James Rudman Novel light-activated antibacterial surfaces by James Michael Rudman A thesis submitted in partial fulfilment for the degree of Doctor of Philosophy in the Department of Chemistry University College London 1 James Rudman Declaration I, James Michael Rudman, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed:_________________________ Date:_1_3_/0_2_/2_0_1_7________ 2 James Rudman Abstract The aim of this project was to covalently link chemically modified organic dyes, such as methylene blue, toluidine blue O, or crystal violet, to the surface of a polymer, via a 1,3-dipolar cycloaddition reaction. The synthesis of variants of methylene blue and toluidine blue O was attempted; unfortunately, we were unable to synthesise any analogues from phenothiazine or from the dyes themselves. The synthesis of a crystal violet analogue via a double Grignard reaction was investigated. We were unable to isolate the desired product in the final step. Instead, two leucocrystal violet analogues were prepared by reacting appropriately functionalised tertiary anilines with Michler’s hydrol. Another leucocrystal violet analogue was prepared by reacting 4-(prop-2-yn-1- yloxy)benzaldehyde with two equivalents of N,N-dimethylaniline. The leucocrystal violet analogues were oxidised to give the corresponding crystal violet analogues, which were incorporated into polyurethane by a dip-coating process. The antibacterial activities of the resultant polymer films were assessed: each displayed differing antibacterial properties when illuminated (no activity was observed in the dark). We were unable to covalently attach any crystal violet analogues to the surface of pre-functionalised silicone, polyurethane, or poly(vinyl chloride) (PVC). Considering the differences that were observed between the antibacterial activities of the crystal violet analogues, the antibacterial activity of silicone incorporated with commercially available ethyl violet was compared with that of the same material containing crystal violet. The superiority of crystal violet over ethyl violet as a photobactericidal agent was demonstrated. Several porphyrins and metalloporphyrins were synthesised and incorporated into silicone. The resultant films were characterised by measuring their UV- Vis, IR, and fluorescence spectra. Unfortunately, due to time constraints, the antibacterial properties of these polymers were not assessed. Finally, we were unable to synthesise polyurethane with covalently attached crystal violet moieties via a polymerisation reaction. 3 James Rudman Table of Contents 1 Introduction ................................................................................................. 9 1.1 Bacterial biofilms: a brief overview ...................................................... 10 1.2 Antibiotics ........................................................................................... 10 1.2.1 Antibiotic lock therapy ................................................................... 11 1.2.2 The impregnation or coating of polymeric medical devices with antibiotics .............................................................................................. 12 1.2.3 Surface-bound antibiotics ............................................................. 14 1.2.4 The future of antibiotics ................................................................ 18 1.3 Non-metallic disinfectants ................................................................... 19 1.3.1 N-Halamines ................................................................................. 19 1.3.2 Quaternary ammonium compounds (QACs) ................................ 21 1.3.3 Quaternary phosphonium compounds (QPCs) ............................. 24 1.3.4 Antimicrobial peptides (AMPs) ..................................................... 25 1.3.5 Nitric oxide (NO) ........................................................................... 26 1.3.6 Crystal violet ................................................................................. 27 1.3.7 Triclosan ....................................................................................... 29 1.3.8 Other non-metallic biocides .......................................................... 30 1.3.9 The future of non-metallic disinfectants ........................................ 31 1.4 Metallic disinfectants ........................................................................... 31 1.4.1 Silver ............................................................................................ 31 1.4.2 Copper .......................................................................................... 35 1.5 Light-activated antibacterial surfaces .................................................. 36 1.5.1 Photocatalytic surfaces ................................................................. 37 1.5.2 Surfaces incorporated with photosensitiser molecules ................. 39 2 Results and discussion ............................................................................. 49 2.1 Preparation of alkyne-functionalised dyes, and their antibacterial activities .................................................................................................... 50 2.1.1 Methylene blue analogues ............................................................ 50 2.1.2 Toluidine blue O analogues .......................................................... 54 2.1.3 Crystal violet analogues via a Grignard reaction .......................... 55 2.1.4 Leucocrystal violet analogues from Michler’s hydrol..................... 59 2.1.5 Leucocrystal violet analogues from aryl aldehydes ...................... 67 4 James Rudman 2.1.6 Oxidation of leucocrystal violet analogues .................................... 71 2.1.7 Physical and biological characterisation of crystal violet- polyurethane samples ........................................................................... 73 2.2 Attempted grafting of alkyne-functionalised dyes to a variety of polymer surfaces .................................................................................................... 76 2.2.1 Synthesis of a mono-amine, mono-azide-terminated PEG linker . 77 2.2.2 Attempted 1,3-dipolar cycloaddition reaction between a PEG linker and an alkyne-functionalised dye .......................................................... 78 2.2.3 Attempted modification of silicone ................................................ 80 2.2.4 Attempted modification of polyurethane ....................................... 83 2.2.5 Attempted covalent attachment of a PEG linker to PVC ............... 84 2.2.6 Modification of PVC with sodium azide......................................... 85 2.2.7 Attempted grafting of an alkyne to an azide-functionalised PVC film via a 1,3-dipolar cycloaddition ............................................................... 91 2.3 Ethyl violet as an alternative to crystal violet? .................................... 94 2.3.1 Preparation and characterisation of dye-incorporated polymer samples ................................................................................................. 95 2.3.2 Antibacterial activity of dye-incorporated polymer samples .......... 97 2.4 Assessment of the antibacterial activity and physical properties of silicone incorporated with different porphyrins .......................................... 98 2.4.1 Synthesis of related analogues of meso-tetraphenylporphyrin (TPP) ..................................................................................................... 98 2.4.2 Preparation and characterisation of porphyrin-incorporated polymer films ..................................................................................................... 104 2.5 Attempted formation of polyurethane with a covalently attached crystal violet analogue via a polymerisation reaction ......................................... 110 2.5.1 Attempted preparation of an amine-functionalised crystal violet analogue.............................................................................................. 110 2.5.2 Preparation of a modified polyurethane film ............................... 112 3 Conclusions and future work ................................................................... 114 4 Experimental ........................................................................................... 117 4.1 Techniques, materials, and instrumentation ..................................... 117 4.2 Procedures for the synthesis of organic compounds and associated data......................................................................................................... 118 5 James Rudman 4.3 Procedures for surface modifications, incorporations, leaching experiments, and other miscellaneous experiments ............................... 142 4.3.1 Incorporation of crystal violet analogues into polyurethane ........ 142 4.3.2 Preparation of a PVC film ........................................................... 142 4.3.3 Modification of PVC with sodium azide....................................... 142 4.3.4 Incorporation of crystal/ethyl violet into medical grade silicone .. 143 4.3.5 The extent of dye leaching from medical grade silicone incorporated with crystal/ethyl violet .................................................... 143 4.3.6 Incorporation of porphyrins into medical grade silicone .............. 143 5 References.............................................................................................. 144 6 James Rudman Acknowledgements Firstly, I would like to thank Mike and Ivan for their support throughout. Their respective doors were always open for frequent chats regarding the direction of my project, which threw its fair share of challenges in my path. I am sure that without their guidance, I would not have reached this stage. I would also like to pay tribute to many fellow PhD students at UCL, past and present. There are surely too many people to list here, but I’ll highlight a few people. Helen and Tom, who were there from start to finish. Chris, Emily, Farzaneh, Oli, and Steve: I enjoyed many fascinating discussions about chemistry as well as other unrelated topics with you all. Kealan, Dave, Natasha, and Alex for being fantastic neighbours in the lab. Will, who helped me with a number of experiments throughout my time at UCL. There are many others who I won’t mention here. It goes without saying that I am sure I’ll stay in contact with you all for a very long time. Without a number of great friends outside UCL, I wouldn’t be the whole and rounded person I am today. I would like to mention Liam and Marv, two of my closest friends and people I can always rely upon when the going gets tough. Finally, without the love and support of my family and Deborah, who I met almost 2 years ago (at the time of writing), I would surely have not got through this. I love you all very dearly and hope you will always remember that, no matter what happens. 7 James Rudman Abbreviations AMP antimicrobial peptide CFU colony forming units DCM dichloromethane DMF N,N-dimethylformamide DMSO dimethyl sulfoxide EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ePTFE expanded polytetrafluoroethylene F TPP meso-tetra(2,4,6-trifluorophenyl)porphyrin 12 F TPP meso-tetra(pentafluorophenyl)porphyrin 20 F TPP meso-tetra(4-fluorophenyl)porphyrin 4 ISC intersystem crossing MDI 4,4’-methylenebis(phenylisocyanate) NBS N-bromosuccinimide PBS phosphate buffered saline PEG poly(ethylene glycol) PVC poly(vinyl chloride) QAC quaternary ammonium compound QPC quaternary phosphonium compound ROS reactive oxygen species TEM transmission electron microscopy THF tetrahydrofuran TPP meso-tetraphenylporphyrin XPS X-ray photoelectron spectroscopy 8 James Rudman 1 Introduction The development of bacterial biofilms on polymeric surfaces is a serious cause for concern, particularly in places such as hospitals, where the chance of immunocompromised patients contracting infections is greatly heightened. Despite the implementation of rigorous cleaning protocols, hospital surfaces are continuously subjected to further bacterial contamination. Moreover, and perhaps more alarmingly, medical implant devices such as catheters are frequently colonised by bacteria, regardless of whether medical staff adhere strictly to proper insertion techniques. This results in the proliferation of malignant infections within the hospital environment, which can lead to the death of patients in some instances. The development of robust antibacterial polymeric surfaces that prevent the attachment of bacteria and subsequent biofilm formation, would therefore appear to be an excellent strategy to combat this problem. In recent years, numerous antibacterial polymeric surfaces have been developed. Broadly speaking, antibacterial surfaces can be divided into three categories: those that kill bacteria, those that resist the adhesion of bacteria, and those that release attached bacteria.1 The main focus of this project was the development of light-activated antibacterial surfaces that kill bacteria; therefore, those that resist bacterial adhesion, or release attached bacteria, will not be discussed here. For more information about surfaces with these properties, the reader is directed towards a number of publications and reviews that discuss a range of topics including quorum sensing inhibition,2,3 the use of biosurfactants to disperse bacterial biofilms,4 surface micropatterning,5 the utility of zwitterionic coatings,6,7 and the development of either super-hydrophobic or highly hydrophilic surfaces.8-11 In addition, there are a number of broader reviews that discuss these topics, and many others, in excellent detail.12-16 In the following sections, the antibacterial properties of a variety of different antibacterial surfaces, prepared in a number of different ways, will be discussed. The utility of light-activated antibacterial surfaces is considered in 9 James Rudman significant detail, as the production of such materials was the main focus of this project. 1.1 Bacterial biofilms: a brief overview A bacterial biofilm is a sessile bacterial community encased within a hydrated polymeric matrix, which consists of proteins, polysaccharides, teichoic acids, and extracellular DNA. The first stage of biofilm formation involves the attachment of planktonic bacteria to a surface by various means, such as with pili, which are proteinaceous outer membrane structures. Once attached to the surface, some bacteria are capable of secreting “slimes” that engulf the bacteria present, and effectively bind them irreversibly to the surface. The bacterial colonies that form within this biofilm matrix are extremely difficult to eradicate: they are up to 1000 times more resistant to biocides than planktonic bacteria. The intermittent release of planktonic bacteria from a mature biofilm means that new biofilms can form elsewhere, or it can result in a persistent infection where indwelling devices, such as catheters, are concerned (Figure 1).17-20 Figure 1. A simplified schematic detailing each phase of biofilm development. 1.2 Antibiotics The most common way of preventing, or combatting, medical device related infections involves the use of antibiotics such as penicillin, rifampicin, minocycline, vancomycin, and cefoxitin. An antibiotic is a compound that is 10