Methylene blue

The in vitro antifungal activity of the optimized fluconazole-loaded nanoemulsions and the FLZ solution (0.3% w/w) as the reference was measured via disk diffusion method using representative Candida strains, as described in Section 2.1 [39 (link)]. The studied fungi were cultured on the Sabouraud dextrose agar plates (BioMaxina, Lublin, Poland) for 24 h at 35 °C, and then, the suspension of each strain was prepared in 0.9% NaCl at a concentration of 0.5 McFarland. The suspensions of the yeasts were spread with a sterile swab on the Mueller–Hinton agar supplemented with 2% dextrose and 0.5 µg/mL methylene blue (BioMaxima, Lublin, Poland). Subsequently, 6 mm diameter paper discs (BioMaxima, Lublin, Poland) were soaked with 10 µL of the prepared fluconazole-loaded nanoemulsions, blank formulations, and the 0.3% drug solution and were placed on plates. After 24 h incubation at 35 °C, the plates were observed for an antifungal inhibition zone around the disks, which was measured using a millimeter ruler. The experiment was repeated three times.

Pluronic F-127, RPMI-1640 medium with the addition of l-glutamine and without
sodium bicarbonate, PBS (phosphate-buffered saline), propidium iodide, and amphotericin B
were purchased from Sigma-Aldrich (USA). Nile red was obtained from Santa Cruz
Biotechnology (USA). Fluconazole and verapamil hydrochloride were obtained from Pol-Aura
(Poland). Sterile, purified water was purchased from Polpharma (Poland).
3-morpholinopropanesulfonic acid (MOPS) was purchased from J&K Scientific (China).
Mueller Hinton LAB-AGAR with glucose and methylene blue were both purchased from Biomaxima
(Poland). Sabouraud dextrose agar with chloramphenicol was purchased from Liofilchem
(Italy). Sabouraud dextrose liquid medium was purchased from Oxoid (Great Britain).
Tryptic soy broth was bought from Biomaxima (Poland). Zymo Research—YeaStar RNA Kit
was obtained from TK Biotech (Poland). The primers were ordered from Genomed (Poland). The
TranScriba Kit and the RT HS-PCR Mix Probe were supplied by A&A Biotechnology
(Poland).