Cationic homopolypeptides: A versatile tool to design multifunctional antimicrobial nanocoatings

Abstract

Postoperative infections are the most common complications faced by surgeons after implant surgery. To address this issue, an emerging and promising approach is to develop antimicrobial coatings using antibiotic substitutes. We investigated the use of polycationic homopolypeptides in a layer-by-layer coating combined with hyaluronic acid (HA) to produce an effective antimicrobial shield. The three peptide-based polycations used to make the coatings, poly(l-arginine) (PAR), poly(l-lysine), and poly(l-ornithine), provided an efficient antibacterial barrier by a contact-killing mechanism against Gram-positive, Gram-negative, and antibiotic-resistant bacteria. Moreover, this activity was higher for homopolypeptides containing 30 amino-acid residues per polycation chain, emphasizing the impact of the polycation chain length and its mobility in the coatings to deploy its contact-killing antimicrobial properties. However, the PAR-containing coating emerged as the best candidate among the three selected polycations, as it promoted cell adhesion and epithelial monolayer formation. It also stimulated nitric oxide production in endothelial cells, thereby facilitating angiogenesis and subsequent tissue regeneration. More interestingly, bacteria did not develop a resistance to PAR and (PAR/HA) also inhibited the proliferation of eukaryotic pathogens, such as yeasts. Furthermore, in vivo investigations on a (PAR/HA)-coated hernia mesh implanted on a rabbit model confirmed that the coating had antibacterial properties without causing chronic inflammation. These impressive synergistic activities highlight the strong potential of PAR/HA coatings as a key tool in combating bacteria, including those resistant to conventional antibiotics and associated to medical devices.

Keywords: Antibiotic substitute; Antimicrobial polypeptides; Hernia mesh implants; Hyaluronic acid; Nanolayer coating.

Graphical abstract

Fig. 1

Construction of (polycation/HA)₇ multilayer films on SiO₂-coated crystal monitored by QCM-D with PAR30 (red), PLL (green), and PLO (blue) as polycations. a. Evolution of the normalized frequency -Δf_ν/ν (for ν = 3) as a function of the number of adsorbed layers is shown. b. Evolution of the estimated thickness as a function of adsorbed layers is shown. HA: hyaluronic acid; PAR: poly(l-arginine), PLL: poly(l-lysine), PLO: poly(l-ornithine). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Normalized pathogen growth of S. aureus as a function of polycation concentration (in mg.mL⁻¹) measured in solution with PAR containing 30, 100, and 200 arginine residues (a), with PLL containing 30, 100, and 250 residues (b) and with PLO containing 30, 100, and 250 residues (c). Minimal inhibitory concentration (MIC, in mg.mL⁻¹) of E. coli, M. luteus, P. aeruginosa, S. aureus and MRSA for polycations, measured in solution for PAR with 30, 100, and 200 residues (d), for PLL with 30, 100, and 250 residues (e) and or PLO with 30, 100, and 250 residues (f). Comparisons of MIC values with PAR, PLL, and PLO containing 30 residues (g). Each polycation was incubated for 24 h at 37 °C in 300 μL of bacterial culture medium with an initial bacterial concentration corrresponding to OD₆₂₀ = 0.001. Each value corresponds to the mean value of three individual experiments (three samples per experiment and condition). All error bars represent standard deviations. PAR: poly(l-arginine), PLL: poly(l-lysine), PLO: poly(l-ornithine).

Fig. 3

Localization of PAR30-TRITC in E. coli-GFP. High resolution image of E. coli-GFP (green channel on the left) and PAR30-TRITC (red channel on the right) with super resolution confocal microscopy. White arrows point a PAR30-TRITC accumulation at membrane of E. coli-GFP. GFP: green fluorescent protein, TRITC: tetramethylrhodamine, PAR: poly(l-arginine), GFP: green fluorescent protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Normalized S. aureus growth in supernatant after 24 h in contact with (polycation/HA)₂₄ multilayer films PAR (a), PLL (b), and PLO (c) with a variable number of residues per chain. Normalized pathogen growth (S. aureus, MRSA, E. coli, P. aeruginosa) in supernatant after 24 h in contact with (polycation30/HA)₂₄(d). Each (polycation/HA)₂₄ multilayer films was incubated for 24 h at 37 °C in 300 μL of bacterial culture medium with an initial bacterial concentration correspondinf to OD₆₂₀ = 0.001. PAR: poly(l-arginine), HA: hyaluronic acid, PLL: poly(l-lysine), PLO: poly(l-ornithine), MRSA: methicillin-resistant Staphylococcus aureus.

Fig. 5

Characterization of the mobility of polycation inside the multilayer films. a. Normalized fluorescence intensity recovery in a photobleached area as a function of [t]^1/2 for each (polycation-FITC/HA)₂₄ film, with PAR30 (red), PLL30 (green), and PLO30 (blue) as polycations. t = 0 corresponds to the end of the photobleaching step. A typical experiment per polycation is shown. b. Corresponding proportion of mobile polycation chain derived from data in part. Three independent experiments for each polycation were performed and error bars correspond to standard deviations. PAR: poly(l-arginine), PLL: poly(l-lysine), PLO: poly(l-ornithine). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Cytotoxicity assays, cell adhesion assays, and NO release quantification. a. Metabolic activity of Balb/3T3 cells incubated for 24 h with supernatant medium extracts from polyelectrolyte multilayer films in contacts during 24 h. Balb/3T3 cells were cultured in presence of different rates of extraction medium in contact with (PAR30/HA)₂₄, (PLO30/HA)₂₄, and (PLL30/HA)₂₄. Normalized metabolic activity higher than 70 % (violet line) indicates the absence of cytotoxicity in the corresponding solutions. b. Deflection AFM images taken in air of epithelial MDCK cells that were cultured on glass, (PAR30/HA)₂₄ and (PLO30/HA)₂₄ multilayer films. c. Schematic representation of the experimental condition, with cells directly in contact with the coating (PAR30/HA)₂₄) and cells with no contact with (PAR30/HA)₂₄ (right). d. NO level quantified through the detection of nitrite in the supernatant in HUVEC. The supernatant was analyzed after 1, 3, and 7 days of culture and mean NO release was obtained from three independent experiments. Error bars correspond to standard deviations. NO: nitric oxide, DMSO: dimethyl sulfoxide, PAR: poly(l-arginine), HA: hyaluronic acid, PLL: poly(l-lysine), PLO: poly(l-ornithine); HUVEC: human umbilical vein endothelial cell, AFM: atomic force microscopy, MDCK: Madin-Darby canine kidney. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Pathogens growth evaluated by CFU number after 24 h in contact with (PAR30/HA)₂₄ and (PAR30/HA)₄₈ multilayer films following the ISO 22196 method. The mean of three independent experiments is shown, error bars correspond to standard deviations. CFU: colony forming unit, PAR: poly(l-arginine), HA: hyaluronic acid.

Fig. 8

Resistance acquisition assay of S. aureus. S. aureus were cultured in the presence of half of the MIC of the antibacterial agent (PAR30 and tetracycline, a conventional antibiotic) for 30 days. The fold changes of MIC values compared with the initial MICs were evaluated at the indicated days and the PAR30 and tetracycline solutions were adjusted accordingly. MIC: minimal inhibitory concentration, PAR: poly(l-arginine).

Fig. 9

Macroscopic outcomes of different implants at euthanasia and quantification of bacteria adhesion on the implants. a. Group #1, (A) Evidence of discrete bulges (arrows) developed under the skin tissue. (B) Implant showing an edematous swelling (dashed line). (C) Implant partially covered by purulent material (*). (D) Implant with severe vascularization and fully covered by purulent material (*). b. Group #2, (A) Evidence of a subcutaneous abscess (dashed line) containing solid purulent material. (B) Detail of a thin fibrous capsule (arrowhead) surrounding an implant. (C) Implant surface with no macroscopic evidence of infection. (D) Implant showing dispersed purulent material (*) restricted to the areas of mesh anchorage. c. Group #3 (A) Evidence of discrete bulges (arrows) developed under the skin tissue. (B) Implant surface with no macroscopic evidence of infection. (C, D) Implants showing dispersed purulent material (*) restricted to the areas of mesh anchorage. d. Quantification of bacterial adhesion to the surface of central mesh fragments collected from the different implants for each animal in each group. CFU: colony forming unit.

Fig. 10

Histological evaluation of the implants from group#1, 2 and 3 (a, b and c). (A, B) Neoformed connective tissue exhibiting accumulation of tissue exudate (Δ) and large abscesses (*) (Masson's trichrome, x50). (C, D) Detail of the inflammatory cells (yellow arrows) surrounding the mesh filaments (f) and the abscesses (hematoxylin eosin, x100). (E, F) Labeled bacteria (black arrows) were located either in the neoformed tissue and within the abscesses (S. aureus immunolabeling, x320). (G, H) Visualization by SEM at high magnification c (x 2000) to check the presence or of bacteria (white arrows) throughout the implant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)