## 4 Surface-Immobilized Peptides on Medical Implants
Conventional antibiotics, such as vancomycin and gentamicin, have been incorporated in antimicrobial coatings for orthopedic implants [91], however they failed to fulfil all of the above-mentioned criteria. The main drawbacks of coatings are the antibiotics limited antimicrobial activity and the release of drugs at levels below the reported MIC, that may evoke bacterial resistance [92; 93]. On the other hand, it is more difficult for bacteria to develop resistance against AMPs, and for example repeated exposure to sub-inhibitory concentrations of melimine (TLISWIKNKRKQRPRVSRRRRRCGGRRRRR), a synthetic peptide produced by combining the active portions of mellitin and protamine, did not induce resistance [94]. Moreover, high doses of antibiotics often harm cell viability and osteogenic activity and may impair osteointegration, which is a vital process for the acceptance of orthopedic implants [95]. Figure 2: Requirements for antimicrobial coatings on medical implants. Several biomaterials have been functionalised with AMPs, including titanium [96; 97; 98; 99], catheter-like surfaces [100; 101; 102; 103] and contact lenses [94]. A challenge for all AMP incorporation in coatings is that the immobilization process on an implant surface should not prohibit the antibacterial activity of the AMP, i.e., the diffusion of the peptides into the bacterial membrane. It has been reported that immobilized antimicrobial drugs often lose their activity, as they do not reach their cellular targets [104]. In addition, altered stereochemistry or orientation of the peptide molecule resulted in lower antimicrobial efficacy compared to when in solution [101; 102]; though increased antibacterial properties have also been reported as a result of tethering AMPs to a surface [105]. Moreover, the concentration of the AMP on the surface as well as its concentration when released, should be appropriate, in order to exert antimicrobial effect. All these parameters have to be considered when developing a new AMP coating. There is a plethora of methods for coating biomaterials, broadly categorized as methods for peptide immobilization, and peptide release from a biomaterial surface. Peptide immobilization methods usually include sophisticated chemical modifications, as producing biomaterial coatings by means of physical adsorption or electrostatic attraction on the surface of the biomaterial is usually unsuitable, due to instability. Otherwise, such coatings will be weak and instable mechanically, during the invasive processes of implant insertion, and at the in vivo biological environment with the biological fluid flow. Thus, a stronger platform is required for the bonding between the AMP and the coating, for example, by means of covalent bonding. Covalent immobilisation would allow for no or very low leakage of the antimicrobial agent at the peri-implant area, allowing for a limited antibacterial effect in the surrounding area. Moreover, a number of biomaterials, such as titanium, have inert surfaces, thus, the surface of such an implant has to be modified to acquire additional properties, such as antimicrobial, bone formation-promoting or other [106]. As a result, harsh chemical conditions and complicated, multiple steps are required--including, for example, coupling with silane agents. On the other hand, for other applications like food packaging coatings, surgical instruments, where release of the antimicrobial agent is undesired, covalent immobilizations offer several advantages by providing stable bonds between the bioactive compound and the functionalised surface [104]. Another strategy offers active release coatings, where the antimicrobial compound coated on the implant is not covalently bound, therefore is released, taking care of peri-implant infections, which often occur due to the dysregulated immune system around the implants [107; 108; 109; 110; 111; 112].