Clove Oil-Based Nanoemulsion Containing Amphotericin B as a Therapeutic Approach to Combat Fungal Infections

Abstract

Background/Objectives: Candidiasis, primarily caused by Candida albicans, and sporotrichosis, mainly caused by Sporothrix schenckii, are skin fungal infections that pose serious threats to global health. The Candida auris is a great concern in immunocompromised individuals, and while Sporothrix brasiliensis cause sporotrichosis, an infection commonly found in cats, this disease can be transmitted to humans through scratches or bites. Existing treatments for these fungal infections often cause problems related to resistance and significant side effects. Consequently, development of alternative therapeutic approaches such as nanotechnology-based topical lipid-based formulations is interesting. Thus, the objectives of this study were to prepare clove oil (CO)-in-water nanoemulsions (NEs) containing amphotericin B (AmB) and characterize them with respect to stability, release profile, and in vitro cytotoxic activity against Candida and Sporothrix strains. As a future alternative for the treatment of fungal skin diseases. Methods: Chemical analysis of clove oil was obtained by GC-MS. The NEs were produced using an ultrasound (sonicator) method with varying proportions of CO, Pluronic^® F-127, and AmB. The NEs were characterized by droplet size, morphology, stability and in vitro release profile. The antifungal and cytotoxic activity against C. albicans, C. auris, S. schenckii, and S. brasiliensis were ascertained employing agar diffusion and colorimetric MTT assay methods. A checkerboard assay was carried out using clove oil and amphotericin B against C. auris. Results: Eugenol was the major compound identified in CO at a concentration of 80.09%. AmB-loaded NEs exhibited particle sizes smaller than 50 nm and a polydispersity index below 0.25. The optimal Ne (NEMLB-05) remained stable after 150 days of storage at 4 °C. It exhibited rapid release within the first 24 h, followed by a slow and controlled release up to 96 h. NEMLB-05 more effectively inhibited C. auris compared to free AmB and also demonstrated greater activity against C. albicans, S. schenckii, and S. brasiliensis. Clove oil and amphotericin B presented synergism inhibiting the growth of C. auris. Conclusions: The selected CO-in-water NEs containing AmB demonstrated promising potential as a topical therapeutic alternative for treating fungal infections.

Keywords: amphotericin B; candidiasis; clove oil; in vitro release studies; nanoemulsion; sporotrichosis.

Figure 1

Key steps in the preparation of nanoemulsions containing amphotericin B.

Figure 2

Release of amphotericin B from the nanoemulsions.

Figure 3

Chemical components of the S. aromaticum essential oil identified by GC-MS: Phenylpropanoid eugenol, β-caryophyllene, and Chavibetol acetate.

Figure 4

Initial experimental design of the nanoformulations without drug demonstrating the effects of concentration in relation to surfactant (Pluronic^® F127) and clove oil (CO) in the particle size: surface graph (A) and Pareto chart (B); and PDI: surface graph (C) and Pareto chart (D).

Figure 5

Experimental design of the nanoformulations with amphotericin B demonstrating the effects of concentration in relation to clove oil (CO) and amphotericin B in the size: surface graph (A) and Pareto chart (B); and PDI: surface graph (C) a Pareto chart (D).

Figure 6

Average particle size (nm) of the nanoemulsions obtained by dynamic light scattering (DLS): NEMLB-06 (A), NESAF-06 (B), NEMLB-05 (C), NESAF-09 (D); NE without amphotericin B (E); NE containing amphotericin B (F) and transmission electron microscopy (TEM) image of the NE containing amphotericin B (NEMLB-05) (G).

Figure 7

Analytical curve of amphotericin B obtained using a UV–VIS spectrophotometer at 412 nm (A); amphotericin B concentrations used to construct the analytical curve and absorption spectra (B).

Figure 8

In vitro release profile of amphotericin B from the nanoemulsion (NEMLB-05) (A); graph obtained after fitting the release data with the Gompertz kinetic model using the DDSolver software (B).

Figure 9

Fungal growth inhibition in the agar diffusion test. NEMLB-05 = 0.035% of drug and 7.5% CO, NEMLB-06 = 0.025% of drug and 5% CO, NESAF-06 = 5% of CO, NESAF-09 = 7.5% CO, AmB 0.035% (drug solution) and Fluconazol = 2.5 mg/mL (standard antifungal). Bar graph with confidence interval (95%) of the means of the inhibition zones of the tested formulations, by fungal strain. The letters indicate significant differences between the formulations for each strain (p < 0.05; Tukey test).

Figure 10

Evaluation of a checkerboard assay in the 96-well microtiter plate. The two rightmost columns contain the positive growth control (pc) and negative growth control (nc) to confirm bacterial growth without CO, AMB, and medium sterility. The red circle shows the clove oil MIC and yellow circle shows the AmB MIC. The green circle indicates the combination that resulted in a synergistic effect on the C. auris strain, for which the FICI calculation should be (0.021875/1.5625) + (0.7/1.5625) = 0.462.