Improving Drug Delivery on Candida Albicans Using Geraniol Nanoemulsion

Abstract

Geraniol (GE) is a monoterpene alcohol with excellent antifungal activity. However, its low solubility and high volatility impair its use. Nanoemulsions (NE) are excellent delivery systems for poorly soluble and volatile drugs, achieving controlled release of the active ingredient. The aim of this study was to improve the delivery of geraniol (GE) incorporated in NE against Candida albicans in order to evaluate the antibiofilm effect and cytotoxicity. Nanoemulsion containing 10% oil phase (cholesterol) (w/w), 10% surfactant (mixture of soy phosphatidylcholine and Brij 58; 1:2) (w/w), and 80% aqueous phase (phosphate buffer) (w/w) was synthesized. Incorporation of GE was carried out by sonication and the final compounds were characterized by hydrodynamic diameter, polydispersity index (PDI), and zeta potential (ZP), in addition to evaluation of physicochemical stability after 6 months and 1 year. The GE-NE effect was evaluated on Candida albicans biofilms and cytotoxic effect was evaluated on immortalized normal oral cell line NOK-Si. The diameter of GE-NE was 232.3 ± 2.7 nm and PDI 0.155 with exhibited homogeneity and stability in solution. GE-NE showed antibiofilm activity at a concentration of 75 μg/mL with reduction of >6.0 log₁₀, and no cytotoxicity against NOK-Si cells at concentrations below 150 μg/mL was observed. GE-NE proved to be a promising candidate for prevention and treatment of fungal diseases.

Keywords: C. albicans; antifungal; biofilm; citotoxity; geraniol; nanoemulsion.

Figure 1

Representative images of nanoemulsion loaded with geraniol after (A) 0 h, (B) 24 h, and (C) 1 month.

Figure 2

Droplet size (A), polydispersity index (B), and zeta potential (C) of geraniol-loaded nanoemulsion at the time of formulation, and after 6 and 12 months following synthesis.

Figure 3

Images obtained by transmission electron microscopy (TEM): (A) blank nanoemulsion; (B) geraniol-loaded nanoemulsion.

Figure 4

Quantification (CFU mL⁻¹) of C. albicans biofilm after treatment: GE, GE-NE, and Nystatin. Different letters indicate statistical differences between groups (one-way ANOVA test, Tukey post test, p < 0.05).

Figure 5

(A) Representative CLSM images of C. albicans biofilm: Nystatin 640 μg/mL, geraniol 600 μg/mL, and geraniol-loaded nanoemulsion 75 μg/mL. Dead cells are stained red and live cells are stained green. (B) Results are expressed as the means ± SD of triplicate assays for three independent experiments (ANOVA/Tukey test, α = 0.05). Different letters mean statistical difference.

Figure 5

(A) Representative CLSM images of C. albicans biofilm: Nystatin 640 μg/mL, geraniol 600 μg/mL, and geraniol-loaded nanoemulsion 75 μg/mL. Dead cells are stained red and live cells are stained green. (B) Results are expressed as the means ± SD of triplicate assays for three independent experiments (ANOVA/Tukey test, α = 0.05). Different letters mean statistical difference.

Figure 6

SEM images of simple biofilms of C. albicans after treatment with geraniol and nanoemulsions (2000× and 5000× magnifications; scale bars represent 10 µm and 1 µm): (A) control; (B) biofilm treated with Nystatin 640 μg/mL; (C) biofilm treated with GE 300 μg/mL; (D) biofilm treated with GE-NE 75 μg/mL.

Figure 7

Cell viability percentage for oral keratinocytes NOK-SI cells after treatment with different concentrations of geraniol. The dotted line represents 70% viability. Equal letters represent statistical similarity between groups (one-way ANOVA test, Tukey post test, p < 0.05).