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Immobilization of antimicrobial peptides onto titanium surfaces Lu, Shanshan 2009

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IMMOBILIZATION OF ANTIMICROBIAL PEPTIDES ONTO TITANIUM SURFACES  by Shanshan Lu  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Materials Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2009  © Shanshan Lu, 2009  ABSTRACT Prosthetic-associated infections are one of the most challenging postoperative complications for orthopedic implants. The consequences that infections may lead to include patient pain, high cost, prolonged hospitalization time, and usually the revision of the implant. Current prophylaxis and therapy utilizing antibiotics are facing an emergency of increasing bacterial resistance; the design of a novel anti-infectious implant surface is therefore required.  Among the potential antimicrobial alternatives are the antimicrobial peptides (AMP). AMPs are a family of natural defense peptides that has not received enough recognition until recently. The complex killing mechanisms of these cationic peptides make them very unlikely to encounter resistant mutants, and their broad-spectrum activity offers them great opportunity in possible clinical applications. In this study, a novel short AMP Tet213 with prominent bactericidal activity was chosen as the antimicrobial candidate and was covalently attached to titanium surfaces through a short bifunctional linker. This designed routine was confirmed with single cysteine before being applied to the 9-mer AMP candidate.  The surface density of the immobilized AMP was determined by detecting its arginine residues after a reaction with 9,10-phenanthrequenon (PHQ). The reaction between arginine and PHQ generates a fluorescent product, by the emission of which the ii  quantity of the arginine-containing peptide can be calculated. The density of the surface-attached Tet213 was measured to be 1.30±0.55 μg/cm2. A relatively large proportion of physically adsorbed Tet213 was also observed, with the net adsorbed quantity to be 0.74±0.20 μg/cm2. The affinity of the cationic AMP to the bare titanium surface is believed to be a result of electrostatic interactions.  Both the covalently immobilized and the physically adsorbed Tet213 showed bactericidal activities of generally > 50% against a Pseudomonas aeruginosa (P. aeruginosa) strain which constitutively expresses luminescence when alive. The inhibition rate was calculated by the luminescence reduction and confirmed by the colony counts of the surviving bacteria. Several parameters were found to be influential to the overall inhibition rate, including the selection of the AMP candidate, the dilution of the bacterial culture and the bacterial incubation time.  iii  TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii TABLE OF CONTENTS ................................................................................................. iv LIST OF TABLES .......................................................................................................... viii LIST OF FIGURES .......................................................................................................... x LIST OF ABBREVIATIONS ........................................................................................ xiii ACKNOWLEDGEMENTS .......................................................................................... xiv CHAPTER 1 INTRODUCTION ..................................................................................... 1 CHAPTER 2 LITERATURE REVIEW ......................................................................... 3 2.1  Bone Implant Infections ................................................................................. 3 2.1.1  Orthopedic Prosthesis ............................................................................. 3  2.1.2  Infections Associated with Orthopedic Implants .................................... 4  2.1.2.1 Prosthetic Joint Infections................................................................... 4 2.1.2.2 Pathogens and Pathogen Resistance ................................................... 5 2.1.3  Treatment of Implant-Associated Infections........................................... 8  2.1.3.1 Surgical Treatment and Antibiotic Therapy ........................................ 8 2.1.3.2 Prophylaxis of Implant Infections ...................................................... 8 2.2  2.3  Antimicrobial Peptides................................................................................... 9 2.2.1  Cationic Antimicrobial Peptides ............................................................. 9  2.2.2  Activity of Tethered Antimicrobial Peptide .......................................... 12  Titanium Surface Modification .................................................................... 14 2.3.1  Modification of Surface Morphology ................................................... 14  2.3.2  Modification of Surface Chemistry ...................................................... 15  2.3.2.1 Adsorption ........................................................................................ 16 2.3.2.2 Surface Coating ................................................................................ 17 2.3.2.3 Covalent Coupling ............................................................................ 18 2.4  Detecting Biomolecules ............................................................................... 21 iv  2.5  Summary ...................................................................................................... 23  CHAPTER 3 SCOPE AND OBJECTIVES .................................................................. 24 CHAPTER 4 MATERIALS AND METHODS ............................................................ 26 4.1  Antimicrobial Peptide Candidates ............................................................... 26  4.2  Immobilization of Peptides to Titanium Surfaces ........................................ 27 4.2.1  Titanium Surface Cleaning and Etching ............................................... 27  4.2.2  Cysteine and Cysteine-Linked Peptide Immobilization ....................... 28  4.2.3  Design of Experimental Groups............................................................ 30  4.2.3.1 Cysteine Groups................................................................................ 30 4.2.3.2 Peptide Groups.................................................................................. 32 4.2.3.2.1 Surface Density Determination and Antimicrobial Experiments .......................................................................................... 32 4.2.3.2.2 Peptide Adsorption with Different Surface Morphologies .... 33 4.3  Determination of the Surface Density of the Immobilized Cysteine and  Peptides ...................................................................................................................... 34 4.3.1  Determination of Immobilized Dansyl-tagged Cysteine ...................... 34  4.3.2  Determination of Immobilized Peptide Density Using a Sensitive  Fluorescent Method ............................................................................................ 36 4.4  Antimicrobial Experiments .......................................................................... 37 4.4.1  Luminescence Inhibition against Pseudomonas aeruginosa strain ...... 37  4.4.2  Bacterial Colony Count......................................................................... 40  4.4.3  Design of Antimicrobial Experiments .................................................. 41  4.4.3.1 Experiment 1: Selection of AMP candidate and dilution of bacterial culture .......................................................................................................... 41 4.4.3.2 Experiment 2: Difference between Linked and Soaked groups ....... 43 4.4.3.3 Experiment 3: Incubation time ......................................................... 43 4.5  Statistical Analysis ....................................................................................... 44  v  CHAPTER 5 RESULTS ................................................................................................. 45 5.1  Titanium Surface Morphologies .................................................................. 45  5.2  Dansyl-tagged Cysteine Immobilization ...................................................... 47  5.3  5.2.1  Fluorescent Analysis of Dansyl-Tagged Cysteine in Solution .............. 47  5.2.2  Cysteine Immobilized on Titanium Surfaces ........................................ 49  5.2.3  Summary ............................................................................................... 53  Peptides Immobilization .............................................................................. 53 5.3.1  Detection of Arginine and Arginine-Containing Biomolecules ............ 53  5.3.1.1 Fluorescent Detection of Single Arginine ......................................... 53 5.3.1.2 Fluorescent Detection of Tet213 and BSA ....................................... 56 5.3.2  Peptides Immobilized on Titanium Surfaces ........................................ 59  5.3.2.1 Immobilization of Tet213 ................................................................. 59 5.3.2.2 Tet213 Adsorption on Titanium with Different Surface Morphologies ............................................................................................... 64 5.4  Antimicrobial Experiments .......................................................................... 67 5.4.1  Experiment 1 ......................................................................................... 67  5.4.2  Experiment 2 ......................................................................................... 71  5.4.3  Experiment 3 ......................................................................................... 73  5.5 Summary .............................................................................................................. 75 CHAPTER 6 DISCUSSION........................................................................................... 77 6.1  Tet213 adsorption on Titanium Surfaces ..................................................... 77  6.2  Surface Density of modified Tet213 on Titanium ........................................ 78  6.3  Antibacterial Activity of Tethered and Adsorbed Tet213............................. 79 6.3.1  Bacterial Inhibitory Effect and the Fluctuation ..................................... 79  6.3.2  Antibacterial Effect of Surface-bound and Adsorbed Tet213 ............... 84  CHAPTER 7 CONCLUSIONS ...................................................................................... 86 CHAPTER 8 RECOMMENDATIONS FOR FUTURE WORK ............................... 88  vi  REFERENCES ................................................................................................................ 89 APPENDIX .................................................................................................................... 101  vii  LIST OF TABLES Table 4. 1 Design of experimental groups in cysteine immobilization. ...............................31 Table 4. 2 Design of experimental groups in peptide immobilization. .................................34 Table 4. 3 Components of Basal Media 2 (BM2) bacterial culture. .....................................39 Table 4. 4 Design of antimicrobial experiments. ..................................................................41 Table 5. 1 The amount of dansyl-tagged cysteine estimated from fluorescent intensities of each group. Group 1: Bare Ti; Group 2: Silanized Ti; Group 3: Silanized Ti with SMP linker; Group 4: Silanized Ti grafted with tagged cysteine through the SMP linker; Group 5: Bare Ti soaked in cysteine solution (average of 5 samples). ...................................................................................51 Table 5. 2 P values of Holm t-test between every two groups. Group 1: Bare Ti; Group 2: Silanized Ti; Group 3: Silanized Ti with SMP linker; Group 4: Silanized Ti grafted with tagged cysteine through the SMP linker; Group 5: Bare Ti soaked in cysteine solution. (Average of 5 samples.) .....................................52 Table 5. 3 The surface density of Tet213 of each group, estimated from the fluorescent intensities. .......................................................................................................62 Table 5. 4 P values of Holm t-test between every two groups of Tet213 sample. ...............62 Table 5. 5 Luminescent inhibition rates of Tet213 groups under different initial bacteria-culture dilutions. ...............................................................................70 Table 5. 6 Inhibition result of Tet213 groups from colony counts after incubation with 1-in-50 initial bacterial dilution. .....................................................................70 Table 5. 7 Luminescent inhibition rates of Tet213 groups after second-round incubation. .71  viii  Table 5. 7 P values of Holm t-test between every two groups on the luminescent inhibition results of Experiment 2. .................................................................72 Table 5. 9 P values of Holm t-test between every two groups on the luminescent inhibition results of Experiment 3, after 4 hr and 24 hr incubation, respectively. ....................................................................................................75  ix  LIST OF FIGURES Figure 2. 1 (a) Major pathogenic species found in orthopedic infections. (b) Proportion of antibiotic-resistant staphylococcal species in clinical analyzed orthopedic infections. (Reprinted from [9] with permission from Elsevier.) .....................7 Figure 2. 2 Typical structures of AMPs. (A) Mixed structure of human β-defensin-2; (B) looped thanatin; (C) β-sheeted polyphemusin; (D) rabbit kidney defensin-1;(E) α-helical magainin-2; (F) extended indolicidin. (Reprinted from [26] with permission from American Society for Microbiology.) .........11 Figure 2. 3 Schematic illustration of methods for implant surface modification of biochemistry. (Reprinted from [42] with permission from Elsevier.) ............16 Figure 2. 4 Schematic representation of the covalent coupling route through silanization [70]..................................................................................................................19 Figure 2. 5 Silane linked to the metal (M) surface and neighbor molecules (Reprinted from [77] with permission from American Chemical Society.) .....................21 Figure 4. 1 Immobilization of cysteine/cysteine-linked peptide via covalent coupling (titanium surface roughness not shown). ........................................................29 Figure 4. 2 Schematic surface chemistry of the designed cysteine groups (titanium surface roughness not shown).........................................................................32 Figure 4. 3 Schematic surface morphology and simplified structure of the designed peptide groups.................................................................................................34 Figure 4. 4 Molecular structure of the dansyl-tagged cysteine [71] .....................................35 Figure 4. 5 PHQ reacts with arginine and arginine residues, generating a stable fluorescent compound [80]. ............................................................................36  x  Figure 5. 1 Left: titanium plate, cleaned; Right: titanium plate, cleaned and etched. ..........45 Figure 5. 2 Scanning electron micrographs of the titanium surfaces. (a): cleaned only, (b): cleaned and etched by 2% HF, and (c): cleaned and etched by 2% HF, followed by the dual-acid etching...................................................................46 Figure 5. 3 Fluorescent excitation and emission spectra of dansyl-tagged cysteine in solution (0.5 µg/ml). .......................................................................................47 Figure 5. 4 (a) Emission spectra of tagged cysteine solutions at different concentrations; (b) Calibration curve drawn from peak intensities in (a). ...............................48 Figure 5. 5 Estimated amount of the dansyl-tagged cysteine on titanium samples after alkali cleavage. Group 1: Bare Ti; Group 2: Silanized Ti; Group 3: Silanized Ti with SMP linker; Group 4: Silanized Ti grafted with tagged cysteine through the SMP linker; Group 5: Bare Ti soaked in cysteine solution (average of 5 samples). .....................................................................50 Figure 5. 6 Fluorescent excitation and emission spectra of arginine in solution (3 µg/ml) after reacting with PHQ. .................................................................................54 Figure 5. 7 Calibration curve of arginine in solution (r = 0.996). .........................................55 Figure 5. 8 Arginine calibration curves tested on different days. .........................................56 Figure 5. 9 Comparison of fluorescent emission spectra of single arginine, Tet213 and BSA. The concentration of arginine, Tet213 and BSA solution is 0.3μg/ml, 2μg/ml and 15μg/ml, respectively. .................................................................57 Figure 5. 10 Calibration curves of Tet213 (linear, r = 0.995) and BSA in solution (first order exponential, r = 0.993). .........................................................................58 Figure 5. 11 Effect of spectrometer slit width on Tet213 calibration curves. .......................59 xi  Figure 5. 12 Comparison of fluorescent emission spectra of Bare Ti Group, Soaked Group, Linked Group and Tet213 solution. ....................................................60 Figure 5. 13 Comparison of the surface density of immobilized Tet213 on Bare Ti, Soaked Group and Linked Group samples (average of 11 samples). .............61 Figure 5. 14 Comparison of surface molecular density of immobilized cysteine and Tet213 groups. ................................................................................................63 Figure 5. 15 (a) Comparison of the surface density of Tet213 on Cleaned Group, Soaked Group, Linked Group and Bare Ti samples. (b) SEM image of titanium surface morphology of Cleaned Group. (c) SEM image of titanium surface morphology of Soaked Group. .......................................................................66 Figure 5. 16 Comparison of luminescence reading from experimental groups after incubation with different dilution of the lux strain: (a) Bac020 groups. (b) Tet213 groups. ................................................................................................69 Figure 5. 17 Luminescent inhibition of the second antibacterial experiment with Tet213 samples (average of 5 samples). .....................................................................72 Figure 5. 18 Comparison of Tet213 surface loading and the antibacterial effect. (a) Estimated surface density of Tet213. (b) Inhibitory rates after 4 hr’s and 24 hr’s incubation. ...............................................................................................74 Figure 6. 1 Comparison of the inhibition rates of Tet213-modified Ti samples after 4 hr’s incubation on different tests. ...........................................................................82  xii  LIST OF ABBREVIATIONS AMP  Antimicrobial peptide  P. aeruginosa  Pseudomonas aeruginosa  S. aureus  Staphylococcus aureus  S.epidermidis  Staphylococcus epidermidis  MIC  Minimum inhibitory concentration  APTES  3-aminopropyl triethoxysilane  SMP  3-maleimidopropionic acid N-hydroxysuccinimide ester  PHQ  9,10-phenanthrenequinone  BSA  Bovine serum albumin  BM2  Basal Medium 2  OD600  Optical density at 600 nm  CFU  Colony forming unit  SS  Stainless Steel  UHMWPE  Ultrahigh molecular weight polyethylene  LPS  Lipopolysaccharide  ECM  Extracellular matrix  HSV  Herpes simplex virus  HIV  Human immunodeficiency virus  CaP  Calcium phosphate  Arg  Arginine  Gly  Glycine  Asp  Aspartic acid  RGD  Arg-Gly-Asp  xiii  ACKNOWLEDGEMENTS I want to thank my supervisor Dr. Rizhi Wang, for granting me this great research opportunity and for the guidance, support and motivation I received throughout the entire study.  I would also like to thank Dr. Kai Hilpert and Dr. Jason Kindrachuk for the assistance in the antimicrobial experiments; Dr. R.E.W. Hancock and Dr. Jayachandran N Kizhakkedathu for valuable suggestions of the whole program, and all my colleagues: Ke Duan, Mehdi Kazemzadeh Narbat, Vincent Ebacher, Allen Tang, Youxin Hu, Menghan Ma and Millie Kwan. I want to express my special thanks to Dr. Ke Duan for his extensive support during this research.  I appreciate the University of British Columbia for offering me the University Graduate Fellowship and Canadian Institutes of Health Research (CIHR) for funding this research.  I want to express my deep gratitude to my friends and my family for their tremendous emotional support. To my forever beloved mother and father, thank you for your infinite love and firm support that has been enlightening my life. I am so grateful being born as the child of yours.  xiv  Chapter 1 Introduction Prosthesis-related infection is among the major causes that lead to orthopedic implant revisions [1]. The current treatment of prosthetic infections is largely dependent on antibiotic therapy [2]; however, traditional antibiotics are facing the increasing challenge of resistant bacterial mutants. A strong need is therefore present to develop effective anti-infectious implants as well as new antimicrobial drugs.  The family of antimicrobial peptide (AMP) is one of the promising candidates for infection prophylaxis and treatment. This group of short cationic peptides is among the first line of innate immune systems in all species of lives. Many of them behave broad-spectrum activity towards Gram-positive and Gram-negative bacteria, viruses, fungi and some parasites [3]. Because of their complex killing mechanisms, the possibility for AMPs to encounter a resistant bacterial strain is much lower than the conventional antibiotics. With the very recent development of a group of short yet potent AMPs [4], the application of AMPs in implant infection prophylaxis has become possible.  Modification of the implant surface with an antimicrobial agent is a potential routine to eliminate periprosthetic infections. Various techniques of immobilizing a biomolecule  1  onto the metal surface have been reported, among which the covalent coupling method has received special attention because of the relatively stable covalent bond created between the biomolecule and the substrate. The grafting strategy is generally applicable to most common substrates and often involves a maleimide linker which specifically reacts with the thiol group in the cysteine residue of the protein/peptide. In this research, we proposed to immobilize a novel AMP with prominent antimicrobial activity onto the surface of titanium, one of the most widely used materials for hard tissue replacement. A short linker was grafted on the titanium surface to immobilize the peptide on the metal surface. The AMP-modified titanium was further tested for their bactericidal activity against a Pseudomonas aeruginosa strain. The goal of this research is to achieve an anti-infectious titanium surface that could potentially be developed into antimicrobial implants.  2  Chapter 2 Literature Review 2.1 Bone Implant Infections 2.1.1 Orthopedic Prosthesis With the aging of population, the demand for orthopedic implants has been climbing with years. More than 500,000 total hip and knee arthroplasties are performed each year in the USA [2]. The CJRR annual report recorded over 70,000 hip and knee replacements in Canada in the year 2006-2007. The number of hospitalization for hip and knee replacement has increased 101% over the past 10 years [1].  Modern total hip and knee replacements were both established in the 1960s [5,6]. A typical hip implant is composed of a hip stem, an acetabular cup and a polymer liner, whereas a total knee replacement generally consists of a femoral component, a tibial component and a polymer patella. Currently, the polymer components in both types of implant are made of ultrahigh molecular weight polyethylene (UHMWPE), while the metallic parts are mostly made of cobalt alloy (Co-Cr-Mo), stainless steel (SS), pure titanium (Ti) or titanium alloy (Ti-6Al-4V) [7].  After the primary replacement, a number of orthopedic implants would have to go through a revision surgery due to implant failure. Among the 0.16–0.2% population in industrial countries who receive a hip replacement arthroplasty, about 10% have to 3  undergo a revision within 10 years after implantation [8]. CJHH reported 13.6% for hip implants and 6.3% for knee implants as the annual proportion of revisions in all the orthopedic prosthesis cases [1]. Various complications can lead to hip and knee revisions, with the mostly reported reasons as aseptic loosening, poly wear and prosthetic-related infections.  2.1.2 Infections Associated with Orthopedic Implants 2.1.2.1 Prosthetic Joint Infections The risk for an implant-related infection after orthopedic surgeries is generally reported to be in the range of 0.3%-5% [2,9]. However, infection rates after a revision surgery can increase significantly up to 10% [10-12]. Considering the significant number of orthopedic implantations each year as well as the difficulty to treat the infected implants, this proportion should never be underestimated. Infection after prosthetic surgeries has been reported as the second major cause of implant failures [13,14], and is among the most devastating complications. Consequences of the prosthetic joint infections include patient trauma and pain, economic cost ($30,000 to $60,000 for each implant [2]), prolonged hospitalization, revision surgery, and sometimes even loss of limb or life [9,15]. The mortality was reported between 1% and 2.7%, whereas a significant increase was recorded for aged patients [16]. Some of the contributing factors to a high infection risk include surgical site infection, wound healing complications, malignant disease, underlying joint disease, prior surgery at the arthroplasty site and 4  previous infections [17,18].  Prosthetic joint infections can be categorized into early infection, delayed infection and late infection [18]. Early infection is usually a complication of an overt wound infection caused by S. aureus and similar virulent microbes, occurring in the first 1 to 3 months after the surgery. Delayed infection can be noticed after several months and up to two years with pain as an indicative symptom. It generally involved less virulent pathogens such as coagulase-negative staphylococci. Hematogenous infection with the origin from skin, dental, respiratory and urinary tract infections [11] can sometimes be observed in late stage (> 2 years) with a stable orthopedic implant, although the risk is generally reported to be low [18].  2.1.2.2 Pathogens and Pathogen Resistance Infections take place with the attachment of microorganism to the implant surface as a primary step. A local immune depression would occur in the surrounding area of the implant, which allows the microbes to form an infection at a much lower initial concentration (<100,000-fold [19]). Most bacteria adsorb to the implant surface through non-specific physical interactions, while some species produce proteins that help to adhere. A number of microorganisms can subsequently grow into a biofilm on the implant surfaces, where self-organized microbes are embedded in an extracellular polymeric matrix. Pathogens in a biofilm are 10-1000 times less susceptible to the host 5  defense system and antibiotics, and an excessive local concentration for antimicrobial agents is usually required to eliminate the biofilm microbes [16].  The most commonly found species in orthopedic implant infections are Gram-positive  bacteria,  especially  staphylococci.  Two  specific  pathogens,  Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S.epidermidis), contribute to approximately 70% of all the infection cases (Figure 2.1 a). Of particular concern of these two species is that both of them have been reported to have high potential of developing resistance towards antibiotics such as penicillin and methicillin/oxacillin (Figure 2.1 b) [9]. A study of 102 hospitals in England showed that 24.3% surgical site infections after total hip replacement involved methicillin-resistant S. aureus, while in the United States this resistant strain was responsible for 31% incisional or organ space infections after orthopedic surgery [20]. These antibiotic-resistant microbes are sometimes referred to as ―superbugs‖, which would cause severe consequences due to lack of effective treating methods.  6  (a)  (b) Figure 2. 1 (a) Major pathogenic species found in orthopedic infections. (b) Proportion of antibiotic-resistant staphylococcal species in clinical analyzed orthopedic infections. (Reprinted from [9] with permission from Elsevier.)  7  2.1.3 Treatment of Implant-Associated Infections 2.1.3.1 Surgical Treatment and Antibiotic Therapy The most widely used surgical treatment to implant infections in North America is a standard two-stage procedure. The revision involves the removal of the implant and infected surrounding tissue, a following 4- to 6-week antibiotic therapy and the final re-implantation. Antibiotic therapy is preferably combined with surgical procedure, as in the two-stage treatment. The selected antimicrobial treatment should be able to achieve high local concentration and to be active on the surface biofilm. Duration of the antibiotic therapy varies depending on individual treatment; prolonged therapy is required if antibiotic-resistant pathogens are discovered. Patients that are advised against revision surgeries are treated with suppressive antibiotic therapy, which usually takes more than one year [21]. Concerns regarding antibiotic treatment include complications, toxicity in long term, allergic reactions, and most of all an acute resistance arouse. A combination of at least two antibiotics is thus required to achieve a complete killing.  2.1.3.2 Prophylaxis of Implant Infections As bacterial adhesion is the first and critical step in prosthetic infections, various methods of modifying implant surfaces have been utilized as a prophylaxis of possible infections. The techniques usually involve the design of a surface with bactericidal activity. Bacterial inhibitory chemicals and biomolecules are usually entrapped in a surface coating by which a slow release is expected; they can also be incorporated into 8  the bulk material at the time of processing or directly immobilized on the surface through covalent bonding. The detailed modification approach of titanium surface is discussed later in Section 2.3. Surface-delivered substance can reach a high local concentration at the targeted area when the device is implanted, which makes this method advantageous over the systemic delivery routines [22].  Some of the antimicrobial substances that have been studied through implant surfaces delivery include metal particles such as silver and copper, ammonium compounds and various antibiotics including vancomycin, gentamicin and ampicillin [23]. While most of them are reported to successfully inhibit the bacteria growth in vitro, cytotoxicity to osteoblasts and possible resistance from bacteria towards antibiotics are the remaining issues.  2.2 Antimicrobial Peptides 2.2.1 Cationic Antimicrobial Peptides Antimicrobial peptides (AMP) are potential candidates as an alternative to traditional antibiotics. AMPs have been found in the immune system of almost all species of life, including bacteria, plants, invertebrates and vertebrates. This group of peptides is generally short (<100 amino acid), cationic and amphiphilic, and can be expressed either constitutively or inducibly by invading pathogens [3]. AMPs are considered to be among the first line in host defense systems, in the sense that they not only can kill microbes 9  directly but also are widely involved in the innate immune response. Up to now, hundreds of AMPs have been isolated from natural organisms, while even more have been synthesized in the laboratory.  Of all the AMPs discovered so far, various three-dimensional structures have been reported (Figure 2.2). The typical structures include α-helices, β-strands, loop structures and extended structures [24]. Despite their various spatial appearances, these peptides are all composed of around 30-50% hydrophobic residues and excessive amounts of cationic residues (lysine, arginine and/or histidine) [25]. The amphiphilic and cationic properties allow the peptides to interact strongly with the bacterial membrane, which is the critical step in the bactericidal process. High proportion of negatively charged lipids on the bacterial membrane surface makes the AMPs more selective for bacteria over the eukaryotic cells [26].  10  Figure 2. 2 Typical structures of AMPs. (A) Mixed structure of human β-defensin-2; (B) looped thanatin; (C) β-sheeted polyphemusin; (D) rabbit kidney defensin-1;(E) α-helical magainin-2; (F) extended indolicidin. (Reprinted from [26] with permission from American Society for Microbiology.)  Besides their immunomodulatory role, many AMPs exhibit excellent antimicrobial activity against a wide variety of pathogens. Several AMPs have been reported to behave antiviral against herpes simplex virus (HSV) and human immunodeficiency virus (HIV) [27-30], and many were proved to be inhibitory against fungi and parasites [31]. The mostly reported characteristic of the AMPs is their broad-spectrum activity against both Gram-positive and Gram-negative bacteria, and even towards antibiotic-resistant microorganisms [32-34].  11  The mechanism of AMPs’ bactericidal activity is still not fully understood. Due to their amphiphilic and cationic characteristics, most AMPs are able to bind to the lipopolysaccharide (LPS) of Gram-negative bacteria or other cell wall components of Gram-positive bacteria [35]. While this interaction is generally observed to be the initial step, the actual killing event varies with different peptides. Many AMPs cause permeabilization of the bacterial cell membrane, which further leads to the cell lysis. However, with some AMPs such as idolicidin and bactenecin, membrane permeabilization was not observed at their minimum inhibitory concentrations (MIC). It is therefore proposed that these peptides act through a different routine by traversing the cell membrane and acting directly on intercellular targets [25,36]. In many cases, more than one killing mechanism can be observed with a single peptide. As AMPs take effect through very complex mechanisms and act on more general targets of the bacteria than conventional antibiotics, the possibility of encountering a resistant mutant is significantly reduced.  2.2.2 Activity of Tethered Antimicrobial Peptide The activity and mechanism of AMPs in solution have been studied for more than 50 years. However, limited attention has been paid to activities of AMPs that are immobilized on a substrate. One of the earliest experiments by Haynie et al reported the antimicrobial activity of resin-tethered AMPs synthesized using a solid-phase strategy at the concentration of >1,000 µg/ml. The covalently-bonded AMPs were able to 12  significantly reduce the number of viable cells and showed broad spectrum activity against pathogens [37].  A recent publication of Hilpert et al focused on the characterization of a group of highly active AMPs synthesized on a cellulose sheet [38]. The peptides from the most active class were found to show an inhibition rate of almost 100% against Pseudomonas aeruginosa (P. aeruginosa), even when they were restricted on the cellulose substrate. It was also observed that the activity of the tethered AMPs does not directly correspond with their analogs in free solution. Therefore, attention should be paid to the selection of AMP candidate when the peptide is delivered on a substrate. A higher surface density for most tethered AMPs was required to kill the pathogens than the non-tethered AMPs. A most possible explanation is that immobilization results in limited mobility of the AMPs, reducing their ability to interact with or penetrate the bacterial membrane. Gabriel et al grafted a less effective AMP LL-37, the human cathelicidin, on a titanium substrate [39]. Antibacterial activity was only observed when the peptide was linked via a flexible poly(ethylene glycol) spacer, which provided improved lateral mobility over direct linking method and short linker coupling. As claimed by Bagheri et al, the most important factors affecting the activity of surface-bound peptide include the length of the spacer and the amount of target-accessible peptide [40]. However, it is speculated that a highly active peptide candidate may be able to compensate the negative parts of a rigid short linker, according to the positive results previously discussed from Hilpert et al. 13  2.3 Titanium Surface Modification The interface between the titanium implant surface and the host tissue has long been of interest to researchers and engineers. By modifying the titanium surface, various goals can be achieved. Two of the most concerned purposes are to improve the bone-implant fixation and to protect the implant from infection.  In general, the modification on implant surfaces can be classified as physicochemical, morphological, or biochemical [41]. Physicochemical modification usually involves alternation of surface energy, surface charge or surface composition, the effect of which, however, is still not well defined [42]. Modifications of the surface morphology and biochemistry are more extensively studied. The surface morphology approach is usually adopted to improve bone-implant fixation, while the biochemistry approach is the most commonly used method today for delivery of bioactive molecules and drugs.  2.3.1 Modification of Surface Morphology It has been hypothesized that the surface morphology plays an important role in bone-implant interaction. Macro-scale porosity can promote bone ingrowth and provide a strong mechanical interlocking for better bone fixation, while micro-scale morphology could regulate the behavior of osteoblast cells [43,44]. In vitro cell culture on micro-rough implant showed improved adhesion, differentiation and calcification of osteoblast cells [45], as well as reduced formation and activity of osteoclasts [46]. In vivo 14  study further confirmed that titanium implants with micro-scale roughness revealed increased pullout strength, indicating a better bone-implant fixation [47].  Macro-scale porosity of the substrate can be achieved at the time of manufacturing, with examples being metal mesh, porous cylinder or sintered particles. For bulk material, surface morphology can be created by mechanical methods, chemical treatment or electrochemical modification. The most commonly used approaches include grid blasting, sandblasting, etching by acid or alkaline, and anodizing. Etching by a dual-acid mixture of either HF/HNO3 or HCl/H2SO4 is reported to be a successful method for removing surface layer and creating micro-scale roughness [48-50]. A combination of two or more treatments is usually adopted in real application for a better outcome.  2.3.2 Modification of Surface Chemistry The modification of implant surface chemistry utilized surface attached molecules for a direct local delivery. The approaches can be divided into adsorption, immobilization and release from a surface coating (Figure 2.3).  15  Figure 2. 3 Schematic illustration of methods for implant surface modification. (Reprinted from [42] with permission from Elsevier.)  2.3.2.1 Adsorption Soaking the implant directly into a solution containing biomolecules is one of the simplest ways to attach the molecules onto titanium surface. In vivo test using the simple adsorption method for alkaline phosphatase delivery showed improved bone formation with the drug-adsorbed titanium implants [51]. Upon contact with air or water, titanium surface is rapidly oxidized with a rigid TiO2 layer, which is hydrophilic and weakly anionic at physiological pH. Proteins and other biomolecules can react with the oxide layer through van der Waals, hydrophobic or electrostatic forces. These interactions, however, are generally based on reversible phase equilibrium, and the adsorbed quantity and the subsequent release profile are largely dependent on the metal surface treatment,  16  the soaking conditions and the external physiochemical environment.  2.3.2.2 Surface Coating Surface coating on titanium implant can serve as a layer of active molecules alone, or can be incorporated with entrapped drugs as a delivery method. Calcium phosphate (CaP) coating is one of the most commonly utilized inorganic coatings because of its chemical similarity with the mineral component of nature bone tissue [52,53]. The mineral coating can be deposited onto implant surfaces by plasma spray, electrolytic deposition or biomimetic dip-coating techniques [45,54-57]. Organic components such as collagen and chitosan are usually co-deposited into the CaP coating to provide a mechanical reinforcement [58-62]. The porous coating can be further incorporated with drugs, proteins or growth factors to achieve different purposes [63].  Collagen and other organic components can be deposited onto titanium surfaces alone, serving as a bioactive layer or a drug delivery vehicle for a controlled release. Collagen is one of the most widely investigated extracellular matrix proteins and has an important role in promoting osteoblast adhesion and differentiation as well as controlling cell progression [64]. Schliephake et al studied the bone formation around a Ti screw coated with collagen to which a cell-adhesive peptide RGD (Arg-Gly-Asp) was linked. Animal test model with dog mandibles showed significantly improved bone contact and increased volume density of the new bones with the drug-collagen coated screws [65]. 17  Other organic coatings are also investigated utilizing different biomolecules. An animal study on rabbit was performed by Bumgardner et al with chitosan-coated titanium pins. The implants were inserted into the tibia of the rabbits, and the pins with chitosan coatings were proved more supportive for bone formation and osteointegration [66]. Poly(D,L-lactide) and politerefate coatings are reported to be potential candidates as well for controlled slow drug release [67].  2.3.2.3 Covalent Coupling Grafting biomolecules on titanium surfaces through covalent coupling provides a stable linkage, which can be retained for several days under physiological conditions [68,69]. This method is expected to retain the surface biomolecules for a longer period than the adsorption and coating delivery routines, and is receiving extensive attention from biomaterial researchers.  Covalent coupling routine starts with the functionalization of the metal surface, usually through silanization. A bifunctional linker is subsequently conjugated onto the surface and links the biomolecules to the surface functional groups. The most commonly used crosslinkers are maleimides, which reacts with the thiol moiety in the cysteine residue more rapidly than with any other groups. This maleimide-involving strategy can be used for cysteine immobilization, and more importantly, the covalent coupling of a bioactive peptide/protein that is linked with a cysteine end (Figure 2.4). 18  Figure 2. 4 Schematic representation of the covalent coupling route through silanization [70].  Silverman et al studied the immobilization of a Dansyl-tagged single cysteine onto Ti-6Al-4V alloys through the maleimide linker [71]. The metal surface was silanized using 3-aminopropyl triethoxysilane (APTES) and coupled with a bifunctional linker, 3-maleimidopropionic acid N-hydroxysuccinimide ester (SMP). The Dansyl-tagged cysteine was immobilized by reacting with the maleimide end in SMP. A constant release of the cysteine was observed with the modified surfaces in pH 7.5 water over 5 days, indicating a relative stable bond between the cysteine and the metal surface.  One of the applications for the covalent coupling strategy is the attachment of Arg-Gly-Asp (RGD), a cell-adhesive peptide to titanium surface for modulating the 19  adhesion of extracellular matrix (ECM) proteins. Xiao et al used three different heterobifunctional linkers to immobilize the RGD-cysteine peptide on silanized titanium surfaces [72]. The silanization step was found to be the key step in controlling the loading reproducibility, and the surface peptide coverage is estimated to be similar regardless of the choice of linker. Ferris et al reported significant increase in new bone thickness and greater pull-out strength in rat femurs with Au-coated titanium grafted with RGD compared with non-RGD implants [73], suggesting that this specific peptide is capable of maintaining its activity when tethered. RGD immobilized on a silicon surface through the same modification routine was also proved to enhance fibroblast adhesion and proliferation [74]. Besides cell-adhesive peptides, attempts have been made to graft antimicrobial molecules on titanium surfaces through covalent bonding as well. Vancomycin covalently bonded to titanium and Ti-6Al-4V alloy is reported to inhibit Staphylococcus aureus colony forming [75,76]. The antibiotic-surfaces were able to retain a stable activity after wash and upon repeated challenges.  One remaining concern about the covalently coupling approach is the control of the silanization process. Hydrolysis of silanol groups and facile polymerization often result in multilayers of surface silane, as the silane molecules not only link to the metal surface but also to the neighbor molecules (Figure 2.5). The multiple silane layers might lead to a variation in surface uniformity as well as the density of immobilized molecules.  20  Figure 2. 5 Silane linked to the metal (M) surface and neighbor molecules (Reprinted from [77] with permission from American Chemical Society.)  2.4 Detecting Biomolecules Detecting biomolecules has been a challenge especially when the quantity is low. Specific reactions are often introduced to fluorescently label certain functional groups of the biomolecule for detection. Arginine residue in peptides and proteins is a common choice for labeling, with the most famous technique known as the Sakaguchi reaction [78]. Most of these reactions, though, are still incapable for detecting arginine residues in the level of sub-microgram.  In 1966, Yamada et al first described a chemical reaction for detecting arginine and similar guanidines using 9,10-phenanthrenequinone (PHQ) [79]. The reaction of PHQ with  arginine  under  alkaline  condition  generates  a  product,  2-amino-1H-phenanthro-(9,10-d)-imidazole, which emits strong fluorescence upon excitation at ultraviolet wavelength. The exact reaction was later described by Magun et al [80] as follows:  21  The fluorescent emission from the product is stronger in acidic condition than in alkaline condition. Two excitation peaks were observed respectively at 318 nm and 365 nm. Comparing with other arginine-specific reactions such as Sakaguchi reaction, this approach provides a stronger fluorescent emission for the same quantity of arginine residues, thus turns out to be much more sensitive for detecting microgram amount of arginine residues. Yamada et al also proved this method to be adoptable for detecting arginine-containing peptides, and the product stable in 1M HCl solution for at least one day. However, a non-linear relationship was observed between the arginine concentration and the fluorescent emission, explained as a result of unreacted PHQ at lower arginine concentrations.  Smith et al [81]modified this method by retaining a relatively small excess of PHQ and a small slit width of the spectrometer. Additionally by utilizing a different excitation wavelength, a linear relationship was established between the arginine concentration and the fluorescent emission. The linear relationship was also observed with various arginine-containing proteins and peptides after prolonged reaction at higher temperature.  22  This provides a sensitive and quantitative determination of arginine not only in free solution but also in intact biomolecules.  2.5 Summary The challenge for treating prosthetic-related infections has raised the demand for a novel antimicrobial agent in infection prophylaxis and management. Recent development by Hilpert et al on a family of highly effective AMPs had indentified potential candidates for the design of an infection-free surface. The most promising peptides in this family are able to show almost complete inhibition against P. aeruginosa even when tethered to a cellulose sheet [38]. These progresses are encouraging and motivated the current study of developing a peptide-immobilized titanium surface with antimicrobial activity. As far as we know, there have been limited studies on AMPs covalently immobilized on metal surfaces and their activity. This study aims at developing a novel titanium surface tethered with a highly active AMP.  23  Chapter 3 Scope and Objectives This study is part of a collaborating project involving the Department of Orthopaedics, the Center for Hip Health and Mobility, the Centre for Blood Research, the Centre for Microbial Diseases & Immunity Research, Urology, and the Biomaterials lab at the Development of Materials Engineering. While the scope of the whole project is to develop an effective anti-infection implant surface through different delivery methods of highly active antimicrobial peptides [82], this particular study focused on the covalent immobilization routine of the proposed AMPs on the titanium surface.  With the exciting progress on the screening of novel AMPs in Dr. Hancock’s Microbiology Lab at the University of British Columbia, a group of short yet effective candidate AMPs were developed as for clinical applications [4,38,83]. This study selected one peptide from the most effective class as the surface delivered substance. The specific selection of a novel AMP over the traditional antibiotics for the whole project is to eliminate the possibility of encountering resistant bacterial mutant, the so-called ―superbugs‖.  The covalent immobilization of biomolecules through a maleimide linker is a mature technique that has been described in textbooks and massively used in biomaterial researches [49,70,84]. The final efficiency of the product, however, is largely dependent 24  on the characteristics of the linker as well as the particular biomolecule candidate. This study is the first test on the immobilization and characterization of the novel short AMP Tet213 on metal surfaces. As the antimicrobial activity of the AMPs is a quantity- and mobility-dependent property, both the surface density of the covalently immobilized AMP and the antimicrobial activity need to be determined in this study.  The specific objectives of this thesis are: 1. To confirm the feasibility of the proposed immobilization technique. 2. To immobilize the proposed AMP Tet213 onto titanium surfaces through a short linker. 3. To quantitatively determine the surface density of the immobilized AMP. 4. To confirm the antimicrobial activity of the AMP-modified titanium surfaces. 5. To study some of the parameters that would have effects on the antimicrobial activity of the immobilized AMP, including the delivery method (covalent coupling versus physical adsorption), the peptide candidate, the dilution of the bacterial culture and the bacterial incubation time.  25  Chapter 4 Materials and Methods 4.1 Antimicrobial Peptide Candidates A family of short antimicrobial peptides (AMP) was provided by R.E.W. Hancock’s Microbiology Laboratory at the University of British Columbia. A previous study discovered a highly active AMP Bac2A with the amino acid sequence of RLARIVVIRVAR-NH2 [85], which is a 12-mer variant of the natural AMP bactenecin (RLCRIVVIRVCR). Based on the sequence of Bac2A, a complete substitution with each amino acid was performed by Dr. Hancock’s lab to generate new AMPs. Some shorter variants were also synthesized based on the activity assumption. The bactericidal activity of these screened peptides was evaluated by testing their Minimum Inhibitory Concentration (MIC) against a Pseudomonas aeruginosa (P. aeruginosa) strain [4].  Among all the variants, a peptide with the highest bactericidal activity both in solution and when tethered to a cellulose substrate was chosen as the candidate for this study. This peptide is named as Tet213, with the sequence being KRWWKWWRR-NH2. The Minimum Inhibitory Concentration (MIC) of Tet213 in solution was determined to be 0.7 µg/ml against P. aeruginosa. Bactericidal activity of tethered Tet213 was assessed at the surface density of either 50 nmol/spot or 200 nmol/spot, with the average spot size of 0.3 cm2. Inhibition rates against P. aeruginosa were determined to be 92±8% and 94  26  ±3%, respectively [38].  In this study, an original variant of Bac2A was chosen as a control peptide in antimicrobial experiments. The variant Bac020 (RRAAVVLIVIRR-NH2) has a moderate antibacterial ability compared with Tet213. The MIC of Bac020 was determined to be 11μg/ml against P. aeruginosa, while the tethered inhibition rates at 50 nmol/spot and 200 nmol/spot were 39±15% and 45±9%, respectively [38].  Both Tet213 and Bac020 used in this research were synthesized with a single cysteine at the C-termini, with the sequences being KRWWKWWRR-C (Tet213-cysteine, MW 1590.9, GenScript Corp.) and RRAAVVLIVIRR-C (Bac020-cysteine, MW 1524.9, GenScript Corp.),respectively. The linkage of a cysteine to the peptide is essential for the grafting strategy described later in Chapter 4.2.2. The thiol side chain of cysteine reacts specifically with a bifunctional linker, creating a covalent bond that links the peptide to the titanium surface.  4.2 Immobilization of Peptides to Titanium Surfaces 4.2.1 Titanium Surface Cleaning and Etching 1cm×1cm square samples were cut from a 0.20 mm thick commercially pure titanium foil (Goodfellow Inc, USA). Organic contaminant on the metal surface was removed by washing in chromic acid (20 g K2Cr2O7 in 200 ml concentrated H2SO4, both 27  from Fisher Scientific) for 5 min, followed by thorough washing with water. Chromic acid is an organic-removing agent that has been widely used in chemistry laboratories to clean glassware and instruments.  Cleaned titanium plates were pre-pickled in 2% hydrofluoric acid (HF, Fisher Scientific) for 10 min and washed with water and distilled water. The plates were then etched for 1 hr in a dual-acid mixture, which contained 25% hydrochloride acid (HCl, Fisher Scientific) and 25% sulfuric acid (H2SO4, Fisher Scientific). After the dual-acid etching, plates were taken out, washed extensively with water and rinsed with distilled water.  The etching steps were expected to result in an enlarged surface area as well as to create a rough surface morphology which is generally agreed to be essential for osteoblast attachment. Surface morphology of the titanium plates was examined using a Scanning Electron Microscope (Hitachi-S3000N SEM, Hitachi Scientific Instruments, Tokyo, Japan). The electron beam energy was set at 20 kV and the working distance was set to 15 mm.  4.2.2 Cysteine and Cysteine-Linked Peptide Immobilization Figure 4.1 schematically shows the immobilization process on titanium surfaces, adapted from [70]. To confirm the feasibility of the method, a single cysteine was 28  immobilized through the proposed routine as a preliminary test. The grafting technique was then applied to the cysteine-linked peptide candidates in order to achieve an antimicrobial titanium surface.  + Step 1: Silanization  + Step 2: Bifunctional Linker Coupling  Cysteine  Cysteine-Peptide  Step 3: Cysteine/Cysteine-Linked Peptide Coupling Figure 4. 1 Immobilization of cysteine/cysteine-linked peptide via covalent coupling (titanium surface roughness not shown).  29  Etched titanium plates were first silanized to convert the surface hydroxyl groups into amino groups (Step 1 in Figure 4.1). Samples were immersed in 3% 3-aminopropyl triethoxysilane (APTES, Sigma-Aldrich) solution in acetone for 1 hr, then washed with acetone (5 times) and cured in 80 ℃ oven for 30 min. The attachment of the heterobifunctional linker was accomplished by treating silanized titanium plates with an acetonitrile  solution  containing  6  mg/ml  3-maleimidopropionic  acid  N-hydroxysuccinimide ester (SMP, Obiter Research) for 1.5 hr (Step 2 in Figure 4.1).  After a thorough wash with acetonitrile and water (5 times each), the samples were immersed in a solution of the target biomolecules (cysteine or cysteine-linked peptide) to allow covalent coupling of the cysteine to SMP (Step 3 in Figure 4.1). Cysteine solution was prepared at the concentration of 2 mM, while peptide was dissolved in phosphate buffer (sodium phosphate, monobasic, pH 7.0, Fisher Scientific) at the concentration of 1mg/ml. The incubation was left in room temperature for 2 hr before the plates were taken out, washed thoroughly with water (at least 10 times) and rinsed extensively with distilled water.  4.2.3 Design of Experimental Groups 4.2.3.1 Cysteine Groups In the preliminary test with cysteine, five experimental groups were prepared. Three control groups were designed which represented each stage of the immobilization steps 30  shown in Figure 4.2. The presence of the control groups was to confirm that the linking strategy would not interfere with the fluorescent detection of cysteine, as discussed later in Chapter 4.3.1.  Group 4 was the covalent-coupling group prepared following the proposed routine as described above. A parallel Group 5 was made by simply soaking the bare titanium plates (cleaned and etched) in 2 mM cysteine solution for 2 hr. This group was neither silanized nor grafted with any linkers, and only physical adsorption was expected on the surface.  The design of experimental groups is summarized in Table 4.1:  Table 4. 1 Design of experimental groups in cysteine immobilization. Experimental Groups  Design  Group 1  Bare Ti (cleaned and etched)  Group 2  Silanized Ti  Group 3  Silanized Ti modified with SMP linker  Group 4  Silanized Ti modified with SMP linker Bare Ti (cleaned and etched) soaked in cysteine solution  Group 5 (physical adsorption)  31  Group 1  Group 2  Group 3  Group 4  Group 5  Figure 4. 2 Schematic surface chemistry of the designed cysteine groups (titanium surface roughness not shown).  4.2.3.2 Peptide Groups 4.2.3.2.1 Surface  Density  Determination  and  Antimicrobial  Experiments A preliminary experiment was performed to test both silanized and SMP-modified Ti using the fluorescent method described later in Chapter 4.3.2. The silanization and linker-modification were proved to have no interference with the peptide detection. Hereafter, only bare titanium was used in the peptide experiments as a control group.  Titanium plates covalently linked to the peptide through the proposed routine were named as the Linked group. A group of untreated titanium plates were immersed in 1 mg/ml peptide solution in phosphate buffer for 2 hr to make the Soaked group. This 32  comparative group showed the physically adsorbed proportion of the cationic peptides on titanium surfaces. Bare Ti group of cleaned and etched titanium plates served as the control, which were not treated by the succeeding silanization or the grafting procedure. This group was not supposed to exhibit any peptide-related characteristics.  The three groups (Linked, Soaked and Bare Ti) described above were used in experiments for surface peptide density and antimicrobial evaluation. The calculated amount of immobilized peptides as well as the antimicrobial effect was compared among the three groups by ANOVA test and t-tests between every two groups.  4.2.3.2.2 Peptide Adsorption with Different Surface Morphologies In order to determine whether the etching steps have any effect on peptide adsorption, an extra group entitled Cleaned group was designed besides the three groups described above. Titanium plates were cleaned with chromic acid but not etched using either 2% HF or dual-acid before being soaked in 1 mg/ml peptide solution, while the Soaked group samples were both cleaned and etched. The adsorption levels of peptide on the two different titanium surfaces were evaluated using the fluorescent method described in Chapter 4.3.2.  In summary, all the peptide groups in this research are described in Table 4.2:  33  Table 4. 2 Design of experimental groups in peptide immobilization. Experimental Groups  Design  Bare Ti group  Cleaned and etched Ti  Linked group  Cleaned and etched Ti; covalently linked with the peptide  Soaked group  Cleaned and etched Ti; soaked in peptide solution  Cleaned group  Cleaned Ti; soaked in peptide solution  Bare Ti Group  Linked Group  Soaked Group  Cleaned Group  Figure 4. 3 Schematic surface morphology and simplified structure of the designed peptide groups.  4.3 Determination of the Surface Density of the Immobilized Cysteine and Peptides 4.3.1 Determination of Immobilized Dansyl-tagged Cysteine Cysteine with a fluorescent dansyl tag attached to the primary amine was purchased from Research Plus Inc. The molecular structure of the tagged cysteine is shown in  34  Figure 4.4.  Figure 4. 4 Molecular structure of the dansyl-tagged cysteine [71]  After immobilization, the attached surface molecules were released into solution for fluorescent measurement. All the titanium samples were treated with 3 ml of 0.01 M NaOH for 3 hr. The silane bond between the titanium surface and the SMP linker was cleaved by the alkali, and the attached molecules were released into the solution. The solution containing the cleaved molecules was subsequently transferred into a quartz cuvette and the fluorescence was measured by a LS-50B Luminescence Spectrometer (Perkin Elmer Ltd, Buckinghamshire, UK).  Fluorescent excitation and emission spectra from a standard tagged cysteine solution were recorded, according to which the characteristic wavelengths were determined. A series of dansyl-cysteine solution with various concentrations were prepared and a calibration curve was plotted from the peak emission intensity at each concentration. The quantity of cysteine from titanium samples was calculated by comparing the measured emission intensity with the calibration curve.  35  4.3.2 Determination of Immobilized Peptide Density Using a Sensitive Fluorescent Method The surface density of peptide immobilized via a covalent linker is generally reported to be too low for most direct detecting methods, such as High Performance Liquid Chromatography (HPLC).  In this research, a sensitive fluorescent reaction using 9,10-phenanthrenequinone (PHQ) was adopted to allow a quantitative and direct estimation of the immobilized peptide quantity [81]. The reaction between PHQ and arginine or arginine residue forms a stable compound which gives out fluorescence upon excitation [79]. This measurement has been reported to be sensitive for the quantitative detection of arginine residues at microgram level.  Figure 4. 5 PHQ reacts with arginine and arginine residues, generating a stable fluorescent compound [80].  The method was validated with arginine solution first, then with solutions of arginine-containing biomolecules including Tet213, Bac020 and bovine serum albumin 36  (BSA). Tet213-titanium samples from the three experimental groups were immersed in 1 ml of 0.01 M NaOH for 3 hr to release the surface molecules into solution for the test.  1  ml  of  each  sample  solution  was  added  to  3  ml  of  3.5  μM  9,10-phenanthrenequinone (Sigma-Aldrich) in absolute ethanol. 0.5 ml of 2 M NaOH was added simultaneously to adjust the reaction pH. The mixture was then incubated in 30 C water bath for 3 hr, before 2.25 ml of 2.4 M HCl was added to stop the reaction. The fluorescence emission from the mixed solution was then measured using the LS-50B Luminescence Spectrometer.  Standard solutions with known amount of free Tet213 were tested together with the Tet213-titanium samples to provide a calibration curve by which the quantity of Tet213 contained in the sample solution can be calculated.  4.4 Antimicrobial Experiments 4.4.1 Luminescence Inhibition against Pseudomonas aeruginosa strain Antibacterial tests in this research were performed in Dr. Hancock’s Microbiology Laboratory at UBC. Most of the following experiments were done with the help of Dr. Jason Kindrachuk.  A novel antibacterial assay was developed in the Hancock Laboratory for fast yet 37  sensitive screening. This assay is based on a P. aeruginosa PAO1 strain H1001, which was isolated in the Laboratory and has a luciferase gene cassette incorporated in the bacterial chromosome. The lux strain constitutively expresses luminescence as long as ATP is provided by the bacterium. When the bacterial activity is inhibited or the bacterium is killed, the ATP level decreases, leading to a decrease in the luminescence emission from the bacteria culture. The change in luminescence emission can be monitored as a direct indicator of the bacterial activity to evaluate the antibacterial effect.  For each antibacterial experiment, lux strain was previously grown in rich media over night. The bacterial culture was diluted before the experiment with fresh Basal Medium 2 (BM2) to an optical density of 0.35 at the wavelength of 600 nm (OD 600). BM2 is a bacterial culture medium that provides only minimal nutrient for the microbe to survive, the chemical composition of which is shown in Table 4.1. The bacteria concentration corresponding to an OD600 = 0.35 is reported to be approximately 7×106 colony forming unit (CFU) per ml. This starting bacterial culture was further diluted to different concentrations as required in the following experiment designs.  38  Table 4. 3 Components of Basal Media 2 (BM2) bacterial culture. For making 1× BM2  For making 10  (ml)  × BM2  10× BM2 salts  25  (NH4)2SO4  9.25 g  H2 O  225  K2HPO4  69.7 g  36% glucose  2.75  KH2PO4  29.9 g  20 mM MgSO4  0.25  H2 O  1L  10 mM FeSO4  0.25  The Linked, Soaked and Bare Ti sample plates were placed each in a well in the 24-well microtiter plate. The plates were then sterilized with 300 µl of 70% ethanol each. Ethanol solution was removed after every 5 min and the sterilization was repeated for 5 times. Titanium plates were then rinsed for another 5 times using 300 µl of BM2 media.  200 µl of the lux strain culture with a designed concentration was added to each well that contained a titanium sample. The microtiter plate was then placed on a shaker at the speed of 190 rounds per minute to provide a homogenous liquid environment for the interaction. Incubation was done in a 37 ℃ room for 4 hr. The bacterial culture was then transferred to a 96-well TECAN® plate and the luminescence emission was examined by a TECAN® Spectrafluor Plus spectrometer (TECAN U.S., Inc.).  A decrease in the luminescence reading is directly related to the inhibition of the 39  bacteria. The inhibition rate was calculated by comparing the luminescence after incubation from the peptide-modified samples with that from the Bare Ti control. An equation below showed the relationship between the parameters: rS   (I C  I S )  100% IC  Where rS is the luminescence inhibition rate of the peptide-modified group (Linked or Soaked), IC is the average luminescent reading from Bare Ti controls, and I S is the average luminescent reading within each peptide-modified group.  4.4.2 Bacterial Colony Count To confirm the bactericidal data, the luminescence inhibition test was followed by a successive colony count measurement. After the luminescence reading was recorded, the bacterial culture was transferred to a new microtiter plate and serially diluted with BM2 medium. 200 μl of the culture at each dilution was plated in triplicate on a Mueller Hinton agar plate and incubated in 37 ℃, allowing individual surviving microbe to form a bacterial colony. The number of CFU at each dilution rate was counted after 24 hr, and the average CFU per ml from all the countable dilutions was calculated. Inhibition rates were estimated using the equation below: rS   (NC  N S )  100% NC  Where rS is the luminescence inhibition rate of the peptide-modified group (Linked or Soaked), NC is the average CFU/ml from Bare Ti controls, and N S is the average CFU/ml 40  within each peptide-modified group.  4.4.3 Design of Antimicrobial Experiments The antimicrobial activity of Tet213-Ti samples were tested in three bactericidal experiments with individual sample batches. These experiments were designed to address different questions, as briefly listed in Table 4.4. Details of each experiment are described in the following sections.  Table 4. 4 Design of antimicrobial experiments. Parameters to study Effect of the selection of AMP candidate; effect of the dilution Experiment 1 rate of the initial bacterial culture. Statistical difference of the antimicrobial activity between Experiment 2 Linked group and Soaked group. Experiment 3  Effect of the bacterial incubation time.  4.4.3.1 Experiment 1: Selection of AMP candidate and dilution of bacterial culture Titanium surfaces were modified with one of the two antimicrobial candidates, Tet213 or the control peptide Bac020, respectively. Peptides were attached to the surfaces either through proposed covalent-linking method or by simple soaking. Bare titanium  41  plates served as a negative control. The five experimental groups were marked respectively as Tet213-Linked, Tet213-Soaked, Bac020-Linked, Bac020-Soaked and Bare Ti groups, with three samples in each group.  The initial lux culture with an OD600 = 0.35 was diluted serially by 12.5, 25 and 50 times. The three samples in each titanium group were incubated with one of the three dilutions individually. After 4 hr’s incubation, the luminescence was examined under the TECAN® fluorescence spectrometer. Inhibition rates were compared among the five groups as well as under different bacterial dilutions. Colony counts were measured subsequently for Ter213-Linked, Tet213-Soaked and Bare Ti groups under 1 in 50 dilution rate.  Tet213 groups and the Bare Ti control were brought to a second-round incubation after thorough washes. Samples from the three experimental groups were incubated with either 150- or 500-times dilution of the initial lux culture (OD600 = 0.35) for 4 hr. Luminescence emission as well as colony counts was measured afterwards. A third-round experiment was repeated on the same samples using a dilution of 1 in 200.  Before each repeating experiment, the samples were sterilized with 70% ethanol and rinsed with BM2 according to the steps described in Chapter 4.4.1.  42  4.4.3.2 Experiment 2: Difference between Linked and Soaked groups This experiment was designed to compare the antibacterial effect of the peptide-modified titanium surfaces at a higher confidence level. The Linked, Soaked and Bare Ti groups were prepared with 5 samples in each group employing Tet213 as the candidate. 200 µl of 200-times diluted lux culture was incubated on every sample at 37 ℃. The luminescence emission after 4 hr’s incubation was read by the TECAN spectrometer and the inhibition rate was calculated.  4.4.3.3 Experiment 3: Incubation time A batch with six titanium plates per group (Linked, Soaked and Bare Ti) was processed with Tet213 as the candidate. The six samples were divided into two equal units for two comparative experiments. The physical loading of attached Tet213 was estimated for three of the samples per group after a reaction with PHQ, while the other three were tested for their corresponding antibacterial activity. Lux strain was cultured on the samples using a 1-in-200 dilution for 4 hr. After the luminescence reading was recorded, the incubation time was further extended to 24 hr and the luminescence was examined again. Results of the surface density and the actual antibacterial activity of the immobilized Tet213 were compared with each other for Linked and Soaked groups.  43  4.5 Statistical Analysis Difference among experimental groups was analyzed using one way ANOVA test and two-tailed t-test. ANOVA test was utilized to compare the average and standard deviation of all the experimental groups, which determined whether any statistical difference among the groups exists. If a statistical difference was suggested, Holm t-tests were then performed between every two groups to identify if these two groups differed from each other. In this research, a confidence level of 0.05 was adopted. A P value of <0.05 indicated difference with a statistical significance.  44  Chapter 5 Results 5.1 Titanium Surface Morphologies It has been generally accepted that rough surfaces can enhance the attachment of osteoblast cells to the implants, which leads to a better in vivo fixation of the implant after orthopedic surgeries. Rough surfaces would also result in a larger surface area and may lead to a higher degree of peptide immobilization. In this research, all the titanium substrates were cleaned and etched by a two-step acid etching method to generate a rough surface morphology.  After being cleaned with chromic acid, titanium plates were first etched by 2% HF and then by a dual-acid mixture (HCl/H2SO4). The etched plate showed a rough and grayish appearance, while the non-etched titanium surface was smooth and shiny (Figure 5.1).  Figure 5. 1 Left: titanium plate, cleaned; Right: titanium plate, cleaned and etched. 45  SEM images showed that 2% HF etching removed metal on the surface layer, leaving a rough morphology from which the grain shape can be seen (Figure 5.2 b). Dual-acid etching created sub-micron dimples on the existing rough morphology and further increased the surface area (Figure 5.2.c). A significant change in roughness can be seen by comparing the SEM images of the non-etched titanium surface with the surface etched by this two-step etching method (Figure 5.2 a and c).  (a)  (b)  (c) Figure 5. 2 Scanning electron micrographs of the titanium surfaces. (a): cleaned only, (b): cleaned and etched by 2% HF, and (c): cleaned and etched by 2% HF, followed by the dual-acid etching.  46  5.2 Dansyl-tagged Cysteine Immobilization 5.2.1  Fluorescent Analysis of Dansyl-Tagged Cysteine in Solution  The excitation and emission wavelengths of dansyl-tagged cysteine were examined using a standard (0.5 µg/ml) dansyl-cysteine solution. The LS-50B luminescence spectrometer scanned the standard solution and recorded the intensity spectra in the range of both excitation and emission wavelengths (Figure 5.3). The wavelength at which peak intensity was recorded was defined as the characteristic wavelength. The characteristic excitation and emission wavelengths were found to be 320nm and 480nm, respectively. This was confirmed by repeating the tests with different concentrations of cysteine solution. While the peak intensity varies with the concentration, the characteristic excitation and emission wavelengths were found to be consistent (Figure 5.4).  Figure 5. 3 Fluorescent excitation and emission spectra of dansyl-tagged cysteine in solution (0.5 µg/ml).  47  (a)  (b)  Figure 5. 4 (a) Emission spectra of tagged cysteine solutions at different concentrations; (b) Calibration curve drawn from peak intensities in (a).  At the excitation wavelength of 320nm, the emission intensity at 480nm increased with the cysteine concentration (Figure 5.4 a). A linear regression was established between these two parameters with an r=0.9993 (Figure 5.4 b). The relationship was determined as follow: y = 1201.9x – 5 Where y is the peak emission intensity (relative unit), and x is the concentration of the tagged cysteine in solution (µg/ml). The intercept in this equation came from the reading at zero point of the spectrometer (-5).  This calibration curve was used in the following tests to calculate the amount of immobilized tagged-cysteine. 48  5.2.2 Cysteine Immobilized on Titanium Surfaces The five cysteine groups were treated with 3 ml of 1 M NaOH each before the fluorescence examination. The alkali solutions containing the cleaved molecules were then examined using the LS-50B luminescence spectrometer. The peak emission intensity was recorded under the excitation at 320nm. The corresponding amount of tagged cysteine in solution was calculated using the intensity-concentration equation established from the calibration curve.  Figure 5.5 shows the amount of attached cysteine for all the five groups. The three control groups that were not treated with cysteine did not show any detectable emission intensities, suggesting the coupling strategy does not have any interference with the fluorescence detection. An average amount of 0.31±0.16 μg/cm2 (0.58±0.29 nmol/cm2) cysteine, which equals approximately three molecules on each square nanometer, was coupled to the sample surface through the proposed routine (Group 4: Silanized Ti grafted with tagged cysteine through the SMP linker). Group 5 (Bare titanium samples soaked in cysteine solution) adsorbed 0.07±0.02 μg/cm2 (0.12±0.04 nmol/cm2) cysteine on the surface (Table 5.1).  49  Figure 5. 5 Estimated amount of the dansyl-tagged cysteine on titanium samples after alkali cleavage. Group 1: Bare Ti; Group 2: Silanized Ti; Group 3: Silanized Ti with SMP linker; Group 4: Silanized Ti grafted with tagged cysteine through the SMP linker; Group 5: Bare Ti soaked in cysteine solution (average of 5 samples).  50  Table 5. 1 The amount of dansyl-tagged cysteine estimated from fluorescent intensities of each group. Group 1: Bare Ti; Group 2: Silanized Ti; Group 3: Silanized Ti with SMP linker; Group 4: Silanized Ti grafted with tagged cysteine through the SMP linker; Group 5: Bare Ti soaked in cysteine solution (average of 5 samples). Quantity of  Molar quantity of  Number of cysteine  cysteine (μg/cm2)  cysteine (nmol/cm2)  molecules (molecule/nm2)  Group 1  0.00±0.00  0.00±0.00  0.0±0.0  Group 2  0.00±0.00  0.00±0.00  0.0±0.0  Group 3  0.00±0.00  0.00±0.00  0.0±0.0  Group 4  0.31±0.16  0.58±0.29  3.5±1.8  Group 5  0.07±0.02  0.12±0.04  0.8±0.2  P value from ANOVA test (Appendix – Table 1) of the five groups was calculated to be 2.42×10-6, suggesting a statistical difference among the groups at 0.05 level. To determine which of the five groups are different from each other, post hoc t-tests were performed for multiple comparisons. P values of Holm t-test between every two groups were listed in Table 5.2. By comparing with the critical p value, the amount of cysteine from Group 4 was confirmed to be statistically different from Group 5 and all the control groups, which indicated the existence of tagged cysteine on the chemically treated surfaces. Although Group 5 had a noticeable quantity of attached cysteine, the Holm t-test result showed that this soaked group was not statistically different from the bare Ti 51  and the other control groups, suggesting that physical adsorption was not an important factor that would affect the total amount of immobilized cysteine.  Table 5. 2 P values of Holm t-test between every two groups. Group 1: Bare Ti; Group 2: Silanized Ti; Group 3: Silanized Ti with SMP linker; Group 4: Silanized Ti grafted with tagged cysteine through the SMP linker; Group 5: Bare Ti soaked in cysteine solution. (Average of 5 samples.) Group 1  Group 2  Group 3  Group 4  Group 5  Group 1  -  0.987  0.984  0.000  0.166  Group 2  -  -  0.997  0.000  0.161  Group 3  -  -  0.000  0.160  Group 4  -  -  0.000  The results suggested that the single amino acid cysteine was successfully grafted onto titanium surfaces through the proposed method (Group 4). Adsorption of the molecule was also observed when the titanium plates were simply soaked in cysteine solution (Group 5). The quantity of adsorbed cysteine was statistically less than that of the covalently grafted one.  52  5.2.3 Summary The successful coupling of dansyl-tagged cysteine through proposed routine was confirmed by the fluorescence emission of the dansyl tag. Estimation of the quantity of cysteine attached to the surface was accomplished by comparing the peak emission intensities with the calibration curve of standard cysteine solutions. A significant amount of cysteine was covalently grafted to titanium surface through the SMP linker, while physical adsorption was not a major contribution as observed from the comparative group. No fluorescent emission was observed for the control groups. This confirmed the viability of the grafting methodology, which was then used to immobilize the cysteine-linked antimicrobial peptides to titanium surfaces.  5.3 Peptides Immobilization 5.3.1  Detection of Arginine and Arginine-Containing Biomolecules  5.3.1.1 Fluorescent Detection of Single Arginine The fluorescent characteristic of the compound after the arginine-PHQ (9,10-phenanthrenequinone) reaction was determined by the LS-50B luminescence spectrometer. A standard arginine solution with the concentration of 3 µg/ml was treated following the steps described in Chapter 4.3.2. After the reaction, the characteristic wavelengths were determined from the excitation and emission spectra.  The highest emission intensity was found at the wavelength of 385nm. Several 53  excitation peaks were observed in the excitation spectrum, among which the highest one was chosen for the maximum emission output. The excitation wavelength in the following experiments was set according to this peak at 260nm. Free arginine after reacting with PHQ formed a compound, of which the fluorescent intensity was found to be a linear function of the initial arginine concentration. This method is so sensitive that the linear relationship can be found with very low arginine concentrations in the range of sub-microgram per milliliter (Figure 5.7).  Figure 5. 6 Fluorescent excitation and emission spectra of arginine in solution (3 µg/ml) after reacting with PHQ.  54  Figure 5. 7 Calibration curve of arginine in solution (r2 = 0.992).  It was observed that the fluorescent intensity of a certain concentration as well as the calibration curve was dependent on the environment and the instrumental setting. Relationship of intensity and concentration from tests on different days differed slightly, as shown in Figure 5.8. The luminescence spectrometer was set to minimize the background noise and to maximize the signal intensity for different assays, and a calibration curve was prepared each time with the samples.  55  Figure 5. 8 Arginine calibration curves tested on different days.  5.3.1.2 Fluorescent Detection of Tet213 and BSA Arginine-containing peptides and proteins required a longer reaction time than pure arginine, possibly because of the complex structure of the big molecules. It was also found that a low concentration of PHQ is essential for stable outputs, as the resultant fluorescence is likely to be interfered by the background noise from the excessive PHQ.  Same characteristic wavelengths were observed for single arginine and arginine-containing biomolecules after the reaction. Figure 5.9 shows the comparison among the emission spectra of arginine, Tet213 and BSA (bovine serum albumin).  56  Figure 5. 9 Comparison of fluorescent emission spectra of single arginine, Tet213 and BSA. The concentration of arginine, Tet213 and BSA solution is 0.3μg/ml, 2μg/ml and 15μg/ml, respectively.  The relationship between the fluorescent intensity and Tet213 concentration was less linear than that observed for the single amino acid arginine. However, the calibration curve can still be considered as linear over a short range of concentrations, especially under 2μg/ml, with an r = 0.995. This is proved in the later chapter as the concentration range in which the titanium samples fell in. The fluorescent intensity of BSA increased in a more exponential way as a function of the concentration.  57  Figure 5. 10 Calibration curves of Tet213 (linear, r2 = 0.990) and BSA in solution (first order exponential, r2 = 0.986).  Similar to what was observed with arginine, the emitted fluorescence from reacted Tet213 also depended on the environmental condition and instrumental setting. The effect of spectrometer slit width, as an example, can be seen from Figure 5.11. A narrow 58  slit helps in cutting down background noise but results in reduced signal level as well. It has been reported in the literature that limiting slit width can also lead to a more linear relationship between fluorescence and concentration. Day-to-day tests were found to vary slightly. As a result, a calibration series was prepared each time together with the samples to give a more accurate estimation on the peptide quantities.  Figure 5. 11 Effect of spectrometer slit width on Tet213 calibration curves.  5.3.2 Peptides Immobilized on Titanium Surfaces 5.3.2.1 Immobilization of Tet213 The fluorescent emission spectra of Bare Ti, Linked Group and Soaked Group were recorded after alkali treatment and reaction with PHQ. All the groups showed highest emission intensity at the same characteristic wavelength as that of single arginine and Tet213. A typical comparison of the emission spectra of Bare Ti Group, Linked Group, 59  Soaked Group and a standard Tet213 solution is shown in Figure 5.12. By comparing the fluorescent intensity with the standard calibration curve, the quantity of Tet213 from each group was determined.  Figure 5. 12 Comparison of fluorescent emission spectra of Bare Ti Group, Soaked Group, Linked Group and Tet213 solution.  Figure 5.13 showed the calculated quantity of Tet213 on titanium surfaces. The estimated surface density of Tet213 grafted through the proposed routine was 1.30±0.55μg/cm2 (0.82±0.34 nmol/cm2). A relatively larger amount of Tet213 was adsorbed onto titanium surfaces, comparing with the single cysteine. The average surface density of adsorbed Tet213 was calculated to be 0.74±0.20 μg/cm2 (0.46±0.13 nmol/cm2).  60  Meanwhile, bare titanium substrates only showed signal at the level of the background noise. The Tet213 value from Bare Ti group plotted in Figure 5.13 is merely due to the calibration error when the curve approaches the origin. Table 5.3 listed the estimated surface density of immobilized Tet213 on Bare Ti, Linked and Soaked Groups. The silanizing and linking strategy did not interfere with the arginine detecting method (data not shown).  Figure 5. 13 Comparison of the surface density of immobilized Tet213 on Bare Ti, Soaked Group and Linked Group samples (average of 11 samples).  61  Table 5. 3 The surface density of Tet213 of each group, estimated from the fluorescent intensities. Quantity of Tet213  Molar quantity of  Number of Tet213  (μg/cm2)  Tet213 (nmol/cm2)  molecules (molecule/nm2)  Bare Ti  0.05±0.06  0.03±0.03  0.2±0.2  Soaked  0.59±0.30  0.37±0.19  2.3±1.1  Linked  1.18±0.59  0.74±0.37  4.4±2.2  ANOVA test of the three groups (Appendix Table 2) resulted in a p value of 3.68× 10-6 and confirmed these three groups to be statistically different at 0.05 level. P values from Holm t-test results are listed in Table 5.4. A statistical difference was observed between every two groups. Bare Ti control group significantly differs from both the Linked Group and the Soaked Group. P value between the Soaked and the Linked groups was calculated to be 0.002, indicating a statistical difference between the two groups with the Linked Group being 98% higher in average peptide density.  Table 5. 4 P values of Holm t-test between every two groups of Tet213 sample. Bare Ti  Soaked Group  Linked Group  Bare Ti  -  0.004  0.000  Soaked Group  -  0.002  62  Comparing with single cysteine, surface molecular density of adsorbed Tet213 was higher, suggesting this cationic peptide has a high affinity towards the negatively charged Ti surface even after thorough wash (Figure 5.14). Statistic difference was observed between the molecular densities of the two with a p value = 0.012. The interaction between Tet213 and the titanium surface is most probably electrostatic interactions. This also explains the higher grafting efficiency of Tet213 than cysteine, as the total amount of Tet213 on titanium surface is contributed by the physical adsorption as well. Because both Linked Group and Soaked Group showed a noticeable signal of attached Tet213, these two groups were tested for their antimicrobial activity and the results were compared.  Figure 5. 14 Comparison of surface molecular density of immobilized cysteine and Tet213 groups.  63  5.3.2.2 Tet213 Adsorption on Titanium with Different Surface Morphologies It was noticed from the preceding results that the etched titanium surface can attract more Tet213 through physical adsorption than cysteine or Bac020, the control peptide (39.8% adsorption compared with covalent bonding, average of 3 samples). This might lead to a new and simple delivery method of this specific cationic peptide by simply soaking the implant into the peptide solution.  It was speculated that the cleaning-and-etching treatments of titanium plates might have an impact on the interaction between the metal surface and the cationic peptide. To confirm this speculation, two types of titanium substrates were designed for peptide adsorption. The Soaked Group were treated following the normal steps: cleaned with chromic acid first and then etched successively by 2% HF and the dual-acid mixture. A Cleaned Group was merely cleaned with chromic acid but not etched, leaving a relatively smooth morphology on titanium surface without dimples. These two groups were then soaked in the Tet213 solution. The quantity of adsorbed Tet213 was determined using the fluorescent detecting method. The results were compared with titanium samples grafted with Tet213 (Linked Group) and Bare Ti control.  Linked Group still showed the highest fluorescent intensities, which corresponds to the highest peptide loading. Bare Ti Group exhibited only background level fluorescence. 64  The Cleaned Group and the Soaked Group, however, did not show significant difference in average emission intensities. The average surface density of Tet213 from the Cleaned group and the Soaked Group were calculated to be 0.39±0.07 μg/cm2 (0.25±0.04 nmol/cm2) and 0.31±0.01 μg/cm2 (0.19±0.01 nmol/cm2). T-test confirmed that the difference between the two groups were not statistically significant with a p value of 0.115.  65  (a)  (b)  (c)  Figure 5. 15 (a) Comparison of the surface density of Tet213 on Cleaned Group, Soaked Group, Linked Group and Bare Ti samples. (b) SEM image of titanium surface morphology of Cleaned Group. (c) SEM image of titanium surface morphology of Soaked Group.  66  Opposite to what was speculated, the etching steps did not result in increased peptide adsorption, as comparing the Cleaned Group with the Soaked Group. The sub-micron scale roughness on etched titanium surface as well as the increased surface area did not reveal a significant effect on the peptide adsorption. The interaction is most probably based on electrostatic force between the negatively charged titanium surface layer and the cationic peptide. This is discussed further in Chapter 6.  5.4 Antimicrobial Experiments Three individual antibacterial experiments were performed with different batches of sample respectively. Experiment 1 was designed to examine the effect of the AMP selection and the dilution rate of initial bacterial culture. Experiment 2 compared the difference between the Linked group and the Soaked group, while Experiment 3 mainly studied the effect of the incubation time. The results are discussed below in detail.  5.4.1 Experiment 1 The first antimicrobial test was designed to compare the killing effect of different immobilized peptides, and to find out how dilution of the initial bacterial culture affects the final inhibition rate of the samples.  After 4 hours incubation with the lux strain P. aeruginosa, Bac020 modified samples did not show any noticeable inhibition (Figure 5.16 a). The luminescence readings from 67  the Bac020-Linked and Bac020-Soaked groups under all three dilutions were not noticeably different from that of the Bare Ti control group. Meanwhile, significant decrease in luminescence reading can be seen with Tet213 samples under all three dilutions (Figure 5.16 b). The inhibition rates of both Tet213-Linked group and Tet213-Soaked group increased when the initial bacterial culture was diluted. A maximum inhibitory rate was observed under the 1-in-50 initial dilution, with an inhibition rate for both Linked and Soaked groups to be approximately 50% (Table 5.5). Colony counts of Tet213-Linked and Tet213-Soaked groups as well as Bare Ti group were also conducted. The inhibitory effect confirmed what was observed from luminescence reading. Inhibition rate was calculated based on the colony counts. The inhibition rates for both Linked group and Soaked group were higher than those from the luminescence results (Table 5.6). This difference between lux reading and colony counts was also observed with later experiments.  68  (a)  (b) Figure 5. 16 Comparison of luminescence reading from experimental groups after incubation with different dilution of the lux strain: (a) Bac020 groups. (b) Tet213 groups. 69  Table 5. 5 Luminescent inhibition rates of Tet213 groups under different initial bacteria-culture dilutions. Soaked Group  Linked Group  1 in 50 dilution  51%  41%  1 in 25 dilution  37%  29%  1 in 12.5 dilution  19%  16%  Table 5. 6 Inhibition result of Tet213 groups from colony counts after incubation with 1-in-50 initial bacterial dilution. Number of initial  Number of colonies after  colonies  incubation  Bare Ti  6×106  8×108  -  Soaked  6×106  1.12×108  86%  Linked  6×106  1.6×108  80%  Inhibition  A repeated bacterial incubation with the same Tet213 samples still showed significant inhibitory effects. A second 4-hour incubation led to approximately 60% reduction in luminescence under a higher dilution of the initial bacterial culture. The inhibition rates under two different dilutions are listed in Table 5.7. Colony counts afterwards revealed difference by two orders of magnitude between the two Tet213-modified groups and the Bare Ti control (data not shown). A third incubation with  70  1-in-200 diluted bacterial culture showed 22% inhibition for Tet213-Soaked group and 11% for Tet213-Linked group. Repeated incubation experiments suggest that the Linked and Soaked samples are relatively stable and can exhibit antibacterial activity even after several incubations. However, the antimicrobial effect of both Tet213 modified groups showed a decreasing trend when reiterated tests were performed.  Table 5. 7 Luminescent inhibition rates of Tet213 groups after second-round incubation.  Soaked Group  Linked Group  1 in 150 dilution  58%  57%  1 in 500 dilution  69%  56%  5.4.2 Experiment 2 While the purpose of this experiment was to obtain a statistical conclusion, the inhibition rate largely decreased from the previous test. Under the 1-in-200 diluting rate, only 18% inhibition was observed with the Tet213-Linked group, whereas Soaked group revealed 6% inhibition rate (Figure 5.17). One-way ANOVA test (Appendix Table 3) resulted in p = 0.00004, suggesting statistical difference among the three groups. Although student test did not confirm the difference between the Soaked Group and the Bare Ti control, Holm t-test for multiple comparisons claimed all the three groups to be  71  statistically different (p values listed in Table 5.8), which indicated the inhibition activity of both sample groups. The Linked group showed a higher inhibition rate than the Soaked group, albeit both were lower than the previous experiment.  Table 5. 8 P values of Holm t-test between every two groups on the luminescent inhibition results of Experiment 2. Bare Ti  Soaked Group  Linked Group  Bare Ti  -  0.036  0.000  Soaked Group  -  -  0.000  Figure 5. 17 Luminescent inhibition of the second antibacterial experiment with Tet213 samples (average of 5 samples).  72  5.4.3 Experiment 3 To further explain the inconsistency between Experiment 1 and Experiment 2, a third experiment was designed to confirm both the peptide loading and the antimicrobial effect. A batch of six samples for each experimental group was prepared, among which three were tested after alkali cleavage and the PHQ reaction while the remained were incubated with the lux strain. The incubation was done first for 4 hr and then extended to 24 hr.  Fluorescent emission at 385 nm after the PHQ reaction confirmed the existence of the arginine-containing peptide Tet213 on titanium surfaces. The calculated surface loading of Tet213 from calibration was 0.31±0.01 μg/cm2 and 1.04±0.05 μg/cm2 respectively for Soaked Group and Linked Group, which is comparable with the previous experiment (Figure 5.18 a). Corresponding lux strain culture revealed 26% and 25.9% inhibition rate for Linked and Soaked groups respectively. After 24 hr incubation, the inhibitory rate was increased to 57.9% for Linked group and 53.5% for Soaked group (Figure 5.18 b). Statistical analysis confirmed the difference among the three groups at 4 hr and 24 hr, with p values calculated from ANOVA test (Appendix Table 4) of 0.00033 and 0.00012 respectively. Holm t-test results are listed in Table 5.9.  73  (a)  (b) Figure 5. 18 Comparison of Tet213 surface loading and the antibacterial effect. (a) Estimated surface density of Tet213. (b) Inhibitory rates after 4 hr’s and 24 hr’s incubation.  74  Table 5. 9 P values of Holm t-test between every two groups on the luminescent inhibition results of Experiment 3, after 4 hr and 24 hr incubation, respectively. 4 hr  Bare Ti  Soaked Group  Linked Group  Bare Ti  -  0  0  Soaked Group  -  -  0.972  24 hr  Bare Ti  Soaked Group  Linked Group  Bare Ti  -  0  0  Soaked Group  -  -  0.484  Physical presence of Tet213 on titanium surfaces was confirmed by the fluorescent detection. The lux strain inhibitory rate for both Tet213 modified groups, however, was comparatively lower than that from the first experiment. It was notice that extension of the incubation time can increase the antibacterial effect, which is possibly due to either a slow release of the surface adsorbed peptide or the prolonged exposure of bacteria to the modified titanium surfaces.  5.5 Summary After the immobilization strategy was confirmed with cysteine, the AMP candidate Tet213 was successfully coupled onto titanium surfaces through a bifunctional linker, as confirmed by the fluorescence detection. A significant quantity of physically adsorbed 75  peptide was observed from the Soaked group, indicating the high affinity of the cationic peptide towards the negatively charged metal surface. Antibacterial results against the P. aeruginosa strain from the luminescence inhibition and the colony count confirmed bacterial inhibitory activity for titanium samples both covalently bonded to and physically adsorbed with Tet213. However, a fluctuation of inhibition rates was observed among different experiments.  76  Chapter 6 Discussion 6.1 Tet213 adsorption on Titanium Surfaces Reports have been made on the adsorption on Ti surfaces of various proteins including serum albumin, fibronectin, immunoglobulin, lysozyme and other large biomolecules [44]. Interaction between the metal surface and amino acids has also been studied and several mechanisms have been proposed. It is generally believed that the physical adsorption on titanium surface is mainly contributed by electrostatic interactions. When titanium is in contact with water of neutral pH, the surface TiO2 layer possesses a slightly negative charge, which is explained by the reaction as below:  Ti - OH  H 2 O  [Ti - O]-  H 3 O   Cysteine, with pI = 5.05, possesses a slightly negative charge in solution of a neutral pH. The electrostatic interaction of cysteine towards the negatively charged titanium surface is therefore expected to be weak under the experimental settings. On the other side, Tet213 which has a sequence of KRWWKWWRR contains a large proportion of arginine (R) and lysine (K), the two cationic amino acids at neutral pH with pI of 10.76 and 9.60 respectively. Stronger electrostatic interaction is expected between the cationic peptide Tet213 and titanium, leading to a larger quantity of surface adsorbed molecules (Figure 5.14).  77  As the adsorption of biomolecules onto titanium surfaces is energetically favorable, the process is considered irreversible in most cases. However, the attached molecules can be replaced by other molecules or ions that have a stronger binding force towards the metal surface.  Chapter 5.3.2.2 discussed the effect of titanium surface morphology on Tet213 adsorption. 2% HF followed by dual-acid etching was proved to result in rough surface morphology and therefore increased surface area in microscale; whereas the increased surface area did not directly lead to an improved peptide adsorption. Considering that the acid etching steps also brought modifications to the surface oxide layer, it can not be concluded the electrostatic interaction is not sensitive to metal surface roughness. However, the above result showed that the acid etching step is experimentally irrelevant with the final outcome of surface adsorbed peptides.  6.2 Surface Density of modified Tet213 on Titanium The average molecular density of Tet213 on titanium surfaces is estimated by the fluorescent emission to be 4.4±2.2 and 2.3±1.1 molecule/nm2 after covalent coupling and physical adsorption, respectively. Statistical analysis confirmed the two groups to be different. Surface hydroxyl group density on pure Ti was reported to be in the range of 9-13 OH per nm2 as contributed by the oxide and chemisorbed water [44,86]. The number is in reasonable agreement with the coupling surface density. 78  It should be noticed that the estimated loading of covalently bound Tet213 is influenced by two factors. APTES-silanization is reported to form a crosslinked polysiloxane layer rather than a single surface film, since APTES can react with both the Ti hydroxyl groups and the siloxane in other APTES molecules. The reproducibility of the siloxane layer on metal surfaces is generally reported to be difficult to control at a very precise scale. Considering the contribution of the multilayer, the density of grafted peptide does not necessarily correspond with the number of surface hydroxyl groups. This leads to the deviation among samples as observed in the fluorescent tests. Another contributing factor is the physically adsorbed portion that added to the total detected quantity. It can be seen by comparison that the molecular densities of both grafted and adsorbed Tet213 were higher than those of single cysteine (Figure 5.4). The cationic molecule Tet213 has higher affinity towards the bare metal surface, which is also a possible contributing factor to the apparent total loading that can be detected from fluorescent emission.  6.3 Antibacterial Activity of Tethered and Adsorbed Tet213 6.3.1 Bacterial Inhibitory Effect and the Fluctuation Based on the lux strain incubation experiments, we can conclude that the Tet213-modified samples of both Linked group and Soaked group demonstrated significant antimicrobial activity. Several factors were found in this study to influence the inhibition rate, including the initial bacterial dilution, the duration of incubation time and 79  the selection of the peptide candidate. It is also speculated that the peptide density/concentration and the flexibility of the linker are essential for the activity of tethered peptide.  Diluted bacterial culture and prolonged incubation time could lead to higher overall inhibition rate. A possible explanation is the improved contact of Ti samples to individual bacterium in the culture media. Different from peptide dissolved in solution, the surface-bound peptides have a limited mobility which restricts their approach to the suspended microbes. Bactericidal process was occurring probably only on or near the substrate surface. Diluted bacterial culture and sufficient incubation time under shaking were thus essential for a more complete killing. The difference between inhibition rates observed by lux inhibition and colony counts could also be explained by this regional difference in killing effect. The apparent luminescence detected by the spectrometer could have been augmented by the emission from the superficial media, while the colony count is a more precise indicator of the elimination of all the bacteria. Nevertheless, this regional difference might be useful as well in clinical applications to prevent the formation of biofilm on the implant surface.  A peptide candidate with a high antimicrobial activity is essential for developing an anti-infectious implant. Although it has been reported that the activity of the tethered peptide does not directly related to its dissolved form, it has also been observed that 80  peptides that are most active in solution are also highly active when tethered [38]. Tet213 is among the most efficient bactericidal peptides that have been developed in Dr. Hancock’s lab, with the MIC of 0.7μg/ml. A comparative candidate Bac020 has the MIC of 11μg/ml which is much higher than that of Tet213. As observed in this study, after incubation with the lux strain, Tet213-modified samples showed significant inhibition, while Bac020-modified Ti plates did not reveal evident killing effect. It suggests that the selection of a highly active peptide candidate is one of the key factors that determine the final antimicrobial activity of the modified implant.  A fluctuation of inhibitory rates was observed among experiments. The first experiment revealed inhibitory rates as high as 51% and 41% respectively for Soaked and Linked groups under 1-in-50 dilution, while the second test resulted in 18% for the Linked group and 6% (not significant) for the Soaked group under 1-in-200 dilution. A comparison of inhibition rates from all the three experiments is shown in Figure 6.1.  81  Figure 6. 1 Comparison of the inhibition rates of Tet213-modified Ti samples after 4 hr’s incubation on different tests.  Besides the influence of peptide density deviation which is possibly caused by the multi-silane layer, another explanation to the fluctuation was also proposed after comparing the effective bactericidal dose and the immobilized quantity of the peptide. Hilpert et al [38] compared the inhibition rates of different antimicrobial peptides. At a fixed peptide density, the standard deviation of the inhibition rates increased when the inhibitory effect decreased. Less effective peptides with an MIC higher than the fixed concentration showed a larger fluctuation in killing rates as compared to those with lower MICs, suggesting a relatively low confidence in the actual inhibition. The stability of the  82  bactericidal results from tethered peptides is concluded to rely on the activity of the peptide as well as the tethered surface density.  Estimated from the fluorescence emission after reacting with PHQ, the surface density of Tet213 from the Linked and Soaked groups were 0.74±0.37 nmol/cm2 and 0.37±0.19 nmol/cm2 respectively. That was approximately 1.5 nmol/sample for the covalently-bound Tet213 and 0.7 nmol/sample for the physically-adsorbed Tet213. A previous trial in Dr. Hancock’s Lab reported 92±8% and 94±3% inhibition rates for Tet213 synthesized on a cellulose sheet at the quantities of 50 nmol/spot and 200 nmol/spot. The amount of Tet213 attached on Ti sample in this study is therefore much less than that of Tet213 directly synthesized on cellulose sheets. It can be speculated that the fluctuation observed in this research is a result of the relatively low surface density through the immobilization routine. To achieve a modified implant with more stable antibacterial activity, it is required that either a peptide candidate with higher bactericidal activity is developed, or a delivering method that can load larger quantity of the peptide is used. However, the current design and processing technique may be still useful, as longer contacting time and lower bacterial concentration in clinical case would probably increase the inhibition rate as well as the antimicrobial confidence of the implants.  83  6.3.2 Antibacterial Effect of Surface-bound and Adsorbed Tet213 It is generally agreed that short linker modification would restrict the mobility of grafted peptide and might compromise the activity of the peptide. Gabriel et al [39] reported grafting a traditional antimicrobial peptide LL-37 onto titanium surfaces. LL-37 is a 37-mer peptide with an MIC against E. coli ranging from 2 to 8 µg/ml in solution [87]. Grafting of the peptide to Ti surface using a direct bifunctional linker did not lead to observable antimicrobial effect, while increasing linker length could result in apparent bacterial inhibition, speculated to be a consequence of improved mobility. Similar phenomena have also been reported in other literature and in recent publications on substrate-bound Bac2A variants.  In this research, similar inhibition rates were observed for the Linked group and the Soaked group although the fluorescent detection suggested that the surface density of Tet213 on Linked samples was higher than that on Soaked samples. It is obvious that the actual killing result of the Linked peptide is compromised by the immobility of the covalent coupling technique. Soaked samples, on the other hand, are expected to release the adsorbed peptide once they are in contact with liquid. The free peptide is much more efficient in eliminating microbes than their tethered analogs, as discussed previously. However, a burst release of the majority of the adsorbed peptide is expected during the first few hours, thus the long-term anti-infectious activity could not be guaranteed. Covalently-bound peptide, although exhibiting compromised activity, is comparatively 84  more stable and lasting on the implant surfaces and should still be the option when designing an anti-infectious implant for clinical applications.  85  Chapter 7 Conclusions A highly effective antimicrobial peptide Tet213 has been successfully attached onto titanium surfaces both through covalent immobilization and by simple physical adsorption. The surface density of the peptide was determined by a sensitive fluorescent detecting method, and the antimicrobial activity of the peptide-attached titanium samples was evaluated after incubation with a Pseudomonas aeruginosa strain. The following conclusions were drawn from this study:  1. The proposed immobilization routine was successfully realized both with single cysteine and the peptide candidate Tet213. The grafting strategy was proved to have no interference with the detection of both biomolecules. Surface density of covalently grafted Tet213-Ti samples was estimated to be approximately 4 molecule/nm2, while the net density of physically adsorbed samples was about 2 molecule/nm2. The densities of these two groups are statistically different from each other.  2. Acid etching using 2% HF and a dual-acid mixture (HCl/H2SO4) resulted in an increased surface roughness; however, the adsorption of Tet213 with a rough morphology was not statistically different from that with a non-etched smooth titanium surface, suggesting that the interaction is not strongly dependent on the substrate roughness at sub-micron level. 86  3. Titanium surfaces covalently bonded to and adsorbed with Tet213 both showed inhibitory activity towards the Pseudomonas aeruginosa strain. Similar inhibition rates were observed for both types of samples despite their difference in surface density. It is speculated that the immobilization through a short linker would restrict the approach of the peptide to the bacterium surface, leading to a compromised antimicrobial activity. However, the covalent coupling strategy was still found to show a relatively more stable antimicrobial activity (Experiment 2).  4. The bacterial inhibition rate fluctuated among different antibacterial experiments, which is probably due to the limited quantity of Tet213 on titanium surfaces. The visible fluctuation indicated a relatively low confidence of bacterial inhibition at a peptide concentration lower than its minimum inhibitory concentration. At the dilution rate of 1 in 200 or lower for an initial bacterial culture with OD 600 = 0.35, an inhibition rate of > 50% can generally be achieved at designed experimental settings.  5. With titanium samples modified through the proposed routine, the final bactericidal result was concluded to depend on several important parameters. Besides the evident influence from the initial bacterial dilution and the incubation time, one of the essential factors in the antimicrobial surface design is the selection of an AMP candidate that is highly active when tethered.  87  Chapter 8 Recommendations for Future Work As established from the achieved results of this study, the following research is recommended for a better anti-infectious surface design utilizing the promising AMPs.  1. Examination of titanium modified through the proposed routine using a smooth substrate to confirm the covalent bonding between the peptide and the substrate surfaces.  2. Refine the silanization and linker-grafting technique for a better peptide density. Modify the surface using a flexible long linker instead of the rigid short linker to study the effect of tethered peptide mobility on the antimicrobial activity.  3. In vitro releasing experiments of peptide-modified samples for determination of the releasing rate of attached biomolecules.  4. Cytocompatibility tests and antimicrobial experiments against other clinically relevant pathogens.  88  REFERENCES 1. Canadian Institute for Health Information. Hip and Knee Replacements in Canada—Canadian Joint Replacement Registry (CJRR) 2008–2009 Annual Report. 2009.  2. Darouiche RO. Current concepts - Treatment of infections associated with surgical implants. N Engl J Med 2004;350:1422-1429.  3. Mookherjee N, Hancock REW. Cationic host defence peptides: Innate immune regulatory peptides as a novel approach for treating infections. Cell Mol Life Sci 2007;64:922-933.  4. Hilpert K, Volkmer-Engert R, Walter T, Hancock REW. 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San Diego: Academic Press, 2008.  99  85. Wu MH, Hancock REW. Improved derivatives of bactenecin, a cyclic dodecameric antimicrobial cationic peptide. Antimicrobial Agents Chemother 1999;43:1274-1276.  86. McCafferty E, Wightman JP. Determination of the concentration of surface hydroxyl groups on metal oxide films by a quantitative XPS method. Surf Interface Anal 1998;26:549-564.  87. Cirioni O, Giacometti A, Ghiselli R, Bergnach C, Orlando F, Silvestri C, Mocchegiani F, Licci A, Skerlavaj B, Rocchi M. LL-37 protects rats against lethal sepsis caused by gram-negative bacteria. Antimicrobial Agents Chemother 2006;50:1672-1679.  100  APPENDIX ANOVA tables  Table 1 ANOVA table for cysteine immobilization. Source  Sum of squares  df  Mean square  F  Between Groups  0.3525  4  0.08813  17.65  Within Groups  0.09988  20  0.004994  Total  0.4524  24  Table 2 ANOVA table for Tet213 immobilization. Source  Sum of squares  df  Mean square  F  Between Groups  6.453  2  3.226  20.63  Within Groups  4.38  28  0.1564  Total  10.83  30  Table 3 ANOVA table for antimicrobial Experiment 2. Source  Sum of squares  df  Mean square  F  Between Groups  0.08135  2  0.04068  25.82  Within Groups  0.01891  12  0.001575  Total  0.1003  14  101  Table 4 ANOVA table for antimicrobial Experiment 3. Top: 4 hr incubation; bottom: 24 hr incubation. Source  Sum of squares  df  Mean square  F  Between Groups  0.1346  2  0.06731  40.30  Within Groups  0.01002  6  0.00167  Total  0.1446  8  Source  Sum of squares  df  Mean square  F  Between Groups  0.6234  2  0.3117  58.31  Within Groups  0.03208  6  0.005346  Total  0.6555  8  102  

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