Investigation of Antimicrobial and Biocompatible Surface Coatings

Investigation of antimicrobial and biocompatible surface coatings

Andrea Rachel Leong BBioMedSc(Hons)

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy

School of Optometry and Vision Science The University of New South Wales, Sydney, Australia and Brien Holden Vision Institute, Sydney, Australia

April 2016

ORIGINALITY STATEMENT

I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.

Andrea Rachel Leong

21st April, 2016

i COPYRIGHT STATEMENT

I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright

Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in

Dissertation Abstract International (this is applicable to doctoral theses only).

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.

Andrea Rachel Leong

30th August, 2016

ii AUTHENTICITY STATEMENT

I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.

Andrea Rachel Leong

30th August, 2016

iii ACKNOWLEDGEMENTS

Many thanks are deserved by all who supported me throughout my PhD journey. My deep gratitude goes to my supervisors: Mark Willcox for his expert guidance, absolute patience and good humour; and Nerida Cole for her mentorship and moral support.

Thanks go to my co-supervisor Naresh Kumar for his expertise and encouragement.

I would also like to take this opportunity to acknowledge the late Brien Holden, a brilliant scientist, committed humanitarian, and inspiration for me throughout my PhD project.

This work would not have been possible without support from staff from outside labs, particularly Sally McArthur and Thomas Ameringer at the Biointerface Lab,

Swinburne, and Bill Gong at the Mark Wainwright Analytical Centre.

Chapters of this thesis were proofread by Judith Flanagan, who I also thank for the tea and solidarity. My arrival at the Institute would have been very different if not for the support and care of Vivienne Miller and Eric Papas, and I would have been lost in the lab if not for the generous assistance of Najat Harmis, Shamil Iskandar and Linda

Garthwaite.

The PhD was rough at times, but I was buoyed by the advice and friendship of my group members Debarun Dutta, Renxun Chen, Kitty Ho and Ajay Kumar; and my optom pals: Carolina Kunnen, late-night study buddy and life advisor; Ling Lee, my clone; and Waleed Al Ghamdi, who always had quality chocolate to offer. I was also spurred on by empathy and baked goods from my housemate Kathryn Baker.

To all my colleagues, friends and family: thank you all. A special mention goes to

Andrea (not myself; the other one) for his proofreading assistance and unwavering support.

This work was made possible by funding made available through the University of New

South Wales, Brien Holden Vision Institute and Vision Cooperative Research Centre.

ABSTRACT

Infection of surgical implants is a costly and life-threatening problem which threatens to become more prevalent as implanted devices become more common. Antibiotic treatment is often unsuccessful due to the formation of biofilms, and is a contributing factor to bacterial resistance to antibiotics. A promising strategy to combat implant infection is anti-infective surface coating technology, and antimicrobial peptides

(AMPs) offer an exciting avenue in this regard.

The ideal implant surface not only resists microbial colonisation, but also encourages host tissue integration. Certain peptide sequences can be employed towards this aim, such as arginine-glycine-aspartic acid (RGD).

This work tests a range of novel peptide sequences, based on the synthetic AMP melimine and in some cases incorporating RGD, for antimicrobial activity and cytotoxicity. The peptides tested also contain a single cysteine residue which allows site-directed attachment through a unique sulfhydryl group. A plasma polymer coating method described here renders any desired surface suitable for peptide coating, by the addition of free amine groups and the use of an amine-to-sulfhydryl maleimide linker.

A highly cationic 18-residue peptide, C-Mel4, was identified as antimicrobial and biocompatible. In soluble form, C-Mel4 was lethal to common device-related bacterial pathogens including a methicillin-resistant Staphylococcus aureus (MRSA) isolate, and was toxic to mammalian cells only at high concentrations. When applied as a surface coating, C-Mel4 reduced adhesion of the same bacterial pathogens by approximately

60% and no toxicity was observed.

The in vitro responses of human dermal fibroblasts and epidermal keratinocytes to surfaces coated with C-Mel4 and other peptides were studied. Cells formed focal adhesions and stress fibres in contact with C-Mel4 and other peptide-coated surfaces.

C-Mel4 is an excellent candidate for further in vitro and in vivo testing as a potential biomaterials coating.

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TABLE OF CONTENTS

ORIGINALITY STATEMENT ...... i COPYRIGHT STATEMENT ...... ii AUTHENTICITY STATEMENT ...... iii ACKNOWLEDGEMENTS ...... iv ABSTRACT ...... vi TABLE OF CONTENTS ...... viii ABBREVIATIONS ...... xi LIST OF FIGURES ...... xii LIST OF TABLES ...... xiv

CHAPTER 1. LITERATURE REVIEW AND INTRODUCTION ...... 1 1.1. Biomaterial-associated infections ...... 1 1.1.1. Infection and immunity at the biomaterial-implant interface ...... 3 1.1.2. Aetiology of biomaterial-associated infections ...... 4 1.1.3. The role of bacterial biofilms in biomaterial-associated infections ...... 7 1.1.4. Treatment and prevention of biomaterial-associated infections ...... 10 1.2. Anti-infective Biomaterials ...... 11 1.3. Cationic antimicrobial peptides ...... 19 1.3.1. Structure and function of cationic antimicrobial peptides ...... 19 1.3.2. Microbial resistance to antimicrobial peptides ...... 23 1.3.3. Toxicity of cationic antimicrobial peptides ...... 27 1.4. Biocompatibility and cytocompatibility...... 28 1.4.1. Integrins and the RGD (Arg-Gly-Asp) motif ...... 29 1.4.2. Other cell-active peptide sequences ...... 31 1.5. Melimine: an engineered antimicrobial peptide ...... 32 1.6. Peptide surface coating strategies ...... 40 1.7. Introduction to the present study ...... 43

CHAPTER 2. CHARACTERISTICS OF SOLUBLE MELIMINE AND DERIVATIVE PEPTIDES ...... 47 2.1. Introduction ...... 47

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2.1.1. Effects of peptide sequence modification on antibacterial activity and cytotoxicity ...... 47 2.2. Aims of the chapter ...... 50 2.3. Materials and Methods ...... 50 2.3.1. Peptide design and synthesis ...... 50 2.3.2. Antibacterial testing ...... 53 2.3.3. Biocompatibility of soluble peptides with mammalian cells ...... 56 2.3.4. Therapeutic index ...... 58 2.4. Results ...... 59 2.4.1. Antibacterial testing ...... 59 2.4.2. Cytotoxicity Testing — Cell Growth Inhibition ...... 61 2.4.3. Therapeutic indices ...... 63 2.5. Discussion ...... 64 2.6. Conclusions ...... 68

CHAPTER 3. PREPARATION AND EVALUATION OF SURFACES COATED WITH MELIMINE AND DERIVATIVE PEPTIDES ...... 71 3.1. Introduction ...... 71 3.1.1. Peptide surface attachment ...... 71 3.1.2. Surface attachment of melimine-based peptides ...... 73 3.2. Aims of the chapter ...... 75 3.3. Materials and Methods ...... 75 3.3.1. Peptides ...... 75 3.3.2. Peptide coating of hydrogel polymer surfaces ...... 75 3.3.3. Peptide coating of fluorinated ethylene propylene surfaces ...... 77 3.3.4. Physicochemical analysis of prepared FEP samples ...... 80 3.3.5. Bacterial strains and culture conditions ...... 83 3.3.6. Cytotoxicity Testing — Direct Contact Assay ...... 86 3.4. Statistical analysis ...... 87 3.5. Results ...... 87 3.5.1. Physicochemical analysis ...... 87 3.5.2. Antibacterial activity ...... 92 3.6. Discussion ...... 100

3.6.1. Physicochemical analysis ...... 100 3.6.2. Antibacterial activity ...... 103 3.6.3. Cytotoxicity ...... 106 3.7. Conclusions ...... 107

CHAPTER 4. BIOCOMPATIBILITY AND CYTOCOMPATIBILITY OF SURFACE-ATTACHED PEPTIDES ...... 109 4.1. Introduction ...... 109 4.1.1. Mechanisms of cell-substrate interaction ...... 110 4.2. Aims of the chapter ...... 113 4.3. Materials and Methods ...... 113 4.3.1. Sample preparation ...... 113 4.3.2. Cell types and culture conditions ...... 115 4.3.3. Attachment and behaviour of cells on peptide-coated surfaces ...... 116 4.3.4. Mechanism of cell attachment to surfaces ...... 118 4.3.5. Statistical analysis ...... 120 4.4. Results ...... 121 4.4.1. Behaviour of cells on peptide-coated FEP surfaces ...... 121 4.4.2. Mechanism of cell attachment to surfaces ...... 126 4.5. Discussion ...... 134 4.5.1. Conclusion ...... 138 CHAPTER 5. CONCLUSIONS AND FUTURE PERSPECTIVES ...... 139 5.1. Conclusions ...... 139 5.2. Perspectives ...... 140 REFERENCES ...... 143

ABBREVIATIONS

AAA amino acid analysis BSA bovine serum albumin CFU colony-forming units DMEM Dulbecco's Modified Eagle Medium DMSO dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid EDC 1-[(3-dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride FBS foetal bovine serum FEP fluorinated ethylenepolypropylene FITC fluorescein isothiocyanate FITC kDa kilodalton MBC minimum bactericidal concentration MIC minimum inhibitory concentration MHB Mueller-Hinton broth MRSA/NORSA methicillin-resistant Staphylococcus aureus / non-multiresistant oxacillin-resistant Staphylococcus aureus OD450 optical density at 450 nm OD660 optical density at 660 nm PBS phosphate buffered saline PBST phosphate buffered saline containing 0.1% Tween-20 pHEMA polyhydroxyethylmethacrylate TSA tryptic soy agar TSB tryptic soy broth XPS X-ray photoelectron spectroscopy

Amino acids

Alanine Ala A Leucine Leu L

Arginine Arg R Lysine Lys K

Asparagine Asn N Methionine Met M

Aspartic acid Asp D Phenylalanine Phe F

Cysteine Cys C Proline Pro P

Glutamic acid Glu E Serine Ser S

Glutamine Gln Q Threonine Thr T

Glycine Gly G Tryptophan Trp W

Histidine His H Tyrosine Tyr Y

Isoleucine Ile I Valine Val V

LIST OF FIGURES

Figure 1.1. Illustration of the stages of microbial biofilm formation and maturation...... 7

Figure 1.2. Models of membrane destabilisation by cationic antimicrobial peptides. ... 21

Figure 1.3. Illustration of mechanisms of bacterial resistance against AMPs...... 24

Figure 1.4. Log reduction of bacterial adhesion on melimine-coated surfaces...... 39

Figure 2.1. Density of L929 murine fibroblasts after incubation with soluble peptides 62

Figure 3.1. Reaction scheme for covalent attachment of cationic peptides to pHEMA. 76

Figure 3.2. Scheme of the plasma polymer reactor used for polyallylamine coating. ... 78

Figure 3.3. Reaction scheme for peptide coating of fluorinated ethylene propylene ..... 79

Figure 3.4. Log reduction in viable bacteria recovered from peptide-coated pHEMA .. 92

Figure 3.5. Bacterial surface coverage determined by microscopy and staining ...... 94

Figure 3.6. Bacterial surface coverage determined by microscopy and staining ...... 96

Figure 3.7. Bacterial surface coverage determined by microscopy and staining ...... 97

Figure 3.8. Differential interference contrast images of L929 murine fibroblasts ...... 99

Figure 4.1. Confocal microscopy images of HaCaT cells on FEP surfaces...... 121

Figure 4.2. Fibroblast and keratinocyte counts on FEP surfaces...... 122

Figure 4.3. Confocal microscopy images of fibroblasts on FEP surfaces...... 123

Figure 4.4. Confocal microscopy images of keratinocytes on FEP surfaces...... 124

Figure 4.5. Proliferation activity of fibroblasts on FEP surfaces...... 125

Figure 4.6. Proliferation activity of keratinocytes on FEP surfaces...... 125

Figure 4.7 Short-term adhesion kinetics of fibroblasts used in this study...... 126

Figure 4.8 Short-term adhesion kinetics of keratinocytes used in this study...... 127

Figure 4.9. Fibroblast adhesion to FEP surfaces in the presence of added Mg2+ ...... 128

Figure 4.10. Keratinocyte adhesion to FEP surfaces in the presence of added Mg2+ .. 128

Figure 4.11. Fibroblast adhesion in the presence of interfering peptide ...... 130

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Figure 4.12. Keratinocyte adhesion in the presence of interfering peptide ...... 130

Figure 4.13. Confocal microscopy images of fibroblasts on FEP surfaces...... 132

Figure 4.14. Confocal microscopy images of keratinocytes on FEP surfaces...... 133

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LIST OF TABLES

Table 1.1. Properties of melittin analogues in relation to the parent peptide...... 34

Table 1.2. Melimine and variants designed and tested by Rasul (2010) ...... 40

Table 2.1. One-letter amino acid sequence of peptides used in this study...... 52

Table 2.2. Bacterial species and strains used in this study and their origins...... 54

Table 2.3a. MICs and MBCs (µg/ml) of peptides for various bacterial strains...... 60

Table 2.4. Therapeutic indices of the peptides tested in this study...... 63

Table 3.1. Amino acid sequences of peptides tested in this chapter...... 75

Table 3.2. Bacterial species and strains used in this study and their origins...... 83

Table 3.3. Cytotoxicity grading scale for direct contact testing as per ISO 10993-5:2009...... 87

Table 3.4. Peptide density on coated FEP as measured by quantitative amino acid analysis...... 88

Table 3.5. Atomic percentages of carbon (C), nitrogen (N), oxygen (O) and fluorine (F) as a percentage of total composition, as detected by XPS on sample surfaces...... 89

Table 3.6. Binding energies and proposed assignments for C1s subpeaks detected by XPS...... 90

Table 3.7. Advancing water contact angles (θ°) of coated and control surfaces...... 91

Table 3.8. Cytotoxicity gradings via direct contact assay for peptide-coated surfaces used in this study ...... 98

Table 4.1. Amino acid sequences of peptides tested in this chapter...... 114

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CHAPTER 1. LITERATURE REVIEW AND INTRODUCTION

1.1. Biomaterial-associated infections

The use of biomedical implants is becoming increasingly common, with millions of people annually receiving implanted devices that are life-saving or enhance quality of life. Approximately 800,000 hip and knee replacement surgeries were performed in the

United States alone in 2006 (Del Pozo and Patel 2009) and this number is projected to increase to 4 million by 2030 (Kurtz et al. 2007; Matar et al. 2010). Vastly more prevalent than the use of orthopaedic devices is vascular catheterisation. Each year in

US hospitals, at least 150 million of these devices are used, including surgically- implanted long-term catheters and ports to provide access for drug administration or dialysis (Maki et al. 2006; Mermel et al. 2009).

Unfortunately, a proportion of implanted devices fail. 0.5–5% of joint replacements require treatment for infection, which can involve replacement of the prosthesis—after which the risk of future prosthesis failure rises to up to 10% (Campoccia et al. 2006). In the Australian context, about 1–4% of joint replacements became infected during a 17- month period of observation at a Sydney hospital (Mitchell et al. 1999). In the US, long- term central venous catheters are associated with a rate of 1.6 bloodstream infections per 1,000 catheter-days, and due to the length of use, this translates to a per-device infection risk of 23% or an estimated 250,000–500,000 catheter-related bloodstream infections each year (Maki et al. 2006). The risk of infection is higher in developing countries: 7.8–18.5 cases per 1,000 catheter-days, according to data gathered from intensive care units (ICUs) in Central and South America, India, Morocco, and Turkey

(Rosenthal et al. 2006). The risk of catheter-associated bloodstream infection increases with longer duration of catheterisation, and as such a Cochrane Review recommends that catheters delivering lipids or blood products should be changed every 24 hours, and other catheters should be changed every 96 hours (Gillies et al. 2005), adding expense to the procedure and occupying healthcare professionals’ time. In the Australian context, ICU patients in NSW hospitals who had a central venous catheter in place for six or more days had a 7% risk of contracting a bloodstream infection (compared with a

0.6% risk for five or fewer days) (Wong et al. 2016). As vascular catheters and ports are used with many patients who are already seriously ill, it can be difficult to attribute the exact mortality rate associated with catheter-related infection; but it has been estimated at 25–28% (Smith et al. 1991; Guggenbichler et al. 2011).

Biomaterial-associated infections (BAIs) are costly to treat as they are often unresponsive to antibiotics, in which case they require replacement or possibly debridement (cleaning), both of which re-expose the patient to the trauma and risks of surgery. In economic terms, a single orthopaedic device infection typically costs approximately $15,000–18,000 (Whitehouse et al. 2002; Lalani et al. 2008; de Lissovoy et al. 2009). The cost associated with central line catheter-associated septicaemia is in the thousands of dollars (Rosenthal et al. 2003). The total annual economic impact of biomaterials infection has been estimated at $3.3 billion annually in the USA alone

(Darouiche 2004).

Along with infection, another challenge for permanent or long-term implanted materials is host tissue integration, which in turn reduces the risk of infection. These outcomes highlight the need for anti-infective and tissue-integrative technologies to improve the

2 success rate of biomedical implants. Realising a surface coating that both protects an implant from infection and renders it readily integrated by the surrounding tissues would enormously lower patient morbidity and health system costs, and reduce mortality, especially in ICUs.

1.1.1. Infection and immunity at the biomaterial-implant interface

The need for anti-infective technology for implanted devices is heightened by the particular susceptibility of tissue at the implant interface to infection—more so than non-implant surgical sites (Zimmerli et al. 1984). Referred to as the “immuno- incompetent zone”, this tissue has undergone the trauma of surgery, and the tissue- implant interface suffers from poor vascular supply (Gristina 1994).

Polymorphonuclear leukocytes and macrophages isolated from the tissues surrounding an implant in a rodent model show reduced activity against bacteria—perhaps due to depletion by efforts to phagocytose the implant or tissue debris (Zimmerli et al. 1984)— and oxygen-dependent killing pathways are likely to be impaired by a degree of hypoxia in the region of the implant (Gristina 1994). Finally, if an initial infection is not cleared, the surface of the implant and the surrounding damaged tissues, unlike healthy tissue, provide a hospitable substrate for bacterial biofilm formation (Gristina 1987).

Upon implantation, glycoproteins from the blood and extracellular matrix quickly coat the surface of a device via simple physicochemical forces. This glycoprotein layer enables bacteria or host cells to attach more permanently through specific interactions between the glycoproteins and cell surface proteins (Campoccia et al. 2013). Bacteria and host cells compete for space in what has been described as a “race for the

3 surface”—the population to colonise the implant surface first is more likely to become established, thus influencing whether tissue integration takes place or the implant becomes infected (Gristina 1987). This has been studied experimentally, with six hours being reported as the critical time period in which one or the other population can become established (Neoh et al. 2012). In this time period, bacteria are capable of forming specific bonds with a substrate, following initial non-specific physicochemical surface interactions (An and Friedman 1998).

1.1.2. Aetiology of biomaterial-associated infections

The organisms most commonly isolated from orthopaedic infections in the US in the past decade were the gram positive Staphylococcus aureus and coagulase-negative staphylococci (Trampuz et al. 2007), of which Staphylococcus epidermidis is the most clinically significant species (Becker et al. 2014). These species are also are prevalent in vascular catheter infections, causing approximately two-thirds of catheter-related bloodstream infections between them (Fletcher 2005). In this regard CoNS shows an increased prevalence over time, while S. aureus shows a corresponding decrease

(Rodríguez-Créixems et al. 2013).

S. aureus colonises 30–50% of healthy adults and is also a common and versatile pathogen, capable of causing disease ranging from localised skin infections to food poisoning to fatal bloodstream infections (Lowy 1998). S. aureus has proved capable of developing extensive antibiotic resistance (Lowy 2003); a worrying trend is the emergence of methicillin-resistant S. aureus (MRSA) strains which have developed resistance to all β-lactam drugs including cephalosporins, and are frequently resistant to antibiotics from other classes such as fluoroquinolones and erythromycin (David and

Daum 2010). The Centers for Disease Control reported that 59.5% of S. aureus isolates from US ICU-onset infections were methicillin-resistant (Cardo et al. 2004). Of

S. aureus strains acquired in Australian hospitals, 30.3% are reported as methicillin- resistant (Coombs et al. 2013). MRSA carriage has been reported as rare in asymptomatic carriers (den Heijer et al. 2013; Esposito et al. 2014), but disease due to community-acquired MRSA has nonetheless become a serious health problem since the

1990s (Klevens et al. 2007). In Australia, methicillin resistance is now seen in 17.9% of community acquired S. aureus infections, compared to 11.5% just over a decade ago

(Coombs et al. 2014)

Like S. aureus, S. epidermidis can be widely isolated from the skin or mucous membranes of the healthy population, but can cause serious illness if it gains entry to deeper tissues (Kloos and Bannerman 1994). S. epidermidis is especially prevalent in

BAIs when the surface of the implant or device is a polymer—for example catheters, cardiac devices and intraocular lens implants (Kloos and Bannerman 1994; Raad et al.

1998; Uçkay et al. 2009)—and has thus emerged as a significant pathogen with the increased use of such devices since the 1980s. Clinical isolates are more likely than commensal strains to be resistant to antibiotics (Cherifi et al. 2013) and show a pattern of multidrug resistance that rivals that of S. aureus (Raad et al. 1998; Uçkay et al.

2009).

The most common gram-negative agents of orthopaedic infection are

Pseudomonas aeruginosa and Escherichia coli, each contributing to about 4% of these infections (Sievert et al. 2013). These two species are also lesser but often-recorded causes of vascular catheter-related infection, and are major causes of nosocomial

5 urinary tract infection (Richards et al. 1999; Hidron et al. 2008; Sievert et al. 2013).

P. aeruginosa is ubiquitous and remains viable in very low-nutrient environments and even in distilled water for up to a month (Boyle et al. 1990). This species is a major cause of hospital-acquired infections, including ventilator-acquired pneumonia (Kollef et al. 2005), and colonises the lungs of approximately 70% of all cystic fibrosis CF patients by adulthood (Konstan et al. 1999).

P. aeruginosa and E. coli display extensive resistance to β-lactam antibiotics due to the acquisition of β-lactamase genes, which encode enzymes that break down these antibiotics (Iredell et al. 2016). In addition, P. aeruginosa is intrinsically resistant to a number of conventional antibiotics, owing to its efflux pumps which remove antibiotics from the cell (Dean et al. 2003). E. coli has been reported to combine efflux pumps with a reduction in outer-membrane porins (passive diffusion channels), which reduces cell permeability (Li et al. 1997). The US National Healthcare Safety Network reported extensive resistance among clinical strains from central line-associated bloodstream infections, including fluoroquinolone resistance in 42% of E. coli isolates and over 31% of P. aeruginosa isolates, and multi-drug resistance (to at least three of the following: extended-spectrum cephalosporins, fluoroquinolones, aminoglycosides, carbapenems, or piperacillin) in 15% of P. aeruginosa isolates from this infection route (Hidron et al.

2008).

The prevalence of antibiotic resistance in hospital-acquired infections is a barrier to preventing and treating BAIs.

1.1.3. The role of bacterial biofilms in biomaterial-associated infections

In addition to antibiotic resistance mechanisms found in planktonic (free-living) bacteria, BAIs are often especially difficult to treat due to the formation of bacterial biofilms. Biofilms are multicellular communities, attached to surfaces and characterised by production of extracellular polymeric substance—a slime which surrounds the bacteria, providing protection from predatory microorganisms or host immune cells, bacteriophages, turbulence and desiccation. The stages of biofilm progression are illustrated in Figure 1.1 (reproduced from Monroe (2007)).

Figure 1.1. Illustration of the stages of microbial biofilm formation and maturation. Reproduced under the Creative Commons Attribution License from Monroe (2007).1: Initial (reversible) association of planktonic bacteria with the substrate. 2: Irreversible bacterial attachment. 3: Formation of early, flat biofilm structures and production of extracellular polymeric substance. 4: Formation of complex biofilm structures including voids, and phenotype variation. 5: Dispersion of planktonic bacteria.

The biofilm matrix allows the exchange of dissolved nutrients and waste products

(Costerton et al. 1995), although the degree of permeability varies. Thick P. aeruginosa biofilms have been shown to obstruct the penetration of antibiotics as well as the

7 disinfectants chlorine and hydrogen peroxide (Hoyle et al. 1992; De Beer et al. 1994;

Suci et al. 1994; Stewart et al. 2000). Permeability of the biofilm matrix might be related to the thickness of the biofilm or be species-specific (or both). Thinner

P. aeruginosa biofilms allowed the passage of hydrogen peroxide (Cochran et al. 2000) and S. epidermidis biofilms were permeable to rifampin and vancomycin (Dunne et al.

1993).

Despite biocidal concentrations of rifampin and vancomycin being measured throughout all regions of an S. epidermidis biofilm, these antibiotics were not effective in sterilising the substrate (Dunne et al. 1993). This lack of antibiotic susceptibility is a common feature of biofilms, and the protective effect is due to a combination of physical, chemical and biological factors.

Antibiotic molecules can be physically excluded by the biofilm matrix and/or enzymatically broken down in the outer regions of the biofilm (Stewart 1996). Catalase activity is implicated in the protection of biofilms from hydrogen peroxide (Stewart et al. 2000).

Physiologically, biofilm bacteria are less metabolically active than their planktonic counterparts (Costerton et al. 1995) and less susceptible to antibiotics which target growing bacteria. It is unclear whether the low oxygen and build-up of waste products in the biofilm matrix (Stewart and Costerton 2001) are the main force in suppressing bacterial metabolism, or whether biofilm bacteria actively modulate their metabolism as part of switching to the biofilm phenotype (for example, P. aeruginosa in biofilms upregulate their anaerobic processes (Sauer et al. 2002)). There is also evidence to show that decreased susceptibility of biofilm bacteria to antibiotics is at least partly an active

8 process, rather than a passive result of slowed metabolism—the survival of

P. aeruginosa biofilms in the presence of antibiotics has been linked to starvation- triggered upregulation of genes that combat oxidative stress (Nguyen et al. 2011a).

These survival mechanisms are due to changes in phenotypic expression and are collectively termed antibiotic tolerance—as distinct from true antibiotic resistance, which is characterised by genomic changes (Sousa et al. 2011). As such, bacteria dissociated from biofilms tend to revert to susceptibility (Williams et al. 1997).

Biofilms might also promote true antibiotic resistance by slowing the transport of larger antibiotic molecules, creating a chemical gradient wherein deeply-buried cells are exposed to sub-inhibitory or sub-lethal concentrations, selecting for resistant mutants

(Jefferson et al. 2005). Biofilms also provide an ideal environment for DNA transfer, allowing the spread of antibiotic resistance genes both within and between species

(Aminov 2011; Savage et al. 2013). This has been observed even amongst the mycobacteria, for which horizontal gene transfer has not traditionally been thought of as a major cause of genetic diversity (Brosch et al. 2001). Mycobacterium smegmatis has been reported to transfer DNA by conjugation in biofilms; this behaviour was not seen in liquid culture, perhaps because the biofilm structure holds the cells in place, allowing time for the necessary cell-to-cell contacts to form (Nguyen et al. 2010).

Due to these antibiotic tolerance and resistance mechanisms, established biofilms are very difficult to treat medically, although antibiotics are often effective in treating the disease caused by planktonic bacteria shed from biofilms. Where an implant provides a reservoir of infection, it must be removed and either cleaned or replaced (Marrie et al.

1982; Vaghela et al. 2003). Revision surgery for orthopaedic devices is associated with

9 poorer surgical outcomes than the primary procedure, which negatively impacts the patient’s quality of life (Whitehouse et al. 2002). Treatment for a catheter-related bloodstream infection or an orthopaedic device infection typically requires two extra weeks of hospitalisation (Fry 2002; Nho and Kwon 2003; Kübler et al. 2012; Xin et al.

2013).

1.1.4. Treatment and prevention of biomaterial-associated infections

Potentially pathogenic bacteria can be isolated from 90% of clean surgical wounds, even in a laminar flow environment (Kaiser 1986), indicating that it is all but impossible to completely avoid bacterial contamination in the operating theatre. The rate of postoperative device infection or catheter-related related bloodstream infections is limited by best practices, including skin disinfection at the surgical site, the use of double sterile gloves (Reichman and Greenberg 2009), adequate ventilation of the operating theatre (Hoffman et al. 2002) and ensuring the patient’s body temperature does not drop below 36°C (NICE, 2008). When choosing between jugular or subclavian access for a central venous catheter, doctors must weigh up a potential reduced risk of infection with jugular access with an increased risk of arterial puncture or catheter malposition (which increases the risk of cardiac complications) (Ruesch et al. 2002;

Safdar et al. 2002).

Further recommendations for orthopaedic implants include administration of perioperative prophylactic antibiotics (Dellinger et al. 1994), with the regimen suited to the common causative agents of implant infection and, ideally, to the resistance profile of common strains within the particular hospital (Zimmerli 2006). A meta-analysis found that antibiotic administration reduces the risk of infection in joint replacement

10 surgery by about 80%, for several classes of antibiotics, whether the antibiotics were administered intravenously on the day of surgery or released from loaded bone cement

(AlBuhairan et al. 2008).

Prophylactic antibiotic administration during catheter insertion has not been shown to affect the rate of catheter-associated infection. For patients with existing catheters, prophylactic antibiotics reduced the rate of symptomatic bloodstream infection by about half, but this approach is not recommended due to the risk of acquiring resistant infections (Merrer et al. 2001; Frasca et al. 2010).

Despite these successes in infection control, even low rates of infection are countered by the sheer number of devices implanted. To further reduce the incidence of implant infection, implanted devices can ideally be engineered with the ability to actively resist infection.

1.2. Anti-infective Biomaterials

The strategies used to engineer materials that resist colonisation are many and varied. A few techniques that have been studied in a biomedical context are discussed here; some others are used in the food and marine industries.

Nonfouling surfaces

A nonfouling surface is resistant to the deposition or attachment of cells and biomolecules. Protein adsorption is the first event that occurs on the surface of an implanted device, and the presence of a protein layer enhances bacterial and mammalian cell adhesion (Tsai et al. 2002; Xu and Siedlecki 2015).

Nonfouling (or low-fouling) surfaces that resist protein adsorption have been achieved by coating substrates with a layer of tetraethylene glycol dimethyl ether (tetraglyme)

(Cao et al. 2006), although the nonfouling properties can be attenuated by the particular substrate material (Lopez et al. 1992). Another successful approach is coating with hydrophilic polymer “brushes”—commonly poly(ethylene glycol) (PEG), or oligo(ethylene glycol) (PEO)—of sufficient length (Roosjen et al. 2004). This method generally allows the flexibility of further functionalisation if desired. PEG and PEO can be attached in dendritic geometries to maximise their density (Hoffmann et al. 2006), but are also effective as a monolayer (Khoo et al. 2009; Lokanathan et al. 2011). PEG coating has been used in conjunction with manipulating surface chemistry, to create a patterned surface which was able to control precisely where bacteria adhered on a surface (Pingle et al. 2015). However, such coatings are not always successful in preventing bacterial colonisation even when they prevent protein fouling, highlighting the adaptability of bacteria (Wei et al. 2003).

Nonfouling surfaces (without further modification) are neither actively antimicrobial nor conducive to tissue integration. A “switchable” antimicrobial-to-nonfouling surface coating, which relies on the hydrolysis of an initial antimicrobial coating to produce a nonfouling coating in the long term, has been described (Cheng et al. 2008). However, while the authors suggest that this property protects against the risk of an inflammatory response to dead bacterial cells remaining on an antibacterial surface, they do not address the fate of the cleaved portion of the coating, which presumably would release dead bacteria into its surrounds.

Antifouling surfaces are well suited to biomedical applications where protein, host cell and bacterial adhesion are unwanted, such as urinary catheters. For many long-term or permanent medical devices, however, it is desirable that the device surface integrates with the host’s tissues as well as resists infection.

Antibiotic release from materials

Biomaterials that release antimicrobial substances to reduce infection have been trialled in clinical practice. Bone cements that deliver antibiotics reduce infection rates similarly to intravenous antibiotic administration (AlBuhairan et al. 2008), without an increase in toxicity and without affecting the physical properties of the cement (Langlais et al.

2006).

Vascular catheter materials loaded with conventional antibiotics (typically rifampin and minocycline) or antiseptics deliver an antimicrobial in soluble form where it is most needed, over the course of hours or days. Such catheters are associated with reduced catheter colonisation in animal models (Sampath et al. 2001) and a lower incidence of bloodstream infection in some clinical settings (Veenstra et al. 1999; Safdar et al. 2002;

Maki et al. 2006). However, the leachable antimicrobial approach cannot protect patients in the long term, as devices can become infected via haemotogenous seeding months or years after implantation, when the antimicrobial agent has dissipated

(Downes 1977).

Besides lack of efficacy, concerns with these coatings include patient allergy to chlorhexidine (Oda et al. 1997) and, for conventional antibiotics, the possibility of promoting bacterial resistance. A material which releases an antimicrobial will, for some length of time, release a sub-inhibitory concentration of the substance. The

13 acquisition of resistance has been observed in vitro in S. epidermidis strains exposed to subinhibitory concentrations of rifampin and minocycline (Tambe et al. 2001), common antibiotics in use for anti-infective vascular catheter coatings. Regarding antiseptics, although a survey of the literature found no reports of chlorhexidine resistance in a hospital setting, resistance of Pseudomonas stutzeri to chlorhexidine has been induced in vitro (Tattawasart et al. 1999).

Vascular catheters to which antibiotics and antiseptics have been covalently attached have also been trialled. The concerns with these coatings are much the same as for biomaterials designed to leach these substances: clinical efficacy, patient allergy, and microbial resistance. A review of anti-infective vascular catheter coatings found a reduction in bloodstream infection of about half when antibiotic- or chlorhexidine-silver sulfadiazine-coated catheters were inserted for less than seven days, but no benefit for longer insertion times (Walder et al. 2002). These materials can perhaps be recommended for high-risk patients only (Maki et al. 1997)

Other leachable antimicrobial strategies

Silver is another candidate for an antimicrobial surface coating. The metal has been used to treat and prevent infection since ancient times (Alexander 2009), but it was only recently discovered that the mechanism of action involved silver ion (Ag+) interference with reduced sulfhydryl groups of cysteine, inactivating vital respiratory chain enzymes, and secondary hydroxyl radical formation and DNA damage (Schreurs and Rosenberg

1982; Gordon et al. 2010). Slow-release silver biomaterials can reduce infection in an animal model (Gordon et al. 2010).

However, silver is not an entirely appropriate material to consider for a leachable antimicrobial in the context of biomaterials; it is toxic only at high concentrations

(Hadrup and Lam 2014), but at subtoxic levels it accumulates in the tissues, causing the rarely-dangerous but unsightly condition argyria in which the skin turns blue (Amber et al. 2014). Silver is currently used topically and in wound dressings, but a Cochrane review failed to find evidence that these interventions prevent infection or aid healing

(Storm-Versloot et al. 2010). This lack of clinical effectiveness could be related to the finding that the in vitro efficacy of silver ions is diminished by the presence of serum proteins or glutathione, a cysteine-containing antioxidant produced by human cells

(Mulley et al. 2014). Some gram-negative rods are able to evade silver toxicity by decreased uptake or by the reduction of Ag+ to metallic silver (Russell and Hugo 1994).

One of the more novel anti-infective strategies is the release of IgG antibodies from pooled human sera. This approach can significantly reduce E. coli adhesion to surfaces in vitro, presumably due to IgG physically interfering with bacterial attachment (Rojas et al. 2000), and had a protective effect against MRSA and P. aeruginosa infection in a rodent model (Poelstra et al. 2002). In host tissue, attachment of IgG to pathogens would be predicted to target bacteria for phagocytosis (opsonisation). However, antibodies must either be purified from human blood products, of which supply is limited, or produced in animals, which is costly. This approach also has drawbacks including risk of infection and potential development of microbial resistance to the animal-derived antibodies.

The release of nitric oxide from a porous material, to aid macrophage generation of oxygen radicals, has been effective in reducing bacterial load in vitro (Pegalajar-Jurado

15 et al. 2015b) and in a rodent study (Nablo et al. 2005). However, the safety of this approach is uncertain—its mechanism of action was to accelerate the production of reactive oxygen species, and the authors also note the possibility of vasodilation in response to nitric oxide in the region of the implant.

Taken together, these outcomes suggest that leachable antimicrobial strategies might be best suited to non-implanted or short-term (days) implants. For long-term and permanent devices, the anti-infective properties should be inherent to the material surface (Desrousseaux et al. 2013).

Substrate and cell physico-chemical characteristics

Data on the effect of substrate hydrophobicity on bacterial adhesion are inconsistent, with some studies indicating that bacteria are either more likely to adhere to hydrophobic surfaces (Zmantar et al. 2010; Gomes et al. 2015); or that bacterial adhesion is similar between hydrophobic and hydrophilic surfaces, but the strength of adhesion is stronger on hydrophilic surfaces (Harkes et al. 1991; Bos et al. 2000). This heterogeneity of results could be explained by the varying surface properties of bacteria between species and strains, as well as differences in methodology (Goulter et al. 2009).

Hydrophobic interactions between bacteria and substrate work alongside electrostatic interactions (Zmantar et al. 2010), which is dependent on the specific surface charge of a hydrophilic surface. On a range of polymer substrates, E. coli adhered in greatest numbers to a cationic polymer, in lesser numbers on a hydrophobic polymer, and least on an anionic polymer, presumably due to electrostatic interactions with the negatively- charged bacterial surface (Harkes et al. 1991; Abrigo et al. 2015a).

It is understood that hydrophopbic implanted biomaterials induce a conformational change in plasma proteins to expose hydrophobic domains and promote protein adhesion (Thevenot et al. 2008). Protein deposition in turn promotes bacterial and tissue adhesion (An and Friedman 1998; Tsai et al. 2002). However, the specifics of the intended biomaterial environment need to be considered, as hydrophobic interactions are not always the dominant force in protein-surface interactions. A study of the adhesion of lysozyme (a relatively inflexible 14 kDa tear protein, cationic at pH 7.4

(Howarth and Lian 1984)) on hydrogels reported the highest lysozyme adhesion on the polymer with the highest density of carboxyl surface groups (negatively-charged) and the lowest lysozyme adsorption on a nonionic hydrogel (Garrett et al. 1999).

Substrate nanotopograhpy

Examining the effect of substrate nanotopography, increased bacterial adhesion has been reported on nano-smooth compared to nano-rough glass surfaces, where contact angle or surface free energy was not altered (Mitik-Dineva et al. 2009). This was perhaps due to nano-scale smoothness allowing closer contact between bacterial cell surface molecules and the substrate (Hsu et al. 2013). A similar effect was seen on titanium, with several-fold greater adhesion of S. aureus and P. aeruginosa on smoother surfaces (Ivanova et al. 2010). While these titanium surfaces were similar in average roughness, the peaks on the smoothest surface were comparatively narrow. The effect of nanotopography is not always clear, though, with one study finding no difference in

E. coli adhesion between nano-smooth or nano-rough substrates coated with various polymers (Pegalajar-Jurado et al. 2015a). A computer modelling approach suggests that the relationship between spacing and amplitude of nanotopographical features is important in bacterial adhesion (Siegismund et al. 2014). Bacterial factors are also

17 important: on of a mesh of polystyrene fibres with varying diameters, E. coli and

P. aeruginosa cells (elongated rods of 1–2 µm length) became distorted and wrapped around fibres of 0.3 µm diameter, and adhered and proliferated maximally on fibres of

0.5 µm diameter (1 and 3 µm-diameter fibres were also tested). On the same substrates,

S. aureus (spherical, approximately 1 µm in diameter) adhered and proliferated maximally on the narrowest (0.3 µm) fibres (Abrigo et al. 2015b).

This species-specific interaction, along with the finding that bacterial surface hydrophobicity correlates with greater bacterial adhesion (van Loosdrecht et al. 1987) highlights the importance of considering the properties of the dominant bacterial species when devising an anti-infective strategy.

The effect of nanotopography on mammalian cells has been studied widely with osteoblastic cells, which must undergo differentiation to form bone tissue. For the

MG63 osteoblast-like cell line, nano-smooth tantalum or titanium was associated with increased cell attachment and spreading, while nano-rough surfaces appeared more conducive to differentiation and ossification (Lincks et al. 1998; Wang et al. 2013;

Wang et al. 2015b). Mesenchymal stem cells displayed similar behaviour on smooth polymethylmethacrylate compared to a substrate with disordered nanopits (Dalby et al.

2007). Titanium nanotube materials are of interest in bone implants because of their favourable mechanical properties (von Wilmowsky et al. 2009) and their increased surface area relative to flat titanium. Their effect on bone-forming cells has been reviewed (Minagar et al. 2013) and evidence suggests that, overall, nanotubes of 15–

30 nm in outer diameter maximally encourage cell adhesion, spreading, and

18 differentiation. This is intuitive given that osteoblastic filopodia are typically ~100 nm wide, and so they can more easily treat surfaces with smaller pits as continuous.

The varying effects of different topographies point to the need to assess the mammalian cell response of candidate biomaterials surfaces.

1.3. Cationic antimicrobial peptides

Cationic antimicrobial peptides (AMPs) are a class of peptide with broad-spectrum antimicrobial activity, and which provide an alternative to conventional antibiotics.

They are often effective against clinically significant antibiotic-resistant strains such as methicillin-resistant S. aureus and vancomycin-resistant Enterococcus, fungi, and in some cases viruses and even tumour cells (Hancock and Diamond 2000).

Cationic AMPs as a component of the immune system were first reported in the cecroporia moth (Hultmark et al. 1980) and peptides carrying out this role have since been discovered in a wide variety of animals, plants, bacteria and fungi (Reddy KV et al. 2004). The field continues to grow, with 104 new AMPs reported in 2014 (Wang et al. 2015a) adding to a growing total of over 2600 (Wang et al. 2016).

AMPs exhibit resilience to extreme pH and temperature (Yoshihiro et al. 1991; Willcox et al. 2008). They retain activity after autoclaving (Willcox et al. 2008; Dutta et al.

2013), presumably due to their simple secondary structure (Rasul et al. 2010).

1.3.1. Structure and function of cationic antimicrobial peptides

AMPs are generally 10-25 amino acids (AAs) in length, with a molecular weight 1–

5 kDa. They commonly exist in one of four conformations: β-sheet peptides stabilised

19 by disulfide bridges, α-helices, loop peptides with one disulfide bridge, and extended chain structures. The activity of many α-helical AMPs depends largely on amphipathicity (having one hydrophobic and one hydrophilic face) and is often enhanced by the presence of bulky hydrophobic residues. These properties and the contributions of specific amino acids to AMP activity are discussed in more detail in the introduction to the following chapter, §2.1.

The net positive charge of cationic AMPs is due to the inclusion of cationic residues

(arginine (Arg), histidine (His) and Lysine (Lys)) and is essential to their activity and selectiveness. In mammalian cell membranes, lipids with negatively charged head groups, e.g. phosphydatlyserine, are located on the inner leaflet, while the outer leaflet has a net neutral charge. In contrast, bacterial cell membranes contain the anionic lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in their outer leaflet, allowing an electrostatic interaction between AMPs and the bacterial membrane

(Teixeira et al. 2012). Although there are important differences between the cell walls of gram-positive and gram-negative bacteria, both have modifications to their outer surfaces that are anionic at physiological pH: the gram-positive cell wall features lipotechoic acids which extend through the peptidoglycan layer and the gram-negative outer membrane includes lipopolysaccharide.

Models of membrane destabilisation

A number of models of membrane destabilisation following AMP adsorption to the microbial membrane have been studied and are illustrated in Figure 1.2 (reproduced from Nyugen, Haynie et al. (2011b)). The barrel-stave model (Ojcius and Young 1991) consists of peptides, either singly or aggregated, inserting into the membrane to form

20 pores lined by the peptide. The toroidal pore model also involves a peptide-lined pore, but instead of the peptide coming into contact with the lipid tails in the bulk of the membrane, the inner and outer leaflets of the bacterial membrane fuse to form a continuous surface, such that the peptide is in contact with the lipid head groups only. In the carpet model, AMPs smother the membrane and form micelles in a detergent-like manner.

Figure 1.2. Models of membrane destabilisation by cationic antimicrobial peptides. Reproduced with permission from Elsevier (Nguyen et al. 2011b). Centre: Membrane adsorption, aided by electrostatic interaction between cationic residues and anionic lipid species. The classical models—barrel-stave, carpet, and toroidal pore—involve membrane poration. Peptides can induce membrane thinning, or clustering of anionic lipids which affects the composition elsewhere in the membrane. Peptides can be incorporated into the bulk of a lipid bilayer and then released from either side, translocating a proportion across the membrane. Oxidised lipid headgroups might present ideal targets for cationic peptides, enhancing their interaction with membranes (Mattila et al. 2008). Electrostatic interactions across the bilayer can lead to loss of anionic species across the membrane, membrane depolarisation, or electroporation allowing passage of the peptides or molecules.

These modes of action are not exclusive—for example, the same AMP might form toroidal pores at physiological concentrations, but tend towards the carpet model at high concentrations (Nguyen et al. 2011b). Other specific mechanisms have also been described, for instance the non-bilayer intermediate model involves the incorporation of peptides into the bulk of the bilayer, surrounded by inverted micelles formed from the outer leaflet of the bilayer. The intermediate collapses according to the surrounding physical forces, releasing the peptides from either side of the bilayer (Haney et al.

2010).

While the classical models of AMP action on bacterial cells rely on destabilisation of the bacterial lipid membrane, other targets for AMPs have been identified. Human defensins have been shown to bind to the peptidoglycan precursor Lipid II (de Leeuw et al. 2010; Sass et al. 2010) and the enzyme undecaprenyl pyrophosphate phosphatase, essential in peptidoglycan production (Kjos et al. 2014), which suggests that the peptides inhibit gram-positive cell wall synthesis. Theta-defensin 1 and theta-defensin 2 from macaque induce an uncontrolled release of bacterial autolysin from staphylococci.

These enzymes normally remodel the bacterial cell wall during cell division, but in excess degrade the cell wall, contributing to cell destruction (Wilmes et al. 2014).

Intracellular targets for AMPs have also been described. Of the microcins (bacterially- derived AMPs), microcin J25 binds RNA polymerase, preventing DNA-to-RNA transcription (Mukhopadhyay et al. 2004). Some insect and mammalian AMPs bind the

70S ribosome subunit of E. coli, inhibiting protein translation (Krizsan et al. 2014;

Mardirossian et al. 2014). Membrane interactions are still essential, though, for internalisation of these peptides that act on intracellular targets. Membrane interactions

22 are also sufficient for the activity of many AMPs. Haynie et al. (1995) reported antimicrobial activity of highly cationic peptides of as few as 10 residues attached to a substrate via a two-carbon linker. To span a typical lipid bilayer, an α-helix must contain 20 residues, while a β-sheet needs to contain 10 residues (Engelman et al.

1986). The short length of the 10-residue active molecules suggests that antimicrobial activity was probably effected through surface interactions only, without insertion into the cytoplasm.

1.3.2. Microbial resistance to antimicrobial peptides

A number of attempts to induce microbial resistance to AMPs by exposure to subinhibitory levels have been unsuccessful (Willcox et al. 2008; Li et al. 2016), or the induced resistance was unstable (Samuelsen et al. 2005), in contrast to chlorhexidine and conventional antibiotics (Young et al. 2011). In clinical practice, a wide range of microbial pathogens (including MRSA) isolated from patients with a history of decade- long topical use of tyrothricin—a mixture of antimicrobial peptides isolated from the soil bacterium Brevibacillus brevis (Dubos 1939)—were found to be tyrothricin- susceptible (Stauss-Grabo et al. 2014).

Bacterial resistance to AMPs in vivo may be limited because pathogens face an array of

AMPs which may act in different ways immediately upon invasion—effectively a form of combination therapy. The primitive, membrane-active nature of AMPs is likely to hinder development of resistance, and the heterogeneous nature of AMPs in terms of primary structure reduces the likelihood of many AMPs containing a common site vulnerable to proteases (Zasloff 2002). Peschel and Sahl (2009) highlighted in their review that AMPs and bacteria have co-evolved for millions of years, and that the

23 unusual prevalence of disulfide bridges and high number of positive charges seen in some AMPs may have been selected for to overcome common bacterial resistance mechanisms. However, some bacteria still employ effective mechanisms to evade killing by AMPs (Figure 1.3).

Figure 1.3. Illustration of mechanisms of bacterial resistance against AMPs. Reproduced with modifications under the Creative Commons Attribution License from Bahar and Ren (2013). (A) Gram-positive bacteria can modify techoic acid and phospholipids to reduce net negative charge. (B) Gram-negative bacteria can modify lipopolysaccharide to reduce net negative charge, or acylate outer membrane lipids to inhibit AMP membrane insertion. (C) Cationic proteins in the outer leaflet of the cell wall can repel cationic AMPs while (D) anionic secreted proteins bind and inactivate them. (E) Efflux pumps expel antimicrobials. (F) Proteases digest AMPs.

Bacterial resistance mechanisms include cell surface modification to evade AMPs. In gram-positive bacteria, the staphylococcal gene mprF allows S. aureus to append the cationic amino acid lysine to its membrane lipids, reducing the net negative charge of the membrane (Peschel et al. 2001). The dlt operon allows bacteria to incorporate the nonpolar amino acid D-alanine into lipotechoic acid, reducing the net negative charge of the cell wall. The antimicrobial peptide sensor (Aps) system of staphylococci triggers the activity of both of these surface-modification systems in the presence of AMPs (Joo and Otto 2015).

Similarly, gram-negative bacteria can reduce their negative surface charge by modifying lipopolysaccharide molecules, for example by appending aminoarabinose to the Lipid A portion (Yan et al. 2007). This technique is used by the genus Burkholderia, often opportunistic pulmonary pathogens and intrinsically resistant to many antibiotics

(Loutet and Valvano 2011). An aminoarabinose gene cluster has been found to be essential for viability of Burkholderia cenocepacia (Ortega et al. 2007). A range of gram-negative bacteria also possess mechanisms to acylate the Lipid A unit of lipopolysaccharide, which inhibits AMP membrane insertion by reducing membrane permeability generally (Guo et al. 1998; Bengoechea et al. 2003; Hittle et al. 2015).

Another bacterial strategy is inactivation of AMPs by secreted proteins. Staphylokinase, secreted by S. aureus, induces the release of human α-defensins (human neutrophil peptides 1 and 2) and then forms a complex with the defensins, rendering them ineffective (Jin et al. 2004).

In addition to evading and inactivating AMPs at their outer surface, bacteria can expel peptides after they have breached the cell membrane. Multi-drug efflux pumps that eject

AMPs and other antimicrobials exist in the cytoplasmic membrane and, in gram- negative bacteria, the outer membrane (Sun et al. 2014). Neisseria gonorrhoeae is able to resist killing by the human cathelicidin AMP LL-37 (but not by the human defensin human neutrophil peptide 2) by the use of its efflux pumps encoded by the mtr (multiple transferrable resistance) system (Shafer et al. 1998). Efflux pumps are also seen widely in P. aeruginosa, and confer resistance to multiple antimicrobials (Poole 2001).

Bacterial proteases can degrade AMPs either inside or outside the cell. The excreted metalloproteinase aureolysin of S. epidermidis and S. aureus is upregulated by AMP- sensing systems. Aureolysin is highly effective against the human AMPs dermicidin

(which is unusual in that it is anionic (Lai et al. 2007)) and LL-37 (Sieprawska-Lupa et al. 2004). The peptidase elastase, secreted by P. aeruginosa, earlier recognised as a cause of host tissue damage (Nicas and Iglewski 1985), plays a similar role in AMP inactivation (Schmidtchen et al. 2002).

There have been grave predictions for what might follow if pathogens become resistant to therapeutic AMPs and gain cross-resistance to endogenous human AMPs (Bell and

Gouyon 2003). This has been demonstrated in vitro to be possible in principle (Habets and Brockhurst 2012), although this study selected for resistant bacterial variants via an increasing twofold concentration, which does not mimic correct antibiotic use at therapeutic doses. It does, however, highlight the need to be vigilant with AMP use as with conventional antibiotics.

Countering resistance to antimicrobial peptides

Non-natural peptide analogues might be an important weapon in the battle against bacterial resistance to AMPs. Artificial dipeptides with “superbulky” hydrophobic side

26 chains that interact with membrane lipids were found to have activity against S. aureus and in fewer cases E. coli, despite their short length (Haug et al. 2004).

Another approach is modification of the peptide backbone structure. One such avenue is the peptoids: very similar in structure to peptides except with the side chain attached to the amide nitrogen, rather than the alpha-carbon, of the peptide backbone (Masip et al.

2005). This structure is resistant to degradation by proteases (Miller et al. 1994).

Peptoid counterparts of AMPs, known as ampetoids, have exhibited broad-spectrum antibacterial activity, and are also resistant to protease degradation due to the specific nature of protease-peptide interaction (Ng et al. 1999; Chongsiriwatana et al. 2008).

Enantiomeric forms of AMPs, synthesised with D-amino acids, are also resistant to protease degradation. These analogues retain their activity, demonstrating that their interaction with membranes is not stereospecific (Hancock and Sahl 2006).

1.3.3. Toxicity of cationic antimicrobial peptides

While the membrane selectiveness of AMPs protects the host, the same cannot be said of AMPs gathered from one species for therapeutic use in another. For example melittin, the major component of bee venom, is not only a potent antimicrobial peptide but also strongly haemolytic (Habermann 1972) and therefore unsuitable for therapeutic use without modification. Polymyxins B and E, derived from gram positive bacteria, are used to treat antibiotic-resistant gram-negative infections, but only as a last line of defence due to their nephrotoxicity (Kelesidis and Falagas 2015). Structure-function studies of polymyxin derivatives may address this toxicity and deliver a safer drug for therapeutic use in the future (Gallardo-Godoy et al. 2016). Pexiganan, a synthetic analogue of the AMP magainin II found in frog skin, is well-tolerated and effective as a

27 topical antimicrobial treatment (Lamb and Wiseman 1998; Lipsky et al. 2008).

Interestingly, pexiganan and polymyxin E appear to have a synergistic effect in a rodent model (Cirioni et al. 2016), perhaps mimicking the multi-pronged attack used by endogenous AMPs as part of the innate immune system.

Other AMPs show a much better safety profile. Melimine, an artificial peptide derived from melittin, showed its first haemolytic activity at 600 times the concentration required to inhibit P. aeruginosa growth (Willcox et al. 2008). HLF1-11, a fragment of the human AMP lactoferrin, has a good safety profile in humans when administered intravenously (van der Velden et al. 2009), as could perhaps be expected for a derivative from an endogenous peptide.

1.4. Biocompatibility and cytocompatibility

The term “biocompatibility” with regards to biomaterials is used to refer to the safety, or lack of cytotoxicity, of an implant. In contrast, close integration between host tissues and an implant may be termed “cytocompatibility”. Good tissue integration is one of the challenges for the biomedical implant surface. Integration promotes long-term success of the implant, not only through acceptance of the foreign object, but also by crowding out bacteria that might otherwise find a niche on the implant surface (Gristina 1987).

The choice of bulk material for medical devices depends largely on mechanical properties and resilience. For instance, titanium is suitable for bone prostheses due to its mechanical properties as well as its inertness. Due to the formation of a layer of non- reactive titanium oxide (TiO2) on the surface upon exposure to air, the material is well tolerated by the host tissue and suffers no degradation. However, TiO2 does not

28 particularly encourage attachment and growth of host cells at the implant site (Van

Noort 1987) and in animal studies promotes formation of a fibrous capsule in soft tissue surrounding the implant (Laing et al. 1967; Jansson et al. 2001; Suska et al. 2008).

Titanium implants are often coated with hydroxylapatite—a mineral found in teeth and bone—to produce a surface more conducive to bone ongrowth (Kilpadi et al. 2001).

A newer approach in inducing integration between biomaterials and host tissue is the addition of biological molecules, such as peptide sequences, to biomaterial surfaces to elicit an active biological response from host cells.

1.4.1. Integrins and the RGD (Arg-Gly-Asp) motif

Integrins are mammalian membrane-spanning receptor proteins involved in cell adhesion, shape, migration, and growth cycle. They are heterodimers consisting of one

α and one β unit. At least eighteen α and eight β subunits—and twenty-four heterodimers with varying specificities—have been described in humans. These proteins are linked to intracellular structures and processes to allow the cell to sense the extracellular environment and act upon it (Takada et al. 2007).

Many integrins recognise and bind to the RGD motif: a three-amino-acid sequence of arginine (Arg, R), glycine (Gly, G) and aspartic acid (Asp, D). The motif first came to attention as the cell binding site of fibronectin, and was subsequently discovered in a number of other blood and extracellular matrix proteins with cell-binding activities

(Pierschbacher and Ruoslahti 1984a; Ruoslahti and Pierschbacher 1987; Ruoslahti

1996; Gabriel et al. 2012). Binding of RGD-sensitive integrins to their ligand triggers

29 recruitment of focal adhesion proteins, actin cytoskeleton remodelling, and cell spreading (Yu et al. 2011).

Soluble short peptides containing RGD can interfere with RGD-dependent processes

(Pierschbacher and Ruoslahti 1984a). The hexapeptide GRGDSP (the context in which

RGD is found in fibronectin) is particularly effective in this regard, and substitution of any one of R, G, or D with close analogues or their D-amino acid counterparts reduced the activity of the interfering peptides. The amino acid directly following RGD also modulates RGD-motif binding. Substitution of the serine (Ser, S) of GRGDSP with proline (Pro, P) (which introduces a rigid constraint to the peptide backbone) diminished the binding ability of the sequence, but substitution with most other amino acids (including arginine) had little effect. The RGD tripeptide alone with no modifications (RGD-COOH) was inactive; however, when the aspartic acid was amidated (RGD-NH2) the peptide was active (although less so than GRGDSP)

(Pierschbacher and Ruoslahti 1987) .

Given the activity of the RGD motif, one strategy for promoting tissue integration is therefore the inclusion of the RGD motif on the biomaterial surface. The presence of

RGD-containing peptides on a surface has successfully enhanced adhesion of MRC5 human lung fibroblasts (Ohno et al. 1999), human bone marrow stromal cells (Zhang et al. 2009; Cao et al. 2012), human osteosarcoma cells (Weng et al. 2010) and B16 mouse melanoma cells in culture; and increased bone formation in vivo (Kim et al. 2007).

Synthetic analogs of RGD have been shown to mimic the biological effects of RGD, including inhibition of platelet aggregation (Boxus et al. 1998) and inhibition of integrin-mediated cell adhesion(Biltresse et al. 2005). This indicates that while the RGD

30 ligand-receptor interaction is specific, it can be replicated with substitutes of similar structure and properties.

The effect of RGD on bacteria

Whole fibronectin has been shown to increase binding of S. aureus, but this effect has been localised to the heparin-binding domain of fibronectin, distinct from the RGD motif (Boeuf et al. 2012). The RGD motif, however, has been shown to favour mammalian cell adhesion while not affecting adhesion of gram-negative or gram- positive bacteria (Harris et al. 2004; He et al. 2009; Boeuf et al. 2012).

1.4.2. Other cell-active peptide sequences

Other peptide sequences have similar pro-cell survival effects to RGD. The PHSRN motif of fibronectin is not active by itself, but acts synergistically with RGD. Co- presentation of PHSRN has been shown to enhance the effect of RGD on human umbilical vein endothelial cells and mouse osteoblasts on a titanium substrate

(Ochsenhirt et al. 2006; Chen et al. 2013). The peptide sequences WQPPRARI from fibronectin (Wilke et al. 1993), YIGSR from laminin and GEFYFDLRLKGDK from collagen IV (Salber et al. 2007) have been shown to promote keratinocyte survival.

Apart from peptide motifs found in blood and extracellular matrix proteins, short homopolymers of cationic peptides also have specific effects on mammalian cells.

These sequences are readily internalised by certain mammalian cells, with peptides of

7–15 arginine residues particularly efficient at entering murine macrophages (Futaki et al. 2001), human T-cells (Mitchell et al. 2000) and human vascular smooth muscle cells

(Uemura et al. 2002). This uptake appears distinct from either the cells’ transport

31 system for free amino acids or endocytosis (Weigel and Oka 1981). The HIV-1 Tat protein contains an arginine-rich domain that is essential for its translocation into mammalian cells. For short polyarginines and Tat, entry into cells appears largely dependent on the presence of glycosaminoglycans (large negatively-charged polysaccharide molecules) on the cell surface (Rusnati et al. 1998; Rusnati et al. 1999;

Suzuki et al. 2002; Fuchs and Raines 2004).

Protamine is another cell-penetrating arginine-rich peptide. It is involved in DNA packing in spermatozoa, replacing histones during spermiogenesis (Alfert 1956) and has been used to stabilise RNA to protect it from RNases (Skold et al. 2015). Protamine complexes are potential therapeutic carriers for small molecules such as siRNA, growth factors and nitric oxide (Umerska et al. 2014; Ishihara et al. 2015; van Lith et al. 2015).

1.5. Melimine: an engineered antimicrobial peptide

Melimine (Willcox et al. 2008) is a synthetic antibacterial peptide, composed of sections of the natural peptides melittin and protamine. Its sequence, comprised of portions of melittin and protamine, is as follows:

Charge + + + + + + + + + + + + + + + + Residue T L I S W I K N K R K Q R P R V S R R R R R R G G R R R R residues 15–26 from melittin residues 11–27 from protamine

Protamine

In addition to its mammalian cell interactions, protamine shows antimicrobial activity

(Brock 1958; Uyttendaele and Debevere 1994; Johansen et al. 1997) and is generally non-toxic to mammalian cells (Sarwar et al. 1989), although one cell line showed a few

32 percent decrease in proliferation in the presence of protamine (Arbab et al. 2004). The

C-terminal portion of protamine is retained as residues 13-29 of melimine. The sequence used in melimine is that reported as the pTP11 variation of protamine from rainbow trout (Jenkins 1979).

Melittin

Melittin, the main component of bee venom, is antimicrobial (Fennell et al. 1968) and also cytotoxic (Habermann 1972). Melittin is a linear cationic α-helical peptide, specifically composed of two α-helices connected by a central hinge region in a “bent rod” formation (Brogden 2005). The peptide self-associates through hydrophobic interactions as a homotetramer with a largely hydrophilic outer surface (Anderson et al.

1980; Tosteson and Tosteson 1981; Terwilliger and Eisenberg 1982b; Terwilliger and

Eisenberg 1982a) and is described as forming toroidal pores in negatively-charged lipid membranes (Yang et al. 2001).

The findings of structure-function studies of melittin are summarised in Table 1.1. Both the haemolytic ability and antimicrobial action of melittin depend on its amphipathic helix structure (Boman et al. 1989; Blondelle and Houghten 1991; Oren and Shai 1997;

Keun Kim et al. 2002). Haemolytic activity requires strict adherence to this amphipathicity, a helix of sufficient length, and a few vital hydrophobic residues. In fact, a nonhomologous analogue of melittin with increased amphipathicity and retention of secondary structure showed enhanced haemolytic properties compared to melittin

(DeGrado et al. 1981).

Table 1.1. Properties of melittin analogues in relation to the parent peptide.

13 is is 13

ngle.

l for haemolysis. for l

Comments is structure helix Amphipathic essentia for important is Sequence structure general haemolysis; antibacterial confer charge and activity amphipathicity as well as Length Leu vital. be may helices of important. particularly some to tolerant is region Hinge a bend in change is charge positive one of Loss activity. to disruptive not charged, negatively to bind Can lipid zwitterionic, not but bilayers.

Haemolytic Haemolytic activity 2 1/25 reduced. Greatly for zero Almost Ile and Leu deletions. ≥1 ≈1 zero Almost

Antibacterial Antibacterial activity tested not 1/2 ≤1 ≈1 ≈1 ≥1

helix helix

of one helix helix one of

Structural features features Structural melittin to compared amphipathicity, Increased secondary of retention tail cationic and structure are helices but tested, Not retained. be to presumed Amphipathicity cases. some in disrupted is angle. bend in Change secondary in change No structure. α reduced Greatly content.

) )

8, Ile 8,

)

Val 5,

)

amino acid acid amino

)

and Blondelle

(

13) 13) 21

–

AA deletion analogues: analogues: deletion AA analogues: deletion AA analogues: deletion AA

- - -

26)

7 or any hydrophobic residue hydrophobic any or 7

–

DeGrado et al. 1981 al. et DeGrado 1989 al. et Boman 19 Houghten and Blondelle 1991 Houghten and Blondelle 1997 Shai and Oren

Peptide / Sequence / Peptide melittin Nonhomologous analogue ( melittin: Shuffled (16 ( Single Lys ( Single region hinge ( Single tail Cationic 1991 Houghten Diastereomer: Val at substitutions Lys and 17 (

Table 1.1. Properties of melittin analogues in relation to the parent peptide. (cont’d).

and and

ytic

eric, with some some with eric,

terminal portion alone is alone portion terminal

terminal helix is insufficient for for insufficient is helix terminal

Comments C The haemol poorly antibacterial. less substantially amphipathicity. Increased N haemolysis. the in structure helical Reduced lipid zwitterionic of presence presence the in not but vesicles, lipids. charged negatively of dim Mostly tetrameric is (melittin monomer monomeric small very a with component)

Haemolytic Haemolytic activity 1/300 1/200 Zero tested not tested not

Antibacterial Antibacterial activity 1/5 ≤1 1/2 tested not tested not

3 and and 3

12 and and 12

13 13

Ala Ala



13 residues are are residues 13 Arg and 12

4. Pro 4.

- -

tibacterial, non tibacterial,

Structural features features Structural melittin to compared Lys melittin, inAs Arg the on located face. hydrophobic Lys Pro with transposed Ala substitution. slightly a is HP Similar. an of protein haemolytic pylori Helicobacter of disruption Moderate zipper leucine potential motif of disruption severe More zipper leucine potential motif

)

12)

) )

–

Asthana Asthana

(

)

Ala Ala



27) 27)

melittin(1

–

alogue: Leu alogue:

–

)

Asthana et al. 2004 al. et Asthana

(

Ala Ala

Ala and Leu and Ala



terminal helix portion only only portion helix terminal with portion terminal

- -



Subbalakshmi et al. 1999 al. et Subbalakshmi 1999 al. et Subbalakshmi 2002 al. et Kim Keun

Peptide / Sequence / Peptide C ( C (12 modification: ( HP(2 Hybrid: ( Leu analogue: Substitution 13 an Substitution 6 2004 al. et

The antibacterial activity of melittin, on the other hand, is tolerant to minor disruptions in helix amphipathicity, and requires the inclusion of sufficient positively charged residues. Some maintenance of structure seems necessary, as substitutions from compact hydrophobic residues chains to bulky phenylalanine (Phe, F) or rigid proline

(Pro, P) diminish both antimicrobial activity and haemolytic activity (de Latour et al.

2010). The C-terminal helix portion alone is 300 times less haemolytic than the complete peptide, while antimicrobial activity is reduced by only a factor of five

(Subbalakshmi et al. 1999). Residues 15–26 from this portion of melittin are retained as residues 1-12 of melimine.

Studies of melimine

Melimine has been shown to be an effective as an antimicrobial in solution, and in reducing microbial colonisation of glass, titanium and polymer surfaces. Structurally, melimine alone adopts a predominantly random coil structure, with an estimated 10%

α-helical content amounting to a “fold”. In comparison, melimine in the presence of micelles mimicking the bacterial membrane adopts a 35–40% α-helical structure (Rasul et al. 2010). Melimine in solution retains its antimicrobial activity after autoclaving.

S. aureus and P. aeruginosa did not readily gain resistance to melimine, showing no increase in the concentration of peptide required to inhibit their growth after 30 consecutive days of culture in medium containing subinhibitory concentrations (Willcox et al. 2008).

Both S. aureus and P. aeruginosa cells examined by electron microscopy showed deformation in the presence of lethal concentrations of melimine (Willcox et al. 2008).

Exposure to soluble melimine resulted in cytoplasmic membrane permeabilisation for

36 both species. However, for P. aeruginosa the extent of permeabilisation correlated with loss of viability, while permeabilisation of S. aureus membranes only slightly affected viability (Rasul et al. 2010).

Melimine attached in a random orientation to a glass substrate was effective in reducing

S. aureus and P. aeruginosa adhesion by 98% and 93%, respectively (Chen et al. 2009).

Site-directed attachment of melimine to surfaces via a disulfide bond made possible by the addition of a cysteine residue had different effects based on cysteine placement.

N-terminal attachment resulted in the most active coating, reducing S. aureus and

P. aeruginosa adhesion by 83% and 70%, respectively, while C-terminal and central peptide attachment led to measureable but lesser activity (Chen et al. 2012). Exposure of the arginine tail might therefore be important for the antibacterial activity of melimine.

Soluble melimine has a good safety profile against sheep erythrocytes, leading to approximately 10% lysis at 2,500 µg/ml and 50% lysis at 5,000 µg/ml, compared with inhibitory concentrations of 4 mg/ml for P. aeruginosa and 125 mg/ml for S. aureus

(Willcox et al. 2008). Polyhydroxyethylmethacrylate (pHEMA) contact lenses covalently coated with melimine showed no toxic effects on mouse fibroblasts in culture (Dutta et al. 2013), nor in 22 days of wear in a rabbit model, or in a 24-hour period of wear in humans (Dutta et al. 2014).

In vitro efficacy of a melimine coating on pHEMA was dose-dependent up to a maximum, with the maximal peptide coating density (38 µg/cm2) showing broad- spectrum efficacy against a range of corneal pathogenic microbes. Surface adhesion of antibiotic-sensitive S. aureus and P. aeruginosa strains was reduced by at least 3 log on

37 the coated vs. uncoated pHEMA. Viable counts of five multi-drug resistant S. aureus strains, four multi-drug resistant strains and one strong biofilm-producing strain of

P. aeruginosa on pHEMA-melimine were reduced by at least 2 log. In addition, colonisation was reduced by 1 log for Serratia marcescens, the fungal species Candida albicans and Fusarium solani, and the protozoan Acanthamoeba castellanii (Dutta et al.

2013).

A melimine coating on titanium was effective in reducing bacterial colonisation in vitro and in a mouse subcutaneous implant infection model (Chen et al. 2016). Covalent incorporation of melimine into silicone hydrogel contact lenses reduced ocular symptoms in rodent models, on both ulcerated corneas challenged with S. aureus and intact corneas exposed to P. aeruginosa (Cole et al. 2010). (Dutta et al. 2014)

The in vitro efficacy of melimine coatings on different substrates are compared in

Figure 1.4. Peptide coverage and activity levels on the rigid substrates were substantial, but lower than for pHEMA. Approximately 1 log reduction in bacterial adhesion was reported for pHEMA coated with 10 nmoles of melimine per cm2, while this order of reduction was achieved by melimine-coated glass and/or titanium at lower coating densities. However, the reported density on pHEMA does not account for the porosity of the polymer (i.e. 1cm2 translates into greater actual surface area) compared to the smooth glass and titanium surfaces.

Overall, the data describe a peptide with broad-spectrum antimicrobial activity, with somewhat greater activity against S. aureus than against P. aeruginosa in its soluble form, and vice versa when the peptide is covalently attached to surfaces.

Figure 1.4. Log reduction of bacterial adhesion on melimine-coated surfaces.  Glass reacted with melimine via 4-aminobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA) linkers (Chen et al. 2009). ▲ Ti reacted with melimine via (3- aminopropyl)-triethoxysilane (Chen et al. 2016).  Poly(2-hydroxyethyl methacrylate) reacted with melimine at the noted concentrations from 0.1 to 5.0 mg/ml, peptide density determined using quantitative amino acid analysis (Dutta et al. 2016).

As peptides can be precisely modified, they offer a platform for functionalisation that can be fine-tuned. There is scope for peptides like melimine to be further modified to encourage mammalian cell integration while retaining its antimicrobial activity, while

39 being utilised as a surface coating. Deletion studies of melimine to find the minimal active sequence produced the truncated peptides melimine 1–4, outlined in Table 1.2

(Rasul 2010). These peptides were covalently coupled to pHEMA substrates and challenged with S. aureus or P. aeruginosa to determine the effect of the peptides on bacterial adhesion and viability. Melimine 1 and melimine 2 performed poorly, while melimine 3 and 4 gave similar results to full-length melimine.

Table 1.2. Melimine and variants designed and tested by Rasul (2010) Peptide Amino acid sequence Melimine TLISWIKNKRKQRPRVSRRRRRRGGRRRR Melimine 1, removal of charged portions TLISWIQRPRVS Melimine 2, removal of C-terminal charged TLISWIKNKRKQRPRVS portion Melimine 3, removal of N-proximal charged TLISWIQRPRVSRRRRRRGGRRRR portion Melimine 4, removal of hydrophobic portions KNKRKRRRRRRGGRRRR

Set-up costs for artificial peptides increase per amino acid, and so there is an economic advantage to using a shorter peptide if the activity is equivalent to a longer one. Thus, the truncated peptide melimine 4 is a promising candidate for further study as a potential coating for biomaterials.

1.6. Peptide surface coating strategies

Covalent attachment is the most suitable method for permanent attachment of active peptide to biomaterial surfaces. Physical adsorption is simple, but it is prone to aggregation, uneven distribution and desorption of peptides. Loading materials with antibiotics, antiseptics and silver ions which are then slowly released into the surroundings allows relatively high doses to be delivered locally, while staying below

40 systemic toxicity levels; however, they have failed to achieve efficacy (Rabindranath et al. 2009).

A range of substrates—from hard, crystalline materials such as titanium and ceramics to polymers such as polyurethane and silicone rubber—are in use as biomaterials. The many successful peptide coating strategies reported have been performed on substrates with varying surface chemistries. Standardising surface chemistry across these substrates would streamline the process for coating various implants for which a certain coating is suitable. A method for standardising surface chemistry on most substrate materials and objects of irregular shape is plasma polymerisation.

Plasma polymerisation

Plasma polymerisation involves polymer deposition onto a substrate by crosslinking of monomer from a vapour phase by electrical discharge. The structure of plasma polymers are typically more highly-crosslinked than their conventional counterparts, and the layers are generally pinhole-free and resistant to extreme environmental conditions (Goodman 1960; Yasuda 1981). The method is able to coat complex

3-dimensional scaffolds (Hawker et al. 2014) while maintaining the bulk properties of the substrate material as it typically deposits a layer in the order of tens of nanometres in thickness.

Plasma polymer films can themselves have favourable cell-attachment or antibacterial properties (France et al. 1998; Pegalajar-Jurado et al. 2014), but as is often the case, these two properties are difficult to achieve at once. Plasma polymerisation can also be used to provide a standardised surface chemistry that is appropriate for further functionalisation.

Covalent peptide attachment methods

Peptides can be anchored to surfaces using compatible linker molecules. Cationic peptides, by their nature, contain positively charged residues. Melimine is arginine-rich in particular, and the primary amines of the arginine side chains can be utilised for attachment to surfaces. However, this also means that attachment via amine groups will result in a random peptide orientation—binding may occur at an unmodified N-terminus or at any exposed amine-containing residue. Attachment at more than one of these sites would constrain the peptide in its orientation, which might reduce its ability to interact with microbial membranes (Onaizi and Leong 2011). Involvement of positively charged residues in attachment will decrease the overall positive charge of the peptide, which could also diminish antimicrobial efficacy.

Among the common amino acids, only cysteine residues contain a sulfhydryl group in their side chain. This sulfhydryl can be used for site-specific attachment of a peptide with a single cysteine in its sequence.

As mentioned, both amine- and cysteine-directed attachment have been successfully used to attach melimine to suitable substrates (Chen et al. 2009; Chen et al. 2012; Dutta et al. 2013).

Visibility of peptide motifs

The presentation of surface-attached peptides can affect their biological effects. The attachment point of melimine affects the peptide’s antimicrobial activity in some cases

(Chen et al. 2012), but N-terminal attachment using short (~1 nm) linker molecules or amine-directed coupling directly to a polymer matrix resulted in active melimine coatings (Chen et al. 2009; Dutta et al. 2013).

Given the large size of mammalian cells in comparison to bacterial cells and peptides, steric hindrance may reduce the ability of cell surface receptors to come into contact with ligands on a rough surface. A spacer arm of ~10 amino acids may mitigate this effect, as shown for platelets in response to RGD-containing short peptides (Beer et al.

1992). In other contexts, though, mammalian cells responded to the minimal peptide

RGDC coupled to a polycaprolactone scaffold via a ~1 nm linker (Zhang et al. 2009).

1.7. Introduction to the present study

The problem to be addressed is the need for a surface coating for implanted biomedical devices which is both anti-infective and promotes host tissue integration. This literature review has examined anti-infective surface modification strategies in use and in development. Peptides offer an exciting avenue, due their ability to be fine-tuned and modified.

Based on previous studies of melimine and the truncated derivative melimine 4, these peptides were used as starting points for peptide design in the current study. Based on the known properties of the RGD peptide motif, peptides containing both melimine- based sequences and RGD were trialled as prospective bifunctional antimicrobial and tissue-integrative peptides.

A consideration in designing these peptides was the variable results for the ability of cells to respond to the RGD motif without a spacer arm, while melimine coatings were effective in several attachment strategies linking the peptide closely to the surface.

Placing the antimicrobial portion of the peptide proximal to the substrate surface, and

43 therefore the RGD-containing portion further out into the medium, might maximise the activity of the RGD domain.

Three experimental chapters follow:

Chapter two examines the antibacterial activity and cytotoxicity of a panel of peptides in soluble form to determine their maximum potential as bifunctional molecules.

Chapter three describes a method for attaching the peptides to any given substrate and examines the in vitro efficacy and safety of these peptides when applied as a coating.

The panel of candidate peptides is refined in this chapter.

Chapter four explores the effect of surface-immobilised peptides on human dermal and epidermal cells, and investigates the mechanisms through which the peptide coatings might exert their effects.

A final conclusions and future perspectives chapter closes this thesis.

CHAPTER 2. CHARACTERISTICS OF SOLUBLE MELIMINE AND DERIVATIVE PEPTIDES

2.1. Introduction

This chapter will examine the in vitro antimicrobial efficacy and biocompatibility of the peptide melimine and its derivatives in solution. The ultimate aim of this project is to advance the development of a peptide surface coating which resists microbial colonisation while promoting mammalian cell adhesion. The study of bacterial and mammalian cell interaction with peptide coated surfaces is the focus of later chapters.

Peptides were first tested in solution to give an indication of maximal activity and toxicity when the peptide is not physically constrained.

The peptides tested in the present chapter were designed based with the antimicrobial peptide melimine (Willcox et al. 2008) as a starting point, with the addition of pro-cell integration properties as the aim. For this purpose, the tripeptide motif arginine-glycine- aspartic acid (RGD) (Pierschbacher and Ruoslahti 1984a) is immediately attractive for its properties which include enhanced adhesion and survival of various mammalian cell types (Yu et al. 2011). RGD was therefore included in several peptide sequences tested in this chapter.

2.1.1. Effects of peptide sequence modification on antibacterial activity and cytotoxicity

Structure-function studies of AMPs have shown that positive charge density (through the inclusion of arginine, lysine, and less often histidine residues) is a vital factor in peptide antimicrobial activity (Falla and Hancock 1997; Taniguchi et al. 2014). Total

47 length is another important factor. For example, deletion studies of rainbow trout cathelicidins found that a highly-cationic 16-mer was less antimicrobial than a 32-mer with fewer cationic resides, despite its greater net positive charge. These peptides were predicted to exist in a random coil structure and had minimal cytotoxicity (Zhang et al.

2015).

The presence of an amphipathic α-helical region, with hydrophobic residues aligned on one face of the helix and hydrophilic residues on the other, is a common feature of

AMPs but is also implicated in cytotoxicity. However, amphipathic helix content is neither necessary nor sufficient for cytotoxicity. Members of the cecropin and magainin

AMP families display low cytotoxicity (Steiner et al. 1981; Hultmark et al. 1982;

Zasloff 1987; Moore et al. 1996) despite a high degree of amphipathic helicity in conditions that mimic the mammalian cell membrane (Steiner 1982; Zasloff 1987; Sipos et al. 1992; Maher and McClean 2006). The cathelicidin NA-CATH displayed similar properties, and an 11-residue portion of this peptide displayed equivalent antibacterial activity and lower haemolytic activity despite increased amphipathic helicity (de Latour et al. 2010), perhaps due to the shorter length of the modified peptide.

Melittin, the major component of bee venom, is a potent broad-spectrum AMP (Fennell et al. 1968) and is also highly haemolytic (Willcox et al. 2008). It comprises two α- helical regions with a central hinge region, and self-assembles into a homotetramer

(Anderson et al. 1980; Terwilliger and Eisenberg 1982b; Terwilliger and Eisenberg

1982a). However, a number of modifications to the sequence of melittin—transposition of the two helical regions (Boman et al. 1989), disruption of the amphipathic helix

(Oren and Shai 1997), or deletion of either of the two helical portions (Subbalakshmi et

48 al. 1999; Keun Kim et al. 2002)—greatly diminish haemolytic activity with a much smaller loss of antimicrobial activity. These changes in function could be due to disruption of tetramer formation—other studies note that self-association of AMPs correlates with greater haemolytic activity and lesser antimicrobial activity (Oren et al.

1999; Chen et al. 2005).

These findings suggest that antibacterial activity of AMP requires sufficient cationic residues; haemolytic activity is unlikely in the absence of an amphipathic helical region; and both functions require a minimum length (or bulk), specific to the peptide.

The antimicrobial peptide melimine is the starting point for this study. Its sequence

(Table 2.1) is derived from portions of melittin (described above) and protamine derived from salmon sperm (Jenkins 1979). Protamines are an arginine-rich family of peptides involved in DNA packing in spermatozoa (Alfert 1956). Salmon protamine shows antimicrobial activity (Brock 1958; Uyttendaele and Debevere 1994; Johansen et al.

1997) with minimal cytotoxicity (Sarwar et al. 1989; Park et al. 2003). The C-terminal portion of protamine is retained as residues 13-29 of melimine.

Melimine in solution has been shown to inhibit the growth of Pseudomonas aeruginosa and Staphylococcus aureus at concentrations well below haemolytic concentrations.

Melimine exhibited 50% lysis of sheep erythrocytes at 5,000 µg/ml, in comparison to melittin which caused a similar level of haemolysis at 30 µg/ml (Willcox et al. 2008).

These bacteria did not acquire resistance to melimine after 30 days of continuous culture in sub-inhibitory concentrations (Willcox et al. 2008). A helical projection for melimine did not indicate any amphipathicity (Rasul et al. 2010).

While a minimal peptide length has been found to be important for antimicrobial activity in some cases, shorter sequences can be desirable because they are less expensive to synthesise. To investigate shorter alternatives to melimine, a truncated peptide retaining the most highly-charged portions was designed, and initial antimicrobial testing was carried out (Rasul 2010). This peptide, designated melimine 4

(Mel4), was found to have similar antimicrobial activity as melimine when adsorbed onto or covalently attached to a poly-2-hydroxyethylmethacrylate (pHEMA) substrate.

Mel4 was therefore considered as a candidate in the present study.

2.2. Aims of the chapter

Candidate peptides were tested to determine their in vitro antimicrobial activity and cytotoxicity in solution. Successful candidate peptides were those that retained antimicrobial activity of the parent peptide melimine (an MIC in molar terms within two-fold was considered equivalent), and had a higher therapeutic index (ratio of MIC- to-cytotoxic concentration).

2.3. Materials and Methods

2.3.1. Peptide design and synthesis

The one-letter amino acid sequence, molecular weight (as per the manufacturer’s documentation) and charge-to-length ratio of all peptides used in this study are listed in

Table 2.1. The C-terminus of all peptides was amidated. C-terminal amidation mimics biological synthesis, can reduce susceptibility to proteases (Maillère et al. 1995;

Brinckerhoff et al. 1999), and may enhance interaction with the bacterial membrane, leading to faster bacterial killing (Kim et al. 2011). Peptides were synthesised by

American Peptide Company (Sunnyvale, CA, USA) using conventional solid-phase synthesis and Fmoc chemistry, and supplied in desiccated powder form with documentation listing the peptide content and purity (>95%) by weight.

The charged regions of melimine (residues 7–11, KNKRK, and residues 18–29,

RRRRRRGGRRRR) were noted in a previous study to be vital for antimicrobial activity (Rasul 2010). However, the peptides tested in that study were designed by deleting these residues, resulting in a peptide with the sequence TLISWIQRPRVS; or by deleting all but these residues, resulting in Mel4. Length alone is noted to influence the antimicrobial activity of peptides and so in the present study, a substitution variant was tested. This variant, designated malamane, is based on melimine with each of the six arginine residues at positions 18–23 replaced with alanine (Ala, A) (Table 2.1).

Successful candidate peptides from this chapter must be amenable to covalent surface attachment. Formation of amide bonds between substrate carboxyl groups and peptide amine groups has been used to obtain a high-density of melimine on a polymer substrate

(Dutta et al. 2013); this method remains an option for surface attachment of melimine derivatives. The amino acid cysteine enables site-directed attachment as it contains a sulfhydryl group unlike other standard genetically-encoded amino acids. A previous study found that N-terminal attachment of melimine resulted in greater antimicrobial activity than either central or C-terminal attachment (Chen et al. 2012). Thus, an

N-terminal cysteine was added to the peptides designed for this study.

length ratio length

Net charge / / charge Net

+17 / 29 = 0.59 = 29 / +17 0.57 = 30 / +17 0.52 = 33 / +17 0.82 = 17 / +14 0.78 = 18 / +14 0.70 = 20 / +14 0.72 = 18 / +13 0.58 = 24 / +14 0.38 = 29 / +11

3.786 3.889 4.217 2.349 2.451 2.779 2.509 3.021 3.275

Molecular Molecular

weight (kDa) weight

R R

R R R R

R G G R

G G G G

G R R R G

R R R R A

R R R D R A

R R R G G A

R R R R G A

R S S R R R R A

S V V R R R R R S

V R R R R R R R V

R P P R R R R R R

P R R R G G D R P

R Q Q G G G G R R

Q K K G R R R K Q

K R R R R R R R K

R K K R R R R K R

K N N R R R R N K

N K K R R R R K N

I I

K R R R R C K

I I

W W R K K K P

W S S K R R R S W

I I

S R K K K D S

I L L K N N N G

L T T N K K K R L

no acid sequence of peptides used in this study. this in used peptides of acidsequence no

letter amino acid sequence acid amino letter

1 T C C K C C C G T

letter ami letter

RGD Mel4

- -

. One .

RGD

melimine melimine Mel4 Mel4 Mel(RGD)4

- - - - -

Table Table Peptide Melimine C C Mel4 C C C GRGDSPC Malamane

The RGD motif was added to peptide sequences with the aim of introducing cytocompatibility. Initially, the motif was introduced at the C-terminus, as this was expected to give the motif the greatest mobility and visibility, given the planned attachment through an N-terminal cysteine (Chen et al. 2012). The resulting peptides were termed C-melimine-RGD and C-Mel4-RGD. Also, the arginine and glycine occurring at positions 12 and 13 of C-Mel4 was exploited; the glycine at position 14 was substituted with an asparagine to produce a peptide with a central RGD sequence, termed C-Mel(RGD)4.

The peptide GRGDSPC (the RGD motif with surrounding residues as found in fibronectin) can influence mammalian cells in a similar manner to fibronectin

(Pierschbacher and Ruoslahti 1984b). To test the activity of a peptide containing both this sequence and C-Mel4 (in their original configurations relative to their cysteine attachment point), GRGDSPC-Mel4 was included in testing.

2.3.2. Antibacterial testing

Bacterial strains used in this study

The bacterial strains used in this study are listed in Table 2.2. Staphylococcus aureus 31 and Pseudomonas aeruginosa 6294 were previously used for melimine testing (Willcox et al. 2008; Dutta et al. 2013). Other strains were clinical isolates from hospital settings and were used to test the broad-spectrum activity of the peptides.

Three strains of Staphylococcus aureus and one strain of Staphylococcus epidermidis were tested as this genus is implicated in approximately two-thirds of orthopaedic and cardiac device infections (Montanaro et al. 2011; Durante-Mangoni et al. 2013; Luk et

53 al. 2014). Two S. aureus isolates from septicaemic patients were tested: one was associated with an infected vascular catheter and the other is methicillin-resistant and non-multiresistant oxacillin-resistant (MRSA-NORSA). Staphylococcus epidermidis strain 013 was isolated from a vascular catheter.

Gram-negative bacteria are a lesser cause of implant associated infections (BAIs) than

Staphylococcus spp., but within the gram-negative pathogens, P. aeruginosa is a major cause of BAIs (Hidron et al. 2008). Escherichia coli is the major cause of catheter- associated urinary tract infections (Cardo et al. 2004). E. coli ATCC 0157, isolated from a urinary catheter, was among the strains tested.

Table 2.2. Bacterial species and strains used in this study and their origins. Bacterial species and strain Isolation site and characteristics Staphylococcus aureus 31 Contact lens-induced peripheral ulcer (School of Optometry and Vision Science, Sydney, Australia) Staphylococcus aureus 004 Vascular catheter associated with septicaemia (Prince of Wales Hospital, Sydney, Australia) Staphylococcus aureus 152 Septicaemia; MRSA/NORSA (St Vincent’s Hospital, Sydney, Australia). Staphylococcus epidermidis 013 Central venous catheter tip (Prince of Wales Hospital, Sydney, Australia) Pseudomonas aeruginosa 6294 Microbial keratitis. Invasive; serogroup O6 (Fleiszig et al. 1994) Escherichia coli ATCC 0157 Bacteriuria. O157 K- (Harkes et al. 1991)

Bacteria were incubated aerobically. Cultures were grown from frozen stocks stored at

−80°C in a 1:1 mixture of glycerol and broth medium. A small amount of material from the frozen vial was streaked onto a chocolate agar plate (Thermo Fisher Scientific

Australia, Scoresby, VIC), and the plate was incubated at 37°C overnight to check purity and obtain single colonies. Material from a single colony was inoculated into

54 tryptone soy broth (TSB; Oxoid, Basingstoke, UK) and incubated for 16-20 h at 37°C, with shaking at 120 RPM, to obtain broth cultures. To minimise variation in phenotypic expression, cultures were not subcultured further before use in experiments.

Minimal inhibitory concentration

The minimal inhibitory concentration (MIC) was determined via a microplate dilution assay in 96-well flat-bottom polystyrene plates (Greiner Bio-One, Kremsmünster,

Austria), chosen for ideal optical properties. The assay medium was Mueller-Hinton broth (MHB; Oxoid) containing 0.2% bovine serum albumin to minimise adhesion of peptides to the well plate surface, and 0.01% acetic acid to reduce precipitation of the cationic peptides (Turner et al. 1998; Hancock 1999). Serial twofold dilutions of the antimicrobial agent were made in a series including 1000 µg/ml (as per a widely- accepted MIC test series (EUCAST 2003)).

The test organism was grown overnight in TSB, and then twice centrifuged and resuspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM

Na2HPO4, 1.8 mM KH2PO, 10 mM Na2HPO4 in H2O, pH 7.4) to remove debris. This suspension was adjusted with PBS to an optical density at 660 nm (OD660) of 0.08 for

P. aeruginosa and E. coli, or 0.12 for Staphylococcus spp., corresponding to approximately 1 × 108 CFU/ml as tested by agar plate counts. This suspension was diluted in sterile assay medium and added to the wells to give a final concentration of

1–2 × 105 CFU/ml (confirmed by retrospective plate count). Blank wells contained sterile medium, and positive growth controls containing no antimicrobial agent were inoculated with bacteria. Wells were prepared in triplicate and the experiment was repeated three times.

The well plates were incubated at 37°C for 24 h with shaking, and the MIC was recorded as the lowest concentration of peptide at which no microbial growth was observed. Inhibition of growth was determined by measuring the OD660 with a spectrophotometer and confirmed by transferring aliquots from wells to a plate of

Mueller-Hinton agar (Oxoid). The plates were incubated at 37°C for 24 h and the resulting colonies were counted to ensure the bacterial density was no greater than the inoculum. Results are reported in both micrograms per millilitre (µg/ml) and micromolar (µM) concentrations to better allow for comparison between peptides.

Minimal bactericidal concentration

The minimal bactericidal concentration (MBC) of peptides was determined within the same assay as the MIC. Aliquots (100 µl) from all wells which were visually growth- free were transferred onto Mueller-Hinton agar. The plates were incubated at 37°C for

24 h and the resulting colonies were counted. The concentration that resulted in a 99.9% from the inoculum was recorded as the MBC (French 2006).

2.3.3. Biocompatibility of soluble peptides with mammalian cells

Cell line and culture conditions

Unless otherwise stated, cell culture materials were purchased from Gibco and solutions were warmed to 37°C before use. Cells were maintained in polystyrene culture flasks

(Greiner Bio-One CELLSTAR®) in a humidified incubator at 37°C with 5% CO2.

Haemolytic activity of AMPs is tested in some cases to investigate the suitability of the peptides for systemic use. As the haemolytic range of melimine is known to be low

(Willcox et al. 2008), and the peptides tested here are intended for use on implant

56 surfaces, their effect on fibroblast viability was chosen as a relevant measure of toxicity.

L929 murine fibroblasts (a standard line for the purpose (International Organisation for

Standardization 2009)) were cultured in Dulbecco's Modified Eagle Medium (DMEM) growth medium supplemented with 10% foetal bovine serum (FBS) and 50 units of penicillin and 50 µg/ml of streptomycin (Life Technologies, Carlsbad, CA, USA).

Medium was changed every two or three days and cells were subcultured when they reached 80–90% confluence. To subculture, cells were rinsed twice with PBS and then dissociated from the flask with 0.05% trypsin in 0.91 mM ethylenediaminetetraacetic acid (trypsin-EDTA) (1 ml per 10 cm2 surface area). Once cells were detached, trypsin was neutralised with three volumes of DMEM containing 30% FBS. Cells in suspension were centrifuged at 500 × g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in fresh medium. Resuspended cells were added to a new flask at a ratio of 1:10 for continued culture.

For experiments, cell density was determined by trypan blue staining: an aliquot of suspended cells was added to an equal volume of 0.4% trypan blue stain (excluded by viable cells), and the cell density counted manually using a Neubauer improved haemocytometer slide and light microscope.

Cytotoxicity Testing

The peptides used in this study were subjected to cytotoxicity testing in accordance with

ISO10993-5:2009 “Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity” (International Organisation for Standardization 2009). Soluble peptides were tested via a cell growth inhibition assay. L929 cells were seeded in a 6-well tissue culture-treated polystyrene plate (Greiner Bio-One) at a density of 5 × 104 cells/cm2.

After 24 h, medium was replaced with 3 ml of fresh DMEM and 1 ml of test solution.

Wells were prepared in triplicate.

Peptides were tested in three-fold dilutions, at final concentrations of 3.7, 11, 33, 100 and 300 mM, chosen to reflect concentrations close to the MICs. Peptides were dissolved in PBS at four times the test concentration and autoclaved before use. Control solutions were PBS (non-cytotoxic control) and ethanol (cytotoxic control, diluted in

PBS to a final concentration of 7.5% (v/v) ethanol). After 48 h incubation with test solutions, all liquid was aspirated. Cells were dissociated with trypsin and stained with trypan blue, and viable (non-stained) cells were counted under a light microscope using a haemocytometer slide and light microscope. Cell numbers were compared to those from wells containing DMEM only, to determine inhibition of proliferation. Inhibition of ≥ 30% indicated cytotoxicity. Cultures in the wells containing blank medium were sub-confluent at this time.

2.3.4. Therapeutic index

The therapeutic index (TI) of each peptide was calculated as its cytotoxic concentration divided by the geometric mean of its MIC values (Chen et al. 2006). This provides an overall measure of suitability as a therapeutic agent as it indicates the size of the therapeutic window: the range of concentrations within which the compound is effective against bacteria without causing cytotoxic effects. A TI greater than that of melimine was considered as an indication that a peptide was a good candidate for further study.

2.4. Results

2.4.1. Antibacterial testing

MICs and MBCs were determined for six bacterial strains. Results are reported in µg/ml in Table 2.3a, and in micromolar concentrations in Table 2.3b. The MIC of the shortened peptide, Mel4, was similar in mass-per-volume terms to that of melimine

(within one dilution). Exceptions were seen with S. aureus 152 and E. coli ATCC 0157, which showed greatly reduced susceptibility to Mel4 in comparison to melimine.

S. epidermidis 013 was consistently more susceptible to melimine-based peptides than were S. aureus strains, which were more susceptible than the gram-negative strains tested. The MRSA-NORSA strain, S. aureus 152, was as susceptible to melimine and its derivatives (with the exception of Mel4) as the non-resistant S. aureus strains.

The addition of cysteine to melimine or Mel4 was generally associated with a slight decrease in MIC and MBC. This effect was pronounced for S. aureus 152 and E. coli

ATCC 0157, for C-Mel4; and for S. epidermidis 013, which was susceptible to

C-melimine and C-Mel4 at 8 µg/ml, a clear reduction from the MICs for melimine and

Mel4 (31 and 63 µg/ml, respectively). The addition of RGD to the N-terminus of C- melimine or C-Mel4 was associated with a moderate increase in MIC. The inclusion of a central RGD motif (in C-Mel(RGD)4) did not alter the MIC from that of C-Mel4. The

MICs of GRGDSPC-Mel4 were higher than for C-Mel4.

0157 0157

2000

> >

/ 213 /

NT NT

63 / 63 / 63 63 / 63 17 / 17 16 / 16 51 / 26 / 50

63 / 125 / 63 180 / 90

250 / 500 / 250 500 / 250 125 / 125 106 331 / 166

500 / 1000 / 500

611 / >611 / 611

coli ATCC coli ATCC coli

2000 / / 2000

E. E.

6294

a 6294 a

2000

/ 32 /

32 32 59 / 59 90 / 45

125 / 125 / 125 250 / 250 500 / 500 500 / 250 500 / 500 250 / 125 264 / 132 213 / 213 204 / 102 180 / 180 331 / 166

500 / 1000 / 500 1000 / 500

>611 / >611 / >611

aeruginosa aeruginosa aeruginos

>2000 / / >2000

P. P.

013 013

2000

NT NT

8 / 31 / 8 16 / 8

31 / 63 / 31 63 / 63 63 / 63 31 / 16 63 / 63 27 / 27 23 / 23 21 / 21

8.4 / 17 / 8.4 11 / 5.8

2.1 / 4.2 / 2.1 6.7 / 3.3

611 / >611 / 611

epidermidis epidermidis epidermidis

>2000 / / >2000

S. S.

ains. ains.

152 152

2000

166

/ 851 /

100

NT NT

/ /

8 / 16 / 8

31 / 63 / 31 34 / 17 51 / 26 90 / 90 0

63 / 125 / 63 125 / 63 / 83

aureus aureus aureus

250 / 250 / 250 250 / 125 500 / 250 42 5

1000 / 2000 / 1000

>611 / >611 / >611

S. S.

>2000 / / >2000

004 004

2000

/ 1 /

NT NT

3 / 63 / 3

8 / 16 / 8

31 / 63 / 31 6 34 / 17 53 / 53 26 / 26 83 / 83

63 / 125 / 63 180 / 90

aureus aureus

125 / 125 / 125 500 / 250 500 / 500 250 / 250 1

>611 / >611 / >611

S. S.

>2000 / / >2000

ml) of peptides for various bacterial strains. strains. bacterial various for peptides of ml)

s 31 s

C (µM) C

/ 5 /

8 / 16 / 8

31 / 63 / 31 63 / 63 34 / 17 59 / 30 27 / 27 51 / 26 90 / 45 25 83 / 42

aureu aureus

63 / 125 / 63 125 / 63 125 / 63

125 / 250 / 125 250 / 125 250 / 125

>611 / >611 / >611

S. S.

>2000 / 2000 / >2000

MIC / MB / MIC

MIC / MBC (µg/ml) (µg/ml) MBC / MIC

RGD Mel4 RGD Mel4

- - - -

a. MICs and MBCs (µg/ MBCs and MICs a. str bacterial various for peptides of (µM) MBCs and MICs b.

RGD RGD

. .3

- -

2 2

melimine melimine Mel4 Mel4 Mel(RGD)4 melimine melimine Mel4 Mel4 Mel(RGD)4

------

Table Table Peptide Melimine C C Mel4 C C C GRGDSPC Malamane tested not NT= Table Peptide Melimine C C Mel4 C C C GRGDSPC Malamane tested not NT=

For malamane, the MIC was observed only for E. coli ATCC 0157, at the highest test concentration of 2000 µg/ml. For P. aeruginosa 6294 a dose-dependent decrease in

OD660 was observed in malamane—from an OD600 of ~1.6 at 0 µg/ml of peptide to

~1.2 at 2000 µg/ml of peptide (CFU was not determined). For all other strains tested, no difference in OD660 was observed between the blank growth medium and up to

2000 µg/ml of malamane.

2.4.2. Cytotoxicity Testing — Cell Growth Inhibition

L929 cells were counted after incubation in each test solution. Cell densities are displayed in Figure 2.1 as a percentage of cell density from wells containing DMEM only, with a density of 70% or less indicating cytotoxicity. The positive cytotoxic control, 7.5% ethanol (EtOH), resulted in 99–100% cell death in all experiments. The blank sample, 25% PBS without peptide, was associated with a 4–19% reduction in viable cell density. Most peptides tested were found to be cytotoxic to L929 cells at

33 mM. Exceptions were Mel4 (Figure 2.1 D), which was cytotoxic at 300 mM; C-Mel4 and GRGDSPC-Mel4 (Figure 2.1 E,G), cytotoxic at 100 mM and above; and malamane

(Figure 2.1 I), which was not cytotoxic at any concentration tested, including 300 mM.

For most peptides, concentrations below the cytotoxic threshold were associated with increased cell density.

For full length melimine, the addition of cysteine and RGD had little effect (Figure 2.1

A–C). For Mel4, however, the cytotoxicity increased with the addition of cysteine and further with the addition of RGD (Figure 2.1 D–F). The profile of C-Mel(RGD)4

(Figure 2.1 H) was similar to melimine, C-melimine and C-melimine-RGD, with increased cell density at 3.7 and 11 µM and minimal cell numbers at 33 µM and above.

Figure 2.1. Density of L929 murine fibroblasts after incubation with soluble peptides as a percentage of density in standard growth medium. A cell density of <70%, indicated by the dashed line, indicates cytotoxicity. Error bars represent the standard deviation of three separate experiments. (A) Melimine. (B) C-melimine. (C) C-melimine- RGD. (D) Mel4. (E) C-Mel4. (F) C-Mel4-RGD. (G) GRGDSPC-Mel4. (H) C-Mel(RGD)4. (I) Malamane.

The profile of GRGDSPC-Mel4 (Figure 2.1 G) was most similar to Mel4 and C-Mel4-

RGD, with no increase in cell density at low concentrations, although at the cytotoxic threshold there was a sudden drop from no measured effect to complete cell destruction at 100 µM, whereas for Mel4 and C-Mel4-RGD a gradual cytotoxic effect was seen with increasing concentration.

2.4.3. Therapeutic indices

TIs for each peptide are shown in Table 2.4. A value above the TI of melimine (1.55) was considered indication of suitability of the peptide for further testing. C-melimine,

C-Mel4 and Mel4 fulfilled this requirement with TIs of 3.65, 5.62 and 4.41, respectively. The therapeutic index for malamane could not be calculated as neither cytotoxic concentration nor MICs for most bacterial species was determined.

Table 2.4. Therapeutic indices of the peptides tested in this study. Values were calculated as the cytotoxic concentration divided by the geometric mean of MICs for each peptide. Therapeutic indices greater than that of melimine are emphasised in bold type. Cytotoxic Geometric mean Therapeutic Peptide concentration of MICs index Melimine 33 21.27 1.55 C-melimine 33 9.05 3.65 C-melimine-RGD 33 42.07 0.78 Mel4 300 84.79 3.54 C-Mel4 100 22.68 4.41 C-Mel4-RGD 33 71.70 0.46 C-Mel(RGD)4 33 39.81 0.83 GRGDSPC-Mel4 100 83.33 1.20

2.5. Discussion

Antimicrobial activity

The greater melimine susceptibility of S. aureus in comparison to P. aeruginosa is consistent with previous findings (Rasul et al. 2010). However, Rasul et al. reported

MICs for melimine of 125 µM (475 µg/ml) for P. aeruginosa 6294 and 3.9 µM

(15 µg/ml) for S. aureus 31. The former is consistent with this study, but the MIC for melimine against S. aureus 31 was found in this study to be 63 µg/ml. This difference could be due to the use of polystyrene plates in this study, in comparison to the polypropylene plates used by Rasul et al. (2010). Although Hancock (1999) reported that the addition of bovine serum albumin (BSA) and acetic acid to the assay medium considerably reduced adhesion of cationic peptides to polystyrene, the MICs for two cationic AMPs were doubled when polystyrene plates were used, compared to when polypropylene plates were used. This effect is likely to be greater at lower dilutions, and so the polystyrene plates used in this study (chosen for optical properties) might underestimate the activity of these peptides in the lower concentration range.

For most strains tested, the MIC for Mel4 was two or three times higher (in molar terms) than for melimine, but for E. coli ATCC 0157 the MIC of Mel4 was nine times higher, and for the MRSA strain S. aureus 152, twenty-five times higher.

The addition of an N-terminal cysteine to Mel4 had little effect on MICs for antibiotic- susceptible S. aureus, but for S. aureus 152 the MIC of C-Mel4 was much lower than for Mel4, similar to the susceptible strains. For S. epidermidis, P. aeruginosa and

E. coli, MICs for either melimine or Mel4 or both was reduced with the addition of an

N-terminal cysteine.

This increase in potency following the addition of a single cysteine might be related to the small increase in peptide length, especially considering that the shorter peptide Mel4 and its variants had MICs higher than their full-length counterparts. Additionally, the added cysteine might be able to form disulfide bonds with other peptide molecules in the medium. Disulfide bond formation in a non-biological environment has been reported in synthetic peptides, and can be investigated with mass spectrometry

(MacColl et al. 2001).

C-melimine and C-melimine-RGD precipitated to some extent to give a cloudy appearance in MHB at concentrations of 125 µg/ml and above. For P. aeruginosa, the

MICs for C-melimine and C-Mel4 were recorded as 125 and 250 µg/ml, respectively; these values may be higher than the true MIC if precipitated peptides were unable to interact with the bacteria. This precipitation was not seen with C-Mel4 or C-Mel4-RGD, and therefore appears dependent on either a minimum peptide length or the inclusion of the (uncharged) resides that exist in full length melimine but not in Mel4.

The increase in MIC seen with the addition of the RGD motif to the N-terminus might be related to a capping effect, with the anionic aspartic acid hindering the interaction between the cationic arginine side chain and the bacterial surface. This is supported by the partial restoration of activity seen when the RGD motif was instead placed centrally with minimal difference to the charge-to-length ratio, as in C-Mel(RGD)4. The addition of RGD near the C-terminus in GRGDSPC-Mel4 was also associated with an increase in MIC despite no capping of the polyarginine tail; however, the charge-to-length ratio for GRGDSPC-Mel4 is the lowest of any tested in this study, so this is a likely factor in diminishing antibacterial activity.

The MBC of all peptides tested was in most cases the same as, or twice, the MIC (with the exception of C-melimine and S. epidermidis 013, which had an MIC of 8 µg/ml and an MBC of 31 µg/ml). This is a common feature of antimicrobial peptides (Liu et al.

2011; Suwandecha et al. 2015) which exert their activity largely through lethal disruption of the bacterial membrane (Hancock and Rozek 2002).

A previous study (Rasul 2010) found that a variant of melimine designed by deleting all charged residues was ineffective against bacteria. The results for malamane indicated more specifically that one or more of the six arginine residues at positions 18–23 of melimine are necessary for antibacterial activity. Substitution of cationic residues of short AMPs with alanine can be highly detrimental to antimicrobial activity (Ryan et al.

2006) but in some cases enhances it (Taniguchi et al. 2014), demonstrating that charge density is only one of a number of factors responsible for antimicrobial activity, and its effect is peptide-specific. For a given peptide, a single amino acid substitution can have different effects on potency against different bacterial strains (Norberg et al. 2011).

Thus, while there appears to be an ideal range of charge density for any particular AMP, instances of unpredictable results highlight the need for empirical testing of any sequence modifications.

Cytotoxicity

The cationic residues not present in malamane appeared to be important for cytotoxicity as well as antibacterial activity—malamane was the only peptide tested to have no measureable cytotoxic activity at 300 µM. Short cationic peptides have been reported to enter mammalian cells, with 7-15 consecutive arginine residues being most efficient

(Mitchell et al. 2000, Uemura et al. 2002). The ten C-terminal arginines of melimine

66 might interact with the mammalian cell membrane in this manner, despite the presence of two glycine residues in the middle of the sequence. Mel4, C-Mel4 and C-Mel4-RGD showed lower cytotoxicity than their full-length counterparts, despite retaining all the arginines of the full-length sequence, suggesting that peptide length and/or inclusion of a certain number or bulk of hydrophobic residues is a factor in the cytotoxicity of melimine variants.

The presence of at least one tryptophan (Trp, W) and a number of arginines towards the

N-terminus, as in melimine, is seen in biological AMPs including tachyplesin from the horseshoe crab (Nakamura et al. 1988), lactoferricin (an active fragment of bovine lactoferrin (Yamauchi et al. 1993)) and indolicidin, from bovine neutrophils (Selsted et al. 1992). In aqueous medium, bulky, non-polar tryptophan has a preference for the lipid bilayer/medium interface. It is energetically favourable for the arginine side chain to be near to tryptophan, and this association is thought to assist arginine insertion into the bacterial membrane after arginine first brings the peptide into close contact with the negatively-charged outer leaflet (reviewed in Chan et al. 2006; Shagaghi et al. 2016).

It also appears that the structure of tryptophan allows for “fine-tuning” of a peptide’s hydrophobic bulk: depending on the peptide, substitution of compact hydrophobic residues with tryptophan has been shown to increase antibacterial activity and reduce haemolytic activity (Bi et al. 2014), or vice versa (Podorieszach and Huttunen-Hennelly

2010). Despite sacrificing any benefits of the tryptophan-arginine combination, C-Mel4 was found to have very similar MICs as melimine against a range of pathogens. This retention of antibacterial activity despite the lack of any hydrophobic side chains may be due to increased charge-to-length ratio. The lesser cytotoxicity of Mel-4 and C-Mel4

67 to fibroblasts is perhaps not surprising, given the importance of amphipathicity in the cytotoxicity activity of some AMPs (Ryge et al. 2004; Wessolowski et al. 2004).

C-Mel(RGD)4 unexpectedly had a similar profile to the longer peptides, enhancing growth at low concentrations and exhibiting toxicity at 33 µM. In contrast to C-Mel4 it contains a central RGD motif, which might enhance its interaction with fibroblasts, and in comparison to C-Mel4-RGD there is no anionic cap at the C-terminus that might interfere with the cell-penetrating ability of the arginine tail. Ma et al. (2014) reported a clear increase in the MIC of an AMP with the deletion of two C-terminal arginine residues, exposing two hydrophobic residues at the C-terminus, but it is difficult to compare this result to the current findings as the peptide modification described by Ma and colleagues reduced the net charge considerably from +6 to +4.

The increased cell density associated with the addition of peptides below their cytotoxic threshold is presumably due to the provision of amino acids enriching the growth medium. Notably, this enhancement of growth was not seen with Mel4, C-Mel4-RGD, or GRGDSPC-Mel4. Possible explanations include that these particular peptides are internalised by the cells to a lesser degree, or are more quickly degraded by proteases.

2.6. Conclusions

C-Mel, C-Mel4 and Mel4 showed similar antibacterial activity to melimine, and similar or lesser toxicity towards fibroblasts. Mel4 has provided insight into the relationship between peptide sequence and effects on bacterial and mammalian cells, but does not contain the C-terminal cysteine necessary for directed attachment. C-Mel, C-Mel4 will be tested in the following chapter to determine their broad-spectrum activity and

68 mammalian cell response when the peptides are attached to surfaces. C-Mel4-RGD will also be tested; although it was a poor antibacterial agent, it was less toxic to fibroblasts than melimine, and it is of particular interest to determine the mammalian call response to a melimine-based peptide containing the RGD motif.

The methicillin-resistant isolate of S. aureus tested was as susceptible to melimine- based peptides as the antibiotic-susceptible strains.

The minimal activity of malamane observed in this study indicated that at least one of the six of the arginine residues at positions 18–23 of melimine is necessary for both cytotoxicity and antibacterial activity.

CHAPTER 3. PREPARATION AND EVALUATION OF SURFACES COATED WITH MELIMINE AND DERIVATIVE PEPTIDES

3.1. Introduction

The results of the previous chapter demonstrated that C-melimine and C-Mel4 have higher therapeutic indices than melimine and maintain a similar MIC. These two peptides also contain a cysteine residue, allowing site-directed attachment, and were therefore considered good candidates for an antimicrobial surface coating. Soluble

C-Mel4-RGD and C-Mel(RGD)4 did not perform as well in this regard, but it was decided to also test these peptides in this chapter as the results for these two peptides could inform later modification steps involving the RGD motif.

The objectives of the studies that form the basis of this chapter were to establish a reliable and practical coating method that can be used to attach melimine-derived peptides to medical device surfaces, and to test the antibacterial properties and cytotoxicity of these peptide-coated surfaces.

3.1.1. Peptide surface attachment

In addition to possessing the desired biological properties, a biomaterial coating should be robust and suitable for industrial production: scalable, easily sterilised, and cost- effective.

Covalent attachment is the most suitable method for attachment of active peptides to surfaces, due to its stability. Physical adsorption from solution is simple, but prone to uneven distribution and desorption, especially for smaller proteins. While a substantial

71 amount of protein can be adsorbed to surfaces, covalent binding becomes more efficient at higher concentrations of available protein (Steffens et al. 2002). Thus, adsorption may be disproportionately inefficient for short polypeptides such as the melimine derivatives tested here. Impregnation of porous materials which then release an active substance at a controllable rate allows relatively high doses to be delivered locally, while staying below systemic toxicity levels. The release of conventional antibiotics from porous surfaces and has achieved success in animal trials (Liu et al. 2010; Spicer et al. 2013), and effective AMP-releasing surfaces have been reported in vitro

(Kazemzadeh-Narbat et al. 2010; Shukla et al. 2010). However, these systems are more suitable as a drug delivery platform than a long-lasting anti-infective surface. Also, the release of a finite amount of an antimicrobial agent necessarily involves exposure to sub-inhibitory concentrations as the agent is depleted (unless the release can be extremely fine-tuned). Although no bacterial resistance to melimine was seen after lengthy exposure to sub-inhibitory concentrations (Willcox et al. 2008), as a precaution this peptide-leaching approach has not been followed.

The range of available covalent attachment methods is dictated by the surface chemistry of the substrate. Common biomaterials are chosen for their ideal mechanical properties and biological inertness: ceramics, titanium and stainless steel for orthopaedics; and silicone rubber or polyurethane for vascular devices (Maki and Ringer 1991; Bhat and

Kumar 2013). The surface chemistry of these materials varies widely, and so attachment methods must vary accordingly unless the coating process involves a step that standardises the surface chemistry.

Anti-infective and pro-mammalian cell functionality has been reported on titanium surfaces modified with multilayer polyelectrolytes (Chua et al. 2008). This technique is mild and straightforward but the longevity of the surface is uncertain. Covalent attachment of antimicrobial and cell-active peptides has been reported on flexible polymers (Biltresse et al. 2005; Mishra et al. 2014) and titanium (Zhang et al. 2008; Chen et al. 2013). However, the specifics of these techniques are dependent on the underlying substrate, and in the case of titanium still require a priming step such as oxygen plasma or harsh chemical treatment to produce reactive surface groups.

Plasma polymerisation is an ideal technique for applying the desired chemistry to the outer surface of medical devices of any material, allowing a single peptide attachment method to be used. In this method, a gaseous monomer is polymerised on the surface of a substrate in the presence of an electric discharge. In comparison to conventional polymers, plasma polymers offer pinhole-free coating of odd-shaped substrates, and greater resistance to physical and chemical perturbation due to a high degree of branching and cross-linking. While specialised equipment is required, the cost of materials is low as a few millilitres of monomer can efficiently coat a large substrate area with a uniform film to a depth of tens of nanometres up to microns (Yasuda 1981).

3.1.2. Surface attachment of melimine-based peptides

Melimine is amenable to covalent surface attachment methods which are chemically mild and can be performed on a large scale (Chen et al. 2009; Chen et al. 2012), and it retains antimicrobial activity after steam sterilisation, either in solution (Willcox et al.

2008) or when covalently attached to a surface (Dutta et al. 2013).

Melimine has previously been covalently attached to surfaces by crosslinking the primary amines of cationic residues to substrate carboxyl groups. These carboxyls were either added to a glass surface via a linker molecule (Chen et al. 2009) or inherent in a polymer structure (Willcox et al. 2008; Dutta et al. 2013). The latter direct coupling method led to a greater than 99% reduction in bacterial adhesion for melimine and more than 90% reduction for Mel4, and provided peptide coverage of approximately

10 nmol/cm2 for both peptides (Dutta et al. 2016). This method was used in the present chapter to assess the activity of a selected subset of peptides.

For C-melimine, directed covalent attachment has been achieved through the provision of surface amine groups, and coupling of the sulfhydryl group of cysteine with surface amines via a maleimide linker. Attachment on a glass surface via an amine-containing linker molecule resulted in an estimated peptide coverage of 3.5–4.0 nmol/cm2 and approximately 95% reduction in bacterial coverage (Chen et al. 2012). Attachment via plasma coating with heptylamine resulted in approximately 90% reduction in bacterial adhesion (Chen 2012). In the present chapter, plasma polymer coating was chosen to provide surface amines, similar to the latter method.

Fluorinated ethylene propylene (FEP) was chosen as a substrate for plasma polymer coating. FEP is a copolymer of hexafluoropropylene and tetrafluoroethylene, and is chemically inert. As such, it provides a worst-case scenario, i.e. if the method can coat

FEP it can be expected to coat any in-use substrate. Although FEP is not the material of choice for long-term devices in contact with the vascular system (di Costanzo et al.

1988; Maki and Ringer 1991), it has been used in drug delivery devices (Rayman and

Wise 1988).

3.2. Aims of the chapter

The aims were to establish a reliable surface attachment method for the peptides used in this study, and to determine the antimicrobial efficacy and cytotoxicity of these surfaces.

3.3. Materials and Methods

3.3.1. Peptides

The peptides tested in experiments of the present chapter are listed in Table 3.1.

Peptides were synthesised by American Peptide Company (Sunnyvale, CA, USA) using conventional solid-phase synthesis and Fmoc chemistry. The peptide purity of supplied sequences was >95% by weight.

Table 3.1. Amino acid sequences of peptides tested in this chapter. Peptide 1-letter amino acid sequence Melimine T L I S W I K N K R K Q R P R V S R R R R R R G G R R R R C-Melimine C T L I S W I K N K R K Q R P R V S R R R R R R G G R R R R C-Mel4 C K N K R K R R R R R R G G R R R R C-Mel4-RGD C K N K R K R R R R R R G G R R R R R G D C-Mel(RGD)4 C K N K R K R R R R R R G D R R R R GRGDSPC G R G D S P C

3.3.2. Peptide coating of hydrogel polymer surfaces

Poly-2-hydroxyethylmethacrylate (pHEMA) was initially used as a substrate for peptide attachment as it has previously been used with melimine and the surfaces shown to be antimicrobial and non-toxic to mouse fibroblasts (Dutta et al. 2013). The polymer structure contains reactive hydroxyl and carboxyl surface groups that allow for covalent

75 attachment of cationic peptides. PHEMA was readily available in the form of Etafilcon

A contact lenses (Johnson & Johnson, Jacksonville, FL, USA). Coupling of amine groups of the peptide (found on cationic residues) to carboxyl groups of the polymer was carried out as per the method of Dutta et al. (2013), shown in Figure 3.1.

Figure 3.1. Reaction scheme for covalent attachment of cationic peptides to pHEMA. pHEMA: poly-2-hydroxyethylmethacrylate; EDC: 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide.

PHEMA lenses from their original packaging were first rinsed three times with sodium acetate buffer, pH 5.0 (36 mM acetic acid, 64 mM sodium acetate in H2O). Lenses were then incubated with 1-[(3-dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride

(EDC; Sigma, MO, USA) at 10 mg/ml in sodium acetate buffer for 20 min to activate carboxyl groups for further modification. Activated lenses were rinsed three times with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,

1.8 mM KH2PO, 10 mM Na2HPO4 in H2O, pH 7.4), and then incubated with peptide solution (0.80 mM in PBS) for 2 h. After peptide coupling, lenses were rinsed three times with PBS, and then soaked for 2 h in 10% (w/v) NaCl to leach residual peptide from the pHEMA matrix. Lenses were finally rinsed three times with PBS.

Process controls were prepared by the same procedure, using blank PBS instead of peptide solution during the coating step. To sterilise, lenses were autoclaved in PBS, in glass vials at 121°C for 20 min. Previous studies have shown that melimine retains activity after autoclaving, either in solution (Willcox et al. 2008) or when covalently attached to pHEMA (Dutta et al. 2013).

3.3.3. Peptide coating of fluorinated ethylene propylene surfaces

Commercially available FEP film (0.25 mm thickness, Goodfellow, Huntingdon, UK) was used as a substrate for cysteine-directed peptide attachment. FEP was cleaned by rinsing in dichloromethane and then ethanol, stored in ethanol, and dried under a stream of nitrogen gas before use.

FEP was coated with a plasma polymer of allylamine (ppAllylamine) at the Biointerface

Lab, a node of the Australian National Fabrication Facility, at Swinburne University of

Technology, Hawthorn, VIC. The purpose of this coating was to provide primary amines for further modification on the sample surface. Coating was carried out in a custom-built stainless steel reactor (scheme shown in Figure 3.2) comprising a cylindrical chamber 20 L in volume with aluminium plate electrodes at each end.

A radio frequency (RF) generator (13.56 MHz, Coaxial Power Systems Ltd., UK) was used to generate a plasma phase; impedance was matched such that reflected power was

<0.2 W. The cathode end included an inlet connected to a glass flask containing allylamine monomer (Sigma). The monomer was freeze-dried under vacuum three times before use to remove dissolved gases. A needle valve allowed fine control of the flow rate of monomer vapour from the flask into the reaction chamber. A Pirani gauge

77 allowed monitoring of pressure inside the chamber. The chamber outlet flowed through a trap cooled by liquid nitrogen to condense monomer vapour.

Figure 3.2. Scheme of the plasma polymer reactor used for polyallylamine coating.

Samples were placed near the anode end. The chamber was evacuated to a pressure of

2.0 × 10-4 mbar. A flow of monomer vapour into the chamber was established at a rate

3 of 1.9–2.0 cm (STP)/min, as per the usual protocols of the laboratory, using an equation derived from the ideal gas law (Gengenbach and Griesser 1998):

F = (dp/dt) ×16172 V/T where F = flowrate(cm3 (STP)/min); p = pressure (mbar); t = time (s); V = volume of the plasma reactor, 20 L; T = temperature, 293°K. Once this flow was established, the total pressure in the chamber was 1.2–1.3 × 10-2 mbar. An RF power of 20 W was discharged for 20 min to achieve plasma activation. After deposition, the flow of

78 monomer was continued for 5 min to quench reactive sites. The chamber was finally evacuated to 2.0 × 10-4 mbar before being re-pressurised and opened.

Peptides were attached to amine-functionalised surfaces using a modified method of

Chen (2012), shown in Figure 3.3.

Figure 3.3. Reaction scheme for peptide coating of fluorinated ethylene propylene (FEP) with C-Mel4 shown as a representative peptide. EDC: 1-[(3-dimethylamino)- propyl]-3-ethylcarbodiimide hydrochloride. SMCC: succinimidyl-4-[N- maleimidomethyl]cyclohexane-1-carboxylate.

The non-water soluble amine-to-sulfhydryl crosslinker succinimidyl-4-[N- maleimidomethyl]cyclohexane-1-carboxylate (SMCC; ProteoChem, IL, USA) was used in place of sulfo-SMCC. PpAllylamine-coated samples were immersed in a solution of

SMCC at 2 mg/ml and EDC at 10 mg/ml, in dimethylsulfoxide (DMSO, Sigma) for 1 h.

Samples were then rinsed three times with DMSO and three times with PBS containing

10 mM EDTA (PBS–EDTA, pH 7.2). SMCC-functionalised surfaces were immersed in peptide solution (0.51 mM in PBS–EDTA) for 2 h. Samples were then rinsed three times with PBS–EDTA and three times with MilliQ ultrapure water, and allowed to dry in air.

The controls prepared were unmodified FEP, ppAllylamine-coated FEP, and a process control of SMCC (exposed to PBS–EDTA containing no peptide for 2 h). FEP samples were disinfected by soaking in 70% ethanol for 20 min and dried in a biosafety cabinet.

This procedure was sufficient to prevent microbial growth when the samples were later incubated in sterile growth medium.

3.3.4. Physicochemical analysis of prepared FEP samples

The composition and condition of samples at each stage of coating was examined by thin-film ellipsometry, x-ray photoelectron spectroscopy, contact angle analysis and quantitative amino acid analysis. Analysis was performed on sterilised/disinfected samples to ensure any effects from the sterilisation/disinfection process were taken into account.

Ellipsometry

Ellipsometry was used to measure the thickness of deposited plasma polymer films.

Silicon wafers were placed in the plasma polymer reactor alongside FEP during the coating process and ellipsometry measurements were performed on the coated wafers.

The Auto-EL III Ellipsometer with software version 3.9 (Rudolph Research Analytical,

NJ, USA) at the Biointerface Lab, Swinburne University of Technology, was used with a 632.8 nm laser source.

Amino Acid Analysis

Samples were subjected to commercial quantitative amino acid analysis (AAA). Testing was carried out at the Australian Proteome Analysis Facility, Macquarie University,

Macquarie Park, NSW, facilitated using infrastructure provided by the Australian

Government through the National Collaborative Research Infrastructure Strategy

(NCRIS). Duplicate samples of FEP–ppAllylamine–SMCC coated with C-melimine,

C-Mel4, or GRGDSPC were analysed using the High Sensitivity AAA service (gas phase hydrolysis). Samples were weighed before gas phase hydrolysis in 6 M hydrochloric acid at 110°C for 24 h. Hydrolysed amino acids were extracted from the samples in 20% acetonitrile and analysed using the Waters AccQTag Ultra chemistry on a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system.

Duplicate samples of FEP coated with C-melimine, C-Mel4 and GRGDSPC were analysed along with process controls (FEP-ppAllylamine-SMCC). The analysis protocol hydrolyses asparagine and glutamine to aspartic acid and glutamic acid, respectively, and destroys cysteine and tryptophan.

X-ray Photoelectron Spectroscopy

X-ray Photoelectron Spectroscopy (XPS) was used to detect the relative abundance of elements on a sample surfaces as well as the chemical states of the elements present after each stage of coating. Allylamine coatings were analysed 1, 10 and 23 days after coating to observe changes in the film composition during short-term storage. Samples were stored in polystyrene Petri dishes sealed with parafilm, either in air or flooded with nitrogen gas.

Theoretical nitrogen-to-carbon (N/C) and oxygen-to-carbon (O/C) values for surface coatings were calculated from the chemical formulas of each coating and therefore assumed a defect-free coating layer greater than the XPS sampling depth (generally 3–

10 nm (Briggs 1998). Practically, this is not the case for all surfaces tested; this is discussed following the XPS results.

XPS was carried out at the Mark Wainwright Analytical Centre at the School of

Chemistry, University of New South Wales, Kensington, NSW, using an ESCALAB

250 Xi (Thermo Scientific, Waltham, MA, USA). Vacuum pressure was ≤ 10-8 mbar.

The X-ray source was monochromated Al Kα (energy 1486.6 eV) with a source power of 150 W. The spot size was 5000 μm in diameter and the photoelectron takeoff angle was 90°. Curve fitting was carried out on XPS spectra using the CasaXPS program

(Casa Software Ltd., Devon. UK). C1s = 285.0 eV for adventitious hydrocarbon was used as a binding energy reference.

Contact angle measurements

Advancing contact angles for FEP surfaces were measured with a contact angle goniometer (Ramé-Hart Inc., Model 200-FI, Succasunna, NJ, USA). Four 5 μl droplets of MilliQ ultrapure water were placed on duplicate samples of each surface using a micro-syringe (droplet volume has been reported to have minimal effect on advancing static water contact angle between volumes of 5 and 25 μl (Miyama et al. 1997; Harsch et al. 2000)). The droplets were photographed with a NET GmBH 1394 digital camera and the angle between each droplet and the surface measured with DROPimage® imaging software. A contact angle of 90° or greater indicates hydrophobicity and acute contact angles indicate hydrophilicity.

3.3.5. Bacterial strains and culture conditions

The bacterial strains used in this chapter are listed in Table 3.2. All cultures were incubated aerobically. The test organism (stored at −80°C in a 1:1 mixture of glycerol and broth medium) was streaked onto chocolate agar (Thermo Fisher Scientific

Australia, Scoresby, VIC) and incubated at 37°C overnight to obtain single colonies.

Material from a single colony was inoculated into tryptone soy broth (TSB; Oxoid) and incubated at 37°C overnight with shaking at 120 RPM. Broth cultures were twice centrifuged and bacterial cells resuspended in PBS to remove medium components, and then diluted with PBS to an optical density at 660 nm of 0.08 for P. aeruginosa and

E. coli, or 0.12 for Staphylococcus spp., corresponding to approximately

1 × 108 CFU/ml (as tested by agar plate counts). Cultures were diluted 1:100 in test medium (as described in the following sections) for testing.

Table 3.2. Bacterial species and strains used in this study and their origins. Bacterial species and strain Isolation site and characteristics Staphylococcus aureus 31 Contact lens-induced peripheral ulcer (School of Optometry and Vision Science, Sydney, Australia) Staphylococcus aureus 004 Vascular catheter associated with septicaemia (Prince of Wales Hospital, Sydney, Australia) Staphylococcus aureus 152 Septicaemia, MRSA/NORSA (St Vincent’s Hospital, Sydney, Australia). Staphylococcus epidermidis 013 Central venous catheter tip (Prince of Wales Hospital, Sydney, Australia) Staphylococcus epidermidis Bacteraemia, slime producer ATCC 35983 (Tennessee, USA) Pseudomonas aeruginosa 6294 Microbial keratitis, invasive, serogroup O6 (Fleiszig et al. 1994) Escherichia coli ATCC 0157 Bacteriuria, O157 K- (Harkes et al. 1991)

Bacterial adhesion to pHEMA surfaces

The test medium for P. aeruginosa was PBS. For all other species the test medium was

10% TSB (diluted in PBS). Preliminary experiments found that in PBS alone

P. aeruginosa adhered to pHEMA in numbers ideal for comparison, while the addition of growth medium led to overgrowth.

1 ml of a 106 CFU/ml suspension was added to wells of a 24-well polystyrene plate

(Greiner Bio-One) each containing a sterilised peptide-coated pHEMA sample or a process control. Samples were incubated at 37°C for 24 h with shaking at 120 RPM.

Following incubation, samples were gently rinsed three times with PBS, allowing 1 min shaking per rinse.

Adhesion of viable bacteria was quantified via viable counts. Rinsed samples were placed in 5 ml polypropylene vials with 2 ml of PBS and a small Teflon®-coated stirring bar, and vortexed at high speed to dissociate adherent bacteria. 1:10 dilutions were made in PBS and aliquots were placed on tryptone soy agar containing 0.1%

Tween-80 and 0.05% lecithin (TSAT) for the neutralisation of antimicrobial agents

(Baker et al. 1984; Cousido et al. 2008) and incubated at 37°C overnight. The resulting colonies were counted to determine the numbers of viable bacteria recovered from the samples.

Aliquots of the supernatant from these experiments were diluted and plated on TSAT to determine any differences in bacterial growth between samples. The experiment was repeated at least three times with duplicate samples.

Bacterial adhesion to fluorinated ethylene propylene surfaces

The test medium was 1% TSB (diluted in PBS) for P. aeruginosa, or 10% TSB (diluted in PBS) with the addition of 0.25% (w/v) glucose for all other genera. Pilot experiments demonstrated that these conditions favoured bacterial adhesion to FEP surfaces.

1 ml of a 106 CFU/ml bacterial suspension was added to wells of a 24-well plate each containing a disinfected FEP sample. Samples were incubated at 37°C for 48 h with shaking at 60 RPM. Medium was aspirated and replenished at 24 h. Following incubation, samples were gently rinsed three times with PBS (no shaking or waiting time between rinses).

Percentage coverage of viable and non-viable bacteria was determined via staining and microscopy. Samples were placed on a microscope slide and 10 μl of LIVE/DEAD®

BacLight™ stain (Molecular Probes, OR, USA) prepared according to the manufacturer’s instructions was placed on each sample. The drop was trapped with a coverslip and samples were incubated for 15 min in the dark before imaging with a laser scanning confocal microscope using a 10× objective lens (FV1000, Olympus, Tokyo,

Japan). For detecting SYTO 9 viable stain and propidium iodide non-viable stain, excitation wavelengths were 473 and 559 nm and emission wavebands were 485–545 and 570–670 nm, respectively. Channels were scanned sequentially to avoid cross-talk.

Bacteria stained with SYTO 9 (green) were assumed to be viable, while bacteria stained with propidium iodide (red) were assumed to be non-viable. Nine fields of duplicate samples were imaged.

Microscopy images were assessed using ImageJ software. Red and green channels were separated and converted to 8-bit images. Images were binarised using the Image >

Adjust > Threshold function, using the same settings per experiment. Bacterial coverage was quantified with the “Area Fraction” option of the Analyze > Measure function.

Aliquots of the supernatant from these experiments were diluted and plated on TSAT to determine any differences in bacterial growth between samples. The experiment was repeated at least three times.

3.3.6. Cytotoxicity Testing — Direct Contact Assay

Tissue integration with the coated surfaces prepared here is the overall aim. However, more straightforward cytotoxicity testing is a prudent first step. Peptide-coated pHEMA and FEP surfaces were screened qualitatively for cytotoxicity via a direct contact assay carried out in accordance with ISO10993-5:2009 “Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity”(International Organisation for

Standardization 2009).

L929 murine fibroblasts were seeded in 6-well plates (Greiner Bio-One CELLSTAR®) as for cytotoxicity testing of soluble peptides (§2.3.3). When the cells were 80% confluent, the test sample (14 mm diameter pHEMA or 1 cm square FEP) was placed on the cell monolayer. Wells were prepared in triplicate. Non-cytotoxic controls were commercially available pHEMA contact lenses (rinsed three times in sterile PBS).

Cytotoxic controls were 14 mm circles of latex cut from powder-free gloves

(Livingston, Rosebery NSW Australia), disinfected by soaking in 70% ethanol and then rinsed three times in sterile PBS.

After 24 h incubation, the cell monolayer was visually inspected using an inverted light microscope at 20× magnification. Cells were graded according to the recommendations

86 set out in ISO 10993–5:2009 (International Organisation for Standardization 2009) based on morphology, where a grade of 3 or 4 is indicative of cytotoxicity (Table 3.3).

While only semi-quantitative, this method is appropriate for screening purposes.

Table 3.3. Cytotoxicity grading scale for direct contact testing as per ISO 10993- 5:2009. Grade Reactivity Condition of culture 0 None Discrete intracytoplasmic granules, no cell lysis, no reduction of cell growth.

1 Slight ≤ 20% of the cells are round, loosely attached and without intracytoplasmic granules, or show changes in morphology; occasional lysed cells are present; only slight growth inhibition.

2 Mild ≤ 50% of the cells are round, devoid of intracytoplasmic granules; no extensive cell lysis; >50% growth inhibition.

3 Moderate ≤ 70% of the cells are round or lysed; cell layers are not completely destroyed; > 50% growth inhibition.

4 Severe Nearly complete or complete destruction of the cell layer(s).

3.4. Statistical analysis

The mean bacterial counts or percentage coverage per surface were compared using one-way ANOVA, and post-hoc comparison via two-tailed T-test. Statistical significance was set at 5%.

3.5. Results

3.5.1. Physicochemical analysis

Ellipsometery

All ellipsometery measurements on silicon wafer substrates indicated ppAllylamine films between 20 and 40 nm thick, depending on where the substrates were placed within the reaction chamber. Coatings were thicker on samples that had been placed

87 nearer the centre of the reactor than on samples that had been placed near the anode.

FEP samples were therefore placed towards the centre of the reactor for ppAllylamine coating to maximise coating thickness for the set of conditions used.

Amino Acid Analysis

The peptide densities on coated FEP-ppAllylamine-SMCC surfaces as estimated by

AAA are reported in Table 3.4. For the three peptide coatings measured (C-melimine,

C-Mel4, and GRGDSPC), ANOVA found no significant difference (p > 0.05) between groups in terms of mass, but a difference was found when molar amounts were compared (p = 0.026). However, an analysis between groups by T-test found no significant difference between the peptide densities on the three surfaces.

Table 3.4. Peptide density on coated FEP as measured by quantitative amino acid analysis. Amount of peptide of interest (mean ± SD) Surface ng per cm2 nmoles per cm2 C-melimine 159.5 ± 50.2 0.041 ± 0.013 C-Mel4 110.4 ± 48.7 0.043 ± 0.019 GRGDSPC 99.6 ± 19.7 0.144 ± 0.028

X-ray Photoelectron Spectroscopy

The atomic percentages of each element on sample surfaces as detected by XPS are listed in Table 3.5, along with calculated and observed nitrogen/carbon ratios and oxygen/carbon ratios. For FEP, relative amounts of carbon and fluorine were close to the expected 1:2 ratio. Low levels (<1%) of nitrogen and oxygen were detected.

Fluorine was not detected on ppAllylamine-coated FEP 24 h after coating, but was detected in low levels (<1%) 10 days after coating and thereafter.

A 2% increase in oxygen was seen after SMCC coupling. After peptide attachment, a

2% increase in nitrogen content was seen while oxygen content was variable.

Table 3.5. Atomic percentages of carbon (C), nitrogen (N), oxygen (O) and fluorine (F) as a percentage of total composition, as detected by XPS on sample surfaces. – : not detected. Calc: calculated value. Obs, observed value. SMCC: succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate

Atomic percentage (mean ± SD) N/C ratio O/C ratio Surface C N O F Calc. Obs. Calc. Obs.

36.09 0.08 0.44 63.28 FEP 0 0.001 0 0.010 ± 0.25 ± 0.6 ± 0.30 ± 0.79 ppAllylamine 84.12 14.54 1.34 – 0.333 0.173 0 0.016 (24 h in high vacuum) ± 0.25 ± 0.19 ± 0.14 ppAllylamine 84.40 14.14 1.46 – 0.333 0.168 0 0.017 (24 h in N2) ± 0.07 ± 0.01 ± 0.08 ppAllylamine 84.17 14.55 1.28 – 0.333 0.173 0 0.015 (24 h in air) ± 0.16 ± 0..32 ± 0.17 ppAllylamine 81.04 12.15 6.15 0.65 0.333 0.150 0 0.076 (10 days in N2) ± 0.12 ± 0.13 ± 0.25 ± 0.22 ppAllylamine 80.14 11.81 7.34 0.72 0.333 0.147 0 0.092 (10 days in air) ± 0.55 ± 0.28 ± 0.23 ± 0.22 ppAllylamine 76.64 10.73 11.88 0.80 0.333 0.140 0 0.155 (23 days in N2) ± 0.65 ± 0.26 ± 0.30 ± 0.33 ppAllylamine 76.59 10.44 12.03 0.94 0.333 0.136 0 0.157 (23 days in air) ± 0.70 ± 0.20 ± 0.45 ± 0.33 74.39 10.01 14.80 0.39 SMCC 0.125 0.133 0.327 0.375 ± 2.07 ± 0.39 ± 0.23 ± 0.00 69.90 11.93 16.98 0.16 C-melimine 0.466 0.171 0.094 0.217 ± 0.82 ± 1.10 ± 1.55 ± 0.22 73.28 11.90 13.52 0.09 C-Mel4 0.579 0.162 0.133 0.200 ± 0.07 ± 1.20 ± 0.54 ± 0.13

Assignments for narrow scans of C1s carbon peaks are given in Table 3.6. The major, secondary and tertiary C1s peaks detected for FEP were attributed to CF2, CF3 and CF, respectively. Following ppAllylamine coating, the dominant C1s peak at 285.0 eV was attributed to C–C/C–H. A peak at 286.5 eV was attributed to C–N bonds, but possibly also represents C–O or C–OH groups. Narrow scans of N1s peaks could not be deconvoluted from a single peak for any samples.

Table 3.6. Binding energies and proposed assignments for C1s subpeaks detected by XPS. C1s Region Surface Binding energy (eV) Assignment % of C1s peak area FEP 285.0 C–C/C–H 2.97 286.6 C–N / C–O 0.60 288.7 O=C–O 0.53 290.5 CF 3.21

292.4 CF2 85.09

294.2 CF3 7.61 ppAllylamine 285.0 C–C/C–H 80.86 (24 h in air) 286.5 C–N (C–O) 18.54 288.2 O=C–N/O=C–O 0.61 ppAllylamine 285.0 C–C/C–H 68.36 (23 days in air) 286.0 C–N 14.88 286.6 C–O 10.49 287.9 O=C–N 3.96 SMCC 285.0 C–C/C–H 64.04 286.3 C–N (C–O) 24.01 287.9 C=O 9.31 288.9 O=C–O 2.64 C-melimine 284.9 C–C/C–H 60.2 286.3 C–N (C–O) 25.8 287.9 C=O 10.7 288.8 O=C–O 3.4 C-Mel4 285.0 C–C/C–H 65.6 286.4 C–N (C–O) 23.1 288.0 C=O 9.1 288.8 O=C–O 2.3

On aged ppAllylamine, as the oxygen content approached that of nitrogen, peaks at

286.0 and 286.6 eV could be separated, and were attributed to C–N and C–O bonds, respectively. After SMCC and peptide coating, a single peak attributed to those species was observed at 286.3–286.4 eV. A minor peak at 287.9 eV on the aged ppAllylamine surface was attributed to N–C=O. On the SMCC surface, the increased prominence of a peak at the same position was attributed to C=O (ketone) groups of SMCC.

It should be noted that for ppAllylamine, no difference in XPS results was seen between the upper side and underside of the samples, indicating that the method is able to coat the underside of these substrates.

Contact Angle Analysis

Contact angles of the sample surfaces are reported in Table 3.7. Untreated FEP was the only surface found to be hydrophobic, with a contact angle of 106.4°. The measured contact angle for SMCC was 58.8°, and 53.2–58.6° on the various peptide-coated surfaces.

Table 3.7. Advancing water contact angles (θ°) of coated and control surfaces. Surface θ (mean ± SD) FEP 106.4 ± 2.7 ppAllylamine 58.0 ± 0.3 SMCC 58.8 ± 2.0 C-melimine 58.6 ± 3.7 C-Mel4 57.4 ± 1.6 C-Mel4-RGD 53.2 ± 0.9 C-Mel(RGD)4 55.4 ± 0.6 GRGDSPC 57.2 ± 1.5

3.5.2. Antibacterial activity

Peptide-coated hydrogel polymer surfaces

Bacterial adhesion to peptide-coated pHEMA surfaces is shown in Figure 3.4. Results are reported as log reductions in adherent viable bacteria recovered from coated pHEMA as compared to process controls.

Figure 3.4. Log reduction in viable bacteria recovered from peptide-coated pHEMA compared to uncoated pHEMA, determined by colony counts (mean ± 95% CI). *p < 0.05 compared to melimine and C-melimine. #p < 0.05 compared to C-Mel4.

On the melimine coating compared to process controls, adhesion of S. aureus strains was reduced by at least 2 log, S. epidermidis strains by at least 3 log (in many cases no viable S. epidermidis were recovered from peptide-coated lenses), and gram negative species slightly less than 2 log. C-melimine was similar to melimine in terms of reduction of bacterial adhesion for all strains tested. C-Mel4 again was similar for most

92 strains tested, but it was less effective (p < 0.028) against S. aureus 004 and E. coli

ATCC 0157, compared to melimine and C-melimine. C-Mel4-RGD was less effective

(p < 0.009) than melimine, C-melimine and C-Mel4 in inhibiting adhesion of all staphylococcal strains, and less effective (p < 0.016) than melimine and C-melimine in inhibiting adhesion of E. coli ATCC 0157. For P. aeruginosa 6294 there was no significant difference in effectiveness between any of the four peptide coatings.

Bacterial density in the diluted growth medium was approximately 108 CFU/ml for all samples, with no significant difference between any pHEMA controls or sample supernatant.

Peptide-coated fluorinated ethylene propylene surfaces

Results for bacterial coverage on FEP samples are presented as percentage surface coverage by both viable and non-viable bacteria, as determined by viability staining, microscopy and automated area determination.

Results for S. aureus 31 and P. aeruginosa 6294 are shown in Figure 3.5. For both strains, a significant reduction in green-staining bacteria was recorded on C-melimine and C-Mel4 compared to each of FEP, ppAllylamine, SMCC, and the unrelated peptide

GRGDSPC.

For S. aureus 31, green-staining bacterial coverage was reduced by 57.9% on

C-melimine (p = 0.015) and 59.5% on C-Mel4 (p = 0.014), compared to the SMCC process control. Coverage on C-Mel4-RGD was reduced by 41.9%, which was significant (p < 0.039) compared to SMCC only and no significant reduction was seen on GRGDSPC.

Figure 3.5. Bacterial surface coverage determined by microscopy and staining (mean ± 95% CI, n ≥ 3). *p < 0.05 compared to FEP, ppAllylamine, SMCC and GRGDSPC.

For P. aeruginosa 6294, green-staining bacterial coverage was reduced by 62.9% on

C-melimine (p = 0.016) and 62.5% on C-Mel4 (p = 0.017), compared to SMCC. No significant reduction was seen on C-Mel4-RGD or GRGDSPC. Given the poor performance of C-Mel4-RGD, this peptide was not tested against the remaining species.

Results for the remaining strains are shown in Figure 3.6 and Figure 3.7. For all strains tested, coverage by green-staining bacteria was significantly lower for both C-melimine and C-Mel4 compared to each of FEP, ppAllylamine and SMCC. The reductions in coverage for C-melimine and C-Mel4 were 64.0% (p = 0.003) and 68.9% (p = 0.002) for S. aureus 004; 55.1% (p = 0.012) and 62.3% (p = 0.008) for S. aureus 152; 59.6%

(p = 0.005) and 73.4% (p = 0.002) for S. epidermidis 013; and 72.6% (p = 0.001) and

56.4% (p = 0.004) for E. coli ATCC 0157 (respectively, compared to the SMCC process control).

Coverage by green-staining bacteria was also significantly lower on C-melimine and

C-Mel4 than on GRGDSPC, with the exception of C-melimine for S. epidermidis 013.

For S. epidermidis 013, green-staining bacterial coverage was also reduced (p = 0.047) on GRGDSPC, compared to FEP, ppAllylamine and SMCC.

For red-staining bacteria, the only significant difference between surfaces was seen with

E. coli ATCC 0157, for which coverage was increased (p = 0.041) on FEP compared to

C-melimine and C-Mel4.

Bacterial density in the diluted growth medium was approximately 108 CFU/ml for all samples and not significantly different between any surfaces for any species tested.

Figure 3.6. Bacterial surface coverage determined by microscopy and staining (mean ± 95% CI, n ≥ 3). *p < 0.05, **p < 0.01 compared to FEP, ppAllylamine and SMCC. #p < 0.05, ##p < 0.01 compared to GRGDSPC.

Figure 3.7. Bacterial surface coverage determined by microscopy and staining (mean ± 95% CI, n ≥ 3). *p < 0.05, **p < 0.01 compared to FEP, ppAllylamine and SMCC. #p < 0.05, ##p < 0.01 compared to GRGDSPC.

Cytotoxicity Testing

All peptide-coated surfaces were found to be non-cytotoxic in this screening assay.

Reactivity grading for all surfaces is listed in Table 3.8. A grade of 1 (slight reactivity) was seen directly under all pHEMA samples, whether unmodified or peptide-coated.

This appeared to be due to shifting of the sample, resulting in physical damage to the cell monolayer. FEP samples, whether unmodified or peptide-coated, were associated with no visible cell damage. Frequently, the cells appeared to be “banked up” around the FEP samples, helping to prevent shifting of the samples. The controls performed as expected, with complete destruction of most cells observed for the latex cytotoxic control and only slight damage observed for the unmodified pHEMA.

Table 3.8. Cytotoxicity gradings via direct contact assay for peptide-coated surfaces used in this study Samples Grade Reactivity No sample 0 None Latex (cytotoxic control) 4 Severe Unmodified pHEMA (non-cytotoxic control) 1 Slight Melimine-coated pHEMA 1 Slight [C-Mel4]-coated pHEMA 1 Slight [C-Mel4-RGD]-coated pHEMA 1 Slight Unmodified FEP 0 None FEP-ppAllylamine 0 None FEP-ppAllylamine-SMCC 0 None FEP-ppAllylamine-SMCC-[C-melimine] 0 None FEP-ppAllylamine-SMCC-[C-Mel4] 0 None FEP-ppAllylamine-SMCC-[C-Mel4-RGD] 0 None

Representative phase contrast microscopy images of cells after incubation with test samples are shown in Figure 3.8. In Figure 3.8(C–F), pHEMA samples were removed before images were taken. In Figure 3.8(G–L), images were taken with FEP samples in place to show cell growth on the well plate surface underneath the sample.

Figure 3.8. Differential interference contrast images of L929 murine fibroblasts after incubation with samples. Inset: 2 × greater magnification. PS: polystyrene well, FEP: fluorinated ethylene propylene sample. (A) No sample. (B) Latex. (C) Unmodified pHEMA. (D) pHEMA-melimine. (E) pHEMA–[C-Mel4]. (F) pHEMA-[C-Mel4-RGD]. (G) FEP. (H) FEP-ppAllylamine. (I) FEP-ppAllylamine-SMCC. (J) FEP-ppAllylamine- SMCC-[C-melimine]. (K) FEP-ppAllylamine-SMCC-[C-Mel4]. (L) FEP-ppAllylamine- SMCC-[C-Mel4-RGD].

3.6. Discussion

3.6.1. Physicochemical analysis

The thickness of the ppAllylamine layer determined by ellipsometry indicated a deposition rate in the order of nanometres per minute, comparable with other studies of ppAllylamine films (Lejeune et al. 2006; Denis et al. 2011).

For surfaces coated with C-melimine or C-Mel4, peptide density was approximately

0.04 nmol/cm2, or one molecule per 2.0 nm (assuming a square lattice arrangement).

The density of GRGDSPC was higher (1.4 nmol/cm2), perhaps due to more efficient attachment of the shorter peptide due to lack of steric hindrance. This is well above reported minimal densities needed to influence mammalian cells that can recognise surface bound ligands; an RGD coating has been reported to influence cell behaviour at a molecular spacing of 440 nm. (Massia and Hubbell 1991; Cavalcanti-Adam et al.

2007). So, although the precise interaction between the peptides tested here and mammalian cells is not yet known, cellular mechanisms exist that allow peptides of well below this density to influence cell behaviour.

The surface peptide densities estimated in the present study are considerably lower than the estimate of 3.5 nmol/cm2 on surfaces produced on glass functionalised with 3- aminopropyltriethoxysilane and coupled to cysteine residues using maleimide chemistry

(Chen et al. 2012). It may be that the ppAllylamine surfaces produced here provided a lower density of free amine groups available for further modification than did the glass method. The advantage of the plasma polymerisation method is the ability to apply a known surface chemistry for further modification to any given substrate material.

100

The contact angle of 106.4° found for FEP was similar to previously reported measurements for the same polymer (Petke and Ray 1969; Li and Neumann 1992).

While the contact angle of 58° after ppAllylamine coating is difficult to compare with other studies due to the unique results obtained with any given plasma reactor, 58° is within a wide range of angles previously reported, from at least 33° to 67°. Contact angles of ppAllylamine that differ immediately after coating, due to varying deposition power and gas pressure, have been observed over a number of days to increase and converge as oxidation proceeds to completion (Kurosawa et al. 1999; Demirci et al.

2014).

Contact angles for SMCC and peptide surfaces were higher than the angles reported elsewhere, of ~40° for both SMCC and an RGD peptide-coated surface (Shoichet 1998), and 44–46° for a melimine-coated surface (Chen 2012). This could have been due to incomplete surface coverage allowing the properties of the underlying ppAllylamine to dominate contact angle results.

X-ray photoelectron spectroscopy

The absence of fluorine detected on newly-deposited ppAllylamine indicate a pinhole- free ppAllylamine coating, while the low levels of fluorine detected at later stages suggest a slight degradation of ppAllylamine or a reorganisation of the polymer chains such that some FEP entered into the XPS sampling depth. Fluorine content was reduced with each subsequent stage of coating, suggesting that the ppAllylamine layer is stable to the peptide coating method and the fluorine signal is attenuated by further coating steps. XPS analysis of antimicrobial surfaces prepared in a similar manner revealed

~4% fluorine on FEP coated with plasma polymer of heptylamine (Chen 2012). This

101 suggests that small defects in the plasma polymer layer are not critical for short-term antimicrobial activity using this coating method. However, the plasma polymerisation parameters used here provided a resilient film that may translate to greater integrity in a biomaterials setting. Use of higher RF powers during deposition has been correlated with less swelling during immersion in liquid, presumably due to a greater degree of cross-linking (Denis et al. 2011), however Denis and colleagues did not examine the effect of coating thickness, which also increased with RF power.

The N/C ratio of ppAllylamine surfaces was lower than expected. It is difficult to compare results between different plasma reactor configurations, but lower deposition powers have been reported to allow incorporation of relatively intact allylamine into ppAllylamine films, while higher deposition powers increase the ratio of C≡N and

(terminal) CH3 species (Lejeune et al. 2006). N/C decreased slightly and O/C increased over 23 days’ storage time. A similar effect was seen over 30 days by Whittle et al.

(2000) who suggested that nitrogen was lost from their ppAllylamine surfaces and replaced with oxygen species.

Although N1s subpeaks were not differentiated, peak binding energy was measured at

399.4 eV for ppAllylamine, 399.8 eV for SMCC and 399.9 eV for peptide surfaces, supporting a shift towards dominance of imine (C=NR) groups over amine (C–NR2) and amide (O=C–NH) groups. No peak at 401.0–402.0 eV (the expected binding energy of guanidinyl nitrogen, N=CN2, present in arginine) was resolved, which is consistent with findings of low peptide surface density.

The increasing percentages of oxygen detected on ppAllylamine over time indicate continuing oxidation over this timeframe, highlighting the importance of continuing the

102 peptide coating steps as soon as possible after plasma polymerisation. Storage in polystyrene containers flooded with nitrogen gas did not appreciably slow this process.

When interpreting these XPS results, it must be kept in mind that the SMCC linker is

0.83 nm long, and so the underlying ppAllylamine will be dominant in XPS results, given a sampling depth of several nanometres. Similarly, peptide coverage on the surface is unlikely to be either continuous or greater than the sampling depth. The minimal volume of a peptide of a given mass can be calculated according to the method of Erickson (2009), assuming a spherical secondary structure. For C-melimine and

C-Mel4, these theoretical spheres are 2.1 nm and 1.8 nm in diameter, respectively. At the other extreme, if these peptides were completely linear, they could be no more than

12.8 and 7.7 nm long (given the typical length of a peptide bond, 0.13 nm. In reality, these peptides are likely to exist in a random coil conformation (given the lack of defined structure found for melimine by circular dichroism (Rasul et al. 2010)) with an intermediate shape. Given the estimated spacing of ~2 nm between peptide molecules, this suggests that the surface-bound peptides occupy less than the entire surface of the peptide-coated samples at less than the full sampling depth, which would account for the mild influence of peptide coating on elemental makeup.

3.6.2. Antibacterial activity

PHEMA substrates

Bacterial adhesion was significantly (p < 0.05) reduced on pHEMA coated with melimine, C-melimine or C-Mel4. C-Mel4-RGD coating was associated with reduced adhesion of S. epidermidis 013 and P. aeruginosa 6294 only.

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Inhibition of both S. aureus 31 and P. aeruginosa 6294 adhesion was about 1 log less than that found by Dutta et al. (2013) using the same method. However, in that study the amount of viable bacteria recovered from the peptide-coated lenses was similar, and the amount of bacteria recovered from the control lenses was lower (personal communication, Dutta, 16 November 2016), perhaps indicating a slight difference in pHEMA material or harsher rinsing methods.

Results for C-melimine indicate that it is suitable for further testing. C-Mel4 was as effective as melimine and C-melimine against more than half the species tested, and had a measureable inhibitory effect on all species, so C-Mel4 was also deemed a suitable candidate for further testing.

A recent study reported that a Mel-4 coating was effective in inhibiting S. aureus 31 and

P. aeruginosa 6294 adhesion to pHEMA, but less effective (~1.5 log less inhibition) than melimine (Dutta et al. 2016). Unlike the present study, Dutta et al. (2016) used the

Mel4 sequence without the N-terminal cysteine. In contrast to Mel4, attachment of

C-Mel4 via its arginine residues leaves a sulfhydryl group of cysteine free to participate in disulfide bonds with another C-Mel4 molecule (as for soluble peptide). This behaviour has been reported to occur in synthetic peptides (MacColl et al. 2001) and could be investigated to determine if this is the reason for the difference in efficacy between Mel4 and C-Mel4.

The addition of RGD at the C-terminus generally diminished antimicrobial activity, whether on pHEMA or FEP. C-Mel4-RGD did not perform to an acceptable standard as an anti-infective coating.

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FEP substrates

For all bacterial strains tested, adhesion was significantly (p < 0.05) reduced on

C-melimine and C-Mel4 coatings compared to the FEP substrates and process controls.

Non-viable E. coli ATCC 0157 adhered in greater numbers to FEP than to any other surface tested (p = 0.01). High coverage overall is consistent with reports that E. coli adhered more readily to hydrophobic than hydrophilic surfaces, while this effect is less pronounced or absent for S. aureus and S. epidermidis (Sousa et al. 2009; Gomes et al.

2015). As for the greater proportion of dead bacteria, the polymer itself might be the cause: Lu et al. (2015) describe hydrogels which become more hydrophobic at low pH

(for example due to bacterial metabolism) and which become bactericidal as they become more hydrophobic, perhaps due to hydrophobic interactions between the polymer and the bacterial membrane. This mechanism offers one possible explanation for the greater proportion of non-viable E. coli on FEP compared to other surfaces.

Karakecili and Gumusderelioglu (2002) found that the bacterial pathogens

S. epidermidis, S. aureus and P. aeruginosa showed reduced adhesion on hydrophobic surfaces, while adhesion of a non-pathogenic strain, Lactobacillus acidophilus, was not influenced by surface hydrophobicity. While the protocol and strains used in this study found similar bacterial adhesion between hydrophobic (FEP) and hydrophilic

(ppAllylamine) surfaces, these findings in the literature suggest that a hydrophilic surface is in general less susceptible to bacterial infection and therefore more appropriate for an implanted biomaterial surface.

No inhibition of bacterial growth was seen in the supernatant for coated pHEMA or

FEP samples in the present study. A previous study observed inhibition of bacterial

105 growth in the supernatant only when the surface area of the antibacterial-coated sample was high in relation to the volume (Chen 2012). The lack of inhibition seen in the present study suggests that no substantial amount of peptide detaches or leaches from the coated surface. However, to achieve an inhibitory concentration of C-melimine for

S. aureus 31 in 1 ml of supernatant, 63 μg or 16 μM would need to be released, whereas less than a nanomole was estimated (by amino acid analysis) to be present on each

1 cm2 sample.

The results of antibacterial testing in this chapter indicate the effectiveness of melimine- based surface coatings against bacterial genera that had not previously been tested. The diminished activity of C-Mel4-RGD is consistent with the results of MIC testing

(§2.4.1).

S. epidermidis, a major cause of biomaterial-associated infection on polymer surfaces, remained more susceptible than S. aureus to melimine-based peptides attached to pHEMA, but this difference was not apparent on coated FEP. This might be due to the likely lower peptide density on the FEP samples than on the pHEMA samples.

3.6.3. Cytotoxicity

The biocompatibility of the peptide-coated surfaces contrasts with the results of cell growth inhibition assays which found cytotoxicity of soluble peptides at 33 mM. This is not surprising for peptide-coated FEP, considering the low peptide density measured on these surfaces of approximately 0.04 nmol per 1 cm2 sample. The non-toxicity of pHEMA samples and lack of bacterial growth inhibition on the surrounding medium

106 indicated that if any peptide leached from the samples, the concentration was below cytotoxic concentrations and MICs of each peptide.

3.7. Conclusions

The results of this chapter indicate that the covalent coating method on FEP substrates results in surface coverage of 0.04 nmol/cm2 for the antibacterial peptides C-melimine and C-Mel4. These surfaces showed a reduction of approximately 60% in adhesion of six bacterial isolates from BAIs, including an MRSA strain, and biocompatibility in an in vitro cell culture system.

C-Mel4 retained the antibacterial activity of the full-length melimine peptide when coupled to an amine-functionalised FEP substrate via its C-terminal sulfhydryl group.

The addition of RGD to the N-terminus of Mel4 severely compromised the peptide’s anti-staphylococcal activity. Given the importance of Staphylococcus spp. in BAIs, this loss of activity is not acceptable for the purposes set out in this thesis.

C-Mel4 was therefore chosen for further testing in terms of its in vitro tissue effects, while alternative cell-integration approaches were under consideration.

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CHAPTER 4. BIOCOMPATIBILITY AND CYTOCOMPATIBILITY OF SURFACE- ATTACHED PEPTIDES

4.1. Introduction

Biocompatibility of biomaterials refers to the safety of a material in contact with tissue and is a minimum requirement for an implanted material. Cytocompatibility encompasses the behaviours of cells and tissue in response to the material, including adhesion, spreading, migration, proliferation and survival. Encouraging these tissue responses is the aim of third-generation implant materials, in contrast to second- generation (biodegradable or generally bio-active) and first-generation (inert) implant materials (Navarro et al. 2008). Some of the cellular responses to the peptide-coated test materials are examined in this chapter.

A nonfouling or purely antimicrobial and biocompatible coating will find widespread application in non-surgical interventions—such as urinary catheters, which are associated with 1 million hospital-acquired urinary tract infections in the U.S. each year

(Frank et al. 2009). In applying surface coating technology to surgically implanted devices, however, the interaction between the implant and the host’s tissues must be considered. Good tissue integration promotes long-term success of the implant, not only through acceptance of the foreign object, but also by crowding out bacteria that might otherwise find a niche on the implant surface, and by allowing the host’s immune system to defend the tissue-implant interface (Gristina 1987).

Cytocompatibility can be measured in terms of cell behaviour, including adhesion (in number and in terms of strength of adhesion), survival, proliferation and migration;

109 physical characteristics of cells, such as extent of individual cell spreading and cellular structure; and biochemical responses such as cytokine release. Differences in the cell response to different substrates can give an indication of the mechanisms underpinning the cell response.

4.1.1. Mechanisms of cell-substrate interaction

Influence of divalent cations

Magnesium is essential for life in animals, microbes and plants (Berkowitz and Wu

1993). The Mg2+ ion plays a role in stabilising both RNA and DNA (Misra and Draper

1998; Davey and Richmond 2002) and, along with other divalent cations, is important in cellular signalling (Normann et al. 1988; Smart et al. 2004) and cell-substrate and cell-cell adhesion (Martz 1980).

Testing in this chapter to determine whether divalent cation ion concentration affected cells’ interactions with melimine based peptides was based on the work of Rasul (2010), which found that the presence of 20 mM of Mg2+ almost completely negated the antibacterial effect of soluble melimine. The presence of 1–20 mM CaCl2 or MgCl2 also negated the antibacterial activity of protamine, which is arginine-rich, against E. coli and Listeria monocytogenes (Johansen et al. 1997).

Specificity of binding

Melimine-based peptides feature an N-terminal polyarginine tail (this feature is capped in Mel4-RGD and interrupted in Mel(RGD)4 with the neutral and basic amino acids glycine (G) and aspartic acid (D)). Polyarginines efficiently enter a number of mammalian cell types (Mitchell et al. 2000; Uemura et al. 2002). It was therefore

110 hypothesised that the polyarginine tail of melimine-based peptides interacts with mammalian cell surface, and that this interaction could be antagonised by adding soluble oligo-arginine peptides to the medium.

Ruoslahti et al. (1982) found the tripeptide sequence arginine-glycine-aspartic acid

(RGD) to be the active binding site of fibronectin: short soluble peptides containing the

RGD motif inhibited mammalian cell inhibition to fibronectin when added to be the medium, and a similar protocol is adopted in this chapter.

Polyarginine interaction with mammalian cells is a well-studied phenomenon (Mitchell et al. 2000; Uemura et al. 2002; Fuchs and Raines 2004). This interaction has been studied with L- and D- enantiomers of arginine polymers, and with model arginine-rich cell-penetrating peptides such as the transactivator of transcription (TAT) peptide from

HIV. TAT comprises a short N-terminal helix section preceding a run of arginines and lysines, and has effectively delivered various cargoes into mammalian cells in vitro and in vivo (Nagahara et al. 1998; Schwarze et al. 1999; Lewin et al. 2000; Shibagaki and

Udey 2002). Internalisation of short TAT peptides by several cell types was heavily diminished when three arginine residues of the cationic tail were deleted (Vivès et al.

1997). Studies with homopolymers of structurally similar amino acids, including lysine, identified the guanidinium headgroup of arginine as vital for cellular uptake (Mitchell et al. 2000; Richard et al. 2003).

Despite the inefficiency of poly-lysines in entering cells (compared to polyarginines) various mammalian cell types have an affinity for surfaces coated with poly-L-lysine or poly-D-lysine (Macieira-Coelho and Avrameas 1972; Mazia et al. 1975; Choi et al.

2015). So, although the conditions for cellular uptake are not well met, there is still a

111 measureable interaction between poly-lysine and the cell surface. While surface- attached melimine-based peptides should not be able to enter cells, interaction between cells and the basic arginine residues might promote cellular adhesion in a similar manner to poly-L-lysine coatings.

Cell signalling and cytoskeletal changes

Cell adhesion, spreading and migration are driven by chemical and physical signals from the environment. Notably, the integrin superfamily of transmembrane receptors communicate with the cell’s actin cytoskeleton through focal adhesions (Burridge and

Fath 1989; Geiger et al. 2009). Mechanically, cell spreading and migration occurs by filamentous actin structures—either broad, flat lamellipodia or spindly filopodia— pushing outwards from the cell body (Gardel et al. 2010). Focal complexes are formed in these structures and mature into focal adhesions through the recruitment of an array of proteins including vinculin, talin, zyxin and paxillin. (Burridge and Mangeat 1984;

Zaidel-Bar et al. 2003). Vinculin plays a vital role in focal adhesions, binding both to the actin cytoskeleton (Geiger et al. 1980; Schlessinger and Geiger 1983) and to talin, which in turns binds to the intracellular portion of integrin (Izzard 1988). Thus, focal adhesions are able to transfer force from the cell to its environment.

Differences in the distribution of vinculin in response to differing substrates is visible by immunostaining in human dermal fibroblasts (Mercier et al. 1996; Sondermann et al.

1999; Dalby et al. 2004; Schwartz et al. 2013) and keratinocytes (Marchisio et al. 1990;

Kaiser et al. 1993; Braga et al. 1995), making this a suitable marker of formation of focal adhesions in these cell types.

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4.2. Aims of the chapter

The aims were to examine the in vitro human tissue response to the surface coatings prepared in previous chapters. The mechanisms underpinning cell behaviour will be investigated by the addition of divalent cations and polyarginine peptides to the medium to determine the effect on cell-surface interactions, and fluorescence immunostaining to visualise the locations of cytoskeletal proteins in surface-attached cells.

4.3. Materials and Methods

4.3.1. Sample preparation

Coated fluorinated ethylene polypropylene (FEP) sample surfaces were prepared as described in §3.3.3. Briefly, FEP was coated with allylamine by plasma polymerisation

(ppAllylamine). This coating was found to be 20–40 nm in thickness and pinhole-free immediately after coating. The amine groups of ppAllylamine were reacted with the amine-to-sulfhydryl crosslinker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate (SMCC). SMCC-functionalised surfaces were reacted with sulfhydryl groups of cysteine-containing peptides. The reaction scheme for this coating method is shown in Figure 3.3.

The amino acid sequences of the peptides tested in this chapter are listed in Table 4.1.

C-Melimine comprises the sequence of melimine (Willcox et al. 2008) with an

N-terminal cysteine to allow sulfhydryl-directed attachment. C-Mel4 is a truncated sequence which was found in chapter 3 of this thesis to be similarly antimicrobial and less cytotoxic than C-melimine. C-Mel4-RGD contains the arginine-glycine-aspartic acid motif, a well-characterised ligand of integrin receptors (Ruoslahti and

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Pierschbacher 1987), placed at the C-terminus to give the motif maximum visibility for the peptide tethered by its N-terminal cysteine (although C-Mel4-RGD was found to be poorly antimicrobial, the mammalian cell response to a range of melimine-based peptides was of interest, and so this peptide was included in further testing).

C-Mel(RGD)4 contains the RGD sequence centrally to determine any difference in effect when the arginine tail of the peptide is exposed. GRGDSPC was included as a known pro-cell survival peptide, also with a cysteine for directed attachment.

GRGDSPC-Mel4 was included as a centrally-attached peptide containing sequences of both the RGD motif and the peptide of interest, Mel4.

Table 4.1. Amino acid sequences of peptides tested in this chapter. Peptide 1-letter amino acid sequence C-Melimine C T L I S W I K N K R K Q R P R V S R R R R R R G G R R R R C-Mel4 C K N K R K R R R R R R G G R R R R C-Mel4-RGD C K N K R K R R R R R R G G R R R R R G D C-Mel(RGD)4 C K N K R K R R R R R R G D R R R R GRGDSPC-Mel4 G R G D S P C K N K R K R R R R R R G G R R R R GRGDSPC G R G D S P C

For use in experiments, discs of 5 mm diameter were punched, or 1 cm squares were cut with a scalpel, from sheets of each prepared surface. Duplicate samples were placed in polystyrene well plates (Greiner Bio-One CELLSTAR®), disinfected by soaking in

70% ethanol for 30 minutes, and then allowed to air-dry in a biosafety cabinet.

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4.3.2. Cell types and culture conditions

All mammalian cells used in this study were adherent cell types and were maintained in polystyrene culture flasks (CELLSTAR®, Greiner Bio-One, Kremsmünster, Austria) in a humidified incubator at 37°C with 5% CO2.

HaCaT spontaneously immortalised human keratinocytes (Boukamp et al. 1988) were a gift from Nick Di Girolamo (School of Medical Sciences, University of New South

Wales, Kensington, NSW). HaCaT cells were maintained in DMEM with 10% FBS,

100 units/ml of penicillin and 100 µg/ml of streptomycin (Life Technologies, Carlsbad,

CA, USA).

Normal human adult primary cells were used to test the in vitro tissue response to peptide-coated surfaces. Dermal fibroblasts and epidermal keratinocytes were purchased from Lonza (Basel, Switzerland). Media used were KGM-gold and FGM-2 (Lonza), respectively, supplemented as per the manufacturer’s directions. Medium was changed every 2 or 3 days and cells were split at 80–90% confluence. Primary cells were used for experiments prior to passage number 10. Serum-free medium was used for experiments.

To subculture, cells were rinsed twice with phosphate-buffered saline (PBS; 137 mM

NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO, 10 mM Na2HPO4 in H2O, pH 7.4), and then dissociated from the flask with 0.05% trypsin-EDTA with 0.05% trypsin in 0.91 mM ethylenediaminetetraacetic acid (trypsin-EDTA). Once cells were detached, trypsin was neutralised with 1.5 volumes of serum-free trypsin inhibiting solution (Lonza). Detached cells were centrifuged at 500 × g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in fresh medium and added to a new

115 flask. When required for experiments, cell density was determined by trypan blue staining; an aliquot of suspended cells was added to an equal volume of 0.4% trypan blue, and unstained (viable) cells were counted manually using a haemocytometer slide and light microscope.

4.3.3. Attachment and behaviour of cells on peptide-coated surfaces

Cell numbers

The readily available HaCaT cell line was used as a screening test. HaCaT cells were seeded on samples in well plates at 1.3 × 104 cells/ cm2. Cells were observed over the following 72 h and then imaged (at which time the cell layer was not confluent on any sample surface). Samples were rinsed three times with PBS and placed on a microscope slide, and 10 μl of LIVE/DEAD® stain for mammalian cells (Invitrogen; prepared according to the manufacturer’s instructions) was placed on each sample. The drop was trapped with a coverslip and samples were incubated for 15 min in the dark before imaging with a laser scanning confocal microscope. For detecting calcein-AM viable stain and ethidium homodimer-1 non-viable stain, excitation wavelengths were 473 and

559 nm and emission wavebands were 485–545 and 570–670 nm, respectively.

Channels were scanned sequentially to avoid cross-talk. Cells for which the cytoplasm stained with calcein-AM (green) only were assumed to be alive, while cells for which the nucleus stained with ethidium homodimer-1 (red) were assumed to be dead.

Following the results found with HaCaT cells, primary epidermal keratinocytes and dermal fibroblasts were exposed to a subset of surfaces: FEP, ppAllylamine, C-Mel4 and GRGDSPC. Duplicate wells were seeded at 1 × 104 cells/cm2 for keratinocytes and

3 × 103 cells/cm2 for fibroblasts. After 48 h, cells were fixed by the addition of 3%

116 paraformaldehyde, stained with Hoechst 33324 nucleic acid stain and images were taken with a confocal microscope (FV1000, Olympus, Tokyo, Japan). Cells were counted in ImageJ software using the Plugins > Analyze > Cell Counter tool.

Examination of cell proliferation

To examine the relative contributions of initial adhesion and subsequent proliferation to cell numbers, proliferation of primary cells on surfaces was examined via enzyme- linked immunosorbent assay (ELISA) measuring 5-bromo-2'-deoxyuridine (BrdU) uptake during DNA synthesis. A kit (Abcam, Cambridge, UK) was used as per the manufacturer’s instructions. Duplicate wells were seeded at 1 × 104 cells/cm2 for keratinocytes and 3 × 103 cells/cm2 for fibroblasts. A blank condition (no BrdU added to the medium) and a background control (medium without cells) were included.

20–21 h after seeding, medium was replaced with medium containing 1 × BrdU solution. Cells were incubated for a further 20–21 h to allow proliferation with the uptake of BrdU. Fixing Solution was added to the wells to fix and lyse cells, and to denature DNA to allow BrdU detection. Wells were washed three times with the provided Wash Buffer. Primary antibody (mouse monoclonal anti-BrdU) was added to wells and plates were incubated for 60 minutes at room temperature. Wells were washed as before. Secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG) was added to wells and plates were incubated for 30 minutes at room temperature.

Wells were washed three times with Wash Buffer and once with MilliQ ultrapure water.

Samples were transferred to fresh wells to avoid the influence of any reagents bound to the experimental wells. The chromogenic peroxidase substrate 3,3',5,5'- tetramethylbenzidine (TMB) was added to all wells. After 15–20 minutes the reaction

117 was stopped with 0.2 M sulfuric acid and optical density at 450 nm (OD450) was read with a platereader (FLUOstar Optima, BMG Labtech, Ortenberg, Germany). The mean of duplicate wells was taken and the mean of the blank wells was subtracted from all values.

4.3.4. Mechanism of cell attachment to surfaces

Kinetics of cell attachment

Prior to testing whether cell attachment could be inhibited by additives to the medium, pilot experiments were performed to determine the length of time taken for each cell type to attach to the test surfaces. Into 24-well plates containing FEP samples, fibroblasts were seeded at 3 × 103 cells/cm2 and keratinocytes were seeded at 1 × 104 cells/cm2. After 15, 30, 60 and 120 minutes, duplicate wells were gently rinsed once with warm medium added with a micropipette down the side of the wells. Adherent cells were counted at each point in time.

Effect of divalent cations or soluble peptides on cell adhesion

For interfering ion/peptide experiments, primary cells were suspended in serum-free medium containing the Mg2+ ion or test peptide, and seeded at a density of

5 × 104 cells/ml. After a short incubation period, wells were rinsed three times with warm medium and imaged to determine numbers of adherent cells.

It was determined through pilot experiments that incubation times of 120 min and

60 min were suitable for keratinocytes and fibroblasts, respectively, as these durations allowed 50-75% adhesion on the permissive ppAllylamine surface.

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2+ For the Mg experiments, both MgCl2 and MgSO4 were tested. Media containing these salts were filter sterilised. Pilot experiments were conducted to test the cytotoxicity of these salts (as per the grading scale used in §3.3.6). Cytotoxicity was first apparent at

125 mM for both salts and so 62.5 mM was chosen as the experimental concentration.

Soluble nona-arginine (RRRRRRRRR; 1423.7 daltons) was tested to determine its inhibitory effect on initial cell adhesion to any surfaces. The hexapeptide GRGDSP

(587.6 daltons) was included as a peptide which is known to inhibit integrin-mediated adhesion (Pierschbacher and Ruoslahti 1984a). GRGESP (601.6 daltons) was included as an inactive control peptide (Pierschbacher and Ruoslahti 1987). Peptides were tested at a concentration of 3 mM, sufficient for GRGDSP to measurably inhibit adhesion

(Pierschbacher and Ruoslahti 1984a). Nona-arginine toxicity was observed at 10 mM with HeLa adenocarcinoma cells (Chakrabarti et al. 2014).

Peptides were synthesised by American Peptide Company (Sunnyvale, CA, USA) using

Fmoc chemistry, and supplied at a peptide purity of >95%.

Detection of integrin-associated proteins by immunostaining

Unless otherwise stated, incubations were performed with a volume of 100 µl, at room temperature, and wells were rinsed three times with PBS containing 0.1% Tween-20

(PBST) between each step in this protocol. Antibodies were purchased from Santa Cruz

Biotech (Dallas, TX, USA). Ideal concentrations of each fluorophore and any cross- reactivity between antibodies was determined by preliminary experiments.

Cells were seeded at 5 × 103 cells/cm2 in 96-well plates containing coated FEP samples.

Cells were incubated for longer than in adhesion protocols—90 min for fibroblasts and

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180 min for keratinocytes—as initial staining experiments showed a lack of cytoskeletal organisation for any samples after 60 or 120 min. Medium was aspirated and (without any rinsing) cells were fixed with 3% paraformaldehyde for 15 min. Cells were permeabilised with 0.5% Triton X-100 for 3 min. Non-specific binding sites were blocked with 5% normal goat serum in PBS for 60 min. Anti-talin antibody (mouse monoclonal) was added at a dilution of 1:200 in 5% normal goat serum and incubated overnight at 4°C. Anti-mouse antibody (raised in goat) conjugated to fluorescein isothiocyanate (FITC) was added at a dilution of 1:200 in PBST and incubated for

60 min. Phalloidin conjugated to iFluor-633 (Abcam, Cambridge, UK), prepared according to the manufacturer’s directions, was added and incubated for 30 min. These samples were mounted on microscope slides under a coverslip, using Vectashield anti- fade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) nucleic acid stain (Vector Labs, Burlingame, CA, USA).

Samples were incubated for 15 min in the dark before imaging with a laser scanning confocal microscope. For detecting DAPI, FITC and iFluor-633, excitation wavelengths were 405, 473, and 635 nm, and emission wavebands were 425–475, 485–545 and 570–

670 nm, respectively. Channels were scanned sequentially to avoid cross-talk.

Autofluorescence was minimal in unstained cells and secondary antibody in the absence of primary antibody did not visibly stain any structures preferentially.

4.3.5. Statistical analysis

Mean cell counts and OD450 nm for the BrdU assay were compared using one-way

ANOVA and post-hoc comparison via two-tailed T-test. Statistical significance was set at 5%.

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4.4. Results

4.4.1. Behaviour of cells on peptide-coated FEP surfaces

Attachment and survival of HaCaT cells

HaCaT cells adhered to and/or proliferated poorly on blank FEP, the SMCC process control, and the GRGDPSC-Mel4 peptide coating. On ppHeptylamine, few cells were present, but there was evidence of small cell colonies 48 h after seeding. Cell coverage was confluent on both GRGDPSC and C-Mel4 (Figure 4.1. The vast majority of the cells were stained green—i.e. live—and so overall the cultures appeared healthy.

Figure 4.1. Confocal microscopy images of HaCaT cells on FEP surfaces. Cells were stained with live/dead stain (green indicates viability). Bar = 100 µm. (A) FEP. (B) ppHeptylamine. (C) SMCC. (D) C-Mel4. (E) GRGDSPC. (F) GRGDSPC-Mel4.

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Attachment and survival of primary cells

Human primary cells were initially cultured on a subset of sample surfaces for 48 h to determine their ability to attach and survive. Both keratinocytes and fibroblasts were seen in the greatest numbers on ppAllylamine (Figure 4.2). Cell numbers on the hydrophobic FEP were variable between cell types: fibroblast numbers were greatly reduced, while 68% as many keratinocytes were seen on FEP compared to ppAllylamine. Cell numbers on C-Mel4 and GRGDSPC were similar, with approximately 80% as many cells seen on either peptide as on ppAllylamine, for both cell types.

Figure 4.2. Fibroblast and keratinocyte counts on FEP surfaces. Mean of three experiments ± 95% CI. *p < 0.05

Primary keratinocytes and fibroblasts were cultured on a wider range of peptide-coated

FEP surfaces for 24 h and then stained with live/dead fluorescent stain. Representative images are shown in Figure 4.3 and Figure 4.4. Primary fibroblasts, like HaCaT cells, adhered poorly to FEP and GRGDSPC-Mel4. Primary keratinocytes, in contrast, were

122 able to adhere to these surfaces. Keratinocyte adhesion appeared similar on all other peptide coatings.

Figure 4.3. Confocal microscopy images of fibroblasts on FEP surfaces. Bar = 100 μm. Cells were live/dead stained (green indicates intact cell membrane) and imaged with a confocal microscope. (A) FEP. (B) ppAllylamine. (C) SMCC. (D) C-Mel. (E) C-Mel4. (F) C-Mel4-RGD. (G) C-Mel(RGD)4. (H) GRGDSPC. (I) GRGDSPC-Mel4.

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Figure 4.4. Confocal microscopy images of keratinocytes on FEP surfaces. Bar = 100 μm. Cells were live/dead stained (green indicates intact cell membrane) and imaged with a confocal microscope. (A) FEP. (B) ppAllylamine. (C) SMCC. (D) C-Mel. (E) C-Mel4. (F) C-Mel4-RGD. (G) C-Mel(RGD)4. (H) GRGDSPC. (I) GRGDSPC-Mel4.

Proliferation of primary cells

Cell proliferation on the various test surfaces was measured by quantifying uptake of the thymidine analogue BrdU. Results are reported in Figure 4.5 and Figure 4.6 as optical density at 450 nm after detection of BrdU by ELISA.

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BrdU uptake for fibroblasts was similar for ppAllylamine, SMCC, and all peptide surfaces tested. BrdU uptake was significantly less (p < 0.022) on the FEP surface.

BrdU uptake for keratinocytes was similar across all surfaces tested, with no differences between surfaces significant at the 0.05 level.

Figure 4.5. Proliferation activity of fibroblasts on FEP surfaces. Optical density at 450 nm after detection by ELISA of BrdU. Means of 3 experiments ± 95% CI. *p = <0.05

Figure 4.6. Proliferation activity of keratinocytes on FEP surfaces. Optical density at 450 nm after detection by ELISA of BrdU. Means of 3 experiments ± 95% CI.

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4.4.2. Mechanism of cell attachment to surfaces

Kinetics of primary cell attachment

Cells were seeded on sample surfaces and removed after 15, 30, 60 or 120 minutes and gently rinsed before counting adherent cells. 60 minutes after seeding, 25–65% of the total number of fibroblasts added were adherent to surfaces, with almost complete adhesion on some surfaces after 120 minutes (Figure 4.7). Keratinocytes adhered more slowly, with 40–70% of cells adherent after 120 minutes (Figure 4.8).

Based on these results, 60 minutes for fibroblasts and 120 minutes for keratinocytes were chosen as durations for short-term adhesion assays, as these time points would allow discernment of increased or decreased adhesion for any given sample.

Figure 4.7 Short-term adhesion kinetics of fibroblasts used in this study. Mean ± SD from triplicate wells of a single experiment.

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Figure 4.8 Short-term adhesion kinetics of keratinocytes used in this study. Mean ± SD from triplicate wells of a single experiment.

Effect of divalent cations on cell adhesion

Adhesion of human primary fibroblasts or keratinocyes to surfaces in the presence of

2+ 62.5 mM Mg (provided by either MgSO4 or MgCl2) is reported in Figure 4.9 and

Figure 4.10, as a percentage of adhesion to the same surface in the absence of added

2+ Mg . There was no significant difference between the two salts, MgSO4 and MgCl2, in their effect on cell binding to any surface tested.

For fibroblasts, 62.5 mM Mg2+ was associated with a 25–59% (p < 0.024) increase in fibroblast adhesion to FEP, and a 6–46% reduction in adhesion to all other surfaces, although the 9–12% reduction on C-melimine was not significant (p > 0.05). For keratinocytes, a 42–43% (p = 0.022) increase in adhesion to ppAllylamine was seen with the addition of Mg2+. Keratinocyte adhesion was not significantly altered on any other surface in the presence of Mg2+.

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Figure 4.9. Fibroblast adhesion to FEP surfaces in the presence of added Mg2+ as a percentage of counts in the absence of Mg2+. Means of 3 experiments ± 5% CI. *p < 0.05

Figure 4.10. Keratinocyte adhesion to FEP surfaces in the presence of added Mg2+ as a percentage of counts in the absence of Mg2+. Means of 3 experiments ± 95% CI. *p < 0.05

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Effect of soluble peptides on cell adhesion

The effect of soluble RGD peptides and nona-arginine on short-term cell adhesion was examined. Fibroblast and keratinocyte adhesion in the presence of peptide at a concentration of 3 mM is shown in Figure 4.11 and Figure 4.12, reported as a percentage of adhesion on the same surface in the absence of added peptide.

Soluble GRGDSP inhibited fibroblast adhesion by 31–37% on the surfaces which contained the RGD motif only: C-Mel4-RGD, C-Mel(RGD)4 and GRGDSPC

(p < 0.045). Nona-arginine was associated with increased adhesion on the control (non- peptide coated) surfaces: FEP, ppAllylamine and SMCC (p < 0.045).

For keratinocytes, the addition of GRGDSP approximately halved initial adhesion to all surfaces and nona-arginine increased (p < 0.045) keratinocyte adhesion to FEP, ppAllylamine and SMCC.

GRGESP did not affect fibroblast or keratinocyte adhesion to any surfaces (p > 0.05).

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Figure 4.11. Fibroblast adhesion in the presence of interfering peptide (cell counts as a percentage of counts in the absence of peptide). Means of 3 experiments ± 95% CI. *p < 0.05 compared to adhesion in the absence of peptide.

Figure 4.12. Keratinocyte adhesion in the presence of interfering peptide (cell counts as a percentage of counts in the absence of peptide). Means of 3 experiments ± 95% CI. *p < 0.05 compared to adhesion in the absence of peptide.

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Detection of integrin-associated proteins by immunostaining

Keratinocytes and fibroblasts on sample surfaces were fixed after a short incubation period and stained to visualise the cell nucleus (blue), actin cytoskeleton (magenta), and the actin- and integrin-associated protein vinculin (green).

Representative images of fibroblasts on each surface are shown in Figure 4.13. Cells were attached and spread on all surfaces including FEP. Green flecks are visible at the cell margins in line with the actin fibres. The blue channel was omitted for clarity as green staining was also visible in the cytoplasm and nuclear region.

Representative images of keratinocytes are shown in Figure 4.14. Cells were visibly spread on each surface and actin filaments visibly stretched between a number of anchor points around the perimeter of the cells. Vinculin was detected as radially-oriented

FITC-stained flecks at these anchor points.

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Figure 4.13. Confocal microscopy images of fibroblasts on FEP surfaces. Cells were stained with anti-vinculin and FITC-conjugated secondary antibody (green); and phalloidin-iFluor™ 633 (magenta). Bar = 50 μm. (A) FEP. (B): ppAllylamine. (C) SMCC. (D) C-Mel. (E) C-Mel4. (F) C-Mel4-RGD. (G) C-Mel(RGD)4. (H) GRGDSPC.

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Figure 4.14. Confocal microscopy images of keratinocytes on FEP surfaces. Cells were stained with DAPI nuclear stain (blue); anti-vinculin and FITC-conjugated secondary antibody (green); and phalloidin-iFluor™ 633 (magenta). Bar = 50 μm. (A) FEP. (B): ppAllylamine. (C) SMCC. (D) C-Mel. (E) C-Mel4. (F) C-Mel4-RGD. (G) C-Mel(RGD)4. (H) GRGDSPC.

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4.5. Discussion

The melimine-derived peptide coatings C-melimine, C-Mel4, C-Mel4-RGD and

C-Mel(RGD)4 were all found to support adhesion and proliferation of HaCaT cells and human primary dermal fibroblasts and epidermal keratinocytes. Both primary cell types also adhered and proliferated on ppAllylamine, SMCC, and (as expected) GRGDSPC surfaces. Keratinocytes adhered and proliferated on FEP and GRGDSPC-Mel4 to a similar extent as on other surfaces, while HaCaT cells and primary fibroblasts adhered poorly to these two surfaces.

The adhesion kinetics experiment and the blank medium controls in interference assays indicated that initial (60 min) adhesion to FEP by fibroblasts was similar to adhesion on ppAllylamine and other coated surfaces. However, very few fibroblasts were present on

FEP 24 h post-seeding. In keeping with this finding, fibroblast uptake of BrdU was significantly (p < 0.048) lower on FEP than on any other surface tested. It can be inferred that an initial interaction exists between FEP and the primary fibroblasts used in this study, but that FEP does not support continued growth of this cell type. This is consistent with findings that human fibroblasts spread more slowly and with a lower adhesion strength on bare FEP as compared to tissue culture polystyrene (Schakenraad et al. 1989; van Kooten et al. 1992), and that adsorption of proteins such as collagen on

FEP enhanced fibroblast adhesion to the surfaces (Gabridge and Gladd 1984).

The poor fibroblast adhesion on observed on GRGDSPC-Mel4 was unexpected, given the permissiveness of both GRGDSPC and C-Mel4 alone. The central attachment point of GRGDSPC-Mel4 implicates possible steric hindrance interfering with the ability of cells to interact with peptide sequences. The effects of varying the attachment point

134 have been reported for melimine; melimine attached to surfaces through a central cysteine residue was less active against S. aureus and P. aeruginosa than melimine attached at either terminus, despite no difference in level of peptide coverage (Chen et al. 2012). Computer modelling could be employed to predict the likely behaviour of the centrally-attached GRGDSPC-Mel4 compared to GRGDSPC and C-Mel4 (modelling of cecropin P1 suggests very different molecular behaviours depending on attachment position (Wang et al. 2014)).

Apart from reduced fibroblast adhesion on FEP and GRGDSPC-Mel4, attachment and proliferation of fibroblasts and keratinocytes on ppAllylamine, SMCC, and all other peptide-coated surfaces in normal medium was similar.

Effect of divalent cations

For fibroblasts, the addition of 62.5 mM Mg2+ to the medium increased adhesion to FEP only, by 25–59% (p < 0.024), and reduced adhesion to all other surfaces tested

(although the reduction on C-melimine was not significant at the 0.05 level). For keratinocytes, the addition of Mg2+ increased adhesion to ppAllylamine only, by 42–

43% (p < 0.022), with no other significant effects.

Integrin binding is regulated by divalent cations, particularly calcium and magnesium

(Ca2+ and Mg2+), and the cation binding sites of integrins vary in their affinities for each cation (Zhang and Chen 2012). Mg2+ is normally required for integrin binding (although

Ca2+ at high concentrations can perform this role of the magnesium ion) (Leitinger et al.

2000; Onley et al. 2000).

135

As Mg2+ in millimolar concentrations promotes integrin-ligand binding, it is not surprising that the addition of 62.5 Mg2+ increased the rate of fibroblast adhesion on

FEP, to which the fibroblasts otherwise adhered poorly. However, a decrease in adhesion was seen on other surfaces (although not statistically significant in all instances), which was not expected.

For keratinocytes, on the other hand, the only change associated with the addition of

62.5 Mg2+ was an increase in adhesion to ppAllylamine, to which keratinocyte adhesion was already highest.

The basis for the different response of the two cell types to the addition of Mg2+ is not yet known, but it is assumed to be an intrinsic fibroblast response and not related to the peptide coatings.

Effect of soluble peptides

For fibroblasts, the addition of 3 mM soluble GRGDSP to the medium inhibited adhesion to RGD-containing peptide coatings only, in line with original findings of the activity of the RGD motif (Pierschbacher and Ruoslahti 1987). It is interesting that the addition of soluble GRGDPSC inhibited adhesion to C-Mel4-RGD and C-Mel(RGD)4, suggesting a specific integrin-peptide association.

The addition of GRGDPSC inhibited keratinocyte adhesion to all surfaces tested. This contrasts with the results for Mg2+ addition, which led to widespread inhibition of fibroblast adhesion. It is similarly assumed that this global inhibition was related to an

RGD-sensitive mechanism of keratinocytes, such as the RGD-dependent α5β1 integrin, which binds fibronectin and vitronectin (Larjava et al.).

136

Fibroblast and keratinocyte adhesion to FEP, ppAllylamine and SMCC surfaces was increased in the presence of 3 mM nona-aginine. This increased affinity might indicate that nona-arginine was adsorbed to these surfaces and promoted attachment in a similar way to surface-adsorbed poly-lysine (Mazia et al. 1975; Choi et al. 2015).

Soluble nona-arginine did not antagonise cell adhesion to melimine-based peptides.

Nona-arginine added at a concentration of 3 mM to cells at a density of 1 × 105 cells/ml leaves each cell free to interact with 30 pmoles of soluble nona-arginine. Given a peptide density of 0.04 nmoles/cm2 (§3.5.1) and a mammalian cell footprint in the order of thousands of square microns, cells would be exposed to 1–2 pmoles of surface-bound peptide when fully spread on these surfaces, but attachment was not affected by the large excess of soluble non-arginine. In contrast, RGD-containing short peptides at a concentration of 3 mM effectively interfered with integrin-mediated cell adhesion; also, nona-arginine toxicity has been reported at 10 mM (Chakrabarti et al. 2014). The toxicity of melimine-based peptides themselves is sub-millimolar. The complementary approach to investigate the potential interaction between cells and the arginine tail of melimine-based peptides would be addition to the medium of a negatively-charged species to antagonise the relevant cell surface component—negatively charged proteoglycans, which are known to promote uptake of arginine-rich peptides (Amand et al. 2012). This might avoid the toxicity problem encountered at high concentrations of cell-penetrating arginine-rich peptides.

Focal adhesion and stress fibre formation

The cell adhesion times were slightly increased for immunostaining in comparison to the adhesion experiments, as pilot experiments with both cells types showed poor

137 cytoskeletal organisation on any surfaces at the shorter time points. This is in line with the finding that human gingival fibroblasts showed minimal cytoskeletal organisation

40–60 minutes after seeding on titanium surfaces, with stress fibres alignment occurring over the following two hours (Oakley and Brunette 1993).

For both fibroblasts and keratinocytes, vinculin can be seen aligned with actin fibres at cell margins, evidencing the formation of integrin-associated focal adhesions (Soong

1987). Identification of the integrins used by cells can help reveal the precise mechanism by which cells interact with the surface (Marciano et al. 2007).

4.5.1. Conclusion

The results of this chapter demonstrate the ability of primary human dermal fibroblasts and primary human epidermal keratinocytes to adhere, proliferate, and form focal adhesions and stress fibres on melimine-based peptide coatings including C-Mel4.

Cell adhesion to melimine–based peptides was not specifically affected by the addition of Mg2+ ions or 3 mM nona-arginine to the medium. The specific mechanisms at work in the interaction between melimine-based peptide coatings and mammalian cells remain to be determined.

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CHAPTER 5. CONCLUSIONS AND FUTURE PERSPECTIVES

5.1. Conclusions

In this study the peptides melimine, C-melimine, Mel4, C-Mel4, C-Mel4-RGD,

C-Mel(RGD)4, GRGDSPC-Mel4 and malamane—of which the latter five were novel— were assessed for antibacterial activity and biocompatibility. C-melimine, Mel4 and

C-Mel4 were each found to have a higher therapeutic index than the parent peptide melimine.

C-melimine and C-Mel4 were covalently coupled to FEP though addition of surface amines by plasma polymer coating with allylamine, maleimide functionalisation, and peptide coupling through the sulfhydryl group of cysteine. This coating method provided a surface peptide density of approximately 0.04 nmoles/cm2.C-melimine and

C-Mel4 surfaces were challenged with biomaterial infection-associated isolates of

Staphylococcus aureus (including a methicillin-resistant strain), Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli. Both surface coatings were found to reduce bacterial colonisation by approximately 60% for all stains tested. This is the first report of melimine-based peptides being effective against S. epidermidis or

E. coli. The notable sensitivity of S. epidermidis to melimine-based peptides places

C-Mel4 as a particularly favourable candidate for anti-infective technology for polymer- based implant-materials, on which S. epidermidis is the major cause of infections

(Becker et al. 2014).

139

These peptide surface coatings were non-toxic to mouse fibroblasts and promoted adhesion of human fibroblasts and keratinocytes. Twenty-four hours after seeding cells, fibroblast and keratinocyte numbers were equivalent on C-Mel4 and GRGDSPC surfaces, with cells appearing healthy as assessed by a viability stain. Cell proliferation between 24 and 48 hrs post-seeding was also similar between all the peptide-coated substrates. Fibroblasts and keratinocytes adherent to the peptide surfaces formed stress fibres and vinculin plaques within 90 minutes (fibroblasts)and 80 hours, respectively.

The nature of the peptide-cell interaction is thought to be based on electrostatic interactions between the cationic arginine resides of the peptides and negatively-charged cell surface molecules. However, the keratinocyte and fibroblast interactions with the peptide surfaces were not specifically sensitive to high concentrations of Mg2+ or 3 mM nona-arginine.

5.2. Perspectives

The precise mechanisms involved in cell-surface interaction with C-melimine and

C-Mel4 remain to be described. Parameters to be investigated include individual cell spreading, as a quantitative measure of the cells’ affinity for the surface.

The encouraging responses observed in this study with epidermal keratinocytes and dermal fibroblasts indicate further pursuit of a subcutaneous and transdermal implant coating surface. The response of other cell types remains to be evaluated—for example, studies of the osteoblastic cell response to melimine-based peptides will indicate the suitability of applying this technology to joint replacement materials, fixation devices

140 and dental implants; and haemocompatibility testing will ensure the suitability of these peptides for vascular devices.

The inflammatory response to melimine-based peptides also stands to be fully described. Melimine-coated pHEMA and titanium have already shown safety and reduction of bacterial colonisation in animal models, and safety in a short-term human contact lens trial (Dutta et al. 2014; Chen et al. 2016). It would however be prudent to study the molecular mechanisms of the immune response to melimine and related peptides.

This study describes mild toxicity of soluble melimine-based peptides, with no toxicity seen for surface-attached peptides. As such, the long-term integrity of the peptide coating should be assessed. PHEMA-melimine contact lenses have retained good activity after three weeks’ continuous wear (Dutta et al. 2014), suggesting good peptide retention using the EDC attachment method (§3.3.2). As C-Mel4 is being proposed here as a candidate for a coating of long-term permanent implants, longer-term testing would be desirable.

The findings of this work put forward C-Mel4 as an excellent candidate warranting further investigation as a potential anti-infective and tissue compatible surface coating for surgical implants.

141

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