**4.1 AMP immobilization methods**

*Peptide Synthesis*

infection [77].

[80, 81].

towards eukaryotic cells [84].

stability of AMPs [87–89].

**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

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

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

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

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

**52**

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

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].

#### *Peptide Synthesis*

The difficulty of these developments is the lack of comparable information, since most of the observed reactivity and antimicrobial activity are found to be specific to the reactant environment, type of materials and AMPs. Fully understanding these structure-function relationships is important to clarify and improve the performance of surface-immobilized AMPs in future developments.

### *4.1.1 Orientation of direct immobilized AMPs*

When a peptide is directly bonded to the material surface, as displayed in **Figure 6**, the immobilized terminus determines its orientation. When α-helical peptides are immobilized at their N-terminus, they will point perpendicular to the material surface (i.e., orientate upstanding), but when immobilized at their C-terminus, they will take on a laying-down orientation. In general, when an AMP has its N-terminus bonded to the surface, it has been shown to have a lower MIC when compared to C-terminus and N-side-chain-immobilized AMPs [102]. The relatively high MIC of C-terminus-immobilized AMPs is likely related to the inhibition of membrane interaction [51]. However, according to the literature reporting the antimicrobial activity dependence of the orientation difference between N-terminus and C-terminus-immobilized peptides, there is also a cell-dependent potency, which indicates that the mode of action of AMPs is not only dependent on their own characteristics but also on the characteristics of the targeted microorganism [103]. Another important parameter is the position of the cationic amino acids. When they were closer to the bonding site, an increased antimicrobial activity was observed, while, when they were positioned in the middle or closer to the N-terminus, a decrease of the antimicrobial activity was observed [100].

However, as shown in **Figure 7**, the orientation of AMPs changes after their initial interaction with bacteria (i.e., the immobilized α-helices bind to the anionic lipid bilayer). Since immobilized AMPs cannot follow the *barrel-stave* or *toroidal model* due to limited mobility, it is suggested that the charge-charge interaction plays a dominant role in the elimination of bacteria [104, 105].

#### **Figure 6.**

*Schematic of (a) N-terminus and (b) C-terminus surface-immobilized α-helical peptide; not drawn to scale or exact structure.*

#### **Figure 7.**

*Schematic of orientation change of N-terminus-immobilized α-helical peptide before (left) and after (right) bacterial contact; not drawn to scale or exact structure.*

**55**

**Figure 8.**

*scale or exact structure.*

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

*Immobilization of AMPs onto numerous functional groups, using different coupling methods; not drawn to* 

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

*Defensin-Like Peptides and Their Antimicrobial Activity in Free-Form and Immobilized… DOI: http://dx.doi.org/10.5772/intechopen.85508*

#### **Figure 8.**

*Peptide Synthesis*

**54**

**Figure 7.**

**Figure 6.**

*exact structure.*

*bacterial contact; not drawn to scale or exact structure.*

*Schematic of orientation change of N-terminus-immobilized α-helical peptide before (left) and after (right)* 

*Schematic of (a) N-terminus and (b) C-terminus surface-immobilized α-helical peptide; not drawn to scale or* 

The difficulty of these developments is the lack of comparable information, since most of the observed reactivity and antimicrobial activity are found to be specific to the reactant environment, type of materials and AMPs. Fully understanding these structure-function relationships is important to clarify and improve

When a peptide is directly bonded to the material surface, as displayed in **Figure 6**, the immobilized terminus determines its orientation. When α-helical peptides are immobilized at their N-terminus, they will point perpendicular to the material surface (i.e., orientate upstanding), but when immobilized at their C-terminus, they will take on a laying-down orientation. In general, when an AMP has its N-terminus bonded to the surface, it has been shown to have a lower MIC when compared to C-terminus and N-side-chain-immobilized AMPs [102]. The relatively high MIC of C-terminus-immobilized AMPs is likely related to the inhibition of membrane interaction [51]. However, according to the literature reporting the antimicrobial activity dependence of the orientation difference between N-terminus and C-terminus-immobilized peptides, there is also a cell-dependent potency, which indicates that the mode of action of AMPs is not only dependent on their own characteristics but also on the characteristics of the targeted microorganism [103]. Another important parameter is the position of the cationic amino acids. When they were closer to the bonding site, an increased antimicrobial activity was observed, while, when they were positioned in the middle or closer to the N-terminus, a decrease of the antimicrobial activity was observed [100].

However, as shown in **Figure 7**, the orientation of AMPs changes after their initial

interaction with bacteria (i.e., the immobilized α-helices bind to the anionic lipid bilayer). Since immobilized AMPs cannot follow the *barrel-stave* or *toroidal model* due to limited mobility, it is suggested that the charge-charge interaction plays a

dominant role in the elimination of bacteria [104, 105].

the performance of surface-immobilized AMPs in future developments.

*4.1.1 Orientation of direct immobilized AMPs*

*Immobilization of AMPs onto numerous functional groups, using different coupling methods; not drawn to scale or exact structure.*

#### **Figure 9.**

*Schematic of spacer-incorporated immobilized AMPs, showing stiff spacers (left) and flexible spacers (right); not drawn to scale or exact structure.*

Additionally, when functionalizing the material's surface with different reactive groups as seen in **Figure 8**, the orientation of immobilized AMPs can be controlled using chemo-selective (i.e., directed immobilization) coupling reactions. In general, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are found to be widely used as activating agents for the coupling of peptides to numerous functional groups by forming amides, which inhibit the formation of carboxylic salts with an amine [98, 106].

### *4.1.2 Spacer-incorporated immobilized AMPs*

Although orientation can be partly controlled with the use of chemo-selective coupling agents, they often do not add enough mobility to increase the probability of membrane permeabilization or polarization. In order to improve the mobility and subsequent activity of AMPs, the use of spacers is found to be useful. As shown in **Figure 9**, there are two types of spacers, stiff and flexible spacers. Stiff spacers (e.g., polyvinyl chloride or polymethyl methacrylate) allow the increase of reach and thus might allow membrane permeabilization, but will restrict sideways mobility by keeping the AMPs pointing in a specific orientation [98]. Flexible spacers such as polyethylene glycol (PEG) also allow the increase of reach and are able to allow sideways mobility; however, orientation cannot be determined due to their flexible chains [107].

However, even if the peptide was linked to a PEG spacer, the random orientation of an immobilized AMP through its C-terminus is found to result in the loss of antimicrobial activity [97]. However, the oriented immobilization of the same AMP through its N-terminus is found to restore the antimicrobial activity. It has also been suggested that the water-swelling property of PEG aids in maintaining the activity of immobilized peptides [108]. Additionally, in the absence of the PEG spacer, the AMP did not show antimicrobial activity. Nevertheless, the utilization of PEG as a spacer is found to present numerous advantages, as it can create non-adhesive surfaces due to its non-fouling characteristics (i.e., inhibition of microorganisms binding to the material surface) [108]. Lastly, the solubility and stability of peptides, against protease digestion or peptide aggregation, can be improved with the use of a spacer [107, 109, 110]. These previously mentioned factors show yet again the complexity of the factors on influencing the antimicrobial activity of immobilized AMPs.

#### **5. Conclusions**

AMPs are demonstrated to show antimicrobial activity at relatively low concentrations, without damaging mammalian cells, being able to utilize several mechanisms against numerous microorganisms similar to defensins in the human body. Immobilization of AMPs improves their lifespan, preserves the mode of action and does not seem to influence the mechanism on the biological level; however, they

**57**

**Author details**

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Marc Bruggeman, Hanieh Ijakipour and Artemis Stamboulis\* Biomaterials Group, School of Metallurgy and Materials, University of Birmingham, Birmingham, United Kingdom

\*Address all correspondence to: a.stamboulis@bham.ac.uk

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

do show reduced antimicrobial activity upon immobilization to material surfaces. Nonetheless, this reduced activity can be partly restored with the use of chemoselective coupling agents and the incorporation of spacers. Whether degradation of the material and coupling agents is desirable or not, understanding the kinetics is of great importance as the decrease of structural integrity and/or release of particles (i.e., molecules, debris, etc.) might result in an adverse biological reaction. Nonetheless, there are significant indications that AMPs are suitable candidates to replace conventional biocides and antibiotics. In addition, they can be utilized to develop the next generation of antimicrobial surfaces as coatings for medical

The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, or other equity interest; and expert testimony or patent-licensing arrangements) in the subject matter or

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

devices and implants.

**Conflict of interest**

materials discussed in this chapter.

*Defensin-Like Peptides and Their Antimicrobial Activity in Free-Form and Immobilized… DOI: http://dx.doi.org/10.5772/intechopen.85508*

do show reduced antimicrobial activity upon immobilization to material surfaces. Nonetheless, this reduced activity can be partly restored with the use of chemoselective coupling agents and the incorporation of spacers. Whether degradation of the material and coupling agents is desirable or not, understanding the kinetics is of great importance as the decrease of structural integrity and/or release of particles (i.e., molecules, debris, etc.) might result in an adverse biological reaction. Nonetheless, there are significant indications that AMPs are suitable candidates to replace conventional biocides and antibiotics. In addition, they can be utilized to develop the next generation of antimicrobial surfaces as coatings for medical devices and implants.
