*4.3. Peptide Attachment to Polydopamine*

The KR12 analogous peptide labelled with 5(6)-FAM at the N-terminal was conjugated to an already pDA-coated Ti6Al4V surface. The presence of the peptides was confirmed by the emitted fluorescence, showing only small variations in the measured fluorescence intensities between the different peptides. The quinone group of pDA is known to undergo a Schiff base reaction or Michael addition without the use of any other reagent or catalyst, allowing the conjugation of biomolecules to pDA [38]. The catechol group of the pDA is known to oxidise to quinone at a pH above 7.5 in excess of oxygen, which is necessary for successful conjugation to nucleophiles [39]. Here, the designed peptides contained two lysine residues that should be able to conjugate to pDA, but the first lysine was presumed to be shielded by the bulky 5(6)-FAM label at the N-terminus. Therefore, it was expected that pDA was conjugated with the second lysine residue relative to the N-terminus. It was observed that the initial burst of peptides released approximately 25–35% of the peptides from the surface after 6 h, and approximately 40–50% was released after 30 days. The fluorescence release profile studies in SBF indicated that 50–60% of the peptides potentially remained conjugated to the pDA after 30 days. The initial burst of peptide release could be attributed to the release of unattached peptides to the pDA that were not successfully removed during the wash step. After the initial burst, the rate of release of the peptides decreased and was only reduced further by roughly 5–10% after the final 30 days. The release profile from the fluorescence was confirmed by HPLC using non-fluorescent peptides. An initial ≈70% burst of cumulative peptide was released in the first 6 h, and a slower subsequent release for the remaining period was observed. These release studies clearly showed that these peptides were conjugated to the pDA-coated Ti6Al4V surface in a very stable manner through the lysine residues.

#### *4.4. Wettability of the Surface*

New implants within the body come into immediate contact with extracellular fluid and moieties, such as proteins, which are some of the first molecules to interact with the surfaces of an implant [40,41]. The optimal water contact angle of a biomaterial reported for bone-forming cells to attach to a surface was reported to be 55◦ [42]. Metallic surfaces are very hydrophobic, resulting in slow integration rates of the implant with the body. A water contact angle study of pDA-coated titanium surfaces

by Nijhuis et al. reported that coated Ti6Al4V surfaces were much less hydrophobic (≈47◦) than the uncoated surfaces (≈75◦) [43], presumably resulting in an improvement of the implant integration [44]. In this paper, we report that the introduction of pDA coating on the surface of Ti6Al4V resulted in a decrease in the water contact angle from 65.4◦ ± 1.6 to 59.0◦ ± 1.2. The experimental values are in agreement with prior research performed by Luo et al. [45], who reported on the water contact angle of a pDA-coated Ti6Al4V surface. The water contact angle was further reduced to approximately 56.8◦ when the pDA coating was decorated with the peptides—the results varied depending on the used peptide, but they all showed a reduction relative to the pDA-coated Ti6Al4V surface.
