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There is concern that PEG-based materials may lose their effectiveness in vivo due to enzymatic oxidation of the hydroxyl end- groups at the side chains into aldehydes and acids (Ostuni et al. , 2001; Herold et al. , 1989). As such, the corresponding methoxy end- functionalized monomer, poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMEMA), has been proposed as a possible solution to this problem (Barbey et al. , 2009).
The bacterial adhesion and biofilm formation on gold substrates modified via surface- initiated ATRP of OEGMA and zwitterionic sulfobetaine methacrylate (SBMA, Figure 7.4(c)) have been compared by Cheng et al. , (2007). Both types of modified surfaces are effective in reducing S. epidermidis and P. aeruginosa accumu- lation on the surface both short- term (3 h) and long- term (24 h or 48 h). These surfaces are more effective than self- assembled monolayers of alkanethiols with shorter- chain oligo(ethylene glycol) or mixed SO 3 − /N + (CH 3 ) 3 terminated groups in resisting bio- film formation, and this is attributed to the thicker coating and higher densities of non- fouling groups in the former. The same group also graft- polymerized another zwitterionic compound, carboxybetaine methacrylate (CBMA, Figure 7.4(d)), on glass, and the poly(CBMA) was found to be comparable to poly(SBMA) in resisting biofilm formation (Cheng et al. , 2009).
Surface- initiated polymerization has also been used to prepare polyacrylamide (PAAM) and poly(methacrylic acid) for inhibiting bacterial adhesion. PAAM brushes on silicone rubber can be prepared by surface- initiated ATRP using a multi- step reac- tion procedure (Fundeanu et al. , 2008). The silicone rubber was first hydrophilized by UV/ozone treatment and then treated with γ -aminopropyltriethoxysilane followed by the anchoring of the ATRP initiator, 4-(chloromethyl) benzoyl chloride, and polymeri- zation of acrylamide (AAM, Figure 7.4(e)) either in N,N -dimethylformamide (DMF) or in water. The PAAM brushes grown in water reduced the adhesion of S. aureus by 58%, Streptacoccus. salivarius by 52% and C. albicans by 77%. The brush coat- ings grown in DMF are thicker and offer slightly better anti- adhesive properties. The PAAM coatings discouraged microbial adhesion even after exposure to phosphate- buffered saline (PBS) and saliva for one month at 37 °C. Poly(methacrylic acid) coat- ing has been formed on titanium surfaces via surface- initiated ATRP of methacrylic acid sodium salt (MAAS, Figure 7.4(f)) with immobilized trichloro(4-(chloromethyl)- phenyl)silane. The functionalized surfaces are highly hydrophilic (contact angle of 8°) and bacterial adhesion is inhibited (Zhang et al. , 2008).
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eluted agents that may diffuse away from the site. Furthermore, when antibiotics are used in eluting systems, their presence at levels below the minimal inhibitory concen- tration for long periods of time may encourage the emergence of resistant organisms.
7.5.1 Antibiotics and antimicrobial peptides
Different classes of antibiotics have different principal mechanisms of action, i.e.
(i) interference with cell wall synthesis such as β -lactams (e.g. penicillin and ampicil- lin) and glycopeptides (e.g. vancomycin), (ii) inhibition of protein synthesis such as aminoglycosides and macrolides, (iii) interference with nucleic acid synthesis such as fluoroquinolones and rifampin, (iv) inhibition of a metabolic pathway such as sulfon- amides and folic acid analogues, and (v) disruption of bacterial membrane structure such as polymyxins and daptomycin (Tenover, 2006). Antibiotics to be covalently attached to surfaces should ideally be those that work on the bacterial cell wall or membrane since immobilization may inhibit access to sites of action inside the bacte- ria. Vancomycin, an antibiotic active against Gram- positive, but not Gram- negative, organisms has been covalently immobilized on titanium alloy surfaces via silane and aminoethoxyethoxyacetate anchors (Jose et al. , 2005; Antoci Jr. et al. , 2008).
The use of a hydrophilic chain spacer allows the vancomycin to extend away from the titanium surface to enter the bacterial cell wall to bind to L-Lys-D-Ala-D-Ala termini and disrupt the biosynthesis of the peptidoglycan layer. S. epidermidis (Gram- positive) colonization was significantly inhibited on the vancomycin- functionalized surface while E. coli (Gram- negative) readily colonized the surface, suggesting reten- tion of the specificity of vancomycin upon immobilization (Antoci Jr. et al. , 2008).
Bisphosphonic acid derivative of daptomycin, a Gram- positive peptide antibiotic, was synthesized and attached on titanium alloy surfaces through the bisphosphonate groups (Chen and Wickstrom, 2010). Flexibility of the attached daptomycin was provided by a tetra(ethylene glycol) spacer. The daptomycin- functionalized surface killed 53% of bacteria suspended in a drop of broth (1.5 × 10 6 cfu/mL) applied to the surface. Zhang et al. , (2007) grafted poly(2-hydroxyethyl methacrylate) on titanium and converted the pendant hydroxyl end groups of the grafted chains into carboxyl or amine groups to allow the coupling of gentamicin and penicillin to form an antibacte- rial surface. Aumsuwan et al. , (2007, 2008) used microwave maleic anhydride (MA) plasma reactions to generate surface active groups and attach penicillin and ampi- cillin to expanded poly(tetrafluoroethylene) (ePTFE) via a PEG linker. Ampicillin, being a broad spectrum antibiotic, resulted in the formation of antimicrobial surfaces effective against Gram-positive S. aureus, Bacillus thuringiensis , and Enterococcus faecalis , and Gram negative E. coli, P. putida , and Salmonella enterica bacteria.
Approximately 10% of the ampicillin was released after immersion in PBS buffered solution for 24 h.
Antimicrobial peptides (AMPs) are produced by a variety of animals, plants, bacteria, fungi and viruses, and comprise a chemically and structurally heterogene- ous family. Three characteristics are shared by almost all known AMPs: (i) 10–25 amino acids in size with molecular weights between 1 and 5 kDa; (ii) highly cationic although with large variations in the net positive charge; (iii) tendency to adopt
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amphipathic structures in non- polar media (Costa et al. , 2011). Although the exact antibacterial mechanism of AMPs has not been clearly elucidated, it is hypothesized that cationic AMPs are electrostatically attracted to the negatively charged bacterial cell membrane and the interaction with the phospholipid membrane can cause cell lysis and subsequent cell death. AMPs have some advantages over antibiotics since they have a broad spectrum of antimicrobial activity, are effective at low concentra- tions, and they rarely promote the rise of bacterial resistance (Yala et al. , 2011). Costa et al. , (2011) has provided an overview of the immobilization strategies employed for AMPs on different types of substrates, and highlighted the parameters that can modu- late the activity of the immobilized AMPs. One such parameter is the lateral mobility and orientation of the conjugated peptide for interacting with the bacterial membrane.
The immobilization of the AMP in a controlled manner to maintain and allow expo- sure of the relevant peptide structural motifs is also preferred over immobilization which involves random groups in the peptide. Examples of the strategies that can be used for controlled immobilization of AMP are given in Figure 7.5 (Costa et al. , 2011). Gabriel et al. , (2006) compared the antibacterial activity of LL37 grafted on titanium surfaces with and without a flexible hydrophilic PEG spacer, and found that only the peptide conjugated to the PEG spacer via its N-terminus is capable of killing
Figure 7.5 Examples of chemical strategies for controlled covalent attachment of AMPs on surfaces: (a) use of thiol- bearing peptides (Cys usually as thiol donor) for covalent immobilization onto thiol-, maleimide- or epoxide- modified surfaces; (b) use of the Huisgen 1,3-dipolar cycloaddition for immobilization of either alkyne- bearing peptides onto azide- modified surfaces or azide- bearing peptides onto alkyne- modified surfaces. Reprinted with permission from Costa et al. , (2011). Copyright 2011 Elsevier.
Surface nanoengineering for combating biomaterials infections 149
E. coli on contact. No killing was observed when bacteria were exposed to titanium with randomly immobilized peptide, both with and without the use of a spacer, or with directly N-terminally bound LL37. The reduction in antimicrobial activity of an amphipathic model KLAL peptide and magainin- derived MK5E with reduction in the PEGylated spacer length was demonstrated by Bagheri et al. , (2009). However, some other studies (Haynie et al. , 1995, Hilpert et al. , 2009) reported that the spacer did not play a significant role in determining the bactericidal properties, and thus the importance of the spacer may be dependent on the peptide’s mode of action.
Glinel et al. , (2009) combined the antibacterial peptide, magainin I, with anti- adhesive polymer brushes to obtain an efficient antibacterial coating. Surface- initiated ATRP was used to prepare copolymer brushes based on 2-(2-methoxyethoxy)ethyl methacrylate (MEO 2 MA) and hydroxyl- terminated oligo(ethylene glycol) methacr- ylate (HOEGMA) from a silicon wafer surface. The poly(MEO 2 MA- co -HOEGMA) brushes of 110 nm thickness effectively prevented the adhesion of two Gram positive bacteria: Listeria ivanovii and Bacillus cereus . Magainin I was modified by attaching a cysteine residue at its C-terminal part, which was then tethered to the polymer brushes via a N -( p -maleimidophenyl) isocyanate heterolinker. In the presence of magainin I on the polymer brushes, some bacterial attachment was observed but the bacteria were predominantly dead as a result of the interaction with the peptide. Since magainin I is grafted to the hydroxyl groups of the polymer brushes, the density of grafted magainin I can be adjusted by changing the copolymer composition. While Glinel et al. , (2009) reported no reduction in bactericidal activity even for low levels of grafted magainin I, Humblot et al. , (2009) hypothesized that at low concentration of surface- immobilized magainin I, the possibility for multiple entries of the peptide into the cell membrane is low, and this results in a bacteriostatic rather than bactericidal effect.
Lysozyme is an antimicrobial enzyme which can hydrolyze the 1,4- β -linkages between N -acetylmuramic acid and N -acetyl-D-glucosamine in the peptidoglycan of bacterial cell wall (Arnheim et al. , 1973). It has been immobilized on PEG chains grafted on stainless steel to render the surface simultaneously anti- adhesive and anti- bacterial (Yuan et al. , 2011). The polymer brushes were grafted from the stainless steel surface via surface- initiated ATRP of poly(ethylene glycol) monomethacrylate and the hydroxyl groups on the side chains of the brushes were then activated with 1,1′-carbonyldiimidazole to covalently bind the lysozyme. The lysozyme remained bound to the surface even after 10 days immersion in PBS. In another application, lysozyme was covalently conjugated to PEO chains of Pluronic F-127 via reductive amination of the aldehyde- functionalized PEO blocks by the amine groups of the lysine residues of lysozyme (Muszanska et al. , 2011). The Pluronic- lysozyme conju- gate adsorbs on a hydrophobic surface via its PPO block and the PEO chains with the attached lysozyme molecules are exposed to the solution. Thus, this conjugate serves as an anti- adhesive and antibacterial bifunctional surface coating.
7.5.2 Bactericidal polymers
Bactericidal polymers usually contain cationic groups, such as alkyl pyridinium or quaternary ammonium, and it is generally thought that these polymers kill bacteria
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by rupturing their cellular membranes. A number of investigators have functionalized surfaces with bactericidal polymers prepared via the polymerization and subsequent quaternization of 2-(dimethylamino)ethyl methacrylate (DMAEMA). By using the appropriate initiator, surface- initiated ATRP of DMAEMA was carried out from polymeric (Huang et al. , 2007) and metallic (Yuan et al. , 2009) surfaces, and RAFT polymerization of DMAEMA was carried out from cellulosic filter paper (Roy et al. , 2008). The modified surfaces are highly effective in killing bacteria. However, poly(DMAEMA) is known to exhibit cytotoxic effects towards different types of mammalian cells (Jiang et al. , 2007; Newland et al. , 2010; Wang et al. , 2011). Thus, more work has to be carried out to ascertain whether poly(DMAEMA) is suitable as an antibacterial coating for medical devices.
It has been shown that N -hexyl, N -methyl- polyethyleneimine ( N -hexyl, N -methyl- PEI) covalently attached to glass or polymer surfaces can effectively kill Gram- positive and Gram- negative bacteria as well as fungi (Klibanov, 2007). The covalent bonding of this polymer to the surfaces requires a number of steps, and a simpler method of ‘painting’ the more hydrophobic polycations on surfaces was developed by the same group. Glass slides were coated with N -dodecyl, N -methyl-PEI by means of painting the surface with the hydrophobic polycation dissolved in an organic solvent, followed by evaporation of the latter. These surfaces exhibited high killing efficiency (≥ 98%) against airborne S. aureus, E. coli and influenza virus (Haldar et al. , 2006).
Other hydrophobic PEI derivatives can be used but the bactericidal efficiency is dependent on the hydrophobicity of the polymer and the structure/molecular weight of the derivatized PEI. Dhende et al. , (2011) used another method in which copoly- mers of hydrophobic N -alkyl and benzophenone containing PEIs were spin- cast or spray- coated on different types of surfaces followed by photochemical grafting of the pendant benzophenones. When the thickness of the polymer layer was > 50 nm, almost all the bacteria sprayed on the surface were killed. Ignatova et al. , (2009) grafted preformed hyperbranched PEI (Mw = 50,000–60,000, relative contents of primary, secondary, and tertiary amines = 1:1:1) onto poly( N -succinimidyl acrylate) which had been electrografted onto stainless steel. The PEI coating was then quater- nized by 1-chlorooctane. The high density of quaternary ammonium groups in the hyperbranched PEI was highly efficient in suppressing bacterial colonization of the surface.
Antibacterial pyridium- type polymers have also been introduced on surfaces of substrates (Tiller et al. , 2002; Klibanov, 2007; Cen et al. , 2003, 2004). A simple two- step surface functionalization technique involving UV-induced surface graft copolymerization of 4-vinylpyridine (4VP), followed by the alkylation of the grafted poly(4-vinylpyridine) with hexylbromide is shown in Figure. 7.6. This technique has been applied to polymeric and cellulosic materials (Cen et al. , 2003, 2004). The func- tionalized PET surface has a high killing efficiency against E. coli when the concentra- tion of pyridinium groups on surfaces is 15 nmol/cm 2 or higher. Since the pyridinium moieties are covalently bonded to the substrate, the antibacterial property is preserved even after the functionalized substrate has been subjected to prolonged weathering under UV irradiation and water spray.
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Chitosan, a cationic polysaccharide with antibacterial properties (Rabea et al. , 2003; Raafat et al. , 2008), and carboxymethyl chitosan (CMCS) have been grafted on titanium surfaces via a dopamine anchor to confer the surface with antibacterial properties (Shi et al. , 2008, 2009; Hu et al. , 2010). An advantage of using chitosan or CMCS is the availability of reactive groups on these molecules for further func- tionalization. RGD (Arg-Gly-Asp) (Shi et al. , 2008), bone morphogenetic protein-2 (BMP-2) (Shi et al. , 2009) and vascular endothelial growth factor (VEGF) (Hu et al. , 2010) have been conjugated to the chitosan or CMCS graft layer on titanium to develop a biointeractive surface that simultaneously decreases bacterial colonization while enhancing osteoblast functions. CMCS has also been grafted on medical grade silicone surface pre- treated with polydopamine (Wang et al. , 2012). The hydrophilic- ity of the CMCS-grafted surface (contact angle < 30 °) is significantly higher than that of pristine silicone (contact angle ~ 107 °), and the adhesion of E. coli and Proteus mirabilis was reduced by ≥ 90%. The antibacterial property was preserved even after aging of the functionalized surfaces for 21 days in PBS, and also after auto- claving at 121 °C for 20 min. No significant cytotoxicity of the chitosan and CMCS functionalized surfaces was observed with mammalian cells. The LbL method can be utilized to assemble chitosan and hyaluronic acid as PEMs on titanium surfaces to combine the bactericidal property of chitosan with the anti- adhesive nature of hyaluronic acid (Chua et al. , 2008a, 2008b). The number of adherent bacterial cells
Figure 7.6 Functionalization of surfaces with pyridinium groups for antibacterial applications.
Reprinted with permission from Cen et al. , Copyright 2003 American Chemical Society.
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on the PEM-functionalized titanium decreased by about 80% relative to that on pris- tine titanium. By chemical cross- linking of the PEMs using carbodiimide, structural stability was improved and the antibacterial properties were preserved even after the prolonged immersion in PBS.