Polymer multilayers loaded with antifungal β-peptides kill planktonic Candida albicans and reduce formation of fungal biofilms on the surfaces of flexible catheter tubes

Abstract

Candida albicans is the most common fungal pathogen responsible for hospital-acquired infections. Most C. albicans infections are associated with the implantation of medical devices that act as points of entry for the pathogen and as substrates for the growth of fungal biofilms that are notoriously difficult to eliminate by systemic administration of conventional antifungal agents. In this study, we report a fill-and-purge approach to the layer-by-layer fabrication of biocompatible, nanoscale 'polyelectrolyte multilayers' (PEMs) on the luminal surfaces of flexible catheters, and an investigation of this platform for the localized, intraluminal release of a cationic β-peptide-based antifungal agent. We demonstrate that polyethylene catheter tubes with luminal surfaces coated with multilayers ~700nm thick fabricated from poly-l-glutamic acid (PGA) and poly-l-lysine (PLL) can be loaded, post-fabrication, by infusion with β-peptide, and that this approach promotes extended intraluminal release of this agent (over ~4months) when incubated in physiological media. The β-peptide remained potent against intraluminal inoculation of the catheters with C. albicans and substantially reduced the formation of C. albicans biofilms on the inner surfaces of film-coated catheters. Finally, we report that these β-peptide-loaded coatings exhibit antifungal activity under conditions that simulate intermittent catheter use and microbial challenge for at least three weeks. We conclude that β-peptide-loaded PEMs offer a novel and promising approach to kill C. albicans and prevent fungal biofilm formation on surfaces, with the potential to substantially reduce the incidence of device-associated infections in indwelling catheters. β-Peptides comprise a promising new class of antifungal agents that could help address problems associated with the use of conventional antifungal agents. The versatility of the layer-by-layer approach used here thus suggests additional opportunities to exploit these new agents in other biomedical and personal care applications in which fungal infections are endemic.

Keywords: Antifungal; Biofilms; Catheters; Controlled release; Polymer multilayers; Surfaces.

Figure 1

(A) Schematic illustration depicting fabrication of PEM films on the luminal surfaces of polyethylene catheter tubes. (B–F) Representative fluorescence micrographs of catheters coated with (PGA/FITC-PLL)_x PEMs with increasing bilayers; x = 0.5 (B), 4.5 (C), 9.5 (D), 14.5 (E), and 19.5 (F). Scale bar = 250 μm. (G) Plot of the average fluorescence intensity as a function of the number of bilayers of PGA/PLL deposited. Data points are the average of measurements in three regions of two independent experiments, normalized to the fluorescence intensity of (PGA/FITC-PLL)_19.5 films; error bars represent standard deviation.

Figure 2

Scanning electron microscopy images of longitudinally-sliced catheter tubes: (A) bare tube (no film/no peptide), (B) a tube coated with a PGA/PLL film 19.5 bilayers thick, (C) a film with a PGA/PLL 19.5 bilayers thick post-loaded with β-peptide; scale bars = 200 μm. The inset in (B) shows a region where a portion of the sliced films lifted off the surface of the tube, which was useful in measuring the cross sectional thicknesses of the films (see text and Figure S2). Inset scale bar = 25 μm.

Figure 3

(A) Schematic illustration depicting the post-fabrication loading of film-coated catheter tubes with β-peptide. (B–D) Representative fluorescence micrographs of (B) an uncoated catheter and (C) a catheter coated with a PGA/PLL film 19.5 bilayers thick after incubation in a solution of β-peptide for 24 hours. (D) Fluorescence micrograph of a film-coated, β-peptide-loaded catheter after incubation in PBS for 120 days; scale bars = 250 μm. (E) Plot showing the release of β-peptide into the lumen of a film-coated, β-peptide-loaded catheter (circles) and a no-film control catheter incubated with β-peptide (squares) incubated in PBS at 37 °C. Data points are the average of three replicates and error bars represent standard deviation.

Figure 4

Antifungal activity of untreated catheters (tube), catheters coated with PGA/PLL films (tube/film), and catheters coated with PGA/PLL films and loaded with β-peptide (tube/film/pep). C. albicans inoculum (10⁶ cfu/mL) was incubated in the tubes for 6 hours at 37 °C. 100-Fold dilutions of the inocula from tubes were plated on solid YPD and colonies were counted after 24 hours. Data points are averages of measurements from three independent experiments of three replicates each and error bars denote standard deviation; (* indicates p < 0.005 by a two-tailed T-test).

Figure 5

Antifungal activity of β-peptide-loaded PEMs following pre-incubation. Untreated catheters (tube; black bars), catheters coated with PGA/PLL films (tube/film; gray bars), and catheters coated with PGA/PLL films and loaded with β-peptide (tube/film/pep; white bars) were pre-incubated with PBS for the indicated time periods. Each tube was then flushed with fresh buffer before inoculation and incubation with C. albicans for 6 hours at 37 °C. XTT was then used to measure differences in cell metabolic activity. Data points are averages of measurements from two independent experiments of three replicates each normalized to the metabolic activity of the tube for each data point Error bars denote standard deviation. For all pre-incubation conditions, reductions in metabolic activity for β-peptide-loaded films (tube/film/pep) were statistically different (p < 0.05 by two-tailed T-test) from untreated controls (tube) under the same conditions.

Figure 6

Inhibition of C. albicans by PEMs loaded with β-peptide. Untreated catheters (tube), catheters coated with PGA/PLL films (tube/film), and catheters coated with PGA/PLL films and loaded with β-peptide (tube/film/pep) were incubated with a C. albicans inoculum (10⁶ cfu/mL) in RPMI containing 5% FBS at 37 °C. After 48 hours, XTT was used to quantify differences in biofilm metabolic activity. Data points are averages of measurements from three independent experiments of three replicates each and error bars denote standard deviation; (* indicates p < 0.005 by two-tailed T-test).

Figure 7

Low (A–B) and high magnification (C–D) scanning electron microscopy images showing the inner surfaces of catheter tubes after incubation with C. albicans in biofilm assays (the catheter tubes were longitudinally-sliced prior to imaging; see text for additional details of these experiments). (A,C) Images of a tube coated with a PGA/PLL film 19.5 layers thick (tube/film; no β-peptide) incubated with C. albicans for 24 hours. (B,D) Images of an otherwise identical film-coated tube loaded with β-peptide (tube/film/pep) incubated with C. albicans for 24 hours. Scale bars = 100 μm (A), 200 μm (B), 30 μm (C), 20 μm (D).

Scheme 1

Structure of the coumarin-labeled, 14-helical β-peptide used in this study.