Fungal infections, frequently caused by opportunistic pathogens, are serious medical problem, especially among immunocompromised patients. These afflictions can be divided depending on the site of occurrence hence superficial, cutaneous, subcutaneous and systemic mycoses can be differentiated [1]. Chronic wounds are highly prone to fungal infections and the highest prevalence is reported for Candida species (C. albicans, C. glabrata, C. tropicalis) [2].
Fluconazole the member of azole group is one of the most frequently chosen drug for fungal infection treatment. Its activity is based on the inhibition of ergosterol biosynthesis which causes impairments in fungal plasma membranes [3].
The idea of using hydrogels loaded with drugs was reported previously [4]. However hydroxyapatite as a filler and drug carrier is a new approach proposed by our team. Hydrogel can provide gradual drug release towards the infection site and enhance wound healing by moisture retaining. Moreover, hydroxyapatite (Ca10(PO4)6(OH)2 (HAp)) naturally occurs in human body as a component of teeth and bone tissues is highly bioactive and biocompatible. It is also known for its regenerative properties and wound healing acceleration [5].
The antifungal activity of pure fluconazole was assessed using C. albicans, C. glabrata, C. tropicalis, Cryptococcus neoformans, Cryptococcus gatti, Rhodotorula mucilaginosa and Rhodotorula rubra reference and clinical isolates. The biofilm production ability (crystal violet staining) of these strains was tested at 28 and 37°C to select the most appropriate conditions for further experiments. Hydroxyapatite was synthesized by the precipitation method and heat treated at 450°C for 3 hours. Hydrogels (6-anhydro-$\alpha$-L-galacto-$\beta$-D-galactan) were prepared in four different forms: pure hydrogel, hydrogel filled with HAp, hydrogel filled with fluconazole and hydrogel filled with both HAp and fluconazole. All materials were lyophilized and physicochemical characterization was performed to assess their properties. The samples were cut into even pieces and incubated with fungal cultures to evaluate how they influence the growth and biofilm formation (confocal laser microscopy with LIVE/DEAD staining) of selected strains.
Fluconazole-loaded materials almost completely inhibited fungal growth of all tested strains, however living cells were spotted on the surface of all tested materials. It can also be seen that fluconazole additive changed the biofilm morphology increasing the cell aggregation and clusters formation whilst nanohydroxyapatite reversed that effect. That observation suggest the potential antibiofilm activity of HAp which protected from fully grown biofilm formation and suggests its beneficial effect as drug-filled hydrogels additive.