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A Microfluidic Approach to the Study of Cellular Responses to Oxidative Stress

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A Microfluidic Approach to the Study of Cellular Responses to Oxidative Stress A Microfluidic Approach to the Study of Cellular Responses to Oxidative Stress Kapiraj Chandrabalan, BSc. Thesis submitted for the Degree of Doctor of Philosophy The University of Hull and the University of York Hull York Medical School September 2015 Abstract The purpose of this study was to develop a method for exposing biological and chemical components to defined levels of (extracellular) oxidative stress, and to study their effects using conventional methods. Photodynamic therapy (PDT)-mimicking extracellular oxidative stress reactions (OSR); microbatch OSR (MBOSR) and microfluidic flow OSR (MFOSR) were carried out using colon carcinoma COLO320 cell line. An instrument was built and used in all the photosensitisation OSR. Several microfluidic chip configurations were designed, fabricated, studied for flow properties and used in the surface treatment reactions. Different types of photosensitisers were successfully covalently immobilised onto surfaces of the microfluidic channels, and their presence was verified using spectroscopic, colorimetric and photo-oxidation reactions. To conduct the MFOSR; syringe-, pressure- and peristaltic-pumping systems were tested to optimise the flow system and cell recovery was improved. Initially MFOSR with immobilised porphyrin were carried out. To determine the optimum OSR conditions, MBOSR were carried out having the photosensitiser, aluminium (III) phthalocyanine tetrasulfonate (AlPcS4) in solution, and the best set of photosensitisation effect inducing conditions were used in conducting the MFOSR with photosensitiser in solution. The treated samples were analysed to determine the levels of LDH released into supernatants, percentages of cells recovered, cell viability, and the modes of cellular disintegrations induced. MFOSR with immobilised photosensitiser showed high variations with no statistically significant differences, whereas MFOSR with AlPcS4 in solution showed significant induction of photosensitisation effects. It was identified from the MBOSR that the induction of photosensitisation effects significantly increased with the concentration of AlPcS4 or the light dose. The MBOSR and MFOSR with photosensitiser in solution were useful in the generation of extracellular oxidative stress environment. These types of reactions mainly induced cellular disintegration through necrosis relative to apoptosis. This system can possibly be adapted to study numerous diseases where oxidative stress is involved. 2 Contents Abstract .......................................................................................................... 2 Abbreviations ................................................................................................. 9 List of tables ................................................................................................. 20 List of schemes ............................................................................................. 20 Acknowledgements ...................................................................................... 21 Author’s declaration ..................................................................................... 22 CHAPTER 1 - Introduction ......................................................................... 23 1.1 Oxidative stress, diseases and photochemistry ................................... 23 1.1.1 Reactive chemical species and oxidative stress ........................... 23 1.1.1.1 Classes of reactive species .................................................... 25 1.1.1.2 Types of reactive oxygen species (ROS) .............................. 27 1.1.1.3 Detecting and measuring common ROS ............................... 33 1.1.2 Generation of ROS using photosensitisation ............................... 35 1.1.2.1 Photochemistry ...................................................................... 35 1.1.2.2 Photosensitisers ..................................................................... 38 1.1.3 Cellular oxidative stress ............................................................... 44 1.1.3.1 Effects of ROS on the cellular components .......................... 46 1.1.3.2 Measuring cellular oxidative stress ....................................... 54 1.1.4 Chemical and biological antioxidants .......................................... 56 1.2 Cancer, PDT, Oxidative stress and Immunology ............................... 58 1.2.1 Cancer .......................................................................................... 58 1.2.1.1 Stages in the development of cancer ..................................... 60 1.2.1.2 Characteristics of cancer ....................................................... 62 1.2.1.3 Cancer treatments .................................................................. 65 1.2.2 Photodynamic therapy (PDT) ...................................................... 67 1.2.2.1 Treating cancer using PDT ................................................... 69 1.2.2.2 Localisation of photosensitisers and targets of photodamage ........................................................................................................... 71 1.2.2.3 Delivering light energy ......................................................... 74 1.2.2.4 The effects of PDT on cancer................................................ 76 1.2.3 Oxidative stress (PDT) induced activation of immune system ... 77 1.2.3.1 Antitumour immunity ........................................................... 78 3 1.2.3.2 Immune response .................................................................. 80 1.2.4 The components used to study the effects of oxidative stress in vitro ....................................................................................................... 82 1.2.4.1 COLO320 cell line ................................................................ 82 1.2.4.2 Measuring the effects of oxidative stress in vitro ................. 83 1.3 Microfluidics, applications and surface treatments ............................ 89 1.3.1 Microfluidics ................................................................................ 89 1.3.1.1 Applications of microfluidics for photochemical reactions .. 90 1.3.1.2 Biological applications of microfluidic approach ................. 92 1.3.2 Fluid dynamics ............................................................................. 93 1.3.3 Microfluidic chip fabrication ....................................................... 97 1.3.4 Surface treatments ....................................................................... 98 1.3.4.1 Silanisation of glass surface .................................................. 99 1.3.4.2 Photosensitisers onto solid support ..................................... 103 1.3.4.3 Magnetic nanoparticles as a solid support .......................... 106 1.4 Research gap ..................................................................................... 107 1.5 Aims ................................................................................................. 108 CHAPTER 2 - Experimental materials and methods ................................. 109 2.1 General information and materials used in the experiments ............ 109 2.2. The instrument developed for oxidative stress reactions ................ 111 2.2.1 Instrument setup ......................................................................... 111 2.2.1.1 One LED array setup ........................................................... 111 2.2.1.2 Two LED array setup .......................................................... 116 2.2.1.3 Components of microfluidic oxidative stress reactions ...... 118 2.2.2 Light energy delivered ............................................................... 120 2.2.2.1 LED arrays as the visible light source................................. 120 2.2.2.2 LED arrays as the white light source .................................. 121 2.2.2.3 Light intensity (illuminance) emitted .................................. 122 2.2.2.4 Light energy (irradiance) emitted........................................ 124 2.2.3 Thermocouple measurements .................................................... 128 2.2.3.1 Temperature fluctuations due to heat from the light ........... 128 2.3 Pumping methods ............................................................................. 131 2.3.1 Syringe pump ............................................................................. 131 2.3.2 Pressure pump ............................................................................ 140 4 2.3.3 Peristaltic pump ......................................................................... 142 2.4 Synthesis, microfluidics and surface treatments .............................. 146 2.4.1 Synthesis of porphyrin derivatives ............................................ 146 2.4.2 Fabrication of glass microfluidic chip ....................................... 148 2.4.3 Microfluidic chip setup .............................................................. 148 2.4.4 Surface treatment of glass chips ................................................ 150 2.4.4.1 Cleaning microfluidic chips ................................................ 150 2.4.4.2 Converting cationic glass surface into anionic.................... 150 2.4.4.3 Silanisation and photosensitiser immobilisation ................. 151 2.4.5 Measurements to verify immobilisation of photosensitisers ..... 156 2.4.6 Cholesterol oxidation reactions ................................................. 158 2.5 Photosensitiser immobilised microfluidic flow
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