Materialia 6 (2019) 100350 Contents lists available at ScienceDirect Materialia journal homepage: www.elsevier.com/locate/mtla Full Length Article Surface immobilization of a short antimicrobial peptide (AMP) as an antibacterial coating Sasmita Majhi, Ankita Arora, Abhijit Mishra ∗ Materials Science and Engineering, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar, Gujarat 382355, India a r t i c l e i n f o Keywords: Polystyrene Antimicrobial peptide KLR CKLR Antibacterial surface a b s t r a c t Bacterial infections associated with commonly encountered abiotic surfaces are common threat to patient safety and health care. Antimicrobial peptides (AMPs) immobilization on such surfaces are considered as suitable ap- proach towards addressing this clinical issue. In this study, we evaluate the antibacterial effect of immobilizing a short, pore-forming antimicrobial peptide, KLR (KLLLRLRKLLRR). KLR is covalently linked to polystyrene (PS) surfaces, via its C-terminal using EDC/NHS chemical coupling, and via its N-terminal using maleimide-thiol cou- pling with a Cysteine added to KLR (CKLLLRLRKLLRR) first. Physicochemical characterization of PS surfaces at each step of modification is carried out using atomic force microscopy, water contact angle measurements, and X-ray photoelectron spectroscopy. Antibacterial efficacy assays demonstrate that immobilized KLR shows excellent antibacterial activity against Gram-negative (E.coli ATCC 25922) and Gram-positive (S.aureus ATCC 25923) bacteria for both the coupling strategies. In addition, the AMP coatings possess no toxicity against mouse fibroblast cells (NIH 3T3). 1. Introduction Bacterial adhesion leading to their colonization on abiotic surfaces can lead to severe problems in human healthcare and industrial en- vironments. Biofilm infections may further lead to the emergence of multi-drug resistant bacteria. Among the most effective ways to pre- vent such spread of infectious diseases is to interfere with adhesion of bacteria and/or its viability on surfaces [1]. Such antibacterial surface coatings have been investigated employing suitable antibacterial agents, including antibiotics [2,3], metal nanoparticles [4–7], Quaternary am- monium compounds (QACs) [8–11], antimicrobial polymers [12–15], and antimicrobial peptides [16–19]. Problems associated with most of the bactericidal coatings include release of active antibacterial com- ponents (viz. antibiotics, nanoparticles, biocides) from surface due to out-diffusion, which may lead to development of microbial resistance and cumulative toxicity with time [20,21]. Contact active antibacte- rial coatings developed by covalently immobilizing antibacterial agents, e.g., antibacterial polymers, antimicrobial peptides that do not leach out from surface, are less likely to develop bacterial resistance [9]. Variable size and charge of antibacterial polymers affect their interaction with pathogens, hence, their use is limited to certain extent [22]. Antimicrobial peptides (AMPs) are short polypeptides associated with host immune system and most often are cationic in nature. Net positive charge and amphipathicity of AMPs, promote their non-specific ∗ Corresponding author. E-mail address: amishra@iitgn.ac.in (A. Mishra). interaction with negatively charged bacterial membrane, which makes them less prone towards developing antimicrobial resistance. AMPs also have a broad-spectrum of antimicrobial activity, while exhibiting low cytotoxicity and low immune response. These in-dwelling features of AMPs make them an ideal candidate to be used in antibacterial coat- ing in preventing bacterial adhesion and/or survival on commonly used surfaces [16,23]. However, a crucial prerequisite for development of AMP-based coating is maintenance of peptide activity and stability af- ter immobilization. In this work, we immobilize an in-house designed antimicrobial pep- tide KLR (KLLLRLRKLLRR) (Fig. 1(a)) on polystyrene (PS) surfaces. PS is a commonly used packaging material in food industry and medical devices, therefore, it would be advantageous to impart antibacterial ac- tivity to it. The peptide, KLR, is derived from C-terminal residues, 32–43, of Human  -Defensin 3 (hBD-3), with all the non-cationic residues be- ing replaced by a highly hydrophobic residue, Leucine, while leaving the cationic residues untouched. This increase in hydrophobicity enhances the antibacterial activity. KLR is covalently immobilized through its C- terminal onto PS using the well-known EDC/NHS coupling (Fig. 2(a)). At each step, the modified surfaces are analyzed by atomic force mi- croscopy, water contact angle measurements, and X-ray photoelectron spectroscopy. The antibacterial efficacy of immobilized KLR is exam- ined against both Escherichia coli (ATCC 25922) and Staphylococcus au- reus (ATCC 25923). Cytotoxic effect of the immobilized peptides against https://doi.org/10.1016/j.mtla.2019.100350 Received 20 February 2019; Accepted 10 May 2019 Available online 13 May 2019 2589-1529/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.