**Conflict of interest**

*Peptide Synthesis*

**Figure 9.**

*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

*Schematic of spacer-incorporated immobilized AMPs, showing stiff spacers (left) and flexible spacers (right);* 

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

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 immobi-

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

inhibit the formation of carboxylic salts with an amine [98, 106].

*4.1.2 Spacer-incorporated immobilized AMPs*

flexible chains [107].

**56**

lized AMPs.

**5. Conclusions**

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 materials discussed in this chapter.
