Immobilization of Lysozyme on Zwitterionic Poly(4-vinylpyridine) Thin Films Enables Antifouling and Antibacterial Surfaces

In recent years, due to the rise of SARS-CoV-2 there is a heightened necessity to develop antifouling and antibacterial coatings that can be used to mitigate pathogenic infections. Antifouling coatings prevent the adhesion and settlement of microorganisms on a surface, slowing their growth. Zwitterionic coatings are an example of an antifouling coating, which repel incoming microorganisms due to the hydration layer. Zwitterionic surfaces are known to be super-hydrophilic due to the strong hydration layer formed from the electrostatic interactions of ions with water and are biocompatible. Antibacterial coatings kill pathogenic bacteria upon contact with the surface. Antibacterial mono-functional coatings are not beneficial for long-term applications due to the accumulation of dead cells and their debris on the surface, thus reducing performance of the coating overtime. Therefore, it is desirable to develop robust multifunctional coatings with strong antibacterial efficiency and fouling resistance for long-term applications.<br/><br/>In this study, we fabricated zwitterionic coatings with antifouling and antibacterial dual function by using a three-step process: vapor-deposition of a coating, vapor-derivatization of the coating to produce zwitterionic moieties, and immobilization of an antibacterial enzyme to lyse bacterial cells. Specifically, poly(4-vinylpyridine-<i>co</i>-pentaflurophenyl methacrylate-<i>co</i>-divinyl benzene) [P4VP-PFPMA-DVB] thin films were synthesized by an all-dry vapor deposition technique, <i>initiated</i> Chemical Vapor Deposition (iCVD). iCVD is a preferable method since it is an environmentally friendly, conformal, and solvent-free technique which prevents any potential contamination of the substrate. Zwitterionic moieties were formed via a vapor-phase reaction with 1,3-propane sultone, after which quaternary ammonium in pyridine and negatively charged sulfonate groups were produced. The formation of sulfobetaine was confirmed by Fourier transform infrared (FTIR) measurements and x-ray photoelectron spectroscopy (XPS).<br/><br/>The antibacterial property was introduced by the immobilization of lysozyme on the surface of the zwitterionic coating. Lysozyme is an enzyme responsible for the hydrolysis of peptidoglycans in cell walls of gram-positive bacteria leading to cell death. The enzyme was immobilized via a nucleophilic substitution reaction, in which an amide bond was formed with the N-terminus of lysozyme, thus making pentafluorophenyl a leaving group. In medical applications where no PFPMA is desired because fluorine is toxic, PFPMA could be removed through the secondary reaction with 2-(2-aminoethoxy)ethanol. The antifouling and antibacterial performance of the novel zwitterionic coating was tested against gram-positive <i>Bacillus subtilis</i> and gram-negative <i>Pseudomonas aeruginosa</i>. An excellent antifouling performance was achieved for both bacterium types with predominance of antibacterial performance over <i>B. subtilis </i>when compared to a conventional poly(vinyl chloride) [PVC] cell culture surface. Hence, this novel dual-function coating can be a promising anti-pathogenic surface with applications in medical devices, surgical tools, and surfaces in hospitals. Future studies will focus on evaluation of enzymatic activity, stability, and structure before and after the enzyme immobilization to evaluate long-term effectiveness of the novel coating.