Co-immobilization of Liposomal amphotericin B and antimicrobial peptides to prevent multi-kingdom infections

Abstract

The drawbacks arising from microbial colonisation of indwelling devices have been well established, as evidenced by a massive body research on antimicrobial coating strategies. Most of these strategies, however, are designed to target bacterial biofilms, being fungal biofilms or mixed biofilms much less taken into account. In real-life settings, fungi will be inevitably found in consortium with bacteria, so the development of antifungal coating strategies to be combined with antibacterial approaches will be pivotal for the fight of biomaterial-associated infections (BAI). This study aimed to engineer an effective strategy for the immobilization of liposomal amphotericin B (LAmB) on polydimethylsiloxane (PDMS) surfaces to be afterwards co-immobilized with antimicrobial peptides (AMP). Immobilization was performed using a two-step mussel-inspired coating strategy, in which PDMS were first immersed in dopamine solution and its self-polymerization leads to the deposition of a thin adherent film, called polydopamine (pDA), which allowed further incorporation of LAmB and/or AMP. Surface characterization confirmed the polymerization of dopamine and further functionalization with only LAmB yielded surfaces with less roughness and more hydrophilic features. It also rendered the surfaces of PDMS with the ability to prevent the attachment of Candida albicans and kill the adherent cells, without toxicity towards mammalian cells. The decreased number of adhered cells observed on these surfaces together with the no cytotoxicity found suggested the release of AmB but not in its free formulation. It was hypothesized that liposomes were released from the surfaces to target fungi cells, being able to cross the cell wall and reach the cell membrane where a higher affinity to ergosterol than the cholesterol of liposomes will allow AmB to be free and promote fungal death. AMP immobilization alone provided PDMS surfaces with contact-killing activity towards Proteus mirabilis in the first 4 h of colonization and no leaching was observed. AMP permanent immobilization comprised, however, a disadvantage for longer periods of exposure which was attributed to the fact that dead adhered cells can mask the antimicrobial features of contactkilling surfaces, providing a surface for new colonization. Co-immobilization of both LAmB and AMP showed that LAmB retained its antifungal activity towards C. albicans, even when challenged by a multi-kingdom consortium. Although AMP immobilization requires further optimization to promote its controlled release from the surfaces, overall results highlight the remarkable antifungal and biocompatible properties obtained with LAmB immobilization. This coating strategy holds, therefore, a great potential to be combined with antibacterial agents in the development of approaches to fight BAI.