Miconazole

The azole-based drugs ketoconazole, posaconazole,
voriconazole, ravuconazole, clotrimazole, miconazole, letrozole, and
anastrozole were purchased from Santa Cruz Biotechnology (Dallas,
TX), and fluconazole was from ICN Biomedicals. VNI and VFV were synthesized
by the Chemical Synthesis Core Facility (Vanderbilt Institute of Chemical
Biology).^{70 (link)} The synthesis of their analogue
LFV was described previously.^{68 (link)} The purity
of the compounds was >95%. The pyridine derivatives UDO and UDD
were
from DNDi;^{46 (link)} the tetrazole-based compounds
VT1161^{47 (link)} and VT1598^{48 (link)} were from Mycovia Pharmaceuticals (Durham, NC). Hydroxypropyl-β-cyclodextrin
(HPCD) was purchased from Cyclodextrin Technology Development (Gainesville,
FL). Q- and CM-Sepharose resins were from GE Healthcare, and Ni²⁺-nitrilotriacetate (NTA) agarose was from Qiagen. The synthesis
of the CYP51 reaction product (3β,5α)-4,4-dimethyl-cholesta-8,14,24-trien-3-ol
(dihydro-FF-MAS) was reported elsewhere.^{71 (link)} All compounds were >95% pure by high-performance liquid chromatography
(HPLC).

Biofilms were seeded on silicone elastomer sections as described above and grown in RPMI-1640 for 24 h at 37°C. The biofilms were then transferred to fresh RPMI-1640 media containing an antifungal, either; Fluconazole, Miconazole, or Nystatin at indicated concentrations. Fluconazole (Santa Cruz Biotechnology, sc-205698) was made as a 50 mg/ml stock solution in ethanol and diluted in RPMI-1640 final concentrations of 128 and 32 μg/ml. Miconazole (Santa Cruz Biotechnology, sc-205753) was made as a 50 mg/ml stock solution in DMSO and also diluted in RPMI-1640 to final concentrations of 32 and 128 μg/ml. Nystatin (Santa Cruz Biotechnology, sc-212431) was made as a 5 mg/ml stock solution in DMSO and diluted in RPMI-1640 to final concentrations of 2 and 8 μg/ml. Drug vehicle controls (0.25% ethanol for Fluconazole, 0.25% DMSO for Miconazole, and 0.15% DMSO for Nystatin) were used to ensure the solvents were not affecting biofilm growth. The biofilms matured in the RPMI-1640 media containing the select antifungal for a further 24 h at 37°C in both 0.03 and 5% CO₂ before proceeding to quantification via the XTT assay. Experiments were performed in biological and technical triplicate.

Biofilms were set up as described in the ‘In vitro biofilm growth assay’ section above and grown in RPMI-1640 for 24 h at 37 °C. The biofilms were then transferred to fresh RPMI-1640 media containing a select antifungal. Four antifungals were tested; Fluconazole, Miconazole, Nystatin, and Caspofungin. Fluconazole (Santa Cruz Biotechnology, sc-205698) was made as a 50 mg/ml stock solution in ethanol and diluted in RPMI-1640 to final concentrations ranging from 8 to 256 µg/ml. Miconazole (Santa Cruz Biotechnology, sc-205753) was made as a 50 mg/ml stock solution in DMSO and also diluted in RPMI-1640 to final concentrations ranging from 8 to 256 µg/ml. Nystatin (Santa Cruz Biotechnology, sc-212431) was made as a 5 mg/ml stock solution in DMSO and diluted in RPMI-1640 to final concentrations ranging from 1 to 8 µg/ml. Caspofungin (Sigma SML0425) was made as a 16 mg/ml stock solution in DMSO and diluted in RPMI-1640 to final concentrations ranging from 0.03125 to 1 µg/ml. Drug vehicle controls (0.5% ethanol for Fluconazole, 0.5% DMSO for Miconazole and Nystatin, 0.1% DMSO for Caspofungin) were used in all cases. The biofilms matured in the RPMI-1640 media containing the select antifungal for a further 24 h at 37 °C in both 0.03 and 5% CO₂ before proceeding to quantification via the XTT assay. Experiments were performed in biological and technical triplicate.