## 4 Surface-Immobilized Peptides on Medical Implants
### Antimicrobial Peptides Immobilised on Titanium  
Silanization (Figure 3) is a low-cost and effective covalent coating method used to modify material surfaces that are rich in hydroxyl groups by introducing silane active groups (e.g., amino group and carboxyl group), which can easily be modified by further grafting. The first study, to the best of our knowledge, which explored the immobilization of the cathelcidin related AMP LL-37 on titanium via silanization was carried out by [PERSON] and colleagues [121]. Interestingly, they showed that a flexible and long spacer containing polyethylenoglycol (PEG) chains is required between the silane and the AMP, in order to maintain its antibacterial effect against _E. coli_[121]. In this study, immobilized LL-37 alone had a significantly lower antimicrobial effect compared to when it was coupled to the flexible spacer chains. Moreover, the use of PEG as spacer additionally led to intrinsic anti-adhesive properties of the treated surface [122]. Silanization has been successfully used for the immobilization of AMPs on titanium in a number of studies (Table 4). The human lactoferrin-derived peptide hLf1-11 anchored to titanium surfaces via silanization exhibited an outstanding reduction in the adhesion and early stages of biofilm formation of the oral cavity pathogens _Streptococcus sanguinis_ and _Lactobacillus salivarius_[123]. Similarly, when using silanization to immobilize the synthetic, broad-spectrum antimicrobial peptide melimine on titanium surfaces, the coating significantly reduced the in vitro adhesion and biofilm formation of _P. aeruginosa_ and _S. aureus_ on the surfaces compared to the non-modified titanium [96]. Notably, it was further demonstrated that the melimine-coated titanium reduced bacterial colonization in vivo, in both mouse and rat subcutaneous infection models and that the coated implants retained activity even after ethylene oxide gas sterilization [96]. As grafting of the AMPs away from the surface using different spacer elements has demonstrated positive impact on activity, there were attempts to develop this concept further. Thus, polymer brushes (Figure 3) have attracted considerable attention as a way to modify the surface properties of materials [136], and in comparison to other surface modification methods (e.g., silanization), polymer brushes have increased density of functional groups on their surface, allowing the conjugation of a higher number of biomolecules, i.e., AMPs [125]. Among techniques used to prepare polymer brushes, the \"grafting from\" approach based on surface-initiated atom transfer radical polymerization offers an experimentally straightforward route to graft polymer brushes on biomaterial, in a controlled and reproducible manner [137]. Polymer brushes, apart from servicing as scaffolds for the attachment of other molecules, like AMPs, exert non-adhesive properties, a characteristic highly desirable for biomaterial. The AMP Tet-20 (Table 4) conjugated to polyacrylamide brushes grafted on titanium surfaces, retained its antimicrobial properties against _P. aeruginosa_ both in vitro and in vivo, in a rat model [98]. Furthermore, increased peptide densities were achieved on the brush layer, compared to direct grafting of peptides on the material surface [97]. Figure 3: Covalent immobilisation of AMPs on titanium surfaces. \begin{table}
\begin{tabular}{l l l l l l l l l l} \hline \hline
**Rounded** & **AMF** & **Sequence** & **Coding Method** & **Rotation** & **In View Training** & **In View Training** & **Rosenceptibility** & **Red. ** \\ \hline \hline \multicolumn{10}{c}{**Inundobilized**} \\ \hline \hline \  
For clinical applications, it would be ideal to achieve the surface modification of titanium implants by simple procedures; for example, spraying a drug-loaded solution onto the implant immediately before the surgery, which can form a stable coating layer that is strongly bound to the implant in situ. The methods discussed so far (silanization, polymer brushes) involve harsh chemical conditions and are more cumbersome.