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 ~ 700 nm 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 ~ 4 months) 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.

Introduction

Candida albicans is the pathogen most responsible for hospital-acquired fungal infections in humans [1]. This opportunistic pathogen causes systemic and often fatal infections, especially in immunocompromised patients including those afflicted with AIDS, undergoing chemotherapy, or receiving organ transplants [2], [3], [4]. It is estimated that about 400,000 cases of candidemia (the presence of C. albicans in the bloodstream) occur annually, and the mortality rate associated with this condition ranges from 30 to 60% [3], [4], [5]. Apart from the human suffering this causes, the annual economic burden of fungal infections is in the billions of dollars in the U.S. alone [6].

It has been estimated that over half of all hospital-acquired infections are associated with the insertion of a medical device [7], [8]. Indwelling medical devices, and catheters in particular, are major risk factors for systemic C. albicans infections [4], [7], [9] because they act as both a point of entry for the pathogen and a substrate for the subsequent growth of fungal biofilms [10], [11] that exhibit resistance to antifungal drugs and provide protection against host defenses [8], [12], [13], [14]. Current methods for the prevention and treatment of catheter-associated C. albicans infections involve periodic replacement of catheters combined with treatments using systemically-administered antifungal drugs [9], [15]. These measures are far from optimal because catheter removal may require surgery and replacement is both expensive and can cause discomfort or other complications [15], [16], [17], [18]. Moreover, the indiscriminate use of systemic antifungal drugs can facilitate the development of evolved resistance [8], [19]. In view of these and other practical and clinical considerations, there is a critical need for the development of both new antifungal agents and new strategies to deliver these agents to sites either experiencing or at high risk for device-associated fungal infections. The work reported here was motivated by these two important challenges.

Most conventional antifungal agents used to combat candidemia act by inhibiting specific enzymes or cell wall or membrane components in C. albicans. As noted above, this pathogen can, evolve mechanisms of resistance than can render agents that act through these mechanisms ineffective [8], [20], [21]. Additionally, several existing antifungal drugs (including amphotericin B, the current gold standard for the clinical treatment of C. albicans infections) are not particularly selective toward fungi and, thus, also exhibit high levels of toxicity in human cells (e.g., nephrotoxicity in the case of amphotericin B) [22], [23]. Antimicrobial peptides (AMPs) and their analogues derived from or modeled upon components of the innate and adaptive immune systems of various host organisms have also been investigated broadly as antimicrobial agents, in part because these agents achieve their potency through membrane-disrupting processes that are less likely to lead to evolved resistance [24], [25], [26], [27]. Unfortunately, while AMPs are promising in this latter context, these peptide-based agents often exhibit low stabilities and activities in physiological environments, such as in the presence of proteases or at physiologic ionic strength [25], [27]. To address these two issues and advance the design of AMPs that are active and selective against fungal pathogens, our groups and others have designed AMP structures based on more stable β-peptide scaffolds that mimic many of the important structural features of antimicrobial α-peptides [28], [29], [30], [31], [32]. Of particular relevance to the work reported here, we have demonstrated that 14-helical β-peptides can be designed to kill C. albicans efficiently and selectively (e.g., at concentrations that exhibit low toxicity to human red blood cells) [28], [29]. These antifungal β-peptide structures can also inhibit the formation of fungal biofilms when administered in bulk solution to populations of planktonic C. albicans or when released slowly from polymer coatings containing embedded β-peptide [28], [29], [33].

In this study, we developed an approach to localize the delivery of antifungal β-peptides from the surfaces of indwelling medical devices; such approaches have the potential to prevent device-associated infection more effectively, and with fewer patient side effects, than approaches based on systemic drug administration. Past studies on surface-based strategies for the prevention or treatment of device-associated microbial infections generally fall into one of two broad categories: (i) the design of surfaces that either prevent microbial adhesion or kill adherent microbes on contact, or (ii) the design of surfaces that passively release agents that possess antimicrobial properties (controlled release approaches) [34], [35], [36], [37], [38], [39], [40], [41]. Examples of release-based approaches include coatings and surface treatments that slowly elute metal ions such as silver or copper [42], [43], [44], antibiotics and antiseptics [45], [46], [47], antimicrobial compounds [48], [49], [50], nitric oxide [51], [52], or inhibitors of bacterial quorum sensing [53], [54]. The majority of past studies in this area have focused on the inhibition of bacterial biofilms; [35], [39], [40], [42], [47], [48], [54] studies on the localized, surface-mediated release of agents that prevent fungal biofilm growth are fewer and more recent [33], [48], [50].

Here, we report new approaches to the post-fabrication loading and sustained release of a potent antifungal β-peptide from thin “polyelectrolyte multilayer” (PEM) films [40], [55], [56], [57], [58] fabricated on the inner surfaces of flexible catheter tubes. We demonstrate that incorporated β-peptide remains potent against planktonic C. albicans upon both short- and long-term incubation, and that these methods prevent or substantially reduce the formation of C. albicans biofilms on the inner surfaces of film-coated catheter tubes in in vitro assays. Finally, we show that these coatings remain potent and exhibit antifungal activity under conditions that simulate intermittent catheter use and microbial challenge for at least three weeks. Overall, our results suggest that β-peptide-loaded PEMs offer a novel and useful approach to kill C. albicans and prevent fungal biofilm formation on surfaces, with the potential to help prevent device-associated infections in indwelling catheters. The versatility of this approach also suggests potential utility in a range of other biomedical and personal care applications in which fungal infections are problematic.