The in-situ structural characterization of layered double hydroxide materials in catalytic and biological applications By Christopher Hobbs Supervisor: Valeria Nicolosi A thesis in fulfilment of the requirements of the degree of Doctor in Philosophy in the School of Physics Trinity College Dublin March 2019 i Declaration I declare that this thesis has not been submitted as an exercise for a degree at this or any other university and it is entirely my own work, except for unpublished collaborative work and assistance acknowledged herein. I agree to deposit this thesis in the University’s open access institutional repository or allow the Library to do so on my behalf, subject to Irish Copyright Legislation and Trinity College Library conditions of use and acknowledgement. _________________________ Christopher Hobbs, March 2019 ii Acknowledgements First and foremost, I would like to sincerely thank my supervisor, Prof. Valeria Nicolosi, for giving me the opportunity to pursue my PhD research studies in the CPAM research group for the last 4 years. The guidance, motivation and support imparted by Prof. Nicolosi is greatly appreciated. Along with that, I would like to thank both current and past members of the Nicolosi CPAM research group. The helpful, hard-working and enthusiastic approach to our group’s scientific work has made much of my studies possible. It has been a great pleasure to work with every member of the Nicolosi group. A particular mention to Dr. Sonia Jaskaniec, who I have had the pleasure of working with throughout the four years. From teaching me how to boil water in the laboratory to designing and implementing full scientific projects, much of my PhD research is credited to the synthesis work of Dr. Jaskaniec. On that note, my sincere gratitude goes to Mr. Dermot Daly, Mr. Clive Downing and Dr. Eoin K. McCarthy for the SEM and TEM instrumentation training as well as their advice and knowledge involving numerous experiments over the last four years. Their support and teachings are owed a massive credit to my development as an electron microscopist. I also acknowledge current and past Advanced Microscopy Laboratory residents that have made for an enjoyable environment to work in every day during my time here. As part of my PhD project, I have been lucky enough to be involved in a diverse range of national and international interdisciplinary collaborations throughout my journey in the CPAM research group. In this regard, I would like to thank Prof. Fergal O’Brien (RCSI & AMBER) and the Tissue Engineering Research Group (TERG), RCSI Dublin. I am grateful of the opportunities they have provided me to learn about the research areas of tissue engineering through their research. In addition, I would like to thank the TERG members for the several samples they have provided to me for my own electron microscopy studies throughout the last four years. I would also like to iii acknowledge Prof. Adriele Prina-Mello and Dr. Dania Movia (Trinity Translational Medicine Institute, TCD & AMBER) for their helpful knowledge and provision of many samples that have been hugely beneficial to my work. My thanks also to Dr. Maurice C.D Mourad and Dr. Karl Mandel for their insightful knowledge and assistance relating to the layered double hydroxide material studies. Last, and by no means least, my sincerest gratitude goes to my family. I cannot thank them enough for their support and guidance not only for my PhD studies, but for providing me with the best opportunities throughout my education to pursue my career as a scientist. I hope I have done you proud. iv List of Publications Gilroy, D. A., Hobbs, C., Nicolosi, V., Buckley, C. T., O’Brien, F. J., & Kearney, C. J. (2017). Development of magnetically active scaffolds as intrinsically-deformable bioreactors. MRS Communications, 7(3), 367–374. https://doi.org/10.1557/mrc.2017.41 Gonzalez-fernandez, T., Sathy, B. N., Hobbs, C., Cunniffe, G. M., Mccarthy, H. O., Dunne, N. J., … Kelly, D. J. (2017). Mesenchymal stem cell fate following non-viral gene transfection strongly depends on the choice of delivery vector. Acta Biomaterialia, 55, 226–238. https://doi.org/10.1016/j.actbio.2017.03.044 Jaskaniec, S., Hobbs, C., Seral-Ascaso, A., Coelho, J., Browne, M. P., Tyndall, D., … Nicolosi, V. (2018). Low-temperature synthesis of high quality Ni-Fe layered double hydroxides hexagonal platelets. Scientific Reports, 8, 4–11. https://doi.org/10.1038/s41598-018-22630-0 Hobbs, C., Jaskaniec, S., Mccarthy, E. K., Downing, C., Opelt, K., Güth, K., Shmeliov, A., Mourad, M.C.D., Mandel, K., Nicolosi, V. (2018). Structural transformation of layered double hydroxides: an in situ TEM analysis. Npj 2D Materials and Applications, 2(4). https://doi.org/10.1038/s41699-018-0048-4 Mahon, O. R., O’Hanlon, S., Cunningham, C. C., McCarthy, G. M., Hobbs, C., Nicolosi, V., Kelly, D.J., Dunne, A. (2018). Orthopaedic implant materials drive M1 macrophage polarization in a spleen tyrosine kinase- and mitogen-activated protein kinase- dependent manner. Acta Biomaterialia, 65, 426–435. https://doi.org/10.1016/j.actbio.2017.10.041 Stauch, C., Hobbs, C., Shmeliov, A., Nicolosi, V., Ballweg, T., Luxenhofer, R., & Mandel, K. (2018). Colloidal Core–Satellite Supraparticles via Preprogramed Burst of Nanostructured Micro-Raspberry Particles. Particle and Particle Systems Characterization, 35(7), 1–9. https://doi.org/10.1002/ppsc.201800096 https://doi.org/10.1557/mrc.2017.41 https://doi.org/10.1016/j.actbio.2017.03.044 https://doi.org/10.1038/s41699-018-0048-4 https://doi.org/10.1016/j.actbio.2017.10.041 https://doi.org/10.1002/ppsc.201800096 v Ryan, E. J., Ryan, A.J., Gonzalez-Vazquez, A., Philippart, A., Ciraldo, F.E., Hobbs, C., Nicolosi, V., Boccaccini, A.R., Kearney, C.J., O’Brien, F.J. (2019). Collagen scaffolds functionalised with copper-eluting bioactive glass reduce infection and enhance osteogenesis and angiogenesis both in vitro and in vivo. Biomaterials 197, 405–416. Gonzalez-fernandez, T, et. al. 4D bioprinting using pore-forming bioinks to enable spatio-temporally defined gene delivery to stem cells (2018), submitted to Nature Biomedical Engineering. Awards Best Student Oral Presentation in the Physical Sciences, Microscopy Society of Ireland Annual Symposium, Dublin, 2016. M&M Student Scholar Award, Microscopy and Microanalysis 2018, Baltimore, MD, USA, 2018. New Researcher Award in Physical Sciences, 19th International Microscopy Congress, Sydney, Australia, 2018. vi Abbreviations  2-D: Two-dimensional  3-D: Three-dimensional  AES: Atomic emission spectroscopy  CCD: Charge couple display  EDX: Energy dispersive x-ray spectroscopy  EELS: Electron energy loss spectroscopy  EFTEM: Energy filtered transmission electron microscopy  EM: Electron microscopy  FEG: Field emission gun  FTIR: Fourier Transform Infrared Spectroscopy  GFP: Green Fluorescent Protein  HIM: Helium Ion microscopy  LDH: Layered double hydroxide  MSC: Mesenchymal stem cell  MMO: Mixed metal oxide  pDNA: plasmid DNA  SAED: Selected area electron diffraction  SEM: Scanning electron microscopy  STEM: Scanning transmission electron microscopy  TEM: Transmission electron microscopy  XRD: X-ray diffraction vii Abstract The overall aim of this PhD research is to understand the physical behaviours of layered double hydroxide (LDH) nanomaterials in applied environments. Two aspects of LDH-based applications were studied. Firstly, the thermal evolution of LDH nanomaterials were investigated using high-end (scanning) transmission electron microscopy ((S)TEM). The LDH nanomaterials underwent morphological, crystallographic and spectroscopic changes during thermal decomposition. The Ni-Fe LDHs were observed to evolve into an array of mixed metal oxides and spinel phases, shown by high resolution TEM (HRTEM), energy filtered TEM (EFTEM) and electron energy loss spectroscopy (EELS). In particular, in-situ TEM revealed the real-time processes of thermal decompositions where a nucleation and growth of an array of Nickel-based particles was observed. This NiO array was found to be embedded throughout a NiFe2O4 matrix. Similar behaviours were also seen ex-situ where STEM- EELS highlighted a segregation of Ni and Fe species upon ex-situ thermal decompositions of Ni-Fe LDH nanomaterials. Secondly, Mg-Al LDH properties were examined as they interacted with DNA based biomolecules and transfected by biological cells. It was found that the Mg-Al LDHs were successfully up-taken by mesenchymal stem cells and A549 cells. After 72 hours, the particles were observed to reside in the cytoplasm regions. Electron diffraction related studies indicated that the LDH particles retained their crystallographic nature. This was corroborated by EFTEM and STEM-EELS studies of the oxygen K edge, whilst maintaining their structural integrity. The associated findings have vital influences on the future LDH applications in the areas of catalysis, flame retardants and drug delivery. Notwithstanding these research areas, our results impact future applications as a whole, where the versatile LDH nanomaterials should be considered as prime candidates across nanotechnology. viii Thesis Summary This PhD thesis begins with the introduction to the world of nanotechnology and more precisely, two-dimensional (2-D) nanomaterials. We explore the areas of research where 2-D materials have been the subject of much scientific interest in recent times. We then focus our attention to layered double hydroxides (LDH). These versatile 2-D nanomaterials are shown to have several applications across nanotechnology such as pharmaceutics, energy storage, nanomedicine and adsorbents. In particular, we discuss in detail the recent progressions LDHs have had in catalysis and drug delivery. We address what is missing from the field from a characterization perspective. There is a clearly a need to fully understand our materials to determine structure-property relationships and to evaluate the roles of LDHs in their applications. Perhaps one of the leading characterization tools in materials science, transmission electron microscopy was our choice to characterize such materials. The instrument and associated analytical techniques that can be performed in the microscope are discussed in Chapter 2. We explain how TEM can reveal both structural and chemical information at high spatial resolutions, right down to the atomic level. The experimental results of this PhD work is presented in three experimental chapters. The first of which is entails a comprehensive TEM characterization of two types of LDHs, namely Mg-Al LDH and Ni-Fe LDH. We identify and compare the morphological and crystallographic features using TEM and electron diffraction. Moreover, the chemical information and electronic structure features of these materials are probed using energy dispersive x-ray spectroscopy and electron energy loss spectroscopy. The aging evolutions of both LDH compositions are also investigated by characterizing them at interval stages of the synthesis procedures. The high energy electron beam was observed to affect the observed LDH features. In light of this, we studied the influence electron beam exposures and imaging parameters have on the morphological (TEM), crystallographic (electron diffraction) and chemical structure ix (EELS) of the Ni-Fe LDH material. These studies provide a platform to build on as we evaluate the LDH properties in applied environments in the following chapters. Chapter 4 brings the LDHs to life at the nanoscale with in-situ TEM. These characterization methods elucidate the thermal decompositions of both Mg-Al LDH and Ni-Fe LDHs. We further show how certain structures nucleate and develop during these thermal decompositions by combining in-situ heating and energy filtered TEM methods. The thermally treated structures were then characterized themselves as an independent material. EFTEM and STEM-EELS highlight the distribution of the Ni and Fe localisations after thermal decomposition studies. High resolution TEM (HRTEM) was performed to identify the crystallographic features at high spatial resolution such as at interfacial regions. The thermal evolution in-situ study was also extended to EELS edge features. The Ni-Fe LDH was subjected to elevated temperatures and the evolution of the O K edge, Ni L2,3 edge and Fe L2,3 edge was investigated. Samples using ex-situ heat treatments were also characterized and compared to that of the in- situ experimentations. We change our tune slightly as we turn to Chapter 5. In this chapter we exemplify the versatility of the LDH materials. The behaviour of Mg-Al LDHs as potential drug delivery vectors to biological cells are studied. Firstly, we investigate how Mg-Al LDHs interact with biomedical therapeutics. The nanoscale structural and chemical properties of the composites are assessed and provide important information in relation to their suitability as gene delivery vectors. Furthermore, the up-take processes and intracellular properties of the Mg-Al LDHs involved in mesenchymal stem cells and A549 lung adenocarcinoma epithelial cells are characterized using advanced TEM methods. In particular, an EFTEM and EELS study of the Oxygen K edge revealed information about the fate of the LDH particles in the A549 cells. In parallel to this, we conducted a time dependent study of the LDH exposures. Cell culture samples were fixed at various timepoints up to 72 hours and subsequently characterized using similar TEM approaches. We conclude our experimental results and discuss impacts on the field in Chapter 6. This also includes a discussion on how the experimentations and results from this x thesis can be used as a springboard from which further strands of study could be taken. Preliminary results of in-situ liquid TEM characterizes the Ni-Fe LDHs in aqueous environments. In parallel to Chapter 3, the effect of the electron beam on the LDH structures in water is observed. In addition, the interaction of LDHs with other structures is briefly studied in both a materials science and biological science related experiments. In the case of the former, we present the formation of LDHs with other layered materials to form nanocomposites. This area has been a hot topic of research in very recent times. We demonstrate how Ni-Fe LDHs can be combined with layered MXenes. The thermal evolution of this composite is preliminarily investigated using our previous in-situ heating protocols. A particular attention is paid to the effect MXenes have, if any, on the Ni-Fe LDHs decompositions, previously studied in Chapter 4. A further nanocomposite with biomedical applications is the construction of collagen- LDH scaffolds. These could provide novel methods of effective gene/drug delivery as well as promoting favourable cell growth for tissue engineering applications. We study the extent of interaction of the Mg-Al LDHs with individual collagen fibrils, highlighted through the application of TEM spectroscopic methods. xi Table of Contents Chapter 1: Introduction ...................................................................................................... 16 1.1 2-D Nanomaterials: A Journey to Flatland ....................................................... 17 1.2 Electron microscopy: Seeing the Nanoscale ..................................................... 18 1.2.1 Electrons: Our eyepiece to the nanoscale ...................................................... 18 1.2.2 Electron beam damage .................................................................................... 23 1.3 The Cell: Uncharted territory for Electron microscopy .................................. 26 1.3.1 Eukaryotic cellular ultrastructure .................................................................. 26 1.3.2 Mechanisms of endocytosis ............................................................................ 28 1.3.3 Cell types ........................................................................................................... 29 1.4 Layered Double Hydroxides .............................................................................. 30 1.4.1 General structure of LDHs .............................................................................. 30 1.4.2 Synthesis procedures of LDHs ....................................................................... 32 1.4.3 Applications of LDHs ...................................................................................... 33 1.4.4 LDHs in catalysis .............................................................................................. 34 1.4.5 LDH as a gene/drug delivery vector ............................................................. 36 1.5 Outline of Thesis ................................................................................................... 43 1.6 Bibliography .......................................................................................................... 45 Chapter 2: Instrumentation and Experimental Techniques ........................................ 57 2.1 Transmission electron microscopy .................................................................... 57 2.1.1 Electromagnetic lens aberrations ................................................................... 60 2.1.2 Electron diffraction .......................................................................................... 61 2.1.3 Contrast in TEM ............................................................................................... 62 xii 2.2 Scanning transmission electron microscopy .................................................... 65 2.3 TEM/STEM: A microanalytical tool. .................................................................. 68 2.3.1 Energy Dispersive X-ray spectroscopy ......................................................... 68 2.3.2 Electron energy loss spectroscopy ................................................................. 69 2.4 In-situ transmission electron microscopy ......................................................... 71 2.5 Scanning electron and Helium Ion microscopy ............................................... 73 2.5.1 Scanning electron microscopy ........................................................................ 74 2.5.2 Helium Ion microscopy ................................................................................... 74 2.6 Sample preparation .............................................................................................. 74 2.6.1 TEM sample preparation for LDH samples ................................................. 75 2.6.2 Cell culture experimental details of LDH exposures .................................. 75 2.6.3 TEM sample preparation of biological cells ................................................. 76 2.7 Bibliography.......................................................................................................... 76 Chapter 3: Characterization of layered double hydroxides nanomaterials ............. 57 3.1 Introduction .......................................................................................................... 79 3.2 Experimental Methods ........................................................................................ 81 3.2.1 TEM sample preparation ................................................................................ 81 3.2.2 TEM characterization experimental details .................................................. 81 3.3 TEM and STEM characterization of Mg-Al and Ni-Fe LDH nanomaterials. .................................................................................................................... 82 3.3.1 Mg-Al LDH nanomaterials ............................................................................. 82 3.3.2 Aging characteristics of Mg-Al LDH nanomaterials ................................... 87 3.3.3 Ni-Fe LDH nanomaterials ............................................................................... 90 3.3.4 The aging properties of Ni-Fe LDHs ............................................................. 99 3.4 Electron beam interaction with LDHs ............................................................. 101 3.4.1 Low Dose Imaging of Ni-Fe LDHs .............................................................. 110 xiii 3.5 Electron beam effect on Ni-Fe LDH EELS spectra ......................................... 114 3.5.1 Energy filtered TEM of Ni-Fe LDHs ............................................................ 117 3.6 Conclusions ......................................................................................................... 120 3.7 Bibliography ........................................................................................................ 121 Chapter 4: Structural characterization of LDH materials in thermal environments ......................................................................................................................... 125 4.1 Introduction ........................................................................................................ 125 4.2 Experimental Details .......................................................................................... 127 4.2.1 TEM sample preparation ............................................................................... 127 4.2.2 In-situ TEM experimental conditions .......................................................... 128 4.2.3 Microanalytical EELS experimental details ................................................ 129 4.3 Experimental Results and Discussion ............................................................. 130 4.3.1 In-situ heating TEM experiments: Mg-Al LDH ......................................... 131 4.3.2 Thermal evolution of a LDH different composition: Ni-Fe LDH ............ 133 4.3.3 (S)TEM and microanalysis of thermally treated Ni-Fe LDHs .................. 136 4.3.4 STEM-EDX/STEM-EELS mapping of thermally evolved Ni-Fe LDHs .. 142 4.3.5 Core loss EELS study of Ni-Fe LDHs in in-situ thermal environments. 145 4.3.6 TEM studies of thermally treated Ni-Fe LDHs: heating ex-situ. ............. 147 4.3.7 TEM characterization of single particle calcined Ni-Fe LDH nanomaterials ................................................................................................................. 153 4.4 Conclusions ......................................................................................................... 161 4.5 Bibliography ........................................................................................................ 163 Chapter 5: Characterization of Mg-Al layered double hydroxide materials as a delivery vector to biological cells ....................................................................................... 125 5.1 LDH materials in bioengineered samples ...................................................... 172 5.2 Experimental Methods ...................................................................................... 176 xiv 5.2.1 Mg-Al LDH synthesis .................................................................................... 176 5.2.2 TEM preparation of cell cultures samples .................................................. 176 5.2.3 TEM characterization experimental details ................................................ 177 5.3 Mg-Al LDH only characterization ................................................................... 178 5.3.1 Mg-Al LDHs of smaller dimensions............................................................ 178 5.3.2 Mg-Al LDH interaction with plasmid DNA structures ............................ 179 5.3.3 Mg-Al LDH interaction with single stranded RNA .................................. 180 5.4 Mg-Al LDH uptake and intra-cellular behaviour in mesenchymal stem cells 182 5.4.1 Microanalysis of Mg-Al LDHs in MSCs ..................................................... 184 5.5 Mg-Al LDH as a deliver agent to A549 cancer cell lines .............................. 186 5.5.1 Mg-Al LDH synthesized in endotoxin free water ..................................... 186 5.5.2 Mg-Al LDH interaction with A549 lung cancer cell lines. ....................... 187 5.5.3 Time study of LDHs in A549 lung cancer cells .......................................... 190 5.5.4 Microanalysis of LDH properties in A549 cells using EFTEM and STEM- EELS techniques ............................................................................................................ 193 5.5.5 EFTEM analysis of LDHs in A549 cell environments ............................... 200 5.6 Conclusions ......................................................................................................... 204 5.7 Bibliography........................................................................................................ 206 Chapter 6: Conclusions & Future Work ....................................................................... 215 6.1 Characterizing Ni-Fe LDH in liquid environments ...................................... 218 6.1.1 Beam induced effects in liquid environments ............................................ 221 6.2 LDH-nanoparticle composites.......................................................................... 224 6.2.1 Ni-Fe LDH and MXene heterostructures .................................................... 224 6.2.2 Mg-Al LDH – Quantum Dot composite material ...................................... 231 6.2.3 Mg-Al LDH incorporated into collagen based scaffolds .......................... 233 xv 6.3 HIM of LDH interactions with A549 cells ...................................................... 236 6.4 In-situ liquid cell TEM analysis of whole cells ............................................... 238 6.5 Bibliography ........................................................................................................ 240 Chapter 7: Appendix ........................................................................................................ 244 7.1 Experimental Chapter 1 ..................................................................................... 244 7.1.1 Physical characterizations of Mg-Al and Ni-Fe LDHs .............................. 244 7.1.2 Electron Diffraction spot intensity analysis ................................................ 244 7.1.3 Localized STEM-EELS studies of Ni-Fe LDH platelets............................. 245 7.1.4 Aberration corrected STEM imaging of Ni-Fe LDH platelets.................. 246 7.2 Experimental Chapter 2 ..................................................................................... 247 7.2.1 In-situ heating experiments of Ni-Fe LDH nanomaterials ....................... 247 7.2.2 TEM characterization of single particle calcined Ni-Fe LDH nanomaterials ................................................................................................................. 248 7.3 Experimental Chapter 3 ..................................................................................... 249 7.3.1 Experimental details of LDH and plasmid DNA complex formation. ... 249 7.3.2 Experimental details of MSCs and LDH exposures .................................. 249 7.3.3 TEM/STEM of Mg-Al LDH and LDH-pDNA complexes ......................... 250 7.3.4 Additional Images of Mg-Al LDHs exposed to MSCs .............................. 252 7.3.5 Electron Beam damages to resin sections ................................................... 255 7.3.6 Additional LDH A549 cell images. .............................................................. 256 7.3.7 Time study of LDHs exposed to A549 cells ................................................ 257 7.4 Conclusions and Future Work ......................................................................... 258 7.4.1 In-situ liquid cell TEM characterization of Ni-Fe LDHs ........................... 258 7.4.2 LDH-MXene composites ............................................................................... 259 7.4.3 STEM imaging STEM-EDX mapping of collagen based scaffolds .......... 262 16 Chapter 1: Introduction Looking back at humanity’s scientific thoughts over the last century, one clear concept has been on the minds of many scientists, ‘small things are perplexing’. Indeed, we do not need to travel far back in history to see this obsession: A lecture series entitled ‘What is Life?’ was given by Erwin Schrodinger at our very own Trinity College Dublin in 1944. As part of his introductory lecture, he asked ‘Why are atoms so small?’, albeit a description of his own naivety to understanding living organisms. Nevertheless, a true interest at such scales was at heart. Moving forward to 1959, Richard Feynman delivered a talk entitled ‘There’s plenty of room at the Bottom’ at the American Physical Society at the California Institute of Technology. He stated, ‘enormous amounts of information can be carried in an extremely small space’. The outlook of making things smaller is also found throughout the industry world. Moore’s law states that the number of transistors doubles every year, halving the costs. This empirical law describes the evolution of the ever-famous transistor reaching small scales, an objective of all modern-day computers and smart phones. At the turn of the new millennium, rock band Blink-182 released their hit single ‘All the Small Things’, which peaked at number two in the UK singles charts. Granted, the influences of punk rock may not extend to science, but these scientific envisages of the nanoscale is shared amongst many researchers today. Several technologies have incorporated materials synthesized at these scales, so-called nanomaterials, have been incorporated into a wide range of technologies today, such as batteries, biomedical devices and even golf balls.1–3 These technologies are made possible by the processing and characterization of materials. Moreover, the associated methods allow us to expand our knowledge, develop new technologies and are truly owed great credit for the world we live in today. One fascinating and applicable aspect of the materials science world are two-dimensional (2-D) materials. 17 1.1 2-D Nanomaterials: A Journey to Flatland Two-dimensional nanomaterials are materials with very high aspect ratios in two dimensions with respect to the third. This is caused by the strong in-plane bonds but have weak out of plane bonds i.e. Van der Waals forces, giving them a ‘flat’ nature. Examples of these 2-D nanomaterials include graphene, boron nitrides, transition metal dichalcogenides, layered oxides and clays.4 They have been in the spotlight of nanomaterials research due to their excessive interesting and useful properties such as accessible surface areas, favourable interactions with other materials.4 These properties have been utilised in a variety of applications such as energy storage, nanomedicine and catalysis.5,6 2-D materials are prepared in a variety of different ways. Firstly, 2-D nanosheets have been found to be directly synthesized using chemical methods in a ‘bottom-up’ approach style. This is done by techniques such as co-precipitation to generate high- quality dispersions of thin nanosheets. This will be the basis of the materials synthesized throughout this thesis and has been thoroughly investigated by previous PhD research of our group, conducted by Dr. Sonia Jaskaniec. It is more often the case that 2-D materials are made by a ‘top-down’ style approach by mechanical exfoliation or liquid phase exfoliation. The former involves ‘ripping’ the layers from precursor bulk materials.7 This is found to produce high quality monolayer and multilayer samples but at the sacrifice of low yields. Thus, it is obvious why this is not an ideal case for high volume demands, most typically required for industrial applications. Pioneered work by our own group and fellow colleagues in the CRANN (TCD) developed methods and samples. This resulted in large scale productions whilst maintaining high quality stable 2-D material dispersions.8,9 Originally starting with graphite crystals, this liquid phase exfoliation method involved placing the material in a suitable solvent and providing energy in the form of ultrasound. This energy was sufficient to break the Van der Waals bonds between the layers resulting in stable colloidal dispersions of graphene.8,10 A breakthrough study led by our own Prof. Nicolosi discovered that this liquid phase exfoliation method could also be extended to a large number of other layered crystals. 18 This flourished the field of 2-D nanomaterials, with the generation of a wide variety of layered materials of interesting physical and chemical properties. This novel idea is today the core concept of our group’s research. The effective processing, characterization and implementation of these layered materials has led to a number of insightful and impactful scientific publications.11–15 1.2 Electron microscopy: Seeing the Nanoscale Certainly, one of the primary objectives of any scientific method is to critically assess the physical and chemical properties of our materials. Many of these procedures rely on our characterizations to effectively ‘observe’ the sample to assess such properties. For example, light microscopy techniques are capable of studying cellular behaviours, high-speed cameras are used to observe animal behaviours and even high-powered telescopes provide ways of analysing the motion of galaxies.16–18 Indeed at any length scale, a major concern is what resolution can we see with our lens system. This is no different at the nanoscale. Optical microscopy techniques simply do not suffice when we wish to characterize materials at smaller length scales. Furthermore, the design, functionality and processing of materials now requires information at high spatial resolutions to determine correct structure-property relationships as well as an optimization of related processes. This resolution gap can be overcome using electrons. A mathematical description of these benefits is described in the next subsection. The concepts and equations of the next section are based from the textbook, ‘Transmission Electron Microscopy: A Textbook for Materials Science’ by D.B Williams and C.B Carter.19 1.2.1 Electrons: Our eyepiece to the nanoscale The use of visible light microscopy, i.e. photons, presents challenges when our desired material feature we wish to characterize are beyond the diffraction limit. This is due to the wavelength of light and the attainable resolution δ is governed by the Rayleigh criterion, 19 𝛿 = 0.61𝜆 𝜇sin (𝛽) Equation 1.1 (1) Where λ is the wavelength and μsin (β) is known as the numerical aperture of the lens and it close to unity for a visible light microscope. So in the in case of visible light, i.e. photons (𝜆 ≈ 550 nm for green light), the resolution is evaluated as δ ≈ 300 nm. To ‘see’ beyond this limit, we need to make use of particles with a significantly smaller wavelength, i.e. electrons. Electron microscopy is a technique using high-energy electrons to characterise materials. The DeBroglie equation relates the wavelength of the electron to its momentum, and hence its mass m0, 𝜆 = ℎ 𝑝 = ℎ 𝑚0𝑣 Equation 1.2 (2) 𝑒𝑉 = 𝑚0𝑣2 2 Equation 1.3 (3) By comparing to the energy relationship of the electron, the wavelength λ of the electron can be related to its accelerating voltage V including a relativistic correction by the equation, λ = h √2m0eV (1 + eV 2m0c2) (4) Equation 1.4 In the transmission electron microscope (an instrument which we will discuss in great detail at a later stage), the ideal convergence angle β is determined by aperture radius r and focal length L as β = 𝑟 𝐿 . In a typical FEI Titan with an aperture diameter of 50 μm and a focal length of 2 mm, β is evaluated as 12.5 mrad. This can be extended to incorporation of spherical aberrations Cs (another topic which will be addressed in a 20 later section) where the optimum convergence angle can be evaluated as β = ( 4𝜆 𝐶𝑆 )0.25 ≈ 10 mrad. We note that for an electron microscope, 𝜇 ≈ 1 for a vacuum and sin(β) ≈ β using small angles approximations. The wavelength of the electron can be determined by the acceleration voltage of our electron source. When a routine voltage of 300 kV is applied, the electron has a wavelength value of approximately 2pm. This results in a diffraction limit δ of 0.1 nm. As the electron interacts with matter, it is subjected to scattering events by the nucleus or other electrons of the atom. This is a very important property for us to ‘see’ the nanoscale. For simplicity, we refine our discussion to the case where the electron interacts with the nucleus of the atom. A visual representation is shown in Figure 1.1. Figure 1.1 Electron scattering from single atom site. The electron is scattered through angle θ into solid angle Ω. Figure is adapted from Williams and Carter.19 The differential cross section describes the angular distribution of scattering from an atom. This is mathematically defined as, dσ dΩ = 1 2πsin (θ) . dσ dθ Equation 1.5 (5) 21 The case of high angle electron-nucleus scattering is described by the total Rutherford cross section as, 𝜎𝑅(𝜃) = 𝑒4𝑍2 16(4𝜋𝜀0𝐸0)2 . dΩ sin4 ( 𝜃 2) Equation 1.6 (6) Where Z is the atomic number of the atomic scattering site, E0 is the incident energy of the electrons and θ is the scattering angle. This cross section gives us an idea of how the electron will scatter from an atom in a material. We can increase the cross section and hence likelihood of scattering by looking at heavier elements or using an electron beam of lower energy. For example, when imaging ‘heavier’ high Z number atoms, say Carbon vs. Gold, the cross section increases by a factor on the order of 100. Similarly, using electrons of lower energy also has the same effect of increasing the cross section. A complimentary approach, incorporating the electrons wave nature, can also be used to depict how scattering from an atom occurs. We introduce the atomic-scattering factor 𝑓(θ), which is a measure of the amplitude of an electron wave as it is scattered from an atom, |𝑓(θ)|2 = dσ(θ) dΩ Equation 1.7 (7) 𝑓(𝜃) = (1 + 𝐸0 𝑚0𝑐2) 8𝜋2𝑎0 . ( 𝜆 sin ( 𝜃 2) ) 2 (𝑍 − 𝑓𝑋) Equation 1.8 (8) where 𝑓𝑋 is the atomic scattering factor for X-rays, 𝑎0 is the Bohr radius of the scattering atom and the other terms are defined as before. We can extend the elastic scattering from a single atomic site to a more realistic situation. This atomic structure factor F is a measure of the amplitude scattered from a 22 unit cell in a crystal. It is related to the atomic scattering amplitude by the relationship.19 F(θ) = ∑ 𝑓i2e2πi(hxi+kyi+lzi) ∞ i Equation 1.9 (9) This amplitude of scattering is hence dependent of the type of atom, positions of the atomic sites (x,y,z) and atomic planes (hkl). Beyond the spatial resolutions provided by electrons, there is a generation of further distinguishable signals upon interaction with matter. Figure 1.2 displays the available signals of this interaction. In addition to using diffraction and scattered signals to create contrast, TEM methods capitalise on X-rays and energy loss electrons to reveal rich chemical information about the specimen. The collection and interpretation of associated data will be discussed in the subsequent chapter. Figure 1.2: Schematic representation of the available signals upon electron-matter interaction. This figure was adapted from Williams & Carter.19 It is noted that even though there are many interesting signals from such interactions, there can also be undesirable effects induced by the incident electrons in the form of specimen modifications and even damages. The next section discusses the potential detrimental effects caused by the beam. 23 1.2.2 Electron beam damage As the electron beam interacts with a specimen, detrimental effects can be introduced. This can result in structural and chemical changes to the sample.1 The damage induced by the beam can be temporary or permanent to the specimen, but in most cases is unwanted.20 In relation to biological samples, this beam-induced damage can cause unwanted modifications of the specimen structure, morphology and composition. Radiolysis: Chemical bonds in samples such as polymers can be broken due to the inelastic scattering. This electron-electron interaction can cause polymer chains to break as well as forming free radicals, hence a change in structure may be induced.1 In particular, it is the covalent and Van Der Waals bonds are strongly affected by radiolytic damage. The inelastic scattering induces an excitation and a resulting de- excitation of electrons within these molecules, causing a potential change in electronic states. This is viewed via the changing of molecular and chemical arrangements, and a loss of crystallinity in certain cases.20 In addition, this electronic structure change can result in mass loss due to bond breakages and also can have an effect on spectroscopic techniques such as electron energy loss spectroscopy. Polymer specimens can also lose crystalline features as a result of this radiation damage.1 Knock-On/Atomic Displacement: As the electron beam penetrates close to the nucleus, energy is transferred from the incident electrons to the atomic nuclei (mass number A). This causes a dislodgment of the atoms and results in lattice defects.1 The energy transferred (E) is related to the incident electron energy (E0), deflection angle (θ) due to scattering and mass number A such that,20 𝐸 = 𝐸𝑚𝑎𝑥sin2 ( 𝜃 2 ) Equation 1.10 (10) 𝐸𝑚𝑎𝑥 = 𝐸0 465.7𝐴 (1.02 + 𝐸0 106 ) (11) 24 Equation 1.11 While the energy transfer for small θ angles is negligible, it is not the case for backscattering (𝜃 > 90°) or high values of incident energy E0.20 If the transferred energy exceeds that of the specimen’s displacement energy Ed, it can displace the atomic nuclei. As a result, properties of the material are prone to damage such as the displacement of nuclei which leads to a degradation of crystalline structures.20 For example, Aluminium has an Ed value of 17 eV, with a corresponding incident energy E0 of 180 keV.21 In particular, modern day TEMs are capable of attaining such E0 values, and hence ‘knock-on’ damage is a concern for specimens, particularly those with low atomic numbers. This damage mechanism can be reduced by lowering the incident energy E0 i.e. lowering the accelerating voltage of the TEM. A further concern of high-angle electron scattering is sputtering. This occurs when the electron beam interacts with an atom at the surface. These surface atoms are free to exit the specimen due to the lack of interstitial sites available within the material.20 The sublimation energy, Es, is the minimum energy required to emit the surface atom from the material. If the incident energy E0 is greater than that of Es, electron sputtering will occur. Egerton et. al presents the sublimation energy’s dependence on atomic number Z. It is evident that the lower-Z atoms sputter at an energy less than 200 keV. This study analyses many low-Z atomic materials and as such electron sputtering must be considered as a damaging mechanism during TEM studies.20 Electrostatic Charging: Charging of an insulating specimen is due to elastic (backscattering coefficient) and inelastic (secondary electrons) scattering. This charging occurs when the rate of incoming electrons onto the TEM specimen exceeds the rate of outgoing electrons. The current balance is presented by Egerton et. al as, I − It + VS RS = Iη(t) + Iδ(VS) Equation 1.12 (12) Where I is the incident current, It is the transmitted electron current, VS is the developed surface potential due to the beam, RS represents the effective resistance between irradiated area and surrounding regions, η(t) is the backscattering coefficient 25 and δ(VS) is an effective secondary electron yield at +VS..20 As I approaches It, as is the case with the absorbed electrons in a TEM thin specimen, VS becomes positive and thus positive charging occurs. Under these conditions, the charge balance is achieved by increasing VS.20 This can lead to an electric field that can cause electrical breakdown and migration of ions. In addition, a mechanical force capable of tearing films within the TEM sample is also possible due to charging.20 Heating: Phonons (collective oscillations of the atoms in the sample) heat the specimen resulting in damage to biological tissues and polymers.1 Heating of the specimen due to the electron beam interaction is pronounced at high current densities, however low current densities may also induce energy transfers and appreciable heating effects to samples such as organic materials and low-Z atoms, which is the case presented by the current study. Under standard TEM conditions, this heating is negligible for good conductors i.e. a high thermal conductivity.1 Conversely, insulators can be subject to substantial heating effects due to their low thermal conductivity.1 For example, when the electron probe is decreased in diameter from 1 μm to 1 nm and the current density increases by a factor of 106, the change in temperature ΔT is only approximately 1 K.20 Reducing beam damage: Ionization and heating damage can be reduced by introducing a cooling mechanism to the sample,35 for example, in-situ cooling of the TEM specimen to liquid nitrogen temperatures. By lowering the temperature, the material sensitivity and atomic mobility is reduced, rather than an alteration to the inelastic cross section. Coating of a TEM sample in a carbon/metal reduces the mass loss damage. This preventative mechanism reduces temperature variations and charging effects of the specimen30. Moreover, radiation beam damage can be attenuated by using a low electron dose. This reduces the number of electron-electron interactions. However, this technique has its drawbacks as it often results in low signal to noise ratios on the CCD cameras in TEM and annular detectors in STEM, which can be corrected for through the use of signal averaging.1 For example, bases of nucleic acids degrade completely at an electron dosage with an order of magnitude of 10-2 C cm-2 at an incident beam energy of less than 100 keV.36 26 1.3 The Cell: Uncharted territory for Electron microscopy Electron microscopy unites the worlds of biology and the nanoscale. The resolutions provided by EM methods allow for the observation and characterization of cellular ultrastructure features.22,23 Although the focus of this thesis is in electron microscopy, we briefly introduce the ultrastructural features and uptake pathways of biological cells. This will greatly assist the reader with essential background information when interpreting future aspects of this thesis. 1.3.1 Eukaryotic cellular ultrastructure Much like atoms make-up materials, cells are the basic building blocks of all living organisms. They too themselves have their own intricate nature. Eukaryotic cells are composed of organelles i.e. small cellular structures of specific function in cells. We list and illustrate the main organelles of these cells. Further details of these structures can be found in the textbook, ‘Essential Cell Biology: Fourth Edition’ by Alberts et. al.24 A schematic of cellular ultrastructures is presented below, courtesy of Encyclopaedia Britannica 2008 ©. 27 Figure 1.3 Schematic of eukaryotic cell ultrastructure. © Encyclopaedia Britannica Inc. Firstly, the nucleus is arguably the most vital organelle of eukaryotic cells. A nuclear envelope, composed of two concentric membranes, encapsulates the genetic information encoded giant chain molecules, known to us as DNA. The cell cytoplasm is the liquid enclosed by the cell membrane, containing water, salts and proteins. In amongst the cytoplasmic regions lies mitochondria. These organelles are responsible for making chemical energy in the cell. This is done by consuming oxygen and releasing carbon dioxide i.e. cellular respirators. The endoplasmic reticulum makes cell membrane-components and materials that are to be exited from the cell. The golgi apparatus acts as a storage mechanism for materials that will undergo cell excretion or transport to another cell region. Lysosomes are small irregular organelles that digest intracellular materials to release nutrients or breakdown unwanted materials. 28 Within the cell itself, there are many features of the cell that are on the micro and nano scales and hence not observable by optical light. The plasma membrane creates a barrier between the cytosol organelles from the outside media environments. The structure of the plasma membrane consists of protein- containing lipid bi-layer of about 5nm in thickness. Although it has a primary role of protecting and restricting escape of the organelles, it can also affect the cell growth, movement and morphology. Moreover, it also plays a key role in the importation and exportation of molecules to and from the cell. The flexible lipid bi-layer generates small vesicles that transport cargoes around the cell cytoplasm. The processes occur in a variety of pathways and are briefly described below. This information will be important for future interpretations of results which will be discussed in following chapters. 1.3.2 Mechanisms of endocytosis Clathrin-mediated endocytosis dominates most of the molecular uptake of eukaryotic cells. This uptake process is initiated from the clustering of endocytic coated clathrin proteins inside the cell cytoplasm. This aggregation forms to the plasma membrane region forming ‘clathrin coated pits’. Scission then occurs in the protein-rich regions and a clathrin coated membrane is created by the assistance of actin proteins. Finally, the vesicle undergoes an uncoating of the clathrin and sent further into the cell.25 Caveolin mediated endocytosis is the formation of cave-like invaginations in the plasma membrane which themselves become internalized by the cell. Larger particles are typically up-taken by phagocytosis. This involves the progressive formation of invaginations surrounding the material and then internalized within the cell.26 Macropinocytosis is a clathrin independent endocytic pathway. It entails actin mediated ruffling of the plasma membrane,27 creating macropinocytic vesicles with no coating and are considerably larger ( >200nm) than clathrin-mediated vesicles. 29 It is also noted that passive uptake processes can occur across the plasma membrane. Molecules may transmit through the lipid by-layer into the cell due to concentration balances i.e. osmosis/diffusion. 1.3.3 Cell types The work of this PhD thesis involves two types of cells. Mesenchymal stem cells (MSCs): MSCs are found in adult tissues including murine and humans. These multipotent cells have the ability to differentiate into a range of cell lines such as osteoblasts, chondrocytes and adipocytes. In addition, these cells are self- renewable, easily accessible and can be culturally expanded in vitro.28,29 Thus, this makes them primary candidates for experiments in areas such as regenerative medicine and tissue engineering. Practically speaking, there has been many successful works from our collaborators in the Tissue Engineering Research Group (RCSI, Dublin) involving mesenchymal stem cells.30–32 The familiarity with these cells lines was also a reason why we elected to use them for our LDH based experiments of this thesis. A549 cells: Scientists are still currently searching safe and efficient targeted treatments of cancer cells in the body. One example are A549 cells, which are human lung adenocarcinoma cells. They are of obvious interest in the area of therapeutic studies as lung cancer remains one of the most common types worldwide.33 The A549 cell line is often used in targeted drug delivery testing as it is based on pulmonary deliveries, with instantaneous absorption into the blood stream.34 The A549 cell line is also well studied and characterized in a variety of targeted therapy applications, making it an ideal cell line to study from our perspective.35–38 Again from a practical outlook, our collaborators in the Trinity Translational Medical Institute (TCD, Dublin) have had extensive experience with A549 epithelial cells involving nanoparticle uptake.39,40 This made for a natural progression to use them for our own LDH-based exposures in cultured in vitro experiments. 30 1.4 Layered Double Hydroxides Reverting our attention back to the world of material science and 2-D nanomaterials, we now discuss, in detail, the flagship material studied in this thesis. Layered double hydroxides (LDH) are a fascinating material of the 2-D nanomaterial family. The literature has shown that these LDH materials are relatively new to the field of mineralogy and materials science, having only been first described by Allman and Taylor in separate studies in 1968 and 1969 respectively.41,42 Having said that, the material ancestors of LDHs have been used for centuries. Talc, a similar material in the form of white magnesium powders was used for medical purposed in the 19th century, having beneficial aspects in cosmetics, pharmaceuticals and plastics in the modern era. Similarly, Mg(OH)2 i.e. brucite, was a mineral first described by mineralogist Archibauld Bruce (1777-1818, USA).43 LDHs, also known as anionic clays or hydrotalcite-like materials, attain a lamellar structure in nature, similar to that of other 2-D materials such as boron nitride and graphite. 1.4.1 General structure of LDHs The general formula of the LDH structure is given by [M1−𝑥 2+M3+ 𝑥(OH)2]𝑥+[A𝑛−]𝑥/𝑛 · mH2O Equation 1.13 (13) Where M2+, M3+ represent the divalent/trivalent cations in the layers, An- depicts the charge compensating anion and x, where an LDH crystal phase is obtained when 0.2 ≤ x ≤ 0.33. The layers themselves are similar to that of brucite, Mg(OH)2.44 In the case of the LDH structure the is a substitution of trivalent M3+ metals, gives rise to a positive surface charge in the cationic layers. Each metal site is coordinated in the centre of edge-sharing octahedrals with OH- groups at their vertices forming infinite sheets. The O-H bonding lies along the three fold axis (i.e. the basal plane) and are directed towards the vacant tetrahedral sites in adjacent layers.45 These ions lie perpendicularly to the plane of the layers and their stacking gives a three-dimensional 31 structure. Charge compensation is achieved in between the metallic layers by intercalation of negatively charged anions. In general, the positive charge is balanced by the presence of water and anionic species in the interlayers. Hence, the layers are held together by electrostatic interactions and hydrogen bonding with the intercalated species giving the LDHs their overall ‘anionic clay’ structure. The LDH material typically adopts a 3R rhombohedral or 2H hexagonal symmetry.46 Figure 1.4 displays a schematic representation of the LDH structure with viewing directions normal to the basal plane (001). Figure 1.4 Crystal schematic of typical LDH structure viewed along the [001] and [010] directions. Schematics were visualised using CrystalMakerTM. Cationic layers composed of M2+ and M3+ metals are octahedrally surrounded by OH groups. The layers are charge compensated by anionic interlayer moieties such as carbonate (CO23-), nitride (NO3-) and water (H2O). There is a wide variety of possible metal combinations within the layers such as Co, Cu, Mg, Mn, Ni, and Zn for the M2+ and similarly Al, Fe and Ga as examples of M3+ sites.47 This tuning of the material composition also extends to M2+/M3+ charge ratios of the cationic sites giving many possible compositions and stoichiometry. The range of LDH structures is also extended by the nature of the anions in the LDH interlayers. In fact, there is are large numbers of compounds that can occupy these regions. Along 32 with water molecules, interlayer inorganic anions such as Br-, Cl-. NO3-. SO42- and CO32- can be occupied within the interlayers. Furthermore, a fascinating feature of LDH materials is their ability to ‘anion exchange’ the interlayer guests, expanding the range of anionic compound that can be intercalated. In this process, LDHs can substitute their interlayer species for alternative anionic compounds.48 The literature shows a vast number of studies investigating the intercalation and subsequent release of both simple and complex anions. For example, organic chains including carboxylates and biomolecules such as amino acids have been seen to be effectively intercalated.49,50 Moreover, there is a wide range of drugs that have been shown to be successfully intercalated, as reviewed by Rives et. al.46 Previous findings have also highlighted the intercalation of coordination compounds and polyoxometalates.51 The interaction occurs as a weak bond between the host layers and guest species. This allows for the varied orientation of the interlayer molecules with their amount being dependent on the positive surface charge availabilities in the host layers.52 Another interesting property in this context is the ability of the LDHs to alter their basal spacing to accommodate a range of interlayer species for exfoliation53 or drug intercalation.54 This expansion of basal spacing were verified using x-ray diffraction and high resolution TEM approaches. More so when it comes to applications, LDH materials span a wide scope of applications across nanotechnology. This is largely due to the tunability of the potential LDH structures. 1.4.2 Synthesis procedures of LDHs A variety of different routes have been developed to prepare materials of this type. The main synthesis procedures are briefly described as follows: Co-precipitation methods: This is the slow addition of a metal containing solution to another solution engulfing the anionic species. This is accompanied by a slow increment in pH by the addition of a base or urea hydrolysis which leads to LDH precipitation.46 Controlled synthesis parameters permits the intercalation of specific anions in the interlayer. This is achieved by as well as conducting the synthesis is carbon-free environments to prevent carbonate contamination in the host galleries.47 33 Reconstruction: Another unique property is the ‘memory-effect’ of LDHs. After calcination procedures, the dehydrated and mixed oxide phases of LDHs can reform their original structures upon contact with water or suitable anionic species.55 This is often exploited to create LDH based structures. Anion Exchange: As previously mentioned, the ability of anion exchange can be utilised as a synthesis method to create. In this process, precipitated LDHs are mixed in solution with an excess of the intended anion to be intercalated.46,56 An initial anionic species is that of nitride as it is easiest to remove using these methods. 1.4.3 Applications of LDHs LDHs can be considered in a class of their own relative to other 2-D nanomaterials. This is credited to the large variety of advantageous properties of LDH nanomaterials, leading to significant interest in nanotechnology. Some of these properties include: low cost of production, simplistic fabrication methods, tunable characteristics, large number of accessible bonding sites, positively charged layers, anion exchange abilities and biocompatibility.57 The versatility of the LDH nanomaterials is shown through many different applications of LDH-based materials in research sectors such as adsorbents,58 energy storage,59 flame retardants,60,61 magnetics,62 medicine, polymer composites,63 sensors,64 and transistors.65 Figure 1.5 Various applications of LDHs. 34 Without doubt, the properties have led to LDHs establishing themselves as powerhouses in the area of 2-D materials and nanotechnology. In context of this thesis work, we will divulge our interest in two fields in which LDHs are applied: catalysis and gene/drug delivery. 1.4.4 LDHs in catalysis Generally speaking, catalysis is heavily involved across a large proportion of the manufacturing and scientific industry. The attention it has received is truly deserved and is undoubtedly important from an environmental, economic and social perspective.66–68 Particular topical energy and chemical industries rely on the design and production of effective heterogeneous catalysts. The activity, selectivity and ultimate performance of such catalysts is due to specific properties of the LDH structures. Firstly, the intrinsic 2-D nature of the nanosheets makes for a large number of highly accessible basic sites used in solid based catalysts. This is accompanied with an even distribution of cations within the layers which results in better catalytic performance. Moreover, the modulation of the size, composition and morphology of the LDHs make them attractive candidates for catalyst based studies. The flexibility of the synthesis can also lead to high catalytic activity, selectivity and stabililty.69–71 These unique LDH properties have resulted in a wide variety of applications within catalysis itself such as electrocatalysis,72 photocatalysis,73 and nanocomposite catalysis.70 Additionally, there are alternative treatments that have been used to enhance the LDH catalytic performance: Exfoliation has proven to increase the number of reactive catalytic sites, enhancing electrocatalytic performance. Wang et. al used a nitrogen and argon plasmas to exfoliate Co-Fe LDHs. They found that these exfoliation processes generated high surface areas, surplus edge and corner sites which contributed to excellent overpotentials.74,75 Similarly, Song et. al exfoliated similar Ni-Co LDH nanomaterials exhibiting favourable OER performances.76 Novel approaches have devised LDH compositions that contribute to the field of electrocatalysis. Ni-V LDHs have been reported to show high catalytic activity for water oxidation compared to the benchmark Ni-Fe LDH composition.77 Recently, the 35 development of ternary metallic LDHs have exhibited by substitution of a third metal cation into the metallic layers has led to enhanced photocatalytic activities.78–82 The catalytic behaviour of LDHs has also been enhanced by their suitable interaction with other nanostructures such as carbon nanotubes, 83 Yttrium particles,84 graphene,85 palladium86 and quantum dots.87–89 The construction of the LDHs to form nanocomposites for enhanced catalytic activities further extends to the favourable interaction with substrates such as Nickel foams and carbon supports.90,91 The thermal decomposition of LDHs results in mixed metal oxide (MMO) fabrications for electrocatalytic applications. The resulting generation of MMO phases has provided further routes of enhancing catalytic behaviour. The thermal treatment of the LDH precursors results in homogenous stable dispersions of MMOs. These hold similar attributes for catalytic studies such as basic site availability of crystal facets, good thermal stability and synergistic interactions.69,70 Strikingly, the calcination of LDH materials has also been a central interest in the catalytic field in the recent literature. Puscasu et. al utilised zinc based LDHs to generate Zn-based oxides which exhibited greater phenol degradations in photocatalysis.92 Yuan et. al reported the use of similar Zn-Al LDHs to fabricate mixed metal oxides for Cr(VI) reduction.93 In addition, Tang et. al synthesized Ni-Fe alloys from Ni-Fe LDH catalysts for CO methanation.94 A fall-back of many of these catalysis studies is the lack of high-end characterization of the associated LDH materials. Much of this literature utilises bulk techniques such as UV-Vis spectroscopy, x-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD) as characterization methods. Electron microscopy has been applied in some cases but is restricted to basic imaging whilst related crystallographic and spectroscopic studies are limited. The single particle chemical composition is overlooked. These studies would be improved by incorporating TEM spectroscopic characterizations. For example, the study of the distribution of metals such as vanadium or tertiary substitutions like Ce, Co and Cu would be of interest from a materials science point of view. 36 Electron microscopy methodologies can be important to determine correct structure- property relationships of LDHs in the fields of catalysis. The high spatial resolutions will help to understand catalytic mechanisms such as the behaviour of catalytic sites, the synergetic relationships and topotactic transformations as a result of catalytic activity. As well as this, EM could impact future designs and optimizations of catalytic materials with an outlook of a greener more efficient world. 1.4.5 LDH as a gene/drug delivery vector Without question, all aspects of healthcare are of central interest in modern society. The effective treatment of diseases and infections as well as the development of diagnostics are continuous areas of concern worldwide today. Interestingly, the roles and responsibilities of materials science in this world has intensified over the last few years from scientific and medical interests. Research today is making great strides in amalgamating the worlds of materials science, nanoscience and biology. This is owed to the ground-breaking developments in techniques and technologies from all aspects of these fields. Perhaps one of the most influential progressions that was recognised was the technique of cryogenic transmission electron microscopy with the awarding of the Nobel Prize for Chemistry in 2017, ‘for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution’. On top of this, there are also advancements that have contributed to the growth of this body of research. One interesting spoke is the application of nanoparticles to biological cells involved in tissue engineering, cancer therapy and antimicrobial treatments. Recent studies have utilised a vast range of nanoparticles such as nanotubes,95 graphene,96 iron oxide nanoparticles,97 quantum dots and minerals across a diverse range of intended purposes in biological applications.98 The fields of nanobiotechnology and nanomedicine are considered as young flourishing research fields, with the potential to produce the next generation of medical solutions. The effective delivery of genes, drugs and other therapeutics to biological cells is of central importance in the fields of regenerative medicine and tissue engineering. Recent advances in non-viral drug delivery revolve around the interaction of nanomaterials and biomaterials, presenting an increased efficiency in the successful 37 delivery of the therapeutic. In addition, the use of nanoparticles as delivery vectors allow them to be imposed as targeted drug delivery vehicles in relation to diagnostic and therapeutic applications. These layered structures provide promising properties in relation to non-viral drug delivery. LDH materials are biocompatible and as such are suitable to be used in cellular environments without a risk of cytotoxic effects. Furthermore, the large accessible positively charged surface areas is a striking feature in relation to cellular transfection. This is favourable in terms of drug delivery as the LDHs are attracted to the negatively charged cell membranes. But perhaps one of the main reasons they have attracted such interest in medical applications is the ability of the LDHs to exchange their anionic interlayer i.e. anion exchange. This process has applications in toxin removal, transition metal intercalation, drug delivery and exfoliation methods.39 The intercalation of drugs and genes has led to many successful applications of LDH materials in the medical and pharmaceutical sectors. For example, the successful intercalation of LDHs with amino acids,99 antiobiotics,100 cancer therapeutics,101,102 and vitamins103 has been previously seen. The intercalation of gene/drugs is illustrated in Figure 1.6. In the majority of these cases, the LDHs are designed to increase the stability and provide protection of the intercalated drugs as they are intended for delivery applications. Moreover, this properties of the LDHs could potentially be used in the treatment of diseases and illness. In relation to drug delivery, the negatively charged anions, e.g. NO3- or Cl-, can be replaced with biological molecules such as negatively charged plasmid DNA (pDNA). This pDNA-LDH complex can store and protect the biomolecules within the cationic layers.104 This intercalation is a very efficient process, where its high efficacy is attributed to the electrostatic and hydrophobic interactions between drug molecules and hydroxide layers, as reviewed by Xu and Lu.104 A further beneficial feature is the increased likelihood of cellular transfection due to its positive surface charge. Also, the chemical instability in the acidic environment of the cell can be considered as a constructive feature of the material, as this biodegradation assists in the delivery of therapeutics to biological cells.105 Furthermore, the morphology of the LDH platelets 38 play an important role in relation to cellular uptake and bio-distribution. Recent studies have shown that smaller particles (≈50nm) tend to have a higher gene capacity than larger particles (≈100nm).106 Figure 1.6: Schematic representation of DNA intercalation within the interlayer of LDH particles. Note that this schematic is not to scale. Various studies have investigated the uptake of DNA/protein related structures into the LDH interlayers. The studies of Choy,107 Gu,108 Masarudin,109 Nakayama110 and Wong111 portray and compare X-ray diffraction (XRD) patterns before and after the intercalation of LDHs with associated DNA structures. Wong et. al presents both XRD and TEM evidence of DNA structure intercalation. However, this work only analyses this layer expansion based on a single case. Furthermore, it is difficult to interpret the d spacing of LDH particles in TEM imaging mode as the orientation of the platelets play a vital role in such calculations. As such, this study looks to analyse the complexes using a spectroscopic electron microscopy approach. Similarly, Gu et. al presents XRD and TEM studies however in contrast to Wong, where the argument claims a morphological variance before and after intercalation synthesis is presented. Whilst these studies conclude a successful 39 intercalation via XRD pattern interpretations, Ladewig presents an indistinguishable comparison between X-ray diffractograms before and after intercalation.112 Instead the studies of Ladewig suggest a ‘wrapping’ of the plasmid DNA structures around the positively charged LDH layers, observed by TEM studies. This interaction mechanism is also postulated by Wong et. al.111 The morphological variation arguments presented by Ladewig et. al and Wong et. al lack corresponding spectroscopic data of their DNA-LDH complexes. In contrast, the work of Xu et al presents the interaction of plasmid DNA with LDH particles using spectroscopic data.113 The STEM-EDX point analysis conducted by Xu highlight a Mg, Al and P peak. The P peak, as suggested, is a result of a plasmid DNA interaction. However, the presence of this peak is open to debate, as it is not significantly above the background counts. Also, the XRD studies13-17 are typically conducted on a bulk scale and as such key nanoscale features of the biofunctionalization processes are overlooked.107–110 This study will investigate how a pDNA structure interacts with the LDH platelets from an EM perspective. In particular, the morphological and spectroscopic characterizations will determine the structures, compositions and assess the capabilities of the nanobiocomposites to be employed as gene delivery vectors. This information is vital in understanding how the structures of the vectors play a role in their cellular uptake and delivery of their therapeutic cargo. It is also a critical feature of non-viral gene delivery vectors to transfect cells i.e. delivery of nucleic acids. It is an utmost requirement that the vectors cross the cell membrane as otherwise the vectors would not be capable of delivering the proposed DNA or therapeutic in question. Chen et al. conclude that smaller LDH particles are more efficient with this delivery. However, the findings presented by these studies were evaluated using light microscopy, flow cytometry and gene expression experiments, whilst an electron microscopy approach was not considered.106 These findings are mirrored in the studies of Chung et al., where the cellular uptake of LDH particles is also investigated.114 By imposing fluorescent tagging on the LDH particles, the cellular uptake can be analysed using confocal microscopy. Chung et al. found sufficient uptake of 50nm and 100nm LDHs with the former producing higher 40 uptake efficiencies. In addition, the work of Oh et al.115 analysed the size dependence of LDHs to be accepted by the cells. The findings indicated that LDH particles of 50nm – 200nm were uptaken by cells. Also, the use of blocking specific endocytic pathways led to the finding that smaller LDH particles are uptaken via clathrin mediated endocytosis. A subsequent vital requirement for the delivery of therapeutics or genes to cells is their successful transport to the cellular nucleus. The intracellular fate of the LDH and DNA particles play a key role in relation to this condition. Factors that influence the LDH intracellular fate are the degradation of the particles, the release of the loaded therapeutic and the successful delivery to the nucleus via endocytic mechanisms. Xu et al. presents the successful delivery of LDH (rod) nanoparticles to the cellular nucleus. This was confirmed by confocal microscopy where a strong green signal is accumulated in the nucleus due to the tagged LDHs with fluorescein isothiocyanate.116 Ladewig et al. also indicates the intracellular behaviour of siRNA when combined with an LDH vector.117 Confocal microscopy confirmed the endosomal escape of the fluorescently tagged siRNA. Moreover, this study establishes that the LDH particles ‘protect’ the intercalated nucleic acids and act as a carrier to the perinuclear regions of the cell.117 In contrast, Tyner et. al 118 and Masarudin et al. investigate the delivery of green fluorescent proteins (GFP) utilising LDH particles as a gene delivery vector.109 A similar mechanism of uptake is presented where these proteins are intercalated into LDH layers, investigated using XRD analysis, where a variation in d spacing was reported.109 The LDH particles themselves were evaluated using TEM. Although a platelet structure was found, a sharp hexagonal shape was not portrayed. The advantage of using this protein is that it provides evidence of a full effective delivery to the cellular nucleus, as the green fluorescence is the result of successful gene expression in the nucleus due to the encoded green fluorescent protein in the attached therapeutic. These present the fluorescence using optical microscopy and in the case of Tyner et al., successful delivery after 24-48 hours was observed with transfection efficiencies of up to 90% in some cell lines.118 Masarudin et al. suggest a variety of approaches to explain the release of the biomolecule from the LDH host particles.109 It is postulated that the plasmid DNA is released via reverse ion exchange where the 41 plasmid DNA is exchanged with another molecule within the cellular bodies. A further postulate is the layer dissolution of the LDHs due to the acidic environment of the cell, thus releasing the therapeutic. These studies rely on the analysis of LDH uptake via optical microscopy with associated fluorescent tagging techniques. This is only suitable however for analysing larger data sets and is primarily a bulk technique. Moreover, optical microscopy is stunted by the diffraction limit and as such cannot resolve the interaction of individual LDHs with cellular membranes. This study aims to utilise EM analysis to investigate how the cells uptake the LDH nanomaterial with a particular emphasis on analysing the interaction of individual nanoparticles as they interact with cellular membranes and their structure once internalized in the cells. Furthermore, the application of EDX and EELS will enhance the characterization and detection of the LDHs in the cellular environment. This allows for the direct analysis of the size dependency of the LDH uptake and also eliminates the use of fluorescent tags, a requirement for optical microscopy. There is an evident gap in the literature in relation to the intracellular behaviour of the LDH gene delivery vector. This is again due to the resolution limits of optical microscopy methods. Thus, EM characterizations can answer many postulates associated with the fate of the LDH material once internalized by the cell. An aspect of this study is to investigate the intracellular localisation of the LDH material. This is of critical importance as it verifies the LDHs capacity to effectively deliver the intended therapeutic. This was reviewed by Kakuthi et. al20 where particles of smaller size are more efficient in relation to cellular acceptance and particles within 200 nm were uptaken via clathrin mediated endocytosis. Moreover, this review also presents an effect of the particle size on the intracellular localizations. Smaller particles (20nm) localize near the cellular nucleus in contrast to larger particles (180nm) which stay in the cytoplasmic regions20. Furthermore, Chen et al. and Ladewig et al. present the effective delivery of siRNA to cells using LDHs as a delivery vector.106,117 However, the techniques presented were limited by optical microscopy and fluorescent tagging. Thus, the local cellular environments involving the LDH material could not be characterized. 42 More interestingly, this study seeks to investigate the stability of the LDH material as it transfects the cell. There are numerous questions to be addressed in this area of study. Firstly, how the LDH maintains its structure as it enters the cellular environment is a primary concern. A breakdown of the crystalline features or platelet structures of the LDH material may occur in the delivery process. Also, the lifetime of the LDH structures throughout the drug delivery process is of certain concern, particularly in relation to cytotoxic effects. It is yet to be evidenced what happens to the LDH materials once the therapeutic is delivered. Moreover, the cellular processes involved with the LDH material, should they exist in the cell, can be accurately characterized using EM methods. The techniques of TEM along with its complimentary spectroscopic techniques of EELS will provide methods to probe these queries related to non-viral drug delivery. The analysis of afore mentioned systems using advanced EM methods presents its own technical challenges. The structural characterizations of layered materials require specific contrast mechanisms to highlight certain features of the materials such as diffraction contrast and Z contrast which will discussed in later chapters. This is also applicable to the related biomaterials of the current study as due to the low Z number and amorphous natures, effective mechanisms must be exploited to provide contrast. The characterizations and analytical methods, particularly those of EDX and EELS, also introduce a detrimental effect in the form of radiation damage. 43 1.5 Outline of Thesis This thesis aims to contribute to these growing areas of research by exploring how state-of-the-art high spatial resolution characterizations can be used to understand the properties of layered double hydroxide nanomaterials. The primary characterization technique used throughout this thesis is transmission electron microscopy. This engulfs advanced techniques within this such as in-situ TEM, energy dispersive x-ray spectroscopy and electron energy loss spectroscopy. There are several important areas where this thesis makes an original contribution to the field. This is addressed in three related research aims. Firstly, we will provide a complete TEM characterization of LDH nanomaterials, which were synthesized from scratch in our own research laboratories. The structural and chemical information is of interest from a materials science perspective but our findings also provide a reference for subsequent aims of this thesis. We next aim to unravel the mechanisms by which LDHs are used as catalytic precursors. The thermal evolutions of the LDH materials take a central focus of our characterizations in this regard. Our final objective is to elucidate behaviour of LDHs in a different application scheme. We are interested in how LDH nanomaterials employed as biomedical agents. In particular, we aim to investigate how LDHs interact with associated biomolecules intended for drug and gene delivery applications. We further seek to understand how LDHs behave in cellular environments, such as how they enter biological cells as well as their intracellular fate as a material. Overall, this thesis is comprised of 6 chapters, including this Introduction chapter. The following chapters of this thesis present experimental techniques and findings of the research carried out during this PhD. Chapter 2 describes the experimental details of the characterization techniques used during the duration of the PhD study. The experimental results and discussions are presented in Chapters 3, 4, and 5. Firstly, Chapter 3 concerns the EM characterization of two types of layered double hydroxide nanomaterials. The role of the electron beam on the associated findings is assessed in this chapter. This is followed by the experimental results from thermal decompositions of the studied LDH materials in Chapter 4. In-situ transmission electron microscopy 44 was used to characterize the associated decomposition behaviour. Chapter 5 describes the experimental results related to the features of LDH nanomaterials employed as biomedical gene delivery vectors. The uptake processes and intracellular fate of the LDH materials is the main focus of this chapter. Finally, Chapter 6 draws conclusions from the results of Chapters 3, 4 and 5. An appendix of additional data is also included. Potential avenues of future work extending from this thesis work is also proposed. 45 1.6 Bibliography 1. Jiang, C., Hosono, E. & Zhou, H. Nanomaterials for lithium ion batteries. Nano Today 1, 28–33 (2006). 2. Chavan, V. & Bartels, D. M. Graphene Core for a Golf Ball. (2017). 3. McNamara, K. & Tofail, S. A. M. Nanoparticles in biomedical applications. Adv. Phys. X 2, 54–88 (2017). 4. 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