Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Ayatallah Ghaffari, Sahand
Doctor of Philosophy - PhD
University of British Columbia
Despite numerous advantages and versatile applications, medical implants suffer from bacterial colonization and biofilm formation that can lead to many complications. Despite a great degree of effort being put into research and development of antimicrobial technologies, there remains an unmet need for a technology that can be incorporated and applied to a variety of polymeric biomaterials in various fields of medicine and in different settings to decontaminate implant surfaces and reduce biofilm formation and further infection. This thesis is an effort to offer a practical alternative antimicrobial technology using surface photosterilization of both silicone-based urinary catheters and methacrylate-based dental resin fillers that are embedded with a ruthenium (Ru) photosensitizer (PS) and are activated using a LED light source. Throughout this work, the synthesis and characterization of the PS and incorporation methods into both polymers are discussed, and a set of experiments are performed to identify the chemical, physical, and mechanical properties of the final biomaterials. Both Ru-embedded polymers underwent rigorous testing to evaluate their antimicrobial efficacy as surface decontaminating agents for both short (15 min) and prolonged (24 h) periods of LED exposure time which resulted in significant reduction in surface-adhered bacteria and biofilm maturation. These findings can pave the way for successful implementation of photosterilization as a technology with great potential to significantly reduce the extent of both catheter-associated urinary tract infection (CAUTI) and caries-inducing oral infections.
Surface Photosterilization of Implantable Biomaterials: Applications in Urology and Dentistry by Sahand Ayatallah Ghaffari B.Sc., Bogazici University, 2016 M.Sc., Bogazici University, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2024 © Sahand Ayatallah Ghaffari, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Surface Photosterilization of Implantable Biomaterials: Applications in Urology and Dentistry submitted by Sahand Ayatallah Ghaffari in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine Examining Committee: Dirk Lange, Associate Professor, Department of Urologic Sciences, UBC Supervisor Neil R. Branda, Professor, Department of Chemistry, Simon Fraser University Co-supervisor Ben H. Chew, Associate Professor, Department of Urologic Sciences, UBC Supervisory Committee Member Jayachandran Kizhakkedathu, Professor, Department of Pathology and Laboratory Medicine, UBC University Examiner David J. Granville, Professor, Department of Pathology and Laboratory Medicine, UBC University Examiner Additional Supervisory Committee Members: Adriana P. Manso, Associate Professor, Faculty of Dentistry, UBC Supervisory Committee Member iii Abstract Despite numerous advantages and versatile applications, medical implants suffer from bacterial colonization and biofilm formation that can lead to many complications. Despite a great degree of effort being put into research and development of antimicrobial technologies, there remains an unmet need for a technology that can be incorporated and applied to a variety of polymeric biomaterials in various fields of medicine and in different settings to decontaminate implant surfaces and reduce biofilm formation and further infection. This thesis is an effort to offer a practical alternative antimicrobial technology using surface photosterilization of both silicone-based urinary catheters and methacrylate-based dental resin fillers that are embedded with a ruthenium (Ru) photosensitizer (PS) and are activated using a LED light source. Throughout this work, the synthesis and characterization of the PS and incorporation methods into both polymers are discussed, and a set of experiments are performed to identify the chemical, physical, and mechanical properties of the final biomaterials. Both Ru-embedded polymers underwent rigorous testing to evaluate their antimicrobial efficacy as surface decontaminating agents for both short (15 min) and prolonged (24 h) periods of LED exposure time which resulted in significant reduction in surface-adhered bacteria and biofilm maturation. These findings can pave the way for successful implementation of photosterilization as a technology with great potential to significantly reduce the extent of both catheter-associated urinary tract infection (CAUTI) and caries-inducing oral infections. iv Lay Summary Currently, the most common solution to treat infections is antibiotics administration. Antibiotics, however, have many side effects; also, pathogens that cause diseases constantly develop resistance mechanisms against antibiotics. Therefore, different treatments are needed that do not rely on releasing any drugs into the body. One such alternative is photosterilization of surfaces that are common places for bacterial attachment, using a compound called photosensitizer which is reactive against a variety of microorganisms when receives light. The main benefits of using this method are the fact that it does not involve release of any drugs or create any by-products and it can kill a variety of microbes with no chance of recurrence. This technique is investigated in this thesis and its applicability to sterilize surfaces of different medical implants such as urinary catheters and dental filling materials is evaluated. v Preface Ethics approval was received from the University of British Columbia for animal studies conducted at the Medical Animal Facility (MAF) at Jack Bell Research Centre (JBRC), Vancouver General Hospital (VGH) under the guidance of Dr. Dirk Lange (UBC animal ethics approval no: A17-0292). This thesis was conducted under the supervision of Dr. Dirk Lange at the JBRC, and Dr. Adriana Manso at the Faculty of Dentistry, at The University of British Columbia, Vancouver, and under Dr. Neil Branda’s supervision at 4D LABS and department of Chemistry, Simon Fraser University. A modified version of Chapter 2 and Chapter 3 has been published together. Ghaffari, S., Amiri, N., Felix, D. F., Abbasidezfouli, S., Franco, F., Beaupré, S. L., Branda, N. R., & Lange, D. (2024). Surface photosterilization of implantable silicone biomaterials: Structural and functional characterization. Colloids and Surfaces, B, Biointerfaces, 233, 113637-113637 (Special issue: Biomaterial-associated infections: their development, characterization, prevention and treatment). I was responsible for concept/experiments design, demonstrating proof of concept and performing screening experiments, photosensitizer synthesis, characterization, polymer incorporation, mechanical stability, bacterial adhesion, imaging studies, data analysis and writing manuscript draft and incorporating edits received from contributors/co-authors. Demian F. Felix, Nafise Amiri, and Dirk Lange conducted animal studies. Fernanda Franco performed singlet oxygen measurements experiment. Samin Abbasidezfouli performed cytotoxicity measurements. Scott L. Beaupré designed the LED light source. Dirk Lange and Neil R. Branda supervised the whole project. vi A modified version of Chapter 4 has been published. Manso, A. P., Leite, M. L., Comeau, P., Dietrich, C., Ghaffari, S., Lange, D., & Branda, N. (2022). Exploring the use of a ruthenium complex incorporated into a methacrylate-based dental material for antimicrobial photodynamic therapy. Journal of Applied Biomaterials & Functional Materials, 20, 228080002211129-22808000221112989. I was responsible for photosensitizer synthesis and characterization, polymer incorporation, and antibacterial activity measurement. Maria L. Leite, Patricia Comeau and I performed degree of conversion and water sorption and solubility experiments. Claudia Dietrich performed flexural and shear bond strength studies. I performed all the experiments, data collection and analysis of the antimicrobial studies as well as FTIR analysis part of degree of conversion experiments, and Patricia Comeau performed the remaining data analysis and demonstration. Adriana Manso and Patricia Comeau wrote the manuscript draft and incorporating edits were received from contributors/co-authors including myself. Adriana Manso, Dirk Lange and Neil R. Branda supervised the whole project. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Symbols .......................................................................................................................... xvii List of Abbreviations ................................................................................................................. xix Acknowledgements ....................................................................................................................xxv Dedication ................................................................................................................................ xxvii Chapter 1: Introduction ................................................................................................................1 1.1 Two major types of infections plaguing the world ............................................................... 1 1.1.1 Healthcare-associated infections (HAI) and catheter-associated urinary tract infection (CAUTI) .................................................................................................................................. 2 1.1.2 Dental caries and associated infections.......................................................................... 3 1.1.3 Sepsis ............................................................................................................................. 3 1.2 Antimicrobial Resistance ...................................................................................................... 4 1.3 Polymeric Medical implants ................................................................................................. 6 1.3.1 Polydimethylsiloxane (PDMS) .................................................................................... 10 1.3.2 Methacrylate (MA) and its derivatives ........................................................................ 12 1.4 Biofilm formation ............................................................................................................... 15 1.5 Antimicrobial surface treatments ........................................................................................ 18 viii 1.6 Antimicrobial photodynamic inactivation (aPDI) .............................................................. 20 1.7 Photosensitizer classifications ............................................................................................ 23 1.8 Light sources in aPDI.......................................................................................................... 24 1.9 Hypothesis, goals, and outlines of the thesis ...................................................................... 26 Chapter 2: Synthesis and characterization of Ruthenium photosensitizer as antimicrobial photosterilization agent and incorporation in PDMS...............................................................28 2.1 Introduction ......................................................................................................................... 29 2.1.1 Ruthenium photosensitizers ......................................................................................... 29 2.1.2 Antimicrobial product development process ............................................................... 35 2.1.3 Critical to Quality trees for medical device product development .............................. 37 2.2 Experimental section ........................................................................................................... 40 2.2.1 Materials ...................................................................................................................... 40 2.2.2 Synthesis of Ru(dpp)3(PF6)2 photosensitizer............................................................... 40 2.2.3 Characterization of chemical and physical properties of Ru(dpp)3(PF6)2 photosensitizer ...................................................................................................................... 41 2.2.4 PDMS preparation and Ru(dpp)3(PF6)2 photosensitizer incorporation in PDMS ....... 42 2.2.5 LED array light source fabrication and calibration...................................................... 43 2.2.6 Leaching of Ru(dpp)3(PF6)2 from PDMS.................................................................... 44 2.2.7 Singlet oxygen detection using dimethylanthracene (DMA) as a molecular quencher 44 2.3 Results and discussion ........................................................................................................ 45 2.3.1 Synthesis of Ru(dpp)3(PF6)2 photosensitizer ............................................................... 45 2.3.2 Characterization of chemical and physical properties of Ru(dpp)3(PF6)2 photosensitizer ...................................................................................................................... 46 ix 2.3.3 PDMS preparation and Ru(dpp)3(PF6)2 photosensitizer incorporation in PDMS ....... 51 2.3.4 LED array light source fabrication and calibration...................................................... 52 2.3.5 Leaching of Ru(dpp)3(PF6)2 from PDMS.................................................................... 54 2.3.6 Singlet oxygen detection using dimethylanthracene (DMA) as a molecular quencher 55 2.4 Conclusion .......................................................................................................................... 57 Chapter 3: Incorporation of Ruthenium photosensitizer into experimental silicon-based urinary catheter and evaluation of physical, chemical, mechanical, antimicrobial, and biofilm formation properties: The case of PDMS as a soft material .......................................59 3.1 Introduction ......................................................................................................................... 60 3.1.1 The urinary system ....................................................................................................... 60 3.1.2 Urinary tract infections ................................................................................................ 61 3.1.3 Urinary implants .......................................................................................................... 63 3.1.4 Catheter-associated urinary tract infections (CAUTI) ................................................. 64 3.2 Experimental section ........................................................................................................... 67 3.2.1 Materials ...................................................................................................................... 67 3.2.2 Preliminary studies for parameter optimization of PDMS•Ru(dpp)3 .......................... 68 3.2.3 Evaluation of physicomechanical properties of PDMS•Ru(dpp)3 ............................... 69 3.2.4 In vitro evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 .......................... 71 3.2.5 Cytotoxicity evaluation of PDMS•Ru(dpp)3 ................................................................ 72 3.2.6 In vivo evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 ........................... 72 3.2.7 Evaluation of biofilm formation on PDMS•Ru(dpp)3 by imaging ....................... 74 3.2.8 Statistical analysis ........................................................................................................ 75 3.3 Results and discussion ........................................................................................................ 75 x 3.3.1 Preliminary studies for parameter optimization of PDMS•Ru(dpp)3 .......................... 75 3.3.2 Evaluation of physicomechanical properties of PDMS•Ru(dpp)3 ............................... 78 3.3.3 In vitro evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 .......................... 86 3.3.4 Cytotoxicity evaluation of PDMS•Ru(dpp)3 ................................................................ 88 3.3.5 In vivo evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 ........................... 90 3.3.6 Evaluation of biofilm formation on PDMS•Ru(dpp)3 by imaging .............................. 99 3.4 Conclusion ........................................................................................................................ 105 Chapter 4: Incorporation of Ruthenium photosensitizer into an experimental dental resin blend and evaluation of its physical, chemical, mechanical, and antimicrobial properties: The case of MA as a hard material...........................................................................................107 4.1 Introduction ....................................................................................................................... 108 4.1.1 Background ................................................................................................................ 108 4.1.2 Oral biofilms and dental caries .................................................................................. 111 4.1.3 Dental materials ......................................................................................................... 113 4.1.4 Antimicrobial Photodynamic Therapy in dental sciences ......................................... 121 4.2 Experimental section ......................................................................................................... 123 4.2.1 Resin blend formulation ............................................................................................. 123 4.2.2 Degree of conversion (DC) ........................................................................................ 124 4.2.3 Flexural strength (FS) ................................................................................................ 125 4.2.4 Shear bond strength (SBS) to dentin .......................................................................... 126 4.2.5 Water sorption (WS) and solubility (SL) ................................................................... 128 4.2.6 In vitro antimicrobial activity .................................................................................... 129 4.2.7 Statistical analysis ...................................................................................................... 131 xi 4.3 Results and discussion ...................................................................................................... 131 4.3.1 Resin blend (RB) formulation and ruthenium photosensitizer incorporation into RB............................................................................................................................................. 131 4.3.2 Degree of conversion (DC) ........................................................................................ 132 4.3.3 Flexural strength (FS) ................................................................................................ 137 4.3.4 Shear bond strength (SBS) to dentin .......................................................................... 139 4.3.5 Water sorption (WS) and solubility (SL) ................................................................... 142 4.3.6 In vitro antimicrobial activity .................................................................................... 143 4.4 Conclusion ........................................................................................................................ 146 Chapter 5. Conclusions and future work .................................................................................148 Bibliography ...............................................................................................................................152 xii List of Tables Table 1.1 List of common polymeric medical implants under CFR by FDA…………………….9 Table 1.2 Summary of photosensitizer classifications and common examples………………….24 Table 1.3 Summary of advantages and disadvantages of different light sources………………..26 Table 2.1. Absorption, emission, and electrochemical data for Ru(bpy)32+ and Ru(dpp)32+.……34 Table 3.1 Wound histology score results for in vivo experiments………………………………98 Table 4.1 Double bond conversion by radical polymerization of methacrylates with different functionality at room temperature ……………………………………………………………..120 Table 4.2 Experimental resin blend (RB) components and their respective concentrations…...123 Table 4.3 Application mode of materials employed in the control and experimental groups for SBS studies………………………………………………………………………………….128 xiii List of Figures Figure 1.1 Quarterly national SIRs for major HAI types in the U.S. between 2019-2021………..3 Figure 1.2 Projections of deaths from antimicrobial resistance…………………………………...5 Figure 1.3 Main mechanisms of antibiotic resistance……………………………………………..6 Figure 1.4 The structure and industrial synthesis reaction of PDMS…………………………….11 Figure 1.5 The structure and free radical polymerization synthesis reaction of PMMA…………13 Figure 1.6 Stages of biofilm formation and attachment………………………………………….17 Figure 1.7 Available antimicrobial technologies for polymeric medical implants………………20 Figure 1.8 Jablonski diagram showing the principles of antimicrobial photodynamic Inactivation(aPDI)………………………………………………………………………………..22 Figure 2.1. Structure of some common mononuclear ruthenium photosensitizers………………32 Figure 2.2. Mechanisms of photodynamic activity of ruthenium photosensitizers……………....35 Figure 2.3. Critical to quality (CtQ) tree for antimicrobial medical implant design……………..38 Figure 2.4. Schematic diagram illustrating the incorporation of PS into PDMS………………...39 Figure 2.5 Synthetic pathway of photosensitizer Ru(dpp)3(PF6)2 (2)……………………………46 Figure 2.6 UV-Vis absorption spectrum of a CHCl3 solution of Ru(dpp)3(PF6)2 (2)……………47 Figure 2.7 1H NMR spectrum (CDCl3) of Ru(dpp)3(PF6)2 (2)……………………………………48 Figure 2.8 19F NMR spectrum (CDCl3) of Ru(dpp)3(PF6)2 (2)…………………………………..49 xiv Figure 2.9 31P NMR spectrum (CDCl3) of Ru(dpp)3(PF6)2 (2)…………………………………..49 Figure 2.10 HRMS (TOF LC/MS - ESI-(+) mode) of Ru(dpp)3(PF6)2 (2)………………………50 Figure 2.11 Photographic images of solid PDMS disks………………………………………….52 Figure 2.12 Circuit diagram and emission spectrum of the LED array…………………………..53 Figure 2.13 UV-vis absorption spectrum of a CHCl3 solution of Ru(dpp)3(PF6)2 (2) and photographic image of the 4 × 6 blue LED array…………………………………………………54 Figure 2.14 Leaching studies of Ru(dpp)3(PF6)2 (2)……………………………………………..55 Figure 2.15 Reaction of singlet oxygen with 9,10-dimethylanthracene (DMA) produces endoperoxide product…………………………………………………………………………….56 Figure 2.16 Singlet oxygen generation studies of Ru(dpp)3(PF6)2 (2)…………………………….57 Figure 3.1 The human urinary system……………………………………………………………61 Figure 3.2 Scope of urologic implants and prosthetics…………………………………………..64 Figure 3.3 Pathogenesis of biofilm formation on urinary catheters during CAUTI Development……………………………………………………………………………………. 66 Figure 3.4. Preliminary in vitro antimicrobial studies of PDMS•Ru(dpp)3 disks (+/-) LED………………………………………………………………………………………...76 Figure 3.5. Changes in viable E.coli counts on PDMS•Ru(dpp)3 disk surfaces…………………78 xv Figure 3.6 Tensile strength studies of PDMS•Ru(dpp)3…………………………………………79 Figure 3.7 Surface roughness studies of PDMS•Ru(dpp)3………………………………………81 Figure 3.8 Surface hydrophobicity studies of PDMS•Ru(dpp)3…………………………………83 Figure 3.9 Surface encrustation studies of PDMS•Ru(dpp)3…………………………………….86 Figure 3.10 In vitro antimicrobial studies for 4 mM PDMS•Ru(dpp)3 with longer exposure time……………………………………………………………………………………………...87 Figure 3.11 Cytotoxicity evaluation of PDMS•Ru(dpp)3………………………………………..90 Figure 3.12 Flowchart and schematics of the in vivo antimicrobial studies……………………..92 Figure 3.13 Photographic images and results of the in vivo antimicrobial studies………………93 Figure 3.14 H&E staining results of in vivo antimicrobial studies………………………………96 Figure 3.15 Higher magnifications of H&E staining results…………………………………….99 Figure 3.16 SEM images of bare PDMS and PDMS•Ru(dpp)3 coupons………………………100 Figure 3.17. Live/dead staining fluorescence images of bare PDMS and PDMS•Ru(dpp)3 coupons after 1 day……………………………………………………………………………………….101 Figure 3.18. Live/dead staining fluorescence images of bare PDMS and PDMS•Ru(dpp)3 coupons after 2 days……………………………………………………………………………………...102 Figure 3.19 Live/dead staining fluorescence images of bare PDMS and PDMS•Ru(dpp)3 coupons after 5 days……………………………………………………………………………………...103 xvi Figure 3.20 2.5D images of Live/dead staining Fluorescence microscopy……………………..104 Figure 3.21 Live bacterial colony count over area of bare PDMS and PDMS•Ru(dpp)3………105 Figure 4.1 The four types of teeth in the oral cavity and different portions of a tooth…………..109 Figure 4.2 The anatomy of a normal tooth………………………………………………………110 Figure 4.3 Stages of dental caries formation and development……………………………….....113 Figure 4.4 Timeline of restorative dental material composites evolution……………………....117 Figure 4.5 Mechanism of photopolymerization initiation by CQ / amine system……………...119 Figure 4.6 UV-Visible absorption spectrum for the experimental resin blend (RB) and 2% CQ/amine as the photoinitiator system…………………………………………………………132 Figure 4.7 Degree of conversion studies of RB•Ru(dpp)3……………………………………...135 Figure 4.8 Flexural strength studies of RB•Ru(dpp)3…………………………………………..138 Figure 4.9 Shear bond strength studies of RB•Ru(dpp)3…………………………………….....140 Figure 4.10 Water sorption and solubility studies of RB•Ru(dpp)3……………………………143 Figure 4.11 Photographic images of light curing process and antimicrobial studies results of RB•Ru(dpp)3…………………………………………………………………………………...145 xvii List of Symbols °C Degree Celsius E Energy ε Molar absorptivity g gram H Radiant exposure h hour J Coupling constant kΩ Kilo Ohm L Liter m/z Mass over charge ratio MHz Megahertz mA milli Ampere mg milligram mL milliliter mM millimolar μg microgram xviii μL microliter nm nanometer ppm parts per million Ra Average surface roughness V Volt θ Contact angle λ Wavelength τ Emission lifetime δ Chemical shifts Φ Quantum yield xix List of Abbreviations 3D Three-dimensional AFM Atomic Force Microscopy AMD Age-related Macular Degeneration AMP Antimicrobial Peptides AMR Antimicrobial Resistance ANOVA Analysis of Variance aPDT antimicrobial Photodynamic Therapy APO Acylphosphine Oxide AU Artificial Urine AUS Artificial Urinary Sphincter BAPO Bisacylphosphine Oxide Bis-EMA Ethoxylated (4) bisphenol-A dimethacrylate Bis-GMA Bisphenol-A glycidylmethacrylate BPH Benign Prostatic Hyperplasia CAUTI Catheter-associated Urinary Tract Infection CBR Centre for Blood Research CDI Clostridioides Difficile Infection CFR Code of Federal Regulations CFU Colony Forming Unit CLABSI Central-Line–Associated Blood Stream Infections CNT Carbon Nano Tube xx COVID-19 Corona Virus Disease CQ Camphorquinone CtQ Critical to Quality DC Degree of Conversion D/C Direct Current DEJ Dentin-Enamel Junction DMA Dimethylanthracene DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid ED Erectile Dysfunction eDNA Extracellular DNA EM Electromagnetic EPS Extracellular Polymeric Substance ESI Electrospray Ionization FBS Fetal Bovine Serum FDA Food and Drug Administration Fr French (used for urinary catheter size classification) FS Flexural Strength FTIR Fourier Transform Infrared Spectroscopy FUM Female Urinary Microbiota GIC Glass Ionomer Cement H&E Hematoxylin and Eosin xxi HA Hydroxyapatite HAI Healthcare-associated Infections HAI Hospital-acquired Infection HDF Human Dermal Fibroblast HDMA 2-Hydroxypropyl Methacrylate HIV Human Immunodeficiency Virus HOMO Highest Occupied Molecular Orbital HRMS High Resolution Mass Spectrometry IC Internal Conversion ICP-MS Inductively Coupled Plasma Mass Spectrometry ICU Intensive Care Unit IS Impact Strength ISC Intersystem Crossing JBRC Jack Bell Research Centre LASER Light Amplification by Stimulated Emission of Radiation LB Lysogeny Broth LBL Layer-By-Layer LC/MS Liquid Chromatography/Mass Spectrometry LCU Light Curing Unit LED Light Emitting Diode LOG Logarithm LPS Lipopolysaccharide LUMO Lowest Unoccupied Molecular Orbital xxii MAF Medical Animal Facility MB Methylene Blue MDR Multidrug Resistant MMA Methyl Methacrylate MRSA Methicillin-Resistant Staphylococcus Aureus MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide NIR Near Infrared NMR Nuclear Magnetic Resonance NO Nitric Oxide OLED Organic Light Emitting Diode PAC Plasma Arc PAMRC Polyacid-modified Resin Composites PBS Phosphate Buffer Saline PDI Polydispersity Index PDMS Polydimethylsiloxane PDT Photodynamic Therapy PI Propidium Iodide PMMA Polymethyl Methacrylate PS Photosensitizer PVC Polyvinylchloride QTH Quartz-Tungsten-Halogen RB Resin Blend RBG Rose Bengal xxiii ROS Reactive Oxygen Species SARS-Cov-2 Severe Acute Respiratory Syndrome Coronavirus 2 SBMP Dentin Commercial Control Group SBS Shear Bond Strength SD Sprague Dawley SDV Standard Deviation SEM Scanning Electron Microscopy SL Solubility (Water) SSI Surgical Site Infections TBAH Tetrabutylammonium hydroxide TBO Toluidine Blue Ortho TCP Tissue Culture Plate TEEGDMA Tetraethyleneglycol Dimethacrylate TEGDMA Triethyleneglycol Dimethacrylate THF Tetrahydrofuran TMS Tetramethylsilane TOF Time of Flight TPO 2,4,6-Trimethylbenzoyldiphenyl Phosphine Oxide U.S. United States UDMA Urethane Dimethacrylate UN United Nations UPEC Uropathogenic Escherichia coli USB Universal Serial Bus xxiv UTS Universal Testing Machine UV-Vis Ultraviolet-Visible VAE Ventilator-Associated Event VAP Ventilator-Associated Pneumoniae VGH Vancouver General Hospital VPC Vancouver Prostate Centre WBC White Blood Cells WHO World Health Organization WS Water sorption XDR Extremely-drug Resistant xxv Acknowledgements This thesis is an embodiment of how true multidisciplinary collaboration in life science research can come to fruition. What humbles me the most about driving this great initiative is the fact that throughout this work, I had the chance to interact with and learn from a team of experts in microbiology and immunology, materials science and chemistry, as well as urology and dentistry. First and foremost, I would like to thank my supervisor Dr. Dirk Lange for his dedicated guidance and unwavering support which made this thesis work possible within its timeline, despite a concurrent global pandemic. I would also like to thank my co-supervisor Dr. Neil Branda for his guidance and valuable feedback in all chemistry and materials science aspects of the work. My committee members, Dr. Ben Chew and Dr. Adriana Manso played pivotal roles in assessing the practicality and functionality of the technology to ensure it addresses the unmet needs of design and evaluation of both antimicrobial urinary catheter and dental resin. A huge thanks goes to all the current and past members of the basic sciences research laboratory at the Stone centre at VGH&UBC. Their helpfulness and sense of cooperation was truly exemplary. I am also grateful to all the members of 4D LABS and in Dr. Branda’s team for their support. Specifically, Dr. Khaled Arafeh offered help and insights into synthesis and characterization of the ruthenium photosensitizer. Nazanin Assempour kindly helped with sample preparations for NMR and HRMS studies. I received great training on SEM from Dr. Xin Zheng from 4D LABS, and I am thankful for his valuable advice and suggestions. Demian Felix provided great help with the in vivo experiments as well as surface encrustation studies. I also thank Dr. Nafise Amiri from ICORD for providing assistance and support with the animal studies and xxvi cytotoxicity experiments. Drs. Claudia Dietrich, Maria Luisa Leite, and Patricia Comeau from UBC dentistry provided invaluable help in designing and validating the physicomechanical properties of the dental resin. My sincere thanks to Dr. Jayachandran Kizhakkedathu from UBC chemistry and centre for blood research (CBR) for the valuable advice on polymeric biomaterials aspect of the work and for facilitating the microscopy and biofilm imaging studies. Dr. Kai Yu from CBR kindly performed the SEM imaging of the surface encrustation studies. Dr. Rizhi Wang from UBC materials engineering kindly provided access to their facility for tensile strength measurements, and Dr. Qiong Wang helped with the UTM testing. Scott Beaupre designed and calibrated the LED light source and Dr. Saeid Kamal and Dr. Gary Leach from 4D LABS kindly allowed for using their facility for photonic flux and emission spectrum measurements of the light source. Dr. Ali Moeen from VPC kindly performed the histology studies and offered great help with analyzing the results. I also greatly appreciate the time and efforts of Dr. Htoo Zarni Oo from VPC pathology for performing blinded histology scoring. I am grateful for the financial support provided through VCHRI-ITR grant (Grant ID: F20-00249) which helped making this thesis work possible. I also thank Dr. Alex Kavanagh from UBC urology for their involvement in this initiative. Many thanks and heartfelt appreciation go to all my family members and friends for their continued support throughout this endeavor. xxvii Dedication I would like to dedicate this thesis: To my parents who have always supported me unconditionally & To my wife who has stood by me through all the challenges & To my son, Ferzan, whose creativity and curiosity always motivated me. 1 Chapter 1: Introduction 1.1 Two major types of infections plaguing the world In medical terminology, infection is referred to as invasion of the body, followed by multiplication and colonization by microorganisms such as bacteria, viruses, fungi, or other pathogens, which would subsequently trigger a physiological response by the immune system in order to control the spread of colonization and keep the body safe. An infection may cause immediate symptoms and be clinically evident, or it may remain subclinical with no apparent sign. Either way, the fast rate of multiplication, and ease of spread of the infection-inducing microorganisms to other parts of the body after they colonize a local site are some of the factors that result in the treatment and management of infections to be a challenging quest. Contracting infection-inducing pathogens can happen in many ways. Germs can easily be picked by touching unclean materials, foods, and surfaces, and then spread through the body via nose, mouth, skin, or eyes. They can also be transmitted from pets to humans or from person to person [1]. Regardless of the mode of transmission, once microorganisms begin colonizing the body, they are immediately encountered by the immune system that works to suppress and eliminate them by triggering an antigen response (from immunology standpoint, all disease-causing entities such as bacteria, viruses, or toxins are referred to as antigens) by means of leukocyte (also known as white blood cells (WBC)) attack [2]. For a healthy individual, the immune response generally results in containment and overcoming of the infection within a short period of time. However, this process is far more cumbersome for immunodeficient individuals whose immunity is at a weaker state due to contributing factors such as chronic diseases, hereditary disorders, ongoing medical treatments, pregnancy, obesity, or age [3]. 2 1.1.1 Healthcare-associated infections (HAI) and catheter-associated urinary tract infection (CAUTI) Among different places where the possibility of acquiring an infection for an immunocompromised patient is very high are hospitals and healthcare facilities, resulting in the emergence of healthcare-associated infections or hospital-acquired infections (HAI), one of the biggest challenges faced by the global healthcare system [4,5]. According to the latest report by the World Health Organization (WHO), 7% of the patients in developed countries and 15% in developing countries acquire at least one HAI during their hospitalization period, resulting in an overall 10% mortality rate [6]. HAIs not only pose a great threat to patient morbidity and mortality, but they also place a significant financial burden on the healthcare system [7]. In the U.S. alone, the five major nosocomial infections—central line-associated bloodstream infections (CLABSI), ventilator-associated pneumonia (VAP), surgical site infections (SSI), Clostridium difficile infection (CDI), and catheter-associated urinary tract infections (CAUTI)— cost about $10 billion annually in 2009 [8]. In fact, if direct, indirect, non-medical, and societal costs of HAIs are considered, this number could be up to about $150 billion annually [9]. Additionally, with the onset of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) and the coronavirus 19 (COVID-19) global pandemic in 2019, there has been an increase in the incidents of all major nosocomial infections as a result of a major surge in hospitalizations. Figure 1.1 illustrates the national standardized infection ratio (SIR) between the first quarter of 2019 and the third quarter of 2021 in the U.S. hospitals, indicating a clear uptrend after the COVID-19 outbreak [10–12]. 3 Figure 1.1 Quarterly national SIRs for major HAI types in the U.S. between 2019-2021. Reprinted from Lastinger et.al. [10] 1.1.2 Dental caries and associated infections Another major global health concern is dental caries (tooth decay), which is a multifactorial disease and directly linked to bacteria. It is estimated that up to 2 billion people are affected by dental caries of the permanent teeth, while over 500 million children experience it with their primary teeth [13]. Apart from physiological and psychological side effects, dental caries places a huge economic burden on the healthcare system via the need for restoration or replacement of the affected tooth in order to prevent the infection from further progression. 1.1.3 Sepsis Another major issue which is closely associated with HAIs is the development of sepsis [14]. Sepsis is a life-threatening medical emergency that develops as a result of the body’s intense immune response to an already developed infection that if left untreated results in septic 4 shock that may lead to organ failure and death [15,16]. In fact, septic shock and sepsis-related mortality rates are believed to account for up to 20% of the global mortality rate [16,17]. 1.2 Antimicrobial Resistance Given the frequency and high probability of HAI developments, and the criticality of sepsis prevention, the clinicians have always tried to search for treatment options to either prevent or control HAIs when a patient is to undergo medical procedures [18]. From etiology standpoint, the risk factors related to HAI development are closely associated with infection control practices at medical facilities, the immune status of the patient, and the prevalence of specific pathogens within a given community. In general, the risk factors for HAI including immunosuppression, age, length of hospitalization, other underlying comorbidities, frequent visits to healthcare facilities, mechanical ventilatory support, recent invasive procedures, indwelling devices, and stay in an intensive care unit (ICU) need to be accounted for in order to improve patient outcomes [19]. Recent advances in medical sciences and healthcare systems management have allowed healthcare providers to develop and rely on a variety of preventive measures and utilize specific guidelines pertaining to environmental, procedural, healthcare provider hygiene, surveillance, and antibiotic stewardship. However, despite all these efforts, the HAI-related infection rates still follow an upward trajectory. The main reason behind this is the fact that the first treatment of choice to battle against the majority of these nosocomial infections as well as late-stage dental caries is antibiotic administration. Although there is data to support the effectiveness of antibiotic administration for controlling HAIs [20], overuse of antibiotics and the emergence of multidrug-resistant (MDR) pathogens capable of developing antimicrobial resistance (AMR) are one of the major concerns in HAI management [21–23]. In fact, the threat of MDR pathogens and AMR due to antibiotic 5 resistance is so severe that multiple reports have warned of the potential of AMR as a global threat and anticipate it to surpass cancer in mortality rates (over 10 million annually) by as early as 2050 (Figure 1.2) [24–26]. Figure 1.2 Projections of deaths from antimicrobial resistance. Reprinted from Review on Antimicrobial Resistance, UK Department of Health, 2016 (www.amr-review.org) The reasons behind the prevalence of AMR and antibiotic resistance lie within the variety of mechanisms that pathogens can develop in order to render the use of antibiotics ineffective. Among multiple resistance pathways commonly developed by drug-resistant bacteria are the enzymatic degradation of antibacterial drugs, alteration of bacterial proteins as antimicrobial targets, changes in membrane permeability to antibiotics by microorganisms, as well 6 as development of efflux pumps to eject antibiotic drugs from bacterial cells [27,28]. Figure 1.3 shows an overview of the main antibiotic resistance mechanisms developed by bacteria. Figure 1.3 Main mechanisms of antibiotic resistance. Reprinted from https://www.lipotype.com/lipidomics-applications/lipidomics-of-multi-resistant-bacteria/ 1.3 Polymeric Medical implants Apart from the healthcare settings and practices that are contributing factors in HAIs, and the AMR mechanisms that constantly undermine the efficacy of antibiotics to control and mitigate infection rates, another major element of concern that plays a role in development and spread of different forms of nosocomial infections is the availability and suitability of additional inanimate surfaces for microorganism attachment when medical implants are to be used. From evolutionary standpoint, human cells have evolved to minimize or inhibit bacterial adhesion by means of developing cellular and tissue receptors that attenuate bacterial adhesion [29,30]. In contrast, biomedical materials do not have such mechanisms and as a result form an attractive nidus for bacterial attachment and biofilm formation. Polymeric biomaterials have found their 7 widespread use in biomedical sciences since the 1900s. According to the U.S. Food and Drug Administration (FDA), a medical device is any instrument, apparatus, implant, or in vitro reagent that is used for diagnosis, mitigation, treatment, or prevention of a disease or is intended to improve functionality of a specific organ of the body [31]. There are numerous reasons for the popularity of such materials in medicine ranging from ideal physical, chemical, electrical, thermal, and mechanical properties, to ease of fabrication and cost effectiveness, as well as their durability and versatility [32–34]. Regardless of the area of application and usage type, all polymeric biomaterials are specifically designed to enhance the quality of life for their users. These products can range from simple hand-made materials such as knee implants, artificial valves, or synthetic blood vessels to intricate devices such as heart pacemakers and defibrillators [31]. Among many different polymers currently used for medical device and implant fabrication are polyethylene, polypropylene, polydimethylsiloxane, parylene, polyamide, polytetrafluoroethylene, poly (methyl methacrylate), polyimide, and polyurethane [31,35]. Moreover, various classes of polymers such as liquid crystalline polymers and nanocomposites have also been employed as medical devices either directly as substrate materials or as auxiliary materials used for medical device packaging. Table 1.1 provides a list of common polymeric medical implants under the code for federal regulations (CFR) by the U.S. FDA. 8 FDA Category Common medical devices Polymer used Anesthesiology • Epidural catheters • Polyethylene • Polytetrafluoroethylene • Polyamide Cardiovascular • Pacemaker • Implantable cardioverter/defibrillator • Mechanical heart valves • Artificial blood vessels • Catheters • Polypropylene • Polyethylene • Polytetrafluoroethylene • Polyamide • Polyethyleneterephthalate • Polydimethylsiloxane • Polyhydroxyalkanoates Dental • Dentures • Dental implants • Polymethylmethacrylate Ear, nose, and throat • Cochlear implants • Stapes implants • Nasal reconstructive implants • Polydimethylsiloxane • Liquid crystal polymer • Silicone • Parylene • Polyethylene Gastroenterology and urology • Penile implants • Sacral nerve stimulator • Foley catheters • Artificial urinary sphincter • Hernia or vaginal mesh • Polydimethylsiloxane • Polyethylene • Polytetrafluoroethylene • Polyamide • Polyhydroxyalkanoates General or plastic surgery • Breast implants • Cheek, jaw, chin, and lip implants • Hip implant • Titanium surgical implants • Polyethyleneterephthalate • Polydimethylsiloxane • Polypropylene • Polytetrafluoroethylene • Silicone Hematology and pathology • Central venous access device • Peripherally inserted central catheter • Polyethylene • Polytetrafluoroethylene • Polyamide 9 Neurology • Implantable pulse generator for deep brain stimulation • Neuroprosthetics • Cognitive prostheses • Catheters • Polydimethylsiloxane • Polyimides • Parylene • Liquid crystal polymers • Polyethylene • Polytetrafluoroethylene • Polyamide Obstetric and gynecologic • Intrauterine device • Intravaginal rings • Etonogestrel-releasing contraceptive implants • Fetal micro-pacemaker • Silicone • Polyurethane • Polypropylene Ophthalmic • Dexamethasone intravitreal implant • Retinal prothesis • Artificial intraocular lens • Glaucoma valve • Fluocinolone ophthalmic implant • Orbital implant • Polymethylmethacrylate • Polyethylene • Polytetrafluoroethylene • Polyamide Orthopedic • Orthopedic implants • Polyethylene • Polyether ether ketone • Polyhydroxyalkanoates Table 1.1 List of common polymeric medical implants under CFR by FDA. Printed with modifications from [31] Among the available polymers with applications as medical implants, polydimethylsiloxane (PDMS) and polymethylmethacrylate (PMMA), which is derived as a result of polymerization of methyl methacrylate (MMA) and free radicals, have versatile usage, with PDMS being used frequently as brain implants in neurology [36,37], as chin and nose bone augmentation implants and spinal implants in plastic surgery [38,39][40,41], as cochlear implants and hearing aids [40,41], as arterial and blood vessels implants and bioelectronic and biosensor 10 patches in cardiology and cardiovascular systems [42,43], and as Foley catheters and ureteral stents in urology [44,45]. PMMA and MA-based derivatives, on the other hand, are used commonly as an implant material in dentistry [46,47] and as a retinal orbital implant in ophthalmology [48,49]. Since both PDMS and MA derivatives are the main compounds involved in the manufacturing of Foley catheters and dental resins, and since these two medical implants play a significant role in the treatment and management of CAUTI and dental caries, it is crucial to understand their properties and the role of bacterial adhesion and biofilm formation on these substrates. This, together with the state of current available antimicrobial technologies accessible to urologists and dentists, would set the stage for the application of surface photosterilization as a viable alternative to prevent contamination and reduce infection in both settings. 1.3.1 Polydimethylsiloxane (PDMS) Polydimethylsiloxane (PDMS), also commonly referred to as dimethylpolysiloxane or dimethicone, is a silicone polymer with a wide range of applications from cosmetics to industrial lubricants to biomedical implants and device manufacturing. It has chemical formula of CH3[Si(CH3)2O]nSi(CH3)3 , where n is the number of repeating units of Si(CH3)2O monomer. The density of PDMS is 0.965 g/cm3. Industrially, PDMS is synthesized from hydrolysis of dimethyldichlorosilane. Since this hydrolysis reaction produces the silanol terminal groups which are reactive, the polymer chain is capped by reacting it with trimethylsilylchloride (Figure 1.4). 11 n Si(CH3)2 Cl2 + (n+1) H2O HO [Si(CH3)2O]n H + 2n HCl (1) 2 Si(CH3)3 Cl + HO [Si(CH3)2O]n H (CH3)3SiO [Si(CH3)2O]n Si(CH3)3 + 2 HCl (2) Figure 1.4 Structure and industrial synthesis reaction of PDMS. Reprinted with modifications from https://en.wikipedia.org/wiki/Polydimethylsiloxane. Because the Si-O makes up the backbone of the polymeric chain in PDMS, it is technically an inorganic polymer [50]. This endows a set of interesting and unique properties to PDMS, including high thermal stability, as well as low surface free energy which arises from having the Si atom substituted while O atom is unsubstituted. This imparts a set of interesting behaviors of the polymeric chain since larger bond angles around the unsubstituted oxygen makes the planar all-trans form of the chain resemble a series of closed polygons, and such intriguing configurational properties ultimately have implications for the way in which the bulk chain of the polymer can pack and stack together. This is also one of the factors that allows for cross-linking between various chains of PDMS in bulk form. PDMS is optically clear, and is inert, non-toxic, and non-flammable, and exhibits unusual rheological (flow) properties. It can also be manufactured with a variety of different polydispersity indices. Polydispersity index (PDI) is a measure of controlling the broadness of 12 molecular weight distribution of polymers. The polymer can also be made using different degrees of viscosity, which allows its usage to be tailored to specific applications. PDMS flexibility is another advantageous feature of the polymer which stems from the rotational capability of the side chains around the Si-O parent chain. Solid PDMS samples will not allow for infiltration of aqueous solvents or alcohols but some organic solvents such as chloroform, diisopropylamine, ether, and tetrahydrofuran (THF) result in swelling the PDMS polymeric network [51]. Another useful property of PDMS is its availability to be used as an elastomer. Elastomers are polymeric chains with weak intermolecular forces and low Young’s Modulus, which gives them specific viscoelasticity properties [52]. It also allows them to undergo cross-linking reactions with other oligomers containing vinyl groups in the presence of a proprietary platinum catalyst [53]. This results in a three-dimensional cross-linking effect, forming Si-CH2-CH2-Si linkages with multiple reaction sites on both the base and crosslinking oligomers. One advantage of this type of addition reaction is that no side products such as water are generated. Apart from biomedical applications as various types of implants, PDMS and its derivatives are also used extensively as defoaming agents and surfactants in chemicals industry [54], radiative cooling [55,56], lithography [57,58], and food industry [59]. 1.3.2 Methacrylate (MA) and its derivatives The second class of materials with versatile applications as medical implants is methacrylate-based compounds such as methyl methacrylate (MMA), ethoxylated (4) bisphenol-A dimethacrylate (Bis-EMA), tetraethylene glycol dimethacrylate TEEGDMA, hydroxypropyl methacrylate (HPMA), and polymethylmethacrylate (PMMA). Derived from polymerization reaction of methyl methacrylate as monomer, PMMA is also known as acrylic, acrylic glass, Crylux and Plexiglass in industrial applications. When used as plexiglass, PMMA has utility as a 13 shatter-resistant alternative to standard glass [60]. Its chemical formula is (C5O2H8)n, and has a melting point of 160 °C and density of 1.18 g/cm3 . With respect to other methacrylate-based derivatives, although their overall physicochemical properties depend on the nature of the substituents on their side chains, they all share certain advantageous properties such as light transparency, good weatherability and resilience, ease of functionalization, optimal biocompatibility, and reasonable production cost. Figure 1.5 shows the structure of some common methacrylate derivatives and free radical polymerization synthesis reaction of poly (MMA). 14 MMA Bis-EMA TEGDMA HPMA 15 PMMA Polymerization Figure 1.5 Structure of methacrylate derivates and free radical polymerization synthesis reaction of PMMA. Reprinted from [66,67] The physicochemical properties of MA-based materials depend heavily on the nature of the substituents on the side chains. The overall interest for polyacrylates and polymethacrylates is due to their light transparency, elasticity, good weatherability and resilience, as well as easy functionalization route, good biocompatibility, and low cost [68]. PMMA is currently being employed in major sectors such as biomedicine and medical sciences (especially in dentistry) [69–71], food industry, environmental engineering [72,73], as well as automotive industry, building construction [74], and electronics [75,76]. 1.4 Biofilm formation The main reason why bacterial adhesion to various polymeric medical biomaterials and implants is of great concern to the patients and healthcare professional is that the initial attachment of bacteria on these surfaces provides a nidus for colonization and maturation of the bacterial colonies which can ultimately lead to biofilm formation [77]. Biofilm is referred to an assemblage of polymicrobial cells that cluster together to form a complex microcosm with intricate 16 mechanisms of nutritional support, maturation, replication, and irreversible adhesion to their host surfaces [78]. They can form on a wide variety of surfaces and environments, including living tissues, indwelling medical devices, industrial or potable water piping systems, or natural aquatic systems. A biofilm-associated bacterial cell can be differentiated from its planktonic (non-adhered) counterparts through excretion of extracellular polymeric substance (EPS), altered growth rate, as well as genetic modification. These attributes, along with the physical characteristics that reinforce biofilm attachment to the surfaces make their management and removal a very difficult task [78,79]. In addition, once a biofilm community is developed, it is capable of resisting antibiotic penetration into its structure, thus protecting the microbial cells that are embedded deep inside the structure, which subsequently makes eradication of biofilms even more challenging [80,81]. The process of biofilm formation can be divided into the following major stages: initial attachment (reversible), irreversible adhesion (microcolony formation), maturation, and dispersion [82,83]. Initial attachment of bacteria to the surface of implants starts within moments of insertion of the device and is facilitated by the formation of a thin layer of conditioning film on the available surface [84]. Conditioning film is the result of adsorption of biomolecules that are available in the surrounding environment and act as an anchor for the bacteria to attach themselves to the implant surface more easily [85]. Following initial attachment, other factors such as the liquid-solid interface interactions, hydrodynamics of the environment, and the material composition of the substratum together with the EPS formation all play role in the formation of the bacterial microcolonies that can no longer be readily removed. Once the first microcolonies are set in place, they start the maturation process via formation of a biofilm matrix that is efficient in providing nutrients to the bacterial cells inside the slimy layer of the biofilm while being a protective shield from the outer environment. The cells within the biofilm colony also develop a 17 set of complex communication methods among themselves by secretion of specific chemicals. This phenomenon is referred to as quorum sensing [86,87]. During the maturation phase, the biofilm matrix reaches its highest level of complexity, producing a variety of biochemical macromolecules including proteins (e.g. fibrin), polysaccharides (e.g. alginate), as well as extracellular DNA (eDNA) to help its community flourish while evading the host defence system. In the last stage, dispersion, fully-grown bacteria migrate from the biofilm matrix, thereby invading and colonizing other parts of the body, which increases the pathogenicity of biofilm. Figure 1.6 shows the stages of biofilm formation. Figure 1.6 Stages of biofilm formation and attachment. Reprinted from [88] 18 Biofilms play a crucial role in the pathogenesis of the diseases, especially in the case of chronic infectious diseases. This is because biofilm-associated microbial cells are far more resilient to both the immune response and antibiotic treatments [89]. In fact, it is estimated that biofilm-associated bacterial cells are up to hundred times more resistant to antimicrobial agents [90]. All together, the extreme virulence and pathogenicity of bacterial biofilms, together with the availability of additional inanimate surfaces that the insertion of medical implants provide to them, in conjunction with the inefficacy of antibiotic administration as the first treatment of choice, all point to the fact that safer and more efficient alternative antimicrobial technologies where pathogenic resistance is no issue are desperately needed. 1.5 Antimicrobial surface treatments Given the urgency in seeking alternative antimicrobial modalities that unlike antibiotics would not result in resistance by pathogens, there has been a great amount of interest in antimicrobial product development for medical implants recently [91]. The overarching goal of these technologies is that they need to show antimicrobial efficacy that is comparable to antibiotics, and they also need to be proven to exhibit significantly lower or no degree of recurrence of pathogens when used. With respect to polymeric medical materials and implants, where insertion of the device can provide a substrate for bacterial adhesion and biofilm formation which would lead to exacerbation of infection or cause the development of new ones, currently there are three major categories of techniques that hold great promise: 19 1. Antiadhesive surfaces 2. Contact killing surfaces 3. Drug eluting surfaces Antiadhesive polymeric surfaces act to repel bacteria and avoid them from making close contact with the implant surface. Such surfaces can either be made by suitably modifying the structure of the native substrate or via application of an anti-adhesive surface agent covering the native biomaterial. Factors that play role in achieving optimal anti-adhesive surfaces include surface free energy, electrostatic interactions, steric repulsion, and extent of surface hydration. Some of the techniques that have already been tested for antiadhesive polymeric implants are antiadhesive polymeric brushes [92–96], antiadhesive hydrogels [97–99], and antiadhesive surface-engineered medical implants [100,101]. Contact killing surfaces are polymeric medical implants that work to eradicate pathogens once they adhere to the surface of the implant. Unlike the first category, contact killing agents rely on initial attachment of the pathogens to the implant surface, followed by triggering the antimicrobial activity. Antimicrobial peptide (AMP) conjugates [102–104], nanopillar array coatings [105,106], and photoactive surfaces are classified under this category of antimicrobial products. The last category of antimicrobial polymeric implants owes their functionality to the release of an antimicrobial agent from the surface of the implant. Antibiotic releasing coatings, AMP releasing coatings, nitric oxide (NO) and nanocomposite releasing polymeric medical implants are among the most common type of alternative technologies to combat bacterial 20 adhesion and biofilm formation on medical devices and implants [100,107]. Figure 1.7 shows the summary of the available antimicrobial technologies for polymeric medical implants. Figure 1.7 Available antimicrobial technologies for polymeric medical implants. (Image created with biorender) 1.6 Antimicrobial photodynamic inactivation (aPDI) Among different contact killing techniques available to inactivate pathogenic microorganisms from the surface of medical implants and prevent further bacterial colonization and biofilm formation is the antimicrobial photodynamic inactivation (aPDI) method. The method is also referred to as surface photosterilization and surface disinfection. The underlying principle 21 by which aPDI works to eliminate pathogens is by an interplay between three elements: Light, a photosensitive compound, and oxygen. Within the therapeutic scope of operation (e.g. cancer therapy), the name commonly used among the medical community is photodynamic therapy (PDT). In principle, both PDT and aPDI work by the generation of reactive oxygen species (ROS) as the photokilling agents. These ROS can either be highly reactive radicals such as hydroxyl radical (OH.), hydrogen peroxide (H2 O2) and superoxide anion (O2.-), as well as reactive singlet oxygen (1O2). The PS molecule is typically in a singlet low energy state prior to light activation (ground state). After receiving the photons of light that correspond to its maximum absorption wavelength, the electrons that were previously located in the highest occupied molecular orbital (HOMO) of the PS molecule get excited for a short period of time and migrate to the lowest unoccupied molecular orbital (LUMO), which is in a singlet excited energy state [108,109]. At this point, various quantum mechanical phenomena can occur including internal conversion (IC) and fluorescence back to ground state, emission and subsequent phosphorescence, as well as intersystem crossing (ISC) of the excited electron from singlet state to triplet state. This ISC electron transfer is crucial for photodynamic action, as the triplet state of the excited PS matches with the triplet state of molecular oxygen in ground state. Therefore, provided that there is accessible O2 in the environment, the molecule can receive energy from the excited PS, flipping the spin of the outermost O2 electron and shifting it into the orbital containing the other electron, which in turn leaves one orbital entirely unoccupied. Termed singlet oxygen (1O2), this form of oxygen is extremely short lived and reactive and is capable of rupturing cell walls of the bacteria in its proximity [110]. The generation of reactive radical species by direct electron transfer from the excited PS is referred to as Type 1 mechanism, while the energy transfer from excited PS to molecular oxygen and generation of singlet oxygen is called Type 2 reaction mechanism within 22 the scope of photodynamic action. Figure 1.8 shows the Jablonski diagram that is used in order to give an account on the electronic configuration of the photodynamic action. Figure 1.8 Jablonski diagram showing the principles of antimicrobial photodynamic inactivation (aPDI). Reprinted from Hamblin et.al. [111] Apart from applications for surface photosterilization and aPDI, photodynamic action has wide usage case as a minimally invasive treatment option for various types of cancer, including different forms of skin [112,113], lung [114], brain [115], bladder [116], bile-duct [117], esophageal [118], and head and neck [119] cancers. In addition to cancer, phototherapy is used for other therapeutic purposes such as in ophthalmology and for treatment of age-related macular degeneration (AMD) [120,121], in dermatology for acne treatment [122,123], and for immunophototherapy [124,125]. 23 1.7 Photosensitizer classifications Following the first porphyrin and hematoporphyrin photosensitizers, referred to as the first-generation PSs, the advancements in organometallic chemistry and the arrival of nanomaterials and nanostructures have allowed for synthesis of many different classes of photosensitive molecules and thus improved the efficiency of the photodynamic phenomena [126,127]. It is important to note that the terms photosensitizer and photosensitization are specific terms in photochemistry that should not be mixed with other terminology such as photoinitiation, photocatalysis, or photopolymerization. Within the context of photosensitization, the main distinction is that upon excitation by light, the photosensitive compound is responsible for enacting a direct physicochemical change in another substance via energy transfer and will return to its ground state and remain unchanged until the next photon of light triggers the same cycle to repeat. With respect to classification of the available photosensitizers for PDT or aPDI application, they can be categorized depending on their organic, inorganic, organometallic, and nanomaterials composition. One major aspect of all the common PS molecules is the presence of highly conjugated π- systems which promotes electron delocalization, and also benefit from smaller band gap between their HOMO and LUMO. When other factors such as absorption wavelength and overall charge on the PS molecule are also considered, they can be classified into five major categories: phenothiazinium dyes, tetra-pyrrole structures, natural PSs, nanostructures, and transition metal-based PSs [128]. The reason why PS charge is an important element is the fact that bacterial cell walls are negatively charged due to having molecules such as lipopolysaccharide (LPS) and teichoic acids linked to either the peptidoglycan or the underlying plasma membrane [129]. Table 1.2 provides a summary of the different classes of photosensitizers along with examples and details for some of the common PSs in each category. 24 Class PS Example Charge Maximum excitation λ Phenothiazinium dyes • Methylene blue (MB) • Toluidine blue ortho (TBO) • Rose Bengal (RBG) Cationic Cationic Anionic 632 nm 660 and 410 nm 532 nm Tetra-pyrrole structures • Porphyrin • Phthalocyanine Cationic Neutral 446 nm 670 nm Transition metal PSs • Organometallic complexes of Ru, Pt, Rh Cationic Varies with metal, Visible to near infrared (NIR) Natural PSs • Curcumin • Flavin derivatives Neutral Cationic 547 nm 450 nm Nanostructures • Fullerenes • Carbon nano tubes (CNT) Neutral Neutral 532 nm Visible to NIR Table 1.2 Summary of photosensitizer classifications and common examples. Reprinted with modifications from [128] 1.8 Light sources in aPDI Selecting a suitable illumination source is crucial for triggering the photokilling action of the photosensitizer in both PDT and aPDI settings, as failure to excite the PS molecule means 25 not enough ROS can be generated which subsequently jeopardizes the efficacy of the treatment. Once the suitable wavelength of light is selected according to the maximum absorption wavelength of the PS, other factors such as exposure time, depth of light penetration into tissue, and photonic energy density would need to be considered in order to yield an optimum light dosimetry [130,131]. Some of the most common light sources used for PDT and aPDI are lasers, lamps, and light emitting diodes (LED). Although many different laser types have been used for PDT, including dye lasers and diode lasers [132], their applications as surface illumination devices are rather limited due to their light being collimated and not readily propagated. For this reason, other light sources such as arc lamps and LEDs have more practical applications in aPDI. Depending on the nature of the illumination site, it is also possible to use fiber optics guided light sources that can be coupled with lasers and LEDs. Some of the advantages and disadvantages of each light source is summarized in Table 1.3. 26 Light source Advantages Disadvantages Laser • High power delivery • Highly monochromatic • Adaptable with various fiber optic types • Expensive • High maintenance • Can be large and heavy Lamp • Low cost • Simple design • Broadband radiation • Large beam divergence • Requires beam filtering • Very broad spectrum LED • Small and adaptable • Low cost • Can be designed with narrow spectrum • Low power • Possible broad-spectrum radiation Table 1.3 Summary of advantages and disadvantages of different light sources. Reprinted with modifications from [130] 1.9 Hypothesis, goals, and outlines of the thesis The hypothesis is that the LED activation of the biomaterial surfaces that are embedded with Ru photosensitizer would trigger a photokilling action and thus reduce bacterial adhesion and biofilm formation when compared with similar biomaterials samples without photosensitizer. The hypothesis around which this thesis work is centred is based on the fact that many of the current available treatment options to mitigate both CAUTI and dental caries formation fail to provide a sustainable solution due to limitations in controlling the spread of infection and preventing biofilm formation. So, an alternative approach needs to be sought after that is capable of greatly preventing 27 bacterial surface attachment and colonization which subsequently results in prevention of biofilm formation. Given the urgent need for conceptualization, development, and testing of novel antimicrobial technologies as alternatives to common antibiotics administration with minimally invasive effects and lower chance of pathogenic recurrence, this thesis work is an endeavor to propose a new technology based on photodynamic principles for sterilisation of implantable biomaterials where adhesion of bacteria and subsequent biofilm formation is a concern. For this purpose, a transition metal-based photosensitizer (PS) is synthesized, purified and further characterized to validate its incorporation capability into various classes of polymeric biomaterials, specifically polydimethylsiloxane (PDMS) as a class of “soft” polymers commonly used in urologic sciences as the precursor for urinary Foley catheter and ureteral stents. An LED light source is also designed and calibrated for triggering the photodynamic action. This information makes up the contents of chapter 2 of this thesis. In chapter 3, the functionality of the incorporated PS+PDMS matrix is further examined by evaluating the physico-mechanical properties of the PS+PDMS assembly such as tensile strength, surface roughness, surface hydrophobicity, and the extent of mineral deposition and surface encrustation. Moreover, the cytotoxicity, in vitro, and in vivo evaluation of the activity of the PS+PDMS system both in presence and absence of the LED light source are presented. Chapter 4 of the thesis is organized to provide an overview of the incorporation, characterization, and evaluation of antimicrobial activity of the PS in an experimental methacrylate-based (MA) restorative dental resin, a class of “hard” polymers commonly used in dentistry for treatment of dental caries. Chapter 5 of the thesis provides the concluding remarks and a summary of the future work which can be done to advance the photosterilization technology in both urologic and dental sciences. 28 Chapter 2: Synthesis and characterization of Ruthenium photosensitizer as antimicrobial photosterilization agent and incorporation in PDMS Synopsis In this chapter, the synthesis and characterization of the ruthenium photosensitizer (PS) is described. A literature review on the current classification of photosensitizers was carried out in chapter 1, and here the choice of a ruthenium-based photosensitizer as the compound of choice for polymer embedding is discussed. The synthetic pathway leading to the formation and purification of the PS is provided, followed by the evaluation of the chemical and physical properties of the compound. Next, the preparation of polydimethylsiloxane (PDMS) as the native polymer used for PS embedding is described, and an account of various molding and casting methods are provided in order to reach the optimal configuration for PS incorporation. An array of LED light sources was designed and calibrated in order to trigger the photodynamic action and reactive oxygen species (ROS) generation on the surface of the PDMS+PS materials. Once ruthenium PS was loaded into the PDMS polymer, leaching studies were conducted to verify that no PS is released from the polymer following incorporation. Finally, a series of studies was conducted to assess the extent of ROS generation by the PDMS+PS system upon LED exposure to confirm the effectiveness of the technology over time. 29 2.1 Introduction 2.1.1 Ruthenium photosensitizers As discussed in Chapter 1, Section 1.7, transition metal-based photosensitizers (PS) are among some of the new categories of PSs which exhibit favourable characteristics including photocatalysis, anticancer, and antimicrobial photodynamic activities [133–137]. Among various transition metal coordination complexes, ruthenium, Ru (II, III), complexes have recently attracted great interest to be incorporated and utilized as PSs for both cancer and antimicrobial PDT. Ruthenium, a transition metal of the platinum group with atomic number 44, electronic configuration [Kr] 4d7 5s1, atomic mass of 101.07 g / mol, is a hard and white rare metal first isolated in 1844 and has a density of 12.27 g / cm3 and electronegativity of 2.2 [138]. Certain properties of ruthenium make it the most widely used transition metal in catalysis and excited state chemistry. Its complexes have been found to have up to eight oxidation states, with +2, +3, and +4 being the most common. The relatively low toxicity of the ruthenium complexes has allowed them to be used in catalytic synthesis of drugs. Also, the fact that π-bonding ligands are common in lower oxidation states of Ru complexes means they can bind to many compounds where reversible conversion can leave them intact. This has implications for redox processes where reversibility is critical [139]. The complexes with +2 oxidation state are diamagnetic and facilitate substitution reactions with retention of geometry. This allows ruthenium to tune the substituent ligands towards electron transfer and desirable reaction kinetics [140,141]. 30 Ruthenium compounds can be used in many different applications. One of the most important areas of use for them is in water oxidation catalysis [142–144]. Also, the coordination chemistry of ruthenium oxide composites could introduce them as ideal candidates for energy storage [145,146]. Some studies have shown the potential of ruthenium-based products in semiconductor and electronics industries [147,148]. The light absorption properties of ruthenium complexes can also make them suitable for solar energy technologies. The photon harvesting capabilities of Ru-based dye-sensitized solar cells could introduce them as low-cost solar cell energy systems [149]. Medical applications of ruthenium can be divided into diagnostic and therapeutic. For diagnostic purposes, ruthenium complexes have versatility in terms of their application as molecular sensing agents to detect oxygen in aqueous solution and in biological media as well as for imaging purposes [150–154]. This is the result of the luminescence observed by ruthenium bipyridyl and terpyridine complexes [155–157]. In terms of the therapeutic application of ruthenium, the most notable use case of Ru-based complexes in medicine is as photosensitive compounds for cancer or antimicrobial PDT. The DNA and protein binding abilities of the Ru-based PSs make them excellent compounds for targeted cancer PDT [137,158]. Moreover, the photochemical, photophysical, and physico-mechanical properties of Ru-based photosensitizers identifies them as suitable choices for antimicrobial PDT agents. Compared to many first-generation PSs such as Rose Bengal (RBG) and phthalocyanines with limitations such as low photostability and poor performance in hypoxic microenvironment, Ru (II, III) complexes can offer a range of advantages to overcome these challenges to improve PDT outcomes [159]. Due to their ability to form multiligand complexes, ruthenium PSs have a high triplet-triplet energy and high electron transfer reaction rates [160,161]. 31 In addition, ruthenium-based PSs can provide high quantum yields, Φ, that can reach near unity, which strengthens their use case as photosensitizers from a chemist’s perspective [162]. This opens the door for Ru PSs to be used as photocatalytic agents where oxygen- and oxygen-independent-mediated environments are available [163]. Perhaps one drawback that early Ru-based PSs had was the photobleaching and reduced molar absorptivity, ε, with some Ru PSs losing 1/e activity after 40-50 J/ cm2 of radiant exposure, H, in a solution [161]. This, however, is not the issue with more recently synthesized Ru PSs based on propyridyl (bpy), phenanthroline (phen), and diphenyl phenanthroline (dpp) moieties. Some of the common mononuclear Ru-based PSs are listed in Figure 2.1. 32 Figure 2.1 Structure of some common mononuclear ruthenium photosensitizers. The synthetic variety of ruthenium complexes can further be exploited to form binuclear, trinuclear or polynuclear complexes [141,164]. These transition metal photosensitizers can also form mixed metal PSs in which a Ru centre is joined by another metal centre such as 33 platinum, cobalt, rhodium or vanadium to form supramolecular photosensitizers [165,166]. The long absorption range of ruthenium complexes is another characteristic that make them desired choices as photosensitizers as their excitation can be triggered with a broad range of light sources. The photochemical and photophysical properties that play a role in selecting a suitable ruthenium complex as a photosensitizer are its characteristic absorption wavelength, coefficient of molar absorptivity, quantum yield, and the degree of stability and photobleaching. The lability of the ruthenium compounds and its unique properties means that hundreds of its derivatives can find applications as PSs; however, for the purpose of achieving the main goals of this work, which is to apply Ru-based PS as an embedded photocatalyst to trigger surface reactivity and sterilization, specific attention was paid to selecting a ruthenium PS with simple and readily scalable synthetic pathway that is also easy to be permanently incorporated into variety of polymeric structures. Table 2.1 lists the characteristic properties of Ru(bpy)32+ and Ru(dpp)32+ as the most widely used ruthenium-based photosensitizers used in PDT. 34 Ru Compound λmax , Abs (nm) λmax , Em (nm) τ m , (ns) E ½ ox (V) Ru(bpy)32+ 452 630 600 1.26 Ru(dpp)32+ 455 636 270 1.68 Table 2.1 Absorption, emission, and electrochemical data for Ru(bpy)32+ and Ru(dpp)32+. [167–169] With: λmax , Abs (nm) : maximum absorption wavelength in nanometer λmax , Em (nm) : maximum emission wavelength in nanometer τ m , (ns) : Emission lifetime in H2O at room temperature in nanosecond E ½ ox (V) : Reduction potential in volt for first oxidation in acetonitrile with 0.1M TBAH as supporting electrolyte The principles of photodynamic action were described in detail in Section 1.6. Every PDT energy transfer sequence involves a combination of photochemical, photophysical, and photobiological steps, and ruthenium photosensitizers can trigger photokilling action by ROS generation via both type I and type II reactions. This is described using a Jablonski diagram on Figure 2.2. 35 Figure 2.2 Mechanisms of photodynamic activity of ruthenium photosensitizers. modified from [138]) (created with Biorender) The cytotoxic species produced through photophysical processes are typically 1O2 , OH. and H2O2 [170,171]. This dual photo reactivity attribute of the Ru-PSs, combined with the short-range action of the ROS they produce is another factor that makes them ideal embedded agents for surface photosterilization to eliminate surface-adhered microorganisms. 2.1.2 Antimicrobial product development process Developing an antimicrobial biomaterial with application in a specific field of medicine requires that a set of multivariate elements be satisfied in order to ensure the efficacy and safety 36 of the technology. To achieve a photoactivatable PS-polymer system to be used as medical implants in different medical settings, the end product needs to be optimized by considering several factors. First, the suitability of incorporating the photosensitizer into the polymeric matrix needs to be tested and verified. This is crucial in order to guarantee that the final antimicrobial product is easy to manufacture and readily scalable and economical. The parameters associated with different antimicrobial surface treatments were discussed in length in Section 1.5. Although a variety of approaches exist to incorporate antimicrobial products into biomaterials for biomedical applications [172,173], there exists the need for simple-to-use technologies that can yield the final product without lengthy and costly synthetic pathways. This is one of the advantages of the current antimicrobial systems presented here as it can be set as an example of a simple two-step method that involves simple synthesis of the PS and simple incorporation into both soft and hard polymers. To evaluate this, both various PS and polymer concentrations, as well as incorporation time for PS-polymer needs to be studied and optimized. Once the PS-polymer system is made, it needs to be tested for ROS generation to ensure it demonstrates antimicrobial activity over extended periods of time. Furthermore, the permanent embedding of PS inside the polymer is vital; therefore, the system needs to be tested for leaching of the material from the polymeric structure. The mechanical, physical, and chemical properties of the new PS-polymer system would also need to remain intact and comparable to similar medical implants already in use. The next major factor is biocompatibility and cytotoxicity evaluation of the system. This is to ensure the system can be safely used near biological tissue without triggering any inflammatory or immune response. For cancer PDT applications, an ideal PS would also need to be validated for its pharmacokinetics, plasma half-life, and clearance rate from biological media [174,175], but in our case, these parameters are not going to be of significance once it is established that no material leaches out of 37 the implant. Finally, the light source used for surface illumination as well as the exposure time and irradiance would need to be considered and evaluated to offer the best antimicrobial activity. 2.1.3 Critical to Quality trees for medical device product development Medical device quality definition describes critical to quality (CtQ) as a set of quantifiable and measurable attributes or characteristics that are essential to the quality of a newly designed medical device. The concept has been borrowed from the principles of six sigma, a method to ensure error and discrepancies minimization in a manufacturing or management process [176], to provide a set of measurable goals by defining the needs, the drivers (wants), and the specific requirements necessary to achieve them in order to obtain customer satisfaction. Within the biomedical materials product development context, the major need is improving patient outcome (satisfaction) [177], which is translated into developing antimicrobial medical implants to help reduce healthcare-associated infections (HAI) for the purpose of this thesis work. The drivers that ensure the final antimicrobial product would exhibit the desired performance are the availability of the PS to be incorporated into both various polymeric biomaterials, the ability of the PS-polymer system to be activated by a light source to initiate photosterilization, and ideal properties of the final product to turn it into medical materials suitable for applications in healthcare settings. Finally, the specific requirements to guarantee the successful implementation of the drivers are factors such as permanent locking of the PS inside the polymeric substrate, ability of the PS-polymer system to be used in conjunction with visible spectrum LEDs as simple and cost-effective light sources, measurable and acceptable physical, chemical and mechanical properties as well as overall safety, scalability, and simplicity of the end product (Figure 2.3). 38 Figure 2.3 Critical to quality (CtQ) tree for antimicrobial medical implant design. The contents of this chapter cover all the experimental procedures and results related to the synthesis and characterization of the ruthenium photosensitizer as well as its incorporation into PDMS as the precursor polymer for urinary catheters. It also covers the design and optimization of the LED light source as the illumination source for triggering photodynamic action. The studies related to ROS generation and detection by a singlet oxygen quenching molecule are also included in this chapter. The evaluation of the physicomechanical properties of the PS-polymer system, designed with characteristics that resemble those of silicone urinary catheters, as Specific requirementsThe wantThe needAntimicrobial medical implantEmbedded PS-polymer systemRu PS can be incorporated into soft and hard polymersPermanent embedding (No PS leaching)PhotosterilizableLED activatedVisible and broad range absorptionIdeal propertiesPhysicomechanicalproperties comparable to available implantsEffectively reduces contamination while being biocompatibleEasy to manufacture and cost-effective39 well as in vitro, in vivo, and cytotoxicity studies of the system, as well as the extent of biofilm formation monitored by various imaging techniques are presented in the next chapter. Figure 2.4 shows the schematic diagram that illustrates the incorporation of Ru PS in PDMS to provide the desired PS-polymer product. Figure 2.4 Schematic diagram illustrating the incorporation of PS into PDMS. (created with Biorender) 40 2.2 Experimental section 2.2.1 Materials All chemicals were used as purchased unless otherwise stated. Ruthenium chloride (RuCl3), ammonium hexafluorophosphate (NH4PF6), dimethylanthracene (DMA), diethylether, ethylene glycol, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). 4,7-Diphenyl-1,10-phenanthroline was purchased from Oakwood Chemical Inc. (Estill, SC, USA). Chloroform and acetone were obtained from ACP Chemicals Inc. Polydimethylsiloxane (PDMS) SYLGARD 184 kit containing the PDMS elastomer base and the curing agent as cross-linker was obtained from Dow Corning Inc. (Midland, MI, USA). 2.2.2 Synthesis of Ru(dpp)3(PF6)2 photosensitizer The mononuclear ruthenium coordination complex Ru(dpp)3(PF6)2 can be synthesized using multiple pathways which is a result of versatility of synthetic routes for bathophenanthroline ligand [178,179]. In our case, the ruthenium photosensitizer (PS) was prepared following the previously reported methods [180,181] with some modifications. A solution of ruthenium chloride (50 mg, 0.24 mmol) in a 10:1 mixture of ethylene glycol (1.4 mL) and H2O (0.14 mL) in a round-bottom flask was treated with 4,7-diphenyl-1,10-phenanthroline (240 mg, 0.72 mmol) at 120 °C. The mixture was heated at reflux for 45 min, then temperature was increased to 165 °C. The mixture was then cooled in an ice bath. Acetone (13 mL) was added to the cooled reaction mixture, which was then filtered. The resulting filtrate containing tris (4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)3Cl2 1) was added to a solution of NH4PF6 (1.63 g, 10 mmol) in water (100 mL). The resulting orange precipitate was isolated by vacuum filtration and washed 41 three times with cold distilled H2O. Recrystallization using 80:20 acetone: water (10 mL) followed by washing with diethyl ether (10 mL) and drying under vacuum at 40 °C afforded 242 mg (76% yield) of Ru(dpp)3(PF6)2 2 as an orange crystalline solid. 2.2.3 Characterization of chemical and physical properties of Ru(dpp)3(PF6)2 photosensitizer 2.2.3.1 Ultraviolet-Visible (UV-Vis) absorption spectroscopy A 1 mM stock solution of Ru(dpp)3(PF6)2 2 in CHCl3 was prepared and stored in a cool, dark place. UV-vis absorption spectroscopy was performed using a Varian Cary 300 Bio spectrophotometer. Measurements were run in a dual beam setting from 800 nm to 200 nm with a scan rate of 600 nm/min and a data interval of 1.0 nm. All measurements were taken in a quartz cuvette with a path length of 1 cm. 2.2.3.2 Nuclear Magnetic Resonance (NMR) spectroscopy 1H, 19F and 31P NMR characterizations of Ru(dpp)3(PF6)2 2 in CDCl3 were performed on a Bruker QNP 400 cryoprobe working at 400.1 MHz. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) using the residual solvent peak as a reference. Coupling constants (J) are reported in hertz. Multiplicities are reported as: s = singlet, d = double, t = triplet, q = quartet, and m = multiplet. 2.2.3.3 High Resolution Mass Spectrometry (HRMS) High Resolution Mass Spectra (HRMS) measurements of Ru(dpp)3(PF6)2 2 were obtained using an Agilent 1200 time of flight (TOF) liquid chromatography/mass spectrometry 42 (LC/MS) system in electrospray ionization (ESI) positive mode. All measurements were obtained at 4D LABS facility by the personnel. 2.2.3.4 Melting Point Melting points were measured using a Gallenkamp melting point apparatus and are reported as uncorrected values upon compound’s decomposition. 2.2.4 PDMS preparation and Ru(dpp)3(PF6)2 photosensitizer incorporation in PDMS Once the Ru PS is obtained, it can be incorporated into different polymer matrices using a variety of techniques, including dip coating and co-casting methods. In Chapter 4, 2 as a photosensitizer and MA as the polymer are co-casted to produce the methacrylate-based dental resin as an example of a hard polymer to evaluate its mechanical, physical, and antimicrobial properties [182]. However, since 2 cannot be readily mixed with the PDMS elastomer, the co-casting method was not possible and instead, solid PDMS disks containing the photosensitizer were easily prepared using 15 mL plastic Falcon™ tubes as the mold. PDMS elastomer base and its curing agent were mixed at a 10:1 volumetric ratio in scintillation vials using a glass rod and the mixture was sonicated for 5 min to remove air bubbles and provide a uniform sample. PDMS disks were cast by carefully pouring the liquid mixture inside 15 mL Falcon™ conical centrifuge tubes. The curing process was completed by curing the samples at 25 °C for 48 h, and disks with 1.5 cm diameter and 0.3 cm thickness were prepared by removing the PDMS from the tubes and cutting the cast polymer using a precision cutter blade. When needed, 1 × 1 cm2 coupons of PDMS with 0.3 cm thickness were made by casting the polymer in a petri dish. Ru(dpp)3(PF6)2 2 was embedded into the PDMS disks or coupons using the ‘swell-encapsulation-shrink’ method. Solutions of compound 2 in CHCl3 with varying concentrations (1, 2, 4 and 8 mM) were prepared 43 in scintillation vials and stored in the dark. Next, PDMS disks were dipped in each solution and allowed to ‘swell’ for 60 min, at which time they were removed from the solutions, washed gently with EtOH (70% v/v) and Phosphate Buffered Saline (PBS) (0.01 M, pH = 7.4), and dried overnight in an oven at 37 °C to remove any residual solvent. A cell phone compatible 25X Macro Lens (Selvim Inc, China) was used to capture optical images of the top and cross-sections of the PDMS disks. 2.2.5 LED array light source fabrication and calibration A 4 × 6 array of blue (470 nm peak wavelength) LED lights was fabricated to allow up to 24 PDMS disks to be tested simultaneously. The experimental setup consisted of the LED array aimed downwards and aligned with a 24-well tissue culture plate (TCP) containing the samples. The array was enclosed in a cardboard ‘box’ to fit around the assay tray blocking any external light during testing. The power delivered to the LEDs was controlled using a variable resistor (0-10kΩ) allowing a maximum of 60 mW (20 mA @ 3.0 V). The power was monitored using a small microcontroller (Adafruit Pro Trinket) and a D/C current sensor (INA219), which was displayed on a small OLED display. An Ocean Optics USB4000-VIS-NIR spectrometer with a wavelength range of 344-1048 nm was used to monitor the output wavelength of the array’s light. The light emitted from a single LED passed through a collection fiber connected to the fiber optic input of the spectrometer, dispersed, and detected by the spectrometer and the plot of photonic intensity over wavelength was obtained at a 100 scans/min rate. The total photonic output power density per LED was obtained using a ROPMLS-810 optical power meter. and calculated to be 10.1 mW/cm2, with the distance between the LED and the bottom of a 24-well TCP being consistently 1.5 cm. 44 2.2.6 Leaching of Ru(dpp)3(PF6)2 from PDMS PDMS•Ru(dpp)3 disks (prepared using a 1 mM solution of 2 in CHCl3 and the ‘swell-encapsulation-shrink’ method) were placed in ddH2O (10 mL) and UV-vis absorption spectra were obtained every 30 min for a total of 4 h. The intensity of absorption at 450 nm corresponding to Ru(dpp)3(PF6)2 2 was recorded. 2.2.7 Singlet oxygen detection using dimethylanthracene (DMA) as a molecular quencher As a ‘proof-of-principle’ for further studies, a piece of a PDMS catheter currently used by the biomedical community was used for this experiment. A piece of a PDMS-based 16Fr silicone catheter material (0.3 × 0.5 cm2) was immersed in a 1 mM CHCl3 solution of Ru(dpp)3(PF6)2 2 for 30 min. After removing the piece of PDMS and drying it overnight, it was briefly rinsed with EtOH to remove any excess ruthenium complex 2 on the piece’s surface. The resulting orange PDMS piece was immersed in EtOH (10 mL) and dimethylanthracene (DMA) was added to make a 0.1 mM concentration. A separate sample of 0.1 mM DMA in EtOH was prepared to be used as a control. UV-vis absorption spectra were recorded for both samples and then they were irradiated with the 470 nm blue light LED array. UV-vis spectra were recorded every 30 min up to a total of 3 h. Changes in the absorption intensity between 350 nm and 400 nm were measured as an indication of the reaction of DMA with photoinduced production of singlet oxygen. 45 2.3 Results and discussion 2.3.1 Synthesis of Ru(dpp)3(PF6)2 photosensitizer Due to availability of 4,7-diphenyl-1,10-phenanthroline (bathophenanthroline) in pure form, the synthesis pathway for ruthenium PS was designed using the compound as a precursor. Reacting bathophenanthroline with ruthenium chloride under reducing conditions produces coordination complex 1, which undergoes an anion exchange reaction without isolating it with ammonium hexafluorophosphate to yield the final hexafluorophosphate salt. This reaction is referred to as salt metathesis or ion exchange reaction, which is a type of double displacement chemical reactions involving exchange of bonds between two reacting species [183]. The counterion exchange species, hexafluorophosphate (PF6-), has non-coordinating anion with high affinity towards organic solvents while exhibiting very low solubility in aquous solution and also has high stability and shows resistance to both acidic and basic hydrolysis [184–186]. This ion exchange is essential for our application as it ensures the final complex has low water solubility and permanently remains in the organic polymeric material. The final compound is conveniently isolated by filtering from the aqueous reaction mixture and purifying by recrystallization. Figure 2.5 Shows the synthetic pathway of the ruthenium photosensitizer Ru(dpp)3(PF6)2 2. 46 Figure 2.5 Synthetic pathway of photosensitizer Ru(dpp)3(PF6)2 (2). The tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) bis(hexafluorophosphate) (Ru(dpp)3(PF6)2) complex as the final PS product used in this thesis has Molecular Formula C72H48RuF12N6P2 and Molecular Weight of 1388.19 g/mol. 2.3.2 Characterization of chemical and physical properties of Ru(dpp)3(PF6)2 photosensitizer 2.3.2.1 Ultraviolet-Visible (UV-Vis) absorption spectroscopy The coordination complex Ru(dpp)3(PF6)2 2 exhibits broad metal-to-ligand charge transfer bands between 350 and 550 nm in the UV-vis absorption spectrum, with maximum absorption at 440-470 nm as reported in the literature [167,168] (Figure 2.6). This suggests that the use of blue light is the most practical choice to generate ROS. 47 Figure 2.6 UV-Vis absorption spectrum of a CHCl3 (1.0 × 10–3 M) solution of Ru(dpp)3(PF6)2 (2). 2.3.2.2 Nuclear Magnetic Resonance (NMR) spectroscopy NMR spectroscopy is a technique frequently used in order to determine chemical, physical, electronic, and structural information about the organic and coordination complex compounds in both solution and solid state. It works based on the physical phenomenon of magnetic field perturbations when atomic nuclei of the molecules are subjected to strong external magnetic fields, with the effect of the adjacent nuclei with non-zero nuclear spins (an odd number of protons and neutrons). For this reason, the nuclei of the elements and their isotopes with odd sum of protons and neutrons such as 1H, 13C, 19F, and 31P can be used for structure elucidation via peak splitting due to dipolar couplings between various nuclei [187]. For Ru photosensitizer, Ru(dpp)3(PF6)2 , the following data presents the chemical shifts (in ppm) for the major peaks as observed by 1H, 19F, and 31P NMR. 48 1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 5.48 Hz, 1H), 8.23 (s, 1H), 7.81 (d, J = 5.49 Hz, 1H), 7.59 (m, 5H) These obtained values match with the reported values in the literature for the same compound with CDCl3 as solvent [178,188]. 19F NMR (376 MHz, CDCl3) δ -72.17 (d, J = 713 Hz) 31P NMR (162 MHz, CDCl3) δ: -144.4 (septet, J = 713 Hz) The reported chemical shift values for 19F and 31P NMR spectra are due to the equivalent fluorine atoms coupling to the singular phosphorus atom [189]. Figures 2.7-2.9 illustrate the 1H, 19F, and 31P NMR spectra for the Ru(dpp)3(PF6)2 photosensitizer. Figure 2.7 1H NMR spectrum (CDCl3) of Ru(dpp)3(PF6)2 (2). 49 Figure 2.8 19F NMR spectrum (CDCl3) of Ru(dpp)3(PF6)2 (2). Figure 2.9 31P NMR spectrum (CDCl3) of Ru(dpp)3(PF6)2 (2). 50 2.3.2.3 High Resolution Mass Spectrometry (HRMS) HRMS is an analytical method for structural determination of a variety of chemicals. In principle, it works by bombarding a sample of the analyte with electrons, resulting in fragmentation of the initial compound, and the measurement of mass to charge (m/z) ratio of the fragments would ultimately provide insights about the structure of the analyte. With HRMS, the m/z can be measured with three to four decimal places, providing great accuracy for chemical structure determination [190]. HRMS (M+H+) expected for Ru(dpp)32+ (C72H48N6Ru): 549.1496. The observed value found is 549.1490. Figure 2.10 shows the HRMS spectra for Ru(dpp)3(PF6)2 photosensitizer. Figure 2.10 HRMS (TOF LC/MS - ESI-(+) mode) of Ru(dpp)3(PF6)2 (2). 51 2.3.2.4 Melting Point Most Ru (II) coordination complexes have melting points higher than 200 °C, with bathophenanthroline compound derivatives reaching melting points of up to 279 °C [191]. In the case of the Ru(dpp)3(PF6)2 photosensitizer, the value obtained was 206-210 °C (decomposed, which means that at temperatures above 200 °C, visible disintegration of the PS compound is observed via destruction of the orange crystals of Ru(dpp)3(PF6)2). 2.3.3 PDMS preparation and Ru(dpp)3(PF6)2 photosensitizer incorporation in PDMS Polydimethylsiloxane (PDMS)-based products have great versatility and practical usage [192–194]. The polymer provides an ideal choice of to show the success of using the photosensitizer to produce singlet oxygen and decontaminate surfaces. PDMS is already being extensively used as a durable polymeric biomaterial, especially as catheters and drainage tubing, micro valves, dialysis membranes, and adaptive optical lenses. This is mainly because the polymer is highly biocompatible, stable and economic [192,193]. Another important feature of PDMS is the ease at which oxygen diffuses through it due to the high flexibility of the siloxane linkages [195], a necessary characteristic in order to access the photosensitizer complex. Also, PDMS is transparent to blue light but has a higher refractive index than water so it may act as a ‘lens’ or ‘waveguide’ to help propagate and simultaneously contain the light [196]. Figure 2.11 Shows photographic images of the top and cross-sections of the PDMS•Ru(dpp)3 disks with various concentrations. PDMS samples of various sizes and shapes are best prepared from the elastomer base and its curing agent by casting into appropriate molds for the experiments and curing at 25 °C for 48 h [197]. Allowing long curing times eliminates any possible changes in the physical properties 52 such as surface roughness or Young’s modulus [197]. Whatever the size or shape of the PDMS sample, Ru(dpp)3(PF6)2 (2) is readily loaded into it using the ‘swell-encapsulation-shrink’ method [198], where the cured PDMS is soaked in an organic solvent (in this case CHCl3) containing the small molecule of choice to expand the polymer matrix and allow the inflow of the molecule into the gel [199]. After removing the sample and evaporating the solvent, the matrix collapses and traps the molecule permanently within the solid matrix (in this case Ru(dpp)3(PF6)2). Figure 2.11 Photographic images of solid PDMS disks (1.5 cm × 0.3 cm) after they have undergone the ‘swell-encapsulation-shrink’ method using 1 mM, 2 mM, 4 mM and 8 mM CHCl3 solutions of Ru(dpp)3(PF6)2 (2). In each case, the cross-section of the disk is shown in the bottom image. 2.3.4 LED array light source fabrication and calibration An array (8 × 12 cm) of 4 × 6 blue light LEDs provides a suitable illumination system to demonstrate the photosensitization of coordination complex Ru(dpp)3(PF6)2 2. The size and configuration of the array were specifically designed to illuminate 24-well tissue culture plates, 53 which are used to contain the polymer samples in our studies. The circuit diagram of the LED light array along with its emission spectrum are shown in Figure 2.12. Figure 2.12 Circuit diagram of the LED array light source (top), and the emission spectrum of the LED array (bottom). Figure 2.13 shows the UV-vis absorption spectrum of a CHCl3 (1.0 × 10–3 M) solution of Ru(dpp)3(PF6)2 2 with the emission spectrum of the blue LED array superimposed on it in blue, which has excellent overlap with the absorption spectrum of Ru(dpp)3(PF6)2 2. The total photonic 54 output power density per LED was obtained using a ROPMLS-810 optical power meter and calculated to be 10.1 mW/cm2, with the distance between the LED and the bottom of a 24-well TCP being consistently 1.5 cm. Figure 2.13 (a) UV-vis absorption spectrum of a CHCl3 (1.0 × 10–3 M) solution of Ru(dpp)3(PF6)2 2 with the emission spectrum of the blue LED array superimposed (blue) (b) Photographic image of the 4 × 6 blue LED array used in these studies. 2.3.5 Leaching of Ru(dpp)3(PF6)2 from PDMS To function as a light-activated decontamination material, it is essential for the photosensitizing coordination complex to remain inside the polymer matrix and not spontaneously leach into the surrounding environment. This is also important to minimize toxicity since cytotoxicity of the transition metal complexes could pose a risk when tested within biological media [200–202]. The high stability of the solid PDMS•Ru(dpp)3 disks and lack of leaching of compound 2 can be demonstrated by immersing the disks (1.5 cm in diameter, 0.3 cm in thickness) of PDMS loaded with Ru(dpp)3(PF6)2 (using a 1 mM initial solution of 2) in water and measuring any changes in the UV-vis absorption spectra of the solution (Figure 2.14). After 4 hours of (a)250 350 450 550 6500.00.51.0λ (nm)absorption(b)emission55 immersion, no increases in bands corresponding to the coordination compound can be observed at 450 nm, illustrating that no observable leaching of 2 occurs. It is expected that the same trend would be observed over extended exposure period as there is no interaction between the polymeric matrix and aqueous media. Figure 2.14 Leaching studies of Ru(dpp)3(PF6)2 2 - (a) Changes in the UV-vis absorption spectrum when a disk (1.5 cm in diameter, 0.3 cm in thickness) containing Ru(dpp)3(PF6)2 (1 mM original solution of 2) was immersed in water (10 mL) at room temperature for a total of 4 h. (b) Changes in the absorption intensity at 450 nm during the 240 min period (corresponding to 2) when a PDMS disk (1.5 cm in diameter, 0.3 cm in thickness) containing Ru(dpp)3(PF6)2 (1.0 mM original solution of 2) is immersed in water (10 mL) at room temperature for a total of 4 h. 2.3.6 Singlet oxygen detection using dimethylanthracene (DMA) as a molecular quencher Prior to commencing a rigorous investigation into the antimicrobial activity of PDMS disks containing the photosensitizer, a simple ‘proof-of-concept’ study was performed using an ‘off-the-shelf’ silicone catheter. A convenient way to demonstrate that the photosensitive coordination complex triggers the generation of singlet oxygen when exposed to blue light is to 56 trap the 1O2 using anthracene derivatives, which are known to undergo [4+2] reactions with this dienophile [203,204]. One example is 9,10-dimethylanthracene, which produces the endoperoxide shown in Figure 2.15. Because the anthracene has characteristic fingerprint bands in its absorption spectrum in the 325-400 nm region, which the endoperoxide form does not, the decrease in the intensity of these bands is a means to easily monitor the photosensitization. Figure 2.15 Reaction of singlet oxygen with 9,10-dimethylanthracene (DMA) produces endoperoxide product. Figure 2.16 shows the effectiveness of Ru(dpp)3(PF6)2 (2) to produce singlet oxygen when exposed to blue light from the LED array. Immediate decreases in the intensity of the absorption bands corresponding to 9,10-dimethylanthracene can be seen in the spectrum of an EtOH solution of it when exposed to light but only when the photosensitizer is present. No changes are observed in the absence of compound 2. 57 Figure 2.16 Singlet oxygen generation studies of Ru(dpp)3(PF6)2 2. Changes in the UV-vis absorption spectra when EtOH solutions (1.0 × 10–1 M) of 9,10-dimethylanthracene were exposed to blue light from the LED array for a total of 3 hours in the absence (a) and presence (b) of a PDMS-based silicone catheter disk of 0.3 cm thickness embedded with starting concentration of 1 mM of 2, showing changes only when the photosensitizer is present. The inset in each case shows the decrease in the absorption intensity during the 3 h period. 2.4 Conclusion In this chapter, the synthesis, characterization of the Ru photosensitizer (Ru(dpp)3(PF6)2) and its incorporation into PDMS native polymer matrix (PDMS•Ru(dpp)3) to provide the necessary polymer-embedded platform to assess efficacy as a functional antimicrobial biomaterial upon exposure to blue LEDs, are discussed. The optical properties of the LED light source were designed to suitably match the emission wavelength of the blue light with maximum absorption wavelength of the PS in order to maximize photocatalytic conversion from the PS excited state into oxygen ground state to produce ROS that would trigger antimicrobial activity. The safety and efficiency of the embedded polymer-PS was also investigated in order to ensure no leaching of the PS occurs from the PDMS matrix and that the embedded system is efficient at generating ROS upon LED exposure over extended periods of time. 58 The next chapter will use PDMS•Ru(dpp)3 as the precursor of a novel photoresponsive antimicrobial biomaterial, and investigates the physico-mechanical, biocompatibility, antimicrobial, and biofilm formation properties of the platform using both in vitro and in vivo models in order to validate its efficacy and potential as a technology to be employed for photosterilization of a urinary Foley catheter as aimplantable biomaterial. This idea is further expanded and tested in chapter 4 using the same Ru PS, incorporated into a methacrylate-based experimental formulation to produce a photoreactive antimicrobial restorative dental resin system. 59 Chapter 3: Incorporation of Ruthenium photosensitizer into experimental silicon-based urinary catheter and evaluation of physical, chemical, mechanical, antimicrobial, and biofilm formation properties: The case of PDMS as a soft material Synopsis In this chapter, further characterization studies of the PDMS+PS (PDMS•Ru(dpp)3) system is carried out in order to investigate the physical and mechanical properties of the embedded PS+ polymer as a modified precursor of silicone-based urinary catheters. The physico-mechanical properties of the PDMS•Ru(dpp)3 including tensile strength, surface roughness, surface hydrophobicity, and surface encrustation for biomaterial samples with varying ruthenium concentrations are evaluated and compared with those of bare PDMS. A set of preliminary in vitro studies is performed to assess the antimicrobial efficacy of the PDMS•Ru(dpp)3 with different PS concentrations by exposing the samples to LED light source for various time points in an effort to obtain the most efficient doses. Next, the degree of biocompatibility of PDMS•Ru(dpp)3 is determined by a series of cytotoxicity evaluation experiments via MTT assays. The most suitable groups of PDMS•Ru(dpp)3 are also used for testing in a rat infection model, followed by histological analysis to provide additional insight into the efficacy of the technology as an antimicrobial biomaterial in a more realistic setting. Finally, various imaging techniques such as SEM and fluorescence microscopy are employed in order to give a comparative account of the degree of biofilm formation for PDMS•Ru(dpp)3 samples exposed to LED compared to bare PDMS as control. 60 3.1 Introduction 3.1.1 The urinary system The urinary system is responsible for filtration and cleansing of blood by means of removing the waste materials by passing them through kidneys, followed by their removal from the body via urination. The organs involved in this process are a pair of kidneys, a pair of ureters, bladder, and urethra. Thousands of metabolic processes are constantly at work in millions of cells in the body, and these processes contribute to the formation of hundreds of different waste products that need to be eliminated from our system. This filtration process starts in the kidneys, a pair of bean-shaped structures the size of an adult fist, where the incoming blood enters microscopic filtering units called nephrons, which remove unwanted waste and minerals as well as surplus water and turn it into urine through a series of filtration, reabsorption, and secretion processes. The urine then passes through ureter and reaches bladder via involuntary contraction of the ureter smooth muscle (peristalsis) and is excreted from the body through urethra by means of micturition once bladder nears its capacity [205]. Apart from filtration, the urinary system is also responsible for regulating the volume, acidity, concentration, salinity, and chemical composition of blood, lymph, and other bodily fluids [206,207]. Urine is composed of around 95% water and 5% other waste products. Some of the waste materials in the urine are nitrogen-based, with urea accounting for 2%, and creatinine, ammonia, and uric acid accounting for 0.2%. The other 2.8% of the waste product are minerals and ions including sodium, potassium, calcium, as well as phosphates and sulphates [208]. The pH level of urine is usually between 6-7.5; however, values between 5-8 as pH range for urine are still considered to be normal [208,209]. Figure 3.1 shows the human urinary system and organs (bold). 61 Figure 3.1 The human urinary system and organs. Reprinted from Pearson Education, Inc at https://images.slideplayer.com/26/8306750/slides/slide_4.jpg 3.1.2 Urinary tract infections As mentioned in Section 3.1, the human urine constitutes multiple components which are associated with urea. Also, urea and its derivatives are known to exhibit antimicrobial properties [210–212]. Therefore, for a long time this had helped establishing a paradigm known as “sterile urine” which basically posited that the urine of a healthy individual would need to be sterile and that any deviation from it would be a sign of infection. This theory, however, has been recently challenged by the discovery of bacteria in healthy female bladder environment [213–215]. The discovery of urinary microbiome in males as well as the female urinary microbiota (FUM) means that the mere presence of bacteria in urine, a condition known as bacteriuria, does not equate to infectious disease anymore, and that scientists and clinicians need to reassess their assumptions of the etiology of urinary tract disorders [216,217]. 62 A urinary tract infection (UTI) in simple terms is an infection that affects the bladders, the kidneys, or any of the ureters and urethra, and can develop through multiple causes and pathways [218]. It is more common among females than males, which is due to differences in anatomy as female urethra is shorter and is also at a closer proximity to rectum. About 50% of the female population experience UTI, and about 25% of them develop recurrent UTIs [219]. Certain conditions that result in obstruction of the urinary tract, such as kidney stone formation or benign prostatic hyperplasia (BPH) as well as urinary hesitancy and incontinence or having a neurogenic bladder can contribute to the accumulation and buildup of pathogens to develop UTI [220–223]. Moreover, conditions such as using contraceptive diaphragms or condoms coated with spermicides, or insertion of a urinary catheter or stent can result in introducing new pathogens from the outside environment into the body, which can ultimately cause UTIs [224–226]. Certain other physiological conditions such as a weakened immune system and diabetes can also be counted as causes of UTI. UTIs are also very common after a kidney transplant surgery. In terms of pathogenicity and progression, UTIs can be classified as complicated and uncomplicated. Uncomplicated UTIs (or lower UTI) refer to the infection of the bladder, also known as cystitis, and associated structures. They can develop when patients exhibit no structural abnormalities or comorbidities such as an immunocompromised state or diabetes. Escherichia coli is the most common pathogen in uncomplicated UTIs, accounting for up to 80% of the cases, followed by Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa [227,228]. The rest of the UTIs which do not conform to the above-mentioned descriptions are classified as complicated UTIs. In brief, infections that develop despite anatomical protective measures or the ones due to presence of abnormalities such as renal calculi (stones), hydronephrosis (pressure buildup in kidneys due to obstruction), or the ones developing as a result of an 63 immunocompromised state such as the case with human immunodeficiency virus (HIV) and diabetic patients or post-chemotherapy treatment are collectively classified as complicated UTIs. Common symptoms of UTI are unexplained fatigue, fever, chills, cloudy and foul-smelling urine, and autonomic dysreflexia as evident from frequent headache and hypertension. Although in some cases UTI can be treated on its own, in majority of cases, and especially with complicated UTIs, it is crucial to seek help from a healthcare professional to assess the signs, symptoms, and perform urinalysis via urine culture, to allow for the administration of proper medication to treat the infection. Frequent hydration and urination are also believed to reduce the possibility of developing UTIs. 3.1.3 Urinary implants The scope of prosthetics in urologic sciences and practice is very broad. Urinary implants can be divided into two major categories: 1. Urinary prosthetics: any device/material that replaces or restores a body part, with its specific function. These devices are developed in order to assist with conditions such as erectile dysfunction (ED) and urinary incontinence and are vital for maintaining the quality of life. Some examples of such materials include penile and testicular implants, artificial urinary sphincters (AUS), and slings [229,230]. 2. Urinary implants: any device/material that is implanted in order to carry out a specific task and is usually removed from the body once the task is completed. Some of the most common urinary implants are double J (J-J) ureteral stents, catheters, urethral stents, meshes, and Urolift systems [229]. 64 Figure 3.2 provides a summary of the scope of the implants and prosthetics in urology. Figure 3.2 Scope of urologic implants and prosthetics. 3.1.4 Catheter-associated urinary tract infections (CAUTI) UTIs that originate from urologic implants are typically caused by introduction of microorganisms from outside environment into the body. This category of infections is referred to as nosocomial or healthcare-associated infections (sometimes referred to as hospital-acquired infections) (HAI) since it gets initiated in hospital settings. Among these, catheter-associated urinary tract infection (CAUTI) is among the most prevalent global health-related illnesses, 65 accounting for up to half of all the hospital-acquired diseases [224,231]. CAUTIs frequently develop as a result of catheter implantation procedures, whereby the catheter tube is inserted into urethra to connect the urine collecting system (drainage bag) to the urinary bladder in order to help patients with urination. The catheters used in this manner are referred to as indwelling catheters. A major factor that plays role in the extent of CAUTI development is the length of catheterization as prolonged use of the indwelling implant frequently results in the formation of biofilm on the surface of the material and although not many patients may show signs of bacteriuria or symptoms of infection, there is always a significant burden on both the patient and healthcare providers as well as the healthcare system due to frequent need for replacement and re-insertion of the device [231–235]. As shown in Figure 3.3, when a catheter is to be inserted, there are multiple pathways for microorganisms to get introduced into the urinary system. The microorganisms in the urethra, periurethral skin or endogenous intestinal flora can migrate around the catheter extraluminally and localise in the bladder, grow inside the drainage bag and migrate to the body by ascending the catheter intra-luminally, or they can be introduced from the environment and surface of the skin at the time of catheterization and as a result of poor decontamination or improper practices (Figure 3.3.a) [236,237]. For the progression of localised colonies into CAUTI, the first step is the formation of a conditioning film on the surface of the urinary catheter, facilitating the initial binding of the pathogens, followed by division and EPS production and ultimate maturation of the biofilm with intricate 3D structures. These biofilm-associated bacteria can then detach themselves from the surface, migrate to other sites and spread the infection to non-colonized sites (Figure 3.3.b) [234,237]. 66 Figure 3.3 Pathogenesis of biofilm formation on urinary catheters during CAUTI development. (Reprinted with permission from Taylor & Francis Group [237]) The intricate and multi-factorial nature of the CAUTIs, both in terms of their development and their side effects and consequent complications make them a critical issue when 67 it comes to providing care for patients who need catheterization. Besides patients with urologic conditions, there are many other common cases where catheterization is required, including for patients who are anesthetized or the patients who are required to stay in intensive care unit (ICU) post surgery [238–240]. In terms of the connection between the catheter dwell time and CAUTI development risk and patient comorbidities, various studies have shown that a 3% to 7% increase in the CAUTI development rate is expected each day after the catheterization, with the risk mounting close to 80% after 21 days post-catheterization [224,241]. Currently, the preferred method of treatment to avoid or control CAUTIs is via prophylactic administration of antibiotics, which in spite of having some success, can result in antimicrobial resistance and added complications [242,243]. Therefore, different treatment modalities are desperately needed to eliminate the concern with MDR and XDR microorganisms and their recurrence, and the contents of this chapter present a successful attempt to provide such an alternative. 3.2 Experimental section 3.2.1 Materials MTT assay solution was purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM) (Gibco™ 15240062), Fetal Bovine Serum (FBS) (Gibco™ 25200056), dimethyl sulfoxide (DMSO), propidium iodide (PI), and SYTO® 9 green dyes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 68 3.2.2 Preliminary studies for parameter optimization of PDMS•Ru(dpp)3 In Chapter 2, some of the relevant experimental parameters such as PS dose, swell time, LED dose, and exposure times were evaluated and the result indicated that a range of PS concentrations between 1 and 8 mM (specifically 1, 2, 4, 8 mM), with 60 min of swell time in chloroform, a 10.1 mW/cm2 of photonic power density delivered at time points ranging from 15 min and up would provide an ideal setting for further evaluation of the antimicrobial activity of the samples in either disk or coupon shape. Swell times of less than 60 min would sometime result in incomplete imbibing of the PS (Ru(dpp)3 (PF6)2) or compound 2 inside the PDMS matrix. Also, lower power density levels would result in dimming of the LED light source which could potentially decrease the efficacy of ROS production via reduced photon count. For this reason, the LED output photonic power was adjusted at the maximum level for the entirety of the experiments. The starting bacterial CFU counts were selected to be between 105 – 106 , which typically results in 104 – 105 CFU of microorganisms adhering to material surface after 4 h of incubation. Disks of PDMS and PDMS•Ru(dpp)3 were used, and minimal amount of glue was applied to attach the samples to the bottom of TCPs. Identical duplicate groups were used for each of the PS-containing samples as well as PDMS as control, with one set being kept in dark at all times to monitor the dark toxicity of the materials, and the other set being exposed to LED at 15, 30, 60, and 120 min intervals. Prior to LED exposure, Uropathogenic Escherichia coli (UPEC) strain (E. coli C1214) was cultured from a freezer stock for 48 h followed by a subsequent 24 h sub-culture prior to experiments by suspending 10 µL of bacterial stock in 25 mL Lysogeny Broth (LB) growth media at 37 C and under aerobic conditions. Next, 990 µL of sterile LB and 10 µL of bacterial inoculum were added to each well, and the samples were incubated at 37 C under aerobic conditions for 4 h. Next, the disks were removed from the plates, washed gently with PBS 69 twice to remove non-adhered bacteria from the surface, and each disk was placed in a new 24-well TCP exactly as mentioned before. Care was taken not to disturb the top surface of the disks during removal, washing, and transfer procedures. The disks were then covered with 1 ml of PBS and placed under an LED light source at an average power density of 10.1 mW/cm2 per well for different periods of time. Once irradiation was over, the disks were removed from the bottom of the wells and transferred to Eppendorf tubes containing 1 ml PBS. The tubes were sonicated for 5 min at 25 C using a digital Ultrasonic bath (Skymen, JP-010S, China) to remove the adhered bacteria from the surface of the disks. The contents of the tubes were vortexed for 10 s, and adherent bacteria were quantified via CFU counts by removing a 10 µL aliquot from each Eppendorf tube for serial dilution followed by incubation in the above-mentioned growth conditions for 18 h. All experiments were performed at least twice, and in triplicates, under identical conditions. The LOG CFU counts for all experimental groups were obtained for all different periods of LED exposure. 3.2.3 Evaluation of physicomechanical properties of PDMS•Ru(dpp)3 3.2.3.1 Tensile strength The mechanical properties of the PDMS•Ru(dpp)3 were studied by measuring the tensile strength using an INSTRON universal testing machine. The dog-bone shape sample materials with bare PDMS as control and PDMS•Ru(dpp)3 with ruthenium concentrations of 1,2,4 mM were prepared using the mold geometries and dimensions according to previously reported (ASTM D412) protocol [244]. The maximum tensile strength applied to stretch the specimens to rupture was recorded at 10 mm/min stretch rate and a tensile strain gauge length of 100 mm. The stress 70 over strain curve for each sample was obtained, and the linear portion of the graph was used to measure Young’s Modulus of elasticity for each group. 3.2.3.2 Surface roughness A Bruker atomic force microscope (AFM) system (Bruker Corp, MA, US) was used to measure the surface roughness of the bare PDMS and PDMS•Ru(dpp)3 coupons with ruthenium concentrations of 1,2,4 mM using tapping mode. 5 × 5 µm2 surface areas on each sample were selected, with a scan rate of 1 Hz. Optical and AFM images were acquired, and the surface height (topography) and average roughness (Ra) for each sample were obtained. 3.2.3.3 Surface hydrophobicity Water contact angle measurements were performed to compare the degree of hydrophobicity of the bare PDMS and PDMS•Ru(dpp)3 coupons with ruthenium concentrations of 1,2,4 mM. A 5 µL water droplet was placed on the surface of the coupons, followed by taking images of the droplets using a digital camera (Retiga 1300, Q-imaging Co) at various spots. The average value of the contact angle was obtained using Northern Eclipse software. 3.2.3.4 Surface encrustation We used a CBR 90 standard CDC Biofilm Reactor® (BioSurface Technologies Corp) to mimic the urinary tract environment for the encrustation assay and used a formulated artificial urine (AU) [245] to simulate the urine composition. The PDMS•Ru(dpp)3 coupons (1,2,4 mM) were suspended inside the CDC bioreactor using sterile suture trends (Ethicon Prolene Suture, blue filament, size 6-0, 18”, BV-1, 9.3mm 3/8c, taper) attached to the top and bottom part of the reactor. The sutures served as a support for the samples to be hanging while the reactor was moving. The CDC bioreactor was then filled with AU and placed on an orbital shaker rotating at 20 rpm and 71 incubated at 37 C for 14 days. All devices and materials were autoclaved at 121 C for 1 h prior to the experiment. Artificial urine was changed every 2 days in a sterile environment to avoid contamination. After 14 days, the samples were aseptically removed from the reactor and gently washed with ddH2O. Two solutions, one containing 0.2% lanthanum chloride and 2% nitric acid, and a second containing 0.2% lanthanum chloride and 5% nitric acid were used to decalcify the samples and dilute the minerals adhered to the samples after sonication. Diluted samples were then used for calcium and magnesium quantification using inductively coupled plasma mass spectrometry (ICP-MS). SEM images of the surface of the coupons were also obtained for qualitative comparison among all groups. 3.2.4 In vitro evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 Uropathogenic Escherichia coli (UPEC) strain C1214 was cultured from a freezer stock for 24 h followed by a subsequent 24 h sub-culture prior to experiments by suspending 10 µL of bacterial stock in 25 mL Lysogeny Broth (LB) growth media at 37 C and under aerobic conditions. PDMS•Ru(dpp)3 disks of PS-embedded PDMS were prepared by submerging the disks in 4 mM PS in CHCl3 solution for 60 min, followed by EtOH (70% v/v) and PBS (0.01 M, pH = 7.4) wash. The disks were then dried in an oven at 40 C overnight and under sterile condition. Disks of PDMS alone were used as control. 24-well TCP was used for in vitro antimicrobial assay experiments. The experimental setup and procedures are identical to the in vitro preliminary studies (Section 3.2.2) with the exception of the increased LED exposure to up to 24 h. 72 3.2.5 Cytotoxicity evaluation of PDMS•Ru(dpp)3 Primary Human Dermal Fibroblast (HDF) cell lines, obtained from newborn circumcision foreskins collected at Vancouver General Hospital (VGH), were cultivated in DMEM with 10% FBS and antibiotic-antimycotic supplement, and incubated at 37 C under 5% CO2 atmosphere to reach a confluency of 80-90% after two passages as confirmed by optical microscopy monitoring. The proliferated cells were then seeded at the bottom of 24-well TCP at a density of 10,000 cells/well, and 1 mL fresh DMEM was added to each well. Next, coupons of PDMS•Ru(dpp)3 with various PS concentrations (1,2,4,8 mM) were placed directly on top of the cells. The TCPs containing cells and coupons were exposed to an LED light source at an average power density of 10.1 mW/cm2 per well for 15 min. A second TCP with identical conditions was used and kept in the dark for the same period. Next, all the plates were incubated at 37 C under a 5% CO2 atmosphere for 24, 48, and 72 h. After the completion of each timepoint, an MTT assay was performed to obtain the percentage of cell viability for all the cells by measuring the optical density per well at 570 nm. 3.2.6 In vivo evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 Next, we investigated the efficacy and biocompatibility of PDMS+Ru in a subcutaneous implant infection model using rats. Healthy female Sprague Dawley (SD) rats (200-300 g,12-18 weeks old) were obtained from Charles River Laboratories. All animal care and experimental procedures were performed in accordance with the University of British Columbia Animal Care Committee's Guide for the Care and Use of Laboratory Animals. The University of British Columbia Animals and Ethics Committee granted clearance for this study (protocol approval ID: A17-0292). To acclimatize to the laboratory environment, the rats were provided with standard 73 laboratory chow, water, and libitum for at least one week before the experiments in a temperature-controlled room. PDMS•Ru(dpp)3 with ruthenium concentrations of 1 and 2 mM were used (labelled PDMS+Ru1 and PDMS+Ru2). Animals were randomly divided into three groups: PDMS+Ru1, PDMS+Ru2, and PDMS-only as a control. Rats were individually anesthetized using 2% isoflurane in oxygen (flow rate of 1.0 L/min) and kept under anesthesia throughout the procedure. Before the surgeries, the coupons (1×1×0.3 cm3) were pre-incubated with E. coli at a concentration of 106 starting CFUs for 5 days to allow for biofilm formation on the disk surfaces. Prior to animal implantation, biofilm formation was confirmed in vitro using optical imaging. The implant groups were further subdivided into LED exposure and dark groups, with LED exposure groups being illuminated for 4 h periods followed by 8 h incubation over the 5-day period. All experimental conditions were kept the same for the dark groups, except for LED exposure. All samples were replenished with fresh LB as the growth medium every 12 h. Pre-infected coupons were then implanted into subcutaneous pockets on the dorsal area of the rats (2 pockets per rat) followed by suturing the implantation site. After 3 days, implants were removed and the number of bacteria on both implants as well as nearby tissue were counted. Tissue colony counts were obtained by collecting a 100 mg tissue sample in direct contact with the infected implant, followed by processing using a Precellys® 24 tissue homogenizer. Sample tissues in contact with each implant as well as from non-infected and non implant contact sites were also collected for histology assessment using H&E staining. 74 3.2.7 Evaluation of biofilm formation on PDMS•Ru(dpp)3 by imaging 3.2.7.1 Scanning Electron Microscopy (SEM) imaging Bare PDMS and PDMS•Ru(dpp)3 coupons with ruthenium concentrations of 1,2,4 mM were inoculated with 106 starting E. coli CFUs and incubated for 5 days to allow biofilm formation on the substrate surfaces, with LED exposure and LB refreshing done identically to the in vivo studies procedure. Bare PDMS coupons were kept in the dark as a control group. On day 5, samples were removed from LB, with surfaces washed very gently not to disturb the biofilm, and fixated for SEM imaging using established protocols, with some modifications [246]. Next, samples were iridium sputtered and SEM images of the coupon surfaces were obtained using a FEI Helios NanoLab 650 system (Field Electron and Ion Company, OR, US), operating at an accelerating voltage of 10.0 kV and under 13 pA current. 3.2.7.2 Live/dead staining and Fluorescence imaging The same procedure, as stated in Section 2.11.1, was used and coupon samples were prepared for live/dead staining assay using fluorescence imaging [247]. A 1 mL solution of fluorescent dyes containing SYTO® 9 (3 μL/mL) and propidium iodide (PI) (3 μL/mL) dissolved in deionized water was gently added to the coupons in a 24-well TCP. After 20 min, the samples were removed and washed very gently with 1 mL of sterile deionized water and allowed to dry in a fume hood. The dehydrated samples were then imaged with a fluorescence microscope (Zeiss Axio observer 7, Carl Zeiss Microimaging Inc). An image analysis toolkit (SW ZEN Toolkit 2D DLic) was further utilized to obtain the viable bacterial cell counts within randomly selected areas of the biofilms on each coupon. 75 3.2.8 Statistical analysis Data were analyzed by Prism GraphPad software (version 9) using ANOVA and Tukey tests. Error bars indicate mean ± standard deviation (SDV) for all experimental groups with n=3, with P-values denoted under graphs indicating the level of significance between independent experiments. 3.3 Results and discussion 3.3.1 Preliminary studies for parameter optimization of PDMS•Ru(dpp)3 Figure 3.4 shows the results of preliminary in vitro studies on the antimicrobial activity of PDMS•Ru(dpp)3 disks containing different amounts of the photosensitizer 2 using UPEC (E. coli C1214). The difference in the number of colony forming units between 15 min and 120 min of blue light provides a ‘killing rate’, which offers a convenient way to decide the best concentration of Ru(dpp)3(PF6)2 (2) to be used for more in-depth studies. Figure 3.4 clearly shows that PDMS disks that do not contain compound 2 have little effect on decreasing the number of bacteria on the disk’s surface even when exposed to blue light. Under similar light exposure, the disks containing the photosensitizer (PDMS•Ru(dpp)3) are much more effective at killing the E. coli at any concentration of Ru(dpp)3(PF6)2. While the highest concentration of 2 (8 mM initial solution) seems to have a larger effect, the fact that it also has activity when kept in the dark (furthest right black bar) suggests it has some dark antibacterial activity. These studies indicate 1, 2, and 4 mM initial concentrations of compound 2 for ‘swelling’ the PDMS disks are the best choices moving forward. 76 Figure 3.4. Preliminary in vitro antimicrobial studies of PDMS•Ru(dpp)3 disks (+/-) LED. Rate of decrease in the number of E. coli when disks (1.5 cm in diameter, 0.3 cm in thickness) without Ru(dpp)3(PF6)2 are exposed to blue light (white bar), when varying amounts of Ru(dpp)3(PF6)2 are stored in the dark (black bar), and when varying amounts of Ru(dpp)3(PF6)2 are exposed to blue light (blue bar). Concentrations of 1, 2, 4, and 8 mM of 2 in CHCl3 were used to prepare the disks using the ‘swell-encapsulation-shrink’ method. Figure 3.5 shows the changes in the number of E. coli (expressed in LOG CFU/mL) between 15 and 120 minutes on the surface of PDMS and PDMS•Ru(dpp)3 disks (1.5 cm in diameter, 0.3 cm thickness) with various concentrations of compound 2 when exposed to LED light. 77 78 Figure 3.5. Changes in viable E.coli counts on PDMS•Ru(dpp)3 disk surfaces. Changes in the number of E. coli between 15 and 120 minutes on the surface of a PDMS disk (1.5 cm in diameter, 0.3 cm thickness) and PDMS disk containing Ru(dpp)3(PF6)2 (1,2,4,8 mM original solution of 2) when stored in the dark (left) and exposed to LED light (right). 3.3.2 Evaluation of physicomechanical properties of PDMS•Ru(dpp)3 3.3.2.1 Tensile strength PDMS has a Young’s modulus of elasticity ranging from 1.5 to 5 MPa depending on the ratio of elastomer to curing agent as well as curing temperatures [248–251]. When ruthenium PS is incorporated into PDMS, there is an observable increase in Young’s modulus with increasing 79 PS concentration. This is mainly due to the increase in molecular weight of PDMS•Ru(dpp)3 due to PS incorporation, which impacts the polymer structure strength. Figure 3.6 shows photographic images of the samples used for tensile strength test as well as stress over strain curves for different experimental groups and the Young’s modulus of elasticity, obtained by calculating the slope of the stress over strain curve in the linear region reported as the average of three measurements for each group. Figure 3.6 Tensile strength studies of PDMS•Ru(dpp)3 - (Top) Photographic images of the dog-bone shaped samples of PDMS and PDMS•Ru(dpp)3 groups with different concentrations of 2 and the INSTRON UTS system. (Bottom Left) Tensile strength measurement of PDMS•Ru(dpp)3 samples compared to bare PDMS as stress over strain curve for a single measurement and (Bottom Right) Young’s modulus of elasticity for PDMS and PDMS•Ru(dpp)3 groups with different concentrations of 2 as an average of three measurements per group. 80 3.3.2.2 Surface roughness Surface roughness (Ra) is a determinant of bacterial adhesion, biofilm formation and encrustation with rougher surfaces providing more surface area and crevices for bacterial and crystal deposition and attachment, and this has been proven to be the case with a variety of medical implants used in different areas such as orthopedics, dental materials and urinary implants [252–256]. Overall, surface roughness was found to increase with the addition of PS as all PDMS•Ru(dpp)3 groups were found to have higher Ra values than bare PDMS, suggesting that the PS resulted in a rougher surface (Figure 3.7). That said, the Ra for PDMS+Ru4 was found to be lower than that of PDMS+Ru1 and PDMS+Ru2, suggesting that a threshold PS concentration exists beyond which the surface becomes smoother again. 81 Figure 3.7 Surface roughness studies of PDMS•Ru(dpp)3 - (a) Surface height topography images (top left: PDMS, top right: PDMS+Ru1, bottom left: PDMS+Ru2, bottom right: PDMS+Ru4) and (b) Surface roughness measures of PDMS•Ru(dpp)3 samples compared to bare PDMS indicated as average roughness (Ra) for PDMS and PDMS•Ru(dpp)3 coupons with different concentrations of 2. (a) (b) 82 3.3.2.3 Surface hydrophobicity Another important determinant of bacterial adhesion and biofilm formation is the hydrophobicity of a given surface, as the adhesive forces between material surfaces and the bacterium are driven by Van der Waals and electrostatic interactions, with more hydrophobic surfaces providing increased bacterial adhesion extent [257–260]. To assess changes in hydrophobicity characteristics of PDMS with the addition of PS, water contact angle measurements were performed. Overall, the results indicated no marked changes in surface hydrophobicity with PS incorporation into the PDMS polymer structure (Figure 3.8), suggesting that the hydrophobicity of PDMS-based surfaces remains intact and that the presence of PS itself should not change the attraction between bacteria and the material. 83 Figure 3.8 Surface hydrophobicity studies of PDMS•Ru(dpp)3 - (a) Water contact angle images on the surface of PDMS and PDMS•Ru(dpp)3 coupons (top left: PDMS, top right: PDMS+Ru1, bottom left: PDMS+Ru2, bottom right: PDMS+Ru4) and (b) Water contact angle measurements to compare surface hydrophobicity of PDMS and PDMS•Ru(dpp)3 coupons with different concentrations of 2. (a) (b) 84 3.3.2.4 Surface encrustation Deposition of mineral salts on urinary catheters and subsequent surface encrustation can result in catheter blockage and inactivation of antimicrobial technologies being used, which can induce further patient complications [261,262]. This is mainly due to the alkaline nature of urine, as well as the presence of uropathogenic microorganisms that lead to the formation and attachment of calcium (Ca) and magnesium (Mg) ammonium phosphate crystals on implant surfaces respectively [263]. To assess the antifouling nature of PS-containing PDMS, we performed separate encrustation assays that quantify encrustation based on the amount of Ca and Mg on the surface. While modest trend towards decreased calcium deposition was observed for PDMS•Ru(dpp)3 coupons with different concentrations of 2, this reduction was much more striking for the deposition of Mg-based crystals. Magnesium is part of struvite crystal structure (NH₄MgPO₄·6H₂O) and crystal encrustation and crystalline biofilm formation, where crystal growth in fact begins from within the biofilm, can take place on indwelling device surfaces and is responsible for formation of infection kidney stones as a result of presence of urease producing uropathogens such as Proteus mirabilis [264]. This was further confirmed by obtaining SEM images of the surfaces of all coupons, indicating a reduction in surface crystal formation as shown in Figure 3.9. While Ca-based encrustation rarely causes a problem for catheterized individuals, Mg-based encrustation caused by urease-producing bacterial species is associated with catheter blockage and failure [265,266]. These results are significant as they suggest that Ru-containing materials may withstand crystal deposition in an infected environment, keeping the device surface clear of debris while allowing for the interaction of singlet oxygen with bacteria once the material is activated by blue light. 85 (c) (a) (b) 86 Figure 3.9 Surface encrustation studies of PDMS•Ru(dpp)3 - (a,b) Ca and Mg quantification on PDMS and PDMS•Ru(dpp)3 coupons with different concentrations of 2. (c) SEM images of encrusted surfaces of the coupons at 100X magnification (top left: PDMS, top right: PDMS+Ru1, bottom left: PDMS+Ru2, bottom right: PDMS+Ru4). (d) Photographic image of the CDC bioreactor chamber with coupons of PDMS and PDMS•Ru(dpp)3 mounted on thread. 3.3.3 In vitro evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 To assess the efficacy of the technology to prevent bacterial adhesion and biofilm formation, I performed an in vitro bacterial adhesion assay using PDMS+Ru4 as it contained the highest PS concentration and was therefore expected to exhibit the highest degree of activity. Overall, it was found that LED exposure of PDMS+Ru4 samples resulted in the greatest reduction in adherent bacteria at all time points, with significant differences being observed in as little as 0.5 h of exposure and a 4-LOG reduction and complete eradication of bacteria being observed at 4 h and 24 h of exposure respectively (Figure 3.10). Interestingly, a modest antibacterial effect in the absence of light was seen for the PDMS+Ru4 samples at 24 h, which is likely due to minimal antibacterial activity of Ru4, however exposure to LED significantly amplified this activity. (d) 87 Figure 3.10 In vitro antimicrobial studies for 4 mM PDMS•Ru(dpp)3 with longer exposure time - (a) Photographic images showing agar plates used for bacterial enumeration during in vitro studies for samples exposed to LED for 24 h (C: Control (bare PDMS), P1: PDMS+LED, P2: PDMS+Ru4, P4: PDMS+Ru4+LED) (b) In vitro evaluation of the antibacterial activity of PDMS+Ru4 compared to PDMS with and without LED exposure. (* P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001) (٭ Below detection limit of 10 CFU/mL) (a) (b) 88 3.3.4 Cytotoxicity evaluation of PDMS•Ru(dpp)3 PDMS, as an innately inert and stable material, shows no adverse effect on cell viability [192,267], and some studies have even reported cell proliferation and modification of gene expression as a result of contact with PDMS [268,269]. To address the degree of biocompatibility of PDMS•Ru(dpp)3 , the cytotoxic effect against HDF cell lines were evaluated by co-culturing disks of PDMS and PDMS•Ru(dpp)3 with various concentrations of 2. (1, 2, 4, 8 mM) and by having experimental groups with and without LED exposure for 24, 48, and 72 h prior to performing the MTT assay. Overall, no significant cytotoxicity was observed for PS containing materials as the percentage of cell viability +/- LED exposure remained close to the acceptable range of 80% after 72 h (Figure 3.11) [270], except for the 8 mM group (PDMS+Ru8). For this reason, the 8 mM concentration of 2 was eliminated from the remaining of studies. The results of these studies also confirmed cell proliferation for PDMS, PDMS+Ru1, PDMS+Ru2, and PDMS+Ru4 groups exposed to LED for 24 h prior to MTT assay. 89 90 Figure 3.11 Cytotoxicity evaluation of PDMS•Ru(dpp)3. Cytotoxicity evaluation of bare PDMS and PDMS•Ru(dpp)3 with different concentrations of 2, after 24 h (top), 48 h (middle) and 72 h (bottom), with (left) and without (right) LED exposure. (* P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001) 3.3.5 In vivo evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 To assess the efficacy of the PS-based approach to prevent infection in a realistic environment, we utilized a subcutaneous implant infection model and tested the efficacy of PDMS+Ru1 and PDMS+Ru2 as these had the lowest toxicity in vitro and these Ru concentrations did not have an antibacterial effect in the absence of LED exposure. Figure 3.12 illustrates the flowchart describing the procedures followed throughout the in vivo experiments as well as the schematic illustrating the process of in vivo rat implantation surgeries. Overall, it was found that both concentrations of Ru to effectively reduce bacterial numbers (99.5% for PDMS+Ru2) on both the implant and in the surrounding tissues following light exposure (Figure 3.13). Considering that neither Ru1 nor Ru2 had natural antibacterial activity in vitro, the significant activity observed 91 in the animal is due to LED-activated ROS production only. This is further supported by the fact that the absence of LED exposure for Ru-containing samples resulted in bacterial levels similar to the PDMS control. Furthermore, histological analysis of tissues from animals where no antimicrobial effect was observed (PDMS, PDMS+Ru1 and PDMS+Ru2 without LED exposure) showed increased inflammation, fibroplasia and edema compared to clean tissues. (a) 92 Figure 3.12 Flowchart and schematics of the in vivo antimicrobial studies - (a) Flowchart describing the procedure for in vivo evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 with 1 and 2 mM concentration of 2 Compared to bare PDMS for both implanted coupons and tissue in contact with the implant and (b) schematic presenting the sample implantation for in vivo studies. LED Exposure prior to implantation (b) 93 Figure 3.13 Photographic images and results of the in vivo antimicrobial studies - (a) Photographic image of the in vivo surgery showing PDMS+Ru2 coupon prior to being implanted into the rat dorsal pocket (b) In vivo evaluation of the antimicrobial activity of PDMS•Ru(dpp)3 with 1 and 2 mM concentration of 2 Compared to bare PDMS for both implanted coupons and tissue in contact with the implant (P<0.05 for all PS containing groups compared to PDMS as control). (a) (b) 94 Figure 3.14 shows the histology results and H&E staining of subcutaneous tissues of animals dissected from clean (non implant contact) and from contact sites with coupons of PDMS, PDMS+Ru1, and PDMS+Ru2 for samples with and without LED exposure, as well as groups in absence of bacteria. 95 Clean site Infection site NO LED PDMS PDMS+Ru1 PDMS+Ru2 Clean site Infection site PDMS LED (a) (b) 96 Figure 3.14 H&E staining results of in vivo antimicrobial studies. H&E staining results show subcutaneous tissues dissected from clean (non implant contact) and from contact sites with coupons of PDMS, PDMS+Ru1, and PDMS+Ru2 for (a) groups without LED exposure (b) groups with LED exposure and (c) groups in absence of bacteria. PDMS+Ru1 PDMS+Ru2 PDMS+Ru disks with no infection PDMS+Ru1 PDMS+Ru2 Non-contact site Implant contact site (c) 97 To compare the impact of the implantation of various groups of PDMS and PDMS•Ru(dpp)3 in vivo with or without E. coli pre-infection and treatment with LED, a series of blinded histopathologic scoring system was developed using established protocols with some modifications [271]. For subcutaneous rat infection model, the most common conditions that provide insights into distinctive variations between a normal, healthy skin tissue and an infected one are inflammation, fibroplasia (formation of fibrous tissue as a result of wound healing), edema (abnormally large volume of fluid in tissue), and necrosis (damage of healthy tissue resulting in cell death) [272–274]. The scoring system would provide a whole number range between zero and three, with zero indicating no adverse effects, one indicating rare or minimal effect, 2 indicating moderate adverse effects, and three indicating severe adverse effects for each of the four conditions. Table 3.1 summarizes the results of histopathologic blinded scoring of the subcutaneous tissues in direct contact with PDMS and PDMS•Ru(dpp)3 implanted coupons. The wound histology score is defined as the sum of all the scores obtained from each of the four categories. A score of zero was assigned to samples obtained from subcutaneous tissues not in contact with the implant. It can be seen that a certain degree of impact is evident for all experimental groups for which tissue samples were obtained from the incision sites regardless of whether the coupons were pre-infected or not. For rats that had PDMS+Ru implants in the absence of bacteria, the average score of 5.75 is assigned; suggesting that implant insertion itself induces a certain degree of foreign body reaction. For all other groups except PDMS, the average score of over 6.5 is assigned, suggesting a lower extent of physiological and histopathological side effects for samples which contained PDMS•Ru(dpp)3 only. It is also crucial to note that making incision for the purpose of coupon implantation contributes to inflammation on its own, which is why the score results for all tissues in contact with implants are greater than zero. Figure 3.15 shows examples of tissue staining 98 results from clean and infected areas to illustrate the presence of edema, fibroplasia, and inflammation. Table 3.1 Wound histology score results for in vivo experiments. 99 Figure 3.15 Higher magnifications of H&E staining results. H&E staining results from clean tissue (top image) and tissue in contact with infected implant (bottom image), indicate development of inflammation and edema (bottom image, zoomed in top right), and fibroplasia (bottom image, zoomed in bottom right) in tissues in contact with infected implant. 3.3.6 Evaluation of biofilm formation on PDMS•Ru(dpp)3 by imaging 3.3.6.1 Scanning Electron Microscopy (SEM) imaging SEM is a powerful method to qualitatively measure the extent of biofilm growth, especially for UPEC biofilms which tend to form well-organized and complex structures [275,276]. 100 Overall, SEM analysis confirmed the in vitro results that increasing concentrations of Ru in the presence of LED exposure result in decreasing bacterial growth, with PDMS+Ru4 showing almost no bacteria. SEM images of bare PDMS and PDMS•Ru(dpp)3 coupons with 1, 2, and 4 mM of 2 exposed to LED intermittently for 5 days are shown in Figure 3.16. The images clearly show the photokilling effect that the combination of surface-bound PS and light can induce, and the resultant reduction in bacterial attachment and biofilm growth. Figure 3.16 SEM images of bare PDMS and PDMS•Ru(dpp)3. Coupons with 1,2, and 4 mM of 2 exposed to LED intermittently for 5 days, imaged at (a) 2000X magnification and (b) 10000X magnification. 3.3.6.2 Live/dead staining and Fluorescence imaging SYTO® 9 (green/live) and PI (red/dead) as nucleic acid dyes can be utilized to test the integrity of the bacterial cell wall membranes and confirm the extent of live/dead cells within the biofilm structure [277,278]. Fluorescence microscopy images of bare PDMS and PDMS•Ru(dpp)3 coupons, inoculated with 106 starting CFUs of UPEC, with 1,2, and 4 mM of 2 exposed to LED intermittently for 5 days imaged after 1, 2, and 5 days are illustrated in Figures 3.17-19. A clear PDMS PDMS+Ru1+LED PDMS+Ru2+LED PDMS+Ru4+LED (a) (b) 101 trend of biofilm disruption and bacterial photokilling can be observed with increasing concentration of 2. PDMS PDMS+Ru1+LED PDMS+Ru2+LED PDMS+Ru4+LED Figure 3.17. Live/dead staining fluorescence images of bare PDMS and PDMS•Ru(dpp)3 after 1 day. Coupons with 1,2, and 4 mM of 2 exposed to LED intermittently for 1 day, imaged at 40X magnification (top row: live, middle row: dead, bottom row: overlay). 102 PDMS PDMS+Ru1+LED PDMS+Ru2+LED PDMS+Ru4+LED Figure 3.18. Live/dead staining fluorescence images of bare PDMS and PDMS•Ru(dpp)3 after 2 days. Coupons with 1,2, and 4 mM of 2 exposed to LED intermittently for 2 days, imaged at 40X magnification (top row: live, middle row: dead, bottom row: overlay). 103 Figure 3.19. Live/dead staining fluorescence images of bare PDMS and PDMS•Ru(dpp)3 after 5 days. Coupons with 1,2, and 4 mM of 2 exposed to LED intermittently for 5 days, imaged at 40X magnification (top row: live, middle row: dead, bottom row: overlay). The results were further evaluated using the 2.5D fluorescence image capturing features of the Zeiss microscope system. Figure 3.20 illustrates these results which clearly distinguish the marked differences between the extent of live/dead biofilm present on the surfaces of the PDMS and PDMS•Ru(dpp)3 coupons exposed to LED intermittently over a 5-day period. PDMS PDMS+Ru1+LED PDMS+Ru2+LED PDMS+Ru4+LED 104 Figure 3.20 2.5D images of Live/dead staining Fluorescence microscopy. Images show E. coli biofilms grown on bare PDMS coupons and PDMS•Ru(dpp)3 coupons with different concentrations of 2. Coupons containing 2 were exposed to LED intermittently over a 5-day period. Top left: PDMS, Top right: PDMS+Ru1, Bottom left: PDMS+Ru2, Bottom right: PDMS+Ru4. Also, the live bacterial cell counts per µm2 area using the Zeiss microscope system confirmed extensive decrease between bare PDMS coupons and PDMS•Ru(dpp)3 coupons with 1, 2, and 4 mM of 2 (Figure 3.21). 105 Figure 3.21 Live bacterial colony count over area of bare PDMS and PDMS•Ru(dpp)3. Scattered plots show live bacterial colony count over area for non-LED exposed bare PDMS and PDMS+Ru coupons exposed to LED intermittently over 5 days. (Top left: PDMS, Top right: PDMS+Ru1, Bottom left: PDMS+Ru2, Bottom right: PDMS+Ru4). 3.4 Conclusion LED exposure of PS-loaded PDMS disks resulted in significant bactericidal activity as evidenced by the number of adherent bacteria being below the level of detection (<10 CFU/mL) after 24 h. Live/dead staining studies using fluorescence microscopy indicated reduction in surface-adhered bacterial growth and biofilm formation. This potent antibacterial activity was verified in vivo, with exposure of contaminated PDMS coupons containing PS to LED prior to implantation resulting in over 99.5% reduction in adherent bacteria compared to controls over the 3-day implantation period. Histological analysis of the implantation site of PDMS+PS samples (in 106 the absence of bacteria) revealed no adverse reactions. This was also confirmed using in vitro cytotoxicity studies. Tensile strength, surface roughness, and encrustation extent of surface-treated groups exhibit either comparable or improved properties compared to bare PDMS. LED-induced ROS production on the surface of materials loaded with Ru as a photosensitizer is an effective approach to sterilize indwelling device surfaces both in vitro and in vivo. This simple, safe, cost effective, and readily scalable technique can be applied to sterilize both biomedical implants as well as other polymeric surfaces in a healthcare setting. This has significant potential to not only reduce implant infections but also other hospital-associated infections when applied to various surfaces around the hospital. The latter is very appealing, as this has the potential to utilize normal room lighting to activate PS-containing materials on a continuous basis thereby reducing the incidence of nosocomial infections. 107 Chapter 4: Incorporation of Ruthenium photosensitizer into an experimental dental resin blend and evaluation of its physical, chemical, mechanical, and antimicrobial properties: The case of MA as a hard material Synopsis Using light sources to photocure dental materials is a widely accepted procedure in dentistry. In addition, a majority of restorative dental resins are polymeric biomaterials that can be conveniently incorporated with other compounds to enhance their functionality. Due to applicability of surface photosterilization technology for PDMS, it is possible to apply a photosensitizer as surface sterilization agent inside dental material polymers to evaluate its efficacy. In this chapter, the incorporation of the ruthenium photosensitizer (PS) into methacrylate-based dental resin and the characterization and evaluation of the antimicrobial activity of the new polymer-photosensitizer system is discussed. The ruthenium photosensitizer, synthesized and purified in powder form as discussed in Chapter 2, was mixed into a resin composite formulation frequently used in dental practices as an adhesive material to treat dental caries. The preliminary dose determination studies, followed by investigations of chemical, physical, mechanical, and antibacterial properties of the new formulation either freshly prepared or after a prolonged aging period were evaluated. The results indicate a high degree of antibacterial activity of the system upon light activation for resin-PS samples under all conditions, suggesting both the stability and functionality of the material. The degree of conversion, flexural strength, shear bond strength, water sorption, and water solubility test results were compared with a commercial resin blend in all the tests. 108 4.1 Introduction Given the severity and complexity of dental caries as a global health concern and following the 2015 united nations (UN) phase down of dental amalgam program due to safety concerns, there has been a great interest in the development of polymeric restorative dental materials with ideal properties. However, to date, little advancement has been made of resin-based dental materials to effectively improve their antimicrobial activity, particularly in long-term. So, there remains a significant need to design a novel, practical, and effective material-based treatment which can assist with the control of this biofilm-dependent disease [279,280]. Studies on this front have primarily focused on dental materials loaded with a single antimicrobial agent incorporated into the dental material, such as antimicrobial metallic nanoparticles [281,282], or antifouling monomers [283]. However, the quest to manufacture an antimicrobial dental resin possibly without the need to release any agents into the body remains a challenge. 4.1.1 Background The oral cavity is considered the initial segment of the digestive system and consists of multiple anatomically different structures such as the teeth, tongue, lips, and salivary glands, which work together effectively as a uniquely complex network to help sustain many aspects of human life including food and water intake, communication, and breathing [284]. The structure of the oral cavity can be divided into the upper (maxillary) and lower (mandibular) dental arches, with each consisting of different types of teeth with a specific function. The different types of teeth, as characterized by their shape and function, are the incisors, canines, premolars, and molars (Figure 4.1). An adult usually has 32 teeth in their oral cavity, including 8 incisors, 4 canines, 8 premolars, and 12 molars. The incisors and canine teeth are responsible for cutting the food and the premolars 109 and molars are used to crush the food to prepare it as a bolus which is ultimately swallowed down the esophagus with the aid of the tongue and saliva [285]. Each tooth is primarily made of three parts: The crown, the neck, and the root. The crown is the portion of the tooth that is visible and is sitting above the gums (gingiva). The neck is the middle portion of the tooth in direct contact with the gums and is also the smallest portion among all. The root is the section of the tooth that is seated entirely inside the gum and holds the tooth in the jaws and is attached to the soft connective periodontal ligament tissue (Figure 4.1). The salivary glands are responsible for production of saliva and are situated either at the back of the lower jaws or on either side of the tongue and under the floor of the mouth. Saliva keeps the oral cavity moist and helps in the digestion process. It is made of 99% water, as well as some enzymes, proteins, electrolytes, and buffering agents, and plays an important role in maintaining a healthy oral environment [286]. Figure 4.1 The four types of teeth in the oral cavity and different portions of a tooth. Reprinted from The Cleveland Clinic at https://my.clevelandclinic.org/health/body/24655-teeth 110 Anatomically, the tooth consists of four main parts: enamel, dentin, cementum, and pulp. Enamel covers the outermost surface of the tooth crown and is the hardest substance in our bodies. This protective layer is composed of 95% carbonated apatite, also known as hydroxyapatite (HA), a hard calcium phosphate mineral [287]. Dentin lies underneath the enamel and is composed of lesser amounts of carbonated apatite (70%), mixed with some collagen-based organic matrix as well as some dentinal fluid that resembles blood plasma [288]. Unlike enamel, the dentin structure harbors microscopic channels in it. The cementum helps protect the root of the tooth along with periodontal tissue, allowing the tooth to be firmly positioned in the jaw. Pulp is the innermost layer of the tooth, consisting of blood vessels, nerves, and some connective tissue. The inner space of the tooth containing the pulp material and connecting it to other blood vessels and nerves within the periodontal ligament is referred to as the root canal (Figure 4.2). Figure 4.2 The anatomy of a normal tooth. Reprinted from Teethtalkgirl at https://www.teethtalkgirl.com/community/tooth-anatomy 111 4.1.2 Oral biofilms and dental caries The oral cavity hosts close to 700 bacterial and fungal species in both planktonic and adhered form [289]. A vast majority of these microorganisms are commensal and initially do not cause any harm, but they can remain dormant on the surface of the teeth or elsewhere within the oral cavity and pose a threat by causing infection when the body is immunocompromised. Another challenge is the formation of oral biofilms, which are highly complex and intricate colonies of microorganisms that originate from non-reversible attachment of planktonic bacteria to available substrates (tooth surface) [290–292]. This is the direct result of formation of salivary pellicle on tooth surfaces, combined with the presence of hundreds of microorganisms in the oral environment. The formation and development of these polymicrobial matrices eventually leads to one of the most challenging global healthcare issues, oral biofilms, due to the unique characteristics they possess [293,294]. An oral biofilm community provides great nutrient access to its single-cell microbial species via formation of polymeric substances (EPS) while simultaneously protecting the internal microbial species from antibiotic or other antimicrobial agents, allowing them to communicate via quorum sensing and thrive efficiently through phenotypic alterations and modification of gene expression pathways within the oral microenvironment [291,295]. The process of oral biofilm formation is staged through demineralization of the enamel which can expose the dentin or the dentin-enamel junction (DEJ) to the outer oral environment. Etiologically, the demineralization process begins when HA on the enamel gets removed because of increase in the acidity of the environment in close proximity of the tooth surface due to microbial metabolic activity. Major bacterial species involved in the process of oral biofilm formation are Pseudomonas, Vibrio, Escherichia, Salmonella, Listeria, Streptococcus, and Mycobacteria [296]. Among these, Streptococcus mutans (S. mutans) is the major cause of dental caries [297–299]. 112 These microorganisms commonly inhabit the oral cavity and can break down sugars, such as lactose, from the food and the subsequent glycan residues formed can contribute to plaque and tartar (cariogenic biofilm) formation through lactic acid production which would ultimately dissolve calcium and phosphate content of the enamel [300]. It is important to note that the demineralization process is naturally accompanied by the remineralization process where saliva neutralizes the pH and brings it up to the critical pH level of greater than 5.5 [301]. The issue arises from the fact that for a tooth surface already compromised by plaque formation, saliva cannot reach the enamel surface and the remineralization step cannot go to completion [300,302]. Dental plaques are the root cause of some of the most prevalent oral health complications: dental caries and periodontal diseases. Dental caries, also known as cavities or tooth decay, is a chronic oral infectious disease which develops when the enamel surface weakens and provides an access route to microbes to reach dentin and inner parts of the tooth. Dental caries is a vastly prevalent disease, to the extent that around 70% of the adults or around 2.5 billion people are affected by it globally [279,303]. It can be identified as one of the most significant chronic human diseases due to its impact being evident on multiple levels, from reducing the quality of life and nutrition [304], to being the precursor to other medical problems such as cardiovascular disease [305], to placing huge economic burden on the healthcare system, estimated to be over US $545 billion in 2015 [279,280,306]. If left untreated, dental caries can result in other oral diseases such as gingivitis (early-stage gum disease) periodontitis (advanced-stage gum infection), pulpitis (pulp infection) and root canal infection, which without proper intervention could ultimately lead to loss of tooth (Figure 4.3). 113 Figure 4.3 Stages of dental caries formation and development. Reprinted from Med Izzy at https://medizzy.com/feed/33319692 4.1.3 Dental materials Dental materials science involves the study of compositions and properties of different materials and their interaction with the environment (the oral cavity). These materials can be implemented in solid or liquid form depending on their application. According to Ullah et.al, dental materials can be classified into three major categories based on their functionality [307]: 114 1. Preventive dental materials: are designed to prevent infections or injuries related to the teeth, gums, and supporting tissues. The most common examples are mouthwashes, fluoride gels, pits and fissure sealants, and mouth guards. 2. Auxiliary dental materials: are the materials commonly used for fabrication of dental prosthesis. Common examples are dental impressions, waxes, gypsum casts, plasters, and polishing materials. 3. Restorative dental materials: are applied in order to restore or replace any missing hard or soft dental tissue. These materials include dental cements and resin-based composite fillers, ceramics, cast metals, dentures, crowns, inlays, and bridges. They may be applied temporarily or permanently as needed. These materials ideally need to exhibit certain properties such as biocompatibility, physical, chemical, and mechanical stability, proper bonding and structure, and suitable aesthetics [308]. 4.1.3.1 Restorative dental materials One major contributing factor to the prevalence of dental caries is the fact that the tooth enamel has no nerve cells or blood vessels, which can delay early-stage detection of cavity formation. A person with dental caries typically becomes alert when the decayed tooth begins to cause pain, and at which point the infection has already progressed to dentin. Moreover, although demineralized enamel can be re-mineralized, it does not have any regrowth ability once degraded and the structure is permanently lost, and this means that the affected tooth requires intervention by means of providing a replacement for the lost enamel using a restorative material. Restorative dental materials have a long history of usage, dating back to around 13,000 years ago, with materials such as beeswax and bitumen being used to fill decayed teeth [309,310]. Later, metal-115 based restorative materials such as gold and dental amalgam (a mix of mercury (Hg), tin (Sn), and silver (Ag)) found their way in common dentistry practices as they provided good physical and mechanical stability. They are still being used today although not extensively, mainly due to toxicity issues (amalgam), poor aesthetics, and high cost (gold) [311,312]. However, the paradigm shift in dental sciences and dental caries treatment emerged with the arrival of composite restorative dental materials, also commonly referred to as resin-based dental materials, in the 1950s. Initially, the major advantage that these materials possessed was their tooth-coloured appearance. In time, other characteristics such as biocompatibility, cost-effectiveness, mechanical strength, physical and chemical stability, drug and mineral incorporation abilities, and excellent adhesion to dentin and enamel further popularised them as one of the most versatile polymeric biomaterials used in modern medicine. Composite restorative materials can be classified according to their inherent properties as well as their use cases for dental practitioners. Based on their composition, Vallittu et al. proposed they could be mainly divided into three categories: polyacid-modified resin composites (PAMRC), glass ionomer cements (GIC), and resin composites [313]. Adhesive resins used as restorative materials are among some of the commonly used dental composites. A conventional resin composite is typically comprised of an organic resin polymer, an inorganic silicate filler, and other coupling and binding agents that enhance its properties. PMMA application dates to the 1960s and it rapidly drew the attention of clinicians due to its straightforward and economic synthetic pathway, but soon it was revealed that PMMA-based resins lack certain ideal properties due to their poor marginal sealing as well as polymerization shrinkage owing to lack of reliable adhesion and filling materials [318]. To address this, additional resin monomers such as bisphenol-A glycidylmethacrylate (bis-GMA) and later 116 urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) and tetraethylene glycol dimethacrylate (TEEGDMA) were introduced in order to enhance the filling properties of the resin matrix. However, the extent of polymerization of these monomers results in the reduction of volume of the overall polymer, so during 1970s, the idea of resin curing began to gain popularity to overcome this shortfall. The curing process, in general, refers to the application of additional compounds as binding agents which would undergo cross-linking bond formation, that ultimately result in increase in volume of the polymeric matrix structure and reduce shrinkage. Throughout much of the decade, various attempts were made using amin-cured epoxy resins [319] and UV-curing photopolymers; however, due to limited penetration depth of UV light and its high energy and safety concerns, they were later replaced with visible-curing alternatives using quartz-halogen bulbs (QTH) [320]. In the 1980s and onward, the focus of dental materials researchers mainly shifted to working on resin composite systems classified based on the size of the fillers within the polymer matrix, coining new terminologies such as macrofill, microfill, hybrid, and nanofill. Since the 1990s and up to now, concurrent to the growth of the size-based classification system, another classification method was introduced and adopted by clinicians which focused on the viscosity of the composites prior to polymerization [313] (Figure 4.4). 117 Figure 4.4 Timeline of restorative dental material composites evolution. The original glass filler particles ranged from 10-50 µm in diameter size. These were referred to as macrofill. Later, microfill particles (40-50 nm) and nanofill (5-100 nm), as well as hybrid filler systems were manufactured. Throughout time, different formulations have been developed which combine a mix of various particle size as well as shape in order to yield the most optimum characteristic. When the final resin composite formulation is complete, the free-radical polymerization reactions among the matrix monomers result in the formation of a three-dimensional (3D) network [321,322]. The initiation step is carried out using an initiator radical. This reactive radical moiety then activates certain functional groups on the nearby organic monomers and oligomers in the 118 system, thereby inducing a cross-linking effect which promotes the curing process. At present, the most widely used initiator system in dentistry is a photoinitiator, which is a compound or a mixture that forms radicals and other reactive species upon irradiation by a certain wavelength of light. There are many different photoinitiator systems which have been utilized in dental sciences [323,324], with acylphosphine oxide (APO), bisacylphosphine oxide (BAPO), camphorquinone (CQ)/tertiary amine, and 2,4,6-trimethylbenzoyldiphenyl phosphine oxide (TPO) being among some of the common examples. Many of the common photoinitiators get activated in the 400-500 nm range. During the propagation step of polymerization, the aggregation of macro radicals results in microgel particle formation inside the polymer network. When the saturation process and build up of the polymer network is complete, the composite reaches the gel point, with the time it takes to get to the gel point being referred to as the gel time, which normally takes a few seconds depending on the monomer type. A complete photopolymerization and cross-linking process usually takes 20-60 s [321]. Figure 4.5 shows the mechanism of photopolymerization of dental composites through radical formation and initiation by camphorquinone /amine, which is the most common photoinitiator system currently being used by dentists [325]. The end result of the photopolymerization curing reaction is the hard resin composite which is the product of the 3D network build-up and is achieved via double bond saturation of the methacrylate monomers by the radicals of CQ as photoinitiator and amino halides as co-initiator. This double bond to single bond conversion is referred to as the degree of conversion (DC) of the composite resins, a measure of the extent of photopolymerization for the entire network, which varies among different methacrylate derivatives (Table 4.1). These variations arise from the fact that not all the double bonds in the polymeric matrix are available for reaction, mainly due to changes in the density of the 3D network. This has implications on the flexibility, viscosity, shrinkage, and hardness of the 119 final resin composite, which can render the materials prone to water sorption as well as reduced mechanical strength and increase in wear. Figure 4.5 Mechanism of photopolymerization initiation by camphorquinone (CQ) / amine system. [321] 120 Monomer Functionality* % Conversion of C=C bond Methyl methacrylate (MMA) 1 99.5 Decanediol dimethacrylate 2 78.0 TEGDMA 2 68.2 Bis-GMA 2 56.0 Trimethylolpropane trimethacrylate 3 41.7 Table 4.1 Double bond conversion by radical polymerization of methacrylates with different functionality at room temperature. [321]  Monomer functionality is the number of bonds formed by the monomer’s repeating units in a polymer. In order to obtain dental resin composite formulation with optimum characteristics and functionality, the synergistic effect of its constituent units is of crucial importance. This is why an ideal formulation is required consisting of a blend of different monomers which can influence its abrasion resistance, flexural strength, and thermal coefficient of expansion, and improve polymerization shrinkage as well as radiopacity [321,326]. 4.1.3.2 Light sources used for photocuring of restorative dental materials The photopolymerization of the resin composites requires activation of a photoinitiator by a light source. These light sources are commonly referred to as the light curing units (LCU) in modern dental practices. A variety of LCUs have been utilized by dentists to cure dental polymers. Some of the examples of the light source types that have been used to activate the photoinitiator 121 systems are UV-curing units, QTH projector bulbs, plasma arc (PAC) lamps, LEDs, and argon ion LASERs [327,328]. These LCUs are normally selected depending on the type of resin and photoinitiator system, and the area where the resin is to be applied. In general, factors such as the emission wavelength, the intensity of the delivered light, and possible thermal effect of the LCUs on the tooth and nearby tissue are crucial. Owing to this, UV-sources and LASERs have potential risks for patients due to their high photonic energies and power intensity. QTH and PAC also have limitations in terms of their ease of application as they sometimes need to reflect light from a parabolic surface (silver reflector) prior to reaching the dental material [328,329]. For these reasons, nowadays LED-based LCUs are the most widely used light sources in dental practices. Also, since most of the photoinitiator systems are activated within the 400-500 nm range, blue light LED LCUs are usually the first choice to facilitate the curing process. LEDs have many advantages over the other LCUs including narrow irradiance bandwidth, cost efficiency, and low heat generation [327,330,331]. It is important to note that all LCUs, regardless of the type, must be calibrated prior to use, maintained on a regular basis, and applied to the surface of the resin properly to deliver constant irradiance and ensure complete and uniform curing [329,331–333]. 4.1.4 Antimicrobial Photodynamic Therapy in dental sciences Even with an optimum dental resin composite that has all the required characteristics and functionalities as a dental material, once the resin is applied to the tooth, its surface provides a suitable substrate for bacterial adhesion and the recurrence of biofilm. This phenomenon and the development of subsequent infection on a filled tooth is known as secondary caries [334,335]. Since the resin composite is an inanimate surface, it provides a suitable anchoring material for the attachment of oral pathogens. Unfortunately, this means that upon development of the secondary caries, the restoration needs to be replaced. That is why developing techniques to fight against the 122 development of secondary caries is of vital importance [336–339]. A breakthrough treatment for dental caries would be developing methods to make the surface of the resin antimicrobial, and extensive amount of research is currently being carried out in this regard [340–344]. That is also the reason why using antimicrobial photodynamic therapy (aPDT) is an excellent choice to control or treat secondary caries formation [345–347]. The theories and mechanisms of aPDT have been discussed in Chapter 1. It is, however, necessary to point out that incorporation of a photosensitizer (PS) in the dental resin formulation can provide synergistic effect by enhancing the rate of photoinitiator radical formation via intersystem crossing (ISC) energy transfer, given that the PS and the photoinitiator absorb different wavelengths of light [348]. The methodology and experimental setup designed as part of this thesis work is an attempt to address the current knowledge gap and experimental work required by investigating the physical, chemical, and antimicrobial properties, immediately after preparation as well as after prolonged aging, of a novel ruthenium-doped methacrylate-based dental resin formulation using aPDT with LED light. Although many different applications of ruthenium photosensitizers as aPDT agents have been reported, at the time of writing this thesis, based on search results from Google, PubMed, and Web of Science, the only published work on the use of ruthenium as an incorporated photosensitizer in a dental resin composite material has been our published article based on the current material that is part of this chapter [182]. 123 4.2 Experimental section 4.2.1 Resin blend formulation A modified experimental resin blend (RB) formulation mixture, containing 50 wt% ethoxylated (4) bisphenol A dimethacrylate (BisEMA; Esstech, Essington, PA, USA), 30 wt% tetraethylene glycol dimethacrylate (TEEGDMA; Esstech), 14 wt% 2-hydroxypropyl methacrylate (HPMA; Esstech), 4 wt% ethanol and 2 wt% photoinitiator system (camphorquinone and ethyl 4-(dimethylamino) benzoate; Sigma–Aldrich, St Louis, USA) was prepared in 20 mL scintillation vial and stored in the dark at 4 ⁰C (Table 4.2). RB Component Wt% of total formulation Bis-EMA 50 TEEGDMA 30 HDMA 14 Ethanol 4 CQ/Amine photoinitiator system 2 Total 100 Table 4.2 Experimental resin blend (RB) components and their respective concentrations. 124 4.2.1.1 Ruthenium photosensitizer incorporation into resin blend formulation Once the experimental RB was prepared, different concentrations of the Ru photosensitizer, the Ruthenium II complex of tris (4,7-diphenyl-1, 10-phenanthroline)ruthenium(II) bis(hexafluorophosphate) (Ru(dpp)3(PF6)2 ) or “Ru” in crystalline powder form, were added to the experimental RB in 17 different concentrations (0.0, 0.07, 0.14, 0.28, 0.56, 1.12, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 and 10% w/w) and kept under magnetic stirring for 24 h in 2 mL scintillation vials at room temperature and in a dark room. The resulting homogeneous mixtures were then stored at room temperature for further use. 4.2.2 Degree of conversion (DC) Each RB+Ru formulation with different Ru concentrations was then analyzed using Fourier Transform Infrared Spectroscopy (FTIR) (IRPrestige-21 FTIR-8400S, Shimadzu, Kyoto, Japan). To do so, a drop of the mixture was placed between two transparent polystyrene sheets of roughly 2 × 2 cm2 area, and the sheets were then secured on a sample holder inside the FTIR instrument. The preliminary reading (baseline) of each uncured sample was recorded in the absorbance spectrum obtained by scanning the samples 36 times over a range of 4000 to 400 cm-1 with a resolution of 4 cm-1. The samples were then photocured for either 30 or 60 seconds with a LED light curing unit (LCU) (Bluephase, Ivoclar Vivadent AG, Schaan, Liechtenstein) operating at 650 mW/cm2. Prior to sample photocuring, the LCU unit was calibrated using a smart radiometer (Bluelight Analytics Inc, Nova Scotia, Canada) to ensure a constant photonic flux is delivered by the LCU throughout all the experiments. CheckUp 2.0 mobile app was used to monitor the output photonic power. The same FTIR procedure was repeated for each sample after photocuring, and the ratio between the aliphatic carbon-carbon double bond absorbance (at 1638 cm-1) and that of 125 the aromatic component (at 1608 cm-1) for the uncured and cured samples was obtained in order to calculate the degree of conversion (DC) (n=6) according to the following equation [349]: % DC = 100 ×[1- (Rcured / Runcured)] where the ratio “R” = Abs (1638cm-1) / Abs (1608cm-1) 4.2.3 Flexural strength (FS) RB containing 0.0, 0.28, 0.56 and 1.12% Ru were selected to evaluate the flexural strength (FS) (n=12). First, FS bars measuring 25 × 2 × 2 mm3 were produced inside a Teflon matrix followed by light curing unit (ValoTM light curing unit, Ultradent Products Inc., South Jordan, UT, USA) with 60 s curing time. The light was repeatedly applied on different directions, at the top and at the bottom of each specimen. The resulting specimens were stored in MilliQ® water at 37 ⁰C for two aging periods, 24 hours and 2 months, with water being refreshed every week. On the test day, the specimens were positioned in a universal testing machine (UTS) across a 20-mm span gap (Shimadzu Corp., Kyoto, Japan) and were subjected to the three-point bend test with a crosshead speed of 1mm/min. The FS (MPa) was then calculated by Trapezium X software (Shimadzu Corp., Kyoto, Japan) using the following equation [350]: 126 FS = 3FL / 2bh2 Where: “F” is the maximum load applied (N) “L” is the length of support spam (mm) “b” is the width of the specimen (mm) “h” is the height of the bar (mm) 4.2.4 Shear bond strength (SBS) to dentin 4.2.4.1 Sample preparation For this experiment, non-defected human third molars with intact crowns were obtained immediately after extraction with the approval of the Research Ethics Board (certificate number #H14-02189). All tissue debris and calculi were removed mechanically with hand instruments to allow for the storage of the teeth in 0.1% thymol solution (Sigma Aldrich) at 4 °C. The occlusal enamel was removed using a metallographic cutter (SYJ-150, MTI Corporation, Richmond, BC, Canada) coupled to a diamond blade (Buehler) to provide a flat mid-coronal dentin surface. Each tooth was embedded in a round polyvinylchloride (PVC) mold with self-cure acrylic resin (Caulk Orthodontic Acrylic Resin, Dentsply, York, PA, USA). In order to obtain a standard smear layer, the flat dentin surfaces were polished for 60 s using 320-grit silicon carbide paper under constant water-cooling in a polishing machine (Unipol-1210 Precision Lapping, CYKY, Henan, China). Then, samples were subjected to an ultrasonic bath in distilled water for 2 minutes to remove any debris [351]. 127 4.2.4.2 Bonding procedure and SBS testing To complete the bonding procedure, the Adper™ Scotchbond™ Multi-Purpose (3M ESPE, St Paul, MN, USA), a three-step etch-and-rinse adhesive system, was selected as a control group of the bonding procedure. Also, RB containing 0.0, 0.28, 0.56 and 1.12 (w/w%) Ru were selected to evaluate the SBS (n=15). All prepared dentin surfaces were treated as recommended by the manufacturer's instructions (for control group) or according to the experimental procedure (Table 4.3). The adhesive layer was photocured using a LED light curing unit (ValoTM light curing unit, Ultradent Products Inc., South Jordan, UT, USA) operating at 650 mW/cm2. The Ultradent jig, consisting of a polytetrafluoroethylene mold insert (2.4 mm internal diameter) and a bonding clamp (Ultradent Products, Inc., South Jordan, UT, USA) was positioned over the pre-treated dentin surface in order to shape the resin composite (Filtek Z350, 3M ESPE) specimen. Each cylinder specimen (2.0 mm height) was photocured for 40 seconds with the same LED light curing unit. Then, the specimen was carefully removed from the mold and a second resin composite specimen was built on the same dentin surface. The samples were stored in MiliQ water (replaced every week) at 37 °C for 24 hours and 2 months before testing. In each period of analysis, one specimen from each dentin surface was randomly selected and tested. The SBS test was performed using Bisco Shear Bond tester (Bisco Inc., IL, USA) at a crosshead speed of 0.5 mm/min. The SBS results (MPa) were given by the values of the maximum stress supported (N) by the dentin/material bond area (mm2) [351]. Table 4.3 shows the application mode of materials employed in the control and experimental groups for SBS studies. 128 Material Composition Treatment Adper Scotchbond Multi-Purpose Adhesive System (3M ESPE) Primer: HEMA, polyalkenoic acid copolymer and water. Adhesive: Bis-GMA, HEMA and initiators Dentin surface was etched with 35% phosphoric acid (15s), followed by rinsing (10s). The excess of water was removed by blotting. Primer was applied actively (10s) and gently dried with jet of air (5s). Adhesive was applied and light cured for 10 s. Experimental resin blend (RB) RB: BisEMA, TEEGDMA, HPMA, etanol and initiators Dentin surface was etched with 35% phosphoric acid (15s), followed by rinsing (10s). The excess of water was removed by blotting. Primer (from Adper Scotchbond Multi-Purpose Adhesive System) was applied actively (10s) and gently dried with jet of air (5 s). RB was applied and light cured for 30 s. Experimental resin blend associated with Ruthenium II complex (RB+Ru) RB+PS: BisEMA, TEEGDMA, HPMA, etanol, initiators and Ruthenium II complex Dentin surface was etched with 35% phosphoric acid (15 s), followed by rinsing (10 s). The excess of water was removed by blotting. Primer (from Adper Scotchbond Multi-Purpose Adhesive System) was applied actively (10 s) and gently dried with jet of air (5 s). RB+Ru was applied, and light cured for 30 s. Table 4.3 Application mode of materials employed in the control and experimental groups for SBS studies. 4.2.5 Water sorption (WS) and solubility (SL) RB+Ru containing 0.0, 0.28, 0.56 and 1.12 (w/w %) Ru were selected to evaluate the WS and SL of the resin disks with 6 mm diameter and 1 mm thickness (n=12). First, specimens made of different RB+Ru formulations and measuring 6 mm in diameter and 1 mm in thickness were produced. They were photocured for 60 seconds at the top and 60 seconds at the bottom using a LED light curing unit (ValoTM light curing unit, Ultradent Products Inc., South Jordan, UT, 129 USA) inside Teflon molds. Immediately after polymerization, the specimens were placed in a desiccator and transferred to an oven at 37 °C. Next, the specimens were weighed on an analytical balance (ATX 124, Shimatzu, Kyoto, Japan) on a daily basis until a constant mass (variation <0.2 mg) was obtained (M1). Thickness and diameter of the specimens were measured using a digital calliper (Mitutoyo, Aurora, IL, USA) in order to calculate the volume (V) of each specimen in mm3. The disks were then individually placed in sealed tubes with 1 mL of Milli-Q® water at 37 °C. At frequent time points over 14 days, the specimens were washed with running water, gently wiped with a soft absorbent paper, weighed (M2) and returned to the tubes containing 1 mL of fresh MilliQ® water. After a final recording of disk mass on day 14, the specimens were stored in a desiccator to dry the samples until a constant mass (variation <0.2 mg) was obtained (M3). Finally, WS and SL after 14 days of water storage were calculated according to the following equations [352]: WS = (M2-M3)/V SL = (M1-M3)/V 4.2.6 In vitro antimicrobial activity The experimental RB containing 0.28, 0.56, and 1.12 (w/w %) of Ru photosensitizer were used to produce the resin discs of 6 mm diameter and 1mm thickness (n=6) following the same protocol for disk production described for water sorption (WS) and solubility (SL) tests (Section 4.2.5). The disks were then randomly distributed in two subgroups for microbiological tests: immediate or 2-month artificial ageing in MiliQ® water. Specimens for each testing time 130 were autoclaved prior to the antimicrobial activity evaluation. A Streptococcus Mutans (S. mutans) strain (ATCC 33535) was used for the evaluation of the antimicrobial activity of the photosensitive RB+Ru disks. The bacterial isolates were cultured and sub-cultured in Lysogeny Broth (LB) as the growth media under anaerobic conditions with 5% CO2, 48 and 24 hours prior to all experiments. Starting colony forming units (CFU) for all the experiments were within 105 – 106 range. On the day of experiments, disks of the Ru-loaded resins at 0.28, 0.56, and 1.12% (w/w %) were glued on one side at the bottom of a 24-well tissue culture plate (TCP) and 1 mL solution containing S.mutans and LB as bacterial feed was added to each well and incubated for 4 hours. The disks were then removed from the wells, washed gently with sterile PBS to remove any non-adherent planktonic bacteria, and glued on the same side at the bottom of a new TCP and topped with 1 mL of fresh sterile PBS. The LED light source, designed to fit the 24-well TCP and capable of specifically activating the Ru photosensitizer, was applied and the disks were irradiated for 15 minutes at an average power density of 10.1 mW/cm2. A second group of Ru-loaded disks was also evaluated under dark (no light applied) conditions. Upon completion of light exposure, the disks were removed from the TCP, transferred to Eppendorf tubes containing 1 mL of sterile PBS, and sonicated for 5 minutes to remove the adherent bacteria from the disk surfaces. Then, the suspension containing adherent bacteria was serially diluted in PBS by factors of 10, and 10 µL aliquots of the dilutions were spot-plated on agar dishes, before placing them under anaerobic conditions with 5% CO2 at 37 ⁰C. After 96 hours, the CFUs were counted for each dilution and the CFU/mL of the viable bacteria was calculated and the LOG of the determined CFU/mL was reported using the following equation: CFU/mL = [(# of counted colonies) ×(1/dilution factor) ×1000 µL/mL) / (10 µL) 131 4.2.7 Statistical analysis All statistical analysis was conducted using IBM® SPSS® Statistics V28 software package. The data was analyzed with a multi-factor univariate general linear model and post-hoc Tukey (α = 0.05). Data was presented as the mean ± one standard deviation. Statistical significance was accepted as p<0.05. 4.3 Results and discussion 4.3.1 Resin blend (RB) formulation and ruthenium photosensitizer incorporation into RB Figure 4.6 shows the UV-Vis spectrum for the RB with 2% CQ/amine photoinitiator system, which matches the literature [353]. CQ has strong absorption both in 200-300 nm as well as a weaker absorption at around 470 nm, which precisely corresponds to the maximum emission wavelength of the LED light source. Over time and throughout the photocuring process, the absorption at 470 nm decreases due to n to π* transitions after irradiation by blue light. This appears to be because of the conversion of one of the carbonyl groups of CQ into a ketyl radical, with subsequent loss of conjugation of the remaining carbonyl group causing a blue shift on the spectrum [354]. Once ruthenium PS is mixed into RB formulations at different concentrations and upon curing, the method of choice selected to perform surface analysis and characterization of a dental material system is FTIR, as it provides convenience due to its ability to test small amounts of sample compared to other spectroscopic techniques [355,356]. 132 Figure 4.6 UV-Visible absorption spectrum for the experimental resin blend (RB) and 2% camphorquinone (CQ)/amine as the photoinitiator system. 4.3.2 Degree of conversion (DC) The initial screening to measure the degree of conversion was performed using all of the 17 original concentrations of ruthenium (0.0, 0.07, 0.14, 0.28, 0.56, 1.12, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 and 10% w/w). The choice of the initial four concentrations here (0.07, 0.14, 0.28, 0.56 %w/v) was according to the four initial concentrations of the PS used in chapter 2 and Chapter 3 (1,2,4,8 mM), and twelve additional concentrations were used to monitor the extent of solubility of the PS in the experimental RB and to measure their DC values after 30 and 60 s of disk photocuring. As seen in Figure 4.7, increasing the ruthenium concentration had a decreasing effect on the DC of the experimental RB, with concentrations above 1.5 % w/w of Ru in RB having a DC below 70% after 60 s of photocuring. For 30 s photocuring, the respective DC values were always less than 60% and reduced with increasing Ru concentrations. According to these results and owing to early sample fabrication observations, three Ru concentrations were selected with optimum DC values of over 70% and with the highest amount of incorporated ruthenium (0.28, 0.56, 1.12 %w/v). These three Ru concentrations were further analyzed for repeated DC 133 calculations by adding a 10 s photocuring time, and repeated experiments validated the DC values for the selected concentration ranges as evidenced by the reduction in the absorption band of C=C bonding stretch of the monomer at 1638 cm-1, which indicates transformation of monomers into polymers and completion of photocuring process (Figure 4.7). The decrease in DC with increasing PS concentration can be justified by the fact that both QC and ruthenium PS have their maximum absorption wavelength at around 470 nm, which means they will be competing for free photons once the sample is irradiated, which leaves less available photons to initiate QC photocuring process. Another important observation is the extent of polymerization and presence of unreacted monomers and free radicals that remain in the environment as the photocuring process progresses since the diffusion of free radicals reduces as the MA polymerization increases [357,358]. 134 (a) (b) 135 Figure 4.7 Degree of conversion studies of MA•Ru(dpp)3 – a) Degree of conversion (DC) for the experimental resin blend as a function of Ru concentration. Data reported as average ± one standard deviation (n=6). *Indicates a statistically significant change from previously reported w/w% of Ru added to the RB (p<0.05). b) Representative FTIR spectra of the experimental resin blend (0.00%Ru-RB) after 0, 30, and 60 s of light curing. c) FTIR spectra of Ru+RB with 0.28, 0.56, and 1.12% w/v Ru with 0,10, 30, and 60 s of light curing. 1635.6361516.0491575.8431579.71666.4971762.9381512.1921577.7711610.5611666.4971762.938-10123450.28%Ru-resin-2% photoinitiator0 sec10 sec30 sec60 sec1579.71608.6331635.6361664.5681514.1211764.867-1-0.500.511.522.50.56% Ru-resin- 2% photoinitiator0 sec10 sec30 sec60 sec1579.71608.6331635.6361514.1211664.5681764.867-1-0.500.511.522.51.12% Ru-resin - 2% photoinitiator0 sec10 sec30 sec60 sec(c) 136 Another possible reason behind the reduction in DC with increasing Ru concentration could be due to the relatively low solubility of Ru in the experimental RB. Potential LED light scattering due to immiscible Ru particles may also contribute to a reduced DC [359]. Moreover, some studies have previously demonstrated the effect of different PS (Riboflavin and Rose Bengal) incorporations into RB to confirm that generally the PS acts as a competing agent with the photoinitiator system [360]. It is important to note that the addition of a PS to photoinitiator systems that have different absorption wavelengths can induce a synergistic effect as the excitation of the PS molecule at a given wavelength can result in energy transfer and subsequent excitation of the photoinitiator [348]. In our case, the presence of the Ru photosensitizer with broad spectrum absorption in the range of 440-470 nm and the fact that the blue LCU can provide a broad-spectrum emission wavelength that covers the same range could provide the aforementioned synergistic effect. Overall, studying the effects of chemically modified RB+Ru formulations on the DC serves a crucial role as the extent of longevity of dental materials and their mechanical strength as well as the extent of their adhesion to dentin have positive correlation with large values of DC [349,361]. As a general rule and depending on the curing times and structure of the monomer and photoinitiator system, a DC range of 40-75 % is believed to be a reliable value [362], which corresponds to the values observed for RB+Ru groups with up to 1.12% w/w Ru and after 60 s photocuring. Therefore, for the remainder of the characterization and evaluation studies in this chapter, only 0.28, 0.56, and 1.12 %w/w Ru are used. 137 4.3.3 Flexural strength (FS) As observed in Figure 4.8, the incorporation of ruthenium PS into experimental RB did not have a significant impact on its degree of flexural strength (FS), despite 1.12 % w/w RB+Ru showing reduction of FS compared to other groups. This trend could be confirmed for both studies performed immediately after sample preparation as well as the ones after prolonged storage. In addition, all groups showed a detectable increase in FS after 2 months of water storage compared to those tested at 24 hours (p<0.001). It can be suggested that the introduction of ruthenium into the polymer matrix could have a positive impact on the overall mechanical strength of the samples. Overall, an average FS of more than 40 MPa for the immediate storage (24 h) and above 60 MPa for 2-month storage samples were observed. This indicates that the materials exhibited good resistance to bending forces over extended time period. It should also be considered that different dental materials have varying flexural strength values. This variability is expected, as different materials have unique compositions and properties that influence their mechanical performance. This is observed when compositional changes influence the bonding strength of different dental materials, with zirconium oxide showing the highest FS of over 500 MPa [363], MA and its derivatives having FS values of around 57 MPa [364], and nanofiber-reinforced experimental resin blend formulations indicating FS values near 100-120 MPa. Although a comprehensive account of mechanical strength of a dental restorative material can be obtained by studying the FS as well as the elasticity modulus and impact strength (IS), recent studies have shown that the encapsulation and incorporation of small molecules as reinforcement agents into the structure of MA-based dental resins generally result in improvement of flexural strength [365,366]. The small molecules used as reinforcement agents can vary but 138 often include substances like nanoparticles, silanes, or other organic compounds. These molecules are chosen for their ability to enhance the mechanical properties of the resin, such as flexural strength. Figure 4.8 Flexural strength studies of MA•Ru(dpp)3 – a) Photographic images of the RB+Ru bars with 0, 0.28, 0.56, and 1.12 %w/v Ru. b) Flexural strength of the resin specimens as a function of Ru concentration and ageing. Data reported as average ± one standard deviation (n=12). Groups that are assigned the same letter or letter combination are not significantly different (p>0.05). (a) (b) 139 4.3.4 Shear bond strength (SBS) to dentin Overall, the shear bond strength of the resin (Figure 4.9) was not detectably impacted by the incorporation of Ru for both ageing periods (p>0.05). However, the addition of the highest concentration of Ru (1.12%) significantly reduced the shear bond strengths of the experimental resin to dentin at 24 hours (p=0.019). The commercial control group (SBMP) shows the highest values of SBS at 24 hours of storage compared to all resins investigated (p<0.001). Meanwhile, the 2-month ageing did not greatly affect the SBS reported for all sample and control groups (p=0.581). After 2-months of storage there was no detected difference between the commercial control (SBMP), the experimental control (0% Ru), and the experimental Ru 0.28% (p>0.05). Both 0.56% and 1.12% Ru added resin blends had detectably lower SBS reported than the commercial control after 2-months storage (p<0.001). 140 1 Figure 4.9 Shear bond strength studies of MA•Ru(dpp)3 – a) Photographic images of the stages of sample preparation for SBS testing. b) Shear bond strength of resin specimens as a function of Ru concentration and ageing. Data reported as average ± one standard deviation (n=15). Groups that are assigned the same letter or letter combination are not significantly different (p>0.05). In terms of the underlying adhesion mechanisms of restorative dental materials, apart from GICs that bind directly and chemically to dental tissue due to immediate ionic bonding 4 5 2 3 (a) (b) 141 between the carboxylate groups and calcium [367], micromechanical interlocking is the primary mechanism of adhesion for the other restorative dental materials including resin composites. Other physicochemical mechanisms by increasing the wettability and bonding surface area also play a role in this process [369]. Similar to many mortar and brick structures in nature, micromechanical interlocking is based on the flow and penetration of the adhesive material into the pores and irregularities on the dental tissue surface. For methacrylate-based resin composites, the structural bonding between two material phases is achieved via infiltration and embedding on the micron or nanometer scale [370]. The incorporation of small molecules into the polymeric matrix structure of resin-based (RB) formulations can lead to varied results in terms of adhesion and changes in shear bond strength (SBS). Some studies indicate that the inclusion of nanoparticles like ZrO2, TiO2, and SiO2 into the formulation can enhance adhesion between the resin and the tooth [371]. Other approaches, such as modifying the resin structure with layered MA-based resins, have also shown to improve adhesion [372]. Thermal curing of the resin, which increases the polymer molecular weight, is known to further enhance adhesion strength to dental tissue [373]. For our RB+Ru system, the presence of the bulky diphenyl substituents around the PS structure could potentially limit the micromechanical interlocking ability of the formulation which could result in the observed reduction in SBS. With that in mind, it is crucial to note that even at the highest PS concentration of 1.12% Ru, the resulting SBS value is within 5 – 10 MPa, which is an acceptable range for a variety of dental and orthodontic applications [374–377]. Furthermore, this study included a commercial control (SBMP) considered a gold-standard material in its category, with several years of laboratorial and clinical data. The evaluation of this product in this specific category allowed us to directly compare our experimental blends, with and without Ru in the formulation, with a commercial product extensively studied [378]. 142 4.3.5 Water sorption (WS) and solubility (SL) The addition of Ru into the experimental resin blends detectably affected WS (p = 0.010), but it did not affect SL (p=0.089) (Figure 4.10). However, the impact of Ru concentration was minimal as the only significant difference found upon analysis in WS was between the 0.28% and 0.56% Ru experimental blends (p=0.007). Water sorption (WS), also known as moisture sorption isotherm, is a measure of increase in the relative extent of humidity or water content of a specimen at a constant temperature and is a way of testing if the composition or amount of the materials changes over time. Water solubility (SL), on the other hand, is a measure of the extent of leaching or disintegration of the material when placed in aqueous media. An ideal dental resin composite is a formulation with very little variations in both WS and SL when stored in water for extended periods of time. Since both WS and SL values depend on the sample size of the specimens under investigation, their values and variations can only be compared among the different groups in each study. Overall, in terms of photocurable resin-based dental composites, factors such as degree of photocuring and complete polymerization play crucial role in minimizing the variations in WS and SL [315,352,379,380]. Another contributing factor to WS and SL variations is presence of ethanol in the final formulation. Although ethanol incorporation leads to increased DC extent of the dental materials, it also contributes to increased hydrophilicity of the resin, thereby increasing the WS and SL degrees. 143 Figure 4.10 Water sorption and solubility studies of MA•Ru(dpp)3 – a) Photographic images of the RB+Ru disks. b) (A) Water sorption and (B) Solubility of resin specimens after 14 days as a function of PS concentration. Data reported as average ± one standard deviation (n=15). Groups that are assigned the same letter or letter combination are not significantly different (p>0.05). 4.3.6 In vitro antimicrobial activity The highest degree of antimicrobial photodynamic action was observed for 1.12% (w/w) Ru-resin group, which resulted in greater than 2 LOGS CFU/ml (over 99%) of photokilling effect compared to the dark controls. For dark toxicity effect, with the exception of the 1.12% (w/w) group tested after two months storage, no other experimental group showed RB + Ru1.12% RB + Ru0.56% RB RB + Ru0.28% (a) (b) 144 any significant reduction in CFU counts compared to the respective dark control. The experiments for both immediate as well as two months storage were repeated at least twice (n>3) (Figure 4.11). The assessment of the antibacterial activity of the experimental RB loaded with Ru demonstrated that 1.12 w/w% Ru addition was necessary for a significant LOG reduction (CFU/mL) of S. mutans under LED irradiation (650 mW/cm2). Also, only 2-month stored 1.12 w/w% Ru-added RB showed antibacterial activity in the dark compared to the respective control group (SBMP). The mechanism of action of ruthenium toxicity to bacteria is associated with their capability to produce singlet oxygen species (1O2) because of their low-energy triplet state when irradiated with a blue LED light, leading to damage of several biomolecules and microbial death [381,382]. Researchers have also evaluated the incorporation of other PS in resin-based dental materials for aPDT applications against S. mutans [360,376,377]. For example, it has been demonstrated that increased concentration of PS produced better antimicrobial results, but relatively poor bonding properties [360]. However, it has also been observed that the highest concentration of PS which maintained a clinically acceptable range of bond strength (6-8 MPa), could also provide an antimicrobial effect against S. mutans after 24 hours and 2 months of storage when exposed to blue LED [376,377], and even in the dark [377]. Unlike the present investigation, which achieved positive aPDT results with a low concentration of PS (1.12% Ru), the aforementioned studies used higher concentrations of other photosensitizers, 7.0% Cur/PLGA/NPs [377], and 7.5% Cur/ZnONPs [376], incorporated into an orthodontic adhesive material. 145 Figure 4.11 Photographic images of light curing process and antimicrobial studies results of MA•Ru(dpp)3 – a) Photographic images of resin curing process using LCU. b) Photographic image of aPDT process on experimental disks using LED light source. c) CFU count as a function of Ru concentration, storage time, and application of light. Data reported as average ± one standard deviation (n=15). Groups that are assigned the same letter or letter combination are not significantly different (p>0.05). (a) (b) (c) 146 4.4 Conclusion The present chapter and research performed demonstrated that 1.12% Ruthenium (II) complex incorporated into an experimental dental RB allowed for ideal adhesive, physical, chemical and antimicrobial properties against cariogenic bacteria. This innovative methacrylate-based dental material, designed to control cariogenic bacteria with the aid of aPDI, could potentially be repeatedly applied intra-orally to control and manage oral pathogenic bacteria linked to dental caries. There is great potential for this novel approach in multiple aspects of Dentistry. For example, the material could be fine-tuned as a potential restorative material, as a bonding agent for direct and indirect restorations and orthodontic brackets, and as a preventive material such as fissure sealants or low viscosity resinous blend to be infiltrated on initial interproximal carious lesions. It should be noted that additional investigations exploring the parameters on the light, the resin blend formulation, and other oral pathogenic microorganisms are needed in this promising area, alongside with their effectiveness on in situ and in vivo studies. The findings of this study demonstrated that the addition Ruthenium (II) complex in low concentrations (0.28, 0.56 and 1.12 w/w %) into a methacrylate-based formulation is a feasible and promising aPDT approach. The experimental formulations presented adequate DC, enhanced FS and stable SBS to dentin over time, no compromises on WS and SL, and an antimicrobial activity in fresh and aged specimens. The study demonstrated that the novel approach of incorporating a photosensitizer compound into a methacrylate-based material is a feasible strategy against oral pathogenic bacteria. The proposed concept opens several opportunities for future studies and further explorations of its use for other dental and medical applications. Given the importance that the aesthetic aspects of restorative dental materials have, and the fact that the RB+Ru formulation is an orange-colored mixture, the present technology, using either ruthenium 147 or other photosensitizers, would need to consider the aesthetic prospects of the acrylic dental resins as an integral necessity [383,384], and might therefore be used as dental filling materials for the interior teeth and areas of the enamel not immediately visible. Another possible benefit of developing dental resins based on aPDT and ruthenium as PS is the availability of blue light teeth whitening (bleaching) products. Some of these mouthpiece products use LED blue light and are becoming increasingly popular as they are safe and also are not classified as a drug which would need to be approved by regulatory bodies such as the American food and drug administration (FDA). The fact that the emission wavelength of these devices corresponds to the absorption wavelength of the RB+Ru system can provide greater advantage for the incorporation and application of this new technology as novel dental filling materials in near future. 148 Chapter 5. Conclusions and future work The findings of this thesis provide an experimental framework for the successful design, incorporation, characterization, and evaluation of antimicrobial activity of a ruthenium-based photosensitizer as surface sterilization agent embedded in two different classes of polymeric biomaterials, the soft PDMS-based polymer and the hard methacrylate-based (MA) resin with versatile applications as medical implants in urology and dentistry. The “end-product” in each case would be a photosensitive silicone-based urinary catheter and a photosensitive methacrylate-based dental resin. The characterization studies that evaluated the chemical, physical, and mechanical properties of the Ru-embedded polymeric matrices indicated properties for these new biomaterials that are comparable to the available Foley silicone catheters and experimental restorative dental resins. In the case of the urinary catheter, the current findings of the thesis are based on the synthesis, purification, and characterization of the properties of both the ruthenium photosensitizer in compound form and the incorporated PDMS•Ru(dpp)3 material as precursor to silicone Foley catheters. The embedded PS-polymer matrix shows no leaching of the PS over time, thus indicating its safety. It is also a potent ROS generator as confirmed by singlet oxygen generation studies using DMA as a molecular quencher. Evaluation of the physicomechanical properties of the PDMS•Ru(dpp)3 system including tensile strength, surface roughness, surface wettability, and surface encrustation showed improved or comparable results to bare PDMS. Also, the in vitro, in vivo, histology, and cytotoxicity experiments all confirm both the antimicrobial activity of the technology as well as its safety. 149 For the restorative dental resin, the studies that indicated the viability of the technology as an effective choice include the evaluation of the degree of conversion of the MA•Ru(dpp)3, followed by calculation of flexural strength, shear bond strength, sorption and solubility as indicators of its physicomechanical suitability. Finally, the antimicrobial activity evaluation of the system further verifies its effectiveness. Although the concepts of photosterilization and photodisinfection have been practiced for a while now, the settings in which they are employed have been for either industrial use in water treatment facilities for water disinfection which use very high photonic power [385], for photodisinfection of textiles [386], or as photodisinfection nasal sprays [387]. There are also many studies that show the efficacy of aPDI as a viable technique for materials sterilization using variety of different PSs and substrates. The novelty in our approach, however, is in simultaneous application of an immobilized ruthenium photosensitizer with the ability to be locked as a surface sterilizing agent inside some of the most widely used polymeric biomaterials as medical implants, as well as its photoactivation by a simple, cost effective and safe light source such as LED operating at mW/cm2 photonic density range. This has implications both in terms of patient safety as well as economic considerations as it is quite feasible to power LED strips or other LED-driven fiber optic-coupled light source operating to deliver such photonic fluence with portable 12V battery packs with 8 standard AA batteries and have it run continuously for multiple hours [388]. The practicality of having such a platform is in its availability to be used in the form of a single product including both the PS-embedded Foley silicone cather as well as the light source and its powering unit. This could be of special interest and usage in settings such as patients undergoing post-operation ICU stay with the need to be catheterized consistently during their recovery period. 150 Concerning the photosensitive urinary catheter system, an aspect of the current platform in need of further exploration is the design and calibration of a light source unit capable of illuminating and activating the ruthenium PS directly from inside the catheter lumen once the PDMS•Ru(dpp)3 Foley catheter is inserted. The primary consideration for this setup is to ensure the light source that is to be used is capable of illuminating the entire external portion of the indwelling device that has been inserted since bacterial adhesion and biofilm formation can happen anywhere on the surface of the implant (even the internal surface of the lumen). Some possible avenues to be explored are using an array of LEDs connected in series, also referred to as LED strips, which can be inserted into the catheter lumen and trigger the photokilling action. One advantage of LED strips is their flexibility. However, a possible drawback could be the size of the strip which may not fit into catheters with smaller lumen diameters. Another possibility is to get help from fiber optics to guide the illuminated light inside the catheter lumen. Although fiber optics materials usually work by delivering light to the other end of the tube and thus illumination is expected to occur only at the tip of the fiber, it is also possible to use side-firing or side glowing fiber optics materials that allow for simultaneous illumination of the entire implant where the fiber optic is guided into [389]. The third possibility is to design a layer-by-layer (LBL) approach to have the illumination source as a flexible organic LED (OLED) sheet being sandwiched between two PDMS•Ru(dpp)3 layers. This could ensure that a uniform illumination is provided throughout the entire surface of the inserted catheter. With regards to the MA•Ru(dpp)3 photosensitive dental resin, the recent advancements in dental materials and current use of blue light curing units (LCU) in dental practices to initiate photopolymerization of the resin could pave the way for photosensitive dental resins to find their place as novel classes of antimicrobial technologies in dentistry with ease and speed. This is indeed 151 subject to validation of both efficacy as well as safety via running in vivo studies and later clinical trials using patients. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Lange, Dirk (Microbiologist)
Branda, Neil R.
Surface photosterilization of implantable biomaterials : applications in urology and dentistry
Text