One of the most widely used strategies to prevent bacterial colonization on a sur- face is via a persistent release of an antibacterial agent from the surface, which kills bacteria in the vicinity of the surface. For this method, an inherent challenge is to design the coating to release the antibacterial agent at the desired rate and for the desired period.
7.3.1 Silver ions and nanoparticles
Silver is one of the most commonly used antibacterial agents, and its inhibitory action is believed to result from the interaction of Ag + ions with thiol and other key functional
Surface nanoengineering for combating biomaterials infections 137
groups in enzymes and proteins (Grier, 1983; Jung et al. , 2008). While silver nanopar- ticles are reported to cause pit formation in bacterial cell wall and increased membrane permeability (Sondi and Salopek-Sondi, 2004), it appears that silver nanomaterials may exert bactericidal activity through the release of Ag + ions (Marambio-Jones and Hoek, 2010). Thus, the efficacy of silver- based surface coatings will be dependent on the rate of release of the Ag + ions. A rapid release will result in a limited period of protection as the supply of silver is depleted, while a release which is too slow may not provide sufficient Ag + ions to kill the bacteria. Silver ions have been loaded into a layer of poly(3-sulfopropylmethacrylate) brushes grown from (‘grafting from’ process – see Section 7.4.3) the surfaces of gold and Si/SiO 2 via atom transfer radical polymerization (ATRP) (Ramstedt et al. , 2007). The brush thickness ranged from a few nanometers to several hundred nanometers and silver ions were coordinated to the anionic sulfonate moieties. It was demonstrated that the leaching of the silver ions from the polymer layer inhibited bacterial growth and biofilm formation by S. aureus and P. aeruginosa .
The incorporation of silver nanoparticles in a surface polymeric matrix has also been used as a method of preparing silver- based coatings. The nanoparticles may be incorporated simultaneously with the preparation of the polymeric matrix or gener- ated within the formed matrix. Plasma polymerization has been shown to be a con- venient method for forming the matrix for silver incorporation and subsequent release (Chen et al. , 2008; Körner et al. , 2010; Vasilev et al. , 2011). Vasilev et al. , (2010) demonstrated that the amount of silver nanoparticles loaded in a n -heptylamine plasma polymer matrix on a glass substrate and the subsequent release of Ag + ions can be controlled to result in complete inhibition of S. epidermidis colonization over 24 h.
Another method for incorporating silver onto surfaces is via the use of polyelec- trolyte multilayers (PEMs) (Lichter et al. , 2009). PEMs are usually constructed using the layer- by-layer (LbL) process (Figure 7.1) which essentially involves spray or dip coating a substrate alternately with positively or negatively charged polyelectrolytes (Decher, 1997). Multiple repetitions of this procedure result in a coherent surface coating held together by electrostatic interactions between the oppositely charged polyelectrolytes as well as other secondary interactions such as hydrogen bonding.
The thickness of the coating typically ranges from tens to hundreds of nanometers (Croll et al. , 2006). An advantage of this method is that it can be readily adapted to different types of surfaces provided that surface charges are present. Incorporation of silver within the PEMs is usually carried out by introduction of silver ions into the multilayers followed by in situ reduction to form the nanoparticles (Wang et al. , 2002;
Shi et al. , 2006; Logar et al. , 2007; Agarwal et al. , 2010). Examples of polyelectro- lyte pairs used for constructing PEMs for this application are poly(ethyleneimine) and poly(acrylic acid), and poly(allylamine hydrochloride) and poly(acrylic acid), which were used by Shi et al. , (2006) and Agarwal et al. , (2010), respectively. The latter varied the amount of silver nanoparticles within the PEMs from ~ 0.4 μ g/cm 2 to ~ 23.6 μ g/cm 2 . Films with as little as ~ 0.4 μ g/cm 2 of silver resulted in 99.9999%
reduction of S. epidermidis in suspensions incubated with the films, and no toxic effects on mammalian cells were observed. PEMs have also been used to embed silver ion- containing liposomes (Malcher et al. , 2008). The liposomes were filled
138 Biomaterials and Medical Device-associated Infections
with an AgNO 3 solution and deposited on a poly(L-lysine)/hyaluronic acid multilayer film, followed by capping with hyaluronic acid/poly(L-lysine)/hyaluronic acid. The release of encapsulated AgNO 3 from this composite coating was triggered at 34 °C. A 4-log reduction in the number of viable E. coli cells was observed after contact with a 120 ng/cm 2 AgNO 3 coating for 120 min. Silver has been incorporated in commercial
Figure 7.1 Polyelectrolyte multilayer assembly on a substrate via layer- by-layer deposition.
Surface nanoengineering for combating biomaterials infections 139
medical products like wound dressing and urinary catheters. However, the clinical efficacy of silver- coated urinary catheters in reducing infections is not well- proven (Desai et al. , 2010; Srinivasan et al. , 2006; Johnson et al. , 2010). Recent reports on the cytotoxicity of silver towards mammalian cells have also raised concern over its use as an antimicrobial (Rai et al. , 2009; Park et al. , 2011). The cytotoxic effects include the generation of reactive oxygen species, DNA damage and inhibition of stem cell differentiation, and these effects depend on cell type, nanoparticle size and concentration (Park et al. , 2011).
7.3.2 Antibiotics and antimicrobial peptides
Antibiotics and antimicrobial peptides have also been entrapped in surface coatings for gradual release, and PEMs have been used for this purpose (Jiang and Li, 2009; Shukla et al. , 2010a; Wong et al. , 2010). Jiang and Li (2009) fabricated PEMs from poly- L-lysine and poly-L-glutamic acid and investigated the loading and release behavior of positively charged and negatively charged antibiotics, gentamicin, and cefazolin, respectively. The loading and release of these drugs were found to be pH-dependent and could be controlled by changing the number of film layers and drug incubation time, and by the application of heat- treatment after film formation. Shukla et al. , (2010b) generated multilayer films by alternating an amine terminated ponericin G1 peptide (an antimicrobial peptide known to be highly active against S. aureus ) which exhibits a net positive charge at physiological conditions, a hydrolytically degradable polycation from a series of poly(ß-amino esters), and a polyanion such as chondroi- tin sulfate, dextran sulfate, or alginic acid. The drug loadings, which ranged from 20 to 150 μ g/cm 2 , and the film composition, in particular the polyanion used, strongly influenced the film growth and degradation properties as well as the incorporation and release characteristics of ponericin G1.
Another method for the incorporation and release of antibacterial agents from surface coating is via the use of hydrogels. The thickness of hydrogels may be in the micron range or larger (DiTizio et al. , 1998; De Giglio et al. , 2011). However, Pavlukhina et al. , (2010) prepared a poly(methacrylic acid) hydrogel of thick- ness of ~41 nm in the dry state on silicon wafers. The silicon was pretreated with poly(ethyleneimine), and either ethylenediamine or adipic acid dihydrazide was used as a cross- linker. A positively charged antibacterial agent, lysozyme or gentamicin or peptide L5, was incorporated by exposing the cross- linked hydrogel to a solution of the antibacterial agent for 20 min. The release characteristics of the antibacterial agent are dependent on pH and salt concentration of the surrounding medium, and the type of crosslinker used.
A number of studies have investigated the antibacterial efficacy of catheters impregnated with antibiotics such as minocycline/rifampicin (Raad et al. , 2008) and nitrofurazone (Desai et al. , 2010; Johnson et al. , 1993, 2010) or with triclosan (Bayston et al. , 2009; Stickler et al. , 2003). The antimicrobial effectiveness is largely dependent on the continuous release of the impregnated agents. Relatively long- term (>28 days) efficacy was reported for the minocycline/rifampicin (Raad et al. , 2008) and rifampicin/triclosan/trimethoprim (Bayston et al. , 2009) impregnated catheters
140 Biomaterials and Medical Device-associated Infections
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.