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but Desai et al. , (2010) reported that nitrofurazone impregnation had a significant effect only for the first five days. One concern regarding the use of antibiotics- impregnated devices is the rise of multidrug- resistant bacterial strains with increased and continued use of antibiotics.
7.4 Surface functionalization with anti- adhesive
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units) block lengths. Pluronic coating has also been shown to reduce the adhesion of C. albicans on polystyrene (Wesenberg-Ward et al. , 2005), and S. aureus and S. epidermidis on silicone rubber (Nejadnik et al. , 2008). However, the inhibition effect of the Pluronic coating appears to be strain dependent since the adhesion, growth and detachment of P. aeruginosa on silicone rubber with the Pluronic coating were not significantly different compared to the pristine surface (Nejadnik et al. , 2008).
Polymer chains can also self- assemble on surfaces via functional end or side groups which act as anchors. Polymer chains with thiol groups can self- assemble on gold, while those with silane groups will self- assemble on metal and silicon/glass surfaces. Self- assembled monolayers of oligo(ethylene glycol) (from HS(CH 2 ) 11 (OC H 2 CH 2 ) 6 OH) on gold- coated glass slides were reported to resist bacterial attachment (Ista et al. , 1996). PEG chains have been modified with peptide sequences with spe- cific affinity for titanium (Khoo et al. , 2009) or polystyrene (Kenan et al. , 2006), and the peptide- modified PEG assembled from dilute aqueous solution onto the selected surface through adsorptive mechanisms. The coated surfaces were effective in block- ing the adsorption of fibronectin and reducing the extent of S. aureus attachment and biofilm formation in vitro . Titanium surfaces can also be coated with a graft copoly- mer comprising a polycationic poly(L-lysine) (PLL) backbone and PEG side chains (PLL- g -PEG) (shown schematically in Figure 7.2(b)) (Blättler et al. , 2006; Kenausis
Figure 7.2 Schematic representation of the self- assembly of (a) a triblock copolymer with hydrophilic end blocks and a hydrophobic center block on a hydrophobic surface, and (b) a polymer with cationic backbone and neutral side chains on a negatively charged surface.
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et al. , 2000; Tosatti et al. , 2003, Harris et al. , 2004). The copolymer, comprising approximately 120 L-lysine units, a PEG side chain of 47 ethylene glycol units and a grafting ratio (number of lysine monomers per PEG side chain) of between 3.3 and 4.5, adsorbed rapidly and strongly on the oxide layer of the titanium surface through electrostatic interactions. This coating reduced the adhesion of S. aureus on smooth and rough (chemically etched) titanium surfaces by 89–93% after expo- sure to S. aureus culture for 1 to 24 h. A comparison of poly(2-methyl-2-oxazoline) (PMOXA) and PEG in inhibiting protein and bacterial attachment has been carried out by Pidhatika et al. , (2008). Both polymers were side- chain grafted onto a PLL backbone, and these graft copolymers spontaneously self- assembled to form monol- ayers on negatively charged surfaces. The PMOXA surface coatings were found to be as efficient as PEG-based coatings in suppressing protein and bacterial adsorption.
However, the minimal number of side chain monomer units per surface area that are needed to obtain fully resistant surfaces was lower for PMOXA than for PEG graft copolymers due to the higher molecular weight of the PMOXA monomer unit. The same group found that for PLL(20 kDa)- g -PMOXA(4 kDa), a PMOXA side chain grafting density (PMOXA/lysine) of 0.33 can be considered as being non- fouling to macromolecular entities in a rather general sense, preventing adsorption of proteins independent of charge, and attachment of E. coli independent of the presence/absence of fimbriae (Pidhatika et al. , 2010).
A disadvantage of the self- assembly method is its lack of stability. For example, the stability of the PLL- g -PEG coating is affected by pH and ionic strength of the aqueous media since interfacial electrostatic binding is compromised when the den- sity of positive charges on the PLL backbone is reduced with increasing pH or when the negative surface charge of the metal oxide is decreased upon lowering the pH.
In high ionic strength media, decreased electrical double layer interactions will also compromise the stability of the PLL- g -PEG layer (Blättler et al. , 2006). Covalent binding of the polymer chains to surface groups (‘grafting to’ process) can be carried out to provide a higher degree of stability, and also to provide a polymer coating on surfaces where electrostatic binding is not possible.
7.4.2 Grafting of polymer chains to surfaces
Anti- adhesive polymers such as PEG can be covalently bonded with complemen- tary groups on surfaces via the “grafting to” method (Figure 7.3(a)). The surface functional groups can be generated by various means including plasma treatment, ozone treatment, wet chemical methods or a combination of methods. Blättler et al. , (2006) created an aldehyde plasma interlayer on inorganic and polymeric substrates for covalent immobilization of a PLL- g -PEG coating via reductive amination of the PLL. Coatings produced in this manner were stable in a 2.4 M salt solution for 24 h, whereas electrostatically adsorbed PLL- g -PEG polymers would desorb to substantial extents under such high salt conditions. The plasma technique has also been used to activate the surfaces of substrates for direct immobilization of PEG. Dong et al. , (2007, 2011) used silicon tetrachloride (SiCl 4 ) plasma to treat the surfaces of polyamide and polyester to generate active surface functional groups
Surface nanoengineering for combating biomaterials infections 143
which subsequently reacted with the OH end group of PEG chains. The inhibition of Salmonella enterica serovar Typhimurium ( S. enterica sv. Typhimurium) attachment and biofilm formation was dependent on the PEG molecular weight, and the highest efficacy was obtained with PEG of molecular weight of 600 and 2000. The substrates grafted with these types of PEG also significantly inhibited biofilm formation by L. monocytogenes . Stability tests showed that after storage under ambient conditions for over two months, the PEG 2000-grafted PET demonstrated reduced antifouling ability. Nevertheless, it still significantly reduced biofilm formation by S. enterica sv. Typhimurium. Radio frequency plasma treatment of stainless steel surfaces in the presence of 1,4,7,10-tetraoxacyclododecane (12-crown-4)-ether and tri(ethylene glycol) dimethyl ether (triglyme) has been used to deposit PEG-like structures (Denes et al. , 2001). Bacterial attachment on the 12-crown-4 plasma- modified and triglyme plasma- modified surfaces decreased by 56% and 82%, respectively compared to the unmodified surfaces. The corresponding decrease in biofilm formation on the respective surface after one day was 72% and 94%. Hyaluronic acid, a linear anionic polysaccharide, has been grafted on polymethylmethacrylate intraocular lenses to effectively inhibit fibroblast and S. epidermidis adhesion (Cassinelli et al. , 2000).
The lenses were first subjected to air plasma treatment followed by treatment with polyethyleneimine. The carboxyl groups of hyaluronic acid were then linked to the amino- functionalized surface by carbodiimide.
Figure 7.3 Modification of a substrate surface with polymer brushes via a (a) ‘grafting to’, and (b) ‘grafting from’ approach. ‘M’ denotes monomer.
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The generation of active surface functional groups for tethering PEG chains can also be carried out using wet chemistry methods. Isocyanate groups were conferred on the surface of polyurethane by grafting with hexamethylenediisocyanate through the allophanate reaction between the urethane proton and the isocyanate in the pres- ence of a catalyst (di- n-butyl tin dilaurate) (Park et al. , 1998). The surface isocyanate groups were then reacted with the terminal groups of the PEG (-OH and -NH 2 ).
The efficacy in reducing bacterial adhesion level was found to depend on the length of PEG chains, the bacteria as well as the media. A multistep process was used by Kingshott et al. , (2003) to graft PEG on PET. Hydroxylation of the PET surface was first carried out by exposure to formaldehyde in acetic acid. This was followed by carboxylation of the hydroxyl groups with bromoacetic acid in sodium hydroxide, amidation by reaction with polyethyleneimine using carbodiimide chemistry, and finally reaction with linear methoxy- terminated PEG-aldehyde in the presence of sodium cyanoborohydride at the lower critical solution temperature (LCST) of the PEG. The PEG layer was estimated to be 4–4.5 nm in thickness, and the coating reduced the level of adhesion of Pseudomonas sp. by between 2 and 4 orders of magnitude for up to 5 h. A much simpler and versatile technique of introducing func- tional groups on biomaterials was developed by Messersmith and co- workers based on catecholic anchors inspired by mussel adhesive proteins (Fan et al. , 2005; Lee et al. , 2007). Dopamine strongly adsorbs on titanium and stainless steel surfaces from aqueous solution via its catechol groups (Fan et al. , 2005). Oxidized dextran can be grafted on dopamine- treated titanium via a reductive amination method to inhibit the adhesion of S. aureus and S. epidermidis (Shi et al. , 2009). Linear monomethoxy- terminated PEGs conjugated to either 3,4-dihydroxyphenylalanine (DOPA) or to the N terminus of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (mPEG-MAPD) have been used as a anti- adhesive layer on titanium (Dalsin et al. , 2003). Besides mussel adhesive proteins, another biomimetic anchor based on the cyanobacterial iron chela- tor, anachelin which has a strong binding affinity for metal oxide, has been used to anchor PEG chains to TiO 2 surfaces (Zurcher et al. , 2006).
Phosphorylated PEG derivatives have been used as anti- adherent coatings for hydroxyapatite, which is widely used in orthopedic and dental applications (Shimotoyodome et al. , 2007). Methacryloyloxydecyl phosphate (MDP)-PEG deriva- tives were prepared from the polymerization of PEG methacrylate, methacrylic acid, and MDP in an aqueous solution containing 2-mercaptoethanol and ammonium per- sulfate. The phosphorylated PEG interacts strongly with hydroxyapatite and renders the surface hydrophilic. The hydroxyapatite pretreated with MDP-PEG prior to saliva incubation reduced salivary protein adsorption and saliva- promoted attachment of Staphylococcus mutans . However, the coating is not as effective when MDP-PEG was coated on saliva- pretreated hydroxyapatite since the adsorption of MDP-PEG was inhibited by salivary components already bound to the hydroxyapatite.
7.4.3 Surface- initiated polymerization
While the ‘grafting to’ method can be readily carried out, the polymer brushes that are attached on the surface pose a steric barrier to approaching polymer molecules as the
Surface nanoengineering for combating biomaterials infections 145
reaction progresses. Thus, it is difficult to achieve a high grafting density, and the film thickness is limited by the molecular weight of the polymer in solution (Edmondson et al. , 2004). The effectiveness of the grafted polymer layer in preventing bacterial adhesion can be expected to depend on the chain length, density and conformation.
An alternative to the ‘grafting to’ method is the ‘grafting from’ technique which is the initiation of polymerization from initiators bound to the surface (Figure 7.3(b)).
In this approach, the monomer diffuses to the propagating chain end, which can be expected to be less susceptible to steric hindrance than the diffusion of a preformed polymer in the ‘grafting to’ approach.
The polymerization can be carried out via conventional free radical polymerization or controlled/‘living’ polymerization techniques such as nitroxide- mediated polym- erization (NMP), reversible addition–fragmentation chain transfer (RAFT) polym- erization, and ATRP. By using controlled/‘living’ polymerization techniques, the functionality, density and thickness of polymer brushes can be controlled with higher precision. Of the controlled/‘living’ polymerization techniques, ATRP has been used most extensively to prepare anti- adhesive polymer brushes. The process involves the immobilization of the ATRP initiator on the substrate surface as the first step followed by the ATRP process. As in the ‘grafting to’ approach, there are different methods for the immobilization of the ATRP initiator. Zhao et al. , (2011) formed a self- assembled monolayer of ω -mercaptoundecyl bromoisobutyrate on gold to serve as the ATRP initiator and polymerized 2-hydroxyethyl methacrylate (HEMA) (Figure 7.4(a)) and hydroxypropyl methacrylate (HPMA) to form coatings with different thicknesses.
Mrabet et al. , (2009) used brominated silane solution to introduce the initiators on glass slides for the surface- initiated ATRP of HEMA. Both poly(HEMA) and poly(HPMA) coatings can reduce bacterial adhesion. In another method, brominated aryl ATRP initiators were grafted on gold- coated silicon wafers via the electro- chemical reduction of a non- commercial diazonium salt, BF 4 − + N 2 -C 6 H 4 -CH(CH 3 ) Br (Mrabet et al. , 2011). The diazonium- modified gold surface served as macroinitia- tors for ATRP of oligo(ethylene glycol) methacrylate (OEGMA, Figure 7.4(b)), which resulted in hydrophilic surfaces resistant to fouling by Salmonella typhimurium .
Figure 7.4 Monomers used in the preparation of anti- adhesive surfaces via ATRP:
(a) 2-hydroxyethyl methacrylate (HEMA); (b) oligo(ethylene glycol) methacrylate (OEGMA); (c) sulfobetaine methacrylate (SBMA); (d) carboxybetaine methacrylate (CBMA); (e) acrylamide (AAM) and (f) methacrylic acid sodium salt (MAAS).
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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).