Ion Implanted ‘Trojan Horse’ surfaces with antimicrobial activity

Abstract

INTRODUCTION: Hospital acquired infections (HAIs) are the most frequently occurring adverse event in healthcare delivery globally and result in prolonged hospitalization. Implantable devices are commonly affected by these infections due to the formation of biofilms. Gallium (Ga) has been shown to prevent biofilm formation due to its disruption of iron-dependent bacterial growth processes. Defensin, a naturally occurring peptide, has also shown significant antimicrobial qualities. Hence, the synergistic effect of the two strategies is proposed for study in the form of a surface modified antimicrobial polymer to prevent biofilm formation on implantable devices. AIMS AND HYPOTHESES: To develop a surface modification technology, which imparts antimicrobial activity and biocompatibility to the implantable polymer-based devices. More specifically, we aimed to prepare, modify and characterize surface properties of a gallium implanted, defensin immobilized biopolymer and assess its ability to prevent biofilm formation, bacterial adhesion and cytocompatibility. MATERIALS AND METHODS: PLA films were optimized and fabricated, after which they underwent plasma treatment in air or Ga implantation. Samples were characterized using AFM, PFQNM, nano thermal analysis and LCR. Following this, samples had defensin immobilized on their surfaces and this was to be confirmed via Tween-20 wash and subsequent PFQNM analysis. Defensin alone was tested in solution via the Disc Diffusion Assay to assess its capability as an antimicrobial agent. The surfaces then were subjected to a Biofilm formation assay for a period of 14 days after which they were stained and imaged using CLSM. The surfaces were also tested for their ability to invoke an inflammatory response via FBGC formation by M0 phenotype macrophages (through immunofluorescence staining and CLSM) and via measurement of pro-inflammatory cytokine IL-1β (using ELISA) by these same cells. The QBlue assay was used to measure their viability. Finally, the surfaces were assessed for the initial attachment and morphology of myoblasts and for their osteogenic differentiation potential of these cells via the ALP assay which was normalized using total protein content (through the BCA assay). RESULTS AND DISCUSSION: Ga implantation resulted in the increase in carbonization of the surface layer and accordingly, the stiffness of the sample. The increasing fluence of Ga ions also resulted in inconsistencies in ion implantation which could have follow on effects on peptide immobilization due to increased polymer crosslinking. Linker-free immobilization of defensin was confirmed on the Plasma treated and Ga treated surfaces. Both gallium implantation and defensin immobilization independently and synergistically proved to impart antimicrobial activity to the surfaces and the antimicrobial effects of hBD-1 appear to be potentiated by surface immobilization. Defensin immobilization however, appeared to be inconsistent with increasing Ga fluence. IL-1β formation expression increased with decreasing Ga concentration suggesting less inflammation with increasing Ga dose and the increased with introduction of De also. FBGC formation was significantly decreased in all Ga and Ga De treated groups in comparison with PLA. Myoblast morphology appeared to be negatively affected by De on the Ga 14 De and Ga 15 De samples, potentially due to the effects of inconsistent surface stiffness and surface charge. There were no significant changes in ALP production between the PLA control and any other treated sample. CONCLUSION: This novel therapy has the potential to prevent biofilm infection and opens avenues for future research such as in vivo studies, and the viability of using other antimicrobial peptides as linkerfree immobilization has been made possible.