The design of antimicrobial LL37-modified collagen-hyaluronic acid detachable multilayers

Abstract

The design of antimicrobial membranes and thin films are critical for the design of biomaterials that can combat bacterial contamination. Since the long-term use of conventional antibiotics can result in bacterial resistance, there is a critical need to incorporate natural antimicrobial peptides (AMPs) that not only prevent a wide range of pathogens from causing infections but can also promote many beneficial outcomes in wounded tissues. We report the design and antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a naturally occurring human AMP. LL-37 was physically adsorbed and chemically immobilized on the surface of PEMs. The antimicrobial and cytotoxic properties of PEMs were tested with Gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. The ability to prevent bacterial adhesion and to neutralize an E. coli layer was investigated as a function of LL-37 concentration. An interesting trend was that even unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, bacterial adhesion was significantly lower on the surface of the films as well as in the surrounding broth. Immobilizing LL-37 resulted in less than 3% bacterial adhesion on the surface due to the presence of the peptide. LL-37 modified PEMs did not result in any cytotoxicity up to input concentrations of 16μM. More importantly, urea and albumin secretion by hepatocytes were unaffected even at high LL-37 concentrations. The COL/HA PEMs can serve as antimicrobial coatings, biological membranes and as in vitro platforms to investigate pathogen-tissue interactions.

Statement of significance: Antimicrobial peptides (AMPs) are emerging as an alternative to conventional antibiotics. We report the antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a human AMP. The antimicrobial and cytotoxic properties were tested with gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. Unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, the sustained release of the active peptide killed planktonic bacteria. Immobilizing LL-37 resulted in less than 3% bacterial adhesion. LL-37 modified PEMs did not result in cytotoxicity up to input concentrations of 16μM. The COL/HA PEMs can serve as antimicrobial coatings and to investigate pathogen-cell interactions.

Keywords: Antimicrobial; Collagen; Hyaluronic acid; LL-37; Polyelectrolyte multilayers.