AGELLAN-BASEDFLUIDGELCARRIERFOR SPRAY DELIVERY by BRITT TER HORST Athesissubmittedto The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Chemical Engineering College of Engineering and Physical Sciences The University of Birmingham May 2019 UNIVERSITYDF BIRMINGHAM University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Autologous cell transplantation is a promising approach to enhance burn wound re- epithelialisation. It was introduced to clinical practice decades ago with current delivery techniques involving spraying autologous cultured or uncultured cells in low-viscosity sus- pensions. This delivery method is limited since it results in an uneven distribution on the wound bed and cell loss as the liquid is not retained on the skin surface. In this thesis, a sprayable gel that solidifies on the surface of the skin has been developed to circumvent this problem. A gellan-based fluid gel system was developed with flexible viscoelastic properties that can be tuned by a biocompatible polymer concentration and ionic strength to facilitate spray delivery. The material liquefies at high shear during spraying with self-healing properties of the gel causing it to solidify on the receiving sur- face. Results present the development and characterisation of a gellan fluid gel for the encapsulation and spray-delivery of cells onto surfaces, aiming for reduced cell spillage, improved spray uniformity and maintenance of cell viability and function. Additionally, the same polymer was used to formulate a gellan film that can be rehy- drated in acetic acid for the treatment of wound infections. Gellan is a versatile polymer as shown by the formulations explored in this study and a promising candidate for cell and drug delivery applications. To my parents, who are always proud of me ACKNOWLEDGEMENTS First and foremost, I would like to express a big thank you to my supervisor, Professor Liam Grover, whose continuous support, valuable scientific advice and patience through- out my PhD has been invaluable. I extend my thanks to the NIHR SRMRC (Trauma Research) team of the University Hospitals Birmingham NHS Foundation Trust that supported me in my role as Clinical Research fellow and for giving me the chance to be involved in clinical research studies, which taught me valuable lessons on the direct and indirect influence of research on pa- tients and their care while giving me the chance to keep connected with patients. IwouldliketoacknowledgetheScarFreeFoundationforfundingthisworkandthe University of Birmingham for providing me with the opportunity to undertake my PhD project. I want to thank all the past and current members of the HTI-lab for the fun times during and after work hours. I would especially like to thank Dr Gurpreet Birdi who started me o↵in cell culture and Mr Richard Moakes for his thorough rheology input. Many thanks to Fenella Halstead for the help with microbiology. As well as the many researchers I have been lucky enough to collaborate with from the University of Glasgow under the supervison of Professor Matthew Dalby. Massive thanks goes to my amazing family for their encouragement through this pro- cess and for keeping me sane. And thanks to my dear friends that had to miss me on many fun occasions and still supported me, I would love to name you all but if you read this you know you are on the list (I’ll make an exception for Pkempen2 as promised)! Finally, Radu, you have been there during the hard times making endless trips to Birmingham, you made me proud of my work and myself. I couldn’t have done this without you. CONTENTS 1 Introduction 1 1.1 Burn injury . 2 1.2 Skin . 3 1.2.1 Epidermis . 3 1.2.2 Dermis and basement membrane . 7 1.3 Woundhealingandkeratinocytes . 8 1.3.1 The role of keratinocytes in wound healing . 8 1.3.2 Pathophysiology of burn injury . 11 1.3.3 Rationale for keratinocyte transplantation . 11 1.4 Cell transplantation technology . 12 1.4.1 Cell source . 12 1.4.2 Historical development of keratinocyte culture . 12 1.4.3 Progress towards xenobiotic free culture techniques . 13 1.4.4 Additionstokeratinocyteculture . 14 1.5 Keratinocyteviabilityaftertransplantation. 16 1.6 Methods of keratinocytes transplantation . 16 1.6.1 Introduction: grafting of burn wounds . 16 1.6.2 Cultured keratinocyte sheets . 17 1.6.3 Autologous keratinocyte transplantation in suspension . 20 1.7 Hydrogels .................................... 26 1.8 Methods of spray deposition . 42 1.8.1 Spray parameters and cell viability . 42 1.8.2 Spray systems . 43 1.9 Potential therapeutic applications . 45 1.9.1 Future approaches keratinocyte transplantation . 45 1.9.2 Future spray cell delivery systems for burns wound care . 45 1.10 Thesis overview . 46 2 Gellan-based fluid gel carrier 48 2.1 Introduction . 49 2.2 Material and methods . 51 2.2.1 Fluidgelpreparationandcharacterisation . 51 2.2.2 Rheological measurements . 51 2.2.3 Microscopyoffluidgels. 53 2.3 Contact angle measurements . 53 2.4 Spray methods . 53 2.5 Spray analysis using water sensitive paper . 54 2.5.1 HDF cell culture . 55 2.5.2 Fluidgelencapsulation. 56 2.5.3 Live/Dead assay . 56 2.5.4 AlamarBlueMetabolicActivity . 57 2.5.5 Statistical Methods . 58 2.6 Results . 58 2.6.1 Fluidgelmaterialandcharacterisation . 58 2.6.2 Biological response to fluid gel spray delivery. 69 2.7 Discussion . 72 2.8 Conclusion . 79 3 Keratinocyte spray delivery 81 3.1 Introduction . 82 3.2 Methods . 83 3.2.1 Fluidgelpreparationandcharacterisation . 83 3.2.2 Keratinocyte culture and encapsulation . 84 3.2.3 Cell sedimentation in fluid gel . 84 3.2.4 Cell viability . 85 3.2.5 Morphology . 86 3.2.6 AlamarBlueMetabolicActivity. 87 3.2.7 In-Cell Western . 88 3.2.8 Scratch assay . 89 3.3 Results . 91 3.3.1 Material characteristics . 91 3.3.2 Cell viability . 96 3.3.3 Morphological changes in sprayed cells . 98 3.3.4 Cell function . 100 3.4 Discussion . 105 3.5 Conclusion . 111 4 Formulation of acetic acid - gellan hydrogel dressing 112 4.1 Introduction . 113 4.1.1 Burn wound infection . 113 4.1.2 AceticAcidinburnwoundtreatment . 113 4.1.3 Acetic acid delivery systems . 115 4.1.4 Study rationale . 115 4.2 Methods . 116 4.2.1 Preparation gellan gel films . 116 4.2.2 Material characterisation gellan patches . 116 4.2.3 Release . 118 4.2.4 Agar disk di↵usion assay . 121 4.3 Results . 121 4.3.1 Visualappearanceofdressings. 121 4.3.2 Thickness . 121 4.3.3 Swelling capacity . 122 4.3.4 Dehydration . 123 4.3.5 Tensile strength . 124 4.3.6 Release . 125 4.3.7 Agarose di↵usion . 126 4.3.8 Porcine wound model . 126 4.3.9 Antimicrobial e↵ect . 127 4.4 Discussion . 128 4.5 Conclusion . 131 5 Conclusions and future work 133 5.1 Conclusions . 133 5.1.1 Chapter 1: Introduction . 133 5.1.2 Chapter 2: Gellan-based fluid gel carrier . 134 5.1.3 Chapter 3: Keratinocyte spray delivery . 134 5.1.4 Chapter 4: Formulation of acetic acid - gellan hydrogel dressing . 134 5.2 Limitations and future investigations . 135 Appendix A: Supplementary material Chapter 3 176 Appendix B: Spray delivery of PRP 180 B.1 Introduction . 181 B.2 Methods . 182 B.2.1 Platelet rich plasma preparation . 182 B.2.2 PRP fluid gel encapsulation . 182 B.2.3 PRP viability . 183 B.2.4 Platelet activation . 184 B.2.5 Platelet aggregation . 184 B.3 Results . 185 B.3.1 PRP preparation . ..