**4. Defensin-like peptides immobilized on material surfaces**

Bacterial adhesion and colonization resulting in biofilm formation on the surface of biomaterials are responsible for most medical device-associated infections and malfunctions [73, 74]. The magnitude of this problem continues to pose a significant problem to health care providers, often resulting in major negative medical and economic consequences [75, 76]. Many of the currently used medical devices are amenable to modification, either by functionalization or coating of the surface of the device, making it possible to combat inflammation and reduce the risk of infection [77].

In general, device-associated infections have been treated by developing material surfaces containing antibiotics and biocides [78]. The use of this approach comes with the risk of cytotoxicity, raising concern as a potential threat to human and environmental health. An alternative to this is the use of antifouling coatings, making it possible to prevent attachment of bacteria, proteins and other microorganisms. This more passive approach makes it possible to prevent antibiotic resistance and leaching of cytotoxic biocides but is not capable of killing already adherent bacteria and makes it hard to avoid infection completely [79]. Therefore, a combination of antifouling and antimicrobial properties is favourable to combat biofilm formation and further reduce the risk of infection [80, 81].

Defensin-like peptides (i.e., antimicrobial peptides, AMPs) exhibit a combination of antimicrobial and antifouling properties, which is why AMPs have received significant attention as an alternative to conventional biocides and antibiotics, showing the ability to overcome and combat medical device-associated infections. However, so far, there is little success in the development of AMPs for therapeutic applications, with only a few AMPs that have been approved for medical use at their initial introduction. After the unsuccessful introduction of the 'first-generation' AMPs, research has been performed on reducing cytotoxicity. It was found that AMPs were increasingly more toxic when having hydrophobic characteristics, sufficient enough to interact with neutrally charged eukaryotic cell membranes [82]. By replacing or interrupting these hydrophobic regions of AMPs, cytotoxicity was reduced and only showed a slight decrease in antimicrobial activity, providing the amphipathic characteristics were maintained [83]. Also, developing narrow-spectra AMPs would decrease the required concentration needed to combat pathogens and additionally prevent the cytotoxic activity towards eukaryotic cells [84].

In order to further reduce the cytotoxicity, immobilization of AMPs onto material surfaces is a potential approach to reduce the concentration needed when in free-form and will also increase their half-life time. The half-life time of AMPs is found to be based on the rate of protease digestion or related to peptide aggregation [85]. In order to compensate for their relatively short half-life time, increased concentrations of AMPs are used; but, this results in increased cytotoxicity and has limited the use of AMPs. Other efforts to increase the half-life time of peptides have been focused on using substitutes for L-amino acids, because unprotected peptides are more rapidly metabolized (i.e., broken down for nutrition) [86]. By substitution of L-amino acids by D-amino acids, the introduction of unnatural β- and ɣ-peptide bonds and modifications of the N- or C-terminus, it is possible to increase the stability of AMPs [87–89].

Overall, it is of great importance to improve the stability of AMPs against degradative mechanisms *in vivo* and increase the bond stability between the AMPs and materials in order to develop almost non-cytotoxic and long-lasting antimicrobial

**53**

*Defensin-Like Peptides and Their Antimicrobial Activity in Free-Form and Immobilized…*

surfaces [90, 91]. Therefore, by limiting the necessity of implant removal due to AMP inactivity caused by degradative mechanisms, patient compliance can be

It is possible to immobilize AMPs through two main pathways, either physically or chemically. A popular physical method is layer-by-layer assembly, in which AMPs are 'sandwiched' between two polyionic polymers, making it possible to integrate a controllable loading of AMPs [92, 93]. However, the interspersed AMPs within the polyionic polymer layers are not able to interact with the surrounding environment, and they will need to diffuse outwards to utilize their antimicrobial activity. Therefore, with the use of physical immobilization, it is difficult to fully utilize the potency of AMPs. Covalent-based immobilization of AMPs has significant advantages, in comparison to physical immobilization, such as the formation of more stable bonds and thus improvement of their relatively short half-life time, while also

Furthermore, the material surface is also of great importance to the stability of AMPs. A polymer surface (i.e., dibromomaleimide polymer substrate) prepared by chemical vapour deposition (CVD) and functionalized with AMPs showed a better antimicrobial stability when compared to a self-assembled monolayer (SAM). When exposed to air, this polymer showed slower detachment of bound AMPs when compared to SAM [95]. Additionally, orientation of AMPs was also retained. These results show the importance of using a non-degradable material surface to improve bond stability of AMPs. Next to that, the surface morphology needs to be well defined as an undefined surface could lead to inhibition of attachment of biological molecules. Also, depending on the density of the functional groups that are present or able to be induced, the number of peptides on the material surface can vary significantly. However, even though AMP concentration is of importance, it does not appear to be the most critical criterion for the improvement of antimicrobial activity [96, 97]. The antimicrobial activity of covalent-based surface-immobilized AMPs is seen to be mainly dependent on the used coupling strategy, spacer specifications and peptide orientation and concentration [98]. However, there are certain limits to improving the antimicrobial activity by increasing the AMP concentration, due to factors such as coupling conditions and steric hindrance (i.e., repulsive forces originating from overlapping electron clouds of neighbouring molecules). Next to that, some microorganisms are found to be insensitive to any further increase after

reaching a certain limit in AMP surface density or exposure time [99].

It is demonstrated that direct-immobilized AMPs also show antimicrobial activity without the use of a spacer. Nevertheless, most potent developments make use of spacers, the length (i.e., the distance between the material surface and peptide) of which is shown to significantly influence the activity of surface-immobilized AMPs, when compared to the AMP surface density [90]. Conversely, the possibility of chain cleavage of the spacer due to polymer degradation reactions could lead to the release of immobilized AMPs. This could be minimized with the use of stabilized polymer spacers [59]. The increased activity seen with the use of spacers is a result of improved mobility of AMPs, increasing probability of membrane permeabilization and subsequent cell death. However, this would only be a correct hypothesis assuming that AMPs demonstrate a similar mode of action to that of their free-form counterparts. According to the literature, there are also AMPs that will depolarize the cytoplasmic membrane and disrupt the electron transport, which subsequently would lead to partial membrane permeabilization and thus cell death [100, 101].

*DOI: http://dx.doi.org/10.5772/intechopen.85508*

**4.1 AMP immobilization methods**

minimizing the possibility of leaching of AMPs [90, 94].

increased.

surfaces [90, 91]. Therefore, by limiting the necessity of implant removal due to AMP inactivity caused by degradative mechanisms, patient compliance can be increased.
