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OPTIMISATION of COMPUTATIONAL, BIOLOGICAL and PHYSICAL METHODS to STUDY the CELLULAR ENTRY of NANOPARTICLES By

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OPTIMISATION of COMPUTATIONAL, BIOLOGICAL and PHYSICAL METHODS to STUDY the CELLULAR ENTRY of NANOPARTICLES By OPTIMISATION OF COMPUTATIONAL, BIOLOGICAL AND PHYSICAL METHODS TO STUDY THE CELLULAR ENTRY OF NANOPARTICLES by PHILIP J SMITH A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Chemistry College of Engineering and Physical Sciences The University of Birmingham September 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Recent advances in nanotechnology have generated great potential for use of nanomate- rials in the manufacturing, cosmetic, pharmaceutical and bio-medical sectors. This has provoked great interest in utilisation of nanomaterials in disease diagnostics and treat- ment, as well as significant concern over the potential for unwanted exposure. Thus, understanding mechanisms by which nanoparticles, structures with at least one dimen- sion <100 nm, interact with cells, gain entry to the intracellular space, and potentially affect cellular functions and viability is relevant and timely. This thesis focuses on current methods for nanoparticle characterisation and anal- ysis of cellular interactions. The specific interaction between HeLa cells and 20 nm carboxylate-modified polystyrene nanoparticles is examined in detail, and the effects of serum proteins in corona formation is analysed. Fluorescence microscopy image acquisi- tion is studied, with particular attention given to colocalisation analysis. An automated program is presented, making use of a novel de-noising algorithm which makes analysis of low signal-to-noise images possible without additional input. This is validated using computer generated images, and data acquired and analysed by hand. This program was used to analyse confocal microscopy images to quantify nanoparticle colocalisation with intracellular protein markers and membrane stains. A protocol is developed to enable use of total internal reflection fluorescence (TIRF) microscopy for the study of nanoparticle internalisation. Finally, innovative TIRF methods that permit identification of areas of local plasma membrane curvature and super-resolution analysis of fluorophore motion in Z are examined, and significant steps are made to combine these into a single imaging system. Thus, this project will develop computational analysis methods, novel biological assays, and physical enhancements to existing hardware with the aim of accelerating the characterisation of nanoparticle-cell interactions. ACKNOWLEDGEMENTS A great number of people and organisations have seen me through this project; mentally, emotionally, financially and spiritually. Thank you all. First and foremost to my supervisors Dr. Joshua Rappoport, Dr. Rosemary Dyson, Prof. Jon Preece and Prof. Ela Claridge. Thank you all for your guidance, knowledge- and most notably your patience! Thank you to everyone in the Rappoport lab for persevering with the painful task of teaching Biology to a Physicist. Special mentions to Abdullah Khan and Dr. Julie Mazzolini for not only expanding my mind, but also my heart. And my muscles. Thank you to Dr. Jochen Kronjaeger for the invaluable suggestions and guidance with the building of the laser unit, and for letting me borrow a lot of expensive equipment to test it with. Thank you to all the staff and students in PSIBS, particularly Alan, Ali and James for their frequent yet welcome distractions from reality. The ESPRC for funding via a studentship through the PSIBS Doctoral Training Centre (EP/F50053X/1), and the Wellcome Trust for their additional grant. Thanks Mum and Dad for having me; I wouldn't be here without you. It's all your fault. And finally thank you to my amazing wife for not only keeping me sane, but also making me go to Australia for a year to write this thesis. CONTENTS 1 Introduction 1 1.1 Nanoparticles . 1 1.1.1 Commercial and therapeutic uses for nanoparticles . 2 1.1.2 Concerns regarding harmful effects of nanoparticles . 5 1.1.3 Environmental exposure to nanoparticles . 7 1.1.4 Protein corona . 9 1.2 Cells . 14 1.2.1 Endocytosis . 15 1.3 Optics . 20 1.3.1 Light . 20 1.3.2 Refraction . 21 1.3.3 Dispersion . 23 1.3.4 Total Internal Reflection . 24 1.3.5 Fluorescence . 26 1.3.6 Lasers . 27 1.3.7 Polarisation . 29 1.4 Microscopy . 31 1.4.1 Fundamentals . 31 1.4.2 Nyquist criterion . 34 1.4.3 Widefield microscopy . 37 1.4.4 Confocal microscopy . 39 1.4.5 Total internal reflection fluorescence microscopy . 41 1.5 Aims and objectives of the thesis . 44 1.5.1 Chapter 2: Cellular entry of nanoparticles . 44 1.5.2 Chapter 3: Development of new computational methods for the analysis of nanoparticle uptake . 44 1.5.3 Chapter 4: Effects of the protein corona on nanoparticle uptake/ Development of new biological methods for analysis of NP internal- isation . 45 1.5.4 Chapter 5: Development of a novel imaging system to study the cellular uptake of nanoparticles . 45 1.5.5 Chapter 6: Conclusions . 46 2 Cellular entry of nanoparticles 47 2.1 Overview . 47 2.2 Introduction . 48 2.3 Aims and Objectives . 48 2.4 Materials and Methods . 49 2.4.1 Cell culture . 49 2.4.2 Plate reader assay . 50 2.4.3 Dynamic and electrophoretic light scattering assay . 50 2.4.4 Cytotoxicity assay . 51 2.4.5 Nanoparticle uptake assay at 37◦C .................. 51 2.4.6 Nanoparticle uptake assay at 4◦C................... 52 2.4.7 Dynasore assay . 52 2.4.8 Dominant negative AP-2 and Caveolin 1 transfection . 52 2.4.9 Transferrin uptake experiments at 37◦C . 53 2.4.10 Transferrin uptake experiments at 4◦C . 53 2.4.11 Sytox R Green (Invitrogen) . 53 2.4.12 Imaging and image analysis . 54 2.4.13 Statistical analysis . 55 2.5 Results and Discussion . 56 2.5.1 The effects of serum on nanoparticles . 56 2.5.2 Endocytosis studies . 68 2.5.3 Nanoparticle-mediated cellular permeabilisation . 73 2.6 Conclusion . 79 3 Development of new computational methods for the analysis of nanopar- ticle uptake 81 3.1 Overview . 81 3.2 Introduction . 82 3.2.1 Requirements . 83 3.2.2 Existing Techniques . 87 3.2.3 Noise reduction methods . 99 3.3 Aims and Objectives . 100 3.4 Materials and Methods . 101 3.4.1 Determining a suitable threshold value: validation of de-noising algorithm . 101 3.4.2 Colocalisation program development . 101 3.4.3 Computationally simulated data for validation . 102 3.4.4 Experimental data for validation . 102 3.5 Results and Discussion . 103 3.5.1 Validation of new noise reduction algorithm . 103 3.5.2 Workflow . 106 3.5.3 Validation of new colocalisation program . 108 3.6 Conclusion . 114 4 Effects of the protein corona on nanoparticle uptake/ Development of new biological methods for analysis of NP internalisation 115 4.1 Overview . 115 4.2 Introduction . 117 4.2.1 Live cell imaging . 117 4.2.2 Endocytic trafficking . 118 4.2.3 Membrane stains . 119 4.3 Aims and Objectives . 120 4.4 Materials and Methods . 121 4.4.1 Nikon A1R imaging and analysis . 121 4.4.2 Cell imaging media . 121 4.4.3 High speed imaging of nanoparticles . 121 4.4.4 Transfection with fluorescently labelled Rab5 . 122 4.4.5 Membrane staining with DiI for confocal microscopy . 122 4.4.6 Olympus IX81 imaging and analysis . 123 4.4.7 Dual wavelength imaging system . 123 4.4.8 Auto-Align . 123 4.4.9 Matrigel . 124 4.4.10 Transfection with fluorescent clathrin . 125 4.4.11 Membrane staining with DiI for TIRF microscopy . 125 4.5 Results and Discussion . 126 4.5.1 Live cell imaging in serum and serum-free conditions . 126 4.5.2 Identifying and quantifying nanoparticle movement . 126 4.5.3 Colocalisation of nanoparticles to intracellular markers . 129 4.5.4 SFM data . 132 4.5.5 Making TIRF suitable for studying nanoparticle uptake: Protocol development . 134 4.5.6 Making TIRF suitable for studying nanoparticle uptake: DiI staining136 4.5.7 SFM data . 141 4.6 Conclusions . 141 5 Development of a novel imaging system to study the cellular uptake of nanoparticles 143 5.1 Overview . 143 5.2 Introduction . 144 5.2.1 Variable Angle TIRF . 144 5.2.2 Polarised TIRF . 148 5.3 Aims and Objectives . ..
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