**Zuzanna Trzci ´nska 1, Marc Bruggeman 1, Hanieh Ijakipour 1, Nikolas J. Hodges 2, James Bowen <sup>3</sup> and Artemis Stamboulis 1,\***


Received: 15 May 2020; Accepted: 19 August 2020; Published: 22 August 2020

**Abstract:** Infections are common complications in joint replacement surgeries. Eradicated infections can lead to implant failure. In this paper, analogues of the peptide KR-12 derived from the human cathelicidin LL-37 were designed, synthesised, and characterised. The designed antimicrobial peptides (AMPs) were attached to the surface of a titanium alloy, Ti6Al4V, by conjugation to a polydopamine linking substrate. The topography of the polydopamine coating was evaluated by electron microscopy and coating thickness measurements were performed with ellipsometry and Atomic Force Microscopy (AFM). The subsequently attached peptide stability was investigated with release profile studies in simulated body fluid, using both fluorescence imaging and High-Performance Liquid Chromatography (HPLC). Finally, the hydrophobicity of the coating was characterised by water contact angle measurements. The designed AMPs were shown to provide long-term bonding to the polydopamine-coated Ti6Al4V surfaces.

**Keywords:** Ti6Al4V; polydopamine; antimicrobial peptides; cathelicidin; KR-12

#### **1. Introduction**

Infection are the most common complications of joint replacement surgery, with nosocomial or hospital-acquired infections ranking as the sixth leading cause of death, presenting a major healthcare challenge [1]. Infection can lead to extended inflammation at the site of the surgery, thus causing the rejection and failure of the implant [2]. Although the administration of antibiotics significantly reduces the risk of postsurgical infections, bacterial biofilm production on the implant surface or untimely administration of antibiotics will reduce their effectiveness [3].

The use of titanium in dental and orthopaedic implants is well established due to titanium's strength, stiffness, and corrosion resistance. Titanium also shows seamless integration with the surrounding tissues due to its excellent biocompatibility [4,5]. The drawback of using titanium implants is their susceptibility to bacterial colonisation on the surfaces of the implants [6]. To combat the formation of biofilms on implant surfaces, the time-controlled release of various antibiotic coatings has previously been investigated [7,8]. However, the release of the antibiotics below the level of the minimum inhibitory concentration (MIC) is known to produce antibiotic-resistant strains of bacteria. Higher levels of antibiotic release have been shown to be toxic to the surrounding tissues. The increase of antibiotic-resistant bacteria has led to the search for an alternative method of antimicrobial protection [9].

Antimicrobial peptides, which are a part of the innate immune system of all living organisms, have broad-spectrum activity against many microorganisms, such as Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi [10–13]. Moreover, they can inhibit biofilm formation and induce its dissolution, as well as attract phagocytes to further induce natural defence mechanisms [14]. The mechanisms of action of antimicrobial peptides (AMPs) against bacteria are not fully understood due to the high diversity of these peptides. Nevertheless, it is widely accepted that bacterial cell death is due to the interaction of cationic AMPs with negatively charged phospholipids on the bacterial membrane, which lead to the loss of membrane structural integrity, and eventually cell death [10–13]. AMPs exhibit a strong preference for specific membrane compositions, allowing them to be selective towards bacterial cell membranes, but not mammalian or plant cells [14]. Currently, only a few AMPs are used clinically due to limiting factors, such as the high cost of peptide synthesis, their susceptibility to proteolytic degradation, and their unknown long-term toxicology profiles [1,14,15]. Here, analogues of the peptide KR-12 derived from the human cathelicidin LL-37 were designed due to its established antimicrobial activity and lack of mammalian cell toxicity, as originally found by Jacob et al. [16].

To introduce the antimicrobial peptides stably on a surface, different types of coatings can be employed. A popular approach is the use of polydopamine (pDA), a strong adhesive mussel-inspired polymer, due to its low cost, simplicity of application, and improved biocompatibility [17–19]. Similarly to mussel adhesive proteins, the adhesive properties of the pDA are owed to quinine and catechol groups, which create chelating structures with metals. Additionally, after polymerisation, pDA can be further functionalised with amine-containing nucleophiles, such as proteins and peptides. This allows the application of a pDA coating to Ti6Al4V, where the in-house designed analogues of the peptide KR-12 are subsequently covalently bonded to the pDA coating.
