that are appropriate for further binding of biological ligands with drugs, and (iii) a reduction in capture by the immune system (Shkilnyy et al., 2010). Therefore, this surfactant is very important in reducing the cytotoxicity of NPs.

Iron oxides can exhibit stable magnetic responses because they are not as sensitive to oxidation as other metals (Tran and Webster, 2010), and therefore iron oxide MNPs have many crystalline polymorphs which are appropriate for biomedical applications, particularly c-Fe2O3 (maghemite) and Fe3O4 (magnetite) (Tran and Webster, 2010, Arruebo et al., 2007, Tuček et al., 2006).

Being a multi-disciplinary ramification of science, nanotechnology consists of various areas of science and technology such as advanced materials science, agricultural sciences, biomedicine, chemistry, environmental sciences, information technology, pharmaceutics, and physics, among many others (Chatterjee et al., 2014). Nanoscale substances are very important for biomedical applications because their sizes are comparable with cells (10 – 100μm), viruses (20 – 450 nm), proteins (5 – 50 nm), and genes (2 nm wide by 10 – 100 nm long). Moreover, nanoparticles can access areas of the body which are otherwise inaccessible through other materials because they are small enough and can travel inside human systems without causing any malfunctions (Medeiros et al., 2011). Apart from their size, the surface characteristics of MNPs are also because adsorbing a different layer of material onto MNPs can enhance their surface properties.

The following coating substances are typically utilised to enhance the surface properties of MNPs (Shubayev et al., 2009): (i) Organic surfactants such as sodium oleate and dodecyl amine; (ii) Organic polymers such as dextran, chitosan, polyethylene glycol (PEG), polysorbate and polyaniline; (iii) Inorganic oxides such as silica and carbon; (iv) Inorganic metals such as gold; and (v) Bioactive structures and molecules such as peptides, liposomes, and ligands/receptors.

Unlike ordinary nanoparticles, coated nanoparticles have many advantages due to their superior characteristics, these include: (i) enhanced conjugation with other bioactive molecules; (ii) less toxicity; (iii) enhanced thermal and chemical stability; and (iv) increased dispersibility and biocompatibility (Chatterjee et al., 2014).

Moreover, the surfactant coating of MNPs improves the stability of a suspension of MNPs in an aqueous solution, as well as preventing the aggregation of NPs due to their magnetic features. Oleic acid is one of the most common surfactants and is considered to be a biocompatible material that does not activate the cells of an immune system (Velusamy et al., 2016). Moreover, the chemical structure of oleic acid, including the carboxylic acid, creates the strongest bond with amorphous MNPs. These functionalised MNPs with oleic acid increases the monodispersing percentage, gives great biocompatibility, reduces toxicity, enhances stability, and becomes hydrophobic (Velusamy et al., 2016). This is why the features of oleic acid attracted us to use it to stabilise the MNPs used in this research.

Depending on their composition, size, shape, electric charge, magnetic and optical characteristics, nanoparticles have varying properties, which means they can be altered through the conjugation of reactive functional groups and cargos. The nature of this interaction between cells and nanoparticles is therefore determined by these characteristics, as is their ability to bind or permeate into other cells and to influence other biological reactions. This means the cellular reaction to these nanoparticles is determined by the nature of these interactions which can be seen in the form of cellular morphology, activity or differentiation (Polak and Shefi, 2015).

1.3.1 Fe3O4 nanoparticles

For more than five decades, iron oxide (γ-Fe2O3 or Fe3O4) particles have been increasingly utilised for in vitro medical diagnostics (Gilchrist et al., 1957), and during the last decade, nano-sized iron oxide particles, especially Fe3O4 (magnetite), have been widely utilised due to their unique physical properties, ability to be manipulated at the molecular level, and their biocompatibility (Kirschvink et al., 2001, Dunlop, 1973, Li et al., 2005). The quantum properties that are manifested due to its size combined with the large surface area of magnetite enhanced its magnetic properties enough to enable it to exhibit superparamagnetism and quantum tunnelling.

Consequently, these properties permit a wide range of biomedical applications such as cell labelling, cell isolation, payload delivery, tissue repair, and magnetic resonance imaging (MRI) contrast enhancement. Moreover, homogeneous ferrofluids can also be formed by dispersing the surface functionalised magnetite nanoparticles into an appropriate solvent (Strömberg et al., 2007, Sahoo et al., 2005), and by using an external magnetic field gradient system, these ferrofluids can be navigated manually to any region of the human (or animal) body to carry out therapeutic procedures (Jordan et al., 2001, Hosseini et al., 2011, Probst et al., 2011).

Ferrofluids are currently being used in clinical applications because their administration inside a subject is comparatively non-toxic (Enochs et al., 1999). The use of magnetic nanoparticles (MNPs) rather than non-magnetic particles has many advantages, including: (i) the visualisation of MNPs using MNI and MRI modalities to ascertain that payloads have been correctly delivered to the required areas; (ii) navigation and positioning of MNPs to any part of a subject using an external magnetic field gradient system which can achieve an accuracy of micrometres, and also eliminates the use of cell-specific antigen-antibody reactions; and (iii) increasing the temperature of MNPs using high-frequency magnetic fields. This fact can be utilised to remove certain tissues (for instance cancer tissues) surgically and can also help in releasing a therapeutic drug.

The controlled release of a drug at a specific region of the body can also be achieved when Fe3O4 is subjected to an external magnetic field. The significance of a controlled release of drugs using Fe3O4 in vitro by subjecting it to AC/DC magnetic fields was highlighted by Mustapić and co-workers in 2016. In fact, magnetic fields have been utilised to control the release of methyl blue to water. Here the researchers used methyl blue instead of drugs in these experiments so the release of methyl blue into water after EMF exposure proved the drug release. Although the application of only a DC field did not result in any release, methyl blue was only released to water when an AC field was applied. Following this, both fields were applied simultaneously, this resulted in a faster release possibly due to viscous friction. When subjected to a DC field, nanoparticles become strictly aligned but when they are subjected to an AC field, they start oscillating. Similar to the controlled release of methyl blue, this research shows a concept of controlled drug delivery where pharmaceutical molecules are first adsorbed onto porous Fe3O4, followed by an appropriate localised magnetic field that releases the

7 drug at a specific target (Mustapić et al., 2016). Because of the advantages of iron oxide particles (unique physical properties, biocompatibility, and high potential for numerous biomedical applications such as payload delivery, tissue repair, and hyperthermia), it was used in this research to investigate the effect of these NPs on cells in culture.

1.3.2 Size of MNPs The factor for determining the approximate half-life of MNPs in the bloodstream is their size (Durán et al., 2008, Esmail, 2010). Studies show that very large MNPs (diameter > 200 nm) are quickly absorbed by the reticuloendothelial system (RES) and are then found in the spleen and liver while small MNPs (diameter < 5.5 nm) are quickly excreted through the renal system (Durán et al., 2008, Veiseh et al., 2010); therefore, to control the targeting of any drug, their size should ideally be between these two extremes.

The size of MNPs also determines the concentration of plasma, the half-life of blood circulation, and its toxicity. Particles greater than 200 nm either accumulate in the spleen or are absorbed by the body’s phagocytic cells which then reduces the concentration of plasma particles, whereas particles with diameters less than 10 nm are excreted primarily by the renal system. Owing to their toxicity and accumulation in the body, particles smaller than 2 nm should not be utilised for such purposes. Particles in the range of 10 – 100 nm are optimal for biomedical applications because they are not absorbed by the reticuloendothelial system (RES) and thus have a longer life inside the bloodstream; and they can also permeate very small blood capillaries (Gupta and Wells, 2004). Research suggests that to penetrate the blood-brain barrier (BBB) MNPs must be smaller than 20 nm for neural stimulation in vivo so that payloads can be delivered to specific regions of the brain as required (Yue et al., 2012). We therefore used 20 nm of spherical Fe3O4 in our research in vitro.

1.3.3 Biomedical applications of MNPs The provision of therapeutic payloads to diseased tissues with high efficacy, solubility, and stability has been the prime concern of medical applications, as well as optimising the biodistribution/bioavailability of therapeutic payloads. The proposition to utilise a delivery technique that could target particular diseased tissue/organism and provide a toxin for that specific disease/organism was first suggested by Estelrich and co-authors

(Estelrich et al., 2015). The term “magic bullet” refers to the method used to deliver therapeutic payloads to a particular area or to eliminate an organism exclusively. To deliver therapeutic drug payloads to particular areas, numerous nanomaterials with differing compositions and biological characteristics have recently been designed (Pison et al., 2006, Brannon-Peppas and Blanchette, 2004, Stylios et al., 2005, Yokoyama, 2005, Schätzlein, 2006), but unlike traditional delivery mechanisms, these nanomaterials have advantages such as: (i) the provision of payloads to targeted regions of the body; (ii) minimisation of the drug (since the drug is delivered to a particular site); and (iii) minimisation of any toxic side-effects (since the drug level is minimised at irrelevant sites). Apart from these applications, nanomaterials also have potential applications in cell imaging, diagnostics, and protein and nucleic acid (DNA/RNA) detection. Of this plethora of nanomaterials, those which are widely utilised include Au nanoparticles, iron oxide nanoparticles, magneto-electric nanoparticles, Poly (lactic-co- glycolic acid) nanoparticles, carbon nanotubes, and mesoporous silica nanoparticles. These materials are summarised in Figure 1.1.

Figure 1.1: (Guduru, 2013) shows advantages and disadvantages of using magnetic nanoparticles for various applications. 9

Figure 1 shows the benefits and drawbacks of utilising MNPs for numerous applications that were summarised by Gunduru in 2013. Magnetic nanoparticles (MNPs) seem to be the most widely utilised nanoscale substances because they consist of a core/shell nanoparticle structure with a magnetic core inside an organic or polymeric coating. In the absence of a coating, MNPs exhibit hydrophobic properties, and have large surface- to-volume ratios and a tendency to agglomerate (Lu et al., 2007).

1.3.4 Biomedical application of Fe3O4 NPs Iron oxide nanoparticles (IONPs) are emerging as the focus of biomedical research because they have numerous applications in the field as well as a low toxic profile and collectability by magnetic fields. The metabolic mechanism of IONPs in the body was used to determine their potential for use as MRI contrast agents; these studies showed that initially, iron oxides were transported to the liver and spleen (Weissleder et al., 1989), but within a week of being administered excess iron was either excreted or utilised by the body (Weissleder et al., 1989) as co-factors such as hemoglobin, etc. Moreover, IONPs may also function as enzymatic co-factors. To slow the degradation of nerve growth factor (NGF) down, Marcus and colleagues combined iron oxide nanoparticles with NGF because after NGF and iron oxide nanoparticles conjugate covalently, they also slow the degradation of NGF better than free NGF (Marcus et al., 2015). Furthermore, conjugated NGF promoted the neurite outgrowth of PC12 cells at higher levels than the free NGF available at the same level of concentration (Marcus et al., 2015).

Chen and his team also exhibited their almost non-invasive technique for remote neural excitation by activating the heat-sensitive capsaicin receptor TRPV1 using Fe3O4 (Chen et al., 2015).

According to Paviolo and colleagues, nanoparticles play a pivotal role in activating several transcription factors that can directly influence the growth and differentiation of neuronal tissue (Paviolo et al., 2013). Prior studies regarding iron oxide particles support this hypothesis. After analysing gene expression, Park and colleagues observed widespread changes in the uptake of Iron oxide nanoparticles uptake on neuronal differentiation (Kim et al., 2011). In fact, several gene changes pertaining to cytoplasm, signalling machinery, growth hormone receptors and ion channels were reported, all of which are essential for the growth and differentiation of neuronal tissue. Several factors 10 that affect this process of growth are certain metal ions that hinder cell to cell adhesion; iron is a good example (Hong et al., 2003).

1.3.5 Neuronal Regeneration through Magnetic Nanoparticles. Neuronal tissue seldom regenerates and therefore trying to recover nerve damage after an injury is very difficult (Fu and Gordon, 1997). Although a great deal of research and various procedures have been carried out trying to achieve neuronal regeneration, thus far it has been a futile exercise because they are therapeutically in applicable. However, medical science has come a long way and new research is being carried out, which means the recent discoveries in magnetic nanotechnology are proving to be very beneficial at regenerating neuronal tissue (Franze and Guck, 2010, Bray, 1979, Bray,

1984, Fass and Odde, 2003). One hypothesis that could explain neurite elongation is that magnetic nanoparticles can apply a mechanical force that will stretch the plasma neuronal membrane. This means that nanoparticles of iron oxide could be beneficial because they have the required properties and their previous usage in such experiments have been fruitful (Blackman et al., 1993a, Ciofani et al., 2009) . This theory was tested by Riggio and co-authors who successfully used Iron oxide nanoparticles, Schwann cells cultures, and SH-SY5Y to migrate without causing any cytotoxicity (Riggio et al., 2012).

The Mergel lab adopted another approach to induce neuronal tissue regrowth using nanoparticles of Conjugated Iron oxide to increase the efficacy of the Fibroblast growth factor (Skaat et al., 2011). The fibroblast growth factor has a half-life of only 3-10 minutes (Bikfalvi et al., 1997) so it can help to repair and regrow injured or damaged tissues in the body (Yun et al., 2010); in fact after incubating conjugated nanoparticles with cells from nasal olfactory mucosa, there was marked increase in cellular growth.

Clearly a better understanding of the relationship between nanoparticles and neuronal regeneration and regrowth could pave the way for a therapeutic remedy for those suffering from neural tissue injuries. Several parameters considered during this therapy are worth mentioning; it is imperative to determine a balanced dose in order to draw a line between a therapeutic dose and a toxic dose, other than this morphology, activity, or drug delivery.

1.3.6 Cytotoxicity of iron oxide nanoparticles (IONPs) on Cell Lines The basic pathophysiology by which metal ions influence gene expression is not very clear, possibly due to the production of reactive oxygen species (ROS) (Oravecz et al., 2002). The coating of NPs helps to protect nanoparticle from agglomeration and minimise the rate of surface oxidation, and particle size can be controlled with a stable coating material during the synthesis process. Nanoparticles without a coating are unstable, more exposed to the immune system, have increased chemical reactivity, and are easily oxidised (Santhosh and Ulrih, 2013). Organic ligands, a natural polymer, synthetic polymers, inorganic molecules, and biological molecules are some of the coating materials used (Carlson et al., 2008). A study to assess the cellular uptake of iron oxide NPs (IONPs) and different cell lines was carried out, and revealed that the efficiency of nanoparticle uptake depended on the surface coating, the cell line did not influence uptake at all (Karlsson et al., 2008). This study established a positive association between the surface coating and the biocompatibility of nanoparticles and also showed that it could decrease the IONP toxicity. An in vitro study was carried out on A3 human T lymphocytes, and revealed that the cytotoxicity of IONPs coated with ligands with terminal carboxylic acid groups was higher than those coated with ligands with terminal amine groups (Buzea et al., 2007). Chen et al. used mouse fibroblast cells (L929 cells), murine macrophage cells (RAW264.7 cells), and a Chinese hamster ovarian cells (CHO-K1 cells) to show that coated Fe3O4 NPs cause less DNA damage than uncoated Fe3O4 NPs (Chen et al., 2012).

The cellular uptake of IONPs depends on the coating characteristics and the size of the nanoparticles (Safi et al., 2010), however, remember that the doses of IONPs with which cells were treated are presented here, but they do not always represent the true amount of internalised iron (Safi et al., 2010). This may cause problems because the amount of internalised IONPs is made with an NP dose, the coating composition and thickness, and the media composition (Safi et al., 2011), all of which may influence the levels of mutagenicity. Therefore, the dose of nanoparticles contributes to but is not solely responsible for the cellular internalisation of IONPs (Galimard et al., 2012).

Moreover, in various types of cells, iron is considered to be essential for their survival, e.g., the chick myotubes degenerated when a proper iron supplementation was not provided to the basal culture medium (Ozawa and Hagiwara, 1982). The important

12 factors used in these studies are explained and presented below by Polack and co- workers. These factors are classified on the basis of their activity, type of nanoparticle, coating, use, type of activity, and toxicity (Polak and Shefi, 2015). Iron oxide is also included in Table 1.1 as a factor of nanoparticles.

Table 1.1: (Polak and Shefi, 2015)The most important factors in the studies, classified on the basis of activity, kind of NPs, coating, use, type of activity, and toxicity are given below:

Iron oxide (average dry Functional Active or Toxicity Activity Use Ref. size coating mediator? ? in nm) Yes (at For enhancing the differentiation of high (Kim et al., (11nm) Active PC12 cells concentr 2011) ation) To stabilize the NGF and Differentiatio Conjugated to (Marcus et al., (23nm) Consequently Improve the neuronal Mediator No n and survival NGF 2015) differentiation Conjugated to basic For improving the outgrowth of (Skaat et al., (15nm) Mediator No fibroblast nasal olfactory mucosa cells 2011) growth factor Application of magnetic tensile forces SH-SY5Y and primary (Riggio et al., Directing (73nm) Mediator No Schwann cell cultures to direct 2012) neuronal them towards predefined directions migration and Application of magnetic tensile growth Conjugated to (Riggio et al., (25nm) forces for Induction of directed Mediator No NGF 2014) neurite sprout in PC12 cells

1.4 SH-SY5Y cells Many researchers use neuronal-like cancer cell lines such as human SH-SY5Y or mouse Neuro2a neuroblastoma cells to provide relatively easy access to a vast number of cells for analysis (Shipley et al., 2016). These cycling cells are commonly differentiated into more neuronal-like cells for functional analyses. To do this, cells can be serum starved and /or treated with an agent such as retinoic acid (RA) or cyclic adenosine-3',5'- monophosphate (cAMP) to promote neuronal differentiation (Shipley et al., 2016).

1.5 Aims

1. To compare the effect of AC and DC Magnetic fields on the cell viability of SH- SY5Y neuroblastoma human cells in vitro (Chapter 3). 2. To compare the influence AC and DC MF on the neurite outgrowth of SH-SY5Y cells with and without retinoic acid in vitro (Chapter 3).

3. To measure the cytotoxic impact of iron oxide nanoparticles (IONPs) Fe3O4 NPs on the cell viability of SH-SY5Y neuroblastoma human cells in vitro (Chapter 4).

Chapter 2 SH-SY5Y cell culture SH-SY5Y neuroblastoma cells (94030304, Sigma) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) / F12 pH 7.4 (Life Technologies, 12500096), supplemented with 10 % foetal bovine serum (FBS, Bovogen), and 1 % penicillin/streptomycin (PS, Life Technologies, 15140-122). The cells were kept in culture for up to 22 passages and maintained at 37 °C in a 5 % CO2 atmosphere; they routinely underwent mycoplasma testing (MycoAlert, Lonza) and were confirmed by STR profiling.

The impact of AC and DC MF on Cell viability The SH-SY5Y cells were plated into 96 – well microplates at 12000 cells per well. The cells were cultured in (DMEM) / F12 pH 7.4 supplemented with 10 % foetal bovine serum and 1 % PS. On the first day the cells were plated a concentration of 12000 / well cells were seeded in the plates. The cells were then treated with AC or DC MF every day for 2 or 3 days, starting 24 hours after plating, depending on the experimental conditions. The cells were cultured for 48 hours without any further treatment with EMF. The fluorescent intensity of the reagent composed from AlamarBlue and the cells was read on the plate reader (BMG - POLARstar Omega) 2 hours after adding the viability reagent to the cells.

The cytotoxicity test of homemade and commercial MNPs The cytotoxicity of iron oxide NPs was assessed by the AlamarBlue assay (ThermoFisher, DAL1100). SH-SY5Y neuroblastoma human cells were subcultured in 96 – well / flat – bottomed (FB) microplates (Interpath Services, 655180X) using DMEM / F12, 1 % PS, and 10 % FBS. On the first day, a concentration of 12000 / well cells were seeded in the plates and on the second day the cells were treated with different concentrations of MNPs, depending on the type of MNPs. These homemade MNPs was dispersed in distilled water (D.W) because it was uncoated NPs (bare), whereas the commercial MNPs were dispersed in ethanol absolute because the NPs were coated with oleic acid (Fatty acid and hydrophobic). There were two ranges of a serial dilution of NPs; the first being (5, 10, 15, 20, 25, 30, 37.5, 45, and 50 µg / ml),

15 and the second being (10, 20, 30, 40, 50, 60, 75, 90, and 100 µg / ml). AlamarBlue reagent (1:100 PBS) was added 48 hours later. The fluorescent intensity of the reagent composed from the AlamarBlue reagent and SH-SY5Y cells was read on the plate reader (BMG - POLARstar Omega) 2 hours after adding the cell viability reagent to the cells.

The effect of AC and DC MF on Neuronal differentiation The SH-SY5Y cells were plated into 96 – well microplates at 5000 cells per well and then cultured for 7 days. To induce differentiation in the cells, the culture media was replaced with differentiation media (DMEM/F12, 1% PS, 1% FBS, 10 µM all-trans retinoic acid (RA, R2625, Sigma)) after 24 hours, followed by 100 % media changes every 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle control for the RA, and the vehicle control medium was also changed every 48 hours. SH-SY5Y cells were treated with different intensities of AC or DC MF (either with or without RA) for 2 or 3 days, starting 24 hours after plating, and the cells were cultured without any further exposure to EMF until Day 7 Figure 2.1 Cell images were collected every day using an IncuCyte Zoom live imaging system.

Figure 1.1: Experimental design and schematic diagram of the neurite outgrowth experiments.

Magnetic Exposure System The magnetic exposure system holder was designed to hold an AC coil and commercial N52 grade rare earth (NdFeB) permanent magnets (DC magnetic) in close proximity in order to obtain a uniform and controlled field gradient by varying the distance between the AC coil and the permanent magnets. The holder was made from acrylic (Perspex) material to ensure safety while applying an AC current to the coil. The AC magnetic field exposure system has one circular copper coil (air core) with 400 turns and 10 layers; it was designed at Coast Electrical Industries Pty Limited to fit inside the coil holder which is connected by a 300 mm long rod. The coil is 15 mm long × 160 mm in diameter, and has an internal diameter of 60mm, as shown in Figure 2.2. The wire used in the coil is 1.25 mm in diameter. The coil is connected to the function generator (GFG 200/2100) that applies the frequencies and power amplifier (Sinocera Piezotronics, INC, model: YE5873H) to deliver an alternating current of up to 3.6 A. Two sizes (Ø6.4×5 and are Ø15.6×3 mm) of N52NdFeB cylinder magnets were purchased from Chongqing Seatrend Technology and Development. Co., Ltd., and the 4 larger and 5 smaller magnets were placed in parallel on the base of the magnetic system holder, as shown in Figure 2.2. The 96 – well microplate placed as a flat surface over the MF source direction (AC and DC sources) to ensure maintaining uniform and high magnetic flux intensity. The perpendicular MF direction on the exposed cell surface area of microplate prevents loses in the MF flux intensity. The strength of MF intensity and frequency for both magnetic fields was assessed using a Gaussmeter (GM – 2, AlphaLab, INC)

Figure 2.2: Magnetic exposure system holder designed using the Solid Material software. It consists of a base which containing two sizes of neodymium cylinder magnets, base, rod, a plate holder, and a coil holder. TEM images of the homemade and commercial MNPs

Transmission electron microscopy (TEM) images of the homemade Fe3O4 NPs were taken by Dean Cardillo, but the TEM images of the commercial Fe3O4 NPs were taken by the supplier company.

Dynamic Light scattering test (DLS test) on homemade MNPs

Serial dilutions of homemade MNPs (uncoated NPs) were prepared to start from (10 µg / ml to 100 µg / ml) in distilled water (D.W.), and then 100 µl of 10 µg/ml concentration were added to one well of 96 – well / flat – bottomed (FB) microplates (Interpath Services, 655180X) and placed inside a particle analyser to check the particle size

18 distribution. The particle size distribution of MNPs was checked using a particle analyser (Malvern – Zetasizer APS2000, IHMRI).

Dynamic Light scattering test (DLS test) on commercial MNPs The same process was carried out on the commercial MNPs (coated NPs), but the coated MNPs were dispersed in (Ethanol Absolute, Ajax FineChem, Catalogue NO: 214, CAS NO: 64175) instead of D.W. because the surfactant is oleic acid (hydrophobic).

Statistics

The data for AC, DC, Fe3O4 NPs are given as the mean ± standard error of the mean (SEM) from at least 3 independent replicates. Statistical differences were identified after confirming the normal distribution by one or two repeated measures using ANOVA with a Tukey post hoc comparison, as appropriate, in Graph Pad Prism software. However, the combined AC / DC data are given as the mean ± standard error of the mean from 3 technical replicates that were analysed by t-test in Graph Pad Prism software. The cell images were analysed by the IncuCyte software to acquire data on neurite length, cell confluency, and neurite branch points. Data from the dynamic light scattering tests were collected as an Excel file and also analysed in the same file.

Chapter 3 Results: The effect of EMF on SH-SY5Y viability and neurite outgrowth

The effect of EMF on SH-SY5Y cells is currently unclear, so to investigate the effect of EMF on neurite outgrowth it was important to establish whether EMF affected cell viability.

To investigate the impact of DC MF on cell viability, different intensities of DC MF (10, 25, 50, 75, 100, 125, and 150mT) were optimised for different treatment times 1, 2, and 4 hours / day over 1, 2, and 3 days. The SH-SY5Y neuroblastoma cells were treated separately with various intensities (1, 5, and 10 mT) and different frequencies (50 and 100 Hz) of AC magnetic fields. These conditions were also optimised for different treatment periods of 1, 2, and 4 hours / day for 1, 2, and 3 days. The findings indicated that only 10 mT with 50 and 100 Hz of AC MF reduced cell viability. There was also no difference between the impact of 50 and 100 Hz while the intensity of the magnetic field was constant (1 mT) after treating the cells for 4 hours / day for 3 days.

A number of optimisation experiments with varying AC and DC MF intensities and durations were also tested outside the incubator, but since the results varied, these conditions were not pursued (data not shown). All the other experiments were carried out inside the incubator and are shown in the following sections.

No significant effect of DC MF on cell viability A 100 mT for 4 hours / day for 3 days proved to be the best conditions for treating cells without any negative impact on cell viability. A paired t-test revealed there was no real difference between cells treated with DC MF compared to the control (t = 0.1471, p = 0.8924, Figure 3.1 A).

No significant impact of AC EMF on cell viability AC MF conditions were optimised and 100 Hz AC MF with a constant magnetic field strength of 1 mT for 4 h/day for 2 days was the best condition for treating cells without any negative impact on cell viability (t = 1.55, p = 0.17; unpaired t-test; Figure 3.1 B). 20

Figure 3.1: (A) Cell viability was assessed by AlamarBlue assay to investigate the impact of 100 mT of DC MF. There was no real difference between the treated samples and the control. (B) cell viability test was also carried out to investigate the impact of treating the cells with 1 mT, 100 Hz of AC MF for 4 hours/day over 3 days. There was no significant difference between the treated cells compared to the control. The influence of combined DC / AC MF on cell viability When the DC and AC MF conditions were optimised separately, one strength field of each type was used to carry out a combined DC and AC test on cell viability. It was assumed that a combined DC / AC might increase cell viability so two experiments were carried out to test the impact of long and short periods of treatment for a combined DC / AC MF. In the first experiment, two plates were prepared for SH-SY5Y cells, and then each plate was treated separately for 2 or 4 hours with 1 mT, 100 Hz of AC, in combination with 100 mT of DC EMF. The cell morphology changed to a spherical shape (rounding up of cells normally associated with cell death) in both plates by the end of the treatment time. An AlamarBlue assay after 48 hours to check cell viability suggested many cell deaths (overall one – way ANOVA findings F(2, 6) = 607.4, p < 0.0001, Figure 3.2 A). A post hoc analysis revealed a large decrease in cell viability compared to the control (p < 0.0001 for both 2 and 4 hours of treatment, Figure 3.2 A). In the second experiment, three plates were prepared for SH-SY5Y cells, each plate was treated separately for 30, 60, or 90 minutes under the same conditions of combined DC / AC MF. Treatment for 60 and 90 minutes changed the cell morphology as much as long periods of treatment. An AlamarBlue assay after 48 hours showed alterations in cell viability (overall one – way ANOVA findings F(3, 8) = 142.8, p < 0.0001, Figure 3.2 B). A post hoc analysis identified a large decrease in cell viability (p < 0.0001 for 60 and 90 minutes of treatment, Figure 3.2 B). However, 30 minutes of treatment had no adverse effect on cell viability, but since there was a dramatic reduction in cell viability caused

21 by a combination of AC and DC treatment, we continued the experiments using only AC or DC treatments alone.

Figure 3.2: (A) Cell viability was assessed by AlamarBlue assay to investigate the impact of both 100 mT of DC, and 1 mT, 100 Hz of AC for 2 and 4 hours on cell viability. There were large differences for both long treatment times. (B) Cell viability was also tested by AlamarBlue assay to explore the effect of 100 mT of DC, and 1 mT, 100 Hz of AC for 30, 60 and 90 minutes on the cell viability. There was no real change after treating the cells for 30 minutes but there were large differences after treating the cell for 60 and 90 minutes under the same conditions of MF. The effects of AC and DC MF on neurite outgrowth SH-SY5Y cells were treated with external magnetic fields under either an AC or DC magnetic field to investigate the impact of EMF on neurite outgrowth and cell viability. A range of magnetic field strengths were tested to identify the optimal conditions for treatment. The conditions outlined below represent the best outcomes; this involved using a magnetic field strength of 1 mT, 100 Hz of AC MF or 100 mT of DC MF. The samples were treated with 1 mT, 100 Hz of AC MF or 100 mT of DC MF for 4 hours/day over 2 or 3 days. Images of the cells were collected every day until Day 7. The effects of external magnetic fields on cells were assessed, included measuring the confluency, neurite length, and neurite branch points of the SH-SY5Y cells following treatments.

(1) Treating cells with DC or AC MF for 4 h / day for 2 days

(i) In the presence of RA: To assess the impact of MF on neurite outgrowth, the cells were grown in a medium containing 1% FBS supplemented with RA every other day for the 7 Day differentiation, and DMSO was used as a vehicle control in these tests.

There was no significant impact (overall two-way repeated measures ANOVA F (3, 8) = 0.4948, p = 0.6959) of either DC or AC MF on cell confluency (Figure 3.3 A). the neurite increased in length for both types of MF (overall two-way repeated measures

ANOVA F (3, 8) = 37.68, p < 0.0001). A post hoc analysis revealed a significant increase in neurite length, starting from Day 3 until Day 7 for both AC and DC (p < 0.01 for Days 3 to 7, Figure 3.3 B). This means that the impact became significant 24 hours after the last treatment. The effect of DC MF + RA on Days 3 – 7 led to a larger increase in neurite length compared to RA alone, than the AC MF + RA treatment. Nevertheless, the effects of AC MF became equal to or higher than the DC MF impact by Days 6-7 (Figure 3.3 B). Moreover, the neurite branch points were increased significantly by DC MF + RA and AC MF + RA, compared to RA alone, also starting from Day 3 until Day 7 (overall two-way repeated measure ANOVA findings, F (3, 8) = 21.54, p = 0.0001); a post hoc analysis identified a significant increase for Days 3 – 7 for both AC MF + RA and DC MF + RA compared to RA alone (p < 0.05 for Days 3 – 7, Figure 3.3 C). The impact of AC MF on Day 3 to 7 had a lower and more varied effect on the neurite outgrowth than the DC MF treatment (Figure 3.3 C).

(ii) In the absence of RA: To assess the effect of MF in the absence of RA, RA was omitted from the cell culture medium and the effects of MF on neurite length and branch points were assessed as shown previously. Both DC and AC MF had similar effects on neurite outgrowth, as shown in Figure 4, even without RA. In these experiments MF was applied to cells cultured in a medium containing 1% FBS, so 1% FBS without MF application was utilised as the control. Cells cultured under 10% FBS were used as a further control and showed reduced/absent neurite extensions (Figure 3.3 D – F). As shown previously, both types of MF had no effect on cell confluency

(overall two-way repeated measure ANOVA findings F (3, 8) = 11.34, p = 0.0030, Figure 3.3 D). However, the neurite length increased significantly with both types of MF compared to control (overall two-way repeated measure ANOVA findings F (3, 8) = 16.92, p = 0.0008). A post hoc analysis identified a significant increase starting from

Day 5 until Day 7 for DC (p < 0.05 for days 5 – 7, Figure 3.3 E), and Day 6 to 7 for AC (p < 0.05 for days 6 to 7, Figure 3.3 E). This means that the effect became significant 4 days after the last treatment day for DC and 5 days for AC. Therefore, DC MF was better at increasing the neurite length than AC MF (had a larger effect earlier in the differentiation process). However, the effects of AC MF were similar to DC MF by Day 7 (Figure 3.3 E). Furthermore, the neurite branch points increased significantly starting from Day 6 to 7 for DC while the impact of AC MF led to greatly enhanced neurite branch points on Day 7 (overall two-way repeated measure ANOVA findings, F (3, 8) = 6.347, p = 0.0165); a post hoc analysis identified a significant increase starting on Day 6 for DC MF (p < 0.05 for Days 6 – 7, Figure 3.3 F), and on Day 7 for AC MF (p < 0.01 for Day 7). The influence of AC MF on the neurite branch points occurred 24 hours later during the differentiation process, unlike the DC MF (Figure 3.3 F).

Figure 3.3: Impact of AC and DC MF treatment for 4 hours/day over 2 days on the neurite outgrowth of SH-SY5Y cells in the presence and absence of RA. The cells were treated separately with 1 mT, 100 Hz of AC MF, and 100 mT of DC MF, for 4 h/day for 2 days. (A – C) shows the effects of AC and DC MF on the neurite outgrowth with RA. There was no real change in cell confluency (A). However, the treatment did increase the neurite length and neurite branch points starting from Day 3 until Day 7 (B – C). (D – F) illustrates the influence of the same conditions of MF on SH-SY5Y cells without RA in 1% FBS containing cell culture medium. There was no real change in cell confluency following either MF treatment (D). DC MF or AC MF treatment increased the neurite length and neurite branch points, but starting from Day 5 for neurite length and Day 6 for neurite branch points (E-F), the differences compared to 1% FBS without RA on the same day are shown *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. In order to identify how treating the cells 4 hours / day for 2 days affected neurite outgrowth, see (Figure 3.4), the impact of the conditions mentioned above on the neuronal differentiation of the cells with and without RA is shown in Images (A – H). These Images are related to the presence of RA, while DMSO was utilised as a vehicle control for RA. DMSO had no effect on the neurite length and neurite branch points, as shown in image (A), but RA did reduce cell confluency and promoted neurite length 25 and neurite branch points compared to A, as shown in image B. Moreover, the effect of DC MF on cell confluency and neurite outgrowth was more effective than AC MF compared to A, as shown in image C, however the second group of images are without RA and 1% FBS was used as a control, see (E - H). The effect of 1% FBS and without DMSO on cell confluency and neurite outgrowth is shown in image (E); here it reduced cell confluency but had no positive impact on neuronal differentiation, see (E). However, 1% FBS had a strong effect when the DMSO was attending, as indicated in image A compared to E. When DMSO and RA, which reduced cell proliferation and induced cell differentiation are absent, and the concentration of FBS is 10%, cell confluency increased significantly, as shown in image F compared to images A, B, and E. However, there was no effect with 10% FBS on the neuronal differentiation, see (F). The impact of DC MF on cell confluency and neurite outgrowth is shown in Image (G). When there is no differentiation reagent, cell confluency will decrease more by treating the cell with DC MF than treating the cells with 1% FBS only, as indicated in image G compared to E. Even though treating the cells with DC and RA reduced cell confluency more than treating the cells with DC and 1%FBS only, the DC MF could induce cell differentiation by reducing cell proliferation, and it could replace a differentiation reagent, as shown in image G compared to C. Moreover, the neurite outgrowth was also significantly enhanced by treating the cells with DC MF, even when RA is absent, as shown in image G compared to C. The impact of AC MF on cell confluency and neurite outgrowth is shown in image (H), where AC MF also reduced cell proliferation. Further, cell confluency with AC MF decreased more than when treating the cells with 1%FBS or (DC + 1% FBS), as shown in image H compared to E and G. This result shows that AC MF could also induce neuronal differentiation by reducing cell proliferation. When treating cells with (AC+1%FBS), cell confluency was still higher than when treating them with (AC+RA), as shown in image H compared to D. However, it does not mean that AC MF did not induce neuronal differentiation or that it did not work as an alternative method for differentiation reagent, as indicated in image H compared to D. AC MF also significantly increased the neurite outgrowth, but it had less impact on neurite outgrowth than DC MF because neurite outgrowth was more effective with DC MF treatment than with AC MF treatment. All the images mentioned above were collected on Day 7 of the experiments. To sum up, DC and AC MF could

26 induce neuronal differentiation by reducing cell confluency and significantly increasing neurite outgrowth.

Figure 3.4: IncuCyte images demonstrating the effects of treating SH-SY5Y cells for 4 hours/day over 2 days with AC and DC MF on neurite outgrowth in the presence and absence of RA. Images (A – D) are related to the presence of RA. Images (E – H) are associated with the absence of RA. All images were collected on Day 7. Yellow refers to the cell body and purple refers to the neurites. Scale bar is 300 µm. 27

(2) Treating the cells with DC or AC MF for 4 h / day for 3 days

(i) In the presence of RA: As stated previously, the cells were cultured in a medium containing 1% FBS supplemented with RA and with or without DC or AC MF treatment, and DMSO was used as vehicle control. There was no significant impact

(overall two-way repeated measure ANOVA findings F (3, 8) = 3.275, p = 0.0798) of either DC or AC MF on the cell confluency (Figure 3.5 A). Neurite length had increased significantly for both types of MF (overall two-way repeated measure

ANOVA findings F (3, 8) = 203.6, p < 0.0001), and a post hoc analysis had identified a large increase starting from Day 3 until Day 7 for both AC and DC (p < 0.01 for Days 3 to 7, Figure 3.5 B) for the 2 day treatments; this means that the impact became significant on the last treatment day. The neurite branch points had also increased, starting from Days 3 – 7 in both types of MF (overall two-way repeated measures

ANOVA findings, F (3, 8) = 37.45, p < 0.0001), and a post hoc analysis identified a large increase starting from Days 3 – 7 for both AC and DC MF (p < 0.05 for Days 3 – 7, Figure 3.5 C).

(ii) In the absence of RA: RA was absent in other experiments however, and that showed how the same conditions of EMF affected neurite outgrowth, as shown in Figure 3.5, where 1% FBS was utilised as the control. Both types of MF had no real impact (overall two-way repeated measures ANOVA findings F (3, 8) = 3.237, p = 0.0818) of on cell confluency, as shown in (Figure 3.5 D), but the neurite length for both types of MF (overall two-way repeated measure ANOVA findings F (3, 8) = 30.58, p < 0.0001) had increased. A post hoc analysis identified a large increase on Day 7 for DC (p < 0.0001 for day 7, Figure 3.5 E), and AC (p < 0.05 for day 7, Figure 3.5 E), which means that the effect became important 4 days after the last treatment day for both types of MF. Furthermore, the neurite branch points had increased starting on Day 6 for DC MF only, while the impact of AC MF did not enhance the neurite branch points on Day 6 or 7 (overall two-way repeated measure ANOVA findings, F (3, 8) = 6.497, p = 0.0155); a post hoc analysis discovered a large promote starting from Days 6 – 7 for DC MF (p < 0.05 for Days 6 - 7, Figure 3.5 F). Together this suggests that the effects of DC MF on neuritogenesis were more effective than AC MF. Moreover, whilst 3 days of treatment rather than 2 days had no further benefit with RA present, treating the cells for 2 days rather than 3 days without RA promoted neurite outgrowth because

28 the ratio of cell confluency for 2 days treatment was higher than for 3 days treatment, as shown in Figure 3.3 D compared to Figure 3.5 D, and the neurite length increased starting on Day 5 for 2 days treatment and started on Day 7 for 3 days treatment, as shown in Figure 3.3 E compared to Figure 3.5 E. Finally, after 2 days of treatment the neurite branch points increased starting on Day 6 for DC MF and on Day 7 for AC MF. After 3 days of treatment the neurite branch points had increased starting on Day 6 for DC MF only, but the impact of AC MF did not enhance the neurite branch points on Day 6 or 7 very much, as shown in Figure 3.3 F compared to Figure 3.5 F.

Figure 3.5: The impact of AC and DC MF treatment for 4 hours / day for 3 days on the neurite outgrowth of SH-SY5Y cells in the presence and absence of RA. The cells were treated separately with 1 mT, 100 Hz of AC MF, and 100 mT of DC MF for 4 hours /day for 3 days. (A – C) illustrates the impact of MF on neurite outgrowth in the presence of RA. There was no real change in cell confluency (A). The treatment increased the neurite length and neurite branch points starting from Day 3 until Day 7 (B – C). (D – F) shows the influence of MF on neurite outgrowth in the absence of RA, but there was no real change in cell confluency (D). The treatment increased the neurite length and neurite branch points, starting from Day 7 for neurite length and Day 6 for neurite branch points (E – F). These real differences compared to 1% FBS without RA on the same day are shown *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. 29

The impact of treating the cells for 4 hours / day for 3 days on the neurite outgrowth with and without RA, with 1% FBS used as the control, is shown in the exemplary images in (Figure 3.6). The effect of the first conditions of MF with RA is shown in images (A – D), but the impact of the aforementioned conditions of MF on the neuronal differentiation when RA is absent is shown in images (E – F). When RA was present, there was no difference between the morphology of cells treated with the first and second sets of conditions, as shown in Figure 3.4 (C and D) compared to Figure 3.6 (C and D), but when RA was absent, there was a huge difference between the morphology of the cell treated with the first and second sets of conditions, particularly the AC MF, as shown in Figure 3.4 H compared to Figure 3.6 H. The morphology of cells treated for 4 hours / day for 3 days of AC MF without RA became spherical and aggregated and is almost dead.

Figure 3.6: IncuCyte images illustrating the impact of treating SH-SY5Y cells for 4 hours/day over 3 days with DC and AC MF on neurite outgrowth in the presence and absence of RA. Images (A – D) are related to the presence of RA. Images (E – H) are associated with the absence of RA. All images were collected on Day 7. Yellow refers to the cell body and purple refers to the neurites. The scale bar is 300 µm.

Comparison of the effect of DC and AC MF on neurite outgrowth

The fold change in neurite length following DC and AC MF treatment with and without RA is shown in Table 3.1. The fold change in neurite branch points following DC and AC MF treatment with and without RA is shown in Table 3.2.

Table 3.1: Comparison between the fold change in neurite length of MF compared to control in the 4 hours / day for 2 days and 4 hours / day for 3 days conditions when RA is present and absent (Mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (n.s.) is non- significant

4 hours / day for 2 days 4 hours / day for 3 days Time Presence of RA Absence of RA Presence of RA Absence of RA

DC MF AC MF DC MF AC MF DC MF AC MF DC MF AC MF Day 1 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Day 2 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Day 3 2.9 ± 0.1*** 3.3 ± 0.3**** n.s. n.s. 4.1 ± 0.4**** 3.9 ± 0.7**** n.s. n.s. Day 4 2.5 ± 0.1**** n.s. n.s. n.s. 2.5 ± 0.1**** 1.4 ± 0.1* n.s. n.s. Day 5 3.1 ± 0.1**** 2.2 ± 0.2** 4.7 ± 0.9* n.s. 3.6 ± 0.4**** 2.5 ± 0.3**** n.s. n.s. Day 6 2.2 ± 0.1**** 2.8 ± 0.7**** 2 ± 0.1*** 1.7 ± 0.2* 2.2 ± 0.2**** 3.2 ± 0.4**** n.s. n.s. Day 7 2.3 ± 0.01**** 2.3 ± 0.3**** 2.9 ± 0.3**** 2.1 ± 0.5**** 2.6 ± 0.2**** 2.3 ± 0.2**** 3.3 ± 0.2**** 2.1 ± 0.5*

Table 3.2: Comparison between the fold change in neurite branch points of MF compared to control in the 4 hours / day for 2 days and 4 hours / day for 3 days conditions when RA is present and absent (Mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0. 0001). (n.s.) is non-significant

4 hours / day for 2 days 4 hours / day for 3 days Time Presence of RA Absence of RA Presence of RA Absence of RA

DC MF AC MF DC MF AC MF DC MF AC MF DC MF AC MF Day 1 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Day 2 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Day 3 3.1 ± 0.2** 3.7 ± 0.5**** n.s. n.s. 4.3 ± 0.4**** 4.1 ± 0.9**** n.s. n.s. Day 4 2.8 ± 0.1**** n.s. n.s. n.s. 2.9 ± 0.1**** 1.6 ± 0.1* n.s. n.s. Day 5 3 ± 0.3**** 2.1 ± 0.3* n.s. n.s. 3.8 ± 0.5**** 2.6 ± 0.3**** n.s. n.s. Day 6 2.2 ± 0.06** 2.8 ± 0.7**** 2.1 ± 0.1* n.s. 2 ± 0.2*** 3.1 ± 0.3**** 2 ± 0.04* n.s. Day 7 2.2 ± 0.1**** 2.3 ± 0.5**** 3.3 ± 0.3**** 2 ± 0.5** 2.5 ± 0.2**** 2.2 ± 0.1**** 3.8 ± 0.4**** n.s.

Chapter 4 Results: The effect of MNPs on the cell viability of SH-SY5Y The impact of homemade and commercial MNPs (Fe3O4 NPs) on cell viability Both types of MF increased the neurite outgrowth of SH-SY5Y cells, but AC MF only enhanced cell viability. MNPs can respond to external magnetic fields so Fe3O4 NPs were tested. MNPs could be guided by EMF in further experiments to exploit the features of EMF and MNPs together, but we needed to test whether MNPs affect cell viability. Since the difference between the impact of coated and uncoated MNPs on the cell viability of SH-SY5Y cells is currently unknown, we first investigated their impact on cell viability.

TEM images of homemade and commercial MNPs

Homemade MNPs were tested (Fe3O4 NPs) first, after being synthesised by team member Dean Cardillo in the Innovation campus of University of Wollongong. The NPs were between 10 – 15 nm and were uncoated (bare NPs), but unfortunately this led to their aggregation so these clusters and aggregated NPs increased to around 50 – 80 nm (Figure 4.1).

Figure 4.1: TEM image of homemade MNPs. The scale bar of A and B image is 20 nm. However, the scale bar of C image is 50 nm.

Because of the issue with aggregation in vitro we also tested commercial MNPs (Fe3O4 NPs) purchased from Blacktrace Holdings Ltd, UK. These NPs were 20.16 ± 0.8 nm. The NPs were in powder form and coated with oleic acid to reduce aggregation and produce monodispersed NPs when dispersed in the solution (Figure 4.2).

Figure 4.2: (A – C) TEM images of the commercial Fe3O4 NPs. (D) particles size distribution of Fe3O4 NPs which indicate that the high ratio of Fe3O4 NPs is around 20 nm. NPs were dispersed in a toluene (organic solvent) to take the TEM images. The scale bar of (A) image is 110 nm, (B) image is 60 nm, and (C) image is 20 nm. DLS test of homemade and commercial MNPs Three records of both types of MNPs were collected from the particle analyser instrument, as shown in Figures 4.3 and 4.4. Because the homemade MNPs were uncoated they aggregated to different size clusters, as shown in Figure 4.3. There were various percentages of NPs between 250 – 1000 nm, some of which were less than 10 % as record 1 shows, however the ratio of NPs around 500 nm was 65 %, as record 2 shows. Also, the percentage of NPs which were less than 500 nm was around 45 %, as shown in record 3.

90 Record 1 80 Record 2 70 Record 3 60 50 40

Intensities Intensities 30 20 10 0 0 500 1000 1500 2000 Particle size / nm

Figure 4.3: DLS test of homemade MNPs which indicate the particle size distribution in distilled water. However, because the commercial MNPs were coated with oleic acid, which is hydrophobic, they were dispersed in ethanol absolute (organic solvent) which destroyed the surfactant of MNPs that caused the NPs to aggregate, as shown in Figure 4.4. The percentage of NPs between 1000 – 1250 nm was 30 %, as record 1 shows, whereas the percentage of NPs of 500 nm was more than 80 %, as record 2 indicates. There were two different percentages of NPs sizes, the first percentage was 15 % related to sizes between 500 – 1000 nm while the second percentage was around 20 % associated to sizes between 5 – 6 µm, as record 3 shows.

90 Record 1 80 Record 2 70 60 Record 3 50 40

Intensities Intensities 30 20 10 0 0 1000 2000 3000 4000 5000 6000 Particle size / nm

Figure 4.4: DLS test of commercial MNPs which indicate the particle size distribution in ethanol absolute.

Cytotoxicity assessment of both types of MNPs

Homemade MNPs The serial dilution of MNPs was assessed using AlamarBlue assay. There was no real effect on cell viability for the different concentrations of NPs, apart from 60 and 75

µg/ml of it (overall repeated measures one – way ANOVA findings F (9, 20) = 5.908, p = 0.0005). A post hoc analysis revealed a significant decrease in cell viability for the 60 and 75 µg / ml (p < 0.05 for 60 µg / ml, and p < 0.01 for 75 µg / ml, Figure 4.5).

Figure 4.5: The cytotoxicity of serial dilution of homemade MNPs. AlamarBlue assay was conducted to assess the cytotoxicity of theses concentrations. There were only two real differences compared to the control (C). The p – value of 60 µg/ml vs. (C) is *p < 0.05 while it was **p < 0.01 for 75 µg/ml vs. C. Commercial MNPs An AlamarBlue assay was also carried out to test the cytotoxicity of commercial MNPs, but since the DLS test indicate that large clusters of NPs had formed because the surfactant for some NPs had been destroyed by the ethanol absolute, we tested the impact that a narrow range of NPs concentrations (5, 10, 15, 20, 25, 30, 37.5, 45, and 50 µg / ml) made on cell viability (overall repeated measures one – way ANOVA findings

F (9, 50) = 0.882, p = 0.5475, Figure 4.6); theses concentrations had no real impact on cell viability. It was therefore suggested that the cytotoxicity of a wide range of MNPs (10, 20, 30, 40, 50, 60, 75, 90, and 100 µg / ml) be tested; there were no real changes in 37 the wide range of NPs (overall repeated measures one – way ANOVA findings F (9, 20) = 0.1332, p = 0.9981, Figure 4.7).

Figure 4.6: The cytotoxicity of narrow range of commercial MNPs concentrations. An AlamarBlue assay was carried out to determine the impact that these concentrations made on SH-SY5Y cell viability; there was no real difference compared to the control (C).

Figure 4.7: The cytotoxicity of wide range of commercial MNPs concentrations. An AlamarBlue assay was carried out to determine how these concentrations would impact on SH- SY5Y cell viability; there was no real difference compared to the control (C).

Chapter 5 Discussion

5.1 AC and DC MF Previous studies have reported that cell proliferation in various cell models has increased because of ELF-EMF exposure (Grassi et al., 2004, Wolf et al., 2005). The results of our cell viability that follow AC MF treatment do not agree with the results reported recently by Ma and co-workers (Ma et al., 2016). Even though eNSC and SH- SY5Y neuronal cells were treated with the same strength field of AC MF (1 mT), Ma et al used lower frequencies (50 instead of our 100 Hz), but the cell proliferation of both types of neuronal cells is changed. Also, in this study the cell proliferation findings following DC MF treatment do not show any significant change.

Both sets of conditions (4 hours / day over 2 or 3 days) increased neuronal differentiation starting on Day 3, and with RA, and the neurite outgrowth (neurite length and neurite branch points) increased from Day 3 to 7 for both types of MF, but there was no increase in neurite length or the number neurite branch points after the treatment expanded from 2 to 3 days with and without RA. However, increasing the time exposure without RA reduced this effect on neurite length and the number of neurite branch points, especially for AC MF. This result occurred because the 3 day treatment by AC MF led to a slight decrease in confluence, as shown in Figure 3.6 H compared to Figure 3.4 H. The impact of MF on reactive oxygen species (ROS) has been described (Wang and Zhang, 2017), but the influence of MF depends parameters that contain the exposure time, frequency, the intensity of MF, and increasing or decreasing the ROS level within the cells (Wang and Zhang, 2017). In this study, when the exposure time increased from 2 to 3 days, the ROS level might increase within the cell culture and reduce the cell activity.

Previous research has also reported that treating eNSCs, PC12 cell, and dorsal root ganglia (DRG) with a very low requency electromagnetic field (ELF-EMF) could increase the neurite outgrowth (Zhang et al., 2006, Macias et al., 2000, Ma et al., 2016), but an 1800 MHz radiofrequency electromagnetic field (RF-EMF) reduced the neurite

39 outgrowth of NSCs (Chen et al., 2014). Therefore, this study focused on the impact that low frequency EMF exposure may have on the neurite outgrowth of SH-SY5Y cells. The findings of this research into the neuronal differentiation of SH-SH5Y cells are compatible with the results of neuronal cells previously reported by (Ma et al., 2016, Zhang et al., 2006, Macias et al., 2000). Our results are compatible with theirs due to the large neurite outgrowth of neuronal cells after exposure to AC MF.

The mechanism for inducing neurite outgrowth by exposure to ELF-EMF has already been reported and discussed (Ma et al., 2016). In a growing brain, Ngn1 and NeuroD which belong to pro-neuronal basic helix-loop-helix (bHLH) factors that organise the neurite outgrowth of NSCs (Imayoshi and Kageyama, 2014, Britz et al., 2006). Ma and co-authors found that the expression for proneural genes NeuroD and Ngn1, which are crucial for neuronal differentiation and neurite outgrowth, had increased after the exposure of NSCs to 1 mT, 50 Hz of AC MF for 4 hours / day over 3 days, but exposure to 1800 MHz (RF-EMF) resulted in down-regulation for pro-neuronal genes NeuroD and Ngn1 thereby decreasing the neurite outgrowth of NSCs (Chen et al., 2014). Previous studies also reported that members of the Mammalian TRPC family contributed to the neuronal differentiation and proliferation of NSCs, and various members of it participated in regulating neurite outgrowth (Li et al., 2012, Greka et al., 2003). TRPC1 enhanced the neurite outgrowth in PC12 cells whereas TRPC5 decreased it (Heo et al., 2012). Furthermore, EMF exposure for NSC largely improved the intracellular Ca+2 coupled by significantly up-regulating the gene expression of TRPC1 (Ma et al., 2016). Ma and co-workers also found that the Tuj1+ neurons which differentiated from NSCs expressed TRPC1, showed that TRPC1 may help to regulate neurite outgrowth (Ma et al., 2016).

Many instruments were used to apply an AC electromagnetic field in the previous studies, however, instruments such as a coil, power amplifier, and function generator are more expensive than permanent magnets, and they can become damaged when connected to electricity. The permanent magnets used in this study are safer than electromagnetic devices because they are not connected to the power source. Therefore, the DC MF source (permanent magnets) used in this study is simpler, safer, and more cost-effective (since consumable-free) than the AC electromagnetic equipment that was previously used. Permanent magnets can also produce stronger magnetic fields than the

AC electromagnetic equipment because it can reach almost 300 mT or higher depending on the size of the magnet. Previous studies also stated that the neuronal differentiation of different cell models was enhanced after treating the cells with a DC electric field (Kobelt et al., 2014, Ariza et al., 2010, Zhao et al., 2015). This study also described a novel method and DC MF source that is simple to apply and very efficient. The impact of DC MF on cell viability matched the culture confluence, with both results showing that DC MF did not enhance the cell proliferation of SH-SY5Y neuroblastoma cells. Moreover, the results of this study associated with the influence of DC MF on neurite differentiation might be compatible with the latest findings reported by Zhao et al. (Zhao et al., 2015). They indicated that a DC electric field can enhance directional migration and stem cell differentiation, suggestively facilitated by calcium influx through DC electric field exposure.

Regarding the mechanism of inducing neurite outgrowth by DC MF, we hypothesize that DC MF would work as AC MF and it might induce the gene expression of pro- neuronal genes such as NeuroD and Ngn1, which are crucial for neuronal differentiation and neuronal extension. DC MF may also increase the gene expression of the Tuj1+ neuron that expressed the TRPC1 which facilitated the Ca+2 influxes.

RA induces neuronal differentiation and limits the proliferation of neuronal-like cell lines and neural progenitors (Janesick et al., 2015), however, long periods of differentiation (beyond 8-10 days) with RA are problematic due to the accumulation of proliferating cells (Encinas et al., 2000). RA efficiency is selective for neuronal cells because it is not as good at differentiating between induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). The efficiency of neural differentiation in iPSCs was much lower than ESCs (Hou et al., 2014), so the neuronal differentiation experiments carried out in this study were tested for up to 7 days. We also found that DC MF can promote the neurite extension of neuroblastoma cells in the absence of RA so DC MF would be the best option to replace RA to induce neuronal differentiation beyond 8 – 10 days. We have thus identified a simple, cost-effective method of differentiating SH-SY5Y cells without using expensive reagents such as RA.

5.2 Fe3O4 NPs AC and DC MF increased the neurite outgrowth of SH-SY5Y cells, whereas AC MF only enhanced cell viability because MNPs can respond to external magnetic fields 41

(Chomoucka et al., 2010), and the iron might include the metabolism of mammalian cells such as DNA replication and electron transport in mitochondria (Laskey et al.,

1988); Fe3O4 NPs was included in this research.

We found that treating SH-SY5Y cells with 60 and 75 µg / ml of homemade Fe3O4 NPs led to a large decrease in their viability; this might belong to the aggregation that happened while preparing the serial dilution in D.W because the MNPs were not coated with any type of surfactant to prevent their aggregation and sedimentation in the cell culture; it might also belong to the cellular uptake of uncoated NPs because the cellular uptake of IONPs depends on the coating characteristics and the size of the nanoparticles (Safi et al., 2010). Remember that the doses of IONPs with which cells were treated are presented, but these values do not always represent the true amount of internalised iron (Safi et al., 2010). This may cause many problems because the amount of internalised IONPs are made with an NP dose, a coating composition, the coating thickness and media composition (Safi et al., 2011), all of which may influence the levels of mutagenicity. It must therefore be established that the dose of nanoparticles contributes to but is not solely responsible for the cellular internalisation of IONPs (Galimard et al., 2012).

Furthermore, these findings are not consistent because there was no mono – distribution for the aggregated NPs among the serial dilutions of uncoated NPs. A non-mono distribution of the clusters will lead to this kind of trend, but there was no significant increase or decrease in cell viability after treating the cells with both ranges of commercial NPs. Nevertheless, the SEM in both ranges of coated NPs concentrations was wide and inconsistent with the various concentrations. A high range of SEM issue can be related to the destroyed surfactant of some NPs because the ethanol absolute causes some clusters and aggregated NPs. These clusters might not be distributed in an inhomogeneous way because the different serial dilutions of NPs caused a wide range of SEM. The coated MNPs had a lower cytotoxic impact on the viability of SH-SY5Y cells than uncoated NPs. Chen and co-workers used a single cell line to show that coated Fe3O4 NPs caused less DNA damage than uncoated Fe3O4 NPs (Chen et al., 2012). The cytotoxic effects of nanoparticles can be (Soto et al., 2007, Gutwein and Webster, 2004), attributed to the formation of reactive oxygen species that cause

42 oxidative stress (Salvi and Holgate, 1999, Shi et al., 2003). However, the mechanism of these toxic effects is yet to be established.

Modern technology and biomedical sciences have taken quite a leap with advancement in production and the use of nanoparticles, but like any other entity, nanoparticles have their fair share of limitations because some studies suggest nanoparticles may have adverse effects on biological cells such as mitochondrial damage, oxidative stress, chromosomal and oxidative DNA damage, changes in cell cycle regulation and denaturation of proteins (Nel et al., 2006, Carlson et al., 2008, Karlsson et al., 2008). The cytotoxic effects of nanoparticles can be (Soto et al., 2007, Gutwein and Webster, 2004), attributed to the formation of reactive oxygen species that cause oxidative stress (Salvi and Holgate, 1999, Shi et al., 2003). The toxicity of nanomaterials when interacting with Biosystems are rather difficult to carry out as there is no adequate characterisation of the materials used in such biological studies (Nunes et al., 2012, Cellot et al., 2010).

A great deal of research has been carried out over the past twenty years on the adverse effects of iron, particularly its potential to induce necrosis in mammalian cells by generating a reactive oxygen species through the Fenton reaction (Bacon and Britton, 1990, Gutteridge, 1986, Kotamraju et al., 2002, Quinlan et al., 2002). The importance of iron for the normal metabolism of mammalian cells is undeniable because it is involved in DNA replication and electron transport in mitochondria (Laskey et al., 1988). For these reasons, iron oxide nanoparticles are the subject matter of this study.

In summary, coated and uncoated Fe3O4 NPs did not enhance the viability of SH-SY5Y cells, but two different concentrations (60 and 75 µg / ml) of uncoated MNPs significantly reduced cell viability. Therefore, coated MNPs combined with Y2O3 NPs are the best option for future research.

5.4 Future direction of the research Further research should be undertaken to test the expression of the proneural genes, NeuroD1 and Ngn1, and the intracellular calcium level to understand the mechanism underlying DC MF and AC EMF on the proliferation and differentiation of SH-SY5Y neuroblastoma human cells. With regards to how a combined DC / AC MF affects cell viability and neurite outgrowth, different conditions for combined DC / AC MF must be

43 optimised. Further research should also be undertaken to examine the cellular up-take of

Fe3O4 NPs or the internalisation of these NPs by using flow cytometry and confocal microscopy methods to determine where the NPs are located because it could help to declare whether the cytotoxicity of NPs occurred within the medium or inside the cells. Moreover, knowing their locations can help us understand the cytotoxicity and mechanism of the toxic effects of MNPs.

In this study we have found that the AC and DC MF increase the neurite outgrowth of

SH-SY5Y cells, and AC MF can enhance cell viability, and the coated Fe3O4 NPs did not cause any significant change in cell viability compared to the uncoated ones.

A magnetic field can induce the neuron cells to increase their viability and neurite outgrowth, and it might also control the migration and direction of the neurites.

Fe3O4 NPs guided by EMF can also help to induce the cells chemically and mechanically, while the iron of MNPs might be involved in the metabolic process of mammalian cells such as DNA replication and electron transport in mitochondria. The mechanical tension via the pull for the membrane of neuron cells by the Fe3O4 NPs and EMF might increase the elongation of the neurites; in fact, both processes would induce the neuron cells chemically and mechanically.

5.5 Conclusion The outcomes of this research have indicated the impact of external magnetic fields (AC and DC) and magnetic nanoparticles (Fe3O4 NPs) on SH-SY5Y neuroblastoma cells. The viability of SH-SY5Y neuroblastoma cells was not significantly changed following AC MF treatment, and there was also no real change following DC MF treatment. SH- SY5Y cells under external magnetic fields (either AC or DC), with or without RA, increased the neurite outgrowth (neurite length and neurite branch points) without negatively affecting cell viability or cell confluency. Without RA, both types of magnetic fields increased the neurite outgrowth after being treated for 4 hours per day for either 2 or 3 days. However, 2 days of treatment for both types of MF was better at promoting neurite outgrowth than treatment for 3 days, and DC MF was better at promoting neurite length and neurite branch points than AC MF. This study also established a positive association between the surface coating and biocompatibility of the nanoparticles.

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Appendix (Preliminary Collaborative Study on application of Yttria ceramic nanoparticles for ROS scavenging)

Introduction

1.1 Yttrium oxide nanoparticles (Y2O3 NPs)

Antioxidant NPs such as Yttrium oxide NPs (Y2O3 NPs) can work as a free radical scavenger to reduce oxidative stress (Ghaznavi et al., 2015, Hosseini et al., 2015). Treating cells with magnetic nanoparticles (MNPs) might cause them to die, especially cells with uncoated MNPs; uncoated MNPs are more cytotoxic than coated MNPs.

Chen et al. used a single cell line to show that coated Fe3O4 NPs caused less DNA damage than uncoated Fe3O4 NPs (Chen et al., 2012).

It has been previously reported that Y2O3 NPs can protect neuron cells. Schubert and co- authors found that yttrium oxide NPs are not toxic to cell culture and can protect a HT22 hippocampal nerve cell line from oxidative stress induced by exogenous glutamic acid (Schubert et al., 2006). Schubert and co-workers observed three different explanations for how Y2O3 NPs protects cells from the oxidative stress: (i) it may work directly as antioxidant NPs; (ii) it may inhibit the production of Reactive oxygen species

(ROS) in HT22 by stopping one step of the programmed cell death pathway; (iii) Y2O3 NPs might directly reduce the production of ROS that quickly excites the ROS defence system before completing the cell death program induced by the glutamate (Schubert et al., 2006).

1.2 Oxidative stress Oxidative stress is a component of numerous neurodegenerative diseases such as Alzheimer’s disease (Querfurth and LaFerla 2010) that can be caused by intracellular and extracellular sources. With intracellular sources, mitochondria are the major source of ROS (Wang and Zhang, 2017). 52

Oxidative stress can be caused by mitochondrial dysfunction and is a result of an abnormal generation of ROS that could result in neuronal death (Querfurth and LaFerla 2010, Huang and Mucke, 2012, Tu et al., 2014, Kamenetz et al., 2003). The oxidative phosphorylation process that occurs inside the mitochondria provides cells with adenosine triphosphate (ATP) to maintain cellular energy homeostasis. The reactive oxygen species (ROS) by-products produced during this procedure can lead to mitochondrial dysfunctions (Federico et al., 2012, Yun and Finkel, 2014, Lin and Beal, 2006, Wang and Zhang, 2017). Peroxisome is another organelle considered to be another major source of intracellular ROS; this because xanthine oxidase in the - membranes of peroxisomes generates hydrogen peroxide and •O2 (Wang and Zhang, 2017). However, since NADPH oxidases (nicotinamide adenine dinucleotide phosphate- oxidase) are one of the extracellular sources of ROS, NADPH oxidases are considered to be one of the family of membrane bound-enzymes that face the extracellular space to produce ROS, especially in some immune system cells such as macrophage and neutrophils cells (Wang and Zhang, 2017, Zhu et al., 2016).

1.3 Detoxification of ROS The elimination for ROS can be assisted by antioxidant enzymes which work as a free radical scavenger or through non-enzymatic molecules such as vitamins (A, C, and E), flavonoids, and glutathione. Most of the many different organisms developed an antioxidant protection system to scavenge extreme ROS. The antioxidant defence system of these living organisms consists of enzymes such as glutathione peroxidase (GSH-Px), glutathione reductase (GSH-R), catalase (CAT), and superoxide dismutase (SOD) or non-enzymatic compounds. SOD, which is one of these antioxidant enzymes, is crucial to eliminate the toxicity of ROS. The glutathione S-transferase (GST), (GSH- Px), glutathione (GSH), and glutathione reductase GSH-R create the glutathione system. GSH helps to reduce the oxidative stress caused by disulfide bonds of cytoplasmic proteins, but oxidative stress can also be reduced by changing the bonds of cytoplasmic proteins to cysteines by GSH, during which GSH will be oxidised to glutathione disulphide (GSSG) (Wang and Zhang, 2017).

1.4 Advanced Multifunctional Materials for site-specific free radical scavenging Alzheimer is a neurodegenerative disease, it is the most common type of dementia and it influences almost 10% of the aged community who are in their sixth or more decade of life. Mitochondrial dysfunction is the result of an abnormal generation of reactive oxygen species (ROS) that can result in neuron death (Querfurth and LaFerla 2010, Huang and Mucke, 2012, Tu et al., 2014, Kamenetz et al., 2003).

The oxidative phosphorylation process which occurs inside mitochondria provides cells with adenosine triphosphate (ATP) to keep cellular energy homeostasis. By- products of the reactive oxygen species (ROS) processed during this procedure can lead to mitochondrial dysfunction (Federico et al., 2012, Yun and Finkel, 2014, Lin and Beal,

2006).

Ceria nanoparticles (CeO2) NPs use their catalase-mimetic activities and superoxide dismutase mimetic to defend cells against the superoxide and hydrogen peroxide that are the two major products of reactive oxygen species (Karakoti et al., 2010, Celardo et al., 2011, Karakoti et al., 2009). Kwon and co-workers synthesized small and positively charged triphenylphosphonium ceria (TPP-ceria) NPs capable of targeting mitochondria in several cell lines. To sum up, in vitro and in vivo TPP-ceria NPs have shown they are biocompatible and can scavenge mitochondrial reactive oxygen species (ROS) capable of decreasing oxidative stress (Kwon et al., 2016).

The research of Lee and co-authors aimed to evaluate the antioxidant activities of mixture nanorods such as Au @ Pt NPs. They found it can block and prevent any radical production could cause oxidative stress in various line cells. In vitro experiments have shown that Au@Pt NPs present a cellular defence against oxidative stress that is encouraged by hydrogen peroxide (H2O2). They assessed in vitro the antioxidant capability of Au@Pt NPs by measuring the cell viability of many different line cells such as Chinese hamster lung fibroblasts and human prostate cancer cells under oxidative stress encouraged by H2O2. In summary, their experiments showed that Au@Pt NPs provides a cellular defence against oxidative stress that is encouraged by hydrogen peroxide (Lee et al., 2014).

Furthermore, cerium and yttrium oxide nanoparticles were tested in the research carried out by Schubert and co-workers; they found that even though these types of nanoparticles had a cytotoxic impact on the cells, it has features which directly act as antioxidant NPs to reduce the volume of ROS and thereby reduce the possibility of cell death. These kinds of NPs can be utilised to limit the oxidative stress caused by ROS in a biological system (Schubert et al., 2006).

Moreover, antioxidant NPs such as Yttrium oxide NPs (Y2O3 NPs) can also work as a free radical scavenger to reduce oxidative stress (Ghaznavi et al., 2015, Hosseini et al., 2015), so Yttrium oxide NPs were used in this study to reduce the extreme ROS level in the cell culture induce by H2O2.

1.5 Aim

To test the antioxidant impact of Yttrium oxide nanoparticles (Y2O3 NPs) on the oxidative stress induced by H2O2 in vitro

The nanoparticles were synthesised and provided by Mr. Dean Cardillo, member of the Nanoceramics for Health Protection (NCHP) group at the Innovation Campus working under the supervision of Group Leader Dr. K. Konstantinov. This study is a part of a broad collaborative research program of the team dedicated to application of functionalised nanoceramics for controlled oxidative stress in biological systems.

2. Experimental works and design SH-SY5Y cell culture SH-SY5Y neuroblastoma cells (94030304, Sigma) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) / F12 pH 7.4 (Life Technologies, 12500096), supplemented with 10 % foetal bovine serum (FBS, Bovogen), and 1 % penicillin/streptomycin (PS, Life Technologies, 15140-122). The cells were kept in culture for up to 22 passages and maintained at 37 °C in a 5 % CO2 atmosphere; they routinely underwent mycoplasma testing (MycoAlert, Lonza) and were confirmed by STR profiling.

The cytotoxicity test of homemade Y2O3 NPs The cytotoxicity of Yttrium oxide NPs was assessed by the AlamarBlue assay (ThermoFisher, DAL1100). The SH-SY5Y neuroblastoma human cells were subcultured in 96 – well / flat – bottomed (FB) microplates (Interpath Services, 655180X) by using DMEM / F12, 1 % PS, and 10 % FBS. On the first day the cells were plated a concentration of 12000 / well cells were seeded in the plates. The Y2O3 NPs were dispersed in a phosphate buffer saline (PBS) prepared at IHMRI by technical officers (TOs). The ingredients of PBS were Potassium Chloride, Astral Scientific, BIOPB0440-500G, Potassium Phosphate, Bio – Strategy, VWRC0781-1KG, Sodium Chloride, ThermoFisher, AJA465-5KG, and Sodium Phosphate, Astral Scientific, BIOS0404-500G. On the second day the cells began treatment with three different concentrations of Yttrium oxide NPs (5, 15, and 25 µg / ml). PBS was used as a control (1:1000 medium), and AlamarBlue reagent (1:100 PBS) was added 48 hours later. The fluorescent intensity of the composed reagent from AlamarBlue reagent and SH-SY5Y cells was read on the plate reader (BMG - POLARstar Omega) 2 hours after adding the cell viability reagent to the cells.

The cytotoxicity test of hydrogen peroxide (H2O2)

The cytotoxicity of H2O2 (Sigma – Aldrich, H1009) was also tested by the AlamarBlue assay (ThermoFisher, DAL1100). SH-SY5Y neuroblastoma human cells were subcultured in 96 – well / flat – bottomed (FB) microplates (Interpath Services, 655180X) by using DMEM / F12, 1 % PS, and 10 % FBS. On the first day the cells were plated, a concentration of 12000 / well cells were seeded and on the second day, the cells were treated with three different concentrations of hydrogen peroxide (0.025, 56

0.05, and 0.075 milliMolar). D.W was used as a control (1:1000 medium), and AlamarBlue reagent (1:100 PBS) was added 48 hours later. The fluorescent intensity of the composed reagent from AlamarBlue reagent and SH-SY5Y cells was read on the plate reader (BMG - POLARstar Omega) 2 hours after adding the cell viability reagent to the cells.

Free radical scavenging test of Y2O3 NPs An AlamarBlue assay (ThermoFisher, DAL1100) was carried out to see whether Y2O3 NPs would work as a free radical scavenger; in this experiment, hydrogen peroxide was used to induce oxidative stress. The three aforementioned concentrations of Y2O3 NPs and H2O2 were evaluated together, but each concentration of Yttrium oxide NPs was assessed by treating it separately with three separate concentrations of hydrogen peroxide. The SH-SY5Y neuroblastoma human cells were subcultured in 96 – well/ flat – bottomed (FB) microplates (Interpath Services, 655180X) using DMEM / F12, 1 % PS, and 10 % FBS. On the first day of plating the cells, a concentration of 12000 / well cells were seeded in the plates, and on the second day the cells were treated with the three different concentrations of Yttrium oxide NPs mentioned above. Each concentration of NPs was treated 1 hour later with the three concentrations of hydrogen peroxide mentioned above. The PBS used a control (1:1000 medium), and AlamarBlue reagent (1:100 PBS) was added 48 hours later. Free radical scavenging test has assessed by the same process of testing the cytotoxicity of Yttrium oxide NPs. The fluorescent intensity of the reagent composed from AlamarBlue reagent and SH-SY5Y cells, was read on the plate reader (BMG - POLARstar Omega) 2 hours after adding the cell viability reagent to the cells.

TEM images of the homemade and commercial MNPs TEM images of the homemade Y2O3 NPs were taken by NCHP team member Dean Cardillo in the Innovation Campus of University of Wollongong.

Statistics

The data for H2O2 and Y2O3 NPs are given as the mean ± standard error of the mean (SEM) from at least 4 independent replicates. Statistical differences were identified after confirming the normal distribution by one repeated measures using ANOVA with a Tukey post hoc comparison, as appropriate, in Graph Pad Prism software.

3. Results

The impact of antioxidant NPs on SH-SY5Y viability Because dispersing iron oxide NPs had some issues, they were difficult to work with in the aqueous solutions required for cell culture. Due to their aggregation, the NPs remained in the culture medium and were not internalised into the cells (data not shown). Therefore, Yttrium oxide NPs (Y2O3 NPs) was used to test the impact that this type of uncoated NP would have on cell viability. To assess whether Y2O3 NPs could protect against oxidative stress, different concentrations of uncoated Y2O3 NPs and

H2O2 were tested on cell viability. Many different concentrations of Y2O3 NPs were optimised before the three different concentrations mentioned below were used to treat the neuron cells (data not shown). Moreover, many different concentrations of H2O2 were also optimised before the three concentrations of hydrogen peroxide mentioned below were chosen to continue with this research (data not shown).

TEM images of homemade Yttrium oxide Y2O3 NPs:

The homemade Y2O3 NPs with sizes in the range of 80 – 120 nm were synthesised by team member Dean Cardillo in the Innovation campus of University of Wollongong, Institute for Superconducting and Electronic Materials (data not shown).

Cytotoxicity of Y2O3 NPs There was no real difference between the concentrations of Yttrium oxide NPs (5, 15, and 25 µg/ml) compared to control, thus showing that Y2O3 NPs have no negative impact on cell viability (overall repeated measures one – way ANOVA findings F (3, 9) = 3.318, p = 0.1514, Figure A.1). We then moved to assess whether the NPs could protect against oxidative stress.

Figure A.1: The cytotoxicity of Y2O3 NPs on cell viability. An AlamarBlue assay was carried out to illustrate the toxic impact of Yttrium oxide NPs on cell viability; there was no real difference between the findings compared to the control (C).

Cytotoxicity of H2O2 The AlamarBlue assay carried out to check the cytotoxicity of hydrogen peroxide indicated that 0.025, 0.05, and 0.075 mM of hydrogen peroxide had negative effects on cell viability (overall repeated measures one – way ANOVA findings F (3, 9) = 8.231, p = 0. 0179, Figure A.2). A post hoc analysis identified large reductions in cell viability for a range of hydrogen peroxide concentrations (p < 0.05 for 0.025 and 0.05 mM, and p < 0.01 for 0.075 mM, Figure A.2).

Figure A.2: The cytotoxicity of H2O2 on cell viability. There were large differences between all the results of different concentrations of hydrogen peroxide compared to the control (C). 59

Free radical scavenging test of Y2O3 NPs To test the anti-oxidant impact of the three different concentrations of Yttrium oxide

NPs, H2O2 was used to induce oxidative stress within the cell culture. Each concentration of Yttrium oxide NPs was treated separately with three different concentrations of hydrogen peroxide (Figure A.3). Interestingly, the only real differences were between the results of (5 µg/ml of Yttrium oxide NPs + 0.025 mM of

H2O2) compared to the same concentration of hydrogen peroxide (0.025 mM) alone

(overall repeated measures one – way ANOVA findings F (3, 12) = 10.74, p = 0.0379, Figure A.3 A). A post hoc analysis identified a large increase in cell viability for (p < 0.01 for 5 µg / ml + 0.025 mM), but no real change in cell viability for (15 µg / ml + 0.025 mM, and 25 µg / ml + 0.025 mM) compared to 0.025 only (Figure A.3 A).

However, when the dose of H2O2 was increased to 0.05 mM, both 5 µg / ml + 0.05 mM, whereas 15 µg / ml + 0.05 mM compared to 0.05 mM caused a large reduction in cell viability (overall repeated measures one – way ANOVA findings F (3, 12) = 7.854, p = 0.0307, Figure A.3 B). A post hoc analysis revealed a large decrease in cell viability (p < 0.05 for 5 µg / ml + 0.05 mM, and p < 0.01 for 15 µg / ml + 0.05 mM, Figure A.3 B). Nevertheless, 25 µg / ml + 0.05 mM, and 0.05 mM did not change cell viability compared to the control (Figure A.3 B). Furthermore, when the dose of hydrogen peroxide was increased to 0.075 mM, the cell viability of all the different samples treated with NPs and H2O2 compared to control, had decreased significantly (overall repeated measures one – way ANOVA findings F (3, 12) = 11.05, p = 0.0207, Figure A.3 C). A post hoc analysis identified a large decrease in cell viability (p < 0.01 for 5 µg / ml + 0.075 mM, p < 0.05 for 15 µg / ml + 0.075 mM, and 25 µg / ml + 0.075 mM, Figure A.3 C), which indicates that the NPs were only able to protect against hydrogen peroxide at low, not high concentrations.

Figure A.3: The anti-oxidant impact of three different concentrations (5, 15, and 25 µg / ml) of Y2O3 NPs for the oxidative stress induced by three different concentrations (0.025, 0.05, and 0.075 mM) of H2O2. (A) Indicates the effect of NPs concentrations on 0.025 mM. (B) Illustrates the influence of NPs concentrations on 0.05 mM. (C) Shows the impact of NPs concentrations on the 0.075 mM. 61

4. Discussion

The ability to reduce the oxidative stress of Yttrium oxide NPs (Y2O3 NPs) induced by

H2O2, was tested by checking the viability of SH-SY5Y cells treated with different concentrations of hydrogen peroxide.

The findings suggested that 5 µg/ml of NPs only worked as free radical scavengers to reduce the oxidative stress induced by 0.025 mM. It did not work with higher concentrations of hydrogen peroxide possibly due to the increase of hydrogen peroxide cytotoxicity after increasing its concentration. The aggregation and sedimentation of NPs reduced the active surface area of NPs which work as free radical scavengers, thereby increasing their cytotoxicity. Since Yttrium oxide is not coated, the aggregation and sedimentation of NPs occurred increased while running the experiment as the concentration of NPs increased; this in turn led to more cytotoxicity than with a lower concentration. Other concentrations of Y2O3 NPs (15 and 25 µg/ml) did not reduce the oxidative stress. The reasons why 15 and 25 µg/ml would not work as free radical scavengers might be related to the contribution of cytotoxicity between the high concentrations of Yttrium oxide NPs and hydrogen peroxide, or to the aggregation and sedimentation of high concentrations of NPs.

Yttrium oxide NPs can work as free radical scavengers to reduce the oxidative stress induced by hydrogen peroxide; this feature could help to cure neurodegenerative diseases such as Alzheimer and Dementia, which are results of mitochondrial oxidative stress. Mitochondrial dysfunction is a result of an abnormal generation of reactive oxygen species (ROS) that could result in neuron death (Querfurth and LaFerla 2010, Huang and Mucke, 2012, Tu et al., 2014, Kamenetz et al., 2003). The oxidative phosphorylation process which occurs inside the mitochondria provides cells with adenosine triphosphate (ATP) to keep cellular energy homeostasis, and the by-products of reactive oxygen species (ROS) produced during this procedure can lead to mitochondrial dysfunctions (Federico et al., 2012, Yun and Finkel, 2014, Lin and Beal, 2006).

There is some evidence that the toxicity of nanoparticles is due to the generation of reactive oxygen species (ROS) (Buzea et al., 2007), so studies are being carried out to examine the causes of these toxicities. The results so far indicate that the interaction of 62 metal oxide nanoparticles cells has caused DNA damage of all sorts i.e. chromosomal aberrations, DNA strand breakage, oxidative DNA damage and mutations (Koedrith et al., 2014). Therefore, the difference between MNPs and antioxidant NPs is that antioxidant NPs can reduce oxidative stress while MNPs can generate it. 5. Future direction of the research

Further research should also be undertaken to examine the cellular up-take of Y2O3 NPs or the internalisation of these NPs by using flow cytometry and confocal microscopy methods to determine where the NPs are located because it could help to declare whether the anti-oxidation feature of NPs occurred within the medium or inside the cells. Moreover, knowing their locations can help us understand the cytotoxicity and mechanism of the toxic effects of Y2O3 NPs.

In this study we have found that the Y2O3 NPs can reduce the oxidative stress induced by small concentrations of H2O2. Also, we have found that coated Fe3O4 NPs did not significantly reduced cell viability. Therefore, coated MNPs combined with Y2O3 NPs are the best option for future research.

Therefore, a combination of Y2O3 NPs and Fe3O4 NPs guided by EMF (AC or DC MF) should be considered in future research because these features can be used to cure neurodegenerative diseases by increasing cell viability, enhancing neurite outgrowth, and reducing oxidative stress. Future research should also exploit all these materials by functionalising coated Fe3O4 NPs with Y2O3 NPs. The idea is to make coated Fe3O4

NPs the core and Y2O3 NPs a shell (Y2O3 @ Fe3O4 NPs) that is guided by an external magnetic field. In this case we hypothesise that neuron cells can be induced magnetically, mechanically, and chemically, all at the same time.

The shell Y2O3 NPs can induce the neuron cells chemically, and Y2O3 NPs can reduce oxidative stress by reducing the extreme ROS level inside the neuron cells.

However, the concentrations of (Y2O3 @ Fe3O4 NPs) should first be optimised, while the cellular uptake of Y2O3 @ Fe3O4 NPs should be studied using confocal microscopy or the flow cytometry technique to determine the location of the NPs.

Even though Y2O3 NPs can reduce the oxidative stress of SH-SY5Y cells by small concentrations of H2O2, we hypothesise that Y2O3 NPs might reduce the oxidative stress induced by high concentrations of hydrogen peroxide if primary neuron cells are used rather than neuroblastoma cells. For these reasons, primary neuron cells should be considered in these kinds of experiments. 6. Conclusion

The outcomes of this preliminary study have indicated the impact of antioxidants NPs on SH-SY5Y neuroblastoma cells. This study showed that Y2O3 NPs can protect neuronal cells against oxidative stress induced by H2O2 causing mitochondrial dysfunction. We found that Yttrium oxide NPs could work as free radical scavengers to reduce the oxidative stress induced by hydrogen peroxide in SH-SY5Y cells culture.