Covalently Immobilized Lysozyme and Nitric Oxide Releasing Biomaterial Surface for Synergistic Broad Spectrum Antimicrobial Applications

Introduction: Hospital acquired infections (HAIs) are a major challenge affecting patients worldwide. About 3% of all patients will contract an HAI each day, while costing hospitals and patients an estimated $45 billion every year. [1] Current treatments primarily involve device removal leading to more procedures that could cause an HAI. Additionally, microbes are evolving rapidly resistances against current treatments, presenting the need for a new solution that does not struggle with these resistances. Nitric oxide (NO) is renowned for its broad-spectrum antimicrobial and antithrombotic properties and short half-life, resulting in less developed resistances. Another endogenously produced antibacterial, Lysozymes, has been independently studied for their efficacy in breaking up bacterial peptidoglycan causing cell lysis. Current antibiotic treatments employ a linear method of killing that is unable to affect biofilm-forming infections. In this work, the varied mechanisms of NO and lysozymes were combined on a biomaterial surface for broad-spectrum, synergistic efficacy against gram-positive, gram-negative, antimicrobial resistant, and fungal microbes. For this the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) was loaded into the polymer, with the lysozyme being covalently immobilized to the surface. The resulting material was then thoroughly characterized and investigated for NO release, lysozyme activity, lysozyme quantification, cytocompatibility, and antimicrobial efficacy.

Materials and

Methods: To synthesize the SNAP-lysozyme material, silicone rubber (SR) films were produced and then modified by solvent swelling and covalent immobilization. Firstly, 8 mm punchouts of SR were fully submerged into SNAP-THF solution (125 mg mL-1) for NO donor impregnation over 24 hours. Films were then dried under a vacuum and washed before undergoing plasma treatment. After plasma treatment, the surface was aminated using APTMS, then submerged in the crosslinker Sulfo-EGS, creating an ideal linking site for lysozyme. Films were then placed in lysozyme solution while rocking for 24 hours, resulting in the covalent linking of lysozyme to SR. Finally, films were lyophilized to remove any remaining moisture and preserve lysozyme activity. The presence of lysozyme was confirmed via static contact angle, FTIR, lysozyme quantification, lysozyme leaching, and lysozyme activity assays. To determine how NO release was affected, samples were submerged in PBS with EDTA in physiological conditions and measured for NO release until levels dropped below physiological levels using a chemiluminescent Nitric Oxide Analyzer. The cytocompatibility of the materials leachates was tested against BJ fibroblasts over 24 hours, with a lysozyme IC50 also being tested. Lastly, the films were analyzed for antimicrobial efficacy against Staphylococcus aureus, Escherichia coli, Methicillin Resistant Staphylococcus aureus (MRSA), and Candida Albicans over 24 hours.

Results, Conclusions, and Discussions: The SNAP-Lyso films exhibited a significant hydrophilic shift and lysozyme FTIR peak confirming the presence of immobilized lysozyme. NO was released at physiological levels for up to 3 days, with minimal SNAP and lysozyme leaching. Furthermore, the SNAP-Lyso films demonstrated > 70% cell compatibility and produced up to a 4-log reduction in viable S. aureus adhesion to the surface (Figure 1). Additionally, SNAP-Lyso films showed significant efficacy in reducing viable E. coli and C. albicans adhesion, proving its wide range of potential applications. The SNAP-Lyso films produced a synergistic antimicrobial surface that showed efficacy against a wide range of both fungal and bacteria strains while maintaining cytocompatibility. This material demonstrates the benefit of combining two different antimicrobial mechanisms to create a more effective, safer material for patients. This process can be easily applied to a breadth of medical devices and requires little adaptation.

Acknowledgements (Optional):

References: 1. K. Gidey., et al., PLOS ONE 2023, 18, e0282141