Clearly, to prevent bacterial organisms from irreversibly attaching to the surface of biomaterial would be preferred; however, bacteria have evolved numerous mechanisms to enable their colonisation of even the most hostile environments (Varghese et al. , 2013). Furthermore, the rapid adsorption of proteins in vivo may conceal the physicochemical properties of the underlying substrate needed for antifouling or contact killing effect (Charville et al. , 2008). To mitigate prolif- eration and biofilm formation by attached cells, several types of bioactive, leach-/
release- based coatings have been developed (Huh and Kwon, 2011; Hetrick and Schoenfisch, 2006).
Surface modification of biomaterials for biofilm control 111
6.7.1 Systemic antibiotics
Controlled spatiotemporal release of systemic antibacterial agents (i.e. traditional antibiotics) directly at the site of implantation, i.e. via controlled release from a polymer or ceramic matrix, requires a significantly lower dose compared to systemic treatment, appreciably reducing the potential for negative side effects. The benefit of using an FDA-approved antibiotic is that its effect on the host is well described, including the interactions between this agent with other medications or treatments the patient might be using. These antibiotics have a very specific cidal activity, in that they are designed to target specific features of bacterial cells – vancomycin selec- tively binds to terminal D-alanyl–D-alanine moieties of the NAM/NAG-peptides in the cell wall of Gram- positive bacteria, interfering with proper cell wall synthesis.
The increasing incidence of antibiotic- resistant nosocomial bacterial infections chal- lenges the use of antibiotic- loaded coatings on two levels. Firstly, the antibiotic may not be active against the coloniser, and there is typically more than one type of pathogenic bacterial species that can infect the implant. Secondly, the preventative use of antibiotics may in fact contribute to the development of more resistant bacte- ria (Forbes et al. , 2013). These considerations have brought the spotlight back onto other, non- conventional antimicrobial agents that may have fallen into the shadows with the discovery of penicillin. These natural and designed materials display a wide variety of mechanisms of antimicrobial activity, such as photocatalytic production of reactive oxygen species that are damaging to cell molecules and processes (e.g. TiO 2 , ZnO and fullerol), damage of the bacterial cell envelope (e.g. peptides, chitosan, car- boxyfullerene, carbon nanotubes, ZnO and silver nanoparticles (nAg)), disruption of cell energy transduction (e.g. nAg and aqueous fullerene nanoparticles (nC60)), and inhibition of enzyme activity and DNA synthesis (e.g. chitosan) (Li et al. , 2008).
Their physico- chemical structure and mechanism of action are unlike those of currently used synthetic antibiotics, potentially eliminating the possibility of cross- resistance.
Some of these materials can be used to supplement the activity of tradi- tional antibiotics. A combination of chitosan/tobramycin was shown to be effec- tive against planktonic cultures of P. aeruginosa (Tré-Hardy et al. , 2008), with chitosan also reported to enhance the antimicrobial activity of sulfamethoxazole against a drug resistant strain of the pathogen (Tin et al. , 2009). It is unclear if the synergistic effect can be sustained in the case of antibiotic/chitosan releas- ing coatings, and whether it will be sufficient to prevent bacterial attachment and biofilm formation at the implant surface. Mildly antibiotic hydrophobic tripeptide
D Leu–Phe–Phe has been used as a self- assembling macroscopic hydrogel at physi- ological pH loaded with a sparingly soluble antibiotic, ciprofloxacin. The resultant hydrogel displayed controlled release, and was highly effective against S. aureus, E. coli , and a clinical strain of Klebsiella pneumoniae , with minimal toxicity to host cells (Marchesan et al. , 2013). Certain combinations of phytochemicals with conventional antimicrobial drugs demonstrated enhanced efficacy against methicil- lin resistant S. aureus (Kyaw et al. , 2012). Secondary metabolites in plants, phyto- chemicals such as tannins, flavonoids, alkaloids, terpenoids, and polyphenols are
112 Biomaterials and Medical Device-associated Infections
effective against both Gram- positive and Gram- negative bacteria and are known to modulate or modify resistance mechanisms in bacteria, however due to higher minimum inhibitory concentrations they can rarely be used as the only agents (Kyaw et al. , 2012).
6.7.2 Antibodies and peptides
Another area of research concerns the use of antibodies as active agents. The approach is based on the observation that the peri- implant space is often immunocompromised due to a combination of tissue damage from surgery, disrupted blood supply, acute inflammation, necrosis, and suboptimal healing. As a result, the available humoural and cellular immune response may not be sufficient to clear the advancing pathogen.
Delivery and controlled release of specific antibodies at the site of implantation may guide the immunity towards bacterial clearance. Hydrogels containing solid dispersed bioactive antibodies, e.g. human immunoglobulin G (IgG), have been investigated as coatings for polymer substrates. The use of antibodies not only significantly reduced the adhesion of E. coli , but also significantly enhanced the killing of the bacteria in an in vitro opsonophagocytic assay (Rojas et al. , 2000). In other words, the presence of antibodies promoted bacterial clearance via natural immune mechanisms before the bacteria had an opportunity to form a protective biofilm. Furthermore, certain antibod- ies can bind to the toxins of pathogenic bacteria, thus reducing their virulence. Since the antibody- mediated clearance has the mechanism of action that is distinct from that of other antibiotic agents, opsonic and recognition specificity against drug resistant bacteria is retained. Other peptides and proteins capable of locally stimulating natural immune mechanisms against pathogens (e.g. GM-CSF, IL-8, IFN- γ ) can also be used.
The physico- chemical structure of the host defence peptides, specifically their cationic nature and amphipathic structure, facilitates electrostatic binding of the pep- tide to the negatively charged microbial surface (Powers and Hancock, 2003; Forbes et al. , 2013). The amphipathic property refers to the ability of the molecule to assume a conformation where clusters of hydrophobic and cationic amino acids are spatially organised in discrete sectors of the molecule (Zasloff, 2002). Once bound, the peptides interfere with the proper membrane function by destabilising and disorganising the phospholipid bilayer ( Figure 6.1 ). Once the membrane is compromised and its perme- ability is sufficiently altered, cell death occurs as a result of osmolysis (Augustyniak et al. , 2012). In addition to pore formation, the translocated peptides have been sug- gested to alter cytoplasmic membrane septum formation, inhibit nucleic acid, and cell wall synthesis, suppress protein synthesis and folding, hinder enzymatic activity, and hamper cell division (Brogden, 2005). Nisin, a peptide produced by Lactococcus lactis subsp. lactis , can not only inhibit bacterial growth through the formation of pores in the bacterial membranes, but it also binds to the peptidoglycan precursor carrier lipid II, thus disrupting the synthesis and regeneration of the cell wall and cell division (Hale and Hancock, 2007). Cationic peptides of Bacillus polymyxa (polymyxin B) and Bacillus brevis (gramicidin S) have been used clinically for resistant Gram- negative and some Gram- positive bacteria. Due to multiple mechanisms of action, the antimicro- bial peptides have a limited potential to induce de novo resistance (Yeung et al. , 2011).
Surface modification of biomaterials for biofilm control 113
The killing efficacy of peptides does not only vary for the individual groups of peptides but also for individual groups of target organisms, which further corrobo- rates the presence of multiple antimicrobial mechanisms (Augustyniak et al. , 2012).
It has been suggested that the clinical application of antimicrobial peptides as a new generation of antibiotics is greatly hindered by limited appreciation of all the mecha- nisms involved (Sahl et al. , 2005). Another significant limitation that impedes clinical applications of natural antimicrobial peptides is their enzymatic proteolysis in vivo (Brogden and Brogden, 2011). Natural peptides composed of all D-amino acids, in place of L-amino acids, have been demonstrated to resist enzymatic proteolysis while maintaining full antimicrobial activity (Zasloff, 2002). Thus by varying the content of D- and L-amino acids, short linear and cyclic amphiphillic peptides with preferential activity against Gram- positive and/or Gram- negative bacteria membranes as opposed to mammalian cells and enhanced membrane permeability can be synthesised (Fernandez-Lopez et al. , 2001; Oren and Shai, 2000; Yin et al. , 2012). Antimicrobial peptides composed of β -amino acids have been constructed for they combine resist- ance to protease degradation, unlike the α -amino acid backbone of conventional pep- tides, with a choice of secondary structures (Hamuro et al. , 1999; Porter et al. , 2000).
As most antimicrobial peptides display only moderate antimicrobial activity, researchers have been trying to synthesise new, more potent peptides, with common features including an amphipathic structure and hydrophilic, hydrophobic
Figure 6.1. Models of membrane disruption following antimicrobial peptide (AMP) adsorption onto the bacterial cytoplasmic membrane. These events are not necessarily exclusive of each other. Reproduced from (Nguyen et al ., 2011) with the permission from Elsevier.
114 Biomaterials and Medical Device-associated Infections
and cationic amino acids as structural components. Significant emphasis has often been placed on increasing the membranolytic efficiency while omitting other relevant targets. Yet, the increased non- specific membranolytic activity can also be detrimen- tal to the host cells, thus limiting utility of these antimicrobials in vivo . Enhancing the activity can be obtained via truncation of amino acids, where the cidal activity of neuropeptide Y (NPY) increased 10-fold as a result of the truncation of amino acid residues at the N terminus (Shimizu et al. , 1998). Target- specific antimicrobial pep- tides have also been designed to specifically target harmful bacteria. Pseudomonas - specific targeting moiety was added to a generally killing peptide novispirin G10, with the resultant chimeric peptide showing enhanced bactericidal activity and faster killing kinetics due to increased binding and penetration of the outer membrane of Pseudomonas sp. cells (Eckert et al. , 2006).
A number of reports have used natural and synthetic peptides as building blocks for self assembly of nanostructures, with the approach based on ionic self- complementary of peptides. Veiga and colleagues used self- assembling β -hairpin peptides rich in arginine to fabricate self- assembling hydrogel materials with strong antibacterial activity against Gram- positive and Gram- negative bacteria, including multi- drug resistant P. aeruginosa (Veiga et al. , 2012). The killing efficacy, host cytocompatibility, bulk rheological properties and stimuli- responsiveness of this type of hydrogel can be tuned by controlling the peptide sequence at the monomer level (Salick et al. , 2007, 2009; Liu et al. , 2013). Highly active against bacteria and fungi, biocompatible hydrogels made from ε -poly-L-lysine- graft-methacrylamide could be ultraviolet- immobilised onto plasma- treated plastic surfaces to form thin highly adherent antimicrobial hydrogel coatings, suitable for encapsulation of medical devices and implants (Zhou et al. , 2011).
For prevention of orthopaedic implant- associated infections, a broad spectrum of antimicrobial peptides were loaded into a thin layer of micro- porous octacalcium phosphate coating, which was electrolytically deposited onto titanium (Kazemzadeh- Narbat et al. , 2010). The structure was not cytotoxic for MG-63 osteoblast- like cells, while displaying strong, sustained antimicrobial activity against both Gram- positive ( S.
aureus ) and Gram- negative ( P. aeruginosa ) bacteria. In addition to loading, immobi- lisation of antimicrobial peptides onto the surfaces of solid implants can be achieved physically, via adsorption and layer- by-layer assembly, and chemically, via covalent bonding of the peptide (Glinel et al. , 2012). In a layer- by-layer assembly, the peptides are trapped between the layers of polyionic polymer films (Shukla et al. , 2010). By varying the number and composition of layers, high controllable loading and release can be achieved. The ease with which the peptides can migrate through the multiple polymer layers will depend on the tortuosity of the diffusion pathway, assembly thick- ness, peptide–polymer interactions, and the accumulation of attached bacteria at the surface (Onaizi and Leong, 2011). Overall, irrespective of which entrapment methodol- ogy is employed, it will influence the bioactivity of the peptide when compared to solu- ble analogue; these effects need to be carefully considered and addressed (Gao et al. , 2012). Furthermore, the stability of these antibacterial structures in vivo is a major con- cern, as leaching, de- lamination, rearrangement and ageing of the coating may result in insufficient long term biofilm resistance of the surface (Vreuls et al. , 2010).
Surface modification of biomaterials for biofilm control 115
6.7.3 Nitric oxide
Nitric oxide, an important mediator in the immune response, has been proposed as an alternative agent to be included into polymer coatings to effect highly localised destruction of bacteria (Slomberg et al. , 2013). In the course of phagocytosis, acti- vated macrophages synthesise large quantities of nitric oxide via arginine- dependent mechanisms (Nichols et al. , 2013). Nitric oxide is toxic to a range of cells, including bacteria, also inhibiting growth and multiplication of the affected cells (i.e. cytosta- sis). The mechanism of toxicity involves reactive intermediates of NO, such as per- oxynitrite and dinitrogen trioxide, putting oxidative and nitrosative stress on the cell molecules and essential functions (Nablo et al. , 2001). Although the cytotoxic and cytostatic effects of NO are not cell- type specific, and thus can potentially damage host cells, the very short half- life of NO in vivo (from less than 1 s to several min- utes depending on the concentration of oxygen and availability of NO scavengers) ensures the effects are localised within the peri- implant space (Nablo et al. , 2005).
Furthermore, therapeutic amounts of NO released from the surface of implant have been shown to hinder foreign body response (e.g. capsule formation) and improve device integration into healthy vascularised tissue (Hetrick et al. , 2007).
Since under normal physiological conditions NO is in gas phase, the spatiotempo- ral release is best achieved by impregnating the carrier matrix with donor molecules know to decompose to NO upon exposure to the in vivo conditions ( Figure 6.2 ).
Nitrosothiols, nitrosamines, diazeniumdiolates, metal complexes, and organic nitrates/
nitrites have been tested for their potential for controlled production of NO at thera- peutic levels. Due to their ability to undergo spontaneous decomposition to NO upon exposure to physiological fluid, N -diazeniumdiolate has been a popular material for the synthesis of NO releasing gels. N -diazeniumdiolate is produced by reacting NO with amines, and the rate of donor decomposition can be varied by controlling the structure of the amine segment. The NO release rate is also dependent on the chemical and physical properties of the ambient fluid to which the NO donor is exposed, e.g. pH and temperature. S-nitrosothiols are also popular donors due to their ability to decom- pose thermally and photochemically, as well as via copper- catalysed decomposition at physiological pH. The rate of NO release can be greatly increased in the presence of gold nanoparticles (Taladriz-Blanco et al. , 2013), with gold- nanoparticle-mediated release of NO becoming the dominant mechanism of release. Since the bond energy of gold thiolate (S–Au) is much greater than the bond dissociation energy of S–N, at 40 kcal mol −1 and 20–32 kcal mol −1 , respectively, the latter bond can be readily cleaved in the presence of nAu to favour S–Au bond formation (Priya et al. , 2012).
The manner in which NO donors are entrapped within the polymer material will affect the release behaviour of these structures in vivo . While physically entrapped, uniformly dispersed in hydrophobic polymers diazeniumdiolates have been shown to effectively produce NO (Zhang et al. , 2002; Seabra and de Oliveira, 2004), these potentially toxic molecules and their amine by- products can leach out from the poly- mer matrix into the peri- implant space. Covalent linking of NO donors to the poly- mer backbone can provide for a better control and biocompatibility of the structure, without compromising their antibacterial efficacy (Nablo et al. , 2001). Organic–inor-
116 Biomaterials and Medical Device-associated Infections
ganic polymer matrices can be fabricated using aminosilane- based sol–gel chemistry, with the properties controlled by the nature and amount of diamine- containing organosilane precursors and sol–gel processing conditions (Nablo et al. , 2001, 2005; Hetrick and Schoenfisch, 2007; Hetrick et al. , 2007). A number of alkyl- and aminosilane precursors have been considered, including methyl-, ethyl-, and Figure 6.2. Common sources of nitric oxide. Structure and NO release mechanism of
(a) diazeniumdiolates, (b) S-nitrosothiol, (c) metal–nitrosyl, and (d) nitrobenzene.
(a−c) reproduced from (Kim et al ., 2014) with permission from Royal Society of Chemistry;
(d) reproduced from (Sortino et al ., 2002) with permission from Wiley and Sons.
Surface modification of biomaterials for biofilm control 117
butyltrimethoxysilances, aminoethylaminomethyl phenethyltrimethoxysilane, N -(2- aminothyl)-3-aminopropyltrimethoxysilane, N -(6-aminohexyl)aminopropyltrimeth- oxysilane, and N -[3-(trimethoxysilyl)propyl]diethylenetriamine (Radha and Ashok, 2010; Storm and Schoenfisch, 2013). Hong and colleagues pointed out several drawbacks of these gels, namely that the sol–gel chemistry is limited to chemistry of material surfaces such as metal oxides, and that the silane coatings produced in this manner are typically thick, with the thickness in the micro- to millimeter range (Hong et al. , 2013).
Self- assembled monolayers have also been considered as platforms for controlled release of NO, where the release is triggered by electrochemical stimuli and light excitation (Hou et al. , 2000; Sortino et al. , 2002). In one example, a thiol tail was attached to the NO donor molecule to facilitate self- assembly on the surface of a thin gold electrode (Hou et al. , 2000). The quantitative NO release on a nanomo- lar scale was achieved by applying electric potential to the electrode. The obvious drawback of this approach is that the method is restricted to noble metal surfaces, e.g. those of implantable electrodes. A material- independent NO immobilisation and release is based on poly(norepinephrine) surface chemistry (Hong et al. , 2013).
Norepinephrine is a small- molecule catecholamine which acts as a neurohormone and neurotransmitter in the body. Catecholamine possesses unique non-specific adhesive properties, with the ability to form coordination bonds with metallic and inorganic surfaces, hydrogen bonds, and π - π aromatic interactions. These molecules poly - merize at alkaline pHs to form thin adherent layers with latent reactivity toward amine and thiol groups, thus enabling subsequent immobilisation of functional molecules.
Thin (~12 nm), smooth coatings of poly(norepinephrine) were first deposited; then, the surface- conjugated diazeniumdiolates groups reacted with the secondary amines on the respective poly(norepinephrine) layers. The presence of 3,4-dihydroxybenzal- dehyde polymerisation intermediate on the surface and the hydroxy group within the poly(norepinephrine) layer is believed to facilitate NO loading and release.
6.7.4 Metal ions
In addition to carbon- based nanomaterials, many microorganisms have displayed sensitivity to metals, including Ag, Al, Au, Cu, Zn, and their oxides, e.g. CuO, TiO 2 , and ZnO (Li et al. , 2008; Huang et al. , 2008; Allaker and Ren, 2008; Mühling et al. , 2009). The mechanisms of their antimicrobial action are diverse, ( Figure 6.3 ) and include photocatalytic production of reactive oxygen species that damage compo- nents of cells and viruses; disruption of the integrity of bacterial cell wall/membrane (e.g. ZnO, nAg); interruption of energy transduction (e.g. nAg); and inhibition of enzyme activity and DNA synthesis (Huh and Kwon, 2011; Li et al. , 2008). Both TiO 2 and ZnO display strong photocatalytic activity, whereby irradiation of the oxide with near-UV light results in the production of reactive oxygen species. These spe- cies are then actively involved in peroxidation of the polyunsaturated phospholipid component of the lipid membrane, inducing major disorder in the cell membrane of the microorganism (Maness et al. , 1999). The resultant loss of cell membrane archi- tecture affects such essential functions as respiratory activity, eventually resulting in