Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides

Surface modifications are usually performed on titanium alloys to improve osteo-integration and surface bioactivity. Modifications such as alkaline and acid etching, or coating with bioactive materials such as hydroxyapatite, have previously been demonstrated. The aim of this work is to develop a peptide with combined titanium oxide and hydroxyapatite binders in order to achieve a biomimetic hydroxyapatite coating on titanium surfaces. The technology would also be applicable for the functionalisation of titanium and hydroxyapatite surfaces for selective protein adsorption, conjugation of antimicrobial peptides, and adsorption of specialised drugs for drug delivery. In this work, functionalisation of Ti6Al4V and hydroxyapatite surfaces was achieved using combined titanium-hydroxyapatite (Ti-Hap) peptides based on titanium peptide binder (RKLPDA) and hydroxyapatite peptide binder (EEEEEEEE). Homogeneous peptide coatings on Ti6Al4V surfaces were obtained after surface chemical treatments with a 30 wt% aqueous solution of H₂O₂ for 24 and 48 h. The treated titanium surfaces presented an average roughness of S_a = 197 nm (24 h) and S_a = 128 nm (48 h); an untreated mirror polished sample exhibited an Sa of 13 nm. The advancing water contact angle of the titanium oxide layer after 1 h of exposure to 30 wt% aqueous solution of H₂O₂ was around 65°, decreasing gradually with time until it reached 35° after a 48 h exposure, suggesting that the surface hydrophilicity increased over etching time. The presence of a lysine (L) amino acid in the sequence of the titanium binder resulted in fluorescence intensity roughly 16% higher compared with the arginine (R) amino acid analogue and therefore the lysine containing titanium peptide binder was used in this work. The Ti-Hap peptide KKLPDAEEEEEEEE (Ti-Hap1) was not adsorbed by the treated Ti6Al4V surfaces and therefore was modified. The modifications involved the inclusion of a glycine spacer between the binding terminals (Ti-Hap2) and the addition of a second titanium binder (KKLPDA) (Ti-Hap3 and Ti-Hap4). The combined Ti-Hap peptide which exhibited the strongest intensity after the titanium surface dip coating was KKLPDAKKLPDAEEEEEEEE (Ti-HAp4). On the other hand, hydroxyapatite surfaces, exhibiting an average roughness of S_a = 1.42 μm, showed a higher fluorescence for peptides with a higher negative net charge.