Anti-infective DNase I Coatings on Polydopamine Functionalized Titanium Surfaces by Alternating Current Electrophoretic Deposition

Implant-associated infections (IAIs) can cause serious problems due to the difficult-to-treat nature of biofilms formed on the implant surface. The biofilm matrix, consisting of polysaccharides, proteins, lipids and extracellular DNA (eDNA), forms a protective environment for the residing bacteria in mature biofilms. Recently, indirect prevention of biofilm growth through the degradation of eDNA using an enzyme, such as deoxyribonuclease (DNase) I, is being considered a promising strategy in the battle against IAIs. In this study, the immobilization of protective DNase I coatings on titanium implant materials was investigated. The effectiveness of alternating current electrophoretic deposition (AC-EPD) as a novel processing route to apply DNase I on titanium using polydopamine (PDA) as an intermediate was examined and compared with the commonly applied diffusion methodology (i.e. classic dipping). The use of AC-EPD significantly increased the amount of deposited protein for the same processing time, yielding homogeneous coatings with a thickness of 12.8 nm and increased average surface roughness, Sa, of ~20 nm. The surface free energy components revealed an increase in γ- from 10.2 to ~20 mJ m-2 upon PDA functionalization and enzyme coating regardless of the coating procedure, indicating monopolar surfaces with increased electron-donor capacity . Furthermore, X-ray photoelectron spectroscopy confirmed the presence of peptide bonds on all DNase-coated substrates. Time-of-flight secondary ion mass spectrometry detected a more dense DNase I layer in the case of AC-EPD, which was selectively deposited on the electrode coupled as anode during the high-amplitude half cycle of AC signal. Disulfide signals did not differ from those obtained from a dipped DNase reference sample, confirming that the tertiary enzyme structure was not influenced by the deposition methodology. The enzyme activity, release kinetics, and shelf life of DNase I coatings were monitored in real-time using a quantitative qDNase assay. DNase I coatings produced using AC-EPD are threefold more active compared to coatings prepared by classic dipping. In line with covalent linking of DNase to PDA, both deposition methods enable a stable attachment of a small fraction of the DNase activity to the surface, while loosely adhering superimposing DNase deposits are released from the surface in a high burst. Interestingly, coatings prepared with AC-EPD exhibit a prolonged, gradual release of DNase activity. The AC-EPD DNase coatings significantly reduce biofilm formation of both Staphylococcus epidermidis and Pseudomonas aeruginosa up to 20 h, whereas DNase coatings prepared by short classic dipping only reduce S. epidermidis biofilm formation, and to a lesser extent. Overall, this study indicates that AC-EPD allows to rapidly concentrate DNase I on PDA-functionalized titanium, while maintaining the enzyme activity and anti-infectivity properties. This highlights the potential of AC-EPD as a time-efficient coating strategy (as opposed to the much slower dip-coating methodologies) for bioactive molecules in a wide variety of biomedical applications.