*4.5. Cell Culture Studies*

All surfaces showed attachment of cells on day 5, with no evidence of toxicity (which is determined by any change of cell morphology or density compared to the plain titanium surface), with one exception for the KR12/32-5911 surface. The cell morphology was generally "flattened" and fibroblast-like, typical of the described HOS morphology in the literature [46]. The density of attached cells increased over time, confirming that cells were viable and able to proliferate. In contrast, there was evidence that the surface of KR12/32-5911 was toxic to the cells, and at day 7 there were no longer any cells.

## **5. Conclusions**

In the present research, KR12 and three new analogous peptides were successfully synthesised and showed promising antimicrobial activity against *E. coli*, *P. aeruginosa*, and *S. aureus*, with MIC values generally being lower than that of their native peptide, the human cathelicidin LL-37. The titanium surfaces were coated with pDA and characterised. Subsequently, the pDA-coated surfaces were decorated with KR12 and the other three designed analogous peptides. The pDA coating provided a long-term linking substrate for the peptides, which was confirmed by fluorescence microscopy and HPLC. Cultured HOS cells showed good attachment and cell growth on the material surfaces, with no visible toxicity towards the cells, except for the KR12/32-5911 peptide. This surface treatment shows potential to provide long-term antimicrobial activity on many metallic and organic material surfaces and could be used in biomedical materials and implants.

**Author Contributions:** Conceptualization, A.S. and Z.T.; methodology, A.S., J.B., N.J.H.; software, M.B.; validation, A.S. and Z.T.; formal analysis Z.T. and A.S.; investigation Z.T.; resources, Z.T., M.B. and H.I.; data curation, Z.T., M.B., H.I., J.B., N.J.H. and A.S.; writing—original draft preparation, Z.T., M.B. and H.I.; writing—review and editing, A.S., N.J.H.; visualization, Z.T., M.B., H.I.; supervision, A.S., J.B. and N.J.H.; project administration, A.S.; funding acquisition, A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project has been partially funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 645749. The project has been also financially supported by a studentship provided by the School of Metallurgy and Materials, University of Birmingham, UK.

**Conflicts of Interest:** The authors declare no conflict of interest.
