Antifungal activity of farnesol incorporated in liposomes and associated with fluconazole

Highlights

• Farnesol has an antifungal activity associated with inhibition of fungal dimorphism.

• The incorporation of farnesol into liposomes significantly increased this activity.

• The association of liposomal farnesol potentiated the action of fluconazole.

Abstract

Candida infections represent a threat to human health. Candida albicans is the main causative agent of invasive candidiasis, especially in immunosuppressed patients. The emergence of resistant strains has required the development of new therapeutic strategies. In this context, the use of liposomes as drug carrier systems is a promising alternative in drug development. Thus, considering the evidence demonstrating that sesquiterpene farnesol is a bioactive compound with antifungal properties, this study evaluated the activity farnesol-containing liposomes against different Candida strains. The IC₅₀ of farnesol and its liposomal formulation was assessed in vitro using cultures of Candida albicans, Candida tropicalis, and Candida krusei. The Minimum Fungicidal Concentration (MFC) was established by subculture in solid medium. The occurrence of fungal dimorphism was analyzed using optical microscopy. The effects on antifungal resistance to fluconazole were assessed by evaluating the impact of combined therapy on the growth of Candida strains. The characterization of liposomes was carried out considering their vesicular size, polydispersion index, and zeta medium potential, in addition to electron microscopy analysis. Farnesol exerted an antifungal activity that might be associated with the inhibition of fungal dimorphism, especially in Candida albicans. The incorporation of farnesol into liposomes significantly increased its antifungal activity against C. albicans, C. tropicalis, and C. krusei. In addition, liposomal farnesol potentiated the action of fluconazole against C. albicans and C. tropicalis. On the other hand, the association of unconjugated farnesol with fluconazole resulted in antagonistic effects. In conclusion, farnesol-containing liposomes have the potential to be used in antifungal drug development. However, further research is required to investigate how the antifungal properties of farnesol are affected by the interaction with liposomes, contributing to the modulation of antifungal resistance to conventional drugs.

Introduction

While Candida species live as commensal organisms in healthy individuals, they do not cause disease. However, as opportunistic pathogens, these microorganisms can cause invasive fungal infections which represent a serious threat to human health. Especially in susceptible individuals, Candida spp. cause chronic diseases, increasing both mortality rates and costs with hospitalization (Lamoth et al., 2018; Barac et al., 2020).

Candida albicans, one of the main causative agents of fungal infections in humans, is commonly associated with severe disease (Kauffman, 2006; Zida et al., 2017), deep mycosis, and infiltrating candidiasis (Sobel, 2007; Cassone, 2015; Pfaller et al., 2019; Hendrickson et al., 2019). As dimorphic microorganisms, Candida species can change their morphology from yeast to hyphal, which significantly contributes to their ability to produce biofilms (Jacobsen et al., 2012; Mayer and Hube, 2013; Tsui et al., 2016). Also, the development of resistance to fluconazole (and many other antifungals), the mainstay of antifungal therapy (Fekkar et al., 2014), has impaired the treatment of several infectious diseases (Zomorodian et al., 2016), indicating the urgent need for new antifungal compounds (Fenner et al., 2006).

In this context, studies have identified farnesol as a bioactive compound present in the essential oils of some plant species (Weber et al., 2008). This sesquiterpene alcohol is also found as a product of the metabolism of yeasts belonging to the genus Candida (Hornby et al., 2001; Ramage et al., 2002a,b; Langford et al., 2009). Consistent evidence has demonstrated that farnesol acts as a virulence-repressing factor in Candida species (Enjalbert and Whiteway, 2005; Bandara et al., 2016) by preventing the morphological transition from yeast to hyphae (Yu et al., 2012).

Previous studies demonstrated that this compound has remarkable antimicrobial activity, with significant inhibitory effects on the pathogenicity of resistant yeasts (Dižová and Bujdáková, 2017). In this context, farnesol (300 μM) caused potent inhibition of biofilm formation by C. albicans, both directly and associated with fluconazole or 5-flucytosine (Xia et al., 2017).

The development of nanoparticles as drug carrier systems has represented an important milestone in drug development, especially in the context of chemotherapy. Studies have shown that the incorporation of drugs into carrier nanoparticles can increase bioavailability, reduce the therapeutic dose and increase the safety and efficiency of these molecules (De Jong and Borm, 2008; Shidhaye et al., 2008; Haider et al., 2020).

In this context, liposomes are highly efficient systems with a wide spectrum of clinical applications (Allen and Cullis, 2013; Johnsen and Moos, 2016). Structurally, liposomes consist of spherical vesicles formed by an internal aqueous nucleus surrounded concentrically by one or more phospholipid bilayers. These nanoparticles are notable for being biodegradable, biocompatible, and non-toxic. Besides, they are versatile, stable and compatible with the incorporation of either hydrophilic, lipophilic and amphiphilic compounds (Allen and Cullis, 2013; Johnsen and Moos, 2016).

Therefore, this study aimed to evaluate in vitro the antifungal activity of farnesol-containing liposomes associated with fluconazole against Candida strains. The study also reports the effects of this in vitro treatment on fungal dimorphism, one of the main virulence factors in the strains under investigation.