Antifungal Assay of Niosomal Amphotericin B

The antifungal assay was performed using broad-spectrum fungicidal drugs and niosomal vesicles i.e. NODNH-16 and NODNH-18 containing encapsulated amphotericin B. Standard fungicidal drugs including miconazole (GSK, Pakistan), nystatin (Wyeth, Pakistan), and amphotericin B (Abbott, Pakistan) were reconstituted at a concentration of 1500 µg/mL for the assay (Magaldi et al., 2004 (link); Colozza et al., 2012 (link); Shah et al., 2021 (link)). Briefly, overnight cultures of C. albicans ATCC 10231, C. galeberata ATCC 20001, M. guilliermondi (MW564205), and S. cerevisiae grown in Sabouraud dextrose broth (Oxoid, U.K) and cells were harvested after centrifugation at 9000 g. Cell suspension equivalent to 0.5 McFarland standard (1.5 × 10⁸ CFU/mL), was prepared in 10 mL sterile phosphate buffer saline solution (PBS). Confluent lawns were prepared onto SDA plates and allowed to dry for 5–10 min. Standard fungicide at a final concentration of 15 µg and the vesicle formulations NODNH-16 and NODNH-18, comprising 60.89% (i.e. 9.13 µg) and 68.63% (i.e. 10.29 µg) encapsulated amphotericin B respectively, was dispensed into the wells (6 mm), bored with help of a sterile cork-borer, and plates were incubated at 28 °C for 24–48 h (Magaldi et al., 2004 (link); Mady et al., 2018 (link)). For multi-cellular fungi, spore suspension (5 × 10⁵ spores/mL) was prepared in PBS from five days old cultures. Spores were inoculated with the help of a sterile swab on SDA and incubated at 25 °C for five to seven days. The antifungal activities were performed in triplicates. The results were interpreted and compared with the zones of inhibition in mm(±SD)produced by carrier vesicles and standard drugs as per CLSI (2012) guidelines (Mady et al., 2018 (link)).