Bioactive coating provides antimicrobial protection through immunomodulation and phage therapeutics

Abstract

Medical implant-associated infections (IAI) is a growing threat to patients undergoing implantation surgery. IAI prevention typically relies on medical implants endowed with bactericidal properties achieved through surface modifications with antibiotics. However, the clinical efficacy of this traditional paradigm remains suboptimal, often necessitating revision surgery and posing potentially lethal consequences for patients. To bolster the existing anti-IAI arsenal, we propose herein a chitosan-based bioactive coating, i.e., ChitoAntibac, which exerts bacteria-inhibitory effects either through immune modulation or phage-directed microbial clearance, without relying on conventional antibiotics. The immuno-stimulating effects and phage-induced bactericidal properties can be tailored by engineering the loading dynamic of macrophage migration inhibitory factor (MIF), which polarizes macrophages towards the proinflammatory subtype (M1) with enhanced bacterial phagocytosis, and Staphylococcal Phage K, resulting in rapid and targeted pathogenic clearance (>99.99%) in less than 8 h. Our innovative antibacterial coating opens a new avenue in the pursuit of effective IAI prevention through immuno-stimulation and phage therapeutics.

Keywords: Bacteriophage; Bioactive coating; Immune modulation; Implant-associated infections; Medical implants.

Graphical abstract

Fig. 1

Characterization of PDMS implants with various surface modifications. (A) An illustration of the methodologies for surface modification of PDMS implants. (B) A representative image of the PDMS implants coated with PDA under optical camera (i) with its microstructure at the frame region examined by AFM (ii). (C) A surface topographical image of PDA-modified PDMS implants. (D) A line chart displays the thickness of PDA coating on the PDMS implants with AFM. (E) Contact angle analysis of the hydrophilicity of the PDMS implants subjected to PDA surface modification for various time points. Subscripts under t denote the reaction time (h). (F) The contact angles for various PDA-coated PDMS implants as a function of reaction time. (G) A computed table summarizes the concentrations of crosslinking agents used for the CMCS crosslinking reactions. (H) The water absorption of ChitoAntibac PDMS sheets crosslinked with various concentrations of EDC and NHS. The dotted line indicates the water absorption of un-crosslinked PDMS sheets. n = 4. Triplicate experiments were performed unless stated otherwise.

Fig. 2

ChitoAntibac PDMS enabled fast release of MIF and induced distant macrophage recruitment. (A) Load efficiency of MIF for ChitoAntibac PDMS implants with various crosslinking conditions was assessed after 2 h of loading. ChitoAntibac PDMS #4 provided the highest MIF loading and therefore was chosen for subsequent experiments. (B) MIF loading efficiency using ChitoAntibac PDMS as a function of time. Maximal loading rate was indicated in the plot. (C) Time-dependent MIF release profile using ChitoAntibac PDMS#4 at physiological temperature (37 °C). (D) Schematic illustrating the experimental setup to investigate the effect of MIF released from the ChitoAntibac PDMS implants on macrophage recruitment. (E) Representative images showed macrophage across different groups migrated towards the other side of the inserts or the cell culture well plates after 24 h incubation. Scale bar = 100 μm. (F) Cell counts of migrated macrophages across different groups. Data are presented as mean ± SD. Triplicate experiments were performed. Statistical significance is indicated in the plots where necessary, with * denotes p < 0.05 and ** denotes p < 0.01.

Fig. 3

ChitoAntibac PDMS loaded with MIF activated M1 polarization to enhance bacterial phagocytosis. (A) Percent of M1 macrophages after treatment with MIF-loaded ChitoAntibac PDMS for various durations. Quantification of M1 macrophage population was performed by counting cells that had CD 80 expression level more than 2-fold of that of the cells in the control group. (B) Representative fluorescent images showed macrophages stained with CD 80 antibody (green) after treatment with MIF-loaded ChitoAntibac PDMS for various durations. Scale bar = 50 μm. (C) Representative fluorescent images showed phagocytosis of S. aureus in macrophages with various pretreatments post 3 h of bacterial inoculation. Macrophages were counterstained with Hoechst 33342 (blue) and Phalloidin (red) with the endocytosed bacteria displayed in green. Scale bar = 20 μm. (D) Number of S. aureus internalized by macrophages with different pretreatments. n = 40. Triplicate experiments were performed. Statistical significance is indicated in the plots where necessary, with * denotes p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

ChitoAntibac PDMS loaded with phage K exhibited rapid and efficient removal of S. aureus in vitro. (A) Optical density measurement showed dose- and time-dependent bacteria-eliminating property of phage K in vitro. (B) Quantification of live bacteria after treatment with different concentrations of phage K. MIC99 was indicated in the plot and used as a baseline to determine the effective antibacterial dose range (highlighted in light brown). (C) Quantification of phage K loaded into ChitoAntibac coated PDMS sheets and Ti alloy. (D) Optical density measurement shows the antibacterial efficacy of phage-loaded ChitoAntibac PDMS and Ti alloy as a function of time. (E) Log reduction of live bacteria (S. aureus) after treatment with ChitoAntibac coated PDMS sheets and Ti alloy. Insets showed optical images of bacterial colonies formed on agar plates that corresponded to respective treatment. (F) Cell viability of mouse neurons after 1-, 3-, 5-, 7-day treatment with ChitoAntibac PDMS loaded with MIF or Phage K. (G) Cell viability of MCC3T3-E1 bone cells after 1-, 3-, 5-, 7-day treatment with ChitoAntibac Ti loaded with MIF or Phage K. Triplicate experiments were performed. Statistical significance is indicated in the plots where necessary, with ** denotes p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)