## 4 Surface-Immobilized Peptides on Medical Implants
### Antimicrobial Peptides Immobilised on Titanium
A simpler approach, using a dopamine coating as a scaffold for the loading of AMPs, has been investigated in several studies [113; 114; 128; 138]. Inspired by the mussel's strong adhesion to virtually any wet surface, attributed to a unique protein with special amino acid composition [139], [PERSON] and colleagues reported a method to form thin multifunctional polymer coatings through simple dip-coating of objects in an aqueous solution of dopamine, as a mimic of the mussel's adhesive protein [140]. The mussel's strong adhesive properties are believed to be related to the overexpression of an amino acid bearing a catechol group (L-3,4-dihydroxyphenylalanine, DOPA) and lysine residues that are in close proximity with the substrate interface [141]. Dopamine combines the chemical properties of both DOPA and lysine, hence, it has been chosen as an analogue of the mussel's adhesive protein. The dopamine-modified surfaces were shown to be potent substrates for further ad-layer deposition of various compounds [140; 142; 143]. The reactivity of the dopamine-coated surfaces relates to the formation of poly-di-hydroxy-indole (polydopamine, PDA) as a thin layer which under high pH conditions, oxidizes to the corresponding poly-ortho-quinone-indole. The catechol side chains of DOPA are believed to form strong charge-transfer complexes to metal oxide surfaces, such as titanium [144; 145]. The resulting film can subsequently interact covalently with various compounds via Schiff-base reactions (amine containing molecules) or Michael type reactions (amine and thiol containing molecules) [140; 143]. PDA coating is attractive for medical implant coating manufacturing, due to its high efficiency of surface conjugation and its convenient property of naturally coating of unmodified substrates. Bacitracin immobilised on a PDA scaffold on titanium retained its antimicrobial activity against Gram-positive bacteria, and furthermore, it was found that this coating enhanced osteogenic differentiation of human bone marrow mesenchymal stem cells and reduced the inflammation both in vitro and in vivo in a rat model [113; 138]. PDA layers have also been used for dual coating of titanium, with both AMP and another molecule, to gain dual functionality. A dual coated titanium with the AMP SESB2V and vascular endothelial growth factor retained the antimicrobial properties of SESBV2 (Table 4) against _Bacillus cereus_ and _E. coli_[128] and enhanced fibroblast proliferation. Furthermore, these dual coated implants were tested in vivo in a rabbit keratinitis model where they prevented peri-implant bacterial infection, compared to raw titanium implants [114]. Another alternative to the conventional titanium chemical immobilization methods is a bio-friendly immobilization strategy utilizing chimeric peptides, comprised of an AMP domain and a specific titanium-binding domain, usually with a flexible linker in between. These bifunctional chimeric peptides are able to bind selectively to the titanium surfaces, while exposing the AMP domain. Among other peptides able to recognise the surface of inorganic material [146], peptide TBP-1 has been isolated by a phage system for its ability to bind on titanium surfaces via electrostatic interactions [147]. Mutational analyses revealed that the N-terminal motif fKLPDA (named minTBP-1) is responsible for binding, with the arginine, proline and aspartate residues being the most important. It has been proposed that the electrostatic interactions take place between the charged residues of minTBP-1 and the charged surface of titanium [148] under physiological conditions [144], which is supported by atomic force microscopy data [149]. Chimeric peptides comprised of titanium-binding domain and AMP connected by a linker have been successfully immobilised on titanium surfaces and retained their antimicrobial activity [127; 129]. In order to maintain the functionalities of both titanium binding and antimicrobial activity, it was shown that a rigid linker is preferable compared to a flexible one [127].