The Design and Assembly of Tailored Oligosaccharides as Polymer Therapeutics for Improved Treatment of Chronic Respiratory Disease A thesis submitted to Cardiff University in partial fulfilment of the requirements for the degree of Doctor of Philosophy February 2019 Joana Stokniene Advanced Therapies Group School of Dentistry Cardiff University United Kingdom ii Acknowledgements I would like to express my sincere gratitude to my supervisors Professor David Thomas, Dr Elaine Ferguson and Dr Katja Hill for the patient guidance, endless encouragement and valuable advice throughout my PhD. I would also like to acknowledge the Research Council of Norway and the biopharmaceutical company Algipharma AS for funding my PhD studentship (Grant number: 228542/O30). I am grateful to all my friends and colleagues with whom I have had the pleasure of working throughout this research project. Special thanks to Dr Mathieu Varache, Dr Lydia Powell and Dr Manon Pritchard for your wonderful support, guidance and invaluable help. I would like to express my very great appreciation to Dr Olav Aarstad (Norwegian University of Science and Technology), Dr Diego Andrey and Dr Owen Spiller (Cardiff University) for your kind help in the laboratory. Finally, I wish to thank my family for their unconditional support and enthusiastic encouragement throughout my PhD study. iii Abstract Healthcare-associated infections affect 4 million patients annually in the EU and result in an estimated 37,000 deaths per year. Of particular concern are the rapidly increasing resistance rates of Gram-negative bacterial pathogens to many, or even all, commonly used antibiotics, with a corresponding decrease in the design/development of new antibiotic compounds. Thus, there remains an urgent need for novel therapies for these ‘hard to treat’ infections. Fortunately, polypeptide antibiotics, such as colistin and polymyxin B, still retain potent antimicrobial activity against multidrug resistant (MDR) Gram- negative pathogens. In addition, polymer therapeutics are finding increasing utility as antimicrobial agents. The aim of this thesis was to generate and characterise an alginate oligosaccharide (“OligoG”)-polymyxin conjugate library to optimise the antimicrobial functions of these last resort drugs. Reproducible conjugation of polymyxin to OligoG was achieved using amide or ester cross-linkers, producing conjugates with 6.1-12.9% (w/w) antibiotic loading and molecular weights of 14,500-27,000 g/mol (relative to pullulan MW standards). TNFα ELISA and MTT assays revealed that OligoG conjugation significantly decreased inflammatory cytokine production and cytotoxicity of colistin (2.2-9.3-fold) and polymyxin B (2.9-27.2-fold) from a human kidney cell line. Minimum inhibitory concentration (MIC) assays and bacterial growth curves demonstrated that antimicrobial activity of the OligoG-polymyxin conjugates was similar to that of the parent antibiotic, but with more sustained bacterial growth inhibition. Importantly, ester-linked conjugates showed full retention of the antibiotic’s antimicrobial activity, while the MIC of the amide- linked conjugates increased by more than 2 log-fold. Confocal laser scanning microscopy revealed that both amide- and ester-linked colistin conjugates significantly disrupted the formation of P. aeruginosa biofilms and induced bacterial death. An in vitro ‘time-to-kill’ experiment using A. baumannii indicated that colistin and OligoG-ester-colistin conjugates reduced viable bacterial counts (~2 fold) after 4 h, with no significant activity observed with OligoG-amide-colistin conjugates. OligoG-induced disruption of the 3- dimensional architecture and clumping of P. aeruginosa and E. coli biofilms was demonstrated using a Transwell diffusion model and biofilm disruption assays, while fluorescent labelling of OligoG confirmed its rapid diffusion and distribution within the whole biofilm structure. These studies confirm that bi-functional polymer therapeutics such as OligoG- polymyxin conjugates have potential benefits in the treatment of MDR Gram- negative bacterial infections. iv Contents Thesis title ........................................................................................................ i Declaration ...................................................................................................... ii Acknowledgements ........................................................................................ iii Abstract .......................................................................................................... iv Contents ......................................................................................................... v List of Figures ................................................................................................. x List of Tables................................................................................................ xiv Abbreviations ............................................................................................... xvi Chapter 1: General Introduction 1.1 Introduction .............................................................................................. 2 1.2 Antibiotics in clinical development ............................................................ 5 1.3 Bacterial cell wall structure....................................................................... 7 1.4 Mechanisms of antibiotic resistance ...................................................... 10 1.4.1 Biofilms ........................................................................................... 14 1.5 Modification of polymyxin ....................................................................... 16 1.6 Nanomedicines as novel antibiotic therapies ......................................... 17 1.6.1 Polymer therapeutics ...................................................................... 20 1.6.2 Enhanced permeability and retention effect .................................... 23 1.7 Polymeric carrier .................................................................................... 24 1.7.1 Alginates ......................................................................................... 26 1.7.2 OligoG CF-5/20 ............................................................................... 28 1.8 Examples of polysaccharide-antibiotic conjugates ................................. 30 1.9 Aims of the project ................................................................................. 31 Chapter 2: General Materials and Methods 2.1 Chemicals .............................................................................................. 33 2.1.1 General chemicals .......................................................................... 33 2.1.2 Alginates ......................................................................................... 33 2.1.3 Antibiotics ....................................................................................... 33 2.1.4 Fluorescent probes ......................................................................... 33 2.1.5 Chemicals for bacterial culture ........................................................ 34 2.1.6 Chemicals for cell culture ................................................................ 34 v 2.2 Cell line .................................................................................................. 34 2.3 Bacterial isolates .................................................................................... 34 2.4. Equipment ............................................................................................. 36 2.4.1 General equipment for bacterial and cell culture ............................. 36 2.4.2 Analytical equipment ....................................................................... 36 2.4.2.1 Gel permeation chromatography (GPC) .................................... 36 2.4.2.2 Fast protein liquid chromatography (FPLC) ............................... 37 2.4.2.3 Spectrophotometry: Ultraviolet/visible (UV/vis) ......................... 37 2.4.2.4 Confocal laser scanning microscopy (CLSM) ............................ 37 2.4.2.5 Miscellaneous equipment .......................................................... 37 2.5 Sterilisation ............................................................................................ 38 2.6 General methods ................................................................................... 38 2.6.1 Purification of OligoG-antibiotic conjugates .................................... 38 2.6.2 Characterisation of OligoG-antibiotic conjugates ............................ 39 2.6.2.1 Characterisation by GPC ........................................................... 39 2.6.2.2 Characterisation by FPLC ......................................................... 39 2.6.2.3 Characterisation by BCA ........................................................... 42 2.6.2.4 Characterisation by ninhydrin assay.......................................... 42 2.6.3 Biological characterisation of OligoG-antibiotic conjugates ............ 45 2.6.3.1 Cell maintenance ...................................................................... 45 2.6.3.2 Defrosting cells .......................................................................... 45 2.6.3.3 Cell passaging ........................................................................... 45 2.6.3.4 Cell counting ............................................................................. 47 2.6.3.5 Characterisation by MTT ..........................................................